Gender disparity in tuberculosis cases in eastern and western provinces of Pakistan.

BACKGROUND: Although globally, the number of notified TB cases is higher for males, a few countries in the Eastern Mediterranean Region (Afghanistan; Lebanon; Iran and Pakistan) of the World Health Organization have a relatively higher number of female cases. Pakistan ranks fifth amongst the highest TB burden countries and poses a rich ground for exploratory research to address the gender differences in TB cases. It is uniquely neighboured by India on the East, having higher number of cases in males than in females, and by Afghanistan and Iran on the West, having higher number of cases in females than in males. The objective is to see whether these gender differences are evenly distributed across the country or vary by geographies, to enable effective targeting of TB control strategies. METHODS: Cross-sectional analysis was carried out on secondary data, obtained from National Tuberculosis Program. Disaggregated at the provincial level, the sex-specific case notification rates (CNR) were calculated and trends over a 10-year span (2001-2010) were examined. Sex-specific differences for the four Pakistani provinces were analyzed using chi-square test and odds ratios with corresponding confidence intervals. Cumulative countrywide sex-specific notification rates were used as the reference group. RESULTS: The trends for 2001-2010 in the western provinces of Pakistan show higher female CNR as compared to those seen in the eastern provinces having slightly higher male CNR. The proportions of female notified TB cases are approximately twice as high in the western provinces when compared to the eastern provinces and Pakistan over all. CONCLUSIONS: These findings suggest that females are particularly affected by TB disease burden in the west parts of Pakistan. This gender disparity requires a coordinated regional and international effort to further explore triggers and moderators of increased acquisition and progression of TB disease among females in the region to guarantee effective TB control.

Effects of physiological concentrations of heavy metals both individually and in mixtures on the viability and function of peripheral blood human leukocytes in vitro.

Among environmental contaminants recognized for their toxicity and global distribution, heavy metals are elements known to exert serious ecological consequences. Published experiments on the immunotoxic effects of metals such as methylmercury (MeHg), cadmium (Cd), and lead (Pb) were often conducted at concentrations higher than those present in the environment or those in human blood. In the present study the in vitro effects on human blood of environmentally relevant concentrations of MeHg (33-200 mug/L), Cd (3.1-16 mug/L), and Pb (75-207 mug/L) were assessed individually and in mixtures on the viability and immune competence of peripheral blood leukocytes (PBLs). At MeHg concentrations of 120 and 200 mug/L both lymphocyte proliferation, as measured by [(3)H]thymidine incorporation, and natural killer (NK) cytotoxity activity, as determined by dioctadecyloacarbocyanine, were suppressed. Our results showed an increase of intracellular thiols in lymphocytes and in monocytes at all the concentrations of metals tested. A decrease in the level of metallothionein (MT) was seen in monocytes in presence of Hg at concentration of 120 mug/L and higher. For lymphocytes, a significant increase of MT in groups containing the lower concentrations of Cd, and Hg was noted. In summary, it appears that Hg represents the most toxic metal at environmentally relevant concentrations on human peripheral mononuclear cells. The effects of Hg exposure were greater on lymphocytes and NK cells than on monocytes.

Immunosuppression in mice fed on diets containing beluga whale blubber from the St Lawrence estuary and the Arctic populations.

In order to assess the immunotoxic potential of naturally relevant mixtures of PCBs and other organohalogens, C57Bl/6 mice were fed on diets in which lipids were replaced by blubber of beluga whales from the highly contaminated population of the Saint-Lawrence River, and the less contaminated population from the Arctic. Different ratios of blubber from both sources were mixed in order to allow a dose-response study. Mice were fed for a period of 90 days at the end of which their immunological status was monitored. For general parameters such as body weight, weight of the spleen and the thymus no significant effect of diets were observed. The immunological endpoints such as the blastic transformation of splenocytes and the spleen NK cell activity were not significantly affected by any of the diets compared to control diets. While the different cell subpopulations of peripheral blood and thymus were not affected by the diets, a significant decrease was noted in the CD8+ T cell population in the spleen of mice fed with most of the diets containing beluga blubber. Moreover, the ability of splenic cells to elicit humoral response against sheep red blood cells as well as the potential of peritoneal macrophages to perform phagocytosis were suppressed by all diets containing beluga blubbers. In summary, there was no differences between the groups fed with a blubber diet with low and high organochlorine contamination. However, a clear immunosuppression was demonstrated when these groups were compared to the group fed with beef oil. Despite the fact that we cannot exclude a possible contribution of the fatty acid composition of the beluga blubber to the immunosupression, these results suggest the sensitivity of mouse immune system towards organohalogens, and point out the toxic potential of contaminant mixtures as found in the less contaminated Arctic population.

