Verônica Coelho

Rheumatic Heart Disease: Proinflammatory Cytokines Play a Role in the Progression and Maintenance of Valvular Lesions

Heart lesions of rheumatic heart disease (RHD) patients contain T-cell clones that recognize heart proteins and streptococcal M peptides. To functionally characterize heart-infiltrating T lymphocytes, we evaluated their cytokine profile, both directly in situ and in T-cell lines derived from the heart (HIL). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 expressions were characterized in 20 heart tissue infiltrates from 14 RHD patients by immunohistochemistry. IFN-gamma-, TNF-alpha-, and IL-10-positive cells were consistently predominant, whereas IL-4 was scarce in the valves. In agreement with these data, the in vitro experiments, in which 13 HILs derived from heart samples of eight patients were stimulated with M5 protein and the immunodominant M5 (81-96) peptide, IL-4 was detected in HIL derived from the atrium (three of six) but not from the valve (zero of seven). IFN-gamma and IL-10 production were detected in culture supernatants in 11 of 13 and 6 of 12 HILs, respectively. The predominant IFN-gamma and TNF-alpha expression in the heart suggests that Th1-type cytokines could mediate RHD. Unlike in reversible myocardium inflammation, the significantly lower IL-4 expression in the valvular tissue (P = 0.02) may contribute to the progression of the RHD leading to permanent valvular damage (relative risk, 4.3; odds ratio, 15.8). The lack of IL-4 in vitro production by valve-derived HIL also emphasizes the more severe tissue destruction in valves observed in RHD.

Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen.

Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.

Exercise training restores the endothelial progenitor cells number and function in hypertension: implications for angiogenesis

Objectives: Aerobic exercise training has been established as an important nonpharmacological treatment for hypertension. We investigated whether the number and function of endothelial progenitor cells (EPCs) are restored after exercise training, potentially contributing to neovascularization in hypertension. Methods: Twelve-week-old male spontaneously hypertensive rats (SHRs, n = 14) and Wistar–Kyoto (WKY, n = 14) rats were assigned to four groups: SHR; trained SHR (SHR-T); WKY; and trained WKY. Exercise training consisted of 10 weeks of swimming. EPC number and function, as well as the vascular endothelial growth factor (VEGF), nitrotyrosine and nitrite concentration in peripheral blood were quantified by fluorescence-activated cell sorter analysis (CD34+/Flk1+ cells), colony-forming unit assay, ELISA and nitric oxide (NO) analyzer, respectively. Soleus capillary/fiber ratio and protein expression of VEGF and endothelial NO synthase (eNOS) by western blot were assessed. Results: Exercise training was effective in reducing blood pressure in SHR-T accompanied by resting bradycardia, an increase in exercise tolerance, peak oxygen uptake (VO2) and citrate synthase activity. In response to hypertension, the amount of peripheral blood-EPC and number of colonies were decreased in comparison with control levels. In contrast, exercise training normalized the EPC levels and function in SHR-T accompanied by an increase in VEGF and NO levels. In addition, oxidative stress levels were normalized in SHR-T. Similar results were found in the number and function of bone marrow EPC. Exercise training repaired the peripheral capillary rarefaction in hypertension by a signaling pathway VEGF/eNOS-dependent in SHR-T. Moreover, improvement in EPC was significantly related to angiogenesis. Conclusion: Our data show that exercise training repairs the impairment of EPC in hypertension, which could be associated with peripheral revascularization, suggesting a mechanism for its potential therapeutic application in vascular diseases.

