[Biological risk in non-clinical biological and chemical laboratories]. / II rischio biologico nei laboratori biologici e chimici non sanitari.

The D.Lgs 626/94, regarding the improvement of workers safety in the workplace, introduces the necessity of the biological hazards assessment. In case of not sanitary chemical and biological laboratories, workers are subject to biological hazards due to potential exposure, because many biological agents could be present in the samples to be analysed, and also for deliberated use of micro organisms. However the assessment of the air and surfaces monitoring results in such environment is still difficult without Guidelines that indicate levels of acceptable exposure and contamination, and reference limits in order to judge "safe" the environment. The following report describes a microbiological monitoring into the Laboratories of HERA SpA and wants to underline the need to produce Guidelines dedicated to these particular workplaces environment, in order to standardize air quality sampling procedures and results assessment.

Analysis of extended genomic rearrangements in oncological research.

Screening for genomic rearrangements is a fundamental task in the genetic diagnosis of many inherited disorders including cancer-predisposing syndromes. Several methods were developed for analysis of structural genomic abnormalities, some are targeted to the analysis of one or few specific loci, others are designed to scan the whole genome. Locus-specific methods are used when the candidate loci responsible for the specific pathological condition are known. Whole-genome methods are used to discover loci bearing structural abnormalities when the disease-associated locus is unknown. Three main approaches have been employed for the analysis of locus-specific structural changes. The first two are based on probe hybridization and include cytogenetics and DNA blotting. The third approach is based on PCR amplification and includes microsatellite or single nucleotide polymorphism (SNP) genotyping, relative allele quantitation, real-time quantitative PCR, long PCR and multiplex PCR-based methods such as multiplex ligation-dependent probe amplification and the recently developed nonfluorescent multiplex PCR coupled to high-performance liquid chromatography analysis. Whole-genome methods include cytogenetic methods, array-comparative genomic hybridization, SNP array and other sequence-based methods. The goal of the present review is to provide an overview of the main features and advantages and limitations of methods for the screening of structural genomic abnormalities relevant to oncological research.

Patterns of K-ras mutation in colorectal carcinomas from Iran and Italy (a Gruppo Oncologico dell'Italia Meridionale study): influence of microsatellite instability status and country of origin.

BACKGROUND: K-ras mutations are a key step in colorectal cancer progression. Such mutations have been widely studied in case series from Western countries but there are few data on the rate and spectrum of mutations in tumors from countries where the epidemiological features of the disease are different. PATIENTS AND METHODS: Tumor samples from 182 Iranian colorectal cancer patients (170 sporadic cases and 12 HNPCC cases) were screened for K-ras mutations at codons 12, 13 and 61 by sequencing analysis. The cases were also characterized for microsatellite instability at mononucleotide repeats by PCR and fragment analysis, and classified according to microsatellite instability status. The frequency and the spectrum of K-ras mutations were compared with those observed in a series of colorectal cancer patients from Italy. RESULTS: K-ras mutations were observed in 68/182 (37.4%) cases. Mutation frequencies were similar in HNPCC-associated, sporadic MSI-H and sporadic microsatellite-stable (MSS) tumors. However, the G13D substitution was more frequent in HNPCC (3/4, 75%) and sporadic MSI-H (7/11, 63.6%) tumors compared to sporadic MSS tumors (11/53, 20.4%) (P <0.01). Comparison of mutations in the two series from Iran and Italy showed a significantly higher frequency of G13D among Italian patients. CONCLUSIONS: While the frequency of K-ras mutations could be similar, the mutational spectrum could be differentially influenced by genetic and environmental factors.

Multicenter comparison of PCR assays for detection of human herpesvirus 8 DNA in semen.

Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors (n = 30) and human immunodeficiency virus (HIV)-infected patients (n = 7) plus positive (n = 4) and negative (n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods.

Latent BK virus infection and Kaposi's sarcoma pathogenesis.

