Absence of NOD2 receptor predisposes to intestinal inflammation by a deregulation in the immune response in hosts that are unable to control gut dysbiosis.

Mutations in NOD2 predisposes to Inflammatory Bowel Diseases. Therefore, we evaluated the role of this innate receptor in the modulation of immunity in face of host microbiota changes. NOD2-/- mice presented higher susceptibility to experimental colitis than WT, with increased CD4 and CD8 T lymphocytes in the spleen. NOD2 deficiency also led to reduced Th17-related cytokines in the colon, with overall augmented IFN-γ in the gut and spleen. Nonetheless, there was increased frequency of CD4+IL-4+ cells in the mesenteric lymph nodes besides elevated CTLA-4 and FoxP3 regulatory markers in the spleen of NOD2-/- mice, although it did not result in more efficient control of gut inflammation. Indeed, these animals also had augmented IL-1ß and IL-5 in the peritoneum, indicating that this receptor may be important to control bacteria translocation too. Microbiota exchanging between cohoused WT and NOD2-/- mice led to colitis worsening in the absence of the receptor, while antibiotic therapy in WT mice abrogated this effect. Then, not only the genetic mutation confers increased susceptibility to inflammation, but it is also influenced by the microbiota harbored by the host. Finally, NOD2-/- mice are more prone to intestinal inflammation due to deregulated immune response and increased susceptibility to colitogenic bacteria.

Lacrimal Cytokines Assessment in Subjects Exposed to Different Levels of Ambient Air Pollution in a Large Metropolitan Area.

BACKGROUND: Air pollution is one of the most environmental health concerns in the world and has serious impact on human health, particularly in the mucous membranes of the respiratory tract and eyes. However, ocular hazardous effects to air pollutants are scarcely found in the literature. DESIGN: Panel study to evaluate the effect of different levels of ambient air pollution on lacrimal film cytokine levels of outdoor workers from a large metropolitan area. METHODS: Thirty healthy male workers, among them nineteen professionals who work on streets (taxi drivers and traffic controllers, high pollutants exposure, Group 1) and eleven workers of a Forest Institute (Group 2, lower pollutants exposure compared to group 1) were evaluated twice, 15 days apart. Exposure to ambient PM2.5 (particulate matter equal or smaller than 2.5 µm) was 24 hour individually collected and the collection of tears was performed to measure interleukins (IL) 2, 4, 5 and 10 and interferon gamma (IFN-γ) levels. Data from both groups were compared using Student's t test or Mann- Whitney test for cytokines. Individual PM2.5 levels were categorized in tertiles (lower, middle and upper) and compared using one-way ANOVA. Relationship between PM2.5 and cytokine levels was evaluated using generalized estimating equations (GEE). RESULTS: PM2.5 levels in the three categories differed significantly (lower: ≤22 µg/m3; middle: 23-37.5 µg/m3; upper: >37.5 µg/m3; p<0.001). The subjects from the two groups were distributed unevenly in the lower category (Group 1 = 8%; Group 2 = 92%), the middle category (Group 1 = 89%; Group 2 = 11%) and the upper category (Group 1 = 100%). A significant relationship was found between IL-5 and IL-10 and PM2.5 levels of the group 1, with an average decrease of 1.65 pg/mL of IL-5 level and of 0.78 pg/mL of IL-10 level in tear samples for each increment of 50 µg/m3 of PM2.5 (p = 0.01 and p = 0.003, respectively). CONCLUSION: High levels of PM2.5 exposure is associated with decrease of IL-5 and IL-10 levels suggesting a possible modulatory action of ambient air pollution on ocular surface immune response.

Moderate acute exercise (70% VO2 peak) induces TGF-ß, α-amylase and IgA in saliva during recovery.

Strenuous exercise promotes changes in salivary IgA and can be associated with a high incidence of upper respiratory tract Infections. However, moderate exercise enhances immune function. The effect of exercise on salivary IgA has been well studied, but its effect on other immunological parameters is poorly studied. Thus, this study determined the effect of moderate acute exercise on immunological salivary parameters, such as the levels of cytokines (TGF-ß and IL-5), IgA, α-amylase and total protein, over 24 h. Ten male adult subjects exercised for 60 min at an intensity of 70% VO2 peak. Saliva samples were collected before ('basal') and 0, 12 and 24 h after an exercise session. The total salivary protein was lower after 12 and 24 h than immediately after exercise, whereas α-amylase increased at 12 and 24 h after exercise compared with basal levels. The IgA concentration was increased at 24 h after exercise relative to immediately after exercise, and there was no difference in the IL-5 while TGF-ß concentration increased in recovery. In conclusion, 70% VO2 peak exercise does not induce changes immediately after exercise, but after 24 h, it produces an increase in salivary TGF-ß without changing IL-5.

