| BackgroundTuberculosis (TB) is a chronic infectious disease, and its infection, immunity, prevention and treatment, etc is full of contradictions and challenges. According to WHO, an estimated two billion people have been infected with the Mycobacterium tuberculosis (MTB). China is one of the highest TB burden countries in the world, ranking the second in terms of total number of patients. Tuberculosis (TB) has become the current serious infectious disease which threatens human’s life and health. Besides being the world’s most widely used vaccine, BCG is the most controversial vaccine in current use. When used to prevent TB in infants and children, the effect of BCG can reach 80%, but poor effect has been found among adult. Previous studies have demonstrated that the efficacy of BCG vaccine at protecting against pulmonary TB in adult and aging populations is highly variable.Meanwhile, studies have found that zinc deficiency is predominant in regions with a high incidence of TB and pulmonary tuberculosis patients have a lower serum zinc level. This phenomenon intrigued all of us speculating that zinc may have some relations to TB. Zinc influences the immune system of human beings in several aspects. The intake, transportation, distribution and supersession of zinc is closely associated with zinc transporters, among them ZIP2 and ZIP8 have some connection with infection and inflammation.As inducer of Th1 response, ESAT-6 (Early secretory antigen target-6) and CFP-10 (Culture filtrate protein-10) exist only in pathogenic m. tuberculosis and m.bovis and have protection against tuberculosis. The above-mentioned has made them become one of the most powerful candidate antigens in tuberculosis diagnosis and vaccine research.The first part:The effect of zinc deficiency on the immune responses to BCG and ESAT-6/CFP-10 vaccination in offspring and adult ratsObjectiveThis work investigated the consequences of feeding zinc deficient and normal zinc diets to rats during gestation, infancy or adulthood on the immune responses to BCG and ESAT-6/CFP-10 vaccination.MethodsOn gestation day 0,32 maternal rats were divided into two groups and fed with diets bellow: control diet ad libitum (30mg zinc/kg diet, NC), zinc deficient diet ad libitum (8mg zinc/kg diet, ZnD). Pup rats were served as experimental subjects. From day 1 of pregnancy upon delivery, maternal rats were given a standard diet (30 mg/kg zinc) or zinc deficient diet (8 mg/kg zinc) respectively.32 adult male 10-week Wistar rats were divided into two groups for 17 weeks of feeding: control diet ad libitum (30mg zinc/kg diet, NC), zinc deficient diet ad libitum (8mg zinc/kg diet, ZnD).Newborn pups of these maternal rats were immunized with BCG or ESAT-6/CFP-10 at postnatal weeks 0 and 2. The third week after the second vaccination, rats were sacrificed for immune analysis.Results1. Decreased zinc content, INF-y and TNF-α production in plasma was observed in ZnD rats. Reduced lymphocytes proliferation, ratios of immune organs to body weight was observed in ZnD pup rats. Together with changes of expression level of ZIP2, ZIP8, NF-κB and IL-6 mRNA in immune organs of ZnD rats.2. The ZIP2 and ZIP8 mRNA level in blood lymphocytes of NC adult rats was lower than NC pup rats. The NF-κB mRNA in blood lymphocytes and spleen of NC adult rats was lower than NC pup rats. The IL-6 mRNA in blood lymphocytes, thymus and spleen tissues of NC pup rats were higher than NC adult rats.3. ZIP2 and ZIP 8 mRNA expression level was higher in thymus and spleen of NC BCG pup rats than NC ESAT-6/CFP-10 pup rats. NF-κB mRNA expression level was higher in blood lymphocytes and spleen of NC BCG pup rats than NC ESAT-6/CFP-10 pup rats. IL-6 mRNA in blood lymphocytes and spleen of ZnD BCG pup rats was higher than ZnD ESAT-6/CFP-10 pup rats. In spleens, IL-6 mRNA level of NC BCG pup rats was higher than NC ESAT-6/CFP-10 pup rats.Conclusion1. Zinc deficiency can weaken the humoral and cell-mediated immune responses to BCG and ESAT-6/CFP-10 vaccination.2. The vaccine immunogenicity of BCG may be affect by the time of vaccination.3. ESAT-6/CFP-10 may be helpful to improve the immunity of rats.The second part: The effect of zinc on the expression of zinc transporters and immune responses to ESAT-6/CFP-10 of A-549 cellsObjectiveExposing A-549 cells to zinc deficiency or excess conditions, to study the effect of zinc deficiency, zinc excessive on cell zinc transporter expression and immune response to ESAT-6/CFP-10.MethodsExposing A-549 cells to different concentrations of ZnCl2 and TPEN respectively for 24h, l0μmol/ml ESAT-6/CFP-10 was added; cells were collected after stimulated for 24h.Transfect A-549 cells with ZIP8 or NF-κB expression plasmid and collect cells after transfection for 48 hours.Results1. Low concentration of ZnCl2 and TPEN had inhibiting effect on A-549 cells.2. In condition of high zinc, ZIP2, ZIP8 and NF-κB mRNA expression levels decreased firstly and increased subsequently; in condition of zinc deficiency, ZIP2 mRNA expression level increased firstly and decreased subsequently, ZIP8 and NF-κB mRNA expression level decreased firstly and increased subsequently.3. In case of antigen stimulation, ZIP8 mRNA expression level changed significantly. In condition of zinc excess, NF-κB mRNA expression level decreased firstly and increased subsequently; ZIP2 mRNA expression level increased firstly and decreased subsequently. In condition of zinc deficiency, ZIP2 mRNA expression level decreased; NF-κB mRNA expression level increased firstly and decreased subsequently.4. In case of antigen stimulation and NF-κB over-expression, ZIP2 mRNA expression level reduced, ZIP8 mRNA and protein increased.ConclusionZinc deficiency and excess inhibited proliferation of A-549 cells and have an effect on the expression of ZIP2, ZIP8 and NF-κB mRNA. Under condition of zinc deficiency, zinc excess and over-expression of ZIP8 or NF-κB the immune response of A-549 cells to ESAT-6/CFP-10 have some differences. |
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