NANOPARTICLES FOR MUCOSAL VACCINE DELIVERY

Influenza is a contagious respiratory disease often disregarded due to the mild symptoms associated with it. The economic burden that flu cases impose on health care systems is substantial, not only due to influenza pandemics, but also to required hospitalizations and the need for yearly revisions of seasonal influenza vaccines. The need for a universal influenza vaccine was already recognized by the Global Vaccine Action Plan. Currently, injection-based vaccination is the most common method for influenza immunization. However, evidence has shown that mucosal immune responses, representing an important first line of defense at these sites, since most pathogens enter the body through mucosal tissues, are most efficiently induced by administration of vaccines onto mucosal surfaces than injected vaccines. From the different mucosal tissues, the gastrointestinal tract is an attractive route to be explored for vaccination; nonetheless, oral influenza vaccines are not available yet. The intestinal homeostasis is tightly controlled by several components of the intestinal barrier, such as the mucus layer, epithelial cells with different functions and an underlying immune system that surveys the gut. Between microorganisms that normally inhabit our gut, food antigens constantly present in our diet and potential pathogens, the intestinal barrier has the difficult task of integrating external and internal signals received by different cells in order to establish the correct response, immunity or tolerance, according to the antigen. Hence, oral vaccines will encounter these same intestinal barrier components and face the same obstacles as any other oral antigen or gut microorganism. The UniVacFlu consortium is currently working to develop a new mucosal universal vaccine against influenza, exploring different immunogens, immunization routes and delivery systems. This study was undertaken to understand the potential of the influenza vaccine candidate CTA1-3M2e-DD and polysaccharidic lipidated nanoparticles (NPL) when immunization occurs through the oral route. We found that, while CTA1-3M2e-DD revealed a poor ability to cross the intestinal epithelium and target intestinal antigen-presenting cells, NPL were found to readily overcome the intestinal barrier and were found associated with both CX3CR1+ macrophages and CD103+ dendritic cells. Two different routes of NPL uptake were identified: one depends on Goblet cell-associated passages that allow the transfer of high amounts of NPL from the lumen to the intestinal lamina propria; the second relies on the direct acquisition of NPL by CX3CR1+ macrophages in Peyer\u2019s patches by extension of trans-epithelial dendrites. Moreover, NPL as an oral vaccine vector was able to deliver the loaded antigen in the intestinal lamina propria and enhanced antigen presentation to CD4+ T lymphocytes in different organs. Despite increasing the availability of antigen, NPL did not induce tolerance towards the formulated antigen and a Th1 immune response was found at the level of the Peyer\u2019s patches. We also identified the contribution of the starvation period in the immune response induced by the NPL formulation in our model of oral immunization. The full potential of NPL as a vaccine vector is currently being further investigated to understand its immunomodulatory properties

OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency

Studies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.We identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations

An Antibody-Based Leukocyte-Capture Microarray for the Diagnosis of Systemic Lupus Erythematosus

The diagnosis of Systemic Lupus Erythematosus (SLE) is challenging due to its heterogeneous clinical presentation and the lack of robust biomarkers to distinguish it from other autoimmune diseases. Further, currently used laboratory tests do not readily distinguish active and inactive disease. Several groups have attempted to apply emerging high throughput profiling technologies to diagnose and monitor SLE. Despite showing promise, many are expensive and technically challenging for routine clinical use. The goal of this work is to develop a better diagnostic and monitoring tool for SLE. We report a highly customisable antibody microarray that consists of a duplicate arrangement of 82 antibodies directed against surface antigens on peripheral blood mononuclear cells (PMBCs). This high-throughput array was used to profile SLE patients (n = 60) with varying disease activity, compared to healthy controls (n = 24), patients with rheumatoid arthritis (n = 25), and other autoimmune diseases (n = 28). We used a computational algorithm to calculate a score from the entire microarray profile and correlated it with SLE disease activity. Our results demonstrate that leukocyte-capture microarray profiles can readily distinguish active SLE patients from healthy controls (AUROC = 0.84). When combined with the standard laboratory tests (serum anti-dsDNA, complements C3 and C4), the microarrays provide significantly increased discrimination. The antibody microarrays can be enhanced by the addition of other markers for potential application to the diagnosis and stratification of SLE, paving the way for the customised and accurate diagnosis and monitoring of SLE