Vaccination with enzymatically cleaved GPI-anchored proteins from Schistosoma mansoni induces protection against challenge infection.

The flatworm Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment. In the search for potential vaccine candidates, numerous tegument antigens have been assessed. As the major interface between parasite and mammalian host, the tegument plays crucial roles in the establishment and further course of schistosomiasis. Herein, we evaluated the potential of a GPI fraction, containing representative molecules located on the outer surface of adult worms, as vaccine candidate. Immunization of mice with GPI-anchored proteins induced a mixed Th1/Th2 type of immune response with production of IFN-γ and TNF-α, and low levels of IL-5 into the supernatant of splenocyte cultures. The protection engendered by this vaccination protocol was confirmed by 42% reduction in worm burden, 45% reduction in eggs per gram of hepatic tissue, 29% reduction in the number of granulomas per area, and 53% reduction in the granuloma fibrosis. Taken together, the data herein support the potential of surface-exposed GPI-anchored antigens from the S. mansoni tegument as vaccine candidate.

Schistosoma mansoni antigens alter the cytokine response in vitro during cutaneous leishmaniasis.

Schistosoma mansoni infection or associated products are able to down-modulate the type 1 CD4+ T cell inflammatory response characteristic of autoimmune diseases. In this study, we evaluated how S. mansoni antigens altered the immune response that was induced by the soluble Leishmania antigen (SLA) from cutaneous leishmaniasis (CL) patients. Cytokines were measured from the supernatants of peripheral blood mononuclear cell cultures stimulated with SLA. This was performed using the sandwich enzyme linked immunosorbent assay technique in the presence or absence of S. mansoni recombinant antigens Sm29, SmTSP-2 and PIII. The addition of S. mansoni antigens to the cultures resulted in the reduction of interferon gamma (IFN-γ) levels in 37-50% of patients. Although to a lesser extent, the antigens were also able to decrease the production of tumour necrosis factor-alpha (TNF-α). We compared patients that either had or did not have reduction in IFN-γ and TNF-α production in cultures stimulated with SLA in the presence of S. mansoni antigens. We found that there was no significant difference in the levels of interleukin (IL)-10 and IL-5 in response to S. mansoni antigens between the groups. The antigens used in this study down-modulated the in vitro proinflammatory response induced by SLA in a group of CL patients through a currently undefined mechanism.

Trichinella spiralis: intranasal immunization with attenuated Salmonella enterica carrying a gp43 antigen-derived 30mer epitope elicits protection in BALB/c mice.

Trichinellosis is a public health problem and is considered an emergent/re-emergent disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects domestic animals such as pigs and horses, as well as wild animals and humans. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Attenuated Salmonella strains are especially attractive live vectors because they elicit mucosal immunity, which is known to be important for the control of Trichinella spiralis infection at the intestinal level and can be administered by oral or intranasal routes. In this study, the autotransporter ShdA was used to display, on the surface of the Salmonella enterica serovar Typhimurium SL3261, the 210-239 amino acid epitope, (designated as Ag30) derived from the 43 kDa glycoprotein of T. spiralis muscle larvae. The fusion protein elicited antibodies in BALB/c mice that were able to recognize the native epitope on the surface of T. spiralis muscle larvae. Mice immunized by intranasal route with the recombinant Salmonella induced a protective immune response against the T. spiralis challenge, reducing by 61.83% the adult burden at day eight postinfection. This immune response was characterized by the induction of antigen-specific IgG1 and of IL-5 production. This study demonstrates the usefulness of Salmonella as a carrier of nematode epitopes providing a surface display system for intestinal parasite vaccine applications.

IL-4Rα-responsive smooth muscle cells contribute to initiation of TH2 immunity and pulmonary pathology in Nippostrongylus brasiliensis infections.

