Human exposure to diesel exhaust induces CYP1A1 expression and AhR activation without a coordinated antioxidant response.

BACKGROUND: Diesel exhaust (DE) induces neutrophilia and lymphocytosis in experimentally exposed humans. These responses occur in parallel to nuclear migration of NF-κB and c-Jun, activation of mitogen activated protein kinases and increased production of inflammatory mediators. There remains uncertainty regarding the impact of DE on endogenous antioxidant and xenobiotic defences, mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) respectively, and the extent to which cellular antioxidant adaptations protect against the adverse effects of DE. METHODS: Using immunohistochemistry we investigated the nuclear localization of Nrf2 and AhR in the epithelium of endobronchial mucosal biopsies from healthy subjects six-hours post exposure to DE (PM10, 300 µg/m3) versus post-filtered air in a randomized double blind study, as a marker of activation. Cytoplasmic expression of cytochrome P450s, family 1, subfamily A, polypeptide 1 (CYP1A1) and subfamily B, Polypeptide 1 (CYP1B1) were examined to confirm AhR activation; with the expression of aldo-keto reductases (AKR1A1, AKR1C1 and AKR1C3), epoxide hydrolase and NAD(P)H dehydrogenase quinone 1 (NQO1) also quantified. Inflammatory and oxidative stress markers were examined to contextualize the responses observed. RESULTS: DE exposure caused an influx of neutrophils to the bronchial airway surface (p = 0.013), as well as increased bronchial submucosal neutrophil (p < 0.001), lymphocyte (p = 0.007) and mast cell (p = 0.002) numbers. In addition, DE exposure enhanced the nuclear translocation of the AhR and increased the CYP1A1 expression in the bronchial epithelium (p = 0.001 and p = 0.028, respectively). Nuclear translocation of AhR was also increased in the submucosal leukocytes (p < 0.001). Epithelial nuclear AhR expression was negatively associated with bronchial submucosal CD3 numbers post DE (r = -0.706, p = 0.002). In contrast, DE did not increase nuclear translocation of Nrf2 and was associated with decreased NQO1 in bronchial epithelial cells (p = 0.02), without affecting CYP1B1, aldo-keto reductases, or epoxide hydrolase protein expression. CONCLUSION: These in vivo human data confirm earlier cell and animal-based observations of the induction of the AhR and CYP1A1 by diesel exhaust. The induction of phase I xenobiotic response occurred in the absence of the induction of antioxidant or phase II xenobiotic defences at the investigated time point 6 h post-exposures. This suggests DE-associated compounds, such as polycyclic aromatic hydrocarbons (PAHs), may induce acute inflammation and alter detoxification enzymes without concomitant protective cellular adaptations in human airways.

Efficacy of 2% mepivacaine and 2% lignocaine in the surgical extraction of mesioangular angulated bilaterally impacted third molars: A double-blind, randomized, clinical trial.

OBJECTIVE: The purpose of this prospective, randomized, double-blind study was to compare the anesthetic efficacy of 2% mepivacaine and 2% lidocaine (both with 1:80,000 epinephrine) for inferior alveolar nerve block in mesioangular bilaterally impacted third molar extraction. STUDY DESIGN: Forty patients with mesioangular bilaterally impacted third molars were taken for the study; either 2% mepivacaine or 2% lidocaine is given in a double-blind manner. Surgery started 5 min after solution deposition. Success was defined as no or mild discomfort (visual analog scale [VAS] recordings) during the surgical procedure. RESULTS: The mean time for onset period 4.2 min and 4.6 min (P = 0.018). The mean duration anesthesia 177.17 min 166.71 min (P = 0.085). No significant difference between the scores of pain reported by the patients by VAS and venovenous bypass treated with mepivacaine and lidocaine (P = 0.000). Slight increased postoperative analgesics required for mepivacaine group (4.000 tablets) and lidocaine group (4.170 tablets) (P = 0.335). The sharp increase of pulse rate with respect to both the solutions at 5 min after postinjection of local anesthetics. However, there was no statically significant difference in systolic and diastolic blood (P = 0.681) and (P = 0.270). CONCLUSION: Lidocaine and mepivacaine with the same vasoconstrictor have similar action and both solutions are effective in surgical procedures. There were also no significant differences between them in relation to the intensity of postoperative pain.

Derailed B cell homeostasis in patients with mixed connective tissue disease.

Mixed connective tissue disease (MCTD) is a systemic autoimmune disorder, characterized by the presence of antibodies to U1-RNP protein. We aimed to determine phenotypic abnormalities of peripheral B cell subsets in MCTD. Blood samples were obtained from 46 MCTD patients, and 20 controls. Using anti-CD19, anti-CD27, anti-IgD and anti-CD38 monoclonal antibodies, the following B cell subsets were identified by flow cytometry: (1) transitional B cells (CD19+CD27-IgD+CD38(high)); (2) naive B cells (CD19+CD27-IgD+CD38(low)); (3) non-switched memory B cells (CD19+CD27+IgD+); (4) switched memory B cells (CD19+CD27+IgD-); (5) double negative (DN) memory B cells (CD19+CD27-IgD-) and (6) plasma cells (CD19+CD27(high)IgD-). The proportion of transitional B cells, naive B cells and DN B lymphocytes was higher in MCTD than in controls. The DN B cells were positive for CD95 surface marker. This memory B cells population showed a close correlation with disease activity. The number of plasma cells was also increased, and there was an association between the number of plasma cells and the anti-U1RNP levels. Cyclophosphamide, methotrexate, and corticosteroid treatment decreased the number of DN and CD27(high) B cells. In conclusion, several abnormalities were found in the peripheral B-cell subsets in MCTD, which reinforces the role of derailed humoral autoimmune processes in the pathogenesis.

