CpG-Induced Th1-Type Response in the Downmodulation of Early Development of Allergy and Inhibition of B7 Expression on T Cells of Newborn Mice

Several differences have been described between neonatal and adult immune responses. The predisposition in early life to Th2-type response or tolerance makes it a susceptible period for infections and allergic sensitization. The aim of this work was to evaluate the effects of CpG-containing oligodeoxynucleotides on neonatal and adult immunization with ovalbumin and Blomia tropicalis extract and compare the CpG effects on B and T cells of neonatal and adult mice. Mice that received CpG showed reduced immunoglobulin E (IgE) antibody production in both neonatal and adult periods, in parallel to increased IgG2a antibody levels. We observed that spleen cells of mice that received CpG in early life produced increased amounts of interferon-gamma upon anti-CD3 stimulation. Negative regulation of IgE response was more pronounced in adult than neonate mice; further, CpG decreased anaphylactic antiovalbumin IgG1 only in adults. Also, an upregulation of toll-like receptor 9 expression was detected in adult B cells, but not in neonatal, upon CpG stimuli. Neonatal B cells showed enhanced interleukin (IL)-10 expression and decreased IL-6 levels than adult B cells in response to CpG. When we analyzed in vitro activation of CD4+ T cells, an increased expression of B7 molecules on T cells in neonates was suppressed by CpG. Altogether, we verified qualitative and quantitative evidences regarding CpG effect on neonatal and adult allergens immunizations, which points to the importance of understanding neonatal immune system to establish immunomodulatory strategies for prevention of allergic diseases.

Familial neuromyelitis optica

Background: Detection of aquaporin-4-specific immunoglobulin G (IgG) has expanded the spectrum of neuromyelitis optica (NMO). Rare reports of familial aggregation have suggested a component of genetic susceptibility but these reports mostly antedated the discovery of the NMO-IgG biomarker and recently updated diagnostic criteria. Methods: We report a case series describing the demographic, clinical, neuroimaging, and NMO-IgG serologic status of 12 multiplex NMO pedigrees with a total of 25 affected individuals. Results: Twenty-one patients (84%) were women. Families were Asian (n = 5), Latino (n = 4), white (n = 1), or African (n = 2). Apparent transmission was either maternal (n = 5) or paternal (n = 2). In 1 family, 3 individuals had NMO; in the others, 2 individuals were affected. Sibling pairs (n = 6), parent-child (n = 4), and aunt-niece (n = 3) pairs were observed. Nineteen patients (76%) were NMO-IgG positive. Twelve (48%) had clinical or serologic evidence of another autoimmune disease. Familial occurrence of NMO occurs in approximately 3% of patients with well-established diagnosis of NMO. Conclusions: A small proportion of patients with NMO have relatives with this condition, but familial occurrence is more common than would be expected from its frequency in the general population. Familial NMO is indistinguishable from sporadic NMO based on clinical symptoms, age at onset, sex distribution, and frequency of NMO-IgG detection. One or 2 generations were affected and affected individuals represented a small fraction of family members. Taken together, these data suggest complex genetic susceptibility in NMO. Neurology (R) 2010;75:310-315

Primary immune deficiency disorders presenting as autoimmune diseases: IPEX and APECED

Background Several primary immune deficiency disorders are associated with autoimmunity and malignancy, suggesting a state of immune dysregulation. The concept of immune dysregulation as a direct cause of autoimmunity in primary immune deficiency disorders (PIDDs) has been strengthened by the recent discovery of distinct clinical entities linked to single-gene defects resulting in multiple autoimmune phenomena including immune dysregulation, polyendocrinopathy, enteropathy and X-linked (IPEX) syndrome, and autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy (APECED) syndrome. Conclusion Reviewing recent advances in our understanding of the small subgroup of PIDD patients with defined causes for autoimmunity may lead to the development of more effective treatment strategies for idiopathic human autoimmune diseases.

Paracoccidioidomycosis: Cells expressing IL17 and Foxp3 in cutaneous and mucosal lesions

We demonstrated and quantified by immunohistochemistry the population of cells expressing IL17 and Foxp3 in cutaneous and mucosal paracoccidioidomycosis lesions, associating these populations of cells with different presentations of granulomatous response. For this purpose, 61 skin biopsies and 55 oral mucosal biopsies were evaluated. Cells expressing IL17 were distributed in the inflammatory infiltrate in both groups of lesions and were found in the vessels` wall too. Foxp3+ expression was limited to the nuclei of lymphocytes in the inflammatory infiltrate. The distribution of IL17 was similar among the groups; however, Foxp3+ cells were increased in mucosal lesions that displayed compact granulomas. The results suggest that IL17 seems to play a role in paracoccidioidomycosis cutaneous and mucosal lesions, probably as secondary cells in the clearance of the fungal antigens. The presence of Foxp3+ cells both in skin and mucosa corroborates some previous researches that suggest the role of this group of cells in the modulation of local immune response. (C) 2011 Elsevier Ltd. All rights reserved.