Nippostrongylus brasiliensis infections generate pulmonary pathologies that can be associated with strong T(H)2 polarization of the host's immune response. We present data demonstrating N. brasiliensis-driven airway mucus production to be dependent on smooth muscle cell interleukin 4 receptor-α (IL-4Rα) responsiveness. At days 7 and 10 post infection (PI), significant airway mucus production was found in IL-4Rα(-/lox) control mice, whereas global knockout (IL-4Rα(-/-)) and smooth muscle-specific IL-4Rα-deficient mice (SM-MHC(Cre) IL-4Rα(-/lox)) showed reduced airway mucus responses. Furthermore, interleukin (IL)-13 and IL-5 cytokine production in SM-MHC(Cre) IL-4Rα(-/lox) mice was impaired along with a transient reduction in T-cell numbers in the lung. In vitro treatment of smooth muscle cells with secreted N. brasiliensis excretory-secretory antigen (NES) induced IL-6 production. Decreased protein kinase C (PKC)-dependent smooth muscle cell proliferation associated with cell cycle arrest was found in cells stimulated with NES. Together, these data demonstrate that both IL-4Rα and NES-driven responses by smooth muscle cells make important contributions in initiating T(H)2 responses against N. brasiliensis infections.

Inducible nitric oxide synthase/CD95L-dependent suppression of pulmonary and bone marrow eosinophilia by diethylcarbamazine.

RATIONALE: The mechanism of action of diethylcarbamazine (DEC), an antifilarial drug effective against tropical pulmonary eosinophilia, remains controversial. DEC effects on microfilariae depend on inducible NO synthase (iNOS). In eosinophilic pulmonary inflammation, its therapeutic mechanism has not been established. We previously described the rapid up-regulation of bone marrow eosinophilopoiesis in ovalbumin (OVA)-sensitized mice by airway allergen challenge, and further evidenced the down-regulation of eosinophilopoiesis by iNOS- and CD95L-dependent mechanisms. OBJECTIVES: We investigated whether: (1) DEC can prevent the effects of airway challenge of sensitized mice on lungs and bone marrow, and (2) its effectiveness depends on iNOS/CD95L. METHODS: OVA-sensitized BALB/c mice were intranasally challenged for 3 consecutive days, with DEC administered over a 12-, 3-, or 2-day period, ending at the day of the last challenge. We evaluated: (1) airway resistance, cytokine (IFN-gamma, IL-4, IL-5, and eotaxin) production, and pulmonary eosinophil accumulation; and (2) bone marrow eosinophil numbers in vivo and eosinophil differentiation ex vivo. MEASUREMENTS AND MAIN RESULTS: DEC effectively prevented the effects of subsequent challenges on: (1) airway resistance, Th1/Th2 cytokine production, and pulmonary eosinophil accumulation; and (2) eosinophilopoiesis in vivo and ex vivo. Recovery from unprotected challenges included full responses to DEC during renewed challenges. DEC directly suppressed IL-5-dependent eosinophilopoiesis in naive bone marrow. DEC was ineffective in CD95L-deficient gld mice and in mice lacking iNOS activity because of gene targeting or pharmacological blockade. CONCLUSIONS: DEC has a strong impact on pulmonary eosinophilic inflammation in allergic mice, as well as on the underlying hemopoietic response, suppressing the eosinophil lineage by an iNOS/CD95L-dependent mechanism.

Diagnosing immune-mediated reactions to drugs.

Drug allergy is a type B adverse drug reaction, which is unpredictable and difficult to prevent or manage. In patients who have a previous history of drug allergy it must be confirmed by laboratorial diagnosis. However, the diagnostic test remains a major problem in clinical practice. Skin testing is validated for some drugs, such as penicillin, but not for others. Provocation test is a confirmatory test but bears the risk of severe reactions. Lymphocyte transformation test is a reliable test but is considered as a research tool. This review addresses the most recent published literature regarding the techniques which have already been developed as well as the new tests that can be promising alternatives for diagnosis of drug allergy.

Glucocorticoids inhibit GATA-3 phosphorylation and activity in T cells.

