Cytokine and LL-37 gene expression levels in Bartonella spp. seropositive and seronegative patients of a rheumatology clinic.

PURPOSE: The variation in the immune response to Bartonella spp. infection in humans remains unclear. The present study compares the expression of selected interleukins, cytokines and cathelicidin (LL-37) in rheumatology clinic patients suffering from musculoskeletal symptoms with healthy blood donors. The patients had previously been tested for the presence of Bartonella henselae antibodies. METHODS: Gene expression of LL-37, interleukin (IL)-2, IL-4, IL-6, IL-12, interferon-(IFN)-γ, and tumor necrosis factor (TNF-α)-α was determined in blood samples using quantitative Polymerase Chain Reaction (qPCR). Statistical analysis was prepared with STATISTICA. RESULTS: Statistically significant differences in the mRNA levels of the tested cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-12; p<0.0001) were observed between the healthy controls and patients; however, no difference was observed for LL37 mRNA (p â€‹= â€‹0.1974). No significant differences in mRNA expression were observed between IgG in anti-Bartonella seropositive and seronegative individuals (p>0.05). The only significant differences between the Bartonella spp. DNA positive and negative patients, indicated by PCR, were observed for TNF-α and IL-12 mRNA (p â€‹= â€‹0.0045 and p â€‹= â€‹0.0255, respectively). CONCLUSION: A broadly similar immune response to the tested cytokines was observed among the participants irrespective of anti-Bartonella spp. IgG seropositivity. However, the Bartonella DNA-positive participants demonstrated significantly lower expression of IL-12 and TNF-α mRNA; this may indicate that these bacteria have a suppressive influence on the immune system.

The seroprevalence of Bartonella spp. in the blood of patients with musculoskeletal complaints and blood donors, Poland: a pilot study.

BACKGROUND: Bartonella spp. can cause a variety of diseases, such as lymphadenopathies, cat scratch disease, and trench fever, but can also give rise to many non-specific symptoms. No data exists regarding the prevalence of Bartonella spp. in patients with musculoskeletal complaints, nor among blood donors in Poland. METHODS: The presence of anti-Bartonella IgM and IgG in the serum of blood donors (n = 65) (Lodz, Poland) and in the patients of the Department of Rheumatology Clinic (n = 40) suffering from musculoskeletal symptoms was tested by immunofluorescence. Blood samples were cultured on enriched media. Epidemiological questionnaires were used to identify key potential risk factors, such as sex, age, contact with companion animals, and bites from insects or animals. RESULTS: Altogether, 27 of the 105 tested subjects were seropositive for Bartonella henselae IgG (23%) and three for Bartonella quintana IgG (2.85%); IgMs against B. henselae were found in three individuals (2.85%), and IgMs against B. quintana were found in one (1.54%). No statistically significant difference was found between the prevalence of B. henselae in the blood of donors or patients and the presence of unexplained musculoskeletal complaints (23% vs 30%). Individuals who had kept or been scratched by cats were not more likely to be B. henselae seropositive (p > 0.01). Tick bites were more commonly reported in patients, but insignificantly (p > 0.01). CONCLUSION: This is the first report of a high seroprevalence of anti-Bartonella IgG in patients with musculoskeletal symptoms and in blood donors in Poland. The obtained results indicate that such seroprevalence may have a possible significance in the development of musculoskeletal symptoms, although it should be confirmed on a larger group of patients. Asymptomatic bacteremia might occur and pose a threat to recipients of blood from infected donors. Hence, there is a need for more detailed research, including molecular biology methods, to clarify the potential risk of Bartonella spp. being spread to immunocompromised individuals. KEY POINTS: • This is the first study presenting high seroprevalence of Bartonella spp. in Poland. • IgG and IgM antibodies against B. quintana were found in blood samples of blood donors.

Innate Immune Response to Viral Infections in Primary Bronchial Epithelial Cells is Modified by the Atopic Status of Asthmatic Patients.

