Footprints of BK and JC polyomaviruses in specimens from females affected by spontaneous abortion.

STUDY QUESTION: Are JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) infections associated with spontaneous abortion (SA)? SUMMARY ANSWER: There is no association of JCPyV or BKPyV with SA. WHAT IS KNOWN ALREADY: A large number of risk factors have been associated with SA. The role of polyomaviruses, including JCPyV and BKPyV, in SA remains to be clarified. STUDY DESIGN, SIZE, DURATION: This is a case-control study including women affected by spontaneous abortion (SA, n = 100, the cases) and women who underwent voluntary interruption of pregnancy (VI, n = 100, the controls). PARTICIPANTS/MATERIALS, SETTING, METHODS: Viral DNAs were investigated by qualitative PCR and quantitative droplet-digital PCR (ddPCR) in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from SA (n = 100) and VI (n = 100). Indirect ELISAs with mimotopes/synthetic peptides corresponding to JCPyV and BKPyV viral capsid protein 1 epitopes were then employed to investigate specific IgG antibodies against JCPyV and BKPyV in human sera from SA (n = 80) and VI (n = 80) cohorts. MAIN RESULTS AND THE ROLE OF CHANCE: JCPyV DNA was detected in 51% and 61% of SA and VI samples, respectively, with a mean viral DNA load of 7.92 copy/104 cells in SA and 5.91 copy/104 cells in VI (P > 0.05); BKPyV DNA was detected in 11% and 12% of SA and VI specimens, respectively, with a mean viral DNA load of 2.7 copy/104 cells in SA and 3.08 copy/104 cells in VI (P > 0.05). JCPyV was more prevalent than BKPyV in both SA and VI specimens (P < 0.0001). In PBMCs from the SA and VI cohorts, JCPyV DNA was detected with a prevalence of 8% and 12%, respectively, with a mean viral DNA load of 2.29 copy/104 cells in SA and 1.88 copy/104 cells in VI (P > 0.05). The overall prevalence of serum IgG antibodies against JCPyV detected by indirect ELISAs was 52.5% and 48.7% in SA and VI groups, respectively, whereas BKPyV-positive sera were found in 80% SA and 78.7% VI samples. LIMITATIONS, REASONS FOR CAUTION: This study did not investigate the presence of viral mRNA and/or proteins, which are indicative of an active viral infection, and these might be taken into consideration in future studies. WIDER IMPLICATIONS OF THE FINDINGS: JCPyV and BKPyV DNA sequences were detected and quantitatively analyzed for the first time by PCR/ddPCR in chorionic villi tissues and PBMCs from SA and VI specimens. Moreover specific immunological approaches detected serum IgG against JCPyV/BKPyV. Statistical analyses, however, do not indicate an association between these polyomaviruses and SA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University of Ferrara, FAR research grants and the University Hospital of Ferrara/University of Ferrara joint grant. No potential conflicts of interest were disclosed.

Biomaterials as bone graft substitutes for spine surgery: from preclinical results to clinical study.

Vertebral fusion is performed in order to stabilize the spine in the presence of degenerative, traumatic or oncological pathologies that alter its stability. The autologous bone, harvested from the patient's iliac crest or from the lamina during surgery, is still considered the "gold standard" for spine fusion due to its osteogenic, osteoinductive and osteoconductive properties. However, several biological and synthetic bone substitutes have been introduced as alternatives for regenerating bone tissue. We have studied in particular the use of ceramic biomaterials prepared from hydroxypatite (HA), starting from in vitro analysis, through an in vivo study on ovine animal model and a post-market surveillance analysis, to finally design and perform a clinical study, which is ongoing in our Department. In the first step, HA-derived biomaterials were tested in vitro in the presence of bone marrow-derived human mesenchymal stem cells (hMSCs) and evaluated for their ability to activate precursor cells. In the second step, the biomimetic bone graft substitute SintLife® putty (MgHA) was evaluated in vivo. A posterolateral fusion procedure was applied on 18 sheep, where a fusion level was treated with MgHA, while the other level was treated with autologous bone. Microtomography and histological/histomorphometric analysis were performed six months of after surgery. In the third step, we reported the results of a post-market surveillance study conducted on 4 independent cohorts of patients (total 115 patients), in which HA-derived biomaterials were used as bone graft substitutes or extenders. Finally, a clinical study has been designed and approved by the Ethics Committee of our Institute and is currently ongoing. This study aims to evaluate the efficacy of the ceramic biomaterial SintLife® putty for bone replacement in patients treated by posterolateral fusion for degenerative spine disorders. HA biomaterials were effective in promoting the in vitro growth of hMSCs and their osteogenic differentiation. In the animal model, SintLife® putty has been effective in generating neo-formed bone tissue with morphological and structural features similar to those of the pre-existing bone. The post-market surveillance analysis has not reported any intra-operative nor early or late post-operative adverse events. Seven patients are currently recruited for the clinical trial designed to evaluate Sintlife efficacy for spine fusion (FU range: 1-7 months). No adverse events have been recorded. The first CT analysis performed at 6 months FU showed a good spine fusion. The study is ongoing. Our results, obtained from in vitro, preclinical and clinical studies, suggest that biomaterials derived from hydroxyapatite could be a valid alternative to autologous bone graft for vertebral fusion. This would potentially avoid or reduce the need of autologous bone harvesting and therefore, the risk of drawback-related side effects.

