Expanding the Clinical and Genetic Spectrum of Human CD40L Deficiency: The Occurrence of Paracoccidioidomycosis and Other Unusual Infections in Brazilian Patients

CD40 ligand (CD40L) deficiency or X-linked hyper-IgM syndrome (X-HIGM) is a well-described primary immunodeficiency in which Pneumocystis jiroveci pneumonia is a common clinical feature. We have identified an unusual high incidence of fungal infections and other not yet described infections in a cohort of 11 X-HIGM patients from nine unrelated Brazilian families. Among these, we describe the first case of paracoccidioidomycosis (PCM) in X-HIGM. The molecular genetic analysis of CD40L was performed by gene sequencing and evaluation of CD40L protein expression. Nine of these 11 patients (82%) had fungal infections. These included fungal species common to CD40L deficiency (P. jiroveci and Candida albicans) as well as Paracoccidioides brasiliensis. One patient presented with PCM at age 11 years and is now doing well at 18 years of age. Additionally, one patient presented with a simultaneous infection with Klebsiella and Acinetobacter, and one with condyloma caused by human papilloma virus. Molecular analysis revealed four previously described CD40L mutations, two novel missense mutations (c.433 T>G and c.476 G>C) resulting in the absence of CD40L protein expression by activated CD4(+) cells and one novel insertion (c.484_485insAA) within the TNFH domain leading to a frame shift and premature stop codon. These observations demonstrated that the susceptibility to fungal infections in X-HIGM extends beyond those typically associated with X-HIGM (P. jiroveci and C. albicans) and that these patients need to be monitored for those pathogens.

The effects of particulate ambient air pollution on the murine umbilical cord and its vessels: A quantitative morphological and immunohistochemical study

Previous studies have shown that particulate matter (PM) compromise birth weight and placental morphology. We hypothesized that exposing mice to ambient PM would affect umbilical cord (UC) morphology. To test this, mice were kept in paired open-top exposure chambers at the same location and ambient conditions but, in one chamber, the air was filtered (F) and, in the other, it was not (NF). UCs were analysed stereologically and by immunohistochemistry to localize isoprostane and endothelin receptors. The cords of mice from NF chambers were smaller in volume due to loss of mucoid connective tissue and decrease in volume of collagen. These structural changes and in umbilical vessels were associated with greater volumes of regions immunostained for isoprostane, ETAR and ETBR. Findings indicate that the adverse effects of PM on birth weight may be mediated in part by alterations in UC structure or imbalances in the endogenous regulators of vascular tone and oxidative stress. (C) 2012 Elsevier Inc. All rights reserved.

Molecular mapping of the regenerative niche in a murine model of myocardial infarction

Adult stem cells are distributed through the whole organism, and present a great potential for the therapy of different types of disease. For the design of efficient therapeutic strategies, it is important to have a more detailed understanding of their basic biological characteristics, as well as of the signals produced by damaged tissues and to which they respond. Myocardial infarction (MI), a disease caused by a lack of blood flow supply in the heart, represents the most common cause of morbidity and mortality in the Western world. Stem cell therapy arises as a promising alternative to conventional treatments, which are often ineffective in preventing loss of cardiomyocytes and fibrosis. Cell therapy protocols must take into account the molecular events that occur in the regenerative niche of MI. In the present study, we investigated the expression profile of ten genes coding for chemokines or cytokines in a murine model of MI, aiming at the characterization of the regenerative niche. MI was induced in adult C57BL/6 mice and heart samples were collected after 24 h and 30 days, as well as from control animals, for quantitative RT-PCR. Expression of the chemokine genes CCL2, CCL3, CCL4, CCL7, CXCL2 and CXCL10 was significantly increased 24 h after infarction, returning to baseline levels on day 30. Expression of the CCL8 gene significantly increased only on day 30, whereas gene expression of CXCL12 and CX3CL1 were not significantly increased in either ischemic period. Finally, expression of the IL-6 gene increased 24 h after infarction and was maintained at a significantly higher level than control samples 30 days later. These results contribute to the better knowledge of the regenerative niche in MI, allowing a more efficient selection or genetic manipulation of cells in therapeutic protocols.

