The impact of pretransplant donor-specific antibodies on graft outcome in renal transplantation: a six-year follow-up study

OBJECTIVE: The significance of pretransplant, donor-specific antibodies on long-term patient outcomes is a subject of debate. This study evaluated the impact and the presence or absence of donor-specific antibodies after kidney transplantation on short-and long-term graft outcomes. METHODS: We analyzed the frequency and dynamics of pretransplant donor-specific antibodies following renal transplantation from a randomized trial that was conducted from 2002 to 2004 and correlated these findings with patient outcomes through 2009. Transplants were performed against a complement-dependent T-and B-negative crossmatch. Pre- and posttransplant sera were available from 94 of the 118 patients (80%). Antibodies were detected using a solid-phase (Luminex (R)), single-bead assay, and all tests were performed simultaneously. RESULTS: Sixteen patients exhibited pretransplant donor-specific antibodies, but only 3 of these patients (19%) developed antibody-mediated rejection and 2 of them experienced early graft losses. Excluding these 2 losses, 6 of 14 patients exhibited donor-specific antibodies at the final follow-up exam, whereas 8 of these patients (57%) exhibited complete clearance of the donor-specific antibodies. Five other patients developed "de novo'' posttransplant donor-specific antibodies. Death-censored graft survival was similar in patients with pretransplant donor-specific and non-donor-specific antibodies after a mean follow-up period of 70 months. CONCLUSION: Pretransplant donor-specific antibodies with a negative complement-dependent cytotoxicity crossmatch are associated with a risk for the development of antibody-mediated rejection, although survival rates are similar when patients transpose the first months after receiving the graft. Our data also suggest that early posttransplant donor-specific antibody monitoring should increase knowledge of antibody dynamics and their impact on long-term graft outcome.

Naturally acquired antibodies to Plasmodium vivax blood-stage vaccine candidates (PvMSP-1(19) and PvMSP-3 alpha(359-798)) and their relationship with hematological features in malaria patients from the Brazilian Amazon

An important step when designing a vaccine is identifying the antigens that function as targets of naturally acquired antibodies. We investigated specific antibody responses against two Plasmodium vivax vaccine candidates, PvMSP-1(19) and PvMSP-3 alpha(359-798). Moreover, we assessed the relationship between these antibodies and morbidity parameters. PvMSP-1(19) was the most immunogenic antigen and the frequency of responders to this protein tended to increase in P. vivax patients with higher parasitemia. For both antigens, IgG antibody responses tended to be lower in patients who had experienced their first bout of malaria. Furthermore, anemic patients presented higher IgG antibody responses to PvMSP-3 alpha(359-798). Since the humoral response involves a number of antibodies acting simultaneously on different targets, we performed a Principal Component Analysis (PCA). Anemic patients had, on average, higher first principal component scores (IgG1/IgG2/IgG3/IgG4 anti-MSP3 alpha), which were negatively correlated with hemoglobin levels. Since antibodies against PfMSP-3 have been strongly associated with clinical protection, we cannot exclude the possibility of a dual role of PvMSP-3 specific antibodies in both immunity and pathogenesis of vivax malaria. Our results confirm the high immunogenicity of the conserved C terminus of PvMSP-1 and points to the considerable immunogenicity of polymorphic PvMSP-3 alpha(359-798) during natural infection. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Oxidized low-density lipoproteins and their antibodies: Relationships with the reverse cholesterol transport and carotid atherosclerosis in adults without cardiovascular diseases

Background: Metabolic predictors and the atherogenicity of oxidized LDL (oxLDL) and the specific antibodies against oxLDL (oxLDL Ab) are unclear and controversial. Methods: In 107 adults without atherosclerotic manifestations, we measured oxLDL and oxLDL Ab, and also the activities of CETP. PLTP, lipases and the carotid intima-media thickness (cIMT). Comparisons were performed for the studied parameters between the lowest and the highest tertile of oxLDL and oxLDL Ab, and the relationships between studied variables were evaluated. Results: Subjects with higher oxLDL Ab present reduced hepatic lipase activity and borderline increased cIMT. In the highest oxLDL tertile, besides the higher levels of total cholesterol, LDL-C and apoB100, we found reduced CETP activity and higher cIMT. A significant correlation between oxLDL Ab and cIMT, independent of oxLDL, and a borderline correlation between oxLDL and cIMT independent of oxLDL Ab were found. In the multivariate analysis, apoAl was a significant predictor of oxLDL Ab, in contrast to regulation of oxLDL by apoB100, PLTP and inverse of CETP. Conclusions: In adults without atherosclerotic disease, the metabolic regulation and carotid atherosclerosis of oxLDLAb and oxLDL groups, characterized a dual trait in oxLDL Ab, as a contributor to carotid atherosclerosis, much less so than oxidized LDL, and with a modest atheroprotective role. (C) 2012 Elsevier B.V. All rights reserved.

