IgA Response to ESAT‐6/CFP‐10 and Rv2031 Antigens Varies in Patients With Culture‐Confirmed Pulmonary Tuberculosis,Healthy Mycobacterium tuberculosis–Infected and Non‐Infected Individuals in a Tuberculosis Endemic Setting,Ethiopia

Little attention has been given to the role of antibodies against Mycobacterium tuberculosis (Mtb) infection. We have compared the levels of IgA and IgG against ESAT‐6/CFP‐10 and Rv2031c antigens in sera of patients with culture‐confirmed pulmonary tuberculosis (PTB), healthy Mtb‐infected and non‐infected individuals in endemic TB settings. Venous blood samples were collected from 166 study participants; sera were separated and assayed by an enzyme‐linked immunosorbent assay (ELISA). QuantiFERON‐TB Gold In‐Tube (QFTGIT) assay was used for the screening of latent TB infection. The mean optical density (OD) values of IgA against ESAT‐6/CFP‐10 and Rv2031 were significantly higher in sera of patients with culture‐confirmed PTB compared with healthy Mtb‐infected and non‐infected individuals (P < 0.001). The mean OD values of IgG against ESAT‐6/CFP‐10 and Rv2031 were also significantly higher in sera of patients with culture‐confirmed PTB compared with healthy Mtb‐infected and non‐infected individuals (P < 0.05). The mean OD values of IgA against both antigens were also higher in sera of healthy Mtb‐infected cases compared with non‐infected individuals. There were positive correlations (P < 0.05) between the level of IFN‐γ induced in QFTGIT assay and the OD values of serum IgA against both antigens in healthy Mtb‐infected subjects. This study shows the potential of IgA response against ESAT‐6/CFP‐10 and Rv2031 antigens in discriminating clinical TB from healthy Mtb‐infected and non‐infected cases. Nevertheless, further well‐designed cohort study is needed to fully realize the full potential of this diagnostic marker.     

Association of ESAT‐6/CFP‐10‐induced IFN‐γ, TNF‐α and IL‐10 with clinical tuberculosis: evidence from cohorts of pulmonary tuberculosis patients,household contacts and community controls in an endemic setting

Mycobacterium tuberculosis (Mtb) early secreted protein antigen 6 (ESAT‐6) and culture filtrate protein 10 (CFP‐10) are among candidate vaccines against tuberculosis (TB). Results of experimental animal models show that these antigens are associated with induction of strong T cell immunity [interferon (IFN)‐γ production], while others report that these proteins as virulent factors involved in pathogenicity of Mtb infection. However, the role of ESAT‐6/CFP‐10 during natural Mtb infections in humans has not been established. In this paper we present results of a longitudinal study from an Mtb‐infected human population from an endemic setting. Whole blood assay was used to determine levels of IFN‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐10 against rESAT‐6/CFP‐10 in TB patients, household contacts and community controls. The levels of IFN‐γ, TNF‐α and IL‐10 against rESAT‐6/CFP‐10 at baseline were significantly higher in patients and community controls than in household contacts. In patients, no significant difference was observed in the level of these cytokines before and after chemotherapy whereas, in contacts, the level of these cytokines increased significantly and progressively over time. The study shows that the levels of IFN‐γ, TNF‐α and IL‐10 against rESAT‐6/CFP‐10 are depressed during Mtb infection or exposure but are elevated during clinical TB. Our findings from a study of naturally infected human population suggest that IFN‐γ, TNF‐α and IL‐10 against rESAT‐6/CFP‐10 are markers for clinical TB but not for protective immunity.     