Leishmania (Viannia) shawi purified antigens confer protection against murine cutaneous leishmaniasis

Leishmania (Viannia) shawi was characterized only recently, and few studies concerning the immunogenic and protective properties of its antigens have been performed. The present study aimed to evaluate the protective potential of the five antigenic fractions isolated from L. (V.) shawi promastigotes in experimental cutaneous leishmaniasis. Soluble antigen from L. (V.) shawi promastigotes was submitted to reverse phase HPLC to purify F1, F2, F3, F4 and F5 antigens. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 mu g protein. After 1 week, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 8 weeks, those same mice were sacrificed and parasite burden as well as the cellular and humoral immune responses were evaluated. F1 and F5-immunized mice restrained lesion progression and parasite load in the skin. However, only the F1 group was able to control the parasitism in lymph nodes, which was associated with low IL-4 and high IFN-gamma production; IgG2a isotype was increased in this group. Immunizations with F2, F3 and F4 antigens did not protect mice. The capability of antigens to restrain IL-4 levels and increase IFN-gamma was associated with protection, such as in immunization using F1 antigen.

Clove and eugenol in noncytotoxic concentrations exert immunomodulatory/anti-inflammatory action on cytokine production by murine macrophages

Objectives The extract and essential oil of clove (Syzygium aromaticum) are widely used because of their medicinal properties. Eugenol is the most important component of clove, showing several biological properties. Herein we have analysed the immunomodulatory/anti-inflammatory effect of clove and eugenol on cytokine production (interleukin (IL)-1 beta, IL-6 and IL-10) in vitro. Methods Macrophages were incubated with clove or eugenol (5, 10, 25, 50 or 100 mg/well) for 24 h. Concentrations that inhibited the production of cytokines were used before or after incubation with lipopolysaccharide (LPS), to verify a preventive or therapeutic effect. Culture supernatants were harvested for measurement of cytokines by enzyme-linked immunosorbent assay. Key findings Clove (100 mg/well) inhibited IL-1 beta, IL-6 and IL-10 production and exerted an efficient action either before or after LPS challenge for all cytokines. Eugenol did not affect IL-1 beta production but inhibited IL-6 and IL-10 production. The action of eugenol (50 or 100 mg/well) on IL-6 production prevented efficiently effects of LPS either before or after its addition, whereas on IL-10 production it counteracted significantly LPS action when added after LPS incubation. Conclusions Clove exerted immunomodulatory/anti-inflammatory effects by inhibiting LPS action. A possible mechanism of action probably involved the suppression of the nuclear factor-kB pathway by eugenol, since it was the major compound found in clove

3,4-Methylenedioxymethamphetamine (Ecstasy) Decreases Inflammation and Airway Reactivity in a Murine Model of Asthma

Objective:3,4-Methylenedioxymethamphetamine(MDMA), or ecstasy, is a synthetic drug used recreationally, mainly by young people. It has been suggested that MDMA has a Th cell skewing effect, in which Th1 cell activity is suppressed and Th2 cell activity is increased. Experimental allergic airway inflammation in ovalbumin (OVA)-sensitized rodents is a useful model to study Th2 response; therefore, based on the Th2 skewing effect of MDMA, we studied MDMA in a model of allergic lung inflammation in OVA-sensitized mice. Methods: We evaluated cell trafficking in the bronchoalveolar lavage fluid, blood and bone marrow; cytokine production; L-selectin expression and lung histology. We also investigated the effects of MDMA on tracheal reactivity in vitro and mast cell degranulation. Results: We found that MDMA given prior to OVA challenge in OVA-sensitized mice decreased leukocyte migration into the lung, as revealed by a lower cell count in the bronchoalveolar lavage fluid and lung histologic analysis. We also showed that MDMA decreased expression of both Th2-like cytokines (IL-4, IL-5 and IL-10) and adhesion molecules (L-selectin). Moreover, we showed that the hypothalamus-pituitary-adrenal axis is partially involved in the MDMA-induced reduction in leukocyte migration into the lung. Finally, we showed that MDMA decreased tracheal reactivity to methacholine as well as mast cell degranulation in situ. Conclusions:Thus, we report here that MDMA given prior to OVA challenge in OVA-sensitized allergic mice is able to decrease lung inflammation and airway reactivity and that hypothalamus-pituitary-adrenal axis activation is partially involved. Together, the data strongly suggest an involvement of a neuroinnmune mechanism in the effects of MDMA on lung inflammatory response and cell recruitment to the lungs of allergic animals. Copyright (C) 2012 S. Karger AG, Basel