HPV-based strategy in follow-up of patients treated for high-grade cervical intra-epithelial neoplasia: 5-year results in a public health surveillance setting.

OBJECTIVE: Human papillomavirus (HPV) DNA testing is used increasingly for measuring the outcome of treatment for high-grade cervical intra-epithelial neoplasia (CIN2+). However, there is no international consensus regarding the number of tests and follow-up visits necessary in the post-treatment surveillance. A negative HPV DNA test result may permit relaxing the intensive post-treatment surveillance, but this possibility has not been standardized by all institutions to date. STUDY DESIGN: In 2008, the surveillance programme covering the Emilia-Romagna region in northern Italy adopted the HPV DNA test as a routine tool in the follow-up of women treated for CIN2+. Data from a prospective 5-year study are reported herein. Three hundred and ten patients treated for CIN2+ with a loop electrosurgical excision procedure underwent HPV DNA testing, cytology and colposcopy at 6 months post treatment. If all three tests were negative, women were tested at 18 months with cytology and colposcopy. If any of the three tests were positive, women were tested at 12, 18 and 24 months with cytology and colposcopy. When appropriate, a colposcopy-directed biopsy or CIN2+ retreatment was performed. After 18-24 months, the patients were tested annually with cytology for 3 years. RESULTS: None of the 172 (55%) women who were HPV negative at 6 months were found to have residual/recurrent CIN2+ during the surveillance period. In contrast, among the 138 (45%) HPV-positive women, 17 cases of residual/recurrent CIN2+ (17/138; 12.3%) were identified between 6 and 24 months. CONCLUSION: HPV DNA testing at six months after treatment for CIN2+ effectively identifies women who are disease free (HPV negative), and for whom a single follow-up at 18 months is sufficient.

Serum antibodies from epileptic patients react, at high prevalence, with simian virus 40 mimotopes.

BACKGROUND AND PURPOSE: It has been demonstrated that inflammation may contribute to epileptogenesis and cause neuronal injury in epilepsy. In this study, the prevalence of antibodies to simian virus 40 (SV40), a kidney and neurotropic polyomavirus, was investigated in serum samples from 88 epileptic children/adolescents/young adults. METHODS: Serum antibodies reacting to specific SV40 peptides were analysed by indirect enzyme-linked immunosorbent assay. Synthetic peptides corresponding to the epitopes of viral capsid proteins 1-3 were used as SV40 antigens. RESULTS: A significantly higher prevalence of antibodies against SV40 was detected in sera from epileptic patients compared to controls (41% vs. 19%). Specifically, the highest significant difference was revealed in the cohort of patients from 1.1 to 10 years old (54% vs. 21%), with a peak in the sub-cohort of 3.1-6 years old (65% vs. 18%). CONCLUSION: Our immunological data suggest a strong association between epilepsy and the SV40 infection.

Tuberculosis of the eye in Italy: a forgotten extrapulmonary localization.

