Tumour cells maintain a local hypoxic and acidic microenvironment which plays a crucial role in cancer progression and drug resistance. Urease is a metallohydrolases that catalyses the hydrolysis of urea into ammonia and carbon dioxide, causing an abrupt increase of pH. This enzymatic activity can be employed to target the acidic tumour microenvironment. In this study, we present the anticancer activities of urease mimetic cobalt (III) complexes on A549 cells. The cells were treated with different doses of cobalt (III) complexes to observe the cytotoxicity. The change in cellular morphology was observed using an inverted microscope. The cell death induced by these complexes was analysed through ATP proliferation, LDH release and caspase 3/7 activity. The effect of extracellular alkalinization by the cobalt (III) complexes on the efficacy of the weakly basic drug, doxorubicin (dox) was also evaluated. This combination therapy of dox with cobalt (III) complexes resulted in enhanced apoptosis in A549 cells, as evidenced by elevated caspase 3/7 activity in treated groups. The study confirms the urease mimicking anticancer activity of cobalt (III) complexes by neutralizing the tumour microenvironment. This study will motivate the applications of transition metal-based enzyme mimics in targeting the tumour microenvironment for effective anticancer treatments. The last few decades have seen an upsurge in cancer research. Many developments and milestones have been achieved in discovering compounds with effective anticancer therapeutic properties. Although a lot of such potent drugs such as doxorubicin and cisplatin were discovered several years ago and are still used clinically to treat different kinds of cancers; they are often limited by severe side effects on healthy cells ,2. RecenRecently, some researchers have focused on targeting the microenvironment surrounding tumour cells ,15,16,17Urease  is widespread in nature and is present in various forms in several bacteria, fungi, and algae . It is c2·6H2O , tris(2–aminoethyl)amine , triethylaminetetramine , and urea  were used as obtained. The complexes were prepared following literature methods Cl (III) and [Co(tren)Cl2]Cl (IV)) was measured from the culture media of the treated and untreated groups. 100 μL of the culture media was withdrawn from treated and untreated groups at fixed time intervals and the urease mimetic activity was measured using the urease activity kit from Merck Sigma-Aldrich, MAK120, following the protocol provided.The urease mimetic activity of the chloro complexes, (Cl (II) and [Co(trien)Cl2]Cl (III). The crystals were analysed by SC-XRD analyses and were found to be structurally similar to the initial compounds. The crystal structures are presented in 2]Cl was similar to the starting material, the [Co(tren)(NO2)2]Cl crystallised as a polymorph in a different space group. The most prevalent polymorph for this compound exists in orthorhombic space group (Pbca), and the only reported monoclinic form (A2) was isolated serendipitously Cl (IV) induced cleavage of urea into ammonia was reported by some of us recently. The increasing alkalinity was monitored spectrophotometrically with the aid of a pH dependent indicator, phenolphthalein Cl (IV) and [Co(trien)Cl2]Cl (III) were found to be slightly basic as compared to the other groups. This prompted us to measure the urease mimetic activity of the chloro complexes under treatment conditions. Therefore, confluent cells in culture dishes were treated with the optimised dose of the cobalt (III) complexes (32 μM) in the presence of urea (2 mM). The results were compared to cells treated with urea alone (2 mM) (untreated group) to negate any background absorbance that may arise due to the culture medium or urea. A total of 100 μL of the culture medium was withdrawn at fixed time intervals, and the urease mimetic activity was measured using the urease activity kit from Sigma Aldrich as per the protocol available with the test kit. The increase in absorbance at 670 nm at fixed time intervals for the cobalt treated groups was compared to the untreated group. The results are presented in 2]Cl (IV) and [Co(trien)Cl2]Cl (III) depicted a gradual increase in absorbance at 670 nm, and after 24 h of treatment, the absorbance, and hence the urease mimetic activity for [Co(tren)Cl2]Cl (IV) was significantly higher than [Co(trien)Cl2]Cl (III). Similar trends have been reported for catalytic studies as well [2]Cl (IV) performing better than [Co(trien)Cl2]Cl (III) for A549 cells.The ammonia generated because of urease mimetic activity in treatment groups was ascertained by pH measurements of the cell culture media after 24 h of treatment of A549 cells with the cobalt (III) complexes. Interestingly, the culture media from the cells treated with Cl (IV) and [Co(trien)Cl2]Cl (III) over 24 h  complexes for MCF7 cells were completely surprising and conflicting. There was no appreciable change in absorbance at 670 nm for the cells treated with [Co(tren)Clver 24 h . The abs2]Cl (III) and [Co(tren)Cl2]Cl (IV) complexes outperformed other Co complexes (2)2]Cl (II) and [Co(trien)(NO2)2]Cl (I) complexes exhibit poor catalytic urease activity as well, owing to the strong affinity and poor lability of the nitro group towards cobalt [The WS1 cells in the control and treated groups were seen to be healthy with no significant difference observed in growth and proliferation . The LDHomplexes A. The [Cs cobalt .2]Cl (III) and [Co(tren)Cl2]Cl (IV), while the nitro complexes were dropped out. To ascertain the urease-like anticancer activity of the cobalt complexes, the cells were treated with the complexes alone and with the addition of urea. The resulting alkalinity of urease activity has been shown to enhance the activity of weakly basic anticancer drugs, such as doxorubicin. Such drugs–enzyme combinations and conjugates are gaining increasing attention in recent times Cl (III) and [Co(tren)Cl2]Cl (IV))–urea combination as well as the combinations with doxorubicin, induced profuse toxicity in cells and reduction of ATP. These findings further support our claim that urease mimetic activity of the cobalt complexes induces anticancer effects by facilitating doxorubicin action, as observed in the LDH assay. The results are presented in A similar trend was observed with ATP of WS1 and A549 cells. No significant ATP proliferation was observed in WS1 cells depicting the safety of complexes against normal cells ; howeverThe A549 cells which were incubated with the cobalt complexes alone and in combination with urea and doxorubicin for 24 h showed distinct morphology in a concentration dependent manner compared to the control. The cells showed signs of cell death, with deformed membrane integrity, losing inter-cellular contact, vacuolation, rounding of cells and detachment from the culture plates . The toxAs observed in LDH and ATP assays, cell viability significantly reduced in A549 cells on treatment with the cobalt complexes and the urea and doxorubicin combinations . The celIII and IV) in combination with urea and doxorubicin. The luminescent signals elevated in these groups compared to control and authenticate the role of caspases in inducing cell death  complexes towards cancer cells. Such altering of the microenvironment can also be manipulated to enhance the activity of weakly basic drugs. Under acidic conditions, basic drugs, such as doxorubicin, tend to sequester in the acidic extracellular environment, thus failing to diffuse into the hydrophobic cell membrane. This often results in loss of therapeutic activity and drug resistance. In this study, we report a combination therapy of the cobalt (III) complexes with doxorubicin. The combination is proposed to be working synergistically, wherein the cobalt (III) complexes hydrolyse urea to generate ammonia, thus neutralizing the acidity in the tumour microenvironment. This subsequently, enhances the diffusion of doxorubicin into the cells, thereby, improving the overall cytotoxic activity. The cobalt complexes do not directly target the cancer cells; rather, they act on the extracellular microenvironment, disrupting the physiological factors that enable tumour growth. Thus, they exert a target specific cytotoxic action controlled by the surrounding pH. This can also be attributed to the low toxicity of the complexes towards normal cells that operate under strictly controlled pH conditions. To further confirm the mechanism of apoptotic action, caspase 3/7 expression was determined in treated groups. Even though the cell death stimulus induced by caspase 3 and 7 differs, they are yet considered universal executioner caspases ,53,54. AUrease activity was long viewed as detrimental, and research was mainly focused on inhibiting the enzyme’s activity; however, the undesirable urease activity can be utilized to neutralize the acidity in tumour microenvironments, thereby leading to enhanced therapeutic efficiency. We have presented the preliminary results of the cytotoxicity induced by urease mimetic cobalt (III) complexes on lung cancer cells. The cytotoxicity results correlate well with the observed urease mimetic activity. The complexes reported in this study are found to be nontoxic towards normal cells, making them an ideal drug candidate for treating cancer cells. To further understand the application of these complexes as anticancer drugs, their dose responses on MCF-7 and HKESC-1 were also studied. With moderate toxicity on MCF-7 but commendable action against HKESC-1 cells, it can be concluded that the active complexes (III and IV) were not cell line specific and further research is warranted to understand the reason behind these specificities. Moreover, synthetic enzyme mimics based on transition metal complexes serve several advantages over the actual enzyme; they are robust, with long shelf life, and often can greatly reduce the cost of manufacturing. The urease, like the action of the cobalt (III) complexes, also improves the therapeutic activity of weakly basic drugs, such as doxorubicin, by inducing apoptosis as seen by the expression of caspase3/7 in treated groups; however, the conflicting results obtained with MCF7 cell lines warrant further investigation using in vivo and clinical models. The concept introduced in this article is worthwhile and deserves further investigation.


ABSTRACT IMPACT: Being explicit about the prevention of falls throughout an older adults’ episode of care may further help reinforce the role of physical therapy providers in falls prevention and improve dissemination of this knowledge. OBJECTIVES/GOALS: The purpose of this study was to determine older adults’ awareness of and perspectives about the role of physical therapy providers for falls prevention and determine potential barriers and facilitators to utilization of preventive rehabilitation services METHODS/STUDY POPULATION: We used a qualitative descriptive phenomenological approach to emphasize participants’ perceptions and lived experiences. Four focus groups were conducted with 27 community-dwelling older adults (average age = 78 years). Focus groups were recorded, transcribed, condensed, and coded using thematic analysis. RESULTS/ANTICIPATED RESULTS: Surveys indicated 37% of participants experienced a fall in the last year and 26% reported suffering an injury. Four main themes and six subthemes surrounding older adults’ perceptions of physical therapy providers’ roles for falls prevention emerged: (1) Awareness of Falls Prevention ; (2) Being Able to Get Up from the Floor; (3) Limited Knowledge about the Role of Physical Therapy Providers in Falls Prevention ; and 4). Barriers to Participating in Preventive Physical Therapy Services . DISCUSSION/SIGNIFICANCE OF FINDINGS: Older adults lack awareness about the role of physical therapy services in falls prevention, perceiving services are only to treat a specific problem or after a fall. Physical therapists should be explicit about the role of physical therapy in falls prevention for all older adults undergoing rehabilitation, regardless of the reason.