We have analyzed by PCR skin lesions from classic, endemic and AIDS-related Kaposi's sarcoma (KS), as well as from KS-derived cell lines, the presence of ubiquitous transforming viruses. BK virus (BKV), a transforming human papovavirus which has been associated with human tumors, was detected in 100% of KS skin lesions and 75% of KS cell lines. KS specimens contained a full-length, intact BKV early region, but minor rearrangements were observed in some tumors. BKV was also detected with a high prevalence (57-67%) in genital tissues and sperm, thus fulfilling the role of a sexually transmitted agent in KS. The closely related JC virus (JCV), which has never been associated with human malignancies, was present in 11-20% of KS specimens and was detected with a low prevalence (0-21%) in genital tissues and sperm. Simian virus 40 (SV40) was not detected in any KS lesions. Herpes simplex virus (HSV) DNA sequences were detected in 20-25% of KS lesions. Malignant human papillomavirus (HPV) types 16 and 18 and benign HPV types 6 and 11 were detected in KS specimens with a similar prevalence of 11-83%, suggesting that the presence of HPV-transforming sequences is not a specific trait of HPV interaction with KS tissue. Furthermore, JCV, SV40, HSV and HPV DNA sequences were not detected in KS cell lines, suggesting that these viruses are not associated to KS neoplastic cells in KS tissue. KS cell lines were also negative for DNA sequences of KS-HV, the novel herpesvirus detected in primary KS lesions. The constant association of BKV DNA with KS lesions and KS cell lines suggests that BKV-transforming functions may participate in the development of KS.

Kaposi's sarcoma-associated herpesvirus DNA sequences in prostate tissue and human semen.

BACKGROUND: Sequences of novel herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), have been indentified in Kaposi's sarcoma tissue, but it is not known whether the virus is transmitted by sexual contact. METHODS: Using the polymerase chain reaction (PCR), we searched for KSHV DNA sequences in ejaculates from 43 healthy men and tissue from the urogenital tract or prostate of 100 immunocompetent adults. RESULTS: In an unblinded analysis, we identified KSHV DNA sequences in 2 of 20 tissue specimens from the urinary tract (10 percent; 15 men and 5 women), 3 of 46 specimens from the female genital tract (6.5 percent), 4 of 18 specimens from the glans or foreskin (22 percent), 7 of 16 specimens from the prostate (44 percent), and 30 or 33 ejaculates (91 percent). By contrast, such sequences were present in 1 of 18 samples of normal skin (5.5 percent) and 1 of 14 samples of peripheral-blood mononuclear cells (PBMCs; 7.1 percent). Ejaculates and PBMC samples from each of 10 study subjects were analyzed in a blinded, coded fashion, along with PBMCs and biopsy specimens of normal skin from 4 and 8 other patients, respectively. This analysis confirmed the presence of KSHV DNA sequences in semen. Viral DNA was not found in the sperm heads but was present in the fraction of the ejaculates that contained urothelial and other types of cells. Point mutations were found in PCR products amplified from both prostate tissue and sperm samples. CONCLUSIONS: KSHV infects a large proportion of healthy adults and is probably transmitted by sexual contact.

Human herpesvirus 8 is present in the lymphoid system of healthy persons and can reactivate in the course of AIDS.

In this study the prevalence of human herpesvirus (HHV) 8 DNA was determined in biopsies from persons with lymphoproliferative disorders and in peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-seronegative and HIV-infected persons. The results show that HHV-8 is present in 10% of biopsies from HIV-seronegative persons: HHV-8 is detected with similar prevalence values in HIV-infected patients with lymphoproliferative diseases, but the virus load is higher. HHV-8 was also found in PBMC. The presence of monoclonal Epstein-Barr virus (EBV) genomes in malignant lymphoproliferations was only infrequently associated with HHV-8 infection. Therefore, HHV-8 is fairly common in the population, and the lymphoid system could represent a reservoir of latently infected cells from which the virus may reactivate in conditions of immunodepression; furthermore, HHV-8 and EBV do not seem to act in conjunction in lymphomagenesis.

