Interferon and Alternative Activation of Monocyte/Macrophages in Systemic Sclerosis-Associated Pulmonary Arterial Hypertension

Objective. To explore the relationship between biomarkers of pulmonary arterial hypertension (PAH), interferon (IFN)-regulated gene expression, and the alternative activation pathway in systemic sclerosis (SSc). Methods. Peripheral blood mononuclear cells (PBMCs) were purified from healthy controls, patients with idiopathic PAH, and SSc patients (classified as having diffuse cutaneous SSc, limited cutaneous SSc [lcSSc] without PAH, and lcSSc with PAH). IFN-regulated and ""PAH biomarker"" genes were compared after supervised hierarchical clustering. Messenger RNA levels of selected IFN-regulated genes (Siglec1 and MX1), biomarker genes (IL13RA1, CCR1, and JAK2), and the alternative activation marker gene (MRC1) were analyzed on PBMCs and on CD14- and CD14+ cell populations. Interleukin-13 (IL-13) and IL-4 concentrations were measured in plasma by immunoassay. CD14, MRC1, and IL13RA1 surface expression was analyzed by flow cytometry. Results. Increased PBMC expression of both IFN-regulated and biomarker genes distinguished SSc patients from healthy controls. Expression of genes in the biomarker cluster, but not in the IFN-regulated cluster, distinguished lcSSc with PAH from lcSSc without PAH. The genes CCR1 (P < 0.001) and JAK2 (P < 0.001) were expressed more highly in lcSSc patients with PAH compared with controls and mainly by CD14+ cells. MRC1 expression was increased exclusively in lcSSc patients with PAH (P < 0.001) and correlated strongly with pulmonary artery pressure (r = 0.52, P = 0.03) and higher mortality (P = 0.02). MRC1 expression was higher in CD14+ cells and was greatly increased by stimulation with IL-13. IL-13 concentrations in plasma were most highly increased in lcSSc patients with PAH (P < 0.001). Conclusion. IFN-regulated and biomarker genes represent distinct, although related, clusters in lcSSc patients with PAH. MRC1, a marker for the effect of IL-13 on alternative monocyte/macrophage activation, is associated with this severe complication and is related to mortality.

Oral tolerance attenuates airway inflammation and remodeling in a model of chronic pulmonary allergic inflammation

We investigated the effects of oral tolerance (OT) in controlling inflammatory response, hyperresponsiveness and airway remodeling in guinea pigs (GP) with chronic allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/mL-OVA group) or normal saline (NS group). OT was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st ovalbumin inhalation (OT1 group) or after the 4th (OT2 group). The induction of OT in sensitized animals decreased the elastance of respiratory system (Ers) response after both antigen and methacholine challenges, peribronchial edema formation, eosinophilic airway infiltration, eosinophilopoiesis, and airways collagen and elastic fiber content compared to OVA group (P < 0.05). The number of mononuclear cells and resistance of respiratory system (Rrs) responses after antigen and methacholine challenges were decreased only in OT2 group compared to OVA group (P < 0.05). Concluding, our results show that inducing OT attenuates airway remodeling as well as eosinophilic inflammation and respiratory system mechanics. (C) 2008 Elsevier B.V. All rights reserved.

Histological evaluation of biocompatibility of lyophilized bovine pericardium implanted subcutaneously in rats

This article aims at investigating in vivo evaluation of lyophilization procedure on the biocompatibility of bovine pericardium treated with glutaraldehyde (GA). The bovine pericardium was fixed with 0.5% glutaraldehyde during 10 days and preserved in 4% formaldehyde (FA). Two groups of samples were prepared from treated membranes: Group 1, nonlyophilized samples and Group 2, lyophilized samples. Male Sprague-Dawley rats (4 weeks after birth) were anesthetized (pentobarbital sodium 25 mg/kg of body weight) and in each one were implanted subcutaneously in the dorsal region a sample from Group 1 and another from Group 2. These samples were explanted after 30 days for histological analysis. No intercurrences took place after the surgery. No differences (P > 0.05) in the calcification, granulomatous reaction, mononuclear infiltration, and granulation tissue development was observed between both groups. The implanted lyophilized samples presented a trend for a reduced inflammatory reaction. Lyophilization of the bovine pericardium does not seem to increase the above listed tissue reaction.