Using whole-genome sequencing and combining genome sequence data derived from national surveillance databases, we identified additional emm12 GAS clones similar to those associated with prior SF outbreaks, emphasizing the need for continued surveillance for epidemic emergence in the USA.Scarlet fever (SF) has recently been associated with group A streptococcal (GAS) strains possessing multidrug resistance and specific streptococcal exotoxins. We screened a local surveillance collection of GAS  All raw genome sequences generated in this study are publicly available from the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA608028. In addition, sequences from BioProjects PRJNA471864, PRJNA395240, PRJNA416675, PRJEB2675 and PRJNA13551 were used in comparative phylogenomics analysis, as indicated in Streptococcus (GAS) was an important cause of morbidity and mortality in children during the 19th and early 20th centuries [emm type 12 (emm12) and harbour two mobile genetic elements (MGE): an integrative and conjugative element (ICE-emm12) encoding tetracycline and macrolide resistance and a prophage (ϕHKU.vir) encoding two exotoxins (SSA and SpeC) and the DNase Sda1 [emm12 SF clone [emm12 SF outbreaks have been identified in additional emm types, including emm1 GAS strains [emm12 background to enhance colonization in a mouse model [emm12 clones is warranted, given their persistence in multiple outbreaks.Epidemic scarlet fever (SF) caused by group A ase Sda1 . SubsequSF clone . MGE ass strains . Several strains . Most reemm12 isolates. Molecular typing of GAS isolates from January 2013–July 2018 was performed to determine emm type. A total 300 emm12 GAS isolates were identified and underwent antimicrobial susceptibility testing to tetracycline and erythromycin using disc diffusion [emm12 isolates (n=22) and any isolate resistant to both erythromycin and tetracycline using an Illumina MiSeq (300 bp PE). Raw sequencing reads were assembled using SPAdes and queried for the presence of exotoxin, DNase and resistance genes using a custom algorithm as previously described [emm12 strains (n=22) and previously sequenced emm12 GAS strains from the Centers for Disease Control and Prevention (CDC) Active Bacterial Core Surveillance (ABCs) , Hong Kong/PR China  and the UK   see .emm12 GAS isolates from Houston, TX screened for antimicrobial resistance, a total of 14 (5 %) were identified with resistance to erythromycin or tetracycline and a single isolate (TSPY1687 – pharyngitis from 2018) was identified to be resistant to both antibiotics. Consistent with previous reports on GAS [emm12 GAS isolates sequenced by the CDC ABCs and discovered three additional isolates with erythromycin and tetracycline resistance genes  demonstrated a close relationship of all four US isolates to groups of strains from previous SF outbreaks in Hong Kong and PR China  and tet(M), and differed by a single prophage that was devoid of any exotoxin or DNase genes  and emm1 (17.1 %) lineages [emm12 GAS strains similar to the original outbreak strains in Asia [emm12 GAS strains more closely related to those from the UK were identified in Australia [emm12 SF-like clone in the USA using our local GAS strain surveillance and shown additional clones circulating within the US population using publicly available national surveillance databases. SF is a notifiable disease in many Asian countries and the UK but not the USA. Interestingly, none of the four emm12 SF-like clones identified in our study were derived from SF cases. TSPY1687 was isolated following a case of pharyngitis in an 8-year-old child in 2018 but lacked rash or other symptoms (e.g. strawberry tongue) associated with SF. Likewise, the three CDC ABCs emm12 SF-like strains were from cases of invasive disease in 2015 but at opposite ends of the country . Unlike in our local surveillance, we are unable to further query electronic medical records for additional signs and symptoms in the CDC ABCs cases. Inasmuch as GAS whole-genome sequencing analyses have shown that invasive strains are derived from the population of pharyngeal strains [emm12 SF clones in the US population. At this time, it is unclear if more robust prospective surveillance for GAS pharyngitis would identify additional cases in the USA. Alternatively, it is also possible that the small number of emm12 SF-like clones identified in our analysis represent individual importation events from outbreak regions. Importantly, our data confirm a worldwide spread of emm12 SF clones and suggest that additional factors apart from GAS strain contribute to the development of SF. In summary, our findings emphasize the role of GAS surveillance, antimicrobial susceptibility profiling and heightened awareness of possible SF in Houston and the USA.A rapid rise in the incidence of SF in the Republic of Korea, PR China and Hong Kong marked the beginning of an epidemic in Asia. Extensive surveillance identified GAS strains of primarily emm12 76. % and em in Asia . Most reustralia . We have strains , our finClick here for additional data file.


Here, we present the results from a population study that evaluated the performance of massively parallel sequencing (MPS) of short tandem repeats (STRs) with a particular focus on DNA intelligence databasing purposes. To meet this objective, 247 randomly selected reference samples, earlier being processed with conventional capillary electrophoretic (CE) STR sizing from the Austrian National DNA Database, were reanalyzed with the PowerSeq 46Y kit (Promega). This sample set provides MPS-based population data valid for the Austrian population to increase the body of sequence-based STR variation. The study addressed forensically relevant parameters, such as concordance and backward compatibility to extant amplicon-based genotypes, sequence-based stutter ratios, and relative marker performance. Of the 22 autosomal STR loci included in the PowerSeq 46GY panel, 99.98% of the allele calls were concordant between MPS and CE. Moreover, 25 new sequence variants from 15 markers were found in the Austrian dataset that are yet undescribed in the STRSeq online catalogue and were submitted for inclusion. Despite the high degree of concordance between MPS and CE derived genotypes, our results demonstrate the need for a harmonized allele nomenclature system that is equally applicable to both technologies, but at the same time can take advantage of the increased information content of MPS. This appears to be particularly important with regard to database applications in order to prevent false exclusions due to varying allele naming based on different analysis platforms and ensures backward compatibility.The online version contains supplementary material available at 10.1007/s00414-021-02685-x. Throughout the past decades, short tandem repeat (STR) loci have become the most important genetic markers in forensics. They can be analyzed at a reasonable cost/time ratio and provide high enough statistical discrimination power to identify individuals in the majority of crime and human identification cases . TraditiHere, we evaluated the performance of an MPS-based STR-typing system consisting of the PowerSeq 46GY panel  analyzed on a MiSeq FGx sequencer  for DNA intelligence databasing purposes using a random subset of the Austrian National DNA Database as an example. The PowerSeq 46GY kit includes all loci required for national and international DNA intelligence databasing in the United States and Europe. We evaluated concordance and backward compatibility to extant CE-based genotypes, stutter display, and heterozygote balance and provide new population data to increase the body of STR variation that is currently being collected and catalogued in various environments e.g., , 10)..10]).All 248 buccal swab reference samples included in this study derived from the Austrian National DNA Database in accordance with the Austrian Data Protection regime. They were analyzed in line with Austrian legislation and with permission of the Austrian Federal Ministry of the Interior. Legal requirements with respect to sample storage  as well as the permission to go back to DNA extracts for re-testing are regulated by the Austrian Federal Security Police Act, which also contains specific legal provisions for scientific purposes to provide biometric data, including DNA data, in anonymized form to universities for research. The samples were randomly selected by executive authorities of the Austrian Federal Ministry of the Interior. The selection criteria were based on male sex, Austrian nationality, and birthplaces. The samples were made anonymous to the analyzing laboratory by using barcode information.DNA was extracted from buccal swab samples using the Chelex 100  method , and stoTo determine the amount of genomic DNA, a real-time quantitative PCR (qPCR) assay targeting specific AluYb8 sequences was used . A spikeSTR analysis was performed using the AmpFlSTR NGM SElect Express kit (TFS)  and the The PowerSeq 46GY kit (Promega) was used to co-amplify 22 aSTRs , 23 Y-STRs (data not shown), and amelogenin. This extended STR panel aims to target forensic markers to comply with the European Standard Set (ESS)  deletion Supplementary file2 (PDF 134 KB)Supplementary file3 (PDF 96 KB)Supplementary file4 (PDF 175 KB)Supplementary file5 (PDF 174 KB)Supplementary file6 (PDF 138 KB)Supplementary file7 (PDF 122 KB)Supplementary file8 (XLSX 1015 KB)Below is the link to the electronic supplementary material.


Tuberous sclerosis, angiomyolipoma and lymphangioleiomyomatosis are a group of diseases characterized by mutation in tuberous sclerosis genes (TSC 1-2). TSC mutation leads to continuous activation of the mTOR pathway that requires adaptation to increased ATP requirement. With limited treatment options, there is an increasing demand to identify novel therapeutic targets and to understand the correlations between mTOR pathway activation and the lack of cell death in the presence of TSC mutation. In the current study, we demonstrate deregulation of p53 controlled and mitochondria associated cell death processes. The study also reveals that treatment of TSC mutant cells with the drug candidate Proxison combined with reduced concentration of rapamycin can increase production of reactive oxygen species (ROS), can modify miRNA expression pattern associated with p53 regulation and can reduce cell viability.The online version contains supplementary material available at 10.1007/s10495-021-01670-4. RecentlIn a previous study of lymphangioleiomyomatosis (LAM)  TSC mutaTSC mutations are particularly disruptive in the phosphoinositide 3 kinase (PI3K)/Protein kinase B (PKB or AKT)/mTOR pathway, as the mTOR pathway is inhibited by the complex of two proteins Hamartin (TSC1) and Tuberin (TSC2). Mutation in the TSC genes allows continuous activation of mTOR resulting in slow growing neoplasms . The mTOBased on the above we theorized that investigation and modulation of p53 and mitochondrial function associated cell death pathways might provide a better understanding of the molecular background of TSC mutation induced diseases and the results might lead to identification of additional targets for therapy.Materials and methods are detailed in the Supplementary material . Briefly, LAM tissue samples were obtained from lung transplant donors for generation of cell lines . ParaffimTOR is downstream of the PI3K and AKT enzymes that transduce signals from growth factor activated receptor kinases . To inveTo understand the significance of the results, a linear regression-based prediction algorithm analysis  was usedTo confirm that deregulation of the pro- and anti-apoptotic pathways are not a unique feature of the AML derived S102 cell line, 4 primary LAM tissue derived cell lines  in TSC2 mutant cell lines revealed a distortion in the pro- and anti-apoptotic balance. The cell death processes are blocked in TSC mutant cells despite p53 activation  combined with the drug candidate Proxison (3 µM) can result in cell death. Although further studies are required, combined inhibition of the mTOR pathway and TRXR can potentially lead to a more effective therapy.Supplementary file1 (DOCX 7450 kb)Below is the link to the electronic supplementary material.


