Bholanath Paul

Immunosuppressive effects of aflatoxin in growing rats

The immunosuppressive potential of aflatoxin B1 (AFB1), the carcinogenic metabolite ofAspergillus flavus, was evaluated in growing rats. The weanling rats were subchronically exposed to 60, 300 and 600 µg AFB1/kg body weight for four weeks on alternate days by oral feeding. Various parameters of cell mediated immunity (CMI) and humoral immunity were assessed in control and treated animals. CMI was evaluated by measuring delayed type of hypersensitivity (DTH) response and humoral by plaque forming cell (PFC) assay. The lymphoproliferative response assay for T- and B-cells was also performed. It was observed that AFB1 selectively suppressed cell mediated immunity in growing rats. AFB1 suppressed CMI at the 300 and 600 µg dose levels only as measured by DTH response assay. It is concluded that continuous low level exposure of aflatoxin to growing host may enhance its susceptibility to infection and tumorigenesis.

IL-6 receptor-mediated lung Th2 cytokine networking in silica-induced pulmonary fibrosis

Pulmonary silicosis is a deadly disease which kills thousands of people every year worldwide. The disease initially develops as an inflammatory response with recruitment of inflammatory cells into the lung controlled by multiple cytokines. The question whether these cytokines exert biological functions through signal transducing pathway remains unanswered along with the potential role of interleukin-6 receptor α (IL-6Rα) in regulating inflammatory cytokines. We aimed to assess the status of signal transducers and activator of transcription (Stat3), suppressor of cytokine signalling 3(Socs3) and inflammatory cytokines in airways of silica-exposed mice, and their relationship with IL-6Rα. Silica-exposed and silica-exposed IL-6Rα gene knockdown Balb/c mice were used in the study. Lung function was measured by plethysmography, mRNA expression of cytokines and signal molecules by qRT2-PCR and lung architecture by histopathology; T helper cell-type 2 (Th2) cytokines in broncho-alveolar lavage fluids were evaluated by ELISA and hydroxyproline in lung by colorimetry. Elevated levels of collagen deposition, signs of lung fibrosis, infiltration of inflammatory cells and presence of exfoliated mucosa in the lung of silica-exposed mice with concurrent increase in methacholine-induced specific resistance of airways were observed on day 60 post-exposure. In parallel, heightened expression of Th2 cytokines (IL-4, IL-5, IL-6) and signal molecules (Stat3 and Socs3) were observed in the airways of silica-exposed mice. Th1 (IL-1β and TNF-α) cytokines are underexpressed in majority of the airways tissues of silica-exposed mice. Silencing IL-6Rα in lung of silica-exposed mice down regulated the hypermorphic mRNA pool of potential Th2 cytokines and signal molecules. Hypermorphic expression of Th2 cytokines and signal molecules in airways of silica-exposed mice are mediated through IL-6Rα.

Proinflammatory and Anti-Inflammatory Cytokine Balance in Gasoline Exhaust Induced Pulmonary Injury in Mice

Proinflammatory and anti-inflammatory cytokine balance and associated changes in pulmonary bronchoalveolar lavage fluid (BALF) of unleaded gasoline exhaust (GE) exposed mice were investigated. Animals were exposed to GE (1 L/min of GE mixed with 14 L/min of compressed air) using a flow-past, nose-only, dynamic inhalation exposure chamber for different durations (7, 14, and 21 days). The particulate content of the GE was found to be 0.635, ± 0.10 mg PM/m3. Elevated levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were observed in BALF of GE-exposed mice, but interleukin 1wβ (IL-1β) and the anti-inflammatory cytokine interleukin-10 (IL-10) remained unaffected. GE induced higher activities of alkaline phosphatase (ALP), γ-glutamyl transferase (γGT), and lactate dehydrogenase (LDH) in the BALF, indicating Type II alveolar epithelial cell injury, Clara-cell injury, and general toxicity, respectively. Total protein in the BALF increased after 14 and 21 days of exposure, indicating enhanced alveolar-capillary permeability. However, the difference in the mean was found statistically insignificant in comparison to the compressed air control. Total cell count in the BALF of GE-exposed mice ranged between 0.898 and 0.813 × 106 cells/ml, whereas the compressed air control showed 0.65 × 106 cells/mL. The histopathological changes in GE-exposed lung includes perivascular, and peribronchiolar cuffing of mononuclear cells, migration of polymorphonuclear cells in the alveolar septa, alveolar thickening, and mild alveolar edematous changes indicating inflammation. The shift in pro- and anti-inflammatory cytokine balance and elevation of the pulmonary marker enzymes indicate toxic insult of GE. This study will help in our understanding of the mechanism of pulmonary injury by GE in the light of cytokine profiles, pulmonary marker enzymes, and lung architecture.