PURPOSE: Tuberculosis (TB) of the eye is a well-known extrapulmonary localization in high-incidence countries. Data on its relevance in developed countries are scanty. We aim to study the epidemiological and clinical pattern of ocular TB in a tertiary care institution of a western country. METHODS: From 2007 to 2010, consecutive patients with a diagnosis of isolated ocular TB or associated to extraocular TB were recruited. Patients with ophthalmological and clinical features of TB were treated with standard antitubercular therapy (ATT) and steroids in case of concomitant severe ocular inflammation. RESULTS: Seventeen cases of ocular and extraocular TB and 45 cases of isolated ocular TB were identified. The proportion of patients with ocular and extraocular TB in our local district was 8.1 %, with a proportion of 10.6 % for the isolated cases. In Cohort 1, only one patient was symptomatic for ocular impairment, and uveitis without inflammation was the most common presentation. On the contrary, in Cohort 2, all patients had visual impairment, mainly with bilateral involvement. 77.8 % of the patients showed an inflammatory pattern. ATT was administered for at least 9 months, in four cases with a short course of systemic corticosteroids. Eight cases in Cohort 2 showed recurrence after 1 year from diagnosis. CONCLUSIONS: TB of the eye should not be forgotten, even in geographical areas not considered among endemic countries. Ocular evaluation is advisable in patients with pulmonary and extrapulmonary TB, as early detection may allow ATT to preserve visual acuity.

The addition of GnRH antagonists in intrauterine insemination cycles: a pilot study.

AIM: This prospective study was designed to assess whether the use of GnRH antagonists can improve the success rate of controlled ovarian stimulation (COS) in intrauterine insemination (IUI) treatments. PATIENTS AND METHODS: Eighty patients were divided into two groups: GnRH antagonist group (Group A, n=40) and control group (Group B, n=40). Patients in Group B underwent COS with recombinant Follicle Stimulating Hormone (r-FSH, 50-75 IU/d) only, while patients in Group A were administered r-FSH (50-75 IU/d) plus cetrorelix (0.25 mg/d, starting when ≥ 2 follicles ≥ 14 mm were detected on ultrasound scan). In both groups a single insemination was performed 36 hours after human Chorionic Gonadotropin (hCG, 250 mcg) administration. The primary outcome was clinical Pregnancy Rate (PR). Secondary outcomes were ongoing PR, incidence of Premature Luteinization (PL), number of follicles with mean diameter ≥ 16 mm and between 11 and 15 mm on the day of hCG administration, miscarriage rate, cycle cancellation rate, total amount of r-FSH used and duration of treatment. Student's t test and Chi-square test were used (p < .05 statistically significant). RESULTS: A total of 146 cycles were performed (Group A: n=72; Group B: n=74). A trend towards higher PR in Group A was detected, although it was not statistically significant (Clinical PR: 18.05% vs 10.81%). The number of follicles ≥ 16 mm was significantly increased in Group A. The incidence of both premature LH surge and premature luteinization (PL) was significantly higher in Group B. No significant differences were found in the duration of the stimulation protocol, and in the total amount of r-FSH administered. CONCLUSIONS: The addition of GnRH antagonist in COS/IUI protocol significantly increases the number of mature follicles. However, this multifollicular recruitment is not linked to a significantly higher PR.

Mesenchymal stem cells from patients to assay bone graft substitutes.

Bio-engineered scaffolds used in orthopedic clinical applications induce different tissue responses after implantation. In this study, non-stoichiometric Mg(2+) ions and stoichiometric apatites, which are used in orthopedic surgery as bone substitutes, have been assayed in vitro with human adult mesenchymal stem cells (hMSC) to evaluate cytocompatibility and osteoconductivity. hMSCs from the bone marrow aspirates of orthopedic patients were isolated and analyzed by flow cytometry for the surface markers Stro1, CD29, CD44, CD71, CD73, CD90, CD105 (positive) and CD45, CD235 (negative). The hMSC were analyzed for self-renewal capacity and for differentiation potential. The hMSC, which were grown on different biomaterials, were analyzed for (i) cytotoxicity by AlamarBlue metabolic assay, (ii) osteoconductivity by ELISA for activated focal adhesion kinase, (iii) cytoskeleton organization by fluorescence microscopy, and (iv) cell morphology which was investigated by scan electron microscopy (SEM). Results indicate that isolated cell populations agree with minimal criteria for defining hMSC cultures. Non-stoichiometric Mg(2+) and stoichiometric apatites, in granular form, represent a more favorable environment for mesenchymal stem cell adhesion and growth compared to the non-stoichiometric Mg(2+) apatite, in nano-structured paste form. This study indicates that different forms of biomaterials modulate osteoconductivity and cellular growth by differential activation focal adhesion kinase.

Methylenetetrahydrofolate reductase gene promoter hypermethylation in semen samples of infertile couples correlates with recurrent spontaneous abortion.