The purpose of this study was to generate a selective radiosensitising effect by the intra-hepatic-arterial infusion of misonidazole (MISO). MISO (10 mg) was infused after transcatheter hepatic-arterial embolisation into the livers of rabbits bearing VX2 liver cancer. This procedure was followed by 15 Gy electron irradiation. Evaluation of tumour volume and histological examination was carried out on the 7th day after treatment. The greatest tumour response was obtained in the group which received MISO followed by radiation and was characterised by extensive fibrosis around the tumour and nearly complete tumour necrosis. Liver cell regeneration was also noted in adjacent liver tissue. The advantages of regional infusion of MISO following hepatic-arterial embolisation are: (1) Selectivity increased radiosensitivity of liver cancer alongside very low drug concentration in the plasma. (2) Reduced or absent deleterious side effects of MISO with higher tumour/normal tissue ratios of drug concentration. (3) Reduced cost due to the lower dosage of MISO required for regional infusion.


ABCB4 mutations and low biliary phospholipid concentration with symptomatic and recurring cholelithiasis. This syndrome is infrequent and corresponds to a peculiar small subgroup of patients with symptomatic gallstone disease. The patients with the LPAC syndrome present typically with the following main features: age less than 40 years at onset of symptoms, recurrence of biliary symptoms after cholecystectomy, intrahepatic hyperechoic foci or sludge or microlithiasis along the biliary tree. Defect in ABCB4 function causes the production of bile with low phospholipid content, increased lithogenicity and high detergent properties leading to bile duct luminal membrane injuries and resulting in cholestasis with increased serum gamma-glutamyltransferase (GGT) activity. Intrahepatic gallstones may be evidenced by ultrasonography (US), computing tomography (CT) abdominal scan or magnetic resonance cholangiopancreatography, intrahepatic hyperechogenic foci along the biliary tree may be evidenced by US, and hepatic bile composition (phospholipids) may be determined by duodenoscopy. In all cases where the ABCB4 genotyping confirms the diagnosis of LPAC syndrome in young adults, long-term curative or prophylactic therapy with ursodeoxycholic acid (UDCA) should be initiated early to prevent the occurrence or recurrence of the syndrome and its complications. Cholecystectomy is indicated in the case of symptomatic gallstones. Biliary drainage or partial hepatectomy may be indicated in the case of symptomatic intrahepatic bile duct dilatations filled with gallstones. Patients with end-stage liver disease may be candidates for liver transplantation.Low phospholipid-associated cholelithiasis (LPAC) is characterized by the association of  Low phospholipid associated cholelithiasisABCB4 gene mutationCholelithiasis with ABCB4 gene mutation-associated cholelithiasisABCB4 mutations with symptomatic and recurring cholelithiasis in young adults (e.g. <40 years).Low phospholipid-associated cholelithiasis (LPAC) is characterized by the association of ABCB4 mutations in the whole population of patients with cholelithiasis.The exact prevalence of LPAC remains unknown. The disease is more common in young adults, the usual age at the onset of the symptoms is typically lower than 40 years. The male to female ratio is estimated at approximately 1:3. However, at present, the literature data and our results did not allow to determine the frequency of Up to 10% of the European and American population carry gallstones, approximately 25% of cases have symptoms and less than 2% present with severe complications (cholangitis or pancreatitis). Based on previous epidemiological data, we consider that LPAC syndrome is infrequent and corresponds to a peculiar subgroup of patients with symptomatic gallstone disease [LPAC presents as a peculiar form of cholelithiasis characterized by cholecystitis, cholangitis and intrahepatic gallstone disease, and/or acute pancreatitis associated with biliary microlithiasis. Most patients report a history of cholesterol gallstones amongst their first-degree relatives. Intrahepatic hyperechoic foci are characteristic sign of the LPAC syndrome. Intrahepatic gallstones or sludge and increased serum gamma-glutamyl transferase (GGT) activity are frequently present. It suggests that the biliary symptoms experienced by these patients are probably caused by cholesterol crystal deposits and bile duct inflammation but not directly and exclusively related to the presence of detectable gallstones. Increased cholesterol saturation index and a defect in the hepatic transport and biliary secretion of phospholipids in patients with an intrahepatic cholesterol gallstone disease or an acute pancreatitis associated with biliary microlithiasis have been shown in another studies ,4.ABCB4 mutations presented also with an history of intrahepatic cholestasis of pregnancy and 14% had fetal complications) [The typical biliary pain in patients with LPAC syndrome  often leads to decision of cholecystectomy. The majority of patients is our series (90%) underwent cholecystectomy but recurrence of the symptoms was observed in half of them, despite cholecystectomy . The onscations) . Prophylcations) . Of noteMDR3 (now referred to as ABCB4) gene, the phosphatidylcholine translocator across the canalicular membrane, are thought to be responsible for progressive intrahepatic cholestasis type 3 (PFIC 3) [Phospholipids are the major carrier and solvent of cholesterol and they exert a protective effect against bile salt-induced biliary mucosa injury. Homozygous and heterozygous mutations in the (PFIC 3) , and het(PFIC 3) -11. DefeABCB4 gene mutations in 60 consecutive adult patients who had been referred to our liver unit because of symptomatic or complicated cholelithiasis and we characterized more precisely the clinical phenotype associated with these mutations [ABCB4 gene analysis because of clinical history compatible with the syndrome that we previously described . Multivariate analysis showed that among patients with cholelithiasis three independent factors were predictive of a mutation at the ABCB4 locus: a recurrence of symptoms after cholecystectomy (adjusted OR = 8.5), intrahepatic hyperchoic material (adjusted OR = 6.1), and age <40 years (adjusted OR = 3.0) [Among the 32 patients suspected of having the syndrome, 18  presented a point mutation at the Finally, more recent data shows that the LPAC syndrome is more frequent in females and that biliary symptoms occur earlier in female who present a nonsense mutation .ABCB4 gene using polymerase chain reaction (PCR) amplification and DNA sequencing of exons 2 to 28 and all splice junctions. We identified 14 heterozygous and homozygous point mutations amongst these 18 patients. None of these mutations was detected in a control panel of 140 chromosomes demonstrating that they did not correspond to polymorphisms (SNPs). Affected patients with heterozygous mutations exhibited 1bp-insertion, 1bp-deletion, nonsense mutations or missense mutations resulting in a frameshift predicted to cause premature messenger RNA termination, a loss of protein function and single-nucleotide substitutions, while affected patients with homozygous mutations demonstrated only missense mutations. Most mutations were localized in the central part of the molecule, close to nucleotide binding domain 1 (NBD1), or in adjacent transmembrane domains and intracellular loops .Patients were screened for mutations in the st intracellular loop and resulted in a substitution in a conserved cluster of four amino-acids at position 169–172, required for the adenosine triphosphatase activity of the molecule. This mutant resulted in a complete loss of drug-induced P-gp ATPase activity. The Phe165Ile mutation was localized in the same part of this intracellular loop and may therefore give rise to a similar defect. The Glu528Asp mutation was localized close to the NBD1. The glutamic acid is included in a small amino-acid cluster localized before the walker B motif which is required for drug-induced ATPase activity. These mutations indeed induced a loss of function of the molecule in vitro. It should be noted that the two unrelated patients in whom the Thr175Ala mutation was identified originated from Northern Europe, so they may have inherited a founder mutation from a shared ancestor. This mutation has also been recently identified in adults with cholangiopathy and intrahepatic cholestasis of pregnancy [The definition of a disease-causing mutation (DCM) is problematic when functional assays to determine the phenotypic effects of specific variants are not available or have not been performed. To date, two of the missense mutations (Glu528Asp and Thr175Ala) detected in patients with the LPAC syndrome had been analyzed previously in a homolog of ABCB4  in yeast ,14. The regnancy . The mutregnancy .ABCB4 sequence alterations leading to predicted premature truncation of the protein, small deletions or insertions and non-conserved missense mutations were considered as potential DCMs. Even if this restriction was taken into account, a certain number of arguments strongly supported a pathogenic role for ABCB4 gene mutations. These mutations are indeed detected at high frequency in patients with LPAC syndrome; they affect only highly conserved amino acids between human and rodent homologues of the gene and no mutation was detected in an independent control panel of 140 chromosomes. In addition, LPAC syndrome was observed in patients exhibiting the same or similar nonsense or missense mutations: in a mother and her elder son with an identical heterozygous nonsense mutation (1327insA); in independent patients from non-consanguineous families with the same homozygous missense mutation (Ser320Phe or Ala934Thr), while their heterozygous parents were asymptomatic; in patients with a similar nonsense mutation (1006-1016insT and 1006-1016delT) and in unrelated patients with the same missense mutations (Pro1161Ser). For all these reasons, these mutations could be considered as causing the LPAC syndrome.Only ABCB4 mutations may lead to cholesterol cholelithiasis when residual ABCB4 activity and subsequent biliary phospholipid secretion fall below a critical threshold which may depend on the type of mutation and on other host or environmental factors, including probably bacterial infection of the bile ducts .These results and data in the literature therefore strongly suggest that ABCB4 gene point mutation, defects in the promoter region or large DNA rearrangements might also be involved in some patients presenting with LPAC syndrome. Alternatively, defects in other regions of the gene or in other genes may also be involved, and some evidence from animal studies has pointed to Abcb 11 , Abcc 2  or Abcg5/Abcg8 as other possible candidate genes underlying susceptibility to cholelithiasis [The mutation screening method used in our two studies was unable to detect major DNA rearrangements, and nor did the analysis include the promoter or other potential regulation regions of the gene. As 44% of LPAC patients did not present ithiasis -19 or phithiasis .in vitro to enhance cholesterol crystallization. This mechanism explains the early and higher prevalence of gallstones in female Mdr2 -/- mice and provide a rationale for substitution of more hydrophobic bile salts with ursodeoxycholic acid in LPAC patients [It has also been shown that substantial amounts of cholesterol are secreted by Mdr2 (-/-) mice on a cholate-supplemented diet and microscopic examination of the gallbladder reveals massive amount of cholesterol crystals and that after 12 weeks, 50% of the Mdr2 (-/-) mice on chow also developed gallstones composed of needlike "anhydrous" cholesterol crystals and mucin ,22. Indepatients .ABCB4 gene mutations and sequence diversity in 34 consecutive patients with primary sclerosing cholangitis. We found no ABCB4 gene mutations and no abnormal SNP frequency in these unselected PSC patients [ABCB4 from that observed in healthy Caucasian controls [ABCB4 genetic variation in the pathogenesis of the PSC, although an implication of ABCB4 cannot be ruled out in a subgroup of these patients.As a recent study reported that Mdr2 KO mice develop liver lesions mimicking sclerosing cholangitis characterized by biliary strictures and dilatations , we alsopatients . These dcontrols . Thus, tCholesterol cholelithiasis was also demonstrated in patients with progressive familial intrahepatic cholestasis-3 (PFIC3) and some of their relatives ,26, and etc.) should remind of this rare monogenic form of cholelithiasis (e.g. LPAC syndrome) for which genetic testing may be offered by several genetic laboratories.In patients with symptomatic cholelithiasis, the presence of additional hepatobiliary manifestations , computing tomography (CT) abdominal scan or magnetic resonance cholangiopancreatography;• Intrahepatic hyperechogenic foci along the biliary tree by US;• Hepatic bile composition ;ABCB4 gene analysis.• Other inflammatory bile duct diseases which may lead to biliary symptoms and/or intrahepatic gallstones .ABCB4 genotyping should be used to confirm the diagnosis of LPAC syndrome in young adults who present with a symptomatic cholelithiasis and should allow familial screening.ABCB4 genotyping confirms the diagnosis of LPAC syndrome in young adults, long-term curative or prophylactic therapy with UDCA should be initiated early to prevent the occurrence or recurrence of the syndrome and its complications. UDCA was shown to up-regulate the expression of the protein at the canalicular membrane, minimize the toxicity of the endogenous hydrophobic bile acids and increase the pool of protective hydrophilic bile acids.Special diet is not required. In all cases where the In the case of associated hypercholesterolemia, statins should be preferred to fibrates which increase the lithogenicity of the bile.Cholecystectomy is indicated in the case of symptomatic gallstones but not when only sludge is present in the gallbladder.Biliary drainage or partial hepatectomy may be indicated in the case of symptomatic intrahepatic bile duct dilatation filled with gallstones.Patients with end-stage liver disease may be candidates for liver transplantation.Drugs that may induce ABCB4 expression.Typically, the clinical course is progressive, with multiple recurrence despite the operative intervention(s) until UDCA treatment is started.The majority of patients with LPAC syndrome described in the literature do not develop recurrence or end-stage liver disease under medical treatment.ABCB4 gene defect in LPAC syndrome and replace it in the context of ABCB4 gene-associated liver diseases in adults including attenuated form of PFIC 3, "atypical intrahepatic cholestasis of pregnancy", LPAC syndrome which might lead to adulthood biliary cirrhosis or bile duct dilatations associated with gallstones. A genetic test based on the present results may permit the molecular diagnosis of LPAC syndrome and the screening of high-risk subjects. Depending on the results, long-term curative or prophylactic UDCA therapy may be initiated early to prevent the occurrence or recurrence of this syndrome and its severe complications.In summary, our results strongly support the role of an


