Christina Arslanian

Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

PURPOSE FTY720 (fingolimod) is an immunomodulatory drug capable of preventing T-cell migration to inflammatory sites by binding to and subsequently downregulating the expression of sphingosine-1 phosphate receptor 1 (S1P(1)) leading in turn to T-cell retention in lymphoid organs. Additional effects of FTY720 by increasing functional activity of regulatory T cells have recently been demonstrated, raising the conversion of conventional T cells into regulatory T cells and affecting the sequestration of regulatory T cells in normal mice. In this study, the action of FTY720 in the ocular autoimmune model in mice was investigated. METHODS Mice were immunized with 161-180 peptide and pertussis toxin and were treated with 1 mg/kg/d FTY720 by gavage (7-21 days postimmunization [dpi]) or left untreated. Spleen cells, harvested 21 dpi, were cultured and assayed for cytokine production. Draining lymph node, spleen, and eye cells 21 dpi were assayed for quantification of T-cell populations. Disease severity was evaluated by histologic examination of the enucleated eyes at 21 and 49 dpi. In addition, anti-IRBP antibodies were analyzed by ELISA. RESULTS FTY720 was effective in suppressing the experimental autoimmune uveitis score. Although there was a reduction in the number of eye-infiltrating cells, FTY did not prevent Treg accumulation at this site. FTY720 leads to a significant increase of CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cell percentages in lymph nodes, suggesting that this site could be the source of Treg cells found in the eye. CONCLUSIONS The data showed that treatment in vivo with FTY720 was able to suppress EAU in mice. These results are indicative of the possible therapeutic use of FTY720 in ocular autoimmune processes.

Transfer of antibodies across the placenta and in breast milk from mothers on intravenous immunoglobulin

We studied the levels of immunoglobulins in colostrum, milk and sera from two common variable immunodeficiency (CVID) mothers (M1 and M2), and in sera from their newborn infants. During pregnancy they continued intravenous immunoglobulin therapy (IVIG). Antibody levels from maternal and cord blood collected at delivery and colostrum and milk, collected on the 3rd and 7th post‐partum days, respectively, were analyzed. Although cord/maternal blood ratios of total immunoglobulins and subclasses, as well as specific antibodies differed between M1 and M2, both showed good placental transfer of anti‐protein and anti‐polysaccharide antibodies, despite lower cord/maternal blood ratios in M2. Anti‐Streptococcus pneumoniae antibody avidity indexes were similar between paired maternal and cord serum. Both mothers’ colostrum and milk samples showed only traces of IgA, and IgM and IgG levels in colostrum were within normal range in M1, whereas M2 presented elevated IgG and low IgM levels, when compared with healthy mothers. The study of colostrum and milk activity showed that they strongly inhibited enteropathogenic Escherichia coli adhesion in vitro. CVID patients must be informed about the relevance of regular IVIG administration during pregnancy, not only for their own health but also for their immune immature offspring. Breast‐feeding should be encouraged as colostra from these CVID patients strongly inhibited E. coli adhesion to human epithelial cells thus providing immunological protection plus nutritional and psychological benefits for the infant.

Elevated levels and different repertoire profile of colostral anti-LPS antibodies may have a significant role in compensating newborn immunity.

A high prevalence of systemic infections caused by enterobacteria such as Escherichia coli is observed during the neonatal period. Lipopolysaccharide (LPS) is one of the major factors responsible for septic shock caused by these Gram‐negative bacteria. We have recently demonstrated the presence of anti‐LPS immunoglobulin (Ig)G antibodies in cord blood with a repertoire identical to that found in maternal serum. In the present study, we analyzed anti‐LPS O111 antibody isotypes in maternal serum and colostrum from mothers and in cord serum from their respective full‐term (n = 30) and preterm (n = 13) neonate infants. The main isotype found in serum samples from mothers of term infants was IgM (range between 28 and 54 mg/l), followed by IgA (1–2 mg/l) and IgG (2–3 mg/l). The range of IgG antibody concentrations in cord blood was between 2 and 3 mg/l, as a result of placental transfer. A novel observation in our study was that the LPS bands recognized by colostral antibodies were completely different from those recognized by IgG in serum. Colostral IgA antibodies recognized several bands not bound by serum IgG antibodies from the respective maternal serum, independently of the antibody quantity. In addition, we verified the pattern of LPS recognition by serum IgA and colostral IgA antibodies was identical, what suggested that the antibody isotype found in serum could probably be derived from differentiated IgA‐positive cells which were homing to the mucosa through the mucosal homing mechanism. Identical pattern of recognition was obtained comparing the IgA and IgM isotypes in colostrum. Slight differences in the pattern of recognition were found between colostral and serum IgM antibodies. The fact that colostral antibodies recognize much more bands than serum antibodies may be important for the host to mount an effective immune response in the intestinal lumen, in order to prevent excessive absorption of LPS, reducing possible systemic effects caused by the molecule.