TLR‐7 and ‐9 Stimulation of Peripheral Blood B Cells Indicate Altered TLR Signalling in Primary Sjögren's Syndrome Patients by Increased Secretion of Cytokines

Primary Sjögren's syndrome (pSS) is a chronic, inflammatory autoimmune disease characterised by lymphocytic infiltrations in the exocrine glands, resulting in destruction of salivary and lacrimal glands. B cells have an important role in the disease, as detection of autoantibodies against SSA/Ro or SSB/La is one of the diagnostic criteria, being found in a majority of the patients. Toll‐like receptors (TLR) are pattern recognition receptors. TLR‐7 and ‐9 are found in endosomes and bind microbial nucleic acids. We have previously shown that pSS patients and healthy controls have similar expression pattern of TLR‐7 and ‐9 in various B‐cell populations. In this study we further analysed the responsiveness of B cells upon TLR stimulation. B cells isolated from peripheral blood of 21 pSS patients and 18 healthy controls were stimulated with TLR‐7 and ‐9 ligands for 24 h before being analysed for the expression of certain surface markers and intracellular cytokine levels by flow cytometry. Secreted cytokines were measured by a multiplex cytokine assay. Patients with pSS had more naïve and less preswitched memory B cells compared to controls in unstimulated as well as via TLR‐7 stimulated cells. Unstimulated and via TLR‐7 stimulated B cells from pSS patients also had fewer IL‐10⁺ preswitched memory B cells. Moreover, TLR‐7 and ‐9 stimulated B cells of pSS patients secreted increased amounts of several cytokines. B cells of pSS patients show a different responsiveness upon stimulation of TLR‐7 and ‐9 compared to controls.     

Immune Responses to 6 and 30-kDa Mycobacterial Antigens in Rheumatoid Patients, and Vβ Usage by Specific Synovial T-Cell Lines and Fresh T Cells

We have investigated both the humoral and the cellular immune responses of patients with juvenile rheumatoid arthritis (JRA) and rheumatoid arthritis (RA) to mycobacterial antigens. The JRA group was not Bacillus Calmette Guerin (BCG) vaccinated whilst the majority of the RA group was. As determined by immunoblotting, 79% of sera from patients with JRA reacted mainly with a 18.6-kDa protein (P18.6), whilst 70% of sera from patients with RA reacted mainly with a 30-kDa protein (P30) of BCG, M. tuberculosis and M. kansasii. In contrast, only a moderate proportion of the control sera (25% of adult and 20% of children) showed reactivity to P30, and none of the samples had significant reactivity with the P18.6 antigen. Furthermore, T-cell proliferation to the P18.6 and P30 antigens was detected in the majority of JRA and RA patients, and was nearly always higher in synovial fluid (SF) than in the peripheral blood (PB). We also investigated the usage of V beta family genes in P18.6 and P30 antigen-specific T-cell lines established from the SF of one patient with active RA. We showed that V beta 2, -4, -5, -6, -7, -14, -17, -18 and V beta 19 were over-represented compared with other known V beta families. We also noted that the proportion of V beta 14 was higher in freshly isolated SF mononuclear cells compared with the blood in this patient and in 2 out of 4 other RA patients examined. Other V beta families such as V beta 6, V beta 8, V beta 16, V beta 18 and V beta 19 were also over-represented in the SF compared with the blood in some patients. Taken together our results provide more information concerning the role of mycobacterial antigens in RA and suggest that there may be an in vivo clonal expansion of T lymphocytes in the synovium.     

Cord Blood Monocytes,Neutrophils and Lymphocytes from Preterm and Full‐Term Neonates Show Multiple Aberrations in Signalling Profiles Measured Using Phospho‐Specific Whole‐Blood Flow Cytometry