Human immature dental pulp stem cells share key characteristic features with limbal stem cells

Objectives: Limbal stem cells (LSC) are self-renewing, highly proliferative cells in vitro, which express a set of specific markers and in vivo have the capacity to reconstruct the entire corneal epithelium in cases of ocular surface injury. Currently, LSC transplantation is a commonly used procedure in patients with either uni- or bilateral total limbal stem cells deficiency (TLSCD). Although LSC transplantation holds great promise for patients, several problems need to be overcome. In order to find an alternative source of cells that can partially substitute LSC in cornea epithelium reconstruction, we aimed at investigating whether human immature dental pulp stem cells (hIDPSC) would present similar key characteristics as LSC and whether they could be used for corneal surface reconstruction in a rabbit TLSCD model. Materials: We used hIDPSC, which co-express mesenchymal and embryonic stem cell markers and present the capacity to differentiate into derivative cells of the three germinal layers. TLSCD was induced by chemical burn in one eye of rabbits. After 30 days, the opaque tissue formed was removed by superficial keratectomy. Experimental group received undifferentiated hIDPSC, while control group only received amniotic membrane (AM). Both groups were sacrificed after 3 months. Results and conclusions: We have demonstrated, using immunohistochemistry and reverse transcription-polymerase chain reaction, that hIDPSCs express markers in common with LSC, such as ABCG2, integrin beta 1, vimentin, p63, connexin 43 and cytokeratins 3/12. They were also capable of reconstructing the eye surface after induction of unilateral TLSCD in rabbits, as shown by morphological and immunohistochemical analysis using human-specific antibodies against limbal and corneal epithelium. Our data suggest that hIDPSCs share similar characteristics with LSC and might be used as a potential alternative source of cells for corneal reconstruction.

Cytokine inhibition and time-related influence of inflammatory stimuli on the hyperalgesia induced by the nucleus pulposus

The symptoms of lumbar disc herniation, such as low back pain and sciatica, have been associated with local release of cytokines following the inflammatory process induced by the contact of the nucleus pulposus (NP) with the spinal nerve. Using an animal experimental model of intervertebral disc herniation and behavioral tests to evaluate mechanical (electronic von Frey test) and thermal (Hargreaves Plantar test) hyperalgesia in the hind paw of rats submitted to the surgical model, this study aimed to detect in normal intervertebral disc the cytokines known to be involved in the mechanisms of inflammatory hyperalgesia, to observe if previous exposure of the intervertebral disc tissue to specific antibodies could affect the pain behavior (mechanical and thermal hyperalgesia) induced by the NP, and to observe the influence of the time of contact of the NP with the fifth lumbar dorsal root ganglion (L5-DRG) in the mechanical and thermal hyperalgesia. The cytokines present at highest concentrations in the rat NP were TNF-alpha, IL-1 beta and CINC-1. Rats submitted to the disc herniation experimental model, in which a NP from the sacrococcygeal region is deposited over the right L5-DRG, showed increased mechanical and thermal hyperalgesia that lasted at least 7 weeks. When the autologous NP was treated with antibodies against the three cytokines found at highest concentrations in the NP (TNF-alpha, IL-1 beta and CINC-1), there was decrease in both mechanical and thermal hyperalgesia in different time points, suggesting that each cytokine may be important for the hyperalgesia in different steps of the inflammatory process. The surgical remotion of the NP from herniated rats 1 week after the implantation reduced the hyperalgesia to the level similar to the control group. This reduction in the hyperalgesia was also observed in the group that had the NP removed 3 weeks after the implantation, although the intensity of the hyperalgesia did not decreased totally. The removal of the NP after 5 weeks did not changed the hyperalgesia observed in the hind paw, which suggests that the longer the contact of the NP with the DRG, the greater is the possibility of development of chronic pain. Together our results indicate that specific cytokines released during the inflammatory process induced by the herniated intervertebral disc play fundamental role in the development of the two modalities of hyperalgesia (mechanical and thermal) and that the maintenance of this inflammation may be the most important point for the chronification of the pain.

Involvement of TSSA (trypomastigote small surface antigen) in Trypanosoma cruzi invasion of mammalian cells

TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca2+-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a similar to 2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.

Dynamic Activation and Repression of the Plasmodium falciparum rif Gene Family and Their Relation to Chromatin Modification

The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the "poised'' mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes.

Serrumab: A human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom

In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNF alpha, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom.