Mother to child transfer of IgG and IgA antibodies against Dermatophagoides pteronyssinus.

There is strong evidence from animal models that placental and/or breast milk‐mediated transfer of maternal allergen‐specific IgG prevents allergic immune responses in the progeny. Both human and animal data also point to IgA as having an important regulatory role. In contrast, little is known about maternal transfer of IgG and IgA specific for respiratory allergens in humans. Dermatophagoides pteronyssinus (Der p) is an indoor allergen that is a major cause of asthma worldwide. We analysed maternal to child Der p‐specific IgG and IgA transfer in a cohort of 77 paired maternal and child samples. We found Der p‐specific IgG and its IgG1, IgG2 and IgG4 subclasses in all cord blood samples. Except for IgG1, cord levels were higher in newborns from atopic mothers (n = 29) compared to non‐atopic mothers (n = 48). Der p‐specific IgA was found in all colostrum samples and levels were independent of maternal atopic status. Notably, anti‐Der p IgG was also found in colostrum and levels were higher in atopic mothers. We believe that our work is a critical first step in the identification of early factors that may impact asthma development and should guide the development of clinical studies that assess whether Der p‐specific IgG and IgA protect children from allergy as demonstrated in animal models.

Passive immunity acquisition of maternal anti-enterohemorrhagic Escherichia coli (EHEC) O157:H7 IgG antibodies by the newborn

Enterohaemorrhagic Escherichia coli (EHEC) strains are among the main causes of haemorrhagic colitis (HC) and haemolytic-uremic syndrome (HUS) in industrialised countries. In Brazil, EHEC have been detected in the faeces of patients with non-bloody diarrhoea, though an association between EHEC and HUS has been detected recently. These observations suggest that there is a pre-existing immunity triggered by the contact with EHEC and other categories of bacteria, such as EPEC, that share similar virulence factors and to which our population is highly exposed. Our aim was to evaluate the placental transfer of IgG antibodies reactive to EHEC O157:H7 antigens. We evaluated 28 paired maternal and cord sera for the presence of IgG against EHEC O157:H7 protein antigens and IgG and IgM to O157 LPS employing ELISA and IB technique. Total IgG and IgM level analyses were also made. Anti-EHEC O157:H7 and anti-LPS O157 IgG antibody levels in cord sera were equivalent to those of their maternal sera. A good correlation between the mothers’ anti-LPS O157 IgM and total IgM levels was found. Anti-LPS O157 IgM levels were higher than anti-LPS O157 IgG levels in the same samples, and anti-LPS IgM antibodies were not detected in cord sera. Identical patterns of recognition of bacterial protein antigens by specific IgG were found in the paired samples and the recombinant purified variable region of γ intimin was specifically recognized by one paired maternal and cord sample. In conclusion, although the antibody profile varied among individuals, all paired cord and maternal serum samples showed an identical recognition pattern, indicating an efficient placental transfer of IgG antibodies reactive to EHEC O157:H7 antigens.

IL-10 and TGF-β Immunoregulatory Cytokines rather than Natural Regulatory T Cells are Associated with the Resolution Phase of Vogt-Koyanagi-Harada (VKH) Syndrome

The pro‐inflammatory cytokines play a critical role in the initiation and propagation of ocular autoimmune diseases. Regulation of these cytokines is generally mediated by the immunoregulatory cytokine such as IL‐10 or TGF‐β. In this study, we investigated the immunoregulatory cytokine profile and frequency of natural regulatory T cells (nTregs) in patients with Vogt‐Koyanagi‐Harada (VKH). We obtained the peripheral blood mononuclear cells (PBMC) from patients with VKH and healthy controls. The cytokine profile from supernatants of PBMC cultured with or without phytohaemagglutinin (PHA) was measured by ELISA, the percentage of CD4+ Foxp3+ and CD25highFoxp3+ T regulatory cells were analysed by flow cytometry, and the transcriptional level of Foxp3 expression was analysed by real‐time quantitative PCR. The immunoregulatory cytokines, TGF‐β and IL‐10, increased in patients with VKH in the inactive stage of the disease. We observed no significant difference in the CD4+ Foxp3+ and CD25highFoxp3+ T cells as well as no reduction in FOXP3 mRNA expression in the patients with VKH when compared to healthy controls. We showed in our work, an increase in IFN‐γ secretion by PBMC of patients with VKH in the active stage of the disease when compared to healthy controls and patients in the inactive stage. Our data suggest that IL‐10 and TGF‐β cytokines, rather than nTregs are associated with the resolution phase of the disease and may have a more relevant role in controlling this disease.