Mesenchymal Stem Cell Properties of Periodontal Ligament Cells From Deciduous and Permanent Teeth

Background: Human postnatal stem cells have been identified in periodontal ligaments (PDLs). In this study, the in vitro biologic properties of CD105(+) enriched cell subsets from PDLs harvested from deciduous (DePDL) and permanent (PePDL) teeth are comparatively assessed. Methods: PDL tissue was obtained from 12 teeth (six primary and six permanent) from which CD105(+) CD34(-) CD45(-) cells were isolated by magnetic cell sorting. To identify and quantitatively compare the stem cell markers, DePDL and PePDL cells were assessed for CD166 surface antigen expression by flow cytometry, real-time polymerase chain reaction, and immunostaining for Stro-1 and Oct-4, osteogenic and adipogenic differentiation, and proliferation rate by trypan blue method. Results: Magnetic cell sorting isolated cell populations containing 23.87% (+/- 11.98%) and 11.68% (+/- 6.27%) of CD105(+) expressing cells from PePDL and DePDL, respectively. Flow cytometric analysis demonstrated a higher proportion of CD105(+) cells coexpressing CD166 surface antigen in PePDL, whereas immunostaining and real-time polymerase chain reaction analysis demonstrated that both cell subsets expressed Stro-1 and Oct-4. DePDL-CD105(+) subsets were more proliferative compared to PePDL subsets, and both cell populations showed multipotential capabilities to differentiate in vitro to osteoblast/cementoblast- and adipocyte-like cells. However, a higher expression of adipogenic-related genes was observed in DePDL cells, whereas PePDL-CD105(+) cell subset presented a more homogeneous osteoblast/cementoblast response. Conclusion: These findings demonstrate that highly purified mesenchymal progenitor cell subsets can be obtained from the PDLs of both deciduous and permanent teeth, and further indicate phenotype dissimilarities that may have an impact on their clinical applications. J Periodontol 2010;81:1207-1215.

TGF-beta and CD23 are involved in nitric oxide production by pulmonary macrophages activated by beta-glucan from Paracoccidioides brasiliensis

Pulmonary macrophages (PM), which are CD11b/CD18(+) and CD23(+), may be involved in the onset of inflammatory events caused by Paracoccidioides brasiliensis in the lungs. In the present study, we measured the nitric oxide (NO) and interleukin in PM production after intratracheal (i.t.) inoculation of an enriched beta-glucan cell wall fraction from P. brasiliensis (Fraction F1). BALB/c and C57/BL6 (B6) mice were i.t. treated with Fraction F1, and their PM were restimulated in vitro with LPS and interferon-gamma up to 14 days after treatment. Macrophages BALB/c mice produced less NO than PM from B6 mice. The lower NO production was caused by higher production of TGF-beta by pulmonary macrophages of BALB/c and was abrogated by anti-TGF-beta MoAb in vitro and in vivo. Other interleukins such as IL-10, IL-4 and a combination of IL-1, TNF-alpha and IL-6 were not involved in NO production induced by Fraction F1. Expression of CD11b increases and expression of CD23 decreases on PM of BALB/c mice after in vivo treatment whereas PM of B6 mice do not show a variation of their phenotype. Moreover, the ability of pulmonary macrophages to induce lymphocyte proliferation was reduced in mixed cultures of CD11b(+) or CD23(+) macrophages but was restored when lymphocytes were cultivated in the presence of NO inhibitor (L-NMMA). Thus, the results presented herein indicate that in BALB/c but not in B6 mice TGF- is strongly induced by Fraction 1 in PM in vivo and suppresses NO production. Low NO production by PM is associated with a change in CD11b/CD23 expression and with a high lymphocyte proliferative response. Thus, CD11b(+)/CD23(+) PM modulate NO and TGF-beta production in the pulmonary microenvironment.

