Production of monoclonal antibodies against Streptococcus mutans antigens

Several studies have been conducted in the last decades aiming to obtain an anti-canies vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, G8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG 2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

Invasin of Yersinia pseudotuberculosis is not a polyclonal activator of mouse B lymphocytes

It is known that the invasin molecule of Yersinia pseudotuberculosis stimulates human peripheral B cells in vitro. In this work we evaluated the in vivo role of invasin as polyclonal activator of B lymphocytes in the mouse experimental model, by comparing strains of Y. pseudotuberculosis expressing invasin and isogenic inv mutants. Swiss mice were infected intravenously with two strains expressing invasin (YpIII pIB1 and an isogenic virulence plasmid-cured strain, YpIII) and with two invasin mutant strains (Yp100 pIB1 and Yp100, plasmid-cured). Spleen cells were sampled on days 7, 14, 21 and 28 after infection. Immunoglobulin (Ig)-secreting spleen cells were detected by protein A plaque assay and specific antibodies were detected in sera by ELISA. The virulent strain YPIII pIB1 (wild type) did not provoke polyclonal activation of B lymphocytes in vivo. In general, fewer Ig-secreting spleen cells of all isotypes were found in the infected animals than in the control animals. Specific IgG antibodies were detected in the sera of animals infected with all strains. The peak response occurred on the 21 st day post-infection, and the Yp100 strain provoked the highest level of these antibodies. We concluded that invasin is not a polyclonal activator of murine B cells.

Missing association between telomerase activity and clinicopathological features in patients with early stage carcinoma of the cervix

Objectives: This study was undertaken to evaluate the association between the telomerase activity in the tumor and clinicopathological findings in patients with stage IB-IIA (FIGO) carcinoma of the cervix. Methods: Thirty-eight patients with carcinoma of the cervix submitted to radical hysterectomy were prospectively from January 1998 to November 2001. Samples from the tumor were taken and analyzed by the telomerase PCR-TRAP-ELISA kit. Clinicopathological characteristics such as age, stage, tumor size, grade of differentiation, lymphatic vascular space invasion (LVSI), parametrial involvement and status of pelvic lymph nodes were also recorded. Results: Patient's mean age was 49.3 ± 1.99 years (29-76 years). The clinical stage (FIGO) was IB in 35 patients (92.1%) and IIA in 3 patients (7.9%). The histological classification identified squamous cell carcinoma in 33 patients (86.8%) and adenocarcinoma in 5 patients (13.2%). There was no association between age, clinical stage, histological classification, tumor size, grade of differentiation and presence of LVSI with tumoral telomerase activity. The telomerase activity was not associated with the presence of vaginal involvement (P = 0.349), parametrium involvement (P = 0.916), pelvic lymph node metastasis (P = 0.988) or tumoral recurrence (P = 0.328) in patients with carcinoma of the cervix. Conclusions: Telomerase activity in the tumor is not associated with clinicopathological findings or tumor recurrence in patients with early stage cervical carcinoma. © 2006 Springer-Verlag.

Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells triggered by conformational changes in viral glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished conformational changes on VSV glycoprotein and the fusion reaction catalyzed by the virus. In the present study, we evaluated whether treatment with DEPC was able to inactivate the virus. Infectivity and viral replication were abolished by viral treatment with 0.5 mM DEPC. Mortality profile and inflammatory response in the central nervous system indicated that G protein modification with DEPC eliminates the ability of the virus to cause disease. In addition, DEPC treatment did not alter the conformational integrity of surface proteins of inactivated VSV as demonstrated by transmission electron microscopy and competitive ELISA. Taken together, our results suggest a potential use of histidine (His) modification to the development of a new process of viral inactivation based on fusion inhibition. © 2006 Elsevier B.V. All rights reserved.

Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1β, IL-6, IL-10 and TNF-α. Pb18 induced higher levels of IL-1β, IL-6, and IL-10 than Pb265. IL-8 and TGF-β1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Clinical and nutritional profile of individuals with Chagas disease

Chagas disease (CD), caused by the protozoan Trypanossoma cruzi, affects approximately 18 million individuals in the Americas, 5 million of which five in Brazil. Most chronic sufferers have either the indeterminate form of the disease, without organic compromise, or the cardiac or digestive forms. Despite the importance of this disease, there is no information on the effect of nutrition on CD evolution. We evaluated the clinical-nutritional profile of individuals with CD treated at the Tropical Diseases Nutrition Out-Patient Clinic of the Botucatu School of Medicine, UNESP.A retrospective cohort study was performed between 2002 and 2006, on 66 patients with serum and parasitological diagnosis of CD. Epidemiological, clinical, nutritional, and biochemical data were collected, including gender, age, skin color, smoking, alcoholism, physical activity, weight, stature, body mass index, abdominal circumference, glycemia, and lipid profile. Fifty-three percent were mate and 47% female; 96% were white skinned. Mean age was 49.6±6.36 years. The predominant form was indeterminate in 71 %; smoking and drinking were recorded in 23% and 17%, respectively. Sedentariness predominated in 83%, and 55% presented increased abdominal circumference. Most, 94%, were overweight or obese. The biochemical exam revealed hyperglycemia in 12% and dyslipidemia in 74%. These findings suggest that the Chagas population presents co-morbidities and risk factors for developing chronic non-transmissible diseases, including cardiovascular diseases, making CD evolution even worse. © 2007 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage

Background: Vaccination of neonates is generally difficult due to the immaturity of the immune system and consequent higher susceptibility to tolerance induction. Genetic immunization has been described as an alternative to trigger a stronger immune response in neonates, including significant Th1 polarization. In this investigation we analysed the potential use of a genetic vaccine containing the heat shock protein (hsp65) from Mycobacterium leprae (pVAXhsp65) against tuberculosis (TB) in neonate mice. Aspects as antigen production, genomic integration and immunogenicity were evaluated. Methods: Hsp65 message and genomic integration were evaluated by RT-PCR and Southern blot, respectively. Immunogenicity of pVAXhsp65 alone or combined with BCG was analysed by specific induction of antibodies and cytokines, both quantified by ELISA. Results: This DNA vaccine was transcribed by muscular cells of neonate mice without integration into the cellular genome. Even though this vaccine was not strongly immunogenic when entirely administered (three doses) during early animal's life, it was not tolerogenic. In addition, pVAXhsp65 and BCG were equally able to prime newborn mice for a strong and mixed immune response (Th1 + Th2) to pVAXhsp65 boosters administered later, at the adult life. Conclusion: These results suggest that pVAXhsp65 can be safely used as a priming stimulus in neonate animals in prime-boost similar strategies to control TB. However, priming with BCG or pVAXhsp65, directed the ensuing immune response triggered by an heterologous or homologous booster, to a mixed Th1/Th2 pattern of response. Measures as introduction of IL-12 or GM-CSF genes in the vaccine construct or even IL-4 neutralization, are probably required to increase the priming towards Th1 polarization to ensure control of tuberculosis infection. © 2007 Pelizon et al; licensee BioMed Central Ltd.

Produção deficiente de citocinas Th1 em camundongos BALB/c jovens

O objetivo do presente estudo foi comparar a produção de IFN-γ, IL-12 e IL-4 entre camundongos jovens (5, 12 e 19 dias de idade) e adultos (30 dias de idade). As avaliações foram feitas por estimulação, in vitro, de células esplênicas com Concanavalina A (ConA) , Staphylococcus aureus (S. aureus) e lipopolissacarídeo (LPS). Diferentes concentrações de cada estímulo foram testadas e os sobrenadantes das culturas foram coletados após 48 horas de incubação e as concentrações de IFN-γ, IL-12 e IL-4 determinadas por ELISA. Células de camundongos jovens e adultos produziram níveis igualmente elevados de IFN-γ após estímulo com ConA. Somente animais adultos produziram IFN-γ em resposta ao estímulo com S. aureus. Em culturas estimuladas com LPS, a produção desta citocina foi baixa e similar nos animais jovens e significativamente elevada nos animais adultos. Somente células de animais adultos estimuladas com S. aureus foram capazes de produzir IL-12. O único estímulo capaz de induzir níveis detectáveis de IL-4 foi ConA, sendo que estes níveis foram mais elevados nos animais com 12 e 19 dias de idade em comparação com animais neonatos e adultos. A diminuição das doses ótimas dos estímulos não mudou o perfil de produção de cada citocina nos animais jovens. Estes resultados permitem concluir que a idade afeta a produção de citocinas: ocorre maior produção de IL-4 em camundongos jovens e maior produção de IL-12 e IFN-γ em animais adultos. Estas informações são importantes devido ao papel destas citocinas na polarização das respostas imunes nos sentidos Th1 e Th2. Palavras-chave: camundongo; citocina; interferon-gama; interleucina-4; interleucina-12.