Titanium dioxide nanoparticles augment allergic airway inflammation and Socs3 expression via NF-κB pathway in murine model of asthma

Titanium dioxide nanoparticles (nTiO2) previously considered to possess relatively low toxicity both in vitro and in vivo, although classified as possibly carcinogenic to humans. Also, their adjuvant potential has been reported to promote allergic sensitization and modulate immune responses. Previously, in OVA induced mouse model of asthma we found high expression of Socs3 and low expression of Stat3 and IL-6. However, a clear understanding regarding the signaling pathways associated with nTiO2 adjuvant effect in mouse model of asthma is lacking. In the present study we investigated the status of Stat3/IL-6 and Socs3 and their relationship with NF-κB, with nTiO2 as an adjuvant in mouse model of asthma. nTiO2 when administered with ovalbumin (OVA) during sensitization phase augmented airway hyper-responsiveness (AHR), biochemical markers of lung damage and a mixed Th2/Th1 dependent immune response. At the same time, we observed significant elevation in the levels of Stat3, Socs3, NF-κB, IL-6 and TNF-α. Furthermore, transient in vivo blocking of NF-κB by NF-κB p65 siRNA, downregulated the expression of Socs3, IL-6 and TNF-α. Our study, thus, shows that nTiO2 exacerbate the inflammatory responses in lungs of pre-sensitized allergic individuals and that these changes are regulated via NF-κB pathway.

Status of Stat3 in an Ovalbumin-Induced Mouse Model of Asthma: Analysis of the Role of Socs3 and IL-6

Background: Stat3, Socs3 and cytokines play an integral role in the coordination and persistence of inflammation. However, a clear understanding of the role played by the Stat3/IL-6 and Socs3 pathway in airway inflammation is lacking. We report the alteration in the status of expression and activation of Stat3 by ovalbumin (OVA), and establish its relationship with Socs3 and IL-6 in the lungs of mice with eosinophilic pulmonary inflammation and airway hyperresponsiveness. Methods: Alterations in the expression of Stat3, Socs3 and IL-6 were determined in a murine model of asthma, where Balb/c mice were sensitized and challenged with OVA (OVA/OVA) and compared with control mice sensitized and challenged with saline (SAL) (SAL/SAL) mice. The OVA/OVA mice were characterized by a moderate increase in methacholine-induced specific airway resistance, the presence of 150 μg/ml of OVA-specific IgG and 8.93 μg/ml OVA-specific IgE antibody and elevated levels of eosinophils and Th2 cytokines (IL-4 and IL-5) in the bronchoalveolar lavage fluid. In contrast SAL/SAL mice had low eosinophils, IL-4 and IL-5 and no OVA-specific IgG and IgE antibodies in the BALF. Stat3 and Socs3 expression profiles were monitored in OVA/OVA and Stat3- and Socs3-silenced OVA/OVA mice. Furthermore, expression of IL-6 in Stat3- and Socs3-silenced mice and the exogenous effect of IL-6 on Stat3 were studied. Results: The results show that expression and activation of Stat3 mRNA and proteins are significantly low in lung of OVA/OVA mice in comparison to SAL/SAL mice following OVA challenge. An increased pool of Socs3 mRNA is observed in OVA/OVA mice with or without OVA challenge and in SAL/SAL mice 24 h after OVA challenge. Transient in vivo blocking of Socs3 gene by Socs3 siRNA restores the expression of IL-6 mRNA and protein in OVA/OVA mice, and nasal administration of recombinant IL-6 to OVA/OVA mice enhanced Stat3 mRNA expression. Conclusions: Our data suggest that airway inflammation is associated with low expression of Stat3 and IL-6 and overexpression of Socs3 genes in a mouse model of asthma. Furthermore, IL-6 is under the influence of the Socs3 gene and may contribute to the negative regulation of Stat3 via IL-6 following a challenge with an allergen during the development of asthma.

Effect of In Utero Exposure to Hexachlorocyclohexane on the Developing Immune System of Mice

Gestational exposure to 10 and 100 mg/kg body weight (m.b.w.) hexachlorocyclohexane throughout the gestation period was done to Swiss albino mice. HCH (alpha, beta and gamma isomers) residue analysis in pups showed a higher contamination in the lymphoid organs (Spleen, Thymus and Kidney) than liver in dose dependent manner. Immune functions of the offsprings of these dams along with the offsprings of vehicle treated or untreated control dams were assessed using selected parameters of both the cellular and humoral immune responses. The delayed hypersensitivity (DTH) response to sheep erythrocytes (SRBC) was significantly higher (p less than 0.01) in pups of the dams exposed to 10 mg/kg b.w. HCH and significantly impaired in pups of the dams exposed to 100 mg/kg m.b.w. HCH as compared to controls. Mitogenic responsiveness of the spleen cells in response to Concanavalin A (Con A) and Lipopolysaccharide (LPS) was almost two fold and eight fold higher respectively and antibody response to SRBC, as measured by plaque forming cells (PFC) assay was two fold higher (p less than 0.001) in pups exposed to 10 mg/kg HCH. However, 100 mg/kg m.b.w. HCH did not affect either mitogenic response or PFC response of the pups. The results, therefore, suggest that lower dose of HCH is capable of modulating the development and function of developing immune system possibly by modulating the functional organization of the T-cell populations.

