CD40/CD40L expression and its prognostic value in cervical cancer

CD40, a member of the tumor necrosis factor receptor (TNFR) family, is known to be involved in immune system regulation, acting as a costimulatory molecule, and in antitumor responses against cancer cells. It is a protein that is expressed in different types of cells, including immune cells and cancer cells (e.g., cervical cancer, breast cancer, melanoma). In this study, we investigated CD40/CD40L transcriptional and protein levels in cervical cancer cell lines and tumors. Higher CD40 expression was observed in cervical cancer cell lines derived from squamous cell carcinomas than from adenocarcinomas. Search of CD40/CD40L expression in cervical cancer tissues in public data sets revealed that about 83% of squamous cell carcinomas express CD40 compared to other cervical tumor subtypes. Moreover, expression of CD40 and CD40L in squamous cervical carcinomas is associated with better overall survival. Therefore, these proteins could be explored as prognostic markers in cervical cancers.

Is there a higher frequency of anal dysplasia and infection by human papillomavirus in Crohn's disease patients?

The aim of this study was to compare the frequency of dysplasia and human papillomavirus (HPV) infection in the anal canal of patients with Crohn's disease (CD) with a control group and assess whether there is a correlation between use of immunosuppressants and anal manifestation of CD. Patients with CD and control individuals were submitted to anal cytology and material collection for polymerase chain reaction (PCR). The cytology was classified as normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), or high-grade (HSIL). PCR was considered positive or negative according to virus presence or absence. A total of 117 patients were included (54 in the control group and 63 in the CD group, being 32 without and 31 with immunosuppressants). ASCUS and LSIL were found in 25.9 and 22.2% of control patients and 28.6 and 39.7% of CD patients. HPV was identified in 14.8% of the control group and 27% of the CD group. In CD patients, HPV was found in 37.5 and 16.1% of those without and with immunosuppressants, respectively. Patients with perianal involvement had 15.6% of PCR positivity. There was no statistical difference in dysplasia and infection by HPV between the groups. Use of immunosuppressants did not influence the result, but anal manifestation was inversely proportional to viral detection.

The contribution of Latin American research to HPV epidemiology and natural history knowledge

Our aim was to review the major contributions of studies conducted in different Latin American (LA) countries to the field of human papillomavirus (HPV) epidemiology, natural history, risk of disease, and prevention strategies, mainly in the uterine cervix. Although cytological screening is established in several countries in LA, incidence and mortality rates from cervical cancer (CC) are still extremely high. Finally, data from large cohort studies conducted in LA countries provided seminal data to propose primary and secondary prevention modalities: the HPV vaccine has been introduced in the national immunization programs of several LA countries and multiple screening experiences using HPV testing are under evaluation in the region.

Impact of HPV infection on the development of head and neck cancer

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is considered to be a distinct clinical entity with better prognosis than the classical tobacco- and alcohol-associated tumors. The increasing incidence of this neoplasia during the last decades highlights the need to better understand the role of HPV in the development of these cancers. Although the proportion of HNSCC attributed to HPV varies considerably according to anatomical site, overall approximately 25% of all HNSCC are HPV-DNA positive, and HPV-16 is by far the most prevalent type. In this review we discuss the existing evidence for a causal association between HPV infection and HNSCC at diverse anatomical head and neck subsites.

Epidemiological and functional implications of molecular variants of human papillomavirus

Human papillomavirus genomes are classified into molecular variants when they present more than 98 percent of similarity to the prototype sequence within the L1 gene. Comparative nucleotide sequence analyses of these viruses have elucidated some features of their phylogenetic relationship. In addition, human papillomavirus intratype variability has also been used as an important tool in epidemiological studies of viral transmission, persistence and progression to clinically relevant cervical lesions. Until the present, little has been published concerning the functional significance of molecular variants. It has been shown that nucleotide variability within the long control region leads to differences in the binding affinity of some cellular transcriptional factors and to the enhancement of the expression of E6 and E7 oncogenes. Furthermore, in vivo and in vitro studies revealed differences in E6 and E7 biochemical and biological properties among molecular variants. Nevertheless, further correlation with additional functional information is needed to evaluate the significance of genome intratypic variability. These results are also important for the development of vaccines and to determine the extent to which immunization with L1 virus-like particles of one variant could induce antibodies that cross-neutralize other variants.