Immaturity of the immune system renders newborns susceptible to infections. We searched for aberrations in leucocyte signalling profiles, using phospho‐specific whole‐blood flow cytometry, in cord blood of nine preterm (two born before 32nd gestational week) and nine full‐term infants, born by caesarean section. Thirteen adults served as reference subjects. Monocyte NF‐κB phosphorylation following tumour necrosis factor (TNF) or bacterial stimulation was higher in preterm neonates than in full‐term neonates or adults, p38 phosphorylation following bacterial stimulation was higher in both preterm and full‐term neonates than in adults, while STAT1 phosphorylation by IFN‐γ or IL‐6, STAT3 phosphorylation by IL‐6 and STAT5 phosphorylation by GM‐CSF were lower in both full‐term and preterm neonates than in adults. Neutrophil STAT1 and STAT3 phosphorylation following IFN‐γ stimulation and STAT5 phosphorylation following GM‐CSF stimulation were lower in newborn neonates than in adults. In both CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes, NF‐κB phosphorylation by TNF was higher and STAT5 phosphorylation by IL‐2 was lower in preterm and full‐term newborns than in adults. STAT6 phosphorylation by IL‐4 was comparable in monocytes and lymphocytes of newborns and adults. The results suggest that innate immune signalling pathways responding to inflammatory stimuli are strongly functional in leucocytes of preterm neonates, which may render these neonates susceptible to inappropriate tissue injury. In leucocytes of both preterm and full‐term newborns, responses needed against intracellular pathogens, and regulatory functions show immaturities, possibly contributing to worse control of infections.     

Occupational exposure to trichloroethylene and serum concentrations of IL‐6, IL‐10, and TNF‐alpha

To evaluate the immunotoxicity of trichloroethylene (TCE), we conducted a cross‐sectional molecular epidemiology study in China of workers exposed to TCE. We measured serum levels of IL‐6, IL‐10, and TNF‐α, which play a critical role in regulating various components of the immune system, in 71 exposed workers and 78 unexposed control workers. Repeated personal exposure measurements were taken in workers before blood collection using 3 M organic vapor monitoring badges. Compared to unexposed workers, the serum concentration of IL‐10 in workers exposed to TCE was decreased by 70% (P = 0.001) after adjusting for potential confounders. Further, the magnitude of decline in IL‐10 was >60% and statistically significant in workers exposed to <12 ppm as well as in workers with exposures ≥ 12 ppm of TCE, compared to unexposed workers. No significant differences in levels of IL‐6 or TNF‐α were observed among workers exposed to TCE compared to unexposed controls. Given that IL‐10 plays an important role in immunologic processes, including mediating the Th1/Th2 balance, our findings provide additional evidence that TCE is immunotoxic in humans. Environ. Mol. Mutagen. 54:450–454, 2013. © 2013 Wiley Periodicals, Inc.     

Cultured Mast Cells from Patients with Asthma and Controls Respond with Similar Sensitivity to Recombinant Der P2‐Induced,IgE‐Mediated Activation

The function of cultured mast cells may depend on genetic or environmental influence on the stem cell donor. This study investigates whether asthma or atopy in the donor influenced the growth and sensitivity of mast cells cultured from patients with asthma and healthy controls under identical conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL‐4 and 80 kU/l recombinant human IgE containing two clones (7% + 7%) specific for mite allergen Der p2. The sensitivity of IgE‐mediated activation of mast cells was investigated as FcεRI‐mediated upregulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum CD63 median fluorescence intensity was 20 456 ± 1640 (SE) for patients with asthma and 22 275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in patients with asthma and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in patients with asthma [?0.4795 log ng/ml ± 0.092 (SE)] and controls (?0.6351 log ng/ml ± 0.083, ns) and in atopic and non‐atopic subjects. When cultured, sensitized and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells appears to depend on culture conditions (IL‐4, IgE) rather than on donor status as atopy and asthma.     

Cytokine Production in the Serum and Spleen of Mice from Day 6 to 14 of Gestation: Cytokines/Placenta/ Spleen/Serum

