MMP-9/RECK imbalance: a mechanism associated with high-grade cervical lesions and genital infection by Human Papillomavirus (HPV) and Chlamydia trachomatis

"Manuscript"

Human papillomavirus (HPV) screening and cervical cancer burden. A Brazilian perspective

This review tackles the issues related to disease burden caused by cervical cancer (CC) and its precursor (CIN) lesions in Brazil. A special focus is given to new technologies with potential to interfere with the development of CC by reducing the high-risk human papillomavirus (hr-HPV)-induced lesions that remain a major public health burden in all developing countries where organized screening programs do not exist. Globally, 85 % of all incident CC and 50 % of CC deaths occur in the developing countries. Unfortunately, most regions of Brazil still demonstrate high mortality rates, ranking CC as the second most common cancer among Brazilian women. Recently, CC screening programs have been tailored in the country to enable early detection of CC precursor lesions and thereby reduce cancer mortality. A combination of HPV testing with liquid-based cytology (LBC) seems to be a promising new approach in CC screening, with high expectation to offer an adequate control of CC burden in this country.

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

Lack of the transcription factor hypoxia-inducible factor (HIF)-1α in macrophages accelerates the necrosis of Mycobacterium avium-induced granulomas

Accepted Manuscript

Evaluation of inflammatory response induced by different types of daily contact lenses

Dissertação de mestrado em Genética Molecular

Active cutaneous leishmaniasis in Brazil, induced by Leishmania donovani chagasi

L.d. chagasi was isolated from active cutaneous leishmaniasis in both human and canine infections in an endemic area in Rio de Janeiro, Brazil. Both isolates were identified by molecular and immunological characterization of the parasite using three different methods: electrophoretic mobility of isoenzymes; restriction endonuclease fragment analysis of kDNA and serodeme analysis using monoclonal antibodies. This seems to be the first well documented case in the New World of a "viscerotropic" Leishmania inducing a case of cutaneous leishmaniasis. This observation emphasizes that the diagnosis of the etiologic agent of human or canine visceral leishmaniasis based solely upon clinical and epidemiological critwria may lead to erroneous conclusions.

Photodynamic therapy with mTHPC and polyethylene glycol-derived mTHPC: a comparative study on human tumour xenografts.

The photosensitizing properties of m-tetrahydroxyphenylchlorin (mTHPC) and polyethylene glycol-derivatized mTHPC (pegylated mTHPC) were compared in nude mice bearing human malignant mesothelioma, squamous cell carcinoma and adenocarcinoma xenografts. Laser light (20 J/cm2) at 652 nm was delivered to the tumour (surface irradiance) and to an equal-sized area of the hind leg of the animals after i.p. administration of 0.1 mg/kg body weight mTHPC and an equimolar dose of pegylated mTHPC, respectively. The extent of tumour necrosis and normal tissue injury was assessed by histology. Both mTHPC and pegylated mTHPC catalyse photosensitized necrosis in mesothelioma xenografts at drug-light intervals of 1-4 days. The onset of action of pegylated mTHPC seemed slower but significantly exceeds that of mTHPC by days 3 and 4 with the greatest difference being noted at day 4. Pegylated mTHPC also induced significantly larger photonecrosis than mTHPC in squamous cell xenografts but not in adenocarcinoma at day 4, where mTHPC showed greatest activity. The degree of necrosis induced by pegylated mTHPC was the same for all three xenografts. mTHPC led to necrosis of skin and underlying muscle at a drug-light interval of 1 day but minor histological changes only at drug-light intervals from 2-4 days. In contrast, pegylated mTHPC did not result in histologically detectable changes in normal tissues under the same treatment conditions at any drug-light interval assessed. In this study, pegylated mTHPC had advantages as a photosensitizer compared to mTHPC. Tissue concentrations of mTHPC and pegylated mTHPC were measured by high-performance liquid chromatography in non-irradiated animals 4 days after administration. There was no significant difference in tumour uptake between the two sensitizers in mesothelioma, adenocarcinoma and squamous cell carcinoma xenografts. Tissue concentration measurements were of limited use for predicting photosensitization in this model.