Suppression of rat and mouse lymphocyte function by urban air particulates (Ottawa dust) is reversed by N-acetylcysteine.

Epidemiology studies have demonstrated increased pulmonary morbidity such as allergy and infection with episodes of high particulate air pollution (size range 0.1-10 microm diameter, PM10), but the mechanism(s) for this association is not yet well defined. The present study was undertaken to evaluate the effects of EHC-93 urban particles (Ottawa dust) on immune functions of peripheral blood mononuclear cells (PBMCs) and splenocytes from male Fischer 344 rats and C57Bl/6 mice. Immune function endpoints evaluated included cell viability, lymphocyte blastogenesis stimulated by T-cell mitogen (concanavalin A, Con A) or B-cell mitogens [lipopolysaccharide (LPS) or LPS/dextran sulfate], intracellular Ca2+ concentration, interleukin 2 (IL-2) production, and expression of receptors for transferrin (TfR) and IL-2 (IL-2R). In addition, the effect of N-acetylcysteine (NAC), an antioxidant, on the toxicity of EHC-93 particles was evaluated. Total EHC-93 particles, water leachate of EHC-93, and washed EHC-93 suppressed proliferation of PBMCs and splenocytes to T- and B-cell mitogens. Treatment of splenocytes with EHC-93 particles did not alter intracellular Ca2+ concentration or mitogen-induced expression of TfR and IL-2R expression, but increased IL-2 production assayed by enzyme-linked immunosorbent assay (ELISA). In spite of an increase in IL-2 production, exogenous IL-2 when added to cultures was able to reverse the suppression of Con A-induced lymphocyte proliferation by EHC-93 particles. Furthermore, the suppressive effect of EHC-93 particles on mitogen-induced lymphocyte proliferation was completely abolished by addition of the antioxidant NAC to cultures, suggesting a possible role of oxidative factors for the toxicity of EHC-93 particles.

Functional responses of human neutrophils to sodium sulfite (Na2SO3) in vitro.

An influx of neutrophils into the airways is a common feature observed during pulmonary inflammation induced by air pollutants, including sulfur dioxide and sulfates. In the present study focusing on the in vitro interactions of sodium sulfite (Na2SO3) with human neutrophils, we confirm results indicating that this sulfite induces superoxide production (O2-) by itself. We demonstrated that this response can occur more rapidly than previously reported (within 5 min), and that Na2SO3 can act as a priming agent, in a concentration-dependent fashion, to the bacterial tripeptide N-formyl-methionine-leucine-phenylalanine (fMLP) by increasing O2-production. In addition, our results show that Na2SO3 induces gene expression in human neutrophils in a concentration-dependent manner as assessed by incorporation of 5-[3H] uridine into total RNA. However, it does not induce cell shape changes. We also demonstrated that Na2SO3 does not modulate neutrophil apoptosis nor reverse the well-known delaying effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on apoptosis. We conclude that Na2SO3 acts rapidly on neutrophil physiology, within a few minutes with respect to superoxide production, and a few hours (4 h) with respect to gene expression without altering a biological process such as the rate of apoptosis evaluated after a long period of incubation (20 h). We further conclude that Na2SO3-induced production of O2does not drive neutrophils to undergo apoptosis, a mechanism known to occur in other conditions. Therefore, the potential toxicity of Na2SO3 during pulmonary inflammation or lung-associated diseases may be related to its ability to induce superoxide production without altering neutrophil apoptosis rate.

Lack of suppressive effects of mixtures containing low levels of methylmercury (MeHg), polychlorinated dibenzo-p-dioxins (PCDDS), polychlorinated dibenzofurans (PCDFS), and aroclor biphenyls (PCBS) on mixed lymphocyte reaction, phagocytic, and natural killer cell activities of rat leukocytes in vitro.