Antigen-presenting cells in draining lymph nodes of goats repeatedly infested by the Cayenne tick Amblyomma cajennense nymphs

Resistance to tick feeding has been previously shown to be an acquired, immunologically mediated phenomenon in goats, associated with cutaneous basophilia to nymphs of Amblyomma cajennense, the Cayenne tick, after repeated infestations. On the other hand, it is well known that antigen-presenting cells (APCs) play an important role in the host immune reaction to tick infestations. The most able APCs for Th cells are the well defined dendritic cells, mononuclear phagocytes and B-lymphocytes. Immunohistochemical analysis of draining lymph nodes of goats repeatedly infested with nymphs of the ixodid tick A. cajennense to search for APCs was done. Pre-scapular lymph nodes draining the tick attachment sites were collected 15 days after both the first and third infestations. Tick infestations resulted in increased number of CD21(+) B lymphocytes in lymph nodes after the tertiary infestation. However, the number of CD11b(+) and CD11c(+) cells were not altered after the successive infestations. Lower numbers of CD11c(+) cells had infiltrated lymph nodes responsible for draining the tick infested skin. These findings suggest that acquired immunity of goats against nymphs of A. cajennense is possibly established by B lymphocytes during the first infestation and that APCs may play a key role in this mechanism.

Modulation of cutaneous inflammation induced by ticks in contrasting phenotypes of infestation in bovines

Tick saliva contains molecules that are inoculated at the site of attachment on their hosts in order to modulate local immune responses and facilitate a successful blood meal. Bovines express heritable, contrasting phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus: breeds of Bos taurus indicus are significantly note resistant than those of Bos taurus taurus. Tick saliva may contain molecules that interfere with adhesion of leukocytes to endothelium and resistant hosts may mount an inflammatory profile that is more efficient to hamper the tick`s blood meal. We show in vitro that adhesion of peripheral blood mononuclear cells to monolayers of cytokine-activated bovine umbilical endothelial cells was significantly inhibited by tick saliva. The inflammatory response to bites of adults of R. microplus mounted by genetically resistant and susceptible bovine hosts managed in the same pasture was investigated in vivo. The inflammatory infiltrates and levels of message coding for adhesion molecules were measured in biopsies of tick-bitten and control skin taken when animals of both breeds were exposed to low and high tick infestations. Histological studies reveal that cutaneous reactions of resistant hosts to bites of adult ticks contained significantly more basophils and eosinophils compared with reactions of the susceptible breed. Expression of the adhesion molecules - intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin - was higher in adult-infested skin of susceptible hosts undergoing low infestations compared to resistant hosts; when host was exposed to high infestations expression of these adhesion molecules was down-regulated in both phenotypes of infestations. Expression of leukocyte adhesion glycoprotein-1 (LFA-1) was higher in skin from susceptible hosts undergoing low or high infestations compared to resistant hosts. Conversely, higher levels of E-selectin, which promotes adhesion of memory T cells, were expressed in skin of resistant animals. This finding may explain the resistant host`s ability to mount more rapid and efficient secondary responses that limit hematophagy and infestations. The expression profiles observed for adhesion molecules indicate that there are differences in the kinetics of the inflammatory reactions mounted by resistant and susceptible hosts and the balance between tick and host is affected by the number of tick bites a host receives. We show that the contrasting phenotypes of infestations seen in bovines infested with R. microplus are correlated with differences in the cellular and molecular composition of inflammatory infiltrates elicited by bites with adult ticks. (C) 2009 Published by Elsevier B.V.

Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice

P>Strongyloides stercoralis is an intestinal nematode capable of chronic, persistent infection and hyperinfection of the host; this can lead to dissemination, mainly in immunosuppressive states, in which the infection can become severe and result in the death of the host. In this study, we investigated the immune response against Strongyloides venezuelensis infection in major histocompatibility complex (MHC) class I or class II deficient mice. We found that MHC II(-/-) animals were more susceptible to S. venezuelensis infection as a result of the presence of an elevated number of eggs in the faeces and a delay in the elimination of adult worms compared with wild-type (WT) and MHC I(-/-) mice. Histopathological analysis revealed that MHC II(-/-) mice had a mild inflammatory infiltration in the small intestine with a reduction in tissue eosinophilia. These mice also presented a significantly lower frequency of eosinophils and mononuclear cells in the blood, together with reduced T helper type 2 (Th2) cytokines in small intestine homogenates and sera compared with WT and MHC I(-/-) animals. Additionally, levels of parasite-specific immunoglobulin M (IgM), IgA, IgE, total IgG and IgG1 were also significantly reduced in the sera of MHC II(-/-) infected mice, while a non-significant increase in the level of IgG2a was found in comparison to WT or MHC I(-/-) infected mice. Together, these data demonstrate that expression of MHC class II but not class I molecules is required to induce a predominantly Th2 response and to achieve efficient control of S. venezuelensis infection in mice.

Genetic Susceptibility Loci in Rheumatoid Arthritis Establish Transcriptional Regulatory Networks with Other Genes

Linkage studies have identified the human leukocyte antigen (HLA)-DRB1 as a putative rheumatoid arthritis (RA) susceptibility locus (SL). Nevertheless, it was estimated that its contribution was partial, suggesting that other non-HLA genes may play a role in RA susceptibility. To test this hypothesis, we conducted microarray transcription profiling of peripheral blood mononuclear cells in 15 RA patients and analyzed the data, using bioinformatics programs (significance analysis of microarrays method and GeneNetwork), which allowed us to determine the differentially expressed genes and to reconstruct transcriptional networks. The patients were grouped according to disease features or treatment with tumor necrosis factor blocker. Transcriptional networks that were reconstructed allowed us to identify the interactions occurring between RA SL and other genes, for example, HLA-DRB1 interacting with FNDC3A (fibronectin type III domain containing 3A). Given that fibronectin fragments can stimulate mediators of matrix and cartilage destruction in RA, this interaction is of special interest and may contribute to a clearer understanding of the functional role of HLA-DRB1 in RA pathogenesis.

Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells

Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection. (C) 2011 Elsevier B.V. All rights reserved.

High expression of AURKA and AURKB is associated with unfavorable cytogenetic abnormalities and high white blood cell count in patients with acute myeloid leukemia

In the present study, we analyzed AURKA and AURKB gene expression in 70 acute myeloid leukemia (AML) patients. There was no difference between leukemic samples and bone marrow mononuclear cells (BMMCs, n = 8) or CD34(+) progenitors (n = 10) from healthy donors. High white blood cells (WBC) counts were observed in the AURKA(+) and AURKB(+) groups, but no significant differences regarding age, gender, platelet counts or frequency of FLT3-ITD mutations. AURKA, but not AURKB, expression was independently associated with high WBC counts (OR: 3.15, 95% CI 1.07-9.24, p = 0.03). Moreover, the majority of cases that overexpressed AURKA and AURKB presented unfavorable cytogenetic abnormalities (p < 0.001). In conclusion, we described a significant association between overexpression of AURKA/B and cytogenetics findings in AML, which may be relevant to new therapeutic approaches, based on Aurora kinase inhibitors. (C) 2010 Elsevier Ltd. All rights reserved.