Glucocorticoid (GC) immunosuppression and anti-inflammatory action involve the regulation of several transcription factors (TFs). GCs inhibit the acute production of T-helper (Th) 1 and Th2 cytokines but ultimately favor a shift toward Th2 phenotype. GCs inhibit the transcriptional activity of T-bet Th1 TF by a transrepression mechanism. Here we analyze GC regulation of GATA-3, the master driver of Th2 differentiation. We found that GCs inhibit GATA-3 transcriptional activity. We demonstrate that this mechanism does not involve physical interaction between the glucocorticoid receptor (GR) and GATA-3 or reduction of GATA-3 binding to DNA, as described previously for T-bet. Instead, GCs inhibit GATA-3 activity by inhibition of p38 mitogen-activated protein kinase induced GATA-3 phosphorylation. GCs also inhibit GATA-3 mRNA and protein expression. Finally, GATA-3 inhibition affects the interleukin-5 gene, a central Th2 cytokine. The IC(50) of dexamethasone is 10 nM with a maximum effect at 100 nM. All inhibitory actions were blocked by the GR antagonist RU38486 (1 uM), proving the specificity of GR action. In view of the crucial role of GATA-3 in T-cell differentiation and inflammation, we propose that the mechanism of GATA-3 inhibition compared with that in T-bet may have relevant implications in understanding and modulating the anti-inflammatory and Th-regulatory properties of GCs.

[IL-4, IL-5 and IL-13 expression during in vitro immune response to fungal allergens in sensitized subjects]. / Expresión de IL-4, IL-5 e IL-13 durante la respuesta inmunitaria in vitro a alérgenos fúngicos en individuos sensibilizados.

BACKGROUND: Determination of T(H)2 citokines profile have arisen interest to understand the pathophysiology of allergic diseases. OBJECTIVE: To analyze the expression of IL-4, IL-5 and IL-13 during the immune response to allergic fungi in sensitized patients with allergic rhinitis. MATERIAL AND METHOD: Mononuclear cells (Ficoll-paque d = 1.077) were isolated from four patients with allergic rhinitis and positive skin prick test to allergic fungi and subsequently activated with fungal allergic extracts. The supernatants were collected and the levels of IL-4, IL-5 and IL-13 were determined by enzyme-linked immune assay. RESULTS: The levels of IL-4 in supernatants were higher than IL-5 levels and IL-13 levels (p < 0.05) during the first 72 hours of challenge with fungal allergens in vitro. The expression of above mentioned cytokines was different attending time course response and fungal species in sensitized patients. CONCLUSIONS: IL-4, but not IL-5 and IL-13 either, showed a sustained expression during 72 hours after the challenge to allergic fungi in vitro. IL-4 could be studied as biomarker to estimate the allergenicity to fungi in sensitized patients.

Humoral and cellular immune responses to Blomia tropicalis and concanavalin A-binding fractions in atopic patients.

Blomia tropicalis, Dermatophagoides pteronyssinus and D. farinae are prevalent house dust mites. Concanavalin A-binding components derived from B. tropicalis (Bt-ConA extract) are highly immunogenic in allergic diseases. The aim of the present study was to evaluate the humoral and cellular immune responses to B. tropicalis in mite-sensitized patients. A total of 137 patients with allergic rhinitis with/without asthma and 109 non-atopic subjects were selected and analyzed by the skin prick test, and for total serum IgE and specific IgE levels to both Bt-total and Bt-ConA extracts, their proliferative response and cytokine (IFN-gamma and IL-5) production by peripheral blood mononuclear cells (PBMC) stimulated with both extracts. Skin prick test showed that 70% of the patients were sensitized to Bt (Bt+) and similar levels of specific IgE to Bt-total and Bt-ConA extracts were demonstrable in Bt+ patients. Significant PBMC proliferation was observed in response to Bt-total extract in Bt+, but not in Bt- patients and non-atopic subjects (P < 0.001). Bt-ConA extract induced increased proliferative responses in all patient groups compared to medium alone (P < 0.05), but these responses were significantly decreased in the presence of the mannopyranoside ConA inhibitor (P < 0.05). Significant IFN-gamma production was observed after Bt-ConA stimulation of Bt+ patients (P < 0.05), while Bt-total extract had no effect. IL-5 production was consistently detected in Bt+ patients after allergen-specific stimulation or with no stimulus, indicating that PBMC from allergic patients are prone to produce Th2 profile cytokines, spontaneously or inductively by allergen restimulation. These data showed that ConA-binding components isolated from B. tropicalis may contain relevant antigens that are involved in both humoral and cellular immune responses. However, without an additional purification procedure to eliminate the residual contamination with ConA, its use in immunotherapeutic procedures cannot be recommended.