Endogenous and exogenously-induced immunomodulation of tumour-host responsiveness

In spite of the availability of multiple effector mechanisms of the immune system to combat tumour growth and metastases, their impairment frequently accompanies the appearance of cancer. Factors contributing to this impairment may be related to properties of the host and/or the tumour itself and may be with respect to their origin -endogenous or exogenour. Based on the unique biological behavior of prostate cancer (PCa), and its apparent escape from immune surveillance in the presence of tumour immuno genicity, continuing investigation of endogenous and exogenous factors thought to be relevant to its pathogenesis have been made. For this purpose further studies of the suggested role of human seminal plasma (SePl) and the synthetic oestrogen, diethylstiboestrol (DES), as representative endogenous and exogenous immunomodulatory factors (IMF) of tumour-host responsiveness, together with evaluation of human prostatic tissue extracts and leuprolide (the luteinizing-hormone-releasing-hormone proposed as an alternate to DES therapy) have been made by evaluating their effect on the lytic activity of natural killer (NK) cells. SePl and prostate extracts significantly suppressed NK cell lysis. Physicochemical studies suggest SePl and prostate IMF to be associated with high and low molecular weight macromolecules; and implicate the participation of transglutaminase and prostaglandins. Comparative study of therapeutic levels of DES vs. leuprolide on NK cell lysis demonstrated significant suppression by DES vs. a negligible effect of leuprolide. Metastases are highly prevalent in PCa, and contribute significantly to its morbidity and mortality. Further knowledge of the range of effects of endogenous and exogenous IMF on effector mechanisms of tumour-host responsiveness, to include suppression of NK cells, and elucidation of their nature, may contribute toward our understanding of the unique biological behavior of tumours of the prostate, in addition to improvement in their clinical management.

An atlas of human gene expression from massively parallel signature sequencing (MPSS).

We have used massively parallel signature sequencing (MPSS) to sample the transcriptomes of 32 normal human tissues to an unprecedented depth, thus documenting the patterns of expression of almost 20,000 genes with high sensitivity and specificity. The data confirm the widely held belief that differences in gene expression between cell and tissue types are largely determined by transcripts derived from a limited number of tissue-specific genes, rather than by combinations of more promiscuously expressed genes. Expression of a little more than half of all known human genes seems to account for both the common requirements and the specific functions of the tissues sampled. A classification of tissues based on patterns of gene expression largely reproduces classifications based on anatomical and biochemical properties. The unbiased sampling of the human transcriptome achieved by MPSS supports the idea that most human genes have been mapped, if not functionally characterized. This data set should prove useful for the identification of tissue-specific genes, for the study of global changes induced by pathological conditions, and for the definition of a minimal set of genes necessary for basic cell maintenance. The data are available on the Web at http://mpss.licr.org and http://sgb.lynxgen.com.

Immunoregulation in human Schistosomiasis by idiotypic interactions and lymphokine-mediated mechanisms

Anti-idiotypic (anti-Id) T cells from schistosomiasis patients or former patients proliferate upon exposure to polyclonal or monoclonal anti-soluble egg antigen (SEA) antibodies. Chloroquine does not inhibit, the response, which is induced by F(ab')2 (but not soluble Fab) fragments of these antibodies. Purified T cells from former patients require macrophages or exogenous IL-1 to respond to anti-SEA Ids and can respond to matrix-bound Fab fragments in the presence of IL-1. These anti-Id T cells recognize the Ids directly. Chronic schistosomiasis patients immunoregulate the production of a non-IL-2 lymphokine that stimulates IL-2 receptor expression on resting T cells. This regulation is reversed upon chemotherapeutic cure.

Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions.

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 microgram of VLP given at weekly intervals to anesthetized mice induced high (>10(4)) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-microgram VLP systemic priming followed by two 5-microgram VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.

In vivo and in vitro effects of somatostatin and insulin on glucagon release in a human glucagonoma.

Inhibition of pancreatic glucagon secretion has been reported to be mediated by glucose, insulin and somatostatin. As no human pancreatic alpha-cell lines are available to study in vitro the relative importance of insulin and glucose in the control of pancreatic glucagon release, we investigated a patient presenting with a malignant glucagonoma who underwent surgical resection of the tumour. Functional somatostatin receptors were present as octreotide administration decreased basal glucagon and insulin secretion by 52 and 74%, respectively. The removed tumour was immunohistochemically positive for glucagon, chromogranin A and pancreatic polypeptide but negative for insulin, gastrin and somatostatin. The glucagonoma cells were also isolated and cultured in vitro. Incubation experiments revealed that change from high (10 mM) to low (1 mM) glucose concentration was unable to stimulate glucagon secretion. A dose-dependent inhibition of glucagon release by insulin was however, observed at low glucose concentration. These findings demonstrate that insulin could inhibit glucagon secretion in vitro in the absence of elevated glucose concentrations. These data suggest, as observed in vivo and in vitro in several animal studies, that glucopenia-induced glucagon secretion in humans is not mediated by a direct effect of low glucose on alpha-cells but possibly by a reduction of insulin-mediated alpha-cell suppression and/or an indirect neuronal stimulation of glucagon release.

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor.

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-gamma secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-gamma production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.