Imbalance of MMP-2 and MMP-9 expression versus TIMP-1 and TIMP-2 reflects increased invasiveness of human testicular germ cell tumours.

The histological classification of testicular germ cell tumours (TGCTs) to seminoma or non-seminomatous germ cell tumours is at present the main criterion for the clinical outcome and selection of the treatment strategy. In view of the need to identify novel prognostic biomarkers for TGCTs, we investigated the expression of the matrix metalloproteinases MMP-2 and MMP-9 in testicular tumour tissues and cell lines of both seminoma and non-seminoma origin. Immunohistochemistry and zymography analysis of tumoural tissues showed significantly higher levels of MMP-2 and MMP-9 compared with normal testis with the active forms detected only in the tumour tissues. Three cell lines representative of the different tumour types, JKT-1 seminoma, NCCIT teratocarcinoma and NTERA2/D1 embryonal carcinoma were also evaluated for their expression of these MMPs using qPCR and zymography and for their invasive properties. The more invasive non-seminomatous teratocarcinoma and embryonal cells expressed considerably more MMP-2 and MMP-9 compared with seminoma cells exhibiting lower invasiveness. Furthermore, an inverse relation was observed between invasiveness and the expression of endogenous inhibitors TIMP-1 and TIMP-2. The MMP inhibitor Marimastat inhibited invasion in all cell lines, the highest inhibition was observed in the more invasive NTERA2/D1 and NCCIT cells, which presented the highest ratio of MMP-2 and MMP-9 vs. TIMP-1 and TIMP-2. These results highlight the importance of MMP-2 and MMP-9 in the invasiveness of testicular tumours and suggest that their levels, vs. those of TIMP-1 and TIMP-2, may represent potential biomarkers for testicular malignancy.

Pathophysiology and pharmacological targeting of tumor-induced bone disease: current status and emerging therapeutic interventions.

Bone disease is a common complication of metastatic solid tumors but also of primary hematological malignancies such as multiple myeloma. Our understanding of the molecular mechanisms underlying the development of bone disease by solid tumors and multiple myeloma has been significantly improved. A complex inter-dependence exists between bone disease and malignant cell growth, creating a vicious cycle of extensive bone destruction and tumor progression. Although myeloma and solid tumors share a number of common molecular pathogenetic mechanisms, they involve distinct pathophysiological pathways, resulting in osteoclastic bone resorption and inhibition of bone formation. In this review, we analyze the molecular mechanisms, involved in tumor-induced bone disease and discuss the current therapeutic approaches and the most recent clinical developments of emerging targeted therapies.

Versican but not decorin accumulation is related to metastatic potential and neovascularization in testicular germ cell tumours.

AIMS: To investigate the expression of versican and decorin in patients with testicular germ cell tumours (GCTs) and to correlate this with the clinicopathological findings. Matrix proteoglycans versican and decorin are frequently overexpressed in various malignancies and are involved in the progression of cancer. METHODS AND RESULTS: Overexpression of versican and decorin was detected in GCTs by immunoblotting. Immunohistochemical staining for proteoglycans was performed on 71 cases of paraffin-embedded tissues. In most of the cases increased decorin and versican stromal staining was demonstrated. In both seminomas and non-seminomatous germ cell tumours (NSGCTs) strong staining of decorin was not found to be related to any of the clinicopathological variables. Accumulation of versican was found to be associated with vascular and lymphatic invasion, nodal metastasis and disease stage in seminomas and NSGCTs and, in addition, with tumour size and distant metastasis only in NSGCTs. Additionally, only the deposition of versican was linearly correlated with the number of microvessels in the tumour stroma in GCTs. CONCLUSIONS: Ectopic versican and decorin expression is a frequent feature in GCTs. Versican but not decorin accumulation in GCTs is related to metastatic potential and neovascularization and might be a useful marker for testicular malignancy.

Specific interactions among odorant-binding proteins of the African malaria vector Anopheles gambiae.

In this report we present results from a comprehensive study undertaken toward the identification of proteins interacting with odourant-binding proteins (OBPs) of the African malaria vector Anopheles gambiae with a focus on the interactions among different OBPs. From an initial screen for proteins that interact with a member of the Plus-C group of OBPs, OBP48, which is primarily expressed in female antennae and downregulated after a blood meal, a number of interacting proteins were identified, which included five classic OBPs and OBP48 itself. The interacting OBPs as well as a number of other classic and Plus-C group OBPs that were not identified in the initial screen, were expressed in lepidopteran cells and subsequently examined for in vitro interactions in the absence of exogenously added ligands. Co-immunoprecipitation and chemical cross-linking studies suggest that OBP48 is capable of homodimerizing, heterodimerizing and forming higher order complexes with those examined examples of classical OBPs identified in the initial screen but not with other classical or Plus-C group OBPs that failed to appear in the screen. The latter OBPs are, however, also capable of forming homodimers in vitro and, at least in the case of two examined classic OBPs, heterodimers as well. These results suggest a previously unsuspected potential of nonrandom combinatorial complexity that may be crucial for odour discrimination by the mosquito.