New meningococcal C polysaccharide-tetanus toxoid conjugate Physico-chemical and immunological characterization.

The polysaccharides (Ps) are thymus-independent 2 (TI-2) antigens and poor immunogens in infants and young children; as a result of this delayed response to Ps antigens during ontogeny, infants and young children are highly susceptible to infections caused by encapsulated bacteria. Meningococcal group C polysaccharide (PsC)-proteins conjugate vaccines have been reported to induce significant serum IgG antibodies and immunologic memory in infants resulting in very effective vaccines. We describe here the obtainment, by a new method, of a neoglycoconjugate intended to immunize against Neisseria meningitidis serogroup C, its characterization by physico-chemical methods, including (1)H NMR and fluorescence spectroscopy methods, as well as the characterization of the immune response induced in mice by such conjugate. Amine groups generated by basic hydrolysis in the PsC were successfully conjugated to carboxyl groups of tetanus toxoid (TT), using carbodiimide-mediated coupling. The specific anti-Ps IgG and anti-Ps IgG subclasses (IgG1 and IgG2a) were measured by ELISA methods, the bactericidal activity in sera and the cytokines response (IFNgamma or IL5) in spleen cell of mice immunized with conjugated and native Ps were evaluated. The (1)H NMR spectra and the result obtained by the fluorescence spectroscopy method showed that the PsC and TT maintained structural identity after conjugation process. Conjugated PsC elicited an increase of anti-PsC IgG responses, anti-PsC IgG subclass (IgG1, IgG2a), an eight-fold increase in bactericidal activity in sera of mice immunized with conjugate compared with native PsC, was also observed. Higher titres of IFNgamma were observed in mice immunized with conjugated Ps. These results indicated that, the PsC and TT maintained its chemical and antigenic structure after the conjugation process. A change in the immunological pattern of responses of PsC, from TI-2 to a thymus-dependent (TD) pattern, was also demonstrated.

An opposite role is exerted by the acarian Myocoptes musculinus in the outcome of Toxoplasma gondii infection according to the route of the protozoa inoculation.

Infection with Toxoplasma gondii leads to a Th1 immune response. Alternatively, the acarian Myocoptes musculinus induces a disease in BALB/c mice that involves Th2 immune mechanisms. In this study, we investigated whether infestation by M. musculinus induces Th2 immune response in C57BL/6 mice and if this response influences the T. gondii-induced Th1 response when mice are inoculated by intraperitoneal or oral route. The animals were infected with M. musculinus and one month later with T. gondii ME-49 strain and the survival and immune response were monitored. The co-infected animals displayed higher mortality rate and the spleen cells showed a decreased IFN-gamma and elevated IL-4 and IL-5 production. These changes were associated with severe pneumonia and wasting condition. On the other hand, when mice were orally infected with 100 T. gondii cysts, co-infection prolonged the survival rates and ameliorated intestinal lesions in association with a significant drop in IFN-gamma levels in sera. These results indicate the interference of Th2 response induced by M. musculinus in a T. gondii-induced Th1 response. Altogether, these data demonstrate the profound interactions between the immune response induced against unrelated organisms T. gondii and M. musculinus, and suggest that this type of interactions may impact clinical disease.

Experimental immunization with Thalassophryne nattereri fish venom: striking IL-5 production and impaired of B220+ cells.

Murine experimental model have been useful to understanding the toxic as well as the pharmacological properties of the Thalassophryne nattereri venom. However, the specific immune response to T. nattereri venom in mice is yet unclear. Our results showed that the venom elicited in BALB/c mice high levels of specific IgG1 and total IgE isotype with high affinity, accompanied by a striking IL-5 production, what point out to a Th2-like response. Meanwhile, the production of IFN-gamma by lymphocytes pool expanded upon mitogen stimulus, suggests that the venom was also able to activate Th1 clones. Elevated number of antigen-presenting cells expressing CD11c or CD11b from day 4 to 6 supported ongoing antigen presentation process in the primary response and efficient T-cell expansion (increase of CD4(+) cells). In contrast, decreased B220 expression was observed, suggesting that the formation of memory long lived cell compartment. In conclusion, T. natterri venom stimulates an association of cytokine of both Th1 and Th2 profile, with a notable IL-5 production and specific IgG1 and total IgE isotypes secretion. Furthermore, our finding showed that T. natterri venom can affect the B cell fate and induce a memory antibody response through the secretion of protective IgG subclasses. Further studies with the venom protein toxins may provide clues to molecular mechanism regulating proliferation and differentiation of antibody-secreting cells in our model. A better understating of how T. natterri venom can modulate immune response could be useful in therapeutic strategies.