Immunization with pVAXhsp65 Decreases Inflammation and Modulates Immune Response in Experimental Encephalomyelitis

Background: A DNA vaccine (pVAXhsp65) containing the gene of a heat-shock protein (hsp65) from Mycobacterium leprae showed high immunogenicity and protective efficacy against tuberculosis in BALB/c mice. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 in the development of experimental autoimmune encephalomyelitis (EAE), a rat model of multiple sclerosis. Methods: Female Lewis rats were immunized with 3 pVAXhsp65 doses by intramuscular route. Fifteen days after the last DNA dose the animals were evaluated for specific immunity or submitted to induction of EAE. Animals were evaluated daily for weight loss and clinical score, and euthanized during the recovery phase to assess the immune response and inflammatory infiltration at the central nervous system. Results: Immunization with pVAXhsp65 induced a specific immune response characterized by production of IgG(2b) anti-hsp65 antibodies and IFN-gamma secretion. Previous immunization with pVAXhsp65 did not change EAE clinical manifestations (weight and clinical score). However, the vaccine clearly decreased brain and lumbar spinal cord inflammation. In addition, it downmodulated IFN-gamma and IL-10 production by peripheral lymphoid organs. Conclusion: Our data demonstrated that this vaccine does not trigger a deleterious effect on EAE development and also points to a potential protective effect. Copyright (C) 2010 S. Karger AG, Basel

ICAM-1 (Lys469Glu) and PECAM-1 (Leu125Val) polymorphisms in diffuse astrocytomas

Cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) play an important role in glioma invasion and angiogenesis. The aim of this study was to investigate whether specific genetic polymorphisms of ICAM-1 and PECAM-1 could be associated with glioma development and progression. Single-nucleotide polymorphism in codon 469 of ICAM-1 and codon 125 of PECAM-1 were examined in 158 patients with astrocytomas and 162 controls using polymerase chain reaction and restriction enzyme analysis. The distribution of PECAM-1 polymorphic genotypes in astrocytomas did not show any significant difference. However, a specific ICAM-1 genotype (G/G, corresponding to Lys469Glu) exhibited higher frequency in grade II astrocytomas compared to controls, grade III, and grade IV astrocytomas; suggesting that this polymorphism could be involved in the development of grade II astrocytomas.

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.

IL-4 regulates susceptibility to intestinal inflammation in murine food allergy

Cardoso CR, Provinciatto PR, Godoi DF, Ferreira BR, Teixeira G, Rossi MA, Cunha FQ, Silva JS. IL-4 regulates susceptibility to intestinal inflammation in murine food allergy. Am J Physiol Gastrointest Liver Physiol 296: G593-G600, 2009. First published January 8, 2009; doi:10.1152/ajpgi.90431.2008.-Allergies involve a state of immediate hypersensitivity to antigens, including food proteins. The mechanism underlying the initiation and development of allergic responses involves IL-4 that directly induces the differentiation of committed effector Th2 lymphocytes. Although it is clear that Th2 responses play a pivotal role in the development of allergic responses, it remains unclear which mechanisms are involved in the development of the intestinal damages observed in food allergy. Accordingly, this work aimed to study the role of Th2/IL-4-dependent responses in the development of food allergy and intestinal pathology. C57BL/6 wild-type (WT) and IL-4(-/-) mice were sensitized with peanut proteins, challenged with peanut seeds, and followed for the development of food allergy and intestinal inflammation. Results demonstrated that exposure to peanut seeds led to weight loss in WT but not in IL-4(-/-) mice that preserved gut integrity with no signs of mucosal inflammation. These animals presented increased levels of IgG2a in sera, suggesting a role for allergic antibodies in the pathogenesis of WT animals. Most importantly, results also showed that lack of IL-4 modulated gut mucosal response in food allergy through diminished expression of TNF-alpha mRNA, increased Th1 IFN-gamma, IL-12p40, regulatory cytokines, and Foxp3, demonstrating their relevance in the control of allergic inflammatory processes, especially in the intestine. Finally, this study highlighted some of the complex mechanisms involved in the pathogenesis of allergic responses to food antigens in the gut, thereby providing valuable tools for directing novel therapeutic or preventive strategies to the control of allergic enteropathy.