Detection of human papillomaviruses in squamous cell carcinomas of the lung.

The aim of this study was to evaluate the possible association of human papillomaviruses (HPV) with the development of squamous cell lung carcinomas (SqCLCs). Tissue material from 52 cases of SqCLCs were studied, and the data were evaluated according to the degree of differentiation, HPV presence and type. Analysis was performed by polymerase chain reaction (PCR) method using consensus primers, and the results were confirmed by subsequent Southern blot hybridization. Overall, the results showed 69% positivity (n=32). Forty-one cases were examined for the presence of specific HPV types (6/11 and 16/18) by hybridization of the PCR products with 32P-labelled probes. HPV 6/11 types were detected in 6 of the 29 positive cases (20.6%). HPV 16/18 types were the most prevalent types, and were detected in 11/29 cases (37.9%: 4/10 of well-differentiated cases, 6/25 of moderately and 1/6 of poorly differentiated carcinomas). Our results confirm the possibility that HPV might play a role in the development of SqCLCs and suggest a possible relation of high-risk HPV16/18 types to tumour differentiation.

Type-specific prevalence of genital human papillomaviruses in benign, premalignant, and malignant biopsies in patients from Greece.

BACKGROUND AND OBJECTIVES: More than 30 different human papillomavirus (HPV) types infect the anogenital mucosa and are responsible for a variety of benign, premalignant, and malignant lesions including cervical cancer. The goal of this study was to determine the distribution of individual HPV types in various grads of cervical precancerous lesions and cervical carcinoma in patients from Greece. STUDY DESIGN: Specimens were analyzed for HPV-DNA sequences by polymerase chain reaction and Southern blot hybridization. Polymerase chain reaction analysis was performed with consensus and type-specific primers. Restriction length fragment polymorphism analysis and/or hybridization of the general primer polymerase chain reaction product were used for HPV typing. RESULTS: In cervical carcinomas HPV-16 was found in 56%, HPV-18 in 23%, and HPV-31 in 6% of the HPV-positive patients. In precancerous lesions HPV-16 was found in 13% of low-grade squamous intraepithelial lesions (LG-SIL) as compared with 41% of high-grade squamous intraepithelial lesions (HG-SIL) patients. HPV-18 was found at similar frequency both in LG-SIL (13%) and HG-SIL (14%). HPV-31 and HPV-33 were detected at moderate levels both in LG-SIL (11%) and in HG-SIL (14%). In addition, HPV-53 and HPV-66 were detected at low frequency in LG-SIL (2%), whereas HPV-51 was found only in HG-SIL (4%). Finally, HPV-6 was associated with 13% of LG-SIL. CONCLUSIONS: Overall, the prevalence rate of the genital HPV types was in the range previously described for many western countries but the HPV-18 positivity was higher than that reported for most European countries.

Genital papillomavirus in Greek women with high-grade cervical intraepithelial neoplasia and cervical carcinoma.

Fifty biopsies from high-grade squamous intraepithelial lesions (HG-SIL) and 14 cervical carcinoma biopsies from Greek women were screened for the presence of human papillomavirus (HPV) DNA sequences by Southern blot hybridization and by the polymerase chain reaction (PCR) for the presence of different HPV types. In high-grade SIL, HPV DNA sequences were detected in 44 of 50 biopsies with the following distribution: 36% HPV 16, 12% HPV 18, 6% HPV 31, 6% HPV 33, 4% HPV 51, and 24% unclassified HPV types. In cervical carcinoma biopsies, 13 of 14 specimens were positive for HPV DNA sequences. Six biopsies were positive for HPV 16, three were positive for HPV 18, and four contained unclassified HPV types. Overall, of the total 64 biopsies, 57 (89%) were positive for HPV DNA sequences. Of these, Southern blot hybridization alone detected HPV DNA sequences in 39 cases, whereas by PCR 18 additional specimens were found to be positive for HPV. Among the HPV 16-positive biopsies, two samples produced a Pstl banding pattern very similar but not identical to that of HPV 16 prototype and were referred to as HPV 16 isolates. One HPV 16 isolate appears to carry a mutation within the carboxy-terminal half of the L2 gene that results in the loss of a Pstl site. The other HPV 16 isolate had a similar Pstl banding pattern to that previously reported as HPV 16 "variant" found in Cape Town [Williamson et al., 1989, Journal of Medical Virology 28: 146-149, 1994, Journal of Medical Virology 43: 231-237.] and in Italy [Li Vigni et al., 1994, 2nd International Congress of Papillomavirus in Human Pathology (Abstracts), p 100.].