Differential effects of antigens from L. braziliensis isolates from disseminated and cutaneous leishmaniasis on in vitro cytokine production.

BACKGROUND: Disseminated leishmaniasis is an emerging infectious disease, mostly due to L. braziliensis, which has clinical and histopathological features distinct from cutaneous leishmaniasis. METHODS: In the current study we evaluated the in vitro production of the cytokines IFN-gamma, TNF-alpha, IL-5 and IL-10 by peripheral blood mononuclear cells (PBMC) from 15 disseminated leishmaniasis and 24 cutaneous leishmaniasis patients upon stimulation with L. braziliensis antigens genotyped as disseminated leishmaniasis or cutaneous leishmaniasis isolates. RESULTS: Regardless of the source of L. braziliensis antigens, PBMC from cutaneous leishmaniasis patients produced significantly higher IFN-gamma than PBMC from disseminated leishmaniasis patients. Levels of TNF-alpha by PBMC from cutaneous leishmaniasis patients were significantly higher than disseminated leishmaniasis patients only when stimulated by genotyped cutaneous leishmaniasis antigens. The levels of IL-5 and IL-10 production by PBMC were very low and similar in PBMCs from both disseminated leishmaniasis and cutaneous leishmaniasis patients. The immune response of each patient evaluated by the two L. braziliensis antigens was assessed in a paired analysis in which we showed that L. braziliensis genotyped as disseminated leishmaniasis isolate was more potent than L. braziliensis genotyped as cutaneous leishmaniasis isolate in triggering IFN-gamma and TNF-alpha production in both diseases and IL-5 only in cutaneous leishmaniasis patients. CONCLUSION: This study provides evidence that antigens prepared from genotypically distinct strains of L. braziliensis induce different degrees of immune response. It also indicates that both parasite and host play a role in the outcome of L. braziliensis infection.

Evaluation of cytokines (MIG, IFN-gamma, TNF-alpha, IL-4, IL-5, and IL-10) during the different evolutive phases of chagasic esophagopathy.

The production of cytokines (MIG, IFN-gamma, TNF-alpha, IL-4, IL-5, and IL-10) was studied in 39 individuals, including 28 with chagasic esophagopathy and 11 nonchagasic patients with gastroesophageal reflux disease. A sandwich enzyme immunoassay employing monoclonal antibody pairs specific for each cytokine was used. IFN-gamma and MIG production was significantly higher in patients with megaesophagus compared to control. Furthermore, in the absence of stimulation TNF-alpha levels were lower in the chagasic group than in the control group. In addition, significantly lower TNF-alpha levels were observed for the advanced form of the disease compared to the nonadvanced form. These results support the hypothesis that, although patients with advanced phase of megaesophagus present low number of CD4+ T lymphocytes, PBMC from this patients are able to respond up specific antigen stimulation.

CD4+ CD30+ T cells perpetuate IL-5 production in Dermatophagoides pteronyssinus allergic patients.

BACKGROUND: Airway allergic diseases are regulated by interleukin (IL)-5, which causes infiltration of eosinophils into the bronchial epithelium, and by IL-4 which increases serum immunoglobulin E (IgE) production and promotes CD30 expression on Th cells. CD30 generates a costimulatory signal involved in apoptosis or cell proliferation, depending on the microenvironment. Our aims were: (i) to analyze if CD4+ CD30+ T cells from allergic patients proliferate in response to Dermatophagoides pteronyssinus, and (ii) if upon stimulation this cell population produces IL-4 and IL-5. METHODS: Peripheral blood mononuclear cell (PBMC) from 17 allergic rhinitis and mild allergic asthma patients and 12 healthy nonallergic individuals were stimulated with allergen in the presence or absence of anti-IL-4, anti-IL-5 or anti-IL-4Ralpha monoclonal antibodies (mAbs). TdT-mediated dUTP nick end-labeling (TUNEL) assay, 7-aminoactinomycin-D (7-AAD) intercalation, and flow cytometry were used to determine the CD4+ CD30+ blasts percentage, cell proliferation, apoptosis, and intracellular cytokines after 7 culture days. RESULTS: Cell proliferation induced with allergen showed that 90% of the allergen-stimulated blasts were CD4+, 50% of which were CD30+. Allergen-stimulated PBMC showed a progressive increase (mean: from 7% to 23%) of CD4+ CD30+IFN-gamma+ and CD4+ CD30+IL-4+ blasts which diminished (mean: 6%) after 5 culture days. In contrast, CD4+ CD30+IL-5+ blasts showed a continuous progression (from 12% to 24%) that maintained after 7 culture days. The vast majority of CD4+ CD30+ blasts were negative to 7-AAD or TUNEL. Additionally, a significant decrease (34%) was observed in the number of CD4+ CD30+ blasts when IL-4 was neutralized. CONCLUSIONS: These data suggest that specific allergen stimulation of PBMC isolated from allergic patients generates a nonapoptotic CD4+ CD30+ blast subset that produces IL-5.

