Involvement of circulating platelets on the hyperalgesic response evoked by carrageenan and Bothrops jararaca snake venom

Background: The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. Objective: To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. Methods: Rats were (i) rendered thrombocytopenic by administration of polyclonal anti-rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. Results: Platelet counts were markedly diminished in rats administered with either ARPI (+/- 88%) or busulfan (+/- 96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre-treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre-incubation with alkaline phosphatase. Conclusions: Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.

Pathogenic role of anti-endothelial cell antibodies in autoimmune rheumatic diseases

Anti-endothelial cells antibodies have been detected in numerous autoimmune and inflammatory diseases, including systemic lupus erythematous, rheumatoid arthritis, vasculitis and sarcoidosis. Anti-endothelial cells antibodies bind to endothelial cell antigens and induce endothelial damage. Their effects on the endothelial cell have been considered responsible, at least in part, by the vascular injury which occurs in these pathological conditions. Lupus (2009) 18, 1233-1238.

Autoimmune bullous dermatoses

Autoimmune bullous dermatoses are diseases in which blisters and vesicles are the primary and fundamental types of skin lesion. Their classification is based on the location of the blister; intraepidermal and subepidermal. Patients produce autoantibodies against self-specific structures of the skin detectable by immunofluorescence techniques, immunoblotting and ELISA. Recent advances in molecular and cellular biology have brought to knowledge these self-antigens, against which patients are sensitized, and which are found in epidermis or in the dermo-epidermal junction. These are low incidence, but high morbidity diseases that may be fatal. The aim of this article is to review and describe the progress of four autoimmune vesiculobullous disorders; endemic pemphigus foliaccous (wild fire), pemphigus vulgaris, bullous pemphigoid and dermatitis herpetiformis.

HLA-DR in Brazilian patients with polyarteritis nodosa (PAN) and microscopic polyangiitis (MPA)

The aim of this study was to evaluate the frequency and clinical associations of HLA-DR alleles in Brazilian Caucasian patients with polyarteritis nodosa (PAN) or microscopic polyangiitis (MPA). We evaluated 29 Caucasian patients with vasculitis classified as PAN or MPA according to the American College of Rheumatology (ACR) 1990 Criteria, Chapel Hill Consensus Conference (CHCC) nomenclature for vasculitis and EULAR recommendations for conducting clinical studies in systemic vasculitis. HLA-DR alleles were typed using polymerase chain reaction-amplified DNA, hybridized with sequence-specific low resolution primers. DNA obtained from 59 Caucasian healthy blood donors were used as control. In order to evaluate if a specific HLA may have influence on the clinical profile of those diseases, we also divided the patients according to Birmingham vasculitis score (BVAS) and Five-Factors Score (FFS) at the time of diagnosis. Increased frequency of HLA-DRB1*16 (p = 0.023) and DRB4*01 (p = 0.048) was found in patients with higher disease activity at the time of diagnosis (BVAS >= 22). Patients with less severe disease (FFS = 0) had a higher frequency of HLA-DRB1*03 (p = 0.011). Patients with gastrointestinal tract involvement had significantly increased frequency of HLA-DRB1*11 or B1*12 (p = 0.046), B1*13 (p = 0.021) and B3 (p = 0.008). In contrast, patients with renal disease, had higher frequency of DRB1*15 or DRB1*16 (p = 0.035) and B5 (p = 0.035). In the subgroup of patients with MPA, increased frequency of HLA-DRB1*15 was found in patients with BVAS >= 22 (p = 0.038) and FFS >= 1 (p = 0.039) suggesting that this allele is associated with more aggressive disease. Antineutrophil cytoplasmic antibodies (ANCA) negative MPA patients had significantly increased frequency of HLA-DRB1*11 or DRB1*12 when compared to ANCA positive patients (p = 0.023). Our results suggest that HLA-DR alleles may influence PAN and MPA clinical expression and outcome and that in MPA they participate in the mechanisms involved in the development to ANCA.

Relationship of adhesion molecules expression with epithelial differentiation markers during fetal skin development

Background: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P-cadherins, integrins beta-1 and -4, CK10, CK14 and INV was assessed in skin fragments of 10 human fetuses (gestational weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P-cadherins were present on epithelial cell membranes in all layers. CK14 and CK10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E-cadherin was detected in all layers, but the cornified stratum and P-cadherin were observed in the lower layers. CK14 was expressed in basal layer, CK10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

Isolated lymphadenitis due to Histoplasma capsulatum diagnosed by fine-needle aspiration biopsy and immunohistochemistry

In the disseminated form of histoplasmosis, isolation and further identification of Histoplasma capsulatum can be performed by several methods, namely, bone marrow aspiration, blood culture, and liver biopsy. Lymph node disease usually is diagnosed by excisional biopsy. Although fungal stains can identify this fungus, detection of specific antigens by immunohistochemistry shows a higher specificity and sensitivity. This approach can use the cell block method when the material is not sent to fungal cultures or fresh staining.