Inhibition of human neutrophil apoptosis by Paracoccidioides brasiliensis: Role of interleukin-8

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production. © 2008 The Authors.

Dermatoses bolhosas auto-imunes

Autoimmune bullous dermatoses are diseases in which blisters and vesicles are the primary and fundamental types of skin lesion. Their classification is based on the location of the blister: intraepidermal and subepidermal. Patients produce autoantibodies against self-specific structures of the skin detectable by immunofluorescence techniques, immunoblotting and ELISA. Recent advances in molecular and cellular biology have brought to knowledge these self-antigens, against which patients are sensitized, and which are found in epidermis or in the dermo-epidermal junction. These are low incidence, but high morbidity diseases that may be fatal. The aim of this article is to review and describe the progress of four autoimmune vesiculobullous disorders: endemic pemphigus foliaceous (wild fire), pemphigus vulgaris, bullous pemphigoid and dermatitis herpetiformis. ©2009 by Anais Brasileiros de Dermatologia.

Lipoperoxidação, perda de viabilidade celular e diminuição da liberação de IL-8 de neutrófilos humanos na presença de corpos cetônicos

Type-1 diabetes patients suffer from frequent episodes of acidosis caused by an increased fatty acid metabolism and consequently increased plasma level of acetoacetate (AcAc) and β-hydroxybutyrate (β-HOB). This article describes a study of the effects of pathological concentrations of AcAc and β-HOB on lipoperoxidation, cell viability and the release of the CXCL8 (IL-8) cytokine by activated neutrophils. Neutrophils from healthy donors were isolated by density gradient (Histopaque® 1077/1119) and incubated with the ketone bodies. Lipoperoxidation was determined as thiobarbituric acid reactive substances (TBARS). The cell viability was evaluated by the release of intracellular lactate dehydrogenase. The release of CXCL8 was measured by ELISA in a 24-h culture of opsonized zymosan-stimulated neutrophils. AcAc, but not β-HOB, provoked a dose-dependent increase in the neutrophil membrane lipoperoxidation (p<0.05; r =0.9915). In the cytotoxicity assay, a dose-dependent release of LDH was observed when the neutrophils were incubated with AcAc in concentrations up to 40 mM (p<0.05). β-HOB was devoid of effect. The release of CXCL8 was inhibited by AcAc and β-HOB in a dose-dependent manner. In conclusion, these results suggest that the accumulation of ketone bodies in diabetic patients could be involved in their usually increased susceptibility to infection.

Measurement of melatonin in body fluids: Standards, protocols and procedures

Background and Purpose: The circadian rhythm of melatonin in saliva or plasma, or of the melatonin metabolite 6-sulfatoxymelatonin (a6MTs) in urine, is a defining feature of suprachiasmatic nucleus (SCN) function, the body's endogenous oscillatory pacemaker. The primary objective of this review is to ascertain the clinical benefits and limitations of current methodologies employed for detection and quantification of melatonin in biological fluids and tissues. Data Identification: A search of the English-language literature (Medline) and a systematic review of published articles were carried out. Study Selection: Articles that specified both the methodology for quantifying melatonin and indicated the clinical purpose were chosen for inclusion in the review. Data Extraction: The authors critically evaluated the methodological issues associated with various tools and techniques (e.g. standards, protocols, and procedures). Results of Data Synthesis: Melatonin measurements are useful for evaluating problems related to the onset or offset of sleep and for assessing phase delays or advances of rhythms in entrained individuals. They have also become an important tool for psychiatric diagnosis, their use being recommended for phase typing in patients suffering from sleep and mood disorders. Additionally, there has been a continuous interest in the use of melatonin as a marker for neoplasms of the pineal region. Melatonin decreases such as found with aging are or post pinealectomy can cause alterations in the sleep/wake cycle. The development of sensitive and selective methods for the precise detection of melatonin in tissues and fluids has increasingly been shown to have direct relevance for clinical decision making. Conclusions: Due to melatonin's low concentration, as well as the coexistence of numerous other compounds in the blood, the routine determination of melatonin has been an analytical challenge. The available evidence indicates however that these challenges can be overcome and consequently that evaluation of melatonin's presence and activity can be an accessible and useful tool for clinical diagnosis. © Springer-Verlag 2010.