4-Fluoro-2-methoxyphenol, an apocynin analog with enhanced inhibitory effect on leukocyte oxidant production and phagocytosis

Apocynin, a methoxy-substituted catechol (4-hydroxy-3-methoxyacetophenone), originally extracted from the roots of Picrorhiza kurroa, has been extensively used as a non-toxic inhibitor of the multienzymatic complex NADPH oxidase. We discovered that the analogous methoxy-substituted catechol, 4-Fluoro-2-methoxyphenol (F-apocynin), in which the acetyl group present in apocynin was changed to a fluorine atom, was significantly more potent as an inhibitor of NADPH oxidase activity, myeloperoxidase (MPO) chlorinating activity and phagocytosis of microorganisms by neutrophils; it was also as potent as apocynin in inhibiting tumor necrosis factor-alpha (TNFα) release by peripheral blood mononuclear cells. We attribute the increased potency of F-apocynin to its increased lipophilicity, which could facilitate the passage of the drug through the cell membrane. The inhibition of MPO chlorination activity, phagocytosis and TNFα release shows that apocynin and F-apocynin actions are not restricted to reactive oxygen species inhibition, but further studies are needed to clarify if these mechanisms are related. Like apocynin, F-apocynin did not show cell toxicity, and is a strong candidate for use in the treatment of inflammatory diseases.

Evaluation of humoral response to heptavalent pneumococcal conjugate vaccine in HIV-infected children

OBJETIVO: A doenca pneumococica invasiva e importante causa de morbi-mortalidade em criancas infectadas pelo HIV. O objetivo do estudo foi avaliar quantitativamente a resposta com anticorpos aos sete sorotipos pneumococicos da vacina em um grupo de criancas infectadas pelo HIV. METODOS: Estudo realizado com 40 criancas infectadas pelo HIV, com idade entre 2 e 9 anos, em seguimento em ambulatorio especializado no municipio de Sao Paulo, em 2002-2003. A dosagem de anticorpos IgG contra os polissacarideos da capsula pneumococica foi realizada por meio de ensaio imunoenzimatico (ELISA). Os anticorpos foram dosados imediatamente antes e um mes apos a aplicacao da segunda dose da vacina. Utilizaram-se dois criterios para avaliar a resposta a vacina: titulos de anticorpos >1,3 µg/mL na sorologia pos-imunizacao e aumento >4 vezes nos titulos da sorologia pos em relacao a pre-imunizacao. RESULTADOS: Para o primeiro criterio (>1,3 µg/mL), 26 (65%) criancas obtiveram resposta sorologica a vacina, 12 (30%) delas apresentaram titulos de IgG pos-imunizacao em niveis de pelo menos 1,3 µg/mL para todos os sorotipos. Para o segundo criterio (incremento >4 vezes nos titulos para quatro sorotipos ou mais), obteve-se resposta sorologica para 15 (37,5%) criancas. CONCLUSOES: A resposta a vacina foi considerada satisfatoria, com aumento estatisticamente significante dos titulos geometricos medios pos-vacinais em relacao aos pre-vacinais para todos os sorotipos estudados.OBJECTIVE Invasive pneumococcal disease is a major cause of death in HIV-infected children. The objective of the study was to assess the quantitative antibody response to the seven pneumococcal serotypes of heptavalent pneumococcal conjugate vaccine in a group of HIV-infected children. METHODS Study comprising 40 HIV-infected children aged between 2 and 9 years followed up in a specialized outpatient clinic in São Paulo, Brazil, between 2002 and 2003. Enzyme immunoassay (ELISA) was used to measure IgG antibody titers against pneumococcus capsule. Antibodies were measured immediately before and 1 month after the second dose of the vaccine. Two response criteria were used: IgG titers >or=1.3 microg/mL in the post-immunization serology and an increase of at least 4or=-fold in post- compared to pre-immunization serology. RESULTS For the first criterion (>or=1.3 microg/mL), 26 (65%) children had serological response to the vaccine, 12 (30%) showed post-immunization IgG titers of at least 1.3 microg/mL for all seven serotypes studied. For the second criterion studied (>or=4-fold increase in post- compared to pre-immunization titers for four serotypes or more), serological response was seen in 15 (37.5%) children. CONCLUSIONS Overall response to the heptavalent pneumococcal conjugate vaccine was adequate, showing a statistically significant increase in the post-immunization geometric mean titers for the seven serotypes studied.