The effects of various doses of bacterial lipopolysaccharide on the expression of CD63 and the release of histamine by basophils of atopic and non-atopic patients.

OBJECTIVE: We tested the effect of various doses of bacterial lipopolysaccharide (LPS, endotoxin) on the expression of CD63 and the in vitro release of histamine by basophils stimulated with ragweed allergen in patients with or without ragweed and mite allergies. METHODS: The peripheral blood of 11 patients with ragweed allergy, 10 patients with mite allergy and 14 control patients was incubated with ragweed allergen extract following pretreatment with varying doses of LPS. The expression of CD63 in basophils was measured by flow cytometry, and the release of histamine was determined by ELISA. RESULTS: In the samples of patients with ragweed allergy that were exposed to specific allergen, only high doses of LPS significantly elevated the expression of CD63 (200 ng/ml; 1,000 EU/ml) and the release of histamine (2,000 ng/ml; 10,000 EU/ml). There was no effect of LPS in any other cases. CONCLUSIONS: Bacterial LPS (endotoxin) concentrations higher than 200 ng/ml (1,000 EU/ml), which rarely occurs in nature, could only activate the basophils from atopic patients whilst in the presence of the specific allergen. Thus, the restoration of the urban, "microbe-poor" milieu with endotoxin (as LPS) can be a promising and harmless approach for allergy prevention.

Immunological reconstitution after autologous stem cell transplantation in patients with refractory systemic autoimmune diseases.

OBJECTIVE: High-dose chemotherapy followed by autologous haematopoietic stem cell transplantation (AHSCT) can be a salvage therapy for patients with severe, refractory systemic autoimmune diseases. The function of the newly rebuilt immune system is important, but little is known about immune reconstitution after AHSCT in autoimmune disorders. Our aim was to investigate the repopulation of different lymphocyte subsets in patients with systemic autoimmune diseases after AHSCT. METHODS: Twelve patients with severe refractory, autoimmune diseases were enrolled in the study: four with rheumatoid arthritis (RA), four with systemic sclerosis (SSc), three with systemic lupus erythematosus (SLE), and one with autoimmune overlap syndrome (myositis and RA). After stem-cell mobilization, CD34+ apheresis was carried out, followed by conditioning and AHSCT. After transplantation, peripheral lymphocyte subsets were regularly assessed by flow cytometry. RESULTS: The follow-up time was 24 months. The overall transplantation-related mortality (TRM) was 16.7% and the transplant-related toxicity was 33% 2 years after AHSCT. Regarding the immune reconstitution, CD56+ natural killer (NK) cells appeared in the earliest phase after transplantation, followed by CD8+ T cells. B cells and CD4+ T cells became normal within 150 days. The ratio of naive cells was low 30 days after AHSCT; however, naive B cells regenerated within 2 months whereas the repopulation of naive T cells took longer. After a short increase, the ratio of memory cells decreased 2 months after transplantation. Regulatory T (Treg) cells did not change significantly in the peritransplant period. Altogether approximately 5-6 months were required for the reconstitution of the peripheral immune network. CONCLUSIONS: AHSCT can be a salvage therapeutic modality in autoimmune patients who are refractory to other conventional therapies.

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome.

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögren's syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)-10 producing T regulatory type 1 (Tr1) cells and CD4(+)CD25(+) regulatory T cells (T(reg)) cells were determined by flow cytometry and serum cytokine levels of IL-4, IL-6, IL-10, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma were evaluated by enzyme-linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of T(reg) cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4(+)CD25(+) T(reg) cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4(+)CD25(+) T(reg) cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4(+)CD25(+) T(reg) cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4(+)CD25(+) T(reg) cells in patients compared to controls. Serum IL-6 and TNF-alpha levels were elevated, while IL-10 was decreased in patients compared to controls. Negative correlation was found between IL-10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.

Regulatory T cells in atopic dermatitis: epidermal dendritic cell clusters may contribute to their local expansion.

BACKGROUND: Regulatory T cells (Tregs) have an essential role in tolerance and immune regulation. However, few and controversial data have been published to date on the role and number of these cells in atopic dermatitis (AD). OBJECTIVES: To investigate the number of CD4+CD25+FOXP3+ Tregs and interleukin 10-producing T regulatory type 1 (Tr1) cells in patients with AD. METHODS: Peripheral blood and skin biopsy samples from atopy patch test (APT)-positive patients with acute- and chronic-phase AD were investigated. Immunohistochemistry was applied to identify CD4+CD25+FOXP3+ Tregs in the skin, while flow cytometry was used to detect CD4+CD25highFOXP3+ Tregs and Tr1 cells in the peripheral blood. RESULTS: In the peripheral blood samples of patients with AD significantly elevated numbers of Tr1 cells were found. Although neither the absolute number nor the percentage of CD4+CD25highFOXP3+ Tregs showed significant alteration in the peripheral blood of patients, increased numbers of FOXP3+ Tregs were detected in skin biopsy specimens. All of the APT-positive skin samples showed epidermal dendritic cell aggregates, morphologically consistent with so-called Langerhans cell microgranulomas, which also contained intermingled FOXP3+ Tregs. CONCLUSIONS: Tr1 cell numbers were elevated in the peripheral blood and increased numbers of CD4+CD25highFOXP3+ Tregs were detected in the skin of patients with AD. The epidermal dendritic cell clusters in APT-positive lesional skin showed a close connection to the FOXP3+ Tregs.