Cardiac myosin-binding protein C mutations and hypertrophic cardiomyopathy haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction

Background-Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3(mut)). Methods and Results-Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c. 2373dupG, n=7; c. 2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3(mut). Protein expression of cMyBP-C was significantly reduced in MYBPC3(mut) by 33 +/- 5%. Cardiac MyBP-C phosphorylation in MYBPC3(mut) samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84 +/- 5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the beta-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3(mut) (20.2 +/- 2.7 kN/m(2)) compared with donor (34.5 +/- 1.1 kN/m(2)). Moreover, Ca2+ sensitivity was higher in MYBPC3(mut) (pCa(50)=5.62 +/- 0.04) than in donor (pCa(50)=5.54 +/- 0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic beta-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca2+ sensitivity between MYBPC3(mut) (pCa(50)=5.46 +/- 0.03) and donor (pCa(50)=5.48 +/- 0.02). Conclusions-Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca2+ sensitivity in MYBPC3(mut) is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction. (Circulation. 2009; 119: 1473-1483.

Prelamin A mediates myocardial inflammation in dilated and HIV-associated cardiomyopathies

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues, yet its impact upon the heart is unknown. Here, we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy, and we show that a potentially novel mouse model of cardiac-specific prelamin A accumulation exhibited a phenotype consistent with inflammatory cardiomyopathy, which we observed to be similar to HIV-associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings (a) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (b) have implications for the management of HIV patients with cardiac disease, suggesting protease inhibitors should be replaced with alternative therapies (i.e., nonnucleoside reverse transcriptase inhibitors); and (c) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy

Immunomodulation of inflammatory leukocyte markers during intravenous immunoglobulin treatment associated with clinical efficacy in chronic inflammatory demyelinating polyradiculoneuropathy

© 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc. Objective: The objective of the study was to profile leukocyte markers modulated during intravenous immunoglobulin (IVIg) treatment, and to identify markers and immune pathways associated with clinical efficacy of IVIg for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) with potential for monitoring treatment efficacy. Methods: Response to IVIg treatment in newly diagnosed IVIg-naïve and established IVIg-experienced patients was assessed by changes in expression of inflammatory leukocyte markers by flow cytometry. The adjusted INCAT disability and Medical Research Council sum scores defined clinical response. Results: Intravenous immunoglobulin modulated immunopathogenic pathways associated with inflammatory disease in CIDP. Leukocyte markers of clinical efficacy included reduced CD185 + follicular helper T cells, increased regulatory markers (CD23 and CD72) on B cells, and reduction in the circulating inflammatory CD16 + myeloid dendritic cell (mDC) population and concomitant increase in CD62L and CD195 defining a less inflammatory lymphoid homing mDC phenotype. A decline in inflammatory CD16 + dendritic cells was associated with clinical improvement or stability, and correlated with magnitude of improvement in neurological assessment scores, but did not predict relapse. IVIg also induced a nonspecific improvement in regulatory and reduced inflammatory markers not associated with clinical response. Conclusions: Clinically effective IVIg modulated inflammatory and regulatory pathways associated with ongoing control or resolution of CIDP disease. Some of these markers have potential for monitoring outcome

Prelamin A mediates inflammation in dilated and HIV associated cardiomyopathies

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues yet its impact upon the heart is unknown. Here we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy and show that a novel mouse model of cardiac specific prelamin A accumulation exhibited a phenotype consistent with ‘inflammatory cardiomyopathy’ which we observed to be similar to HIV associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings: (1) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (2) have implications for the management of HIV patients with cardiac disease suggesting protease inhibitors should be replaced with alternative therapies i.e. non-nucleoside reverse transcriptase inhibitors; and (3) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy

Similaridade genética de acessos de pinha por meio de marcadores ISSR.

Este estudo teve por objetivo caracterizar e avaliar a diversidade genética entre 19 acessos de pinha, sendo 9 clones e 10 progênies, pertencentes ao BAG de Fruteiras Nativas da Embrapa Meio-Norte, Teresina, PI, por meio de marcadores moleculares ISSR (Inter Simple Sequence Repeat)