Staining of the Internal Limiting Membrane with the Use of Heavy Brilliant Blue G

Background: Brilliant blue G (BBG) is frequently used in chromovitrectomy to facilitate internal limiting membrane (ILM) peeling. A study was initiated to evaluate if heavy BBG is safe and effective in staining the ILM. Methods: We studied 30 eyes, 23 with idiopathic macular holes and 7 of patients with diabetic macular edema. Removal of the ILMs was assisted by heavy BBG staining. In cases with histopathological correlation the ILMs were evaluated with hematoxylin and eosin, Masson's trichrome, periodic acid-Schiff and glial fibrillary acidic protein staining. In addition, immunohistochemistry was also performed using specific antibodies for vimentin, neuron-specific enolase, factor VIII and CD68. Using the Image-Pro Plus software of Media Cybernetics Co. we found an average thickness in ILMs. Results: Of the ILM specimens sent, 19/30(63.33%) could not be processed properly because of the limited sample material, recognizing only fragments of dispersed fibrillar material. In macular hole ILMs we found an average thickness of 1.3 +/- 0.65 mu m, and in diabetic macular edema ILMs an average thickness of 6.2 +/- 1.4 mu m. Conclusions: In heavy BBG-assisted ILM peeling we observed no intraoperative or postoperative complications after a mean follow-up of 12 months. Heavy BBG could be an effective and safe vehicle for staining the ILM. Copyright (C) 2012 S. Karger AG, Basel

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Abstract Background Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs.

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.

Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.

Antinuclear antibodies testing as a routine screening for systemic lupus erythematosus in patients presenting first-episode psychosis

Aims: This report discusses the use of antinuclear antibody (ANA) detection as a screening test for neuropsychiatry systemic lupus erythematosus (NPSLE) in patients presenting a first-episode psychosis. Methods: We reviewed the medical records of 85 patients admitted to an emergency service due to first-episode psychosis, during a 1-year period, for whom ANA detection was performed through an IFI HEp2 cell assay. ANA-positive patients were subsequently evaluated for autoantibodies and neuroimaging exams. Results: Three patients presented as ANA positive in the initial screening and further investigation confirmed NPSLE in two patients. The patients were treated with antipsychotics and cyclophosphamide pulses with satisfactory outcomes. Conclusion: Even though ANA detection is not specific, it is a low-cost procedure and could be an important screening test for NPSLE in the early-onset psychosis.

Anti-aquaporin-4 antibodies in the context of assorted immune-mediated diseases

Background and purposes: Anti-aquaporin 4 antibodies are specific markers for Devics disease. This study aimed to test if this high specificity holds in the context of a large spectrum of systemic autoimmune and non-autoimmune diseases. Methods: Anti-aquaporin-4 antibodies (NMO-IgG) were determined by indirect immunofluorescence (IIF) on mouse cerebellum in 673 samples, as follows: group I (clinically defined Devic's disease, n = 47); group II [ inflammatory/demyelinating central nervous system (CNS) diseases, n = 41]; group III (systemic and organ-specific autoimmune diseases, n = 250); group IV (chronic or acute viral diseases, n = 35); and group V (randomly selected samples from a general clinical laboratory, n = 300). Results: MNO-IgG was present in 40/47 patients with classic Devic's disease (85.1% sensitivity) and in 13/22 (59.1%) patients with disorders related to Devic's disease. The latter 13 positive samples had diagnosis of longitudinally extensive transverse myelitis (n = 10) and isolated idiopathic optic neuritis (n = 3). One patient with multiple sclerosis and none of the remaining 602 samples with autoimmune and miscellaneous diseases presented NMO-IgG (99.8% specificity). The autoimmune disease subset included five systemic lupus erythematosus individuals with isolated or combined optic neuritis and myelitis and four primary Sjogren's syndrome (SS) patients with cranial/peripheral neuropathy. Conclusions: The available data clearly point to the high specificity of anti-aquaporin-4 antibodies for Devic's disease and related syndromes also in the context of miscellaneous non-neurologic autoimmune and non-autoimmune disorders.

Prevalence of non-organ-specific autoantibodies in a rural community from northeastern Brazil: a population-based study

Non-organ-specific autoantibodies (NOSA) are well-recognized diagnostic markers of autoimmune hepatitis (All-l) and primary biliary cirrhosis (PBC), but can also be observed in patients with viral hepatitis as well as in healthy subjects. The aim of this study was to evaluate the prevalence of NOSA in subjects living in a rural community in Brazil and to correlate their occurrence with the presence of liver disease. Seven hundred twenty-five apparently healthy subjects were randomly selected for assessment of antinuclear (ANA), anti-smooth muscle (SMA), antimitochondrial (AMA), anti-liver/kidney microsome type 1, and anti-liver cytosol type 1 antibodies. Subjects with those NOSA were evaluated for the presence of AIH, PBC, and viral hepatitis. Reactivities for all NOSA, SMA, ANA, and AMA were detected, respectively, in 14, 10, 4, and 0.1% of subjects, with a mean titer of 1:40. NOSA-positive subjects were significantly older and more frequently females. No correlation was observed between the occurrence of NOSA and PBC. AIH, or viral hepatitis. The prevalence of NOSA in Brazilians was 14%. They were usually low titer. NOSA were more frequently observed in females and older subjects and their presence was not correlated with the presence of AIH, PBC, or viral hepatitis. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.