Exogenous purines M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations  M) and markedly potentiating its effect at higher concentrations. The ability of hypoxanthine (HX) to modulate the effects of two antifolates-ICI 198583 (an inhibitor of thymidylate synthetase) and piritrexim -was investigated using cultured mouse leukaemic cells, L1210. HX (10(-4) M) was found to potentiate only the cytotoxicity of DHFR inhibitors (MTS and PTX), increasing cell kill by 20-70 fold to the level achieved by an equivalent concentration (10(-5) M) of ICI 198583 alone. Agarose gel electrophoresis of DNA extracted from cells exposed to antifolates for 24 h demonstrated that the chromatin was cleaved into multimers of 200 base pairs. This pattern of DNA cleavage indicates cell death via apoptosis. The degree of DNA fragmentation was found to be closely linked to cytotoxicity. DNA fragmentation increased from 50% in cells treated with 10(-5) M MTX or PTX to 70% when HX was added with the drugs, a level achieved by 10(-5)M ICI 198583 alone. HX potentiation of cytotoxicity was correlated with a substantial increase in dATP in conjunction with low dTTP pools. The specific potentiation of DHFR inhibitors by HX may be due to their inhibition of purine synthesis with a concurrent rise in PRPP levels. Addition of HX with MTX substantially raised intracellular purine levels via the salvage pathway as indicated by ribonucleotide pool measurements. ICI 198583, on the other hand, stimulated de novo purine synthesis with or without added HX. Treatment with MTX plus HX or ICI 198583 (with or without HX) caused a reduction of dTTP pools to 8% of untreated control and excess dATP accumulation. The subsequent elevation (to 300% of control) of the dATP pool may provide a signal for endonucleolytic fragmentation of DNA and subsequent cell death.


Eighty-four patients with breast cancer at high risk of bone metastases were investigated with magnetic resonance imaging (MRI) of the thoracolumbar spine. Of 58 patients with normal limited skeletal surveys (LSS) and bone scans (BS), 4 (7%) had MR images compatible with malignant infiltration. Fourteen patients had abnormal bone scans with normal or non-diagnostic plain films; 7 of these patients (50%) had MR images compatible with malignant infiltration. Twelve patients had single or multiple wedge collapses of uncertain aetiology on plain film; MR demonstrated metastatic disease as the cause of wedge collapse in 7 (58%). MRI may define a group of patients with extra-osseous relapse who have occult metastatic disease. Although the detection rate in patients with primary breast cancer is low (4/45), MRI is of value in determining the cause of wedge collapse in postmenopausal women with breast cancer and may elucidate the cause of an abnormal bone scan with normal or non-diagnostic plain films.


Between January 1991 and December 1992 a phase I trial of superficial photodynamic therapy (PDT) using topical application of 5-aminolaevulinic acid (ALA) was undertaken to treat Bowen's disease, superficial basal cell carcinomas (BCCs) and metastatic skin secondaries from breast (adenocarcinoma) or pinna (squamous cell carcinoma). Promising results were obtained with 36 areas of Bowen's disease, with a complete response rate of 89% at a median follow-up of 18 months. The treatment of BCCs was less successful, with 50% complete responses in 16 lesions at a median follow-up of 17 months. Metastatic nodules responded poorly. The treatment was well tolerated and discomfort during light irradiation could be reduced by prior application of 'Emla' cream. Lesions wept for 1-2 weeks following treatment and healed over a period of approximately 2 months. For large areas of Bowen's disease, particularly in anatomically difficult areas and in elderly patients, PDT using ALA may constitute a single simple alternative outpatient treatment to existing therapies. Further work is required to improve the results with BCCs.


The oncogenicity of Duck hepatitis B virus (DHBV) is unclear since hepatocellular carcinomas (HCCs) have been reported only in domestic ducks in Qidong, an area of China where hepatitis B virus (HBV) and aflatoxin B1 (AFB1) are risk factors for liver cancer in man. In order to better define the association between DHBV infection, AFB1 and HCC we analysed a series of 16 duck liver samples collected from local farms in Qidong. HCC was found in eight and cirrhosis in one of these samples. Furthermore bile duct proliferation, characteristic of AFB1 exposure in ducks and other animal species, was found in these ducks. Integration of DHBV DNA into cellular DNA was observed in only one out of four DHBV positive HCCs, indicating that viral integration is not prerequisite for tumour development. In four remaining HCCs the polymerase chain reaction (PCR) failed to show any DHBV DNA suggesting that liver tumours do occur in polymerase chain reaction (PCR) failed to show any DHBV DNA suggesting that liver tumours do occur in these ducks in the absence of DHBV infection. In addition, AFB1-DNA adducts were detected by hplc-immunoassay in one such DHBV-negative tumour. In summary we demonstrate that risk factors other than DHBV, including AFB1 exposure, may be important in duck liver carcinogenesis in Qidong.


Electrophoretic isoenzyme separation provides much more precise information than measurement of alkaline phosphatases (AP). Use of this technique for 83 patients with Hodgkin's disease revealed that the presence of the alpha 1 fraction  was very significantly correlated with the stage of disease extension (P less than 0.01) and above all with the presence of general symptoms (P less than 0.001). Repeat measurements performed during patient follow-up demonstrated a close association between presence of alpha 1 AP and existence of progressive disease. While the mechanism of appearance of this abnormal alpha 1 AP fraction is not linked to Hodgkin-specific liver lesions, this test provides much more interesting data than classical measurement of total alkaline phosphatases (TAP).