Fatal septic shock due to a disseminated chronic form of paracoccidioidomycosis in an aged woman

Once rare, septic shock (SS) due to disseminated fungal infections has been increasingly reported due to a growing number of immunocompromised patients, but remains rare in non-immune-compromised individuals. In paracoccidioidomycosis, it has been described in only three patients with the severe, acute form of the disease. We describe the development of a refractory, fatal septic shock due to a severe disseminated chronic form of paracoccidioidomycosis in an older woman without any other microbial insults. A striking event in the evolution of her case was the severe depletion of lymphocytes from the peripheral blood and lymphoid organs. Lymphocyte depletion due to apoptosis is described in the late phase of sepsis and can contribute both to immunosuppression and the progression of SS. The possible mechanisms involved in the induction of SS in the chronic form of paracoccidioidomycosis are discussed.

Radiologic findings of osteoarticular infection in paracoccidioidomycosis

To report the radiological abnormalities of osteoarticular involvement in paracoccidioidomycosis (PCM). After institutional board approval, the medical records and conventional radiology findings of 19 patients with osseous PCM were retrospectively reviewed. Number, distribution, and lesion characteristics were evaluated in consensus by two experienced musculoskeletal radiologists. The mean age of patients was 16.1 years (range 4-49 years), 11 male and eight female. MSK involvement was the only or the primary presentation of the disease in eight of 19 patients (42.1%). In total, 51 focal bone lesions were detected, being 41 in long bones. In long bones lesions, 19 of 41 (46.4%) were metaphyseal, 12 of 41 (29.3%) meta-epiphyseal, and 12 of 41 (29.3%) diaphyseal. The most common presentation was a geographic osteolytic bone lesion (62.7%), without marginal sclerosis (82.4%) and without periosteal reaction (90.2%). Articular involvement was present in six of 19 patients (31.6%), being two cases of primary arthritis. All encountered bone lesions were osteolytic. Metaphyseal or meta-epiphyseal osteomyelitis of a long bone was the most prevalent osteoarticular manifestation of paracoccidioidomycosis. PCM osteoarticular involvement could be solitary or multifocal, occurs almost exclusively in the acute/subacute clinical form, and it is more common in children and in juvenile patients. Axial skeleton involvement, arthritis, or a disseminated osseous pattern of infection may occasionally occur in this fungal disease.

Cannabidiol, a non-psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of acute lung injury: Role for the adenosine A(2A) receptor

Acute lung injury is an inflammatory condition for which treatment is mainly supportive because effective therapies have not been developed. Cannabidiol, a non-psychotropic cannabinoid component of marijuana (Cannabis sativa), has potent immunosuppressive and anti-inflammatory properties. Therefore, we investigated the possible anti-inflammatory effect of cannabidiol in a murine model of acute lung injury. Analysis of total inflammatory cells and differential in bronchoalveolar lavage fluid was used to characterize leukocyte migration into the lungs; myeloperoxidase activity of lung tissue and albumin concentration in the bronchoalveolar lavage fluid were analyzed by colorimetric assays; cytokine/chemokine production in the bronchoalveolar lavage fluid was also analyzed by Cytometric Bead Arrays and Enzyme-Linked Immunosorbent Assay (ELISA). A single dose of cannabidiol (20 mg/kg) administered prior to the induction of LPS (lipopolysaccharide)-induced acute lung injury decreases leukocyte (specifically neutrophil) migration into the lungs, albumin concentration in the bronchoalveolar lavage fluid, myeloperoxidase activity in the lung tissue, and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) 1, 2, and 4 days after the induction of LPS-induced acute lung injury. Additionally, adenosine A(2A) receptor is involved in the anti-inflammatory effects of cannabidiol on LPS-induced acute lung injury because ZM241385 (4-(2[7-Amino-2-(2-furyl)[1,2,4] triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol) (a highly selective antagonist of adenosine A(2A) receptor) abrogated all of the anti-inflammatory effects of cannabidiol previously described. Thus, we show that cannabidiol has anti-inflammatory effects in a murine model of acute lung injury and that this effect is most likely associated with an increase in the extracellular adenosine offer and signaling through adenosine A(2A) receptor. (C) 2012 Elsevier B. V. All rights reserved.