Human IgG but not IgM Antibodies can Protect Mice from the Challenge with Live O6 Escherichia coli

We evaluated the ability of human anti‐lipopolysaccharide (LPS) O6 immunoglobulin G (IgG) and IgM antibodies to protect mice challenged with Escherichia coli serotype O6:K2ac. Purified whole IgG, commercial gammaglobulin, whole IgM‐effluent, pool of normal human serum (NHS), agammaglobulinaemic serum (test groups) or phosphate‐buffered saline (control group) was injected into adult male 18 h before a challenge with viable O6 E. coli. The mortality rate was assessed over a period of 72 h. To determine the opsonic and phagocytic activity of the antibody isotypes, we incubated peritoneal macrophages from the control and test groups collected at different times after challenge with the live bacteria with acridine orange for fluorescent analysis. Tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 were quantified in serum of both the test and control groups. All mice that received commercial gammaglobulin or NHS survived. Purified whole IgG (containing 1.1 mg/l of anti‐LPS O6 IgG antibodies) protected 87.5% of the animals tested in this experiment, while whole IgM‐enriched effluent with 1.5 mg/l of anti‐LPS O6 IgM antibodies protected only 12.5%. The agamma serum showed no protective capacity compared with PBS (serving as control). The minimal concentration of anti‐LPS O6 IgG antibodies able to protect 50% of animals was 0.137 mg/l of purified whole IgG. Whole IgM‐enriched effluent showed no protective capacity independently of the concentration tested (0.048–17.0 mg/l of anti‐LPS O6 IgM antibodies). Fluorescent analysis of peritoneal macrophages from animals pretreated with purified whole IgG showed no bacteria at 8 h after the challenge. By contrast, whole IgM effluent showed an increasing number of live bacteria at the same time. Mice that had received whole IgM effluent (1.5 mg/l of anti‐LPS O6 IgM antibodies) before the challenge with LPS O6 presented 20.5 µg/l of IL‐6 and 1.5 µg/l of TNF‐α. Serum from animals pretreated with purified IgG did not present any detectable pro‐inflammatory cytokine. Our findings suggest that IgG but not IgM antibodies protect animals from a challenge with E. coli O6 serotype.

Early Exposure to Respiratory Allergens by Placental Transfer and Breastfeeding

The relationship between allergen exposure and the onset of or protection from allergic diseases remains unclear. Many factors could be related to immunological responses, such as the age when the exposure occurs, type of allergen, timing, dose, and allergen route. In this study, we investigated whether exposure to respiratory allergens could occur in pregnancy or early life. In particular, we assessed whether Der p 1 and Blo t 5, as well as specific antibodies against these allergens, could be detected in 90 paired cord blood and colostrum samples. Der p 1 was detected in 58.6% of colostrum and 29% of cord blood samples, whereas Blot 5 was positive in 41.3% and 9.6% of the samples, respectively. Similar to specific IgA, which could be detected in all samples for both mites, specific IgG was found in a high number of colostrum samples, 93.5% and 94.8% for Dp and Bt, respectively. Although allergens were not detected in all cord blood samples, a high percentage of them (≥95%) were positive for specific IgM to both mites in cord blood samples, suggesting that neonates can be exposed and sensitized to airborne allergens during pregnancy. Many studies have attempted to correlate allergen exposure or its prevention in early infancy with the onset of or protection from allergic diseases. However, conflicting and inconsistent data do not show a clear correlation with or suggest a way to prevent allergen sensitization. Nevertheless, these unconvincing results could be better understood if the relationship with many aspects of allergen exposure after pregnancy could be clarified. Thus, it is necessary to address basic issues related to allergen exposure, including the development of reproducible, standardized and reliable methods, and to determine how and where the exposure occurs.