For the exceptional self-renewal capacity, regulated cell proliferation and differential potential to a wide variety of cell types, the stem cells must maintain the intact genome. The cells under continuous exogenous and endogenous genotoxic stress accumulate DNA errors, drive proliferative expansion and transform into cancer stem cells with a heterogeneous population of tumor cells. These cells are a common phenomenon for the hematological malignancies and solid tumors. In response to DNA damage, the complex cellular mechanisms including cell cycle arrest, transcription induction and DNA repair are activated. The cells when exposed to cytotoxic agents, the apoptosis lead to cell death. However, the absence of repair machinery makes the cells resistant to tumor sensitizing agents and result in malignant transformation. Mismatch repair gene defects are recently identified in hematopoietic malignancies, leukemia and lymphoma cell lines. This review emphasizes the importance of MMR systems in maintaining the stem cell functioning and its therapeutic implications in the eradication of cancer stem cells and differentiated tumor cells as well. The understanding of the biological functions of mismatch repair in the stem cells and its malignant counterparts could help in developing an effective novel therapies leaving residual non-tumorigenic population of cells resulting in potential cancer cures. Gurdon first introduced the concept of stemness state of the cells while successfully achieving the generation of all cell lineages of a living tadpole after transferring the nuclei from intestinal epithelial cells of feeding Xenopus tadpoles to activated enucleated eggs ,2. TheseThe multipotency of stem cells to maintain tissue homeostasis and its differentiation into mature cell types is under a tightly controlled system and is associated with restricted expression profile. The expression of transport proteins – ABC (ATP-binding cassette) transporter proteins and multidrug resistant proteins that protect cells against toxins and are associated with the efflux of xenobiotic toxins, low rate of cell division and active DNA repair are the innate properties of normal development of stem cells. Besides hormonal stimulation, DNA damage is one of the key factors for stem cell activation. DNA accumulates errors either from environmental factors, which could be the exposure to radiation, chemicals or drugs, viruses and bacteria or DNA replication errors . This woCancer stem cells (CSCs) were first identified in 1990s in hematological malignancies, mainly acute myelogenous leukemia (AML) and also in other subtypes like AML M0, M1, M2, M4 and M5 and chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) and multiple myeloma . CSCs arIn the hematopoietic and solid tumors side population (SP) containing CSCs constitute the fraction of total population of cells that shows that CSCs is a common phenomenon for all the malignancies. SP can be identified by the efflux of fluorescent dyes by ABC transporter proteins which are the major multidrug resistant genes. In the absence of DNA repair capacity, the drug resistant pluoripotent cells accumulate mutations and show increased cell survival .To maintain the genomic integrity and normal functioning of stem cells, several DNA repair pathways come into interplay and provide the robust defense to the cell. Absence of these repair processes have a great impact on the diminution of stem cells and increased chance for stem cell differentiation and malignant transformation due to altered gene expression.AGT repair is a DNA repair protein encoded by MGMT (O6-methylguanine DNA methyltransferase) removes DNA adducts from O6 methylguanine. The failure in the repair system results in G to A transition and DNA strand break observed in hematopoietic tumors, breast, ovarian and retinoblastomas ,14.In NER, the damaged DNA bases are recognized by XPC (xeroderma pigmentosum complementation group C) and RAD23B whereas XPA, RPA (replication protein A), RNA polymerase II transcription factor 11A and XPG excise the fragment of 27–30 nucleotides surrounding the damaged bases. The gap is further restored by DNA polymerases and ligases and replaces it with the correct sequence. Defective NER leads to xeroderma pigmentosum (skin cancer) .It comprises of short and long patch pathways where DNA glycosylases generate apurinic/apyrimidinic (AP) followed by its 5' excision by AP endonucleases (APE-1) and remove damaged bases. Double-stranded breaks have the potential to be the most disruptive form of DNA damage ,17.Double-stranded breaks have the potential to be the most disruptive form of DNA damage. The DNA double strand breaks (DSBs) produced by free radicals generated during oxidative respiration, ionizing radiation, DNA replication, malfunction of recombination activating gene complex during V(D)J  recombination in T and B lymphocytes are brought in closed proximity by Ku70-Ku80 heterodimer and kinase activity by DNA dependent protein kinase catalytic subunit (DNA-PKcs) of DNA-PK complex followed by its ligation by XRCC4-DNA ligase IV, SCID (Severe Combined Immunodeficiency) is associated with defective NHEJ repair system .The DSBs are repaired by misalignment, deletions, and rearrangement. ATM, a PI-3 Kinase binds DNA and phosphorylates multiple proteins. BRCA1 activated by ATM (ataxia telangiectasia) facilitates BRCA2 and RAD51 binding of the overhang followed by the attraction of RAD52/RAD54 with the help of BLM/WRN proteins. The homologous recombination is facilitated by large protein complexes at the DSBs. High incidence of leukemia, breast-ovarian, Werner's and Bloom's syndrome with severe premature aging and cancer is seen in defective HR repair system ,19.It is a genome surveillance system in bacteria, yeast and mammals by maintaining genomic integrity. The MMR pathway is the form of DNA repair responsible for the elimination of specific mismatched and/or unmatched bases and insertion-deletion loops (IDLs) where DNA synthesis is liable to errors, either as a part of DNA replication before cell division, or as part of DNA repair (unscheduled DNA synthesis) . AlthougMismatch in eukaryotic DNA is recognized by two heterodimeric complexes of Mut S related proteins – MSH2/GTBP (Mut Sα) and MSH2/MSH3 (Mut Sβ) . Mut Sα Proliferating cell nuclear antigen (PCNA) has been identified as DNA polymerase processivity factor and has a role in repair at or prior to excision steps as a strand discriminating factor . Bi – diDNA exonuclease and helicase unwind, nick and degrade the error – containing strand. After dissociation of MMR complex, PCNA, which still binds at the end of error containing primer strand, recruits replication complex and thus re-initiation starts with the help of DNA polymerase.The mutations characterized by point mutations, insertions or deletions in the length of DNA microsatellite repeat sequences throughout the genome resulting in microsatellite instability (MSI) is a hallmark of defective DNA MMR system which result in malignant transformation .The maintenance of MMR system decreases the error rates by 100 to 1000 fold during DNA replication. Since it has tremendous importance in the carcinogenesis, this review presents a discussion on the role of DNA mismatch repair system as a therapeutic potential in eliminating the cancer stem cells.The maintenance and longevity of stem cell phenotype is characterized by the presence of efficient MMR system, which result in accurate DNA replication, restore normal DNA after its damage and remove replication defects at microsatellite sequences by repairing the DNA polymerase slippage products.-/- and Mlh2-/- phenotype, mice develop stem cell-derived-lymphoid malignancies and all lymphoid tumors show MSI [-/- primary murine hematopoietic cells with wild type into lethally irradiated mice with temozolomide, a methylating agent. MMR deficiency as evident by MSI was observed in early progenitor colony forming unit (CFU) and Sca+Kit+Lin- derived clones, which explain the alteration in growth and survival of hematopoietic stem cells and its long-term repopulation capacity because of accumulation of genomic instability [-/-, Msh2+/+, Msh2-/+ mouse embryonic stem cells following prolonged low level radiation treatment. The cells deficient in active MMR system survive promutagenic genomic insults by alkylating and oxidating agents that contribute to neoplastic transformation as in hereditary non-polyposis colorectal cancer (HNPCC) [The MMR deficiencies because of the mutations in wild type MMR allele with Msh2show MSI . The sertability . In anot (HNPCC) . The six (HNPCC) .The loss of MMR genes directly or indirectly alters signal pathways, loss of strictly regulated expression of cytokine receptors, transcription factors and cell cycle regulation . Unlike The mitotic homologous recombination can result in chromosomal translocations, deletions or inversions with deleterious consequences in the adult or during development. Loss of MMR adds up an additional level of genetic instability in a form of chromosomal rearrangements commonly observed in hematological malignancies. High incidence of lymphoma is reported in Msh2 deficient mouse embryonic stem cells .-/- mouse ES cells after exposure to temozolomide but not cisplatin and contributed to failed repair mechanism and apoptosis in a p53 dependent manner [The Msh2 low and deficient mouse ES cells showed poor or complete elimination of repair function and demonstrated the resistance against the toxic effects of an ethylating agent, N-ethyl-N-nitrosourea (ENU). The ENU-induced mutagenicity under the fully or partially deficient conditions accelerated the lymphomagenesis . Toft ett manner .-/- mouse embryonic stem cells showed two-fold resistance to the cytotoxic effects of cisplatin as compared to wild type cells [In a study by Fink et al., Msh2pe cells . On the pe cells . Howeverpe cells , increasIn most of the malignancies CSCs constitute less than 1% of total cell population. They are in quiescent state and most of the conventional chemotherapeutic drug therapies are effective in actively dividing cells, so for complete eradication of CSCs, the therapeutic agents either should kill cells independently of the cell cycle or selectively induce cycling of cancer cells. Moreover, therapy should target pathways uniquely used by CSCs. CSCs express various drug transporter proteins which efflux the cytotoxic drugs and protect them from damage. The inhibitors to these transporters make the CSCs sensitive to chemotherapy. Since normal stem cells also express some of these transporters so non selective inhibition of these multidrug resistant genes also kill normal stem cells. The characterization of stem cells markers would help in identification of CSCs from normal stem cells have important implications in elimination of CSCs by specifically targeting the tumor cells . Table 2The wild type MMR expression blocks the homologous recombination between the diverged DNA sequences throughout the genome and thus prevent the stem cell to accumulate further genetic instability. Hematological malignancies and lymphoma arise due to chromosomal rearrangements because of loss of MMR proteins . The effMost of the human cancers are identified having mismatch repair deficiency that result in defect in cell cycle and altered growth as in case of hematological malignancies. The defective MMR status in a damaged cell directly contributes to the resistant phenotype against the cytotoxic effects of chemotherapeutic drugs and thus these CSCs survive chemotherapy Fig . The actThe maintenance of genomic stability is the prime requirement for the stem cell phenotype and its normal functioning. The increased mutation rate and absence of MMR may give rise to stem cell failure, a proliferative advantage and cancer stem cells formation. The cancer stem cells having MMR deficiencies make them insensitive to the treatments against the cytotoxic agents and would increase the risk of relapse and metastasis. The importance of MMR in designing the therapeutic strategies specifically targeting the tumor cells is being explored and pursued in the clinical trials.The author(s) declare that they have no competing interests.The manuscript was written and finalized by the author.


Ploidy and cell proliferation determined by flow cytometry were assessed on colorectal cancers from patients admitted to two Italian cancer research centres. A total of 181 patients were followed prospectively for 4 years at the Istituto Regina Elena (IRE) of Rome and at the Istituto Nazionale Tumori (INT) of Milan. Fresh (at the IRE) or frozen (at the INT) tumour material and similar procedures were used for subsequent sample preparation. Similar frequencies of aneuploid tumours (63% vs 66%) and superimposable median DNA indices (1.6) were observed for the two case series. In both series, DNA ploidy was generally unrelated to clinico-pathological factors, except for a higher frequency of aneuploid tumours in Dukes' D (88%) than in Dukes' A stage (33%) in the IRE experience. DNA ploidy was a weak prognostic indicator at 3 years but not at 4 years in the IRE case series, and it never exhibited a clinical relevance in the INT experience. Conversely, multiploidy was an indicator of worse relapse-free and overall survival at 4 years in the IRE and INT case series.


Some environmental factors of possible aetiological importance for primary liver carcinoma (PLC) in males were analysed in a case-control study including 83 cases of hepatocellular carcinoma (HCC), 15 cases of intrahepatic cholangiocellular carcinoma (CC), 3 cases of haemangiosarcoma and 1 case of unspecified sarcoma in the liver--102 cases in total. Two matched controls were used in each case. One case with haemangiosarcoma was exposed to polyvinyl chloride. The case with unspecified soft-tissue sarcoma was exposed to phenoxy acids. A 4-fold increase in the risk of HCC was seen in alcoholics, and regular drinking gave a 3-fold increase in the risk. Exposure to organic solvents gave a 2-fold increase in the risk of HCC. No increased risk was observed for cases exposed to various other chemicals. Three cases of HCC had a previous diagnosis of porphyria acuta intermittens (PAI), versus no control. Six cases of HCC had a previous diagnosis of porphyria acuta intermittens (PAI), versus no control. Six cases with PLC had polyphyria cutanea tarda (PCT) which in 4 cases was related to alcoholism and in one case to haemochromatosis.


Cervical intraepithelial neoplasia (CIN) grades I to III lesions (n = 94) and squamous cell carcinomas of the uterine cervix (n = 27) were analysed for MHC class I and II expression and presence of HPV genotypes. MHC class I and II expression was studied by immunohistochemistry and HPV typing was performed by general primer- and type-specific primer mediated PCR (GP/TS PCR). Both techniques were performed on paraffin embedded tissue sections. Results show disturbed MHC class I heavy chain expression in CIN I to CIN III, as well as in cervical carcinomas. Upregulated MHC class II expression on dysplastic epithelial cells was also found in the different CIN groups and carcinomas. Prevalence of HPV genotypes increased with the severity of the lesion, mainly due to the contribution of the HPV types 16 and 18. No correlation could be established between the presence of specific HPV genotypes and any MHC expression pattern in the different CIN groups or cervical carcinomas. In some cases these data were confirmed by RNA in situ hybridisation showing HPV 16 E7 transcripts in the same dysplastic/neoplastic cells from which MHC status was determined. The results indicate that local differences may exist in the type of cellular immune response to HPV induced lesions.