Non-Human Leukocyte Antigen Antibodies Reactive with Endothelial Cells Could Be Involved in Early Loss of Renal Allografts

Preformed donor-specific human leukocyte antigen (HLA) antibodies have been associated with allograft dysfunction and failure. However, recipients of HLA-identical kidneys can develop acute humoral rejection, implicating putative pathogenic antibodies that are directed against non-HLA antigens. We investigated the presence of endothelial cell reactive antibodies in 11 patients who experienced early loss of their transplanted kidneys owing to humoral rejection and 1 loss from renal venal thrombosis. We examined the potential efficacy of intravenous immunoglobulin to block the binding of these antibodies, as previously suggested for anti-HLA antibodies.

Paracoccidioidomycosis: Cells expressing IL17 and Foxp3 in cutaneous and mucosal lesions

We demonstrated and quantified by immunohistochemistry the population of cells expressing IL17 and Foxp3 in cutaneous and mucosal paracoccidioidomycosis lesions, associating these populations of cells with different presentations of granulomatous response. For this purpose, 61 skin biopsies and 55 oral mucosal biopsies were evaluated. Cells expressing IL17 were distributed in the inflammatory infiltrate in both groups of lesions and were found in the vessels` wall too. Foxp3+ expression was limited to the nuclei of lymphocytes in the inflammatory infiltrate. The distribution of IL17 was similar among the groups; however, Foxp3+ cells were increased in mucosal lesions that displayed compact granulomas. The results suggest that IL17 seems to play a role in paracoccidioidomycosis cutaneous and mucosal lesions, probably as secondary cells in the clearance of the fungal antigens. The presence of Foxp3+ cells both in skin and mucosa corroborates some previous researches that suggest the role of this group of cells in the modulation of local immune response. (C) 2011 Elsevier Ltd. All rights reserved.

Demonstration of epithelial-mesenchymal transition in kidney - The contribution of a coupled histochemical and immunohistochemical staining with Periodic Acid-Thionin Schiff

Traditional Periodic Acid Schiff has been extensively used, coupled with immunohistochemistry for epithelia or mesenchymal cells, to highlight renal tubular basement membrane (TBM). We recently tried to perform such technique in a 5/6 nephrectomy model of progressive renal fibrosis to demonstrate TBM disruption as an evidence for epithelial-mesenchymal transdifferentiation. Despite excellent basement membrane staining with traditional fuchsin-Periodic Acid Schiff, the interface between epithelial and mesenchymal cells was frequently blurred when revealed with 3`3 diaminobenzidine tetrachloride-peroxidase. Also, it was inadequate when revealed with alkaline phosphatase-fast red. We devised a triple staining method with Periodic Acid-Thionin Schiff to highlight basement membrane in blue, after double immunostaining for epithelium and mesenchymal cells. Blue basement membrane rendered a brisk contrast and highlighted boundaries between epithelial-mesenchymal interfaces. This method was easy to perform and useful to demonstrate the TBM, yield a clear demonstration of the very focal TBM disruption found in this model of progressive renal fibrosis.

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-gamma) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-gamma-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-gamma and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-gamma and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.

CpG-ODN combined with Neospora caninum lysate, but not with excreted-secreted antigen, enhances protection against infection in mice

CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens, but there is limited information concerning their use in immunization protocols against neosporosis. This study aimed to evaluate the potential of CpG-ODN combined with Neosporar lysate antigen (NLA) or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninum infection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals with NLA, NLA+CpG, NcESA, NcESA+CpG, CpG (adjuvant control) or PBS (infection control). Serological assays showed an increased specific IgG2a response in animals immunized with either antigen plus adjuvant and elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferative responses upon antigen stimulation were higher in groups immunized with NLA OF NcESA combined with CpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA+CpG demonstrated higher IFN-gamma levels and IFN-gamma/IL-10 ratio. After lethal challenge, mice immunized with NLA+CpG or NLA had lower Morbidity score and body weight changes in comparison to other groups, and animals did not succumb during acute infection. In contrast, NcESA+CpG or NcESA groups exhibited the highest morbidity scores, body weight impairment and mortality rates, associated with greatest brain parasite burden and inflammation. In conclusion, CpG-ODN was able to induce a Th1-type humoral immune response with predominant IgG2a levels for either NLA or NcESA, but resulting in an effective Th1-driven cellular immune response and total Protection only when combined with NLA. Vaccination with NcESA alone or combined with CpG resulted in a strong cellular immune response associated with high levels of IFN-gamma and inflammation, rendering mice more susceptible to parasite challenge. (C) 2009 Elsevier Ltd. All rights reserved.

Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis

Background: Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drug-resistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. Methods: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime-boost with subcutaneous BCG and intramuscular DNAhsp65. Results: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime-boost strategies. Conclusion: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine.

Phenotypic and functional characterization of pulmonary macrophages subpopulations after intratracheal injection of Paracoccidioides brasiliensis cell wall components

A shift in the activation of pulmonary macrophages characterized by an increase of IL-1, INF-alpha and IL-6 production has been induced in mice infected with Paracoccidioides brasiliensis. It is still unclear whether a functional shift in the resident alveolar macrophage population would be responsible for these observations due to the expression of cell surface molecules. We investigated pulmonary macrophages by flow cytometry from mice treated with P. brasiliensis derivatives by intratracheal route. In vivo labeling with the dye PKH26GL was applied to characterize newly recruited pulmonary macrophages from the bloodstream. Pulmonary macrophages from mice inflamed with P. brasiliensis derivatives showed a high expression of the surface antigens CD11b/CD18 and CD23 among several cellular markers. The expression of these markers indicated a pattern of activation of a subpopulation characterized as CD11b(+) or CD23(+), which was modulated in vitro by IFN-gamma and IL-4. Analysis of monocytes labelled with PKH26GL demonstrated that CD11b(+) cells did infiltrate the lung exhibiting a proinflammatoni pattern of activation, whereas CD23(+) cells were considered to be resident in the lung. These findings may contribute to better understand the pathology of lung inflammation caused by P. brasiliensis infection. (C) 2010 Elsevier GmbH. All rights reserved.

Indirect immune detection of ecdysone receptor (EcR) during the formation of DNA puffs in Bradysia hygida (Diptera, Sciaridae)

Gene amplification occurs in Bradysia hygida salivary glands, at the end of the fourth larval instar. The hormone 20-hydroxyecdysone (20E) triggers this process, which results in DNA puff formation. Amplified genes are activated in two distinct groups. The activity of the first group is dependent on high levels of 20E, while the second group needs low hormone levels. Consequently, the salivary glands of B. hygida constitute an interesting biological model to study how 20E, and its receptors, affect gene amplification and activity. We produced polyclonal antibodies against B. hygida EcR (BhEcR). In western blots a polypeptide of about 66 kDa was detected in salivary gland extracts. The antibodies were also used for indirect immune-localization of BhEcR in polytene chromosomes. RNA-polymerase II was also immune-detected. We did not detect the receptor in chromosome C where the first and second groups of DNA puffs form during DNA puff anlage formation, but it was present during puff expansion. During the active phase of both groups of DNA puffs, RNA polymerase II co-localized with BhEcR. After puff regression, these antigens were not detected. Apparently, EcR plays a direct role in the transcription of amplified genes, but its role in gene amplification remains enigmatic.

Influence of Biliary Drainage on the Repair of Hepatic Lesions in Biliary Fibrosis

Background. Bilioduodenal (BD) and biliojejunal (BJ) derivation induce enterobiliary reflux and bile stasis. Decompression of the excluded loop of the Roux-en-Y (BJD) was proposed to minimize these effects. The aim of this study was to compare the influence of these three modalities of biliary bypass on hepatic lesion repair in rats with secondary biliary fibrosis. Materials and Methods. Rats with 15 d of biliary obstruction underwent BD, BJ, and BJD drainage and were compared with a group submitted to simulated operation (SO) and biliary obstruction (CBO). The serum values of total and fractional bilirubin, alkaline phosphatase (ALP), and aminotransferases (AST and ALT), as well as hepatobiliointestinal excretion determined with (99m)Tc-Disida, were used for comparison. In addition, we used morphometric analyses to estimate the mass of the hepatocytes, bile ducts, and liver fibrosis. We also counted hepatic stellate cells (SC). Results. For each of the three modalities of biliary drainage, there were significant reductions in bilirubin, AST, ALP, and the number of SCs. The recovery of the estimated mass of all histologic components occurred only after BJ and BJD; in the BD group, the estimated hepatocyte mass was reduced compared with the SO group. The residual hepatic radioactivity of (99m)Tc-Disida was greater in the BJD group than in the SO group. Conclusions. The interposition of the jejunal loop between the biliary tree and the intestine may slow hepatobiliary clearance of radioactivity, even though it provides the resolution of cholestasis and is effective in recovering from hepatic lesions. (C) 2011 Elsevier Inc. All rights reserved.