DNA rearrangements impairing BK virus productive infection in urinary tract tumors.

Eighty-nine tissue specimens from the urinary tract and prostate were analyzed for the presence and physical state of BK virus (BKV) DNA. Large T antigen gene sequences were amplified by PCR from prostate, kidney, ureter, and bladder with prevalences ranging from 50 to 83%. Sequence analysis of PCR products from the high variable BKV regulatory region showed that these tissues contained a new BKV strain (URO1). URO1 presents a duplication of part of the 68- and 39-bp elements of the viral enhancer, and a 68-bp deletion spanning part of the 39- and 63-bp enhancer elements. Six neoplastic specimens (11.5%), but none of the control tissues, contained viral DNA in amounts detectable by Southern blot hybridization (P < 0.05). The tumors positive by Southern blot hybridization harbored rearranged and/or integrated DNA sequences whose size was apparently incompatible with assembly into a viral particle. A full-length, macroscopically intact BKV early region was amplified from these tumors by PCR. The restriction pattern of the rearranged sequences was simple, suggesting that tumors were clonal and that DNA rearrangement occurred at an early stage of neoplastic initiation or progression.

Herpesvirus-like DNA sequences in non-AIDS Kaposi's sarcoma.

The pathogenesis of Kaposi's sarcoma (KS) is suggested to be related to an infectious agent transmitted by sexual contact. Recently, DNA sequences homologous to gamma herpesviridae have been identified in AIDS-associated KS, but no information is available concerning non-AIDS-associated KS. Five classic, 12 endemic, and 17 AIDS-associated KS skin lesion specimens were analyzed and all were positive for these novel DNA sequences. Twenty-four specimens from normal skin were negative. These novel sequences were also found in lymph nodes from human immunodeficiency virus-positive patients with AIDS or lymphadenopathy syndrome but not in peripheral blood mononuclear cells from normal subjects. Therefore, the data support the view that a novel gamma herpesvirus might be specifically associated with KS etiopathogenesis.

Chimeric BK virus DNA episomes in a papillary urothelial bladder carcinoma.

BK virus (BKV) DNA sequences were identified in a papillary urothelial bladder carcinoma by Southern blot hybridization. The carcinoma contained both integrated and extrachromosomal DNA. Integrated sequences had a clonal restriction pattern, suggesting that BKV was integrated at some early stage of neoplastic initiation or progression. Viral episomes consisted of a population of covalent polymers based on a high-molecular-weight DNA unit, about 11-12 kb in size. DNA sequences non-homologous to the BKV genome were encompassed within DNA episomes, suggestive of acquisition of cellular sequences by viral DNA replication at the integration site. Extrachromosomal, chimeric DNA molecules were present at an average level of about 50 copies per cell, but their size, apparently incompatible with viral assembly, showed that BKV productive infection was impaired. The data suggest that infected cells underwent reversible changes affecting autonomous BKV DNA replication.

Activation of eukaryotic transcriptional promoters by the bovine papillomavirus E1-replication factor.

It has been suggested that the bovine papillomavirus type 1 E1 replication factor may inhibit E2-conditional gene expression from viral promoter P89. To study the possible role of the E1 protein in gene expression, HeLa cells were transfected with E2-conditional or E2-independent reporter plasmids and with vectors expressing the E1 and E2 open reading frames. The data show that: (i) replication factor E1 stimulates gene expression from a variety of eucaryotic transcriptional promoters; (ii) activation of gene expression by E1 also occurs in the absence of viral activator E2-TA; (iii) expression of a recombinant plasmid containing the human papillomavirus type 18 origin of DNA replication is stimulated, but not repressed, by E1, and (iv) E1-dependent activation of gene expression does not reflect the amplification of transfected plasmids.