STUDY QUESTION: Is the methylation status of the methylenetetrahydrofolate reductase (MTHFR) promoter region in semen samples associated with 'recurrent spontaneous abortion' (RSA)? SUMMARY ANSWER: MTHFR promoter hypermethylation is more frequent in semen samples from RSA couples than in semen samples from infertile couples with no history of RSA (NRSA) and affects the whole sperm population significantly more often. WHAT IS KNOWN ALREADY: Modifications to the MTHFR gene such as polymorphisms and promoter methylations are associated with male infertility. STUDY DESIGN, SIZE AND DURATION: Retrospective cohort study of semen samples from 20 RSA couples, 147 NRSA couples and 20 fertile men between 2011 and 2012. MATERIALS, SETTING AND METHODS: DNA from the semen samples of RSA, NRSA and fertile men were analyzed by methylation-specific PCR amplification using primers which anneal to the methylated or unmethylated cytosine-phosphodiester bond guanine (CpG) islands within the promoter region of MTHFR. The specificity of the PCR products was assessed by DNA sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: The methylated MTHFR epigenotype (including samples where it co-existed with unmethylated MTHFR epigenotypes) was detected in 75% of RSA men, 54% of NRSA men and 15% of fertile men. MTHFR methylation was observed in the whole sperm population in semen samples from 55% of RSA men compared with 8% in NRSA men (P < 0.05) and 0% in fertile men (P < 0.05). DNA sequencing analysis was fully concordant with the PCR results and revealed that when MTHFR methylation occurred, CpG islands within the promoter region were 100% methylated (hypermethylation of MTHFR promoter). LIMITATIONS, REASONS FOR CAUTION: The relatively small sample size of RSA infertile couples. WIDER IMPLICATIONS OF THE FINDINGS: The hypermethylation of the MTHFR gene promoter should be taken into consideration as a novel putative risk factor in RSA etiology. STUDY FUNDING/COMPETING INTEREST(S): Our institution has received an FAR research grant from the University of Ferrara, Ferrara, Italy. No competing interests declared.

Human mesenchymal stem cells and biomaterials interaction: a promising synergy to improve spine fusion.

PURPOSE: Spine fusion is the gold standard treatment in degenerative and traumatic spine diseases. The bone regenerative medicine needs (i) in vitro functionally active osteoblasts, and/or (ii) the in vivo induction of the tissue. The bone tissue engineering seems to be a very promising approach for the effectiveness of orthopedic surgical procedures, clinical applications are often hampered by the limited availability of bone allograft or substitutes. New biomaterials have been recently developed for the orthopedic applications. The main characteristics of these scaffolds are the ability to induce the bone tissue formation by generating an appropriate environment for (i) the cell growth and (ii) recruiting precursor bone cells for the proliferation and differentiation. A new prototype of biomaterials known as "bioceramics" may own these features. Bioceramics are bone substitutes mainly composed of calcium and phosphate complex salt derivatives. METHODS: In this study, the characteristics bioceramics bone substitutes have been tested with human mesenchymal stem cells obtained from the bone marrow of adult orthopedic patients. RESULTS: These cellular models can be employed to characterize in vitro the behavior of different biomaterials, which are used as bone void fillers or three-dimensional scaffolds. CONCLUSIONS: Human mesenchymal stem cells in combination with biomaterials seem to be good alternative to the autologous or allogenic bone fusion in spine surgery. The cellular model used in our study is a useful tool for investigating cytocompatibility and biological features of HA-derived scaffolds.

Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations.

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.

FHIT oncosuppressor gene expression profile in human anal cancers.

The FHIT gene, a member of the histidine triad gene family, is a tumor suppressor gene exhibiting deletions in the majority of human cancers. Aberrant transcripts of this gene have been found in about 50% of esophageal, stomach and colon carcinomas. Little is known about the molecular mechanisms involved in malignant transformation of the lining cells of the anus. In this study FHIT gene expression was investigated in this particular kind of human cancer. FHIT expression was comparatively analyzed at the mRNA level, by RT-PCR, in squamous anal cancers, normal anal tissue and peripheral blood samples. cDNA analyses showed variability in FHIT transcripts, without apparent effects on the predicted amino acid sequence. These different FHIT mRNAs could represent transcripts from an alternative splicing event. Our data indicate that the FHIT mRNA detected in anal cancers and in normal samples is heterogeneous. Immunohistochemical data suggest that the Fhit protein is expressed only in a fraction of the tumor cells, while it is strongly expressed in the epithelial cells of glands of the normal anal mucosa. The absence or poor expression of the Fhit protein in anal cancers suggests a role for this tumor suppressor gene product, as a risk factor, in the onset of this human cancer, as reported before for other human gastrointestinal tumors.