Correlation of Cytokines and Mobility in Mice with Arthritis and During Therapy with Swertia chirayita

We attempt to derive a correlation between pro-inflammatory cytokines and mobility in arthritic mice and after treatment with Swertia chirayita plant extract. Tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1), interleukin-6 (IL-6) and interleukin-10 (IL-10) were measured by solid phase sandwich ELISA and the different parameters of mobility [distance traveled (DT), resting time (RT), ambulatory time (AT) and stereotypic time (ST)] was monitored and recorded by an Auto track system. Joint circumference of the right hind limb was monitored using a Vernier caliper. Our preliminary findings revealed that DT and RT correlated well with IL-1, TNF-a and IL-10 levels in the joint homogenates of arthritic mice and after therapy with Swertia chirayita extracts. AT and ST showed poor correlation with the cytokines. Thus we demonstrate here the anti-inflammatory property of Swertia chirayita and its usage in the prevention of arthritis in the broader aspect in the management of associated cytokines.

Human Hemoglobin Shares Bioactivities Ascribed to Human Tumor Necrosis Factor‐α

Hemoglobin mediated cytotoxicity and apoptosis has been evaluated in Tumor necrosis factor‐α (TNF‐α) sensitive cell line, U937 and compared with TNF‐α. Both species of hemoglobin, Hemoglobin A2 and Hemoglobin A0 induced apoptosis and cytotoxicity in U937 cell as measured by flow cytometry and 3‐(4,5‐dihydro‐6‐(4‐(3,4‐dimethoxybenzooyl)‐1‐piperazinyl)‐2(1H)‐quinoline (MTT) assay respectively. Different concentration of Hemoglobin A0 (4 ng/mL to 4000 ng/mL) induced apoptosis ranging from 9% to 16% in U937 cells. 4000 ng/mL hemoglobin A0 showed maximal apoptotic cells. TNF‐α showed 87% apoptotic U937 cells at concentration of 1 pg/mL. HbA0 displayed cytotoxicity in U937 cell line at higher concentration in comparison to TNF‐α. 4000 ng/mL of hemoglobin A0 showed optimal cytotoxic response in U937 cells. A dose response curve was also observed with varying doses of hemoglobin A0. U937 cells pretreated with serum activated LPS for 1 hr and incubated with different concentration of hemoglobin or human TNF‐α for 24 h reduced the cytotoxic effect on U937. Dexamethasone treatment of U937 cells helped in protecting the HbA0 and HbA2 mediated cytotoxicity and anti‐TNF‐α antibody neutralized the hemoglobin mediated apoptosis and cytotoxicity. It is therefore apparent that human hemoglobin shares some of the bioactivities previously ascribed to TNF‐α. Sharing of bioactivities of TNF‐α by hemoglobin is interesting and suggests that cell free hemoglobin can mimic TNF‐α functionally.

A Study on the B Cell Activity in Protein Deficient Rats Exposed to Methyl Isocyanate Vapour

The effect of MIC on the humoral immunity of the malnourished (protein deficient) subjects has been investigated. A single exposure of MIC (1.60 mg/l) on protein deficient rats showed no significant change in the body weight and mortality rate compared with the normal but the serum protein levels were found significantly low (P less than 0.01) in the protein deficient diet fed control (PDC) ones. Both PDC and protein deficient MIC exposed (PDMIC) rat showed diminished B-cell proliferation with the optimal dose of LPS, compared to the NDC and normal diet fed MIC exposed (NDMIC) group. Furthermore significant suppression (P less than 0.01) in the B-cell activation by LPS was observed in the PDMIC compared to PDC. The total IgM level in PDMIC was 43% less while 26% higher in NDMIC compared to NDC. The total IgG level in PDMIC and NDMIC was higher (20%) compared to NDC, while 25% less in PDC. The antigen specific B-cell immunity was affected in PDMIC, PDC and NDMIC. As the terminal differentiation process of B-cells were found equally affected in both PDC and NDMIC, it appears that MIC has no synergistic effect on the humoral immunity of the protein malnourished host.

Modulation of IgM to IgG class switch by protein A.

The Fc binding property of soluble protein A (SpA) from Staphylococcus aureus has been utilized to form IgG-SpA complexes which enabled an increase or decrease of IgG from the host depending on the dose of SpA administered. When 5 micrograms SpA was administered the IgG-SpA complexes were rapidly catabolized and, hence, low plasma IgG levels were observed. In contrast 25 micrograms SpA resulted in a significant increase in the IgG level in the host plasma. Based on these observations, the present investigation attempted to study the effect of IgG depletion/increase on the primary and secondary B-cell response to sheep erythrocyte (SRBC) antigen in Balb/c mice. Introduction of 5, 10 and 25 micrograms SpA at the time of the primary antigenic challenge inhibited both the primary IgM and the secondary IgM and IgG responses in a dose-dependent manner. Administration of 5 and 10 micrograms SpA at the time of the secondary antigenic challenge enabled the host to maintain the otherwise depressed secondary IgM response equivalent to the normal primary response. In contrast, 25 micrograms SpA at the time of the secondary antigenic challenge inhibited both the IgM and IgG PFC responses. These results extend our understanding of the mechanism of switch in immunoglobulin class expression during antigen driven maturation of the B-cell response.