Evaluation of tissue reaction, cell viability and cytokine production induced by Sealapex Plus

The aim of this study was to investigate the effects of mineral trioxide aggregate (MTA), Sealapex, and a combination of Sealapex and MTA (Sealapex Plus) on the reaction of subcutaneous connective tissue of rats, and on cell viability and cytokine production in mouse fibroblasts. The tissue reaction was carried out with dentin tubes containing the materials implanted in the dorsal connective tissue of rats. The histological analysis was performed after 7 and 30 days. Millipore culture plate inserts with polyethylene tubes filled with materials were placed into 24-well cell culture plates with mouse fibroblasts to evaluate the cell viability by MTT assay. ELISA assays were also performed after 24 h of exposure of the mouse fibroblasts to set material disks. Histopathologic examination showed Von Kossa-positive granules that were birefringent to polarized light for all the studied materials at the tube openings. No material inhibited the cell viability in the in vitro test. It was detected IL-6 production in all root-end filling materials. MTA and Sealapex Plus induced a slight raise of mean levels of IL-1β. The results suggest that Sealapex Plus is biocompatible and stimulates the mineralization of the tissue.

Biologic and genetics aspects of chagas disease at endemic areas

The etiologic agent of Chagas Disease is the Trypanosoma cruzi, transmitted through blood-sucking insect vectors of the Triatominae subfamily, representing one of the most serious public health concerns in Latin America. There are geographic variations in the prevalence of clinical forms and morbidity of Chagas disease, likely due to genetic variation of the T. cruzi and the host genetic and environmental features. Increasing evidence has supported that inflammatory cytokines and chemokines are responsible for the generation of the inflammatory infiltrate and tissue damage. Moreover, genetic polymorphisms, protein expression levels, and genomic imbalances are associated with disease progression. This paper discusses these key aspects. Large surveys were carried out in Brazil and served as baseline for definition of the control measures adopted. However, Chagas disease is still active, and aspects such as host-parasite interactions, genetic mechanisms of cellular interaction, genetic variability, and tropism need further investigations in the attempt to eradicate the disease. Copyright 2012 Marilanda Ferreira Bellini et al.

Three-year follow-up of Interleukin 6 and C-reactive protein in chronic obstructive pulmonary disease

Background: Past studies have shown that mean values of Interleukin-6 (IL-6) and C-reactive protein (CRP) do not change significantly in COPD patients over a one-year period. However, longer period follow-up studies are still lacking. Thus, the aim of this study is to evaluate plasma CRP and IL-6 concentration over three years in COPD patients and to test the association between these inflammatory mediators and disease outcome markers. Methods: A cohort of 77 outpatients with stable COPD was evaluated at baseline, and 53 (mean FEV1, 56% predicted) were included in the prospective study. We evaluated Interleukin-6 (IL-6), C-reactive protein (CRP), six-minute walking distance (6MWD), and body mass index (BMI) at baseline and after three years. Plasma concentration of IL-6 was measured by high sensitivity ELISA, and CRP was obtained by high sensitivity particle-enhanced immunonephelometry. Results: IL-6 increased significantly after 3 years compared to baseline measurements [0.8 (0.5-1.3) vs 2.4 (1.3-4.4) pg/ml; p < 0.001] and was associated with worse 6MWD performance. In the Cox regression, increased IL-6 at baseline was associated with mortality [Hazard Ratio (95% CI) = 2.68 (0.13, 1.84); p = 0.02]. CRP mean values did not change [5 (1.6-7.9) vs 4.7 (1.7-10) pg/L; p = 0.84], although eleven patients (21%) presented with changes >3 mg/L in CRP after 3 years. Conclusions: The systemic inflammatory process, evaluated by IL-6, seems to be persistent, progressive and associated with mortality and worse physical performance in COPD patients. Trial registration: No.:NCT00605540. © 2013 Ferrari et al; licensee BioMed Central Ltd.