Pregnancy, like most biologic phenomena, involves the action of cytokines. These proteins have a short half-life and are believed to exert their effect close to their site of production, where diagnostic tests cannot be easily performed. Here we show that the cytokine content in the maternal serum reflects cytokine production and secretion from maternal spleen cells, which also correlates with production from decidual cells. We show that GM-CSF, IL- 3, and IL-10 are present in the serum at specific time intervals during the first half of murine pregnancy, which correlates with their production from maternal spleen cells. Purified GM-CSF and IL-3 from spleen-cell-culture supernatants are biologically active molecules, able to stimulate placental-cell proliferation. Furthermore, TNF-0, which has been identified in many cases of fetal rejection as well as in labor, is shown to be naturally produced during the second half of pregnancy. Additionally, within the limits of the sensitivity of the technique we have used, the detection of IL-4 and the absence of detectable levels of IL- 2 in the maternal serum strongly comforts the hypothesis that pregnancy is a Th2-dependent phenomenon. The results presented in this paper show that the cytokine profile during pregnancy can be monitored by simple blood tests, which may be of relevance both in the followup of a physiological human pregnancy and to the diagnosis of recurrent abortions due to cytokine imbalance.     

Increased Frequency of Cells Secreting Interleukin-6 and Interleukin-10 in Peripheral Blood of Patients with Primary Sjögren''s Syndrome

Sjögren's syndrome (SS) is a chronic autoimmune disease of exocrine glands. There is increasing evidence that interferon-γ (IFN-γ) plays a role in the pathogenesis of SS. It has also been suggested that other type 1 cytokines, as well as interleukin-6 (IL-6), IL-10 and transforming growth factor-β, are important in the induction and/or maintenance of SS. The aim of this study was to investigate the type 1/type 2 cytokine pattern in peripheral blood of primary SS patients. The enzyme-linked immunospot (ELISPOT) assay was performed to quantify the number of mononuclear cells (MNC) secreting IFN-γ, IL-6 and IL-10 in peripheral blood samples from 33 patients with primary SS and 12 healthy controls. The mean number of cells secreting IFN-γ was 9/10⁵ MNC in the SS patient group, and 4/10⁵ MNC in the control group (P = 0.73). Fifteen of the SS patients had anti-Ro 52 kDa antibodies in serum. In this patient group the mean number of cells secreting IFN-γ was 4/10⁵ MNC, while in the patient group without such antibodies the mean number of cells secreting IFN-γ was 14/10⁵ MNC (P = 0.04). The mean number of cells secreting IL-6 was 12 000/10⁵ MNC in the SS patient group, and 5000/10⁵ MNC in the control group (P = 0.01). The mean number of cells secreting IL-10 was 270/10⁵ MNC in the SS patient group, and 180/10⁵ MNC in the control group (P = 0.04). The SS patients had a significantly higher number of cells secreting IL-6 and IL-10 in peripheral blood than the healthy controls, which may facilitate B-cell activation and production of autoantibodies.     

Allele frequencies of polymorphisms of TNFA,IL‐6, IL‐10 and IFNG in an Italian Caucasian population

Polymorphisms in the regulatory and intronic regions of several cytokines have been associated with differential cytokine production. In this paper we genotyped, using the polymerase chain reaction–sequence‐specific primers (PCR‐SSP) method, a series of 363 healthy Italian Caucasians with the aim of obtaining a reference population for further studies on the role of cytokines in the inflammatory and immune responses. We also compared the results to those for other populations. The polymorphisms analysed were those of tumour necrosis factor alpha (TNFA), interleukin 6 (IL‐6), interleukin 10 (IL‐10) and interferon gamma (IFNG). We found that the frequency of allele TNFA*1 at position ?380 was 87.7% and that of TNFA*2 was 12.4%, significantly different from those of the UK and Japanese populations but not different from that of a population in Gambia. For IL‐10 the frequencies of alleles ?1082A and ?1082G were 63.0% and 37.0% and those of alleles ?819C, ? 819T, ?592C and ?592A were 70.8, 29.2, 70.8 and 29.2%, respectively, significantly different from those observed in south‐east England, in Manchester and in an Oriental population from southern China. The frequencies of IL‐6 alleles ? 174C and ?174G were 29.0 and 71.0%, respectively; for IFNG polymorphisms at position ?874, in the population under evaluation, the alleles ?874T and ?874A were present in 44.7 and 55.3% of the subjects, respectively. Genotype frequencies of IL‐6 were significantly different from those observed in populations from Germany and from the UK. The analysis carried out by our group indicates that there is heterogeneity in the frequencies of the cytokine polymorphisms among the different Caucasian populations, and this underlines the importance of a ‘local’ reference population when evaluating the clinical relevance of cytokine gene polymorphisms.     