Cremophor EL, a pharmacologically inactive solubilising agent, has been shown to reverse multidrug resistance (MDR). Using flow cytometric evaluation of equilibrium intracellular levels of daunorubicin (DNR), we found that eight other surface active agents will also reverse MDR. All the active detergents contain polyethoxylated moieties but have no similarities in their hydrophobic components. The properties of three polyethoxylated surfactants that showed the lowest toxicities, Cremophor, Tween 80 and Solutol HS15, were examined in more detail. The concentrations of Tween 80 and Solutol required to reverse DNR exclusion were 10-fold lower than for Cremophor. However while concentrations greater than or equal to 1:10(2) of the former two surfactants resulted in breakdown of cells, even 1:10 of Cremophor did not lyse cells. Studies of the effects of Cremophor on the uptake and efflux of DNR in normal and MDR cell types showed that Cremophor increases intracellular DNR primarily by locking the rapid efflux from the cells. This blockage of drug efflux may be mediated by a substantial alteration in the fluidity of cell membranes induced by Cremophor, as shown by decreased fluorescence anisotropy of a membrane probe. Consistent with these data, coinjection of adriamycin plus Cremophor into mice carrying a multidrug resistant P388 transplantable tumour significantly increased the survival time of the mice compared with adriamycin treatment alone.


Human papillomavirus (HPV) typing and quantitation by polymerase chain reaction was performed on exfoliated cells from 133 women referred for colposcopy because of an abnormal smear. High levels of HPV 16 correctly predicted cervical intraepithelial neoplasia (CIN) grade II-III in 93% of its occurrences, but only 59% of cases of CIN III were associated with high levels of this type. Eighty-four per cent of CIN III lesions contained high levels of at least one of HPV types 16, 18, 31, 33 and 35, but the other types were less specific for CIN III than HPV 16. Overall HPV testing compared favourably with cytology for predicting high-grade CIN lesions, but it would appear that some combination of the two modalities will produce better performance than either alone. In particular, HPV testing appears to be helpful in determining which women with mildly abnormal smears have high-grade underlying lesions in need of immediate referral for colposcopy.


The chemosensitising effect of the nitroaromatic radiosensitiser misonidazole (MISO) on the alkylating agent cyclophosphamide (CTX) has been investigated in a C3H mammary carcinoma in CDF1 mice. The selective cytotoxicity against aerobic and hypoxic cells was measured indirectly, using a local tumour control (TCD50) assay. The hypoxic fraction was calculated from the dose difference between the TCD50S for tumours irradiated either in air or under clamped conditions. The relative survival of tumour cells after drug therapy was expressed as a surviving fraction (SF). CTX (100 mg kg-1) was found to be considerably more toxic towards hypoxic than aerobic cells (SF 4% versus 52%). MISO (1000 mg kg-1) was almost exclusively toxic to hypoxic cells (SF 22%). When MISO and CTX were administered simultaneously a decrease in the surviving fraction was observed. The effect on aerated cells was found to be 10-fold more than expected from addition of toxicities, suggesting a chemosensitising effect on these cells by MISO when used in combination with CTX. No synergistic effect was found on radiobiologically hypoxic cells. The exact role of hypoxia for the development of chemosensitisation seems to be complex and requires additional research in the future.


A large body of theoretical work suggests that analyses of variation at the maternally inherited mitochondrial (mt)DNA and the paternally inherited non-recombining portion of the Y chromosome (NRY) are a potentially powerful way to reveal the differing migratory histories of men and women across human societies. However, the few empirical studies comparing mtDNA and NRY variation and known patterns of sex-biased migration have produced conflicting results. Here we review some methodological reasons for these inconsistencies, and take them into account to provide an unbiased characterization of mtDNA and NRY variation in chimpanzees, one of the few mammalian taxa where males routinely remain in and females typically disperse from their natal groups. We show that patterns of mtDNA and NRY variation are more strongly contrasting in patrilocal chimpanzees compared with patrilocal human societies. The chimpanzee data we present here thus provide a valuable comparative benchmark of the patterns of mtDNA and NRY variation to be expected in a society with extremely female-biased dispersal. Human societies show significant variation in post-marital residence practices. About 70% of human societies practice some form of patrilocality, with men remaining in and women migrating from their natal household, clan, lineage, village, or other cultural unit subsumed within a larger group of people sharing a common culture and language, often termed a `tribé in traditional societies One study of Hill tribes of northern Thailand showed the predicted differences between patrilocal and matrilocal tribes in patterns of mtDNA and NRY variation At least three factors may contribute to the discrepant findings of prior research comparing contemporary patterns of genetic variation and post-marital residence practices. First, analyses of mtDNA and NRY variation have been performed at a broader scale of social organization than that at which sex-biased migration actually takes place. For example, the different patterns of mtDNA and NRY diversity shown by matrilocal and patrilocal tribes in Thailand versus India may occur because only in Thailand do members of the migrating sex sometimes move out of their own tribe to join another. Migration outside the tribe is actually a rare event in traditional societies, as different tribes can have very different languages and cultures A second factor complicating attempts to compare patterns of mtDNA and NRY variation is that estimates of genetic differentiation between populations are sensitive to variables that reduce the level of within-group variation, with decreased variation resulting in larger genetic differentiation. For example, an early study of global genetic variation found that genetic differentiation among continents was larger for the NRY than the mtDNA Finally, a third possible reason for the contradictory findings of prior research is that we do not know for how long and how consistently each of the sampled societies has practiced their particular form of post-marital residence. One of the societies in the Central Asian study has practiced its contemporary form of post-marital residence for several centuries In contrast to the complex situation in human populations, patterns of sex-biased migration in animals are often well known from long-term behavioral observations. The currently available genetic studies of animal populations, however, furnish limited insights into how sex differences in migration affect mtDNA and NRY variation. Long-term behavioral observations indicate that migration out of bonobo groups is extremely female-biased Here we examine patterns of mtDNA and NRY variation in four groups of wild chimpanzees located in Uganda and separated by 10–165 km. Chimpanzees live in communities consisting of 20–150 individuals, and as a result of over 180 total years of human observation at multiple field sites, their migration patterns are well understood ST based genetic differentiation estimates, we use the recently developed standardized measure of genetic differentiation We compare the patterns of variation in chimpanzees with those inferred from published data on patrilocal human tribes belonging to five different larger cultural/geographic regions, hereafter termed ‘tribal groups’  P = 0.008, two-tailed)  . Chimpanzees thus have reduced levels of NRY diversity relative to mtDNA diversity compared with patrilocal humans.We found that the average NRY haplotype diversity of the four chimpanzee communities  was significantly lower than that of the 20 patrilocal human tribes   . However-tailed) , suggestWe found extensive sharing of mtDNA variants among chimpanzee communities that strongly contrasted with complete community specificity of NRY haplotypes . In contStandardized genetic differentiation produced broadly similar results, with chimpanzees showing significantly larger standardized NRY genetic differentiation than three of five patrilocal human tribal groups . HoweverThe more similar standardized than unstandardized NRY/mtDNA genetic differentiation ratios between chimpanzees and patrilocal human tribal groups suggest that in addition to differences in the extent of female-biased migration, other factors which reduce NRY relative to mtDNA within-group variation have affected chimpanzees more strongly than the patrilocal human tribal groups. One possibility is that the effective population size of males relative to females may be smaller in chimpanzees than it is in humans. To test this hypothesis, we used parentage assignments in the chimpanzee communities and published human data sets to compare sex differences in effective population size in both species. We found that the ratio of male to female variance in lifetime reproductive success (LRS), an important factor influencing sex differences in effective population size, is actually similar in chimpanzees and in humans living in traditional societies . Taken tWhatever the reason for the discrepancy between unstandardized and standardized NRY/mtDNA genetic differentiation ratios, standardized NRY and mtDNA genetic differentiation were still more strongly contrasting in chimpanzees than in patrilocal human tribal groups. This suggests that migration is more female-biased between chimpanzee communities than between patrilocal human tribes within a tribal group. Additional analyses of patrilocal humans which are conducted on the scale over which sex-biased migration actually occurs, e.g., villages within a tribe rather than tribes within a tribal group, will be necessary to test this hypothesis. Alternatively, the recent history of patrilocality in most human populations may have prevented the build-up of such extremely contrasting patterns of mtDNA and NRY variation. For humans, the absence of higher NRY than mtDNA genetic differentiation at the continental and global scale may indicate that migration is not female-biased at these broad geographical distances www.ncbi.nlm.nih.gov) accession numbers for the chimpanzee mtDNA sequences are EU077270-EU077418. Supplementary We examined mtDNA and NRY variation in four communities of chimpanzees (number of mtDNA/NRY-chromosomes sampled in parentheses): the Sonso (28/16) community of Budongo Forest Reserve, the Ngogo (94/41) and Kanyawara (20/10) communities of Kibale National Park, and the Mugiri (7/6) community of Semliki National Park. We also analyzed previously published data from five patrilocal Dravidian tribes from India (Vanne (32/23), Pokanati (59/25), Panta (37/21), Kapu (22/16) and Akhutota (32/21)), five patrilocal Mundari tribes from India , Munda (23/23), Kharia (21/13), Bhumij (40/39) and Asur (30/28)), three patrilocal Hill tribes from Thailand (Akha (91/21), Chiang Rae Lisu (53/9) and Mae Hong Son Lisu (42/22)), four patrilocal tribes from the lowlands of Western New Guinea (Awyu (12/10), Citak (40/28), Mappi (18/10) and Muyu (10/6)), and three patrilocal tribes from the highlands of Western New Guinea (Dani (21/12), Ketengeban (22/19) and Una (51/46). Subjects were sequenced at the first hypervariable control region of the mtDNA and genotyped at microsatellite loci on the NRY. The chimpanzees were sequenced at 473 bases and genotyped at 9 microsatellite loci following procedures described elsewhere U tests to compare average mtDNA haplotype diversity, NRY haplotype diversity, and the ratio of NRY/mtDNA haplotype diversity between the chimpanzee communities (N = 4) and the patrilocal human tribes (N = 20).We used Arlequin 3.10 Unstandardized genetic differentiation was calculated in an AMOVA framework, with the number of different alleles used as the distance metric for both mtDNA and NRY. We examined the overlap in the 95% confidence limits generated by bootstrapping genetic differentiation values in a locus-by-locus AMOVA to evaluate whether (a) genetic differentiation between the chimpanzees and each of the five patrilocal human tribal groups were significantly different and (b) genetic differentiation of NRY and mtDNA were significantly different within chimpanzees and patrilocal human tribal groups.a and b at a locus, in one of the populations these alleles were recoded as c and d), and calculated an AMOVA FST for this transformed data set . This transformation does not affect within-population variation, but maximizes between-population variation. We then calculated standardized genetic differentiation by dividing the original unstandardized AMOVA FST by the AMOVA FST obtained for this transformed dataset. We included only variable loci when calculating both unstandardized and standardized genetic differentiation. The inclusion or exclusion of non-variable loci does not affect the unstandardized AMOVA, but including non-variable loci when calculating the AMOVA for the transformed data set results in AMOVA values that are determined more by the ratio of variable to non-variable sites, and less by the levels of variability of the variable loci. This would push maximum genetic differentiation possible towards 1, and result in standardized genetic differentiation values that are nearly identical to their unstandardized counterparts. To determine 95% confidence intervals for the standardized genetic differentiation, we divided the lower 95% confidence limit of the original unstandardized genetic differentiation value by the upper 95% confidence limit of the transformed genetic differentiation, and divided the upper 95% confidence limit of the original unstandardized genetic differentiation by the lower 95% confidence limit of the transformed genetic differentiation. For all tests, loci had to have less than 5% missing data to be included in calculations.To calculate standardized genetic differentiation, we first transformed the data such that each population had its own set of unique alleles  and females (IF). IM and IF are commonly used to estimate variance in lifetime reproductive success when the sampling of male and female reproductive output is incomplete and the data indicate the sexes have different mean lifetime reproductive success, when in reality they must be equal To estimate variance in lifetime reproductive success, we calculated the variance in the number of offspring divided by the square of the mean number of offspring, for both males Click here for additional data file.


