MSX2 copy number increase and craniosynostosis: copy number variation detected by array comparative genomic hybridization

Fleury Group

Low-Level Laser Therapy Influences Mouse Odontoblast-Like Cell Response in Vitro

Objective: The purpose of this study was to analyze the influence of two different irradiation times with 85mW/cm(2) 830nm laser on the behavior of mouse odontoblast-like cells. Background data: The use of low-level laser therapy (LLLT) to stimulate pulp tissue is a reality, but few reports relate odontoblastic responses to irradiation in in vitro models. Methods: Odontoblast-like cells (MDPC-23) were cultivated and divided into three groups: control/nonirradiated (group 1); or irradiated with 85mW/cm(2), 830nm laser for 10 sec (0.8 J/cm(2)) (group 2); or for 50 sec (4.2 J/cm(2)) (group 3) with a wavelength of 830 nm. After 3, 7, and 10 days, it was analyzed: growth curve and cell viability, total protein content, alkaline phosphatase (ALP) activity, calcified nodules detection and quantification, collagen immunolocalization, vascular endothelial growth factor (VEGF) expression, and real-time polymerase chain reaction (PCR) for DMP1 gene. Data were analyzed by Kruskall-Wallis test (alpha = 0.05). Results: Cell growth was smaller in group 2 (p < 0.01), whereas viability was similar in all groups and at all periods. Total protein content and ALP activity increased on the 10th day with 0.8 J/cm(2) (p < 0.01), as well as the detection and quantification of mineralization nodules (p < 0.05), collagen, and VEGF expression (p < 0.01). The expression of DMP1 increased in all groups (p < 0.05) compared with control at 3 days, except for 0.8 J/cm(2) at 3 days and control at 10 days. Conclusions: LLLT influenced the behavior of odontoblast-like cells; the shorter time/smallest energy density promoted the expression of odontoblastic phenotype in a more significant way.

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

Background: Leishmania (Viannia) shawi parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from L. (V.) shawi promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained. Methods: F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 mu g of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated. Results: The F1 fraction induced a high degree of protection associated with an increase in IFN-gamma, a decrease in IL-4, increased cell proliferation and activation of CD8(+)T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4(+) central memory T lymphocytes and activation of both CD4+ and CD8(+) T cells. In addition, F1-immunized groups showed an increase in IgG2a levels. Conclusions: The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-alpha in the induction of endothelin system genes

Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice. CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100 mg/kg) once a day, starting from the day when arthritis was clinically detectable. CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student's t test. Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1 beta, TNF alpha and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNF alpha upregulated ET system genes. These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNF alpha.

Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.

Anti-IL-2 Treatment Impairs the Expansion of T-reg Cell Population during Acute Malaria and Enhances the Th1 Cell Response at the Chronic Disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T-reg) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T-reg cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4+ T cell activation and IFN-gamma production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-alpha and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T-reg cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.

Low Sensitivity of NS1 Protein Tests Evidenced during a Dengue Type 2 Virus Outbreak in Santos, Brazil, in 2010

In 2010, a large outbreak of dengue occurred in Santos, Brazil. The detection of the NS1 antigen was used for diagnosis in addition to the detection of IgG, IgM, and RNA. A large number of NS1 false-negative results were obtained. A total of 379 RNA-positive samples were selected for thorough evaluation. NS1 was reactive in 37.7% of cases. Most of the cases were characterized as a secondary infection by dengue 2 virus. Sequencing of NS1 positive and negative isolates did not reveal any mutation that could justify the diagnostic failure. Use of existing NS1 tests in the Brazilian population may present a low negative predictive value, and they should be used with caution, preferentially after performing a validation with samples freshly obtained during the ongoing epidemic.