In vitro Monocyte IL‐6 Secretion Levels Following Stimulation with Autologous Spheroids Derived from Tumour or Benign Mucosa Predict Long‐term Survival in Head and Neck Squamous Cell Carcinoma Patients

MCP‐1/IL‐6 in vitro monocyte secretion upon coculture with autologous fragment spheroids was studied in relation to patient 5‐ and 10‐year overall survival rates in head and neck squamous cell carcinoma (HNSCC) patients (n = 65) diagnosed between 1998 and 2005, nine of whom had an human papilloma virus (HPV) tumour infection. The spheroids were harvested from malignant or benign tissue during primary surgery. Two weeks following surgery, freshly isolated autologous monocytes and benign or malignant spheroids were cocultured 24 h in vitro. The IL‐6 secretion was expressed as a fraction of the lipopolysaccharide (LPS) response from the same batch of monocytes. HPV status was obtained by employing PCR analyses of primary diagnostic blocks. IL‐6/MCP‐1 response levels were not found to be dependent on HPV infection status. MCP‐1 secretion did not predict prognosis, nor did in vitro IL‐6 monocyte background or LPS‐stimulated IL‐6 secretion. At 5‐year observation, dichotomized IL‐6 levels following monocyte coculture, with both malignant and benign spheroids, showed a strong trend towards predicting survival, that is a low monocyte malignant coculture response showed a survival of 31 ± 17 versus 58 ± 17% with a high such response (P = 0.057). When studying monocyte IL‐6 coculture responses evaluating benign and malignant spheroid results statistically together, a prediction of survival up to 10 years was found (hazard ratio = 0.48; confidence interval = 0.24–0.96; P < 0.05) with double low IL‐6 responses. This survival prediction was also present after an adjustment for HPV tumour infection status. In conclusion, monocyte IL‐6 in vitro secretion in cocultures with autologous spheroids/serum from HNSCCs predicted 5‐ and 10‐year survivals, both with and without tumour HPV tumour adjustment.     

ORIGINAL ARTICLE: Expression of IL‐6, IL‐8, TNF‐α, IL‐10, HSP‐60, Anti‐HSP‐60 Antibodies,and Anti‐sperm Antibodies,in Semen of Men with Leukocytes and/or Bacteria

Citation Martínez‐Prado E, Bermúdez MIC. Expression of IL‐6, IL‐8, TNF‐α, IL‐10, HSP‐60, anti‐HSP‐60 antibodies, and anti‐sperm antibodies, in semen of men with leukocytes and/or bacteria. Am J Reprod Immunol 2010; 63: 233–243 Problem Different cellular and biochemical markers have been proposed as indicators of infection‐inflammation of male genital tract. Method of study Semen samples from 80 men attending an andrologic clinic were evaluated to determine the presence of leukocyte, bacteria, antibodies against Chlamydia trachomatis, levels of IL‐6, IL‐8, IL‐10, and TNF‐α, HSP‐60, anti‐HSP‐60 antibodies, and anti‐sperm antibodies. Results Leukocytes in semen significantly correlated with an increase in IL‐6, IL‐8, and TNF‐α. The simultaneous presence of pathogens and leukocytes was associated with high levels of IL‐8 and TNF‐α, whereas IL‐6 was more associated with the presence of leukocytes. Anti‐HSP‐60 antibodies positively correlated with IL‐6 and IL‐8. The presence of anti‐sperm antibodies highly associated with an increase in anti‐HSP‐60 antibodies. Conclusions The type of cytokines present in the semen will depend on the single or simultaneous presence of leukocytes and/or pathogens. Chronic male genital tract infections could be associated with the development of anti‐HSP‐60 antibodies and anti‐sperm antibodies.