Hormone-regulated expression and distribution of versican in mouse uterine tissues

Abstract Background Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. Methods Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. Results In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. Conclusion These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co- glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice.

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

Background:The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. Methods Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. Results and Discussion We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. Conclusion Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.

Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

The dystrophin gene, located at Xp21, codifies dystrophin, which is part of a protein complex responsible for the membrane stability of muscle cells. Its absence on muscle causes Duchenne Muscular Dystrophy (DMD), a severe disorder, while a defect of muscle dystrophin causes Becker Muscular Dystrophy (DMB), a milder disease. The replacement of the defective muscle through stem cells transplantation is a possible future treatment for these patients. Our objective was to analyze the potential of CD34+ stem cells from umbilical cord blood to differentiate in muscle cells and express dystrophin, in vitro. Protein expression was analyzed by Immunofluorescence, Western Blotting (WB) and Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR). CD34+ stem cells and myoblasts from a DMD affected patient started to fuse with muscle cells immediately after co-cultures establishment. Differentiation in mature myotubes was observed after 15 days and dystrophin-positive regions were detected through Immunofluorescence analysis. However, WB or RT-PCR analysis did not detect the presence of normal dystrophin in co-cultures of CD34+ and DMD or DMB affected patients' muscle cells. In contrast, some CD34+ stem cells differentiated in dystrophin producers' muscle cells, what was observed by WB, reinforcing that this progenitor cell has the potential to originate muscle dystrophin in vitro, and not just in vivo like reported before.

The effects of long-term endurance training on the immune and endocrine systems of elderly men: the role of cytokines and anabolic hormones

Abstract Background a decline in immune and endocrine function occurs with aging. The main purpose of this study was to investigate the impact of long-term endurance training on the immune and endocrine system of elderly men. The possible interaction between these systems was also analysed. Results elderly runners showed a significantly higher T cell proliferative response and IL-2 production than sedentary elderly controls. IL-2 production was similar to that in young adults. Their serum IL-6 levels were significantly lower than their sedentary peers. They also showed significantly lower IL-3 production in comparison to sedentary elderly subjects but similar to the youngs. Anabolic hormone levels did not differ between elderly groups and no clear correlation was found between hormones and cytokine levels. Conclusion highly conditioned elderly men seem to have relatively better preserved immune system than the sedentary elderly men. Long-term endurance training has the potential to decelerate the age-related decline in immune function but not the deterioration in endocrine function.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1β maturation

Abstract Background Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1β and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1β was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Cough and dyspnea during bronchoconstriction: comparison of different stimuli

Abstract Background Bronchial challenge tests are used to evaluate bronchial responsiveness in diagnosis and follow-up of asthmatic patients. Challenge induced cough has increasingly been recognized as a valuable diagnostic tool. Various stimuli and protocols have been employed. The aim of this study was to compare cough and dyspnea intensity induced by different stimuli. Methods Twenty asthmatic patients underwent challenge tests with methacholine, bradykinin and exercise. Cough was counted during challenge tests. Dyspnea was assessed by modified Borg scale and visual analogue scale. Statistical comparisons were performed by linear mixed-effects model. Results For cough evaluation, bradykinin was the most potent trigger (p < 0.01). In terms of dyspnea measured by Borg scale, there were no differences among stimuli (p > 0.05). By visual analogue scale, bradykinin induced more dyspnea than other stimuli (p ≤ 0.04). Conclusion Bradykinin seems to be the most suitable stimulus for bronchial challenge tests intended for measuring cough in association with bronchoconstriction.

Immunohistochemical features of a papillary squamous cell carcinoma of the endometrium with transitional cell differentiation

An 84-year-old woman underwent hysterectomy due to a friable endometrial mass infiltrating almost half way through the myometrial wall. The tumor consisted of papillary structures with thin fibrovascular cores covered by several layers of pleomorphic cells. The deeply located neoplastic cells were ovoid with a pale eosinophilic cytoplasm resembling urothelial cells. A diagnosis of papillary squamous cell carcinoma of the endometrium with transitional cell differentiation was made. Although she recovered well after surgery, she died one year later because of disseminated disease. In an attempt to obtain new insights into the physiopathology of this very rare tumor, an immunohistochemical panel with 32 markers was performed. The neoplastic cells were positive for cytokeratin 5, vimentin, p63, p21, VEGF, Ki67, BAG1, and bcl-2. The expression of BAG-1 and bcl-2 may suggest that anti-apoptotic stimuli are preponderant in this neoplasm.