A retrospective analysis was performed of 18 patients with primary malignant germ cell tumours of the mediastinum treated with platinum-based chemotherapy between 1977 and 1990. All seven patients with pure seminoma were treated initially with chemotherapy and four of these patients received additional mediastinal radiotherapy. Only one patient relapsed; his initial therapy had included radiotherapy and single-agent carboplatin and he was successfully salvaged with combination chemotherapy. With a follow-up of 11 to 117 months (median 41 months) all seven patients with seminoma remain alive and disease free giving an overall survival of 100%. Eleven patients had malignant non seminoma; following chemotherapy eight of these had elective surgical resection of residual mediastinal masses. Complete remission was achieved in nine (82%) patients, however, one of these patients died from bleomycin pneumonitis. With a follow-up of 12 to 113 months (median 55 months) eight of 11 (73%) patients with malignant mediastinal teratoma remain alive and disease free.


The past ten years parallels have been drawn between the biology of cancer and pulmonary fibrosis. The unremitting recruitment and maintenance of the altered fibroblast phenotype with generation and proliferation of immortal myofibroblasts is reminiscent with the transformation of cancer cells. A hallmark of tumorigenesis is the production of new blood vessels to facilitate tumor growth and mediate organ-specific metastases. On the other hand several chronic fibroproliferative disorders including fibrotic lung diseases are associated with aberrant angiogenesis. Angiogenesis, the process of new blood vessel formation is under strict regulation determined by a dual, yet opposing balance of angiogenic and angiostatic factors that promote or inhibit neovascularization, respectively. While numerous studies have examined so far the interplay between aberrant vascular and matrix remodeling the relative role of angiogenesis in the initiation and/or progression of the fibrotic cascade still remains elusive and controversial. The current article reviews data concerning the pathogenetic role of angiogenesis in the most prevalent and studied members of ILD disease-group such as IIPs and sarcoidosis, presents some of the future perspectives and formulates questions for potential further research. The interstitial lung diseases (ILDs) are a heterogeneous group of diffuse parenchymal lung diseases comprising different clinical and histopathological entities that have been broadly classified into several categories ,2 includThe scope of this review article is to summarize the current state of knowledge regarding angiogenic and angiostatic activity in the most important and prevalent members of ILD disease-group such as IIPs and sarcoidosis, discuss its pathogenetic role and present some of the future perspectives and limitations based on authors' assessment or originated from the statements of original authors.de novo formation of blood vessels from angioblasts or endothelial progenitor cells, process that mostly occurs during embryogenesis [Angiogenesis is the process of new capillary blood vessels growth and is instrumental under both physiologic and pathologic conditions. Physiologic conditions include embryogenesis, growth, tissue repair after injury and the female reproductive cycle whereas pathologic angiogenesis can occur in chronic inflammatory and fibroproliferative disorders and tumorigenesis of cancer. Angiogenesis is similar to but distinct from vasculogenesis which describes the ogenesis . On the 2-terminus a three amino-acid motif (ELR) such as IL-8/CXCL8, epithelial neutrophil activating protein (ENA)-78/CXCL5, growth-related genes , granulocyte chemotactic protein (GCP)-2/CXCL6 and neutrophil activating protein (NAP)-2/CXCL7, originally promote angiogenesis [Molecules that originally promote angiogenesis include members of the CXC chemokine family, characteristically heparin binding proteins which on structural level have four highly conserved cysteine amino acid residues, with the first two cysteines separated by one nonconserved amino acid residue. CXC chemokines display unique diverse roles in the regulation of angiogenesis resulting from dissimilarity in structure. Therefore, members that contain in the NHogenesis ,12. Therogenesis ,12. Anotogenesis ,14. Its ogenesis . Finallyogenesis . In addiogenesis -19.By contrast, other members of the CXC chemokine family that do not contain the angiogenic ELR motif (ELR-) behave as potent inhibitors of angiogenesis. Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit aberrant angiogenesis. Furthermore, the angiostatic ELR- members of the CXC chemokine family include the interferon (IFN)-γ inducible protein (IP)-10/CXCL10, monokine induced by IFN-γ (MIG)-2 and IFN-γ-inducible T-cell a chemoattractant (ITAC)/CXCL11 . The latSeveral transcription factors play instrumental role in promoting angiogenesis and sensing the environmental cues that drive this process. Strieter et al.  identifiThe past ten years parallels have been drawn between the biology of cancer and pulmonary fibrosis. The unremitting recruitment and maintenance of the altered fibroblast phenotype with generation and proliferation of immortal myofibroblasts is reminiscent with the transformation of cancer cells -37. A haThere is increasing evidence supporting the notion that vascular remodeling in fibroproliferative disorders appears to be regulated by an imbalance between angiogenic and angiostatic factors. Seminal observation implicating angiogenic activity as an important aspect of progressive fibrosis was originally made by Turner-Warwick in 1963, when she demonstrated the presence of anastomoses between the systemic and pulmonary microvasculature in lungs of patients with IPF . DespiteKeane and colleagues were the first addressing this crucial issue. They demonstrated increased angiogenic activity in a large number of IPF lung specimens ,42 and sOriginal attempt to prove an association between abnormal vasculature and regional heterogeneity characterizing IPF was performed by Renzoni and coworkers . Fueled Lappi-Blanco et al. addressed this crucial issue ,54. TheyThere is a great lack of knowledge regarding this issue which has been largely ignored. Renzoni et al.  were theTowards this direction, Simler et al.  performeA second attempt to correlate local and systemic expression of angiogenic mediators with clinical biomarkers of disease severity and activity was recently published by Pignatti et al . They inFinally, Strieter et al.  recentlyThe vascular remodeling phenomenon has been also described in the experimental model of bleomycin-induced pulmonary fibrosis. The role of neovascularization during the pathogenesis of experimental pulmonary fibrosis was originally raised by Peao and coworkers . In lineFueled by the prospect that anti-angiogenic treatment could be beneficial for pulmonary fibrosis, Hamada et al.  tested tThe latter results have clear therapeutic implications since inhibition of angiogenic mediators or administration of angiostatic chemokines reduced lung collagen deposition and attenuated the exaggerated matrix remodeling. On the basis of this concept, neutralization of proangiogenic environment should be pursued. However, the latter statement should be treated with caution for the following reasons: 1) Findings derived from the bleomycin model of pulmonary fibrosis may not be applicable to human disease since pathogenetic components seen in bleomycin-induced pulmonary fibrosis do not demonstrate areas compatible with fibroblastic foci, the leading edge of human fibrosis. In addition, there are clear limitations to this model in terms of its self-limiting nature, the rapidity of its development and the close association with inflammation that accompanies the lung injury . Regardi2 terminus. While CXCR3/CXCR3 ligands inhibit angiogenesis, CXCR3 ligands play a pivotal role in orchestrating Th1 cytokine-induced cell-mediated immunity via the recruitment of mononuclear and CD4+ T-cells expressing CXCR3 and consequently via the granuloma formation (1). So far, there are only few studies in the literature implicating angiogenesis in the immunomodulatory cascade of sarcoidosis and correlating its immunopathogenesis with members of the angiostatic group of CXC chemokines. These studies are discussed in the following lines.Recent immunological advances on sarcoidosis have revealed a T helper 1 (Th1) and T helper 2 (Th2) paradigm with predominance of the Th1 response in its immunopathogenesis ,68. The The concept of disparate activity of the IFN-γ-induced CXC chemokines in the context of Th1-like immune disorders, such as sarcoidosis, was originally raised by Agostini et al.  who docuThe aforementioned studies substantiate the assertion that IFN-γ-induced CXC chemokines are strongly involved in the immunomodulatory cascade of sarcoidosis implicating angiostasis with Th1 immune response. However, there are several arguments that should be addressed. The majority of the studies cited above have investigated the ability of CXCR3 ligands to promote Th1-dependent immunity and not to inhibit angiogenesis. Studies have shown that angiostatic CXC chemokines are more likely to contribute to the granuloma formation through their chemotactic rather than angiostatic properties. The contention of "immunoangiostasis" (promotion of Th1 response and at the same time inhibition of angiogenesis) as it has been coined out by Strieter et al.  may possIIPs are a heterogeneous group of diffuse parenchymal disorders resulting from damage to the lung parenchyma by varying patterns of inflammation and fibrosis. On the other hand several patients with sarcoidosis develop irreversible lung damage and pulmonary fibrosis which culminates to a fatal outcome. Several theories and mechanisms have been delineated regarding the pathogenesis of fibrotic lung disorders. Recent evidence support the concept that inflammation is subsequent to injury and that fibrosis occurs as a polarization of the Th2 immune response of the body to repeated injury to the lung ("multiple hits" hypothesis) ,73.Putting the aforementioned data together, we tentatively present the three current theories regarding the role of aberrant vascular remodeling in the fibrogenic process. The first hypothesis is based on the idea that the hypervascularity observed in the least fibrotic areas has a role in the regeneration of the alveolar septa damaged by the fibrotic process and is a compensatory response to the decreased vascularity seen within the fibroblastic foci Figure . In thisAlternative hypothesis regarding the role of vascular remodeling during the process of ECM remodeling has also emerged. This theory supports the premise that newly formed microvessels enhance the exaggerated and dysregulated ECM deposition, support fibroproliferation and inhibit normal epithelial repair mechanisms . Human ,52 and aThe third theory supports the notion that the role of angiogenesis in the pathologic process of pulmonary fibrosis is overestimated and that aberrant vascular remodeling is just a bystander or a consequence of fibrogenesis. The latter idea is based on the assertion that CXC chemokines may exert their anti-fibrotic activities through pathogenetic pathways different from those of angiogenesis . The afoBased on the above data we can state that although several study groups have investigated aberrant vascular remodeling in the pathogenesis of pulmonary fibrosis, the relative roles played by new vessel formation and vascular regression in IPF and subsequently in other fibrotic lung disorders are still elusive and controversial. However, to address this issue further investigation in the context of large prospective multicenter studies using highly standardized techniques is sorely needed. The emergence of massive genome screening tools (DNA microarrays)  coupled Several lines of research have been proven inadequate to demystify the relative role of angiogenesis in the etiopathogenesis of chronic fibroproliferative disorders. The question originally raised still remains unanswered: "A pathogenetic hallmark of just a bystander?" However, the status of knowledge regarding the contribution of newly formed vessels in the initiation and/or progression of the sequential events of abnormal injurious response, paradoxical apoptosis and exaggerated matrix remodeling has been greatly elevated by several studies. So far, a number of investigations give credence to the view that a chemokine imbalance favoring angiogenesis supports fibroproliferation and inhibits normal repair mechanisms. Alternatively, the regional vascular heterogeneity in IPF can be explained as a compensatory response (vascular regression) to the striking hypovascularity described in areas of active fibrosis. Currently, angiogenesis represents one of the most fruitful applications in the therapeutic minefield of fibrotic lung disorders. The lack of an effective treatment option challenges chest physicians to think beyond conventional therapeutic strategies and apply fresh approaches. Blockage of multiple angiogenic mediators may provide a way forward. Whether our hopes will be fulfilled or disproved remains to be seen.Basic fibroblast growth factor (bFGF)Bronchiolitis obliterans-organizing pneumonia (BOOP)Bronchiolitis obliterans syndrome (BOS)Cryptogenic organizing pneumonia (COP)Epithelial neutrophil activating protein (ENA)-78Extracellular matrix (ECM)Farmer's lung disease (FLD)Granulocyte chemotactic protein (GCP)Growth-related genes (GROs)Hypoxia inducible factor-1a (HIF-1a)High resolution computed tomography (HRCT)Idiopathic pulmonary fibrosis (IPF)Interferon-γ (IFN-γ)(IFN)-γ inducible protein (IP)-10(IFN)-γ inducible T cell-a chemoattractant (ITAC)Idiopathic interstitial pneumonias (IIPs)Interstitial lung diseases (ILDs)Matrix metalloproteinases (MMPs)Macrophage inflammatory protein (MIP)-2Monokine induced by IFN-γ (MIG)-2Natural killer (NK) cellsNeutrophil activating protein (NAP)-2Nuclear factor-κB (NF-κB)Non-specific interstitial pneumonia (NSIP)Platelet factor-4 (PF-4)Pigment epithelium-derived factor (PEDF)Systemic sclerosis (SSc)Usual interstitial pneumonia (UIP)Vascular endothelial growth factor (VEGF)von Willebrand factor (VWF)The author(s) declare that they have no competing interests.AT and DB were involved with the study conception. AT and SA performed the data acquisition and interpretation. AT prepared the manuscript. DB was involved in revising the article for important intellectual content. All authors read and approved the final manuscript.