Inflammatory events during murine squamous cell carcinoma development

Background: Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. In SCC, tumour development is accompanied by an immune response that leads to massive tumour infiltration by inflammatory cells, and consequently, local and systemic production of cytokines, chemokines and other mediators. Studies in both humans and animal models indicate that imbalances in these inflammatory mediators are associated with cancer development. Methods: We used a multistage model of SCC to examine the involvement of elastase (ELA), myeloperoxidase (MPO), nitric oxide (NO), cytokines (IL-6, IL-10, IL-13, IL-17, TGF-β and TNF-α), and neutrophils and macrophages in tumour development. ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment. Results: ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment. Significantly higher levels of IL-6 and lower levels of IL-10 were detected at 4 weeks following 7,12-Dimethylbenz(a)anthracene (DMBA) treatment. Similar levels of IL-13 were detected in the precancerous microenvironment compared with control tissue. We identified significant increases in the number of GR-1+ neutrophils and F4/80+/GR-1- infiltrating cells in tissues at 4 and 8 weeks following treatment and a higher percentage of tumour-associated macrophages (TAM) expressing both GR-1 and F4/80, an activated phenotype, at 16 weeks. We found a significant correlation between levels of IL-10, IL-17, ELA, and activated TAMs and the lesions. Additionally, neutrophil infiltrate was positively correlated with MPO and NO levels in the lesions. Conclusion: Our results indicate an imbalance of inflammatory mediators in precancerous SCC caused by neutrophils and macrophages and culminating in pro-tumour local tissue alterations.

A retrospective study of oral manifestations in patients with paracoccidioidomycosis

South American blastomycosis, paracoccidioidomycosis (Pb mycosis) or Lutz disease is an endemically fungal infection in Latin America. It is caused by the dimorphic fungus Paracoccidioides brasiliensis and may cause oral mucosal lesions. The incidence of Pb mycosis oral lesions was evaluated in patients assisted at a Brazilian Dental School's Specialized Oral Diagnosis Service with special focus on the different clinical forms of these lesions, its location, patients' occupation, deleterious habits, and diagnosis methodology. Students' and professionals' initial diagnoses were compared with the definitive diagnosis. Lesions were detected 31 cases (18 patients). The results show that 88.8% of the patients were male with a mean age of 50 years and 39% work(ed) with activities related to agriculture. As much as 88.9% were smokers and 72.2% were alcohol users. Exfoliative cytology was performed in 66.6% of the patients. Oral mucosa (30%), gingiva (16.6%) and lips 16.6% were the most common sites of Pb mycosis oral lesions. Comparing the initial with the definitive diagnosis made by the professionals their accuracy was 33% (6 out of 18 patients). Students' diagnosis was more accurate demonstrating 72.5% of initial correct diagnosis (13 out of 18). Statistical analysis by ANOVA (α=0.05, SPSS WIN) demonstrated a significant difference between the diagnosis of Pb mycosis made by students and professionals when considering initial diagnosis and final diagnosis (after histopathological analysis) (p=0.25). Incisional biopsy and exfoliate cytology are efficient for an early diagnosis of this disease in mouth. Students' training in diagnosis of oral pathologies to recognize lesions is urgent to improve public health.

New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

Abstract Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.