Malignant mesothelioma in 2004: How advanced technology and new drugs are changing the perspectives of mesothelioma patients. Highlights from the VIIth Meeting of the International Mesothelioma Interest Group.

Malignant mesothelioma (MMe) is a seemingly uncommon tumour whose incidence has in fact increased steadily and progressively over the last 30 years. Indeed, an actual "epidemic" is expected in the next 20 years, with over 1300 new cases a year till 2020 at least. Despite unquestionable improvement in the diagnostic methods at our disposal and the availability of new treatment strategies, the prognosis of MMe patients remains dramatically poor. For all the above reasons, translational research is the key to success; indeed, ever increasing knowledge of the molecular mechanisms underlying MMe pathogenesis could lead (and is actually leading) to new, hopefully more active, treatment options. To foster discussion among investigators working in this field, and to exchange different viewpoints concerning the newest advances in MMe pathogenesis and treatment, the VII International Mesothelioma Interest Group (IMIG) meeting was held in Brescia (Italy) between 24 and 26 June 2004 in cooperation with the Italian Group for the Study and Therapy of MMe (GIMe). The aim of this report is to summarize the most significant advances in the different disciplines applied to MMe presented and discussed during the IMIG meeting and how these advances will be changing the perspective of patients with MMe.

Maternal MTHFR variant forms increase the risk in offspring of isolated nonsyndromic cleft lip with or without cleft palate.

The pathogenesis of cleft lip with or without cleft palate (CL/P) is complex; its onset could be due to the interaction of various genetic and environmental factors. Recently MTHFR functional polymorphisms were found to increase the risk of this common malformation; however, this finding is still debated. We investigated 110 sporadic CL/P patients, their parents and 289 unrelated controls for c.665C>T (commonly known as 677C>T; p.Ala222Val) and c.1286A>C (known as 1298A>C; p.Glu429Ala) polymorphism in the MTHFR gene. Transmission disequilibrium test (TDT) showed no distortion in allele transmission. Nevertheless, association studies revealed significant differences in allele frequencies between mothers of CL/P patients and controls. This work supports the hypothesis that a lower MTHFR enzyme activity in pregnant women, mostly related to the c.665C>T variant form, is responsible for a higher risk of having CL/P affected offspring.

Classification of oral clefts by affection site and laterality: a genotype-phenotype correlation study.

OBJECTIVES: The aim of this study was to classify the phenotypes found in a series of patients with non-syndromic cleft lip (CL) with or without cleft palate (CP) and isolated cleft palate. Additionally, the frequency distribution of cases belonging to families linked to markers on chromosomes 6 and 2 within these phenotypic patterns were estimated. DESIGN: A retrospective examination of all the available affected cases collected in Italy. SETTING AND SAMPLE POPULATION: Ninety-seven affected subjects aged 5-18 years belonging to 38 families were considered. Patterns were identified by variance of the cleft (lip, primary palate, secondary palate) and stratified according to the side of occurrence (right, left, or bilateral). Latent class analysis was used as main statistical tool for carrying out the results. RESULTS: Three homogenous classes were identified (P < 0.0001) by means of latent class analysis. Individuals were assigned to the most suited class. All three variables (lip, primary and secondary cleft palate) generated a specific class. Optimal findings were reported in cases having 'any isolated cleft lip' (class 1); 'secondary CP with or without bilateral/right primary cleft palate + bilateral/right cleft lip' (class 2); and 'left primary cleft palate + left/bilateral cleft lip with or without secondary CP' (class 3). Correspondence to the evidence of linkage to chromosome 6 showed that 9 of 10 cases presenting with 'right primary CP + right CL with secondary cleft palate' (class 2) belonged to a linked family. The same combination, but occurring on the left side (class 3), revealed that only three of nine cases belong to families linked to chromosome 6 (P-value = 0.02). The two patterns (right and left) never occurred in the same family. Three reliable groups were identified based on laterality and the presence of a cleft. A single right sided pattern displayed a statistically different distribution of linkage to chromosome 6 when compared with the homologous left side. CONCLUSION: Non-syndromic CL with/without CP can be classified according to laterality that can be under genetic control.