Rat splenocyte mixed leukocyte reaction (MLR), splenic natural killer (NK) cell activity, and phagocytic activities of splenic, peritoneal, and peripheral blood leukocytes (PBLs) were evaluated in vitro to determine the immunotoxicity of mixtures containing low levels of methylmercury (MeHg), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and Aroclor polychlorinated biphenyls (PCBs). The mixtures were based on the concentrations of the chemicals in fish flesh. Leukocytes from male Fischer rats were exposed to MeHg (0.1-2 microg/ml), PCDD/PCDF mixtures (1-15 pg/ml) of three PCDDs (2,3,7,8-tetrachlorodibenzo-p-dioxin, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin) and two PCDFs (2,3,7,8-tetrachlorodibenzofuran and 1,2,3,7,8-pentachlorodibenzofuran), three Aroclor PCB (Aroclor 1242, 1254, and 1260) mixtures (0.01-0.5 microg/ml), or combinations of MeHg/PCB/PCDD/PCDF mixtures for 24 or 72 h before immunological assays. Phagocytosis and NK cell cytotoxicity were evaluated with a flow cytometer, and MLR of Fischer rat responder splenocytes cultured with mitomycin C-treated Long-Evans splenocytes by [3H]thymidine uptake. Exposure to MeHg (2 microg/ml) alone or with PCB/ PCDD/PCDF resulted in significant cytolethality in rat splenocytes, peritoneal leukocytes, and PBLs at 24 h exposure. Treatment with Aroclor PCB mixtures, PCDD/PCDF mixtures, 0.1 microg MeHg/ml (noncytolethal), or PCB/PCDD/PCDF mixtures with 0.1 microg MeHg/ml caused no suppression of splenocyte MLR response, splenic NK cell-mediated lysis of Yac-l cells, or phagocytosis of fluorescent beads by splenic, peritoneal, and peripheral blood phagocytic cells. The results indicate that in vitro exposure of rat leukocytes to low levels of MeHg, Aroclor PCB mixtures, PCDD/PCDF mixtures, or MeHg/PCB/PCDD/PCDF mixtures had no suppressive effects on the immune functions assayed, and thus produced no additive immunotoxicity. However, in order to predict the potential risk of these chemical mixtures to the human immune system, in vivo animal studies with blood (tissue) levels compatible with the levels of MeHg, PCBs, and PCDDs/PCDFs in exposed human populations should be evaluated.

Immunomodulatory and protective effects of N-acetylcysteine in mitogen-activated murine splenocytes in vitro.

N-Acetylcysteine (NAC) is a pro-glutathione drug used to treat chronic lung disorders and because of its anti-AIDS virus activity in vitro, has been proposed for AIDS therapy. The effect of NAC on mitogen-activated-lymphocyte blastogenesis in C57B1/6 mouse splenocytes and ability of NAC to protect lymphocytes against mitogen-induced cytotoxicity was examined in vitro. NAC increased splenocyte proliferation in the presence of optimal and suboptimal concentrations of concanavalin A (Con A) and lipopolysaccharide (LPS). Stimulatory and costimulatory effects of NAC on mitogen-induced responses were also evident. The dose-response relationship describing the effects of NAC on lymphocyte proliferation with Con A-induced responses were enhanced in a dose-dependent manner, whereas the corresponding LPS-induced responses increased to a maximum level followed by decline in responses at higher concentrations of NAC. When splenocytes were incubated with inhibitory supraoptimal concentrations of Con A (10 microg/ml) or LPS (150 microg/ml), NAC partially enhanced the Con A-induced response but completely prevented the inhibitory effect of supraoptimal concentrations of LPS on splenocyte blastogenesis. Optimal and supraoptimal concentrations of Con A caused activation-induced cell death in the splenocytes whereas comparable concentrations of LPS did not produce a similar effect. Splenocyte cell death produced by the optimal mitogenic concentrations of Con A was completely blocked by the addition of NAC to cultures. Immunomodulation and protective effects of NAC were observed in mitogen-activated lymphocytes in vitro.