PReS-FINAL-2357: Effects of anti-melanocyte stimulating hormone in murine pristine-induced lupus

Introduction Alfa-melanocyte stimulating hormone (α-MSH) has a variety of biological functions such as downregulation of pro-inflammatory pathways, reduction of skin delayed-type hypersensitivity and blockage of leukocyte migration. Inhibition of experimental disease models development including inflammatory bowel disease and rheumatoid arthritis has been shown, however the immunomodulatory and anti-inflammatory effects of α-MSH on murine lupus remain undetermined. Objectives To evaluate the effect of α-MSH analogue (NDP α-MSH) on pristane-induced murine lupus. Methods Thirty-five BALB/c mice were injected with 0.5 ml intraperitoneal (IP) pristane for lupus-like model induction and 5 age/gender matched control mice were given saline. Pristane-induced lupus animals received daily IP saline (n = 5) or treatments with 3.1 mg/kg/d chloroquine (n = 10), 1.25 mg/kg/d NDP α-MSH (n = 10) or 2.5 mg/kg/d NDP α-MSH (n = 10). Prior and 180 days after induction, clinical and laboratorial lupus-like parameters were examined. Sera ANA was tested by IF using Hep2 cells. Statistical analysis was performed by Mann-Whitney and Fisher test and P < 0,05 considered significant. Results Arthritis in both hind legs and large amounts of lipogranulomas in peritoneal cavity were observed in all lupus-like animals in contrast to all controls. By visual observation, all lupus animals treated with both doses of α-MSH had significant less amount and lower size lipogranulomas. Mean arthritis score in 5 untreated mice, 9 animals treated with chloroquine and 8 with α-MSH 2.5 mg/kg/d was 5.2, 3.33 and 3.1 respectively. Remarkably, mean arthritis score of animals treated with α-MSH 1.25 mg/kg/d was 1.6, significantly lower than untreated mice (1.6 vs 5.2, p = 0.0291). ANAs were negative in sera from all 40 animals before pristane lupus injection; 180 days after induction, ANAs remained negative in normal mice but became positive in all 5 (100%) untreated lupus animals, 7 (77%), 4 (50%) and 3 (35%) lupus models treated with chloroquine, α-MSH 2.5 mg/kg/d and α-MSH 1.25 mg/kg/d (100% vs 35%, p = 0,0256), respectively. Before the end of the experiment, by day 150, 3 animals died: 1 treated with chloroquine and 2 with higher doses of α-MSH. Conclusion NDP α-MSH promoted improvement of clinical and serological parameters in pristane-induced murine lupus suggesting a potential role for this drug in human SLE.

STAT3 and SOCS3 expression patterns during murine placenta development

Signal transducers and activators of transcription 3 (STAT3) has been identified as an important signal transducer in the invasive phenotype of the trophoblasts cells in in vitro studies. However, the in situ distribution and patterns of expression of this molecule in trophoblast cells during the development of the placenta are still under-elucidated. Mice uteri of gestational ages between 7 and 14 days of pregnancy (dop) were fixed in methacarn and processed with immunoperoxidase techniques for detection of STAT3 and its phosphorylation at serine (p-ser727) residues, as well as the suppressor of cytokine signaling 3 (SOCS3) expression. STAT3 was observed at 7 through 9 dop in both the antimesometrial and mesometrial deciduas, while continued immunoreactivity between 10 and 13 dop was seen only in the mesometrial decidua. In the placenta, STAT3 was detected in the cytotrophoblast cells of labyrinth and giant trophoblast cells between 10 and 14 dop. Immunoreactivity for STAT3 was also seen in trophoblast cells surrounding the maternal blood vessels. On days 10 and 11 of pregnancy, p-ser727 was detectable in the mesometrial decidua and in giant trophoblasts, while during 12-14 dop in the spongiotrophoblast region. In addition, SOCS3 was immunodetected in maternal and placental tissues, principally in the giant trophoblast cells during the whole period of the study. The present in situ study shows the distribution of STAT3, its serine activation and SOCS3 in different maternal and fetal compartments during murine placental development, thus further supporting the idea that they play a role during physiological placentation in mice.

In pulmonary paracoccidioidomycosis IL-10 deficiency leads to increased immunity and regressive infection without enhancing tissue pathology

BACKGROUND: Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM. METHODOLOGY/PRINCIPAL FINDINGS: Wild type (WT) and IL-10(-/-) C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10(-/-) mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10(-/-) and WT mice were i.t. infected with 1×10(6) Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10(-/-) mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10(-/-) mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4(+) and CD8(+) T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10(-/-) mice. CONCLUSIONS/SIGNIFICANCE: Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.