Modulation of mucosal immunity in a murine model of food-induced intestinal inflammation

Background Hypersensitivity or uncontrolled responses against dietary antigens can lead to inflammatory disorders like food allergy and current models reflect a variety of causes but do not reveal the detailed modulation of gut immunity in response to food antigens after breakdown in mucosal tolerance. Objective To develop and characterize a murine model for food-induced intestinal inflammation and to demonstrate the modulation of gut immune response by dietary allergenic antigens. Methods C57BL/6 mice were sensitized with peanut proteins, challenged with peanut seeds and their sera and gut segments were collected for subsequent analyses. Results Sensitization and challenged with peanut seeds led to alterations in gut architecture with inflammatory response characterized by oedema in lamina propria and cell infiltrate composed mainly by eosinophils, mast cells, phagocytes, natural killer and plasma cells, together with low percentage of gamma delta(+) and CD4(+)CD25(+)Foxp3(+) cells in Peyer`s patches. These animals also presented high levels of specific IgE and IgG1 in sera and modulation of mucosal immunity was mediated by increased expression of GATA-3, IL-4, IL-13 and TNF-alpha in contrast to low IFN-gamma in the gut. Conclusion A murine model for food-induced intestinal inflammation was characterized in which modulation of gut immunity occurs by peanut antigens in consequence of T-helper type 2 (Th2) allergic response and failure of regulatory mechanisms necessary for mucosa homeostasis, resembling food allergy. This work shed some light on the understanding of the pathogenesis of gastrointestinal disorders and intolerance in the gut and supports the development of therapies for food-related enteropathies like food allergy, focusing on gut-specific immune response.

Multi-system neurological disease is common in patients with OPA1 mutations

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal `dominant optic atrophy plus` variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

Human immature dental pulp stem cells` contribution to developing mouse embryos: production of human/mouse preterm chimaeras

In this study, we aimed at determining whether human immature dental pulp stem cells (hIDPSC) would be able to contribute to different cell types in mouse blastocysts without damaging them. Also, we analysed whether these blastocysts would progress further into embryogenesis when implanted to the uterus of foster mice, and develop human/mouse chimaera with retention of hIDPSC derivates and their differentiation. hIDPSC and mouse blastocysts were used in this study. Fluorescence staining of hIDPSC and injection into mouse blastocysts, was performed. Histology, immunohistochemistry, fluorescence in situ hybridization and confocal microscopy were carried out. hIDPSC showed biological compatibility with the mouse host environment and could survive, proliferate and contribute to the inner cell mass as well as to the trophoblast cell layer after introduction into early mouse embryos (n = 28), which achieved the hatching stage following 24 and 48 h in culture. When transferred to foster mice (n = 5), these blastocysts with hIDPSC (n = 57) yielded embryos (n = 3) and foetuses (n = 6); demonstrating presence of human cells in various organs, such as brain, liver, intestine and hearts, of the human/mouse chimaeras. We verified whether hIDPSC would also be able to differentiate into specific cell types in the mouse environment. Contribution of hIDPSC in at least two types of tissues (muscles and epithelial), was confirmed. We showed that hIDPSC survived, proliferated and differentiated in mouse developing blastocysts and were capable of producing human/mouse chimaeras.

CCR5-dependent regulatory T cell migration mediates fungal survival and severe immunosuppression

Paracoccidioidomycosis, a debilitating pulmonary mycosis, is caused by the dimorphic fungus Paracoccidioides brasiliensis. The infection results in the formation of granulomas containing viable yeast cells that are the fungal sources for disease reactivation. Because CD4(+)CD25(+) regulatory T cells (Tregs) are in the lesions of patients with paracoccidioidomycosis, the migration of Treg cells is dependent on the axis chemokine-chemokine receptors, and CCR5 ligands are produced in P. brasiliensis-induced lesions, we investigated the role of CCR5 in the control of the infection. The results showed that CCR5(-/-) mice are more efficient in controlling fungal growth and dissemination and exhibited smaller granulomas than wild-type (WT) mice. In the absence of CCR5, the percentage of CD4(+)CD25(+) T cells expressing Foxp3, glucocorticoid-induced TNFR (GITR), CD103, CD45(low), and CTLA-4 in the granulomas was significantly decreased. Interestingly, P. brasiliensis infection resulted in an absence of T cell proliferation in response to Con A in WT but not CCR5(-/-) mice that was abrogated by anti-CTLA-4 mAb and anti-GITR mAb. Moreover, the adoptive transfer of CD4(+)CD25(+) but not CD4(+)CD25(-) T cells from infected WT to infected CCR5(-/-) mice resulted in a significant increase in fungal load. Overall, CCR5 is a key receptor for the migration of Treg cells to the site of P. brasiliensis infections leading to down-modulation of effector immune response and the long-term presence of the fungus in the granulomas. Thus, a tight control of Treg cell migration to the granulomatous lesions could be an important mechanism for avoiding exacerbation and reactivation of the disease.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.