PURPOSE: In order to gain an insight into determinants of reported variability in immune responses to respiratory viruses in human bronchial epithelial cells (HBECs) from asthmatics, the responses of HBEC to viral infections were evaluated in HBECs from phenotypically heterogeneous groups of asthmatics and in healthy controls. METHODS: HBECs were obtained during bronchoscopy from 10 patients with asthma (6 atopic and 4 non-atopic) and from healthy controls (n=9) and grown as undifferentiated cultures. HBECs were infected with parainfluenza virus (PIV)-3 (MOI 0.1) and rhinovirus (RV)-1B (MOI 0.1), or treated with medium alone. The cell supernatants were harvested at 8, 24, and 48 hours. IFN-α, CXCL10 (IP-10), and RANTES (CCL5) were analyzed by using Cytometric Bead Array (CBA), and interferon (IFN)-ß and IFN-λ1 by ELISA. Gene expression of IFNs, chemokines, and IFN-regulatory factors (IRF-3 and IRF-7) was determined by using quantitative PCR. RESULTS: PIV3 and RV1B infections increased IFN-λ1 mRNA expression in HBECs from asthmatics and healthy controls to a similar extent, and virus-induced IFN-λ1 expression correlated positively with IRF-7 expression. Following PIV3 infection, IP-10 protein release and mRNA expression were significantly higher in asthmatics compared to healthy controls (median 36.03-fold). No differences in the release or expression of RANTES, IFN-λ1 protein and mRNA, or IFN-α and IFN-ß mRNA between asthmatics and healthy controls were observed. However, when asthmatics were divided according to their atopic status, HBECs from atopic asthmatics (n=6) generated significantly more IFN-λ1 protein and demonstrated higher IFN-α, IFN-ß, and IRF-7 mRNA expressions in response to PIV3 compared to non-atopic asthmatics (n=4) and healthy controls (n=9). In response to RV1B infection, IFN-ß mRNA expression was lower (12.39-fold at 24 hours and 19.37-fold at 48 hours) in non-atopic asthmatics compared to atopic asthmatics. CONCLUSIONS: The immune response of HBECs to virus infections may not be deficient in asthmatics, but seems to be modified by atopic status.

Winter ambient training conditions are associated with increased bronchial hyperreactivity and with shifts in serum innate immunity proteins in young competitive speed skaters.

INTRODUCTION: Regular training modulates airway inflammation and modifies susceptibility to respiratory infections. The impact of exercise and ambient conditions on airway hyperreactivity and innate immunity has not been well studied. We aimed to assess exercise-related symptoms, lung function, airway hyperresponsiveness and innate immunity proteins in relation to meteorological conditions and exercise load in competitive athletes. MATERIAL AND METHODS: Thirty-six speed skaters were assessed during winter (WTP) and summer (STP) periods. The control group comprised 22 non-exercising subjects. An allergy questionnaire for athletes (AQUA) and IPAQ (International Physical Activity Questionnaire) were used to assess symptoms and exercise. Meteorological parameters were acquired from World Meteorological Organization resources. Serum innate immunity proteins were measured by ELISA. RESULTS: Exercise-associated respiratory symptoms were reported by 79.4% of skaters. Despite similar exercise load and lung parameters during both periods, positive methacholine challenge was more frequent during winter (p = 0.04). Heat shock protein HSPA1 and IL-1RA were significantly decreased during STP compared to WTP and controls. During WTP, IL-1RA was elevated in skaters reporting exercise-induced symptoms (p = 0.007). sCD14 was elevated in athletes versus controls in both periods (p < 0.05). HSPA1 was significantly higher in WTP compared to STP irrespective of presence of respiratory tract infections (RTIs). IL-1RA in WTP was elevated versus STP (p = 0.004) only in RTI-negative athletes. Serum IL-1RA negatively correlated with most meteorological parameters during WTP. CONCLUSIONS: Ambient training conditions, but not training load, influence bronchial hyperreactivity and the innate immune response in competitive athletes assessed during winter. The protective effect of regular exercise against respiratory infections is associated with a shift in serum innate immunity proteins.

IgE-mediated 15-hydroxyeicosatetraenoic acid (15-HETE) generation by peripheral blood leukocytes: its association with basophil activation.

INTRODUCTION: Allergen-induced basophil activation has been associated with the release of several mediators and with an increased expression of CD203c molecules on basophils. AIM: To assess the influence of specific allergens on the generation of 15-hydroxyeicosatetraenoic (15-HETE) from peripheral blood leukocytes in relation to basophil activation, on the basis of CD203c molecule expression and histamine release. MATERIAL AND METHODS: The study included 15 patients with clinical symptoms of birch pollen allergy confirmed by a positive skin prick test with the birch allergen, and 6 healthy controls. Leukocytes isolated from peripheral blood were incubated with 3 concentrations of the birch pollen allergen (Bet v 1), anti-IgE or with ionophore A23187. RESULTS: In vitro challenge of leukocytes from allergic patients with 1 ng/ml of allergen induced a significant increase in 15-HETE generation. An increase above 30% was observed in almost half the allergic patients, with mean values ranging from 40% to 46%, but not in healthy controls. Anti-IgE antibodies increased 15-HETE generation in 5 patients (termed IgE+), and the allergen induced a significant increase in 15-HETE in all patients who reacted to anti-IgE. The mean CD203c expression on basophils of the allergic patients increased after allergen challenge, but a significant increase (> 30%) was observed only in patients who demonstrated an increased expression after anti-IgE exposure. A significant correlation was seen between 15-HETE generation and histamine release induced by the highest concentration of the allergen (r = 0.95; p < 0.01). CONCLUSIONS: Allergen-induced, IgE-mediated activation of basophils is associated with a significant increase in 15-HETE generation.