Infection with human papillomaviruses of sexual partners of women having cervical intraepithelial neoplasia

Epidemiological studies show that human papillomaviruses (HPV) are strongly related to cervical cancer and cervical intraepithelial neoplasias (CIN). Unlike the case for women, there are no consistent data on the natural history of HPV in the male population even though these viruses are prevalent in males. We carried out a prospective study to assess the prevalence of HPV in males as well as the factors that determine such infections in 99 male sexual partners of women with CIN. The genitalia of the males were physically examined and subjected to peniscopy for the collection of scrapings which were subjected to the polymerase chain reaction and restriction fragment length polymorphism to detect HPV. Of the 99 males sampled, 54 (54.5 percent) were positive for HPV DNA, 24 percent of whom presented normal peniscopy, 28 percent presented evident clinical lesions and 48 percent isolated lesions consistent with subclinical infection. In the HPV-negative group, 53 percent showed normal peniscopy, 4 percent presented evident clinical lesions and 42 percent isolated lesions consistent with subclinical infection. The study detected a statistically significant association (P < 0.02, Pearson chi-square test) between HPV infection and both the mean number of sexual partners which a male had during his life and the mean number of sexual partners in the year prior to testing. Viral types 6 and 11 were most frequently encountered. The study shows that infection with HPV was frequent in male sexual partners of women with CIN.

Estrogen and progesterone receptors in human papilloma virus-related cervical neoplasia

Estrogen (ER) and progesterone (PR) receptors in the normal uterine cervix, cervical intraepithelial neoplasia and invasive carcinoma were studied in consecutive samples from Hospital do Câncer, São Paulo, between 1996 and 1997. Tissue was collected by removing a fragment of the tumoral area using a 5-mm diameter biopsy punch, followed by removal of a macroscopically normal area as close as possible from the tumor. Histopathological confirmation was obtained for all specimens analyzed. A total of 24 normal tissues, 17 cases of cervical intraepithelial neoplasia and 7 of invasive carcinomas were studied. The ER/PR ratio was determined by immunohistochemistry using monoclonal antibodies specific for each receptor. Adjacent tissue slides were submitted to generic PCR for human papillomavirus (HPV) DNA detection followed by typing by dot blot hybridization. About half (45.8 percent) of the tumors were HPV DNA positive while 29.1 percent of the patients were also HPV positive in their respective normal tissue. ER was negative in the tumoral epithelium of 11 HPV-positive patients (P = 0.04). There was a trend in the ER distribution in normal tissue that was opposite to that from lesions, but it was not statistically significant (P = 0.069). No difference in ER distribution in stromal tissues was observed between HPV-positive and HPV-negative tissues. PR staining was negative in the epithelium of all cases studied. The results obtained from this small number of cases cannot be considered to be conclusive but do suggest that factors related to viral infection affect the expression of these ER/PR cervix receptors.

Human papillomavirus prevalence among women with cervical intraepithelial neoplasia III and invasive cervical cancer from Goiânia, Brazil

This study estimated the prevalence and distribution of human papillomavirus (HPV) types among women with cervical intraepithelial neoplasia (CIN) grade III and invasive cervical cancer from Goiás (Brazil Central Region). Seventy-four cases were analyzed and consisted of 18 CIN III, 48 squamous cell carcinomas, 4 adenocarcinomas, 1 adenosquamous carcinoma and 3 undifferentiated carcinomas. HPV-DNA sequences were examined in formalin-fixed and paraffin-embedded tissues using primers from L1 region GP5+/GP6+. Polymerase chain reaction products were typed with dot blot hybridization using probes for HPV 16, 18, 31, 33, 45, 54, 6/11, 42/43/44, 51/52, 56/58. The prevalence of HPV was estimated to be 76 percent (56/74). HPV 16 was the most frequently found type, followed by HPV 33, 18 and 31. The prevalence of untyped HPV was 6 percent; 79 percent percent of the squamous cell carcinoma cases and 61 percent percent of the CIN III were positive for HPV and the prevalence rate of HPV types was the same for the total number of cases. According to other studies, HPV type 16 is the most prevalent virus in all Brazilian regions, but there is variation regarding to other types. Type 18 is the second most prevalent HPV in North, Southeast and South Brazil regions and types 31 and 33 are the second most prevalent HPV in Northeast and Central Brazil, respectively

TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required

Low frequency of p53 mutations in cervical carcinomas among Brazilian women

Human papillomavirus (HPV) infections of the high-risk types are strongly linked to the development of cervical carcinoma. The HPV oncoproteins E6 and E7 are thought to play a crucial role in this process through their interactions with the p53 protein and the retinoblastoma susceptibility gene product pRb, respectively. E6 binds to p53 protein promoting its degradation. This is considered to contribute to the oncogenesis of HPV-associated anogenital cancer. On the other hand, in HPV-negative cervical carcinoma, p53 mutations are thought to have a role in the transformation process. A total of 122 HPV-positive cervical carcinoma tissue samples were evaluated for the presence of mutations in exons 5-8 of the p53 gene by single-stranded conformation polymorphism analysis and DNA sequencing. Only four missense point mutations were detected. These findings suggest that other mechanisms independent of p53 inactivation may play a role in the genesis of cervical carcinomas.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100 per cent of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 106 leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 106 leukocytes, the limiting dilution assay had sensitivity of 100 per cent, specificity of 92 per cent, a positive predictive value of 43 per cent and a negative predictive value of 100 per cent for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

Genetic susceptibility to HPV infection and cervical cancer

Squamous cell carcinoma of the cervix (SCCC) is one of the leading causes of death in developing countries. Infection with high-risk human papillomavirus (HPV) is the major risk factor to develop malignant lesions in the cervix. Polymorphisms of the MHC and p53 genes seem to influence the outcome of HPV infection and progression to SCCC, although controversial data have been reported. MHC are highly polymorphic genes that encode molecules involved in antigen presentation, playing a key role in immune regulation, while p53 is a tumor suppressor gene that regulates cell proliferation. The HPV E6 protein from high-risk types binds p53 and mediates its degradation by the ubiquitin pathway. The role of these polymorphisms in genetic susceptibility to HPV infection and to SCCC remains under investigation

Detection of point mutations by non-isotopic single strand conformation polymorphism

We have developed a procedure for nonradioactive single strand conformation polymorphism analysis and applied it to the detection of point mutations in the human tumor suppressor gene p53. The protocol does not require any particular facilities or equipment, such as radioactive handling, large gel units for sequencing, or a semiautomated electrophoresis system. This technique consists of amplification of DNA fragments by PCR with specific oligonucleotide primers, denaturation, and electrophoresis on small neutral polyacrylamide gels, followed by silver staining. The sensitivity of this procedure is comparable to other described techniques and the method is easy to perform and applicable to a variety of tissue specimens

A direct method for cDNA cloning employing translation released mRNA

1. A new cloning procedure is described for cDNA synthesis from mRNA released by in vitro translation of polysomes in a cell-free amino acid incorporating system. The usefulness of the method lies in the feasibility of employing nanogram amounts of mRNA. 2. Complementary DNA is synthesized derectly in the translation mixture simply by adjusting the concentration of some components and removing ribosomes by boiling and centrifugation. 3. As an example, we report here the construction and characterization of cDNA clone corresponding to chick alfa (1) procollagen starting from a collagen-synthesizing polysome fraction obtained from chick embryos

As tecnicas envolvidas em engenharia genetica: suas perspectivas e dificuldades. / The involved technics in genetic engineering: its perspectives and difficulties

A essencia da Engenharia Genetica esta na juncao de fragmentos de DNA ou genes de diferentes organismos com o DNA de vetores plasmidiais ou virais, a fim de serem introduzidos e propagados em bacterias ou outras celulas hospedeiras. Com o objetivo de analisar a estrutura genica, os processos envolvidos em sua regulacao e expressao ou, ainda, para produzir proteinas especificas de interesse medico ou comercial, uma serie de tecnicas vem sendo descritas. Este artigo resume a maioria dos metodos empregados, conhecidos em conjunto como a tecnologia do DNA recombinante

Insulina humana: obtencao de uma bacteria contendo a informacao para a sua sintese. / Human insulin: construction of a bacteria containing the information for its synthesis

Empregando a tecnologia de DNA recombinante (clonagem molecular), um clone bacteriano contendo sequencias de DNA complementares a insulina humana foi construido. O RNA mensageiro utilizado para a sintese do DNA complementar foi isolado a partir de um insulinoma de pancreas humano. O plasmideo recombinante pl2, que abriga uma insercao de 400 pares de bases, contem toda a informacao para proinsulina, alem de se estender por 5 a 10 pares de bases na regiao do pre-peptideo. Essas investigacoes deverao beneficiar o estudo da regulacao genica de insulina, seja no individuo normal, seja no estado diabetico. Outrossim, poder-se-a desenvolver um sistema biologico alternativo para a producao deste hormonio em larga escala