Lower numbers of natural killer T cells in HIV-1 and Mycobacterium leprae co-infected patients

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes that recognize antigens presented by CD1d and have attracted attention because of their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on peripheral blood mononuclear cells. We demonstrated that patients co-infected with HIV-1 and M.leprae have significantly lower NKT cell frequencies [median 0.022%, interquartile range (IQR): 0.0070.051] in the peripheral blood when compared with healthy subjects (median 0.077%, IQR: 0.0320.405, P < 0.01) or HIV-1 mono-infected patients (median 0.072%, IQR: 0.0300.160, P < 0.05). Also, more NKT cells from co-infected patients secreted interferon-? after stimulation with DimerX, when compared with leprosy mono-infected patients (P = 0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and M.leprae co-infected patients compared with HIV-1 mono-infected patients alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1-infected subjects, a concurrent mycobacterial infection probably hyper-activates and lowers circulating NKT cell numbers.

Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes

Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity. (C) 2012 Elsevier Ltd. All rights reserved.

Living Invisible: HTLV-1-Infected Persons and the Lack of Care in Public Health

Introduction: Human T-cell lymphotropic virus type 1 (HTLV-1) infection is intractable and endemic in many countries. Although a few individuals have severe symptoms, most patients remain asymptomatic throughout their lives and their infections may be unknown to many health professionals. HTLV-1 can be considered a neglected public health problem and there are not many studies specifically on patients' needs and emotional experiences. Objective: To better understand how women and men living with HTLV-1 experience the disease and what issues exist in their healthcare processes. Methods: A qualitative study using participant observation and life story interview methods was conducted with 13 symptomatic and asymptomatic patients, at the outpatient clinic of the Emilio Ribas Infectious Diseases Institute, in Sao Paulo, Brazil. Results and Discussion: The interviewees stated that HTLV-1 is a largely unknown infection to society and health professionals. Counseling is rare, but when it occurs, focuses on the low probability of developing HTLV-1 related diseases without adequately addressing the risk of infection transmission or reproductive decisions. The diagnosis of HTLV-1 can remain a stigmatized secret as patients deny their situations. As a consequence, the disease remains invisible and there are potentially negative implications for patient self-care and the identification of infected relatives. This perception seems to be shared by some health professionals who do not appear to understand the importance of preventing new infections. Conclusions: Patients and medical staff referred that the main focus was the illness risk, but not the identification of infected relatives to prevent new infections. This biomedical model of care makes prevention difficult, contributes to the lack of care in public health for HTLV-1, and further perpetuates the infection among populations. Thus, HTLV-1 patients experience an "invisibility" of their complex demands and feel that their rights as citizens are ignored.

Liposuction induces a compensatory increase of visceral fat which is effectively counteracted by physical activity: a randomized trial

Context: Liposuction is suggested to result in long-term body fat regain that could lead to increased cardiometabolic risk. We hypothesized that physical activity could prevent this effect. Objective: Our objective was to investigate the effects of liposuction on body fat distribution and cardiometabolic risk factors in women who were either exercise trained or not after surgery. Design, Setting, and Participants: Thirty-six healthy normal-weight women participated in this 6-month randomized controlled trial at the University of Sao Paulo, Sao Paulo, Brazil. Interventions: Patients underwent a small-volume abdominal liposuction. Two months after surgery, the subjects were randomly allocated into two groups: trained (TR, n = 18, 4-month exercise program) and nontrained (NT, n = 18). Main Outcome Measures: Body fat distribution (assessed by computed tomography) was assessed before the intervention (PRE) and 2 months (POST2), and 6 months (POST6) after surgery. Secondary outcome measures included body composition, metabolic parameters and dietary intake, assessed at PRE, POST2, and POST6, and total energy expenditure, physical capacity, and sc adipocyte size and lipid metabolism-related gene expression, assessed at PRE and POST6. Results: Liposuction was effective in reducing sc abdominal fat (PRE vs. POST2, P = 0.0001). Despite the sustained sc abdominal fat decrement at POST6 (P = 0.0001), the NT group showed a significant 10% increase in visceral fat from PRE to POST6 (P = 0.04; effect size = -0.72) and decreased energy expenditure (P = 0.01; effect size = 0.95) when compared with TR. Dietary intake, adipocyte size, and gene expression were unchanged over time. Conclusion: Abdominal liposuction does not induce regrowth of fat, but it does trigger a compensatory increase of visceral fat, which is effectively counteracted by physical activity. (J Clin Endocrinol Metab 97: 2388-2395, 2012)