The effects of the major human serum bile acid, glycochenodeoxycholic acid (GCDC), as well as unconjugated chenodeoxycholic acid (CDC), on the MCF-7 human breast cancer cell line have been studied in vitro under oestrogen and bile acid deprived culture conditions. GCDC increased the growth of the breast cancer cells over the range 10-300 microM. At concentrations in excess of the bile acid binding capacity of the medium cell growth was prevented. In contrast 10 microM CDC tended to reduce cell growth. Oestrogen (ER) and progesterone (PgR) receptors, pS2 and total cathepsin D were quantified by monoclonal antibody based immunoassays. Ten to 100 microM GCDC and 10 microM CDC down-regulated ER protein and this was accompanied by induction of the oestrogen-regulated proteins PgR, pS2 and possibly cathepsin D, including increased secretion of the latter two proteins into the culture medium. All these changes were quantitatively similar to those observed with 10 nM oestradiol. The bile acid effects on ER and PgR were not due to interference with the assay procedures. Cells incubated with 50 microM GCDC or 10 microM CDC had higher pmolar concentrations of the bile acids than controls. This study suggests that naturally occurring bile acids influence the growth and steroid receptor function of human breast cancer cells.


Somatostatin (SMS), a naturally occurring peptide is known to inhibit the production of certain protein molecules and to diminish the ability of peripheral blood mononuclear cells to proliferate. We tested the effects of three forms of SMS on the bioactivity of both lymphotoxin (LT) and tumour necrosis factor (TNF). We also tested the effects of these agents on production of cytotoxins by peripheral blood mononuclear cells. We found the 28 amino acid form of SMS significantly enhanced the bioactivity of both LT and TNF (10(-9) M concentration) when tested in mouse L cells. The 14 amino acid form of SMS enhanced LT (10(-9) M concentration) activity but not TNF activity. The first 14 amino acid form of SMS-28  did not affect bioactivity of the cytotoxin. In contrast, the naturally occurring 14 amino acid form of SMS (10(-8) M concentration) significantly diminished production of cytotoxin by human peripheral blood mononuclear cells. Cytotoxin produced by the latter was shown to be a combination of both LT and TNF. Similarly after SMS exposure, the cytotoxin produced remained a mixture of LT and TNF in roughly similar proportions. It thus appears that certain forms of SMS can enhance the bioactivity of cytotoxins, but at the same time decrease the production of these cytotoxins.


Alterations in the p53 gene were analysed in 39 patients with locally advanced breast cancers (LABCs) (stage III-IV) with inflammatory signs in most cases (UICC stage T4d = 32 patients) by molecular and immunohistochemical (IHC) approaches. All patients were included in the same therapy protocol. Using polymerase chain reaction (PCR) and a single-strand conformational polymorphism migration technique (SSCP), the presence of mutations in exons 2-11, covering the entire coding sequence of the p53 gene, was evaluated. Using the mouse specific anti-human p53 monoclonal antibody (PAb 1801), we also looked for overexpression of the p53 protein in tissue sections. In 16 cases shifted bands were reproducibly identified by PCR-SSCP, and all but one  were in exons 5-8, the usual mutational hotspots. Fifteen of these 16 samples were sequenced and 14 of the suspected mutations (36%) were confirmed. Most of them (12) were single nucleotide substitutions, and transitions were more frequent (eight cases) than transversions (four cases). Fourteen of the tumour samples were positively stained with the monoclonal antibody PAb 1801, 11 with nuclear staining only, two with mixed cytoplasmic and nuclear staining and one with cytoplasmic staining only. Staining patterns were very heterogeneous in terms of the percentage of positive cells (10-75%) and their distribution in the tissue section (isolated foci or dispersed cells). In 11 of the 14 mutated cases a positive immunostaining was observed. The presence of a p53 mutation was significantly associated with larger tumour diameter  and the presence of clinical metastases (stage IV) . A non-statistically significant trend of association was observed between p53 mutation, negative oestrogen receptors and lower response rate to therapy. Our results in this group of patients and the heterogeneity of the staining of tumour cells in tissue sections suggest that p53 mutations could be a late event in this non-familial form of breast cancer.


We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a neuronal direction through the establishment of four extraosseous ES cell lines and by in vitro stimulation with dibutyryladenosine cyclic monophosphate (db-cAMP) of eight ES lines. All except one of the lines expressed the molecule defined by 5C11, the antibody specifically reactive with ES. Two ES lines expressed a 200 kilodalton (kD) neurofilament protein (NFP) although their original tumours were negative for NFP. Elongation of cytoplasmic processes and increased NFP expression were observed after db-cAMP treatment of these lines and microtubules in the cytoplasmic processes were ultrastructurally demonstrated. Six lines were NFP negative, but three lines changed their morphology after induction of 200 kD NFP expression by db-cAMP treatment. The other three showed no definitive differentiation after db-cAMP treatment. Chromosomal analysis of the new ES lines showed the typical t in one line and a +der(22) in two lines. No correlation was observed between the chromosomal abnormality and the differentiation capability. We conclude that ES is a heterogeneous group of tumours with respect to capability of differentiation into the neuronal lineage, but it is clearly distinguished from peripheral primitive neuroectodermal tumours by its 5C11 reactivity.


We report the natural killer (NK) and lymphokine activated killer (LAK) cell activities in peripheral blood lymphocytes (PBL) from untreated patients with Hodgkin's disease (HD) and from healthy donors. The frequency of LAK cell precursors was also studied using limiting dilution analysis (LDA). About 75% of the HD patients had normal NK activity. There was a higher percentage of low NK responders  in patients with lymphocyte depletion histologic grade of the disease and those who were in clinical stage IV, suggesting a correlation of decrease in NK activity with poor prognosis. We found efficient LAK activity against the NK-sensitive K562 cells and NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumour targets in both low and normal NK responder HD patients, irrespective of the histopathological grade and clinical stage of the disease. In concordance with their good LAK cell activity, HD patients showed a frequency distribution of LAK cell progenitors in the PBL comparable to that of healthy donors.


Intra-tumoural heterogeneity of proliferation has been assessed by taking multiple biopsies from 30 colorectal cancers. Following in vivo IUDR labelling, dual parameter flow cytometry was used to measure tumour DNA index (DI) and labelling index (LI) and to derive DNA synthesis time (Ts) and potential doubling time (Tpot). Heterogeneity was seen for all parameters under investigation. Overall coefficients of variation (CV) and logarithmic transformation of Ts and Tpot (due to their non-gaussian distributions) indicate that LI (CV 25%) was the most variable parameter. Intra-tumoral heterogeneity in Tpot (lnTpot CV = 22%) was less than inter-individual variation (CV = 63%), suggesting that this variation should not be a limitation to the possible usefulness of this technique as an independent prognostic indicator. Correlations of Tpot values were examined between the shortest, the median and the value for a pooled homogenate sample from a single tumour. Using an homogenate, it was possible to accurately predict classification of tumour Tpot values as being below the median ('fast tumours') in 15 of 19 cases (79%). The data suggest that assaying an homogenate may allow a more rapid analysis of a multiply sampled tumour.