Locally Produced Survival Cytokines IL-15 and IL-7 may be Associated to the Predominance of CD8+ T cells at Heart Lesions of Human Chronic Chagas Disease Cardiomyopathy

Human chronic Chagas disease cardiomyopathy (CCC) is an inflammatory‐dilated cardiomyopathy occurring years after infection by the protozoan Trypanosoma cruzi. The heart inflammatory infiltrate in CCC shows a 2:1 predominance of CD8+ in relation to CD4+ T cells, with a typical Th1‐type cytokine profile. However, in vitro expansion of infiltrating T cells from heart biopsy‐derived fragments with interleukin‐2 (IL‐2) and phytohaemagglutinin leads to the outgrowth of CD4+ over CD8+ T cells. We hypothesized that survival cytokines, such as IL‐2, IL‐7 and IL‐15 might be differentially involved in the growth and maintenance of heart‐infiltrating and peripheral CD8+ T cells from CCC patients. We found that IL‐7 and IL‐15 were superior to IL‐2 in the expansion and viability of CD8+ T cells from both PBMC and heart‐infiltrating T‐cell lines from CCC patients, and the combination of the three cytokines showed synergic effects. Heart‐infiltrating CD8+ T cells showed higher expression of both IL‐15Rα and γc chain than CD4+ T cells, which may explain the improvement of CD8+ T‐cell growth in the presence of IL‐2 + IL‐7 + IL‐15. Immunohistochemical identification of IL‐15 and the higher mRNA expression of IL‐15Rα, IL‐7 and γc chain in CCC heart tissues compared with control individuals indicate in situ production of survival cytokines and their receptors in CCC hearts. Together, our results suggest that local production of IL‐7 and IL‐15 may be associated with the maintenance and predominance of CD8+ T cells, the cells effecting tissue damage in CCC hearts.

Restricted heterogeneity of T cell receptor variable alpha chain transcripts in hearts of Chagas’disease cardiomyopathy patients

The role of autoimmunity in the pathogenesis and progression of heart lesions in the chronic phase of Chagas’disease is controversial. In the absence of parasites in situ, the T cell infiltrate seen in heart lesions may be the primary determinant of tissue damage ultimately leading to heart failure and death. We used the polymerase chain reaction to amplify each known T cell receptor (TCR) Vα and Vβ subfamily‐specific sequence in transcripts derived from heart samples obtained from Chagas’cardiomyopathy patients. The average number of TCR Vα. subfamilies (7·1 per tissue sample) was significantly lower than that for TCR Vβ subfamilies (15·1 per sample). The average percentage of tissue samples positive per TCR Vα. and Vβ subfamily was respectively 39·6% vs. 73·5%. These data suggest that, in Chagas’heart lesions, the detectable TCR Vα. repertoire is significantly narrower than TCR Vβ repertoire. On the other hand, in normal heart tissue, diversity of Vα. and Vβ TCR is similar among the scarce circulating T cell population. Such evidence of restricted TCR V region repertoire has been described in experimental and human autoimmune diseases. Our results are consistent with the possibility that T cells responsible for heart damage in chronic Chagas’cardiomyopathy may be recognizing a few heart‐specific antigenic targets.

Cellular autoreactivity against heat shock protein 60 in renal transplant patients: peripheral and graft-infiltrating responses

Autoreactivity to heat shock protein 60 (Hsp60) has been implicated in the pathogenesis and regulation of chronic inflammation, especially in autoimmune diseases. In transplantation, there is a lack of information regarding the cytokine profile and specificity of cells that recognize self‐Hsp60 as well as the kinetics of autoreactivity following transplantation. We studied the cellular reactivity of peripheral and graft‐infiltrating lymphocytes against Hsp60 in renal transplant patients. Cytokine production induced by this protein in peripheral blood mononuclear cells indicated a predominance of interleukin (IL)‐10 during the late post‐transplantation period, mainly in response to intermediate and C‐terminal peptides. Patients with chronic rejection presented reactivity to Hsp60 with a higher IL‐10/interferon (IFN)‐γ ratio compared to long‐term clinically stable patients. Graft‐infiltrating T cell lines, cocultured with antigen‐presenting cells, preferentially produced IL‐10 after Hsp60 stimulation. These results suggest that, besides its proinflammatory activity, autoreactivity to Hsp60 in transplantation may also have a regulatory role.

Rethinking the multiple roles of B cells in organ transplantation.