A BALB/c mouse model shows that liver involvement in dengue disease is immune-mediated

In the present study, BALB/c mice were used to develop a model for the hepatic injury associated to dengue infection. Histological analysis after subcutaneous inoculation with a low viral dose of dengue-2 virus showed Kupffer cell hyperplasia and an increased inflammatory cellular infiltrate next to the bile ducts on days 5, 7 and 14 post-inoculation, mainly characterized by the presence of mononuclear cells. The liver mRNA transcription level of IL-1 beta was highest on the 5th day post-infection (p.i.) and decreased by the 21st day, TNF-alpha showed a peak of mRNA transcription after 14 days p.i. coinciding with the regression of cellular infiltrates and elevated expression of TGF-beta mRNA. Serum AST and ALT levels were slightly elevated at 7 and 14 days post-infection. Dengue-2 RNA levels were undetectable in the liver on any of the days following inoculation. Our observations suggest that, as it is true for humans, the animals undergo a transient and slight liver inflammation, probably due to local cytokine production and cellular infiltration in the liver. (C) 2010 Elsevier Inc. All rights reserved.

TNF microsatellite alleles may confer protection against the development of lipodystrophy syndrome in Brazilian HIV patients

P>The aim of this study was to evaluate the frequency of TNFa-e microsatellites and the promoter region (TNF-308 and TNF-238) in HIV/AIDS-infected patients presenting or not lipodystrophy syndrome (LS). The design is the genetic case-control association study. Microsatellite and the TNF promoter region polymorphisms were amplified by PCR and submitted to polyacrylamide gel electrophoresis. The genotypes and allele frequencies for 67 HIV-positive patients with lipodystrophy were compared with 50 HIV-positive patients with no evidence of lipodystrophy and with 131 healthy HIV-negative individuals. The presence of the TNFa5 allele could provide HIV/AIDS patients with protection against developing LS. The presence of TNF-308G allele, as well as of its homozygote TNF-308GG, were associated with susceptibility to developing LS. In addition, the presence of the haplotype TNFe3-d3-238G-308A-c1-a5-b7 suggests protection against developing that syndrome. This study highlights that polymorphic sites spanning the region nearby the TNF locus are associated with LS development in HIV/AIDS patients.

Effect of stem cells seeded onto biomaterial on the progression of experimental chronic kidney disease

Different routes for the administration of bone marrow-derived cells (BMDC) have been proposed to treat the progression of chronic renal failure (CRF). We investigated whether (1) the use of bovine pericardium (BP) as a scaffold for cell therapy would retard the progression of CAF and (2) the efficacy of cell therapy differently impacts distinct degrees of CRF. We used 2/3 and 5/6 models of renal mass reduction to simulate different stages of chronicity. Treatments consisted of BP seeded with either mesenchymal or mononuclear cells implanted in the parenchyma of remnant kidney. Renal function and proteinuria were measured at days 45 and 90 after cell implantation. BMDC treatment reduced glomerulosclerosis, interstitial fibrosis and lymphocytic infiltration. Immunohistochemistry showed decreased macrophage accumulation, proliferative activity and the expression of fibronectin and alpha-smooth muscle-actin. Our results demonstrate: (1) biomaterial combined with BMDC did retard the progression of experimental CRF; (2) cellular therapy stabilized serum creatinine (sCr), improved creatinine clearance and 1/sCr slope when administered during the less severe stages of CRF; (3) treatment with combined therapy decreased glomerulosclerosis, fibrosis and the expression of fibrogenic molecules; and (4) biomaterials seeded with BMDC can be an alternative route of cellular therapy.