A combined indirect ELISA and immunoblotting for the detection of intrathecal herpes simplex virus IgG antibody synthesis in patients with herpes simplex virus encephalitis.

A combined indirect ELISA and immunoblotting assay was used for the detection of intrathecal synthesis of IgG antibodies to herpes simplex virus (HSV) in patients with HSV encephalitis (HSVE). By using these two assays as well as three markers for blood-brain barrier, leakage can be easily excluded. A total of 21 sera and 24 cerebrospinal fluid (CSF) samples from 11 patients with HSVE were examined. For seven patients more than one pair of serum and CSF were available. For one patient IgG antibodies began to be detectable in CSF after the sixth day from the onset of the disease. In the other 10 patients the intrathecal synthesis of HSV IgG antibodies was detected later than the sixth day and reached high optical density (OD) values after the 10th day from the onset of disease, at the earliest. In contrast, intrathecal HSV antibody synthesis was not found in specimens taken from 20 patients with acute meningitis who composed our negative control group. The use of a combined indirect ELISA and of an immunoblotting assay on a single dilution of serum and CSF for HSV IgG synthesis in the central nervous system (CNS) allowed the diagnosis of HSVE after the first week of disease.

Typing of human papillomaviruses in condylomata acuminata from Greece.

DNA samples from recurrent condylomata acuminata biopsies of Greek males and females were examined for the presence of human papillomavirus (HPV) DNA using high-stringency Southern blot hybridization analysis. Of the twenty-six biopsies, 25 were positive for the HPV 6/11-related DNA sequences, and when further analyzed with the polymerase chain reaction (PCR) the HPV-negative biopsy was also positive for HPV 6/11 DNA. Nineteen specimens were further characterized based on their Pstl restriction endonuclease hybridization pattern. Twelve biopsies were positive for HPV 6a, one biopsy was positive for HPV 11a, and one biopsy was positive for HPV 6c DNA. Three specimens contained HPV 6/11 related DNA that gave an unusual Pstl pattern, and one specimen appeared to represent a multiple HPV infection containing HPV 6/11- and HPV 31/35/39-related sequences. Finally, one sample contained a mixture of HPV 6a DNA and an HPV 6a-like genome. Biopsies were also taken from adjacent apparently normal tissue, 0.5 cm away from the lesion, in 19 of the patients. Only one of these was found to be positive for HPV 6a DNA by Southern blot analysis.

Evolution of human papillomavirus type 18: an ancient phylogenetic root in Africa and intratype diversity reflect coevolution with human ethnic groups.

Papillomaviruses are an ideal model system for the study of DNA virus evolution. On several levels, phylogenetic trees of papillomaviruses reflect the relationship of their hosts. Papillomaviruses isolated from remotely related vertebrates form major branches. One branch of human papillomaviruses (HPVs) includes an ape and two monkey papillomaviruses, possibly because the diversification of the viruses predated the separation of the infected-primate taxa. This hypothesis predicts that the root of the evolution of some if not all HPV types should point to Africa, since humans evolved from nonhuman primates in this continent. We tested this hypothesis and compared the genomic sequences of HPV type 18 (HPV-18) isolates from four continents. Diversity within HPV-18 correlates with patterns of the evolution and spread of Homo sapiens: HPV-18 variants, just like HPV-16 variants, are specific for the major human races, with maximal diversity in Africa. Outgroup rooting of the HPV-18 tree against HPV-45, which is closely related to HPV-18, identifies African HPV-18 variants at the root of the tree. The identification of an African HPV-45 isolate further reduces the evolutionary distance between HPV-18 and HPV-45. HPV-18 variants from Amazonian Indians are the closest relatives to those from Japanese and Chinese patients and suggest that a single point mutation in the phylogenetically evaluated genomic segment represents at least 12,000 years of evolution. We estimate that diversity within HPV-18 and probably within other HPV types evolved over a period of more than 200,000 years and that diversity between HPV types evolved over several million years.

Naturally occurring antibodies against nerve growth factor in human and rabbit sera: comparison between control and herpes simplex virus-infected patients.

Antibodies against nerve growth factor (NGF) in sera were detected by enzyme-linked immunosorbent assays (ELISA), by their isolation after passage of sera through NGF immunoadsorbent columns and by their specificity to bind and immunoprecipitate mouse NGF as well as to stain by immunohistochemical methods cellular sites of NGF synthesis. Increased levels of anti-NGF antibodies were found in sera of herpes simplex virus (HSV)-infected patients but not in HSV-inoculated rabbits. As HSV latency is known to be promoted by NGF in vitro, these results may suggest that anti-NGF antibodies modulate the cytokine function of NGF and thus might play a role in HSV infection. The biological function of circulating antibodies against NGF, in general, is now open to future investigation.