Cancer testis antigens expression in mesothelioma: role of DNA methylation and bioimmunotherapeutic implications.

Recent evidences suggest that malignant mesothelioma may be sensitive to immunotherapy; however, little is known about malignant mesothelioma-associated tumour antigens. Focusing on cancer/testis antigens, the expression of well-characterised immunogenic tumour-associated antigens was investigated in malignant mesothelioma cells. At variance with MAGE-4 and NY-ESO-1, malignant mesothelioma cells frequently expressed MAGE-1, -2 and -3, GAGE 1-2, GAGE 1-6, SSX-2 and SSX 1-5, and distinct malignant mesothelioma cells concomitantly expressed at least four cancer/testis antigens. Additionally, the tumour-associated antigens RAGE-1 was expressed at high levels in both benign and malignant mesothelial cells. Lastly, treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the expression of the cancer/testis antigen examined in malignant mesothelioma cells. Overall, these findings strongly suggest that cancer/testis antigens-based immunotherapy may represent a suitable therapeutic approach to malignant mesothelioma, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemo-immunotherapeutic strategies in malignant mesothelioma patients.

SV40 replication in human mesothelial cells induces HGF/Met receptor activation: a model for viral-related carcinogenesis of human malignant mesothelioma.

Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation.

Association of SV40 with human tumours.

SV40 was discovered as a contaminant of poliovirus vaccines that were inadvertently administered to millions of people in Europe and the United States between 1955 and 1963. Shortly afterwards, SV40 was proven to be oncogenic in rodents and capable of transforming human and animal cells in vitro. The possibility that SV40 might cause tumours in humans thus became a subject of scientific and public interest and scrutiny. However, largely due to a lack of significant epidemiological evidence, interest in assessing SV40's potential carcinogenic role in humans diminished. Recently, many laboratories have reported the presence of SV40-like DNA in a high proportion of human mesotheliomas, ependymomas and osteosarcoma (the three main types of tumours caused by virus in hamsters), renewing the question whether SV40 might be a human tumour virus. Molecular data from these studies are reviewed to re-evaluate the potential role of SV40 as a human carcinogen.

Investigation of the simian polyomavirus SV40 as a potential causative agent of human neurological disorders in AIDS patients.

Neurological diseases and a variety of neoplasms frequently occur in AIDS patients. Human JC and BK polyomaviruses have been associated with neurological disorders in such patients. SV40 polyomavirus sequences have been detected in human brain tumours, other neoplasms and normal tissues. JCV, BKV and SV40 DNA sequences were investigated in cerebrospinal fluid (CSF) samples from 12 AIDS patients affected by different neurological disorders, by PCR assay and filter hybridisation with specific internal oligoprobes, and DNA sequencing. Three of the 12 CSF samples were positive for JCV (one sample) or SV40 (one) DNA, or both (one). No sample was positive for BKV DNA. JCV- and SV40-specific genomic regions were confirmed by DNA sequencing. CSF samples from the two patients diagnosed clinically as having progressive multifocal leukoencephalopathy (PML) contained either JCV (one sample) or SV40 (one) DNA. The CSF found to contain both JCV and SV40 DNA originated from a patient with a cerebral mass lesion of unknown aetiology. These results suggest that SV40 may be involved in the aetiology of PML in AIDS patients, and raise the possibility that SV40 and JCV may act synergically in vivo to enhance their pathogenicity.

C677T variant form at the MTHFR gene and CL/P: a risk factor for mothers?

Maternal folic acid supplementation in early pregnancy has been suggested to play a role in the prevention of nonsyndromic orofacial cleft, i.e., cleft lip with or without cleft palate (CL/P). Moreover, some authors demonstrated association of the C-->T mutation (C677T), converting an alanine to a valine residue in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, with other congenital anomalies such as neural tube defects (NTDs). Because of MTHFR's involvement in the metabolism of folate, we investigated 64 CL/P patients and their parents for C677T MTHFR mutation. No linkage disequilibrium was found using the transmission disequilibrium test (TDT). However, a significantly higher mutation frequency was detected in mothers of CL/P patients compared to controls. The odds ratios calculated for mothers having CT or TT genotype, compared to the normal CC genotype, were 2.75 (95% confidence interval 1.30-5.57) and 2.51 (1.00-6.14), respectively. These results support the involvement of the folate pathway in the etiology of CL/P, and indicate an effect of the maternal genotype, rather than influence of the embryo's genotype.