Genital Manifestation of Graft-vs.-Host Disease: A Series of Case Reports

Introduction. After hematopoietic stem cell transplantation (HSCT), many patients present genital graft-vs.-host disease (GVHD) that can culminate with sexual problems, which are poorly dimensioned. Aim. We hope to draw attention to the need to perform genital biopsy to diagnose genital GVHD, and thus to call attention to the need to incorporate careful attention to sexual health in the treatment of these patients. Methods. Five allogeneic stem cell transplant recipients complaining of coital pain after HSCT were clinically diagnosed for genital GVHD. Genital biopsies were given for histological analysis, and microphotographs of the corresponding marked field in the slide were taken. Specimens were evaluated by the site pathologist and then sent to a reference pathologist, each blinded to the histological findings. A literature search was performed in PubMed/MEDLINE (1966-2009) for cross-sectional and cohort studies or trials related to genital GVHD. Expert opinions peer reviews and case reports were also considered. Main Outcome Measures. HSCT, genital GVHD, genital biopsy. Results. The biopsy showed evidence of dilated apoptotic cells in the basal layer and detachment of the epithelial lining of the mucosa, hyalinization and thickening of collagen fibers, capillary ectasia, and mononuclear inflammatory infiltrate of the submucosa. Three patients presented vulval lesion such as leucoplasia and ulcer on the large lip. Histological analyses showed evidence of epithelial hyperplasia and influx of inflammatory cells to the epithelial surface, intercellular edema and spongiosis, apoptotic bodies on the basal layer of the epithelium, spongiosis, and nuclear vacuolization. A common treatment based on corticotherapy resulted in complete remission of coetaneous or mucous genital lesions in all five patients. Conclusion. Genital biopsy is important to differentially diagnose GVHD and secondary symptoms due to hypoestrogenism. Prevention is the most important step in controlling the evolution GVHD in the vagina to prevent vaginal obstruction and sexual dysfunction. da Silva Lara LA, de Andrade JM, Mauad LMQ, Ferrarese SR, Marana HRC, Tiezzi DG, and de Sa Rosa e Silva ACJ. Genital manifestation of graft-vs.-host disease: A series of case reports. J Sex Med 2010;7:3216-3225.

Dorsal root ganglionectomy for the diagnosis of sensory neuropathies. Surgical technique and results

Background: Inflammatory diseases stand out among sensory neuronopathies because, in their active phase, they can be treated with immunosuppressive agents. Immunosuppressive therapy may present severe adverse effects and requires previous inflammatory activity confirmation. Sensory neuronopathies are diagnosed based on clinical and EMG findings. Diagnostic confirmation and identification of inflammatory activity are based on sensory ganglion histopathological examination. We describe the surgical technique used for dorsal root ganglionectomy in patients with clinical/EMG diagnosis of sensory neuronopathies. Methods: The sensory ganglion was obtained from 15 patients through a small T7-T8 hemilaminectomy and foraminotomy to expose the C7 root from its origin to the spinal nerve bifurcation. In 6 patients, the dural cuff supposed to contain the ganglion was resected en bloc; and in 9 patients, the ganglion was obtained through a longitudinal incision of the dural cuff and microsurgical dissection from the ventral and dorsal roots and radicular arteries. All ganglia were histopathologically examined. Results: No ganglion was found in the dural cuff in 2 patients submitted to en bloc removal, and the ganglion was removed in all patients who underwent microsurgical dissection. All but 2 patients that had ganglion examination presented a neuronopathy of nerve cell loss, 3 with mononuclear inflammatory infiltrate. These patients underwent immunosuppressive therapy, and 2 of them presented clinical improvement. No surgical complications were observed. Conclusions: Microsurgical dorsal root ganglionectomy for diagnosing inflammatory sensory ganglionopathies was effective and safe. Although safe, en bloc resection of the proximal dural cuff was not effective for this purpose. (c) 2008 Published by Elsevier Inc.

Effect of BCG stimulus on proinflammatory cytokine production in laryngeal cancer

Background Evaluate the production of TNF and IL-6 in the supernatant of peripheral blood mononuclear cell (PBMC) cultures of patients with supraglottic laryngeal cancer before and after surgical treatment. Materials and methods Adherent cell cultures were stimulated with LPS and BCG. Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cytokine concentration was determined by ELISA in supernatants of mononuclear cell cultures. Results In non-stimulated cultures, lower TNF cytokine levels were detected during the late postoperative (LP) period compared to control (P = 0.02). LP TNF and IL-6 levels were high in cultures stimulated with LPS compared with the preoperative period (PREOP) (P = 0.007; P = 0.008, respectively). Stimulation with BCG led to increased levels of TNF and IL-6 during the LP period compared to control (P = 0.001; P = 0.04, respectively). Conclusions BCG is able to modulate the immune response of patients with advanced supraglottic laryngeal cancer in vitro, increasing the secretion of TNF and IL-6 by macrophages during the postoperative period.