Purpose of reviewTo discuss the B-cell diverse functions in organ transplantation, highlighting the emerging debate on the role of regulatory B cells (Bregs). We underscore the need to re-examine and integrate data on B-cell functional activities, aiming to discriminate their regulatory (REG) and inflammatory (INFLAMMA) functions and to translate this knowledge for the development of novel immunomodulatory therapeutic strategies and to rethink the current ones. Recent findingsData from both experimental models and clinical trials point that B cells of various phenotypes have immunoregulatory activity and play an important role in controlling graft inflammation. Data on the state of operational tolerance, in kidney transplantation, suggest the relevance of preserving a healthy B-cell compartment – in numbers and in the Breg capacity to activate the CD40/STAT3 signalling pathway – for achieving and maintaining homeostasis. Moreover, autoantibodies also comprise transplant immunobiology and it seems that not all alloantibodies are deleterious. SummaryThe role of B cells, in organ transplantation, can no longer be taken as mere generators of plasma cells, which produce alloantibodies deleterious to the graft. B cells also seem to integrate a complex immunoregulatory network in organ transplantation, with Bregs of various phenotypes and possibly also antibodies. The functional discrimination of REG/INFLAMMA B-cell roles needs to be considered in the clinical setting.

CD4+CD25+Foxp3+ Indirect Alloreactive T cells from Renal Transplant Patients Suppress Both the Direct and Indirect Pathways of Allorecognition

Alloreactive T cells recognize donor antigens by two routes: direct and indirect pathways of allorecognition. Although the direct pathway is reported to be dominant in allograft rejection, indirect allorecognition also plays an important role. Indirect alloreactivity is also observed in renal transplant patients irrespective of rejection. Previously we showed a predominance of interleukin (IL)‐10 induced by indirect allorecognition of donor human leucocyte antigen (HLA)‐DR peptides, suggesting the existence of indirect alloreactive T cells displaying regulatory activity. In the present work, our objective was to characterize these regulatory T cells. We detected indirect alloproliferation of peripheral blood mononuclear cells (PBMC) from renal transplant patients, induced by donor HLA‐DR peptides, dependent on IL‐4 or IL‐10, suggesting regulatory activity as part of the alloreactive T‐cell repertoire. PBMC‐derived indirect alloreactive T‐cell lines were established and produced both inflammatory and regulatory cytokines. We showed that two of these T‐cell lines which were able to inhibit both direct and indirect alloproliferation of another T‐cell line from the same patient presented a CD4+CD25+Foxp3+ T‐cell population. These data support the idea that indirect alloreactive T cells may also have regulatory activity and may contribute to the maintenance of the human renal allograft.

Heat-shock proteins: Inflammatory versus regulatory attributes

The workshop on “Heat-shock proteins: inflammatory versus regulatory attributes” was held in August 2007 at the 13th International Congress of Immunology (ImmunoRio2007), Rio de Janeiro, Brazil. This report contains a summary of the presentations, the discussions during the workshop, and the ideas exchanged with experts as well as among presenters before the meeting.

Treatment with Encapsulated Hsp60 Peptide (p277) Prolongs Skin Graft Survival in a Murine Model of Minor Antigen Disparity

The increased expression of heat shock protein (Hsp)60 in different kinds of graft tissues has been associated with a proinflammatory role and rejection. However, there are very few reports in which treatment with Hsp60 delays skin allograft rejection. The aim of this work was to evaluate the capacity of encapsulated human Hsp60‐derived peptide p277 to delay graft rejection in two murine models of skin transplantation with minor antigen disparities. Briefly, BALB/c mice and C57BL/6 were intranasally pre‐treated with five doses of Hsp60 p277 peptide encapsulated in polylactide‐co‐glycolide acid microspheres (PLGM), and received skin grafts from DBA2 mice and 129/B6 (F1) mice respectively. The treatment with the peptide increased skin graft survival more than 20 days in both the mouse strains, mainly in C57BL/6 recipients (P < 0.05). Also, p277‐treated BALB/c and C57BL/6 mice showed IL‐10 and IFN‐γ production, induced by p277 peptide. For the first time, a mucosal schedule using the Hsp60 C‐terminal peptide p277 encapsulated in PLGM showed some survival prolongation of skin grafts bearing minor antigen disparities. Our results suggest a potential role for Hsp60‐based therapy and the mucosal route as a useful tool to control the inflammatory response to allografts.