Modulation of the induction of lung and airway allergy in the offspring of IFN-gamma-treated mother mice.

Recent studies have highlighted the influence of fetal/maternal interactions on the development of asthma. Because IFN-gamma reduces Th2-mediated allergic responses, we assessed its capacity to modulate asthma in the offspring when injected into mothers during pregnancy. IFN-gamma was injected in CD1 female mice on day 6.5 of gestation. Immediately after birth, male newborns were housed in cages with interchanged mothers: the offspring from IFN-gamma-treated mothers were breastfed by normal mothers (IFN/nor), and those from normal mothers were breastfed by IFN-gamma-treated (Nor/IFN) or normal mothers (Nor/nor). Immediately after weaning, the spleen cells from IFN/nor and Nor/IFN mice produced less IL-4 and more IFN-gamma than Nor/nor mice when stimulated with Con A. At the age of 6-7 wk, mice were immunized with OVA on days 0 and 7. From day 14 to 16, they were exposed to aerosolized OVA. The bronchoalveolar lavage fluid from Nor/nor mice showed eosinophilia, a large number of these cells being present in perivascular and peribronchial regions of lung tissues. IFN/nor or Nor/IFN mice showed greatly reduced eosinophil numbers in bronchoalveolar lavage fluid. In addition, lung sections from IFN/nor, but not Nor/IFN mice showed almost normal histology. In OVA-sensitized IFN/nor and Nor/IFN mice, the production of IFN-gamma, IL-4, and IL-5 by spleen cells was significantly reduced as compared with cells from the OVA-sensitized Nor/nor group. IgE and anaphylactic IgG1 were also reduced in plasma of IFN/nor mice. In conclusion, the presence of IFN-gamma during pregnancy confers to the fetus a protection against allergenic provocations in the adult life.

Effect of mitomycin C on the secretion of granulocyte macrophages colonies stimulating factor and interleukin-5 in eosinophilic nasal polyps stromal culture.

UNLABELLED: The research involving tissue factors, such as granulocyte macrophage colonies stimulating factor (GM-CSF) and interleukin 5 (IL-5), leads to the mechanisms involved in the maintenance of eosinophilia, which is essential for the pathogenesis on eosinophilic nasal polyps. Mitomycin C has been successfully used in otolaryngology. AIM: The objective of this study was to evaluate the effect of mitomycin C in secretion of GM-CSF and IL-5 on eosinophilic nasal polyps. STUDY DESIGN: Case-control. MATERIAL AND METHOD: This is a comparative and auto-matched experimental study, performed with fragments of polyps which had been obtained from biopsy of patients with eosinophilic nasosinusal polyposis. The fragments of the experimental group were treated with mitomycin C (400 microg/ml) for 5 minutes and then washed in RPMI substrate. At time zero, 12 and 24 hours, the surface material was taken to determination of its GM-CSF levels in 22 patients and of IL-5 levels in 19 patients, by ELISA method. RESULTS: Reduction in GM-CSF expression on the experimental group at time 24 h (p< or = 0.05). The treated group presented significant GM-CSF expression between zero time and 12 h time (p= 0.013) showing the culture viability such as in the non-treated group. Tendency to decreasing IL-5 levels on the treated groups at 24 hours. CONCLUSION: This study showed that mitomycin C was efficient in inhibiting GM-CSF synthesis with reduction of IL-5 secretion, but this fact needs complementary studies.