In vitro behaviour of endothelial cells on a titanium surface

Abstract Background Endothelial cells play an important role in the delivery of cells to the inflammation site, chemotaxis, cell adhesion and extravasation. Implantation of a foreign material into the human body determines inflammatory and repair reactions, involving different cell types with a plethora of released chemical mediators. The evaluation of the interaction of endothelial cells and implanted materials must take into account other parameters in addition to the analysis of maintenance of cell viability. Methods In the present investigation, we examined the behavior of human umbilical vein endothelial cells (HUVECs) harvested on titanium (Ti), using histological and immunohistochemical methods. The cells, after two passages, were seeded in a standard density on commercially plate-shaped titanium pieces, and maintained for 1, 7 or 14 days. Results After 14 days, we could observe a confluent monolayer of endothelial cells (ECs) on the titanium surface. Upon one-day Ti/cell contact the expression of fibronectin was predominantly cytoplasmatic and stronger than on the control surface. It was observed strong and uniform cell expression along the time of α5β1 integrin on the cells in contact with titanium. Conclusion The attachment of ECs on titanium was found to be related to cellular-derived fibronectin and the binding to its specific receptor, the α5β1 integrin. It was observed that titanium effectively serves as a suitable substrate for endothelial cell attachment, growth and proliferation. However, upon a 7-day contact with Ti, the Weibel-Palade bodies appeared to be not fully processed and exhibited an anomalous morphology, with corresponding alterations of PECAM-1 localization.

A light in the shadow: the use of Lucifer Yellow technique to demonstrate nectar reabsorption

Abstract Background Nectar reabsorption is a widely known phenomenon, related to the strategy of resource-recovery and also to maintain the nectar homeostasis at the nectary. The method currently performed to demonstrate nectar being reabsorbed involves the use of radioactive tracers applied to the nectary. Although this method works perfectly, it is complex and requires specific supplies and equipment. Therefore, here we propose an efficient method to obtain a visual demonstration of nectar reabsorption, adapting the use of Lucifer Yellow CH (LYCH), a fluorescent membrane-impermeable dye that can enter the vacuole by endocytosis. Results We applied a LYCH solution to the floral nectary (FN) of Cucurbita pepo L., which is a species known for its ability of nectar reabsorption, and to the extrafloral nectary (EFN) of Passiflora edulis Sims which does not reabsorb the secreted nectar. In all tests performed, we observed that LYCH stained the nectary tissues differentially according to the reabsorption ability of the nectary. The treated FN of C. pepo presented a concentrated fluorescence at the epidermis that decreased at the deeper nectary parenchyma, until reaching the vascular bundles, indicating nectar reabsorption in the flowers of the species. In contrast, treated EFN of P. edulis presented fluorescence only at the cuticle surface, indicating that nectar is not reabsorbed by that particular tissue. Conclusion LYCH is an efficient marker to demonstrate nectar reabsorption.

HPV type infection in different anogenital sites among HIV-positive Brazilian women

Abstract Objectives To evaluate the prevalence of human papillomavirus (HPV) types, and risk factors for HPV positivity across cervix, vagina and anus, we conducted a study among 138 women with human immunodeficiency virus (HIV). Goal Compare the prevalence of different HPV types and the risk factors for HPV positivity in three sites. Results The most frequently detected HPV types in all sites were, in decreasing order, HPV16, 53, 18, 61 and 81. Agreement between the cervix and vagina was good (kappa 0.60 – 0.80) for HPV16 and 53 and excellent (Kappa > 0.80) for HPV18 and 61. HPV positivity was inversely associated with age for all combinations including the anal site. Conclusion In HIV positive women, HPV18 is the most spread HPV type found in combinations of anal and genital sites. The relationship of anal to genital infection has implications for the development of anal malignancies. Thus, the efficacy of the current HPV vaccine may be considered not only for the cervix, but also for prevention of HPV18 anal infection among immunossuppressed individuals.

The influence of human papillomavirus type and HIV status on the lymphomononuclear cell profile in patients with cervical intraepithelial lesions of different severity

Abstract Background Immunological alterations are implicated in the increased prevalence of high-grade squamous intraepithelial lesions (HG-SIL) and persistent human papillomavirus (HPV) infection. This study evaluated the expression of CD4, CD8, CD25 (IL-2Rα) and CD28 antigens from SIL biopsies, stratified by HIV status and HPV-type. Biopsies specimens from 82 (35 HIV+) women with a normal cervix, low-grade (LG-SIL) or high-grade lesions (HG-SIL) were studied. CD molecule expression was evaluated by immunohistochemistry and HPV detection/typing performed using PCR techniques. Results CD4 stromal staining was increased in patients with HPV18. Women with HPV16 infection showed decreased: a) CD8 and CD25 stromal staining, b) CD25 staining in LG-SIL epithelium and in HG-SIL stroma. In HIV- women samples, CD28 epithelial staining and CD8 stromal staining surrounding metaplastic epithelium were less intense and even absent, as compared to HIV+ women. Both epithelial and stromal CD8 staining was more intense in the HG-SIL/HIV+ group than in the HG-SIL/HIV- group. Positive correlations were observed between CD4/CD25, CD4/CD28 and CD25/CD28 in the stroma and CD25/CD28 in the epithelium. Conclusion HIV status and HPV-type may influence the lymphomononuclear cell profile present in the spectrum of cervical lesions. The knowledge of the infiltrating cell profile in cervical tumours may help the development of specific anti-tumoural strategies.