Human parainfluenza virus type 3 (HPIV3) induces production of IFNγ and RANTES in human nasal epithelial cells (HNECs).

BACKGROUND: Human parainfluenza virus type 3 (HPIV3), while infecting lower airway epithelial cells induces pneumonia and bronchiolitis in infants and children, and may lead to asthma exacerbations in children and adults. Respiratory viruses invading the airway epithelium activate innate immune response and induce inflammatory cytokine release contributing to the pathophysiology of upper and lower airway disorders. However, the effects of HPIV3 infection on nasal epithelial cells have not been well defined. The aim of this study was to evaluate the effect of the HPIV3 infection on cultured human nasal epithelial cells (HNECs) and the release of interferon gamma and other cytokines. METHODS: RPMI 2650, a human nasal epithelial cell line was cultured into confluence and was infected with HPIV3 (MOI of 0.1, 0.01 and 0.001). The protein release into supernatants and mRNA expression of selected cytokines were assessed 24, 48 and 72 h after infection. Cytokine concentrations in supernatants were measured by ELISA and expression of cytokine mRNA in RPMI 2650 cells confirmed by real time RT-PCR analysis. RESULTS: HNECs infection with HPIV3 did not induce cytotoxicity for at least 48 hours, but significantly increased IFN-γ protein concentration in the cell supernatants at 24 h and 48 h post infection (by 387% and 485% respectively as compared to mock infected cells). At 24 h a significant increase in expression of mRNA for IFNγ was observed. RANTES protein concentration and mRNA expression were significantly increased at 72 h after infection (mean protein concentration: 3.5 ± 1.4 pg/mL for 0.001 MOI, 10.8 ± 4.6 pg/mL for 0.01 MOI and 61.5 ± 18.4 pg/mL for 0.1 MOI as compared to 2.4 ± 1.3 pg/mL for uninfected cells). No measurable concentrations of TNF-α, IL-10, TSLP, IL-8, GM-CSF or eotaxin, were detected in virus infected cells supernatants. CONCLUSIONS: HPIV3 effectively infects upper airway epithelial cells and the infection is associated with induction of IFN-γ and generation of RANTES.

Association of serum Clara cell protein CC16 with respiratory infections and immune response to respiratory pathogens in elite athletes.

BACKGROUND: Respiratory epithelium integrity impairment caused by intensive exercise may lead to exercise-induced bronchoconstriction. Clara cell protein (CC16) has anti-inflammatory properties and its serum level reflects changes in epithelium integrity and airway inflammation. This study aimed to investigate serum CC16 in elite athletes and to seek associations of CC16 with asthma or allergy, respiratory tract infections (RTIs) and immune response to respiratory pathogens. METHODS: The study was performed in 203 Olympic athletes. Control groups comprised 53 healthy subjects and 49 mild allergic asthmatics. Serum levels of CC16 and IgG against respiratory viruses and Mycoplasma pneumoniae were assessed. Allergy questionnaire for athletes was used to determine symptoms and exercise pattern. Current versions of ARIA and GINA guidelines were used when diagnosing allergic rhinitis and asthma, respectively. RESULTS: Asthma was diagnosed in 13.3% athletes, of whom 55.6% had concomitant allergic rhinitis. Allergic rhinitis without asthma was diagnosed in 14.8% of athletes. Mean CC16 concentration was significantly lower in athletes versus healthy controls and mild asthmatics. Athletes reporting frequent RTIs had significantly lower serum CC16 and the risk of frequent RTIs was more than 2-fold higher in athletes with low serum CC16 (defined as equal to or less than 4.99 ng/ml). Athletes had significantly higher anti-adenovirus IgG than healthy controls while only non-atopic athletes had anti-parainfluenza virus IgG significantly lower than controls. In all athletes weak correlation of serum CC16 and anti-parainfluenza virus IgG was present (R = 0.20, p < 0.01). In atopic athletes a weak positive correlations of CC16 with IgG specific for respiratory syncytial virus (R = 0.29, p = 0.009), parainfluenza virus (R = 0.31, p = 0.01) and adenovirus (R = 0.27, p = 0.02) were seen as well. CONCLUSIONS: Regular high-load exercise is associated with decrease in serum CC16 levels. Athletes with decreased CC16 are more susceptible to respiratory infections. Atopy may be an additional factor modifying susceptibility to infections in subjects performing regular high-load exercise.