Increasing Intensity of TENS Prevents Analgesic Tolerance in Rats

Transcutaneous electrical nerve stimulation (TENS) reduces hyperalgesia and pain. Both low-frequency (LF) and high-frequency (HF) TENS, delivered at the same intensity (90% motor threshold [MT]) daily, result in analgesic tolerance with repeated use by the fifth day of treatment. The current study tested 1) whether increasing intensity by 10% per day prevents the development of tolerance to repeated TENS; and 2) whether lower intensity TENS (50% MT) produces an equivalent reduction in hyperalgesia when compared to 90% MT TENS. Sprague-Dawley rats with unilateral knee joint inflammation (3% carrageenan) were separated according to the intensity of TENS used: sham, 50% LF, 50% HF, 90% LF, 90% HF, and increased intensity by 10% per day (IF and HF). The reduced mechanical withdrawal threshold following the induction of inflammation was reversed by application of TENS applied at 90% MT intensity and increasing intensity for the first 4 days. On the fifth day, the groups that received 90% MT intensity showed tolerance. Nevertheless, the group that received an increased intensity on each day still showed a reversal of the mechanical withdrawal threshold with TENS. These results show that the development of tolerance can be delayed by increasing intensity of TENS. Perspective: Our results showed that increasing intensity in both frequencies of TENS was able to prevent analgesic tolerance. Results from this study suggest that increasing intensities could be a clinical method to prevent analgesic tolerance and contribute to the effective use of TENS in reducing inflammatory pain and future clinical trials. (c) 2012 by the American Pain Society

Dose and latency effects of leucine supplementation in modulating glucose homeostasis: opposite effects in healthy and glucocorticoid-induced insulin-resistance states

Dexamethasone (DEXA) is a potent immunosupressant and anti-inflammatory agent whose main side effects are muscle atrophy and insulin resistance in skeletal muscles. In this context, leucine supplementation may represent a way to limit the DEXA side effects. In this study, we have investigated the effects of a low and a high dose of leucine supplementation (via a bolus) on glucose homeostasis, muscle mass and muscle strength in energy-restricted and DEXA-treated rats. Since the leucine response may also be linked to the administration of this amino acid, we performed a second set of experiments with leucine given in bolus (via gavage) versus leucine given via drinking water. Leucine supplementation was found to produce positive effects (e. g., reduced insulin levels) only when administrated in low dosage, both via the bolus or via drinking water. However, under DEXA treatment, leucine administration was found to significantly influence this response, since leucine supplementation via drinking water clearly induced a diabetic state, whereas the same effect was not observed when supplied via the gavage.

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.

Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion

Background Split-hand/foot malformation (SHFM)-also known as ectrodactyly-is a congenital disorder characterised by severe malformations of the distal limbs affecting the central rays of hands and/or feet. A distinct entity termed SHFLD presents with SHFM and long bone deficiency. Mouse models suggest that a defect of the central apical ectodermal ridge leads to the phenotype. Although six different loci/mutations (SHFM1-6) have been associated with SHFM, the underlying cause in a large number of cases is still unresolved. Methods High resolution array comparative genomic hybridisation (CGH) was performed in patients with SHFLD to detect copy number changes. Candidate genes were further evaluated for expression and function during limb development by whole mount in situ hybridisation and morpholino knock-down experiments. Results Array CGH showed microduplications on chromosome 17p13.3, a locus previously associated with SHFLD. Detailed analysis of 17 families revealed that this copy number variation serves as a susceptibility factor for a highly variable phenotype with reduced penetrance, particularly in females. Compared to other known causes for SHFLD 17p duplications appear to be the most frequent cause of SHFLD. A similar to 11.8 kb minimal critical region was identified encompassing a single gene, BHLHA9, a putative basic loop helix transcription factor. Whole mount in situ hybridisation showed expression restricted to the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knock down of bhlha9 in zebrafish resulted in shortening of the pectoral fins. Conclusions Genomic duplications encompassing BHLHA9 are associated with SHFLD and non-Mendelian inheritance characterised by a high degree of non-penetrance with sex bias. Knock-down of bhlha9 in zebrafish causes severe reduction defects of the pectoral fin, indicating a role for this gene in limb development.