The effects of iron deficiency and iron overload on cell-mediated immunity in the mouse.

The influence of Fe status on cell-mediated immunity was studied in weanling mice fed on Fe-deficient (7 mg Fe/kg), Fe-sufficient (120 mg Fe/kg) and high-Fe (3000 or 5000 mg Fe/kg) diets for 7 weeks. The contact sensitivity (CS) response to dinitrofluorobenzene (DNFB), the in vivo delayed-type hypersensitivity (DTH) response to sheep erythrocytes (SRBC) and the ability of primed spleen cells to transfer DTH response to naive normal mice were suppressed in mice consuming the Fe-deficient diet. High-Fe diets (3000 or 5000 mg Fe/kg) selectively suppressed the CS response to DNFB, but the DTH response to SRBC or the transfer of DTH response by primed spleen cells to naive normal mice remained normal. Spleen cell functions associated with the expression of class II major histocompatibility (MHC) surface antigens, concanavalin A-induced interleukin-2 (IL-2) secretion or the antigen-presenting cell (APC) ability to stimulate antigen-dependent proliferation of an SRBC-specific helper T-lymphocyte clone were not altered by Fe status. However, consistent with the suppressed DTH response in the Fe-deficient mice was the suppressed concanavalin A-induced T-lymphocyte blastogenesis and the interferon-gamma (INF-gamma) production by spleen cells from mice fed on the Fe-deficient diet. Spleen cells from mice fed on excess levels of Fe in the diet secreted less INF-gamma than the control mice, although T-lymphocyte proliferation remained unaffected. Suppression of the cellular immune response associated with Fe deficiency may be related in part to impaired T-lymphocyte proliferation and INF-gamma secretion rather than to deficits in IL-2 secretion or APC function.

Effect of iron status on endotoxin-induced mortality, phagocytosis and interleukin-1 alpha and tumor necrosis factor-alpha production.

The host susceptibility to endotoxin (lipopolysaccharide, LPS), production of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) or phagocytosis in resident peritoneal macrophages were examined in iron-deficient and iron-loaded mice. Four groups of weanling male CD-1 mice were fed diet containing 7, 120, 5000 or 8000 ppm iron for 7 w. Body weight gain or hematocrit was not affected by iron consumption except for a lower weight gain in mice fed the 8000f1p4 iron diet. Iron-deficient and loaded diets produced a marked decrease and increase in liver iron concentration, respectively (P < 0.05). When challenged with an ip lethal dose of LPS, mortality was enhanced in iron-deficient and loaded mice (P = 0.035). The production of TNF-alpha and IL-1 alpha was assessed in the peritoneal macrophages stimulated by LPS in vitro. The production of IL-1 alpha and TNF-alpha was not altered in macrophages from iron-deficient mice. In contrast, macrophages from the 2 iron-loaded groups of mice produced more TNF-alpha (150% of control) without altering IL-1 alpha production. However, the total peritoneal leukocyte cell yield was not different among the treatment groups. Phagocytosis in the peritoneal macrophages determined by in vitro uptake of yeast cells was lower in the iron-deficient or loaded mice. This study indicates that iron deficiency and overload enhance LPS toxicity and impair phagocytosis, whereas excess iron also increases TNF-alpha production by macrophages.

The IgM and IgG antibody responses in iron-deficient and iron-loaded mice.

The humoral immune response was evaluated in male CD-1 mice fed the iron deficient (7 ppm Fe), iron sufficient (120 ppm Fe), and high-iron diets (3000 or 5000 ppm Fe) for 54 d. The IgM and IgG antibody responses against sheep erythrocytes (SRBC) determined by hemolytic plaque assay were suppressed by 65.4 and 51.2%, respectively, in the iron deficient mice. Subclinical iron deficiency was manifested by a marked reduction in hepatic iron concentration without any changes in hematocrit or body weight gain. In contrast, consumption of high-iron diets caused a marked accumulation of iron in the liver and a twofold reduction in the IgM antibody response without alteration in the IgG response. The suppression of the IgG antibody response in the iron deficient mice, however, did not result in a compensatory increase in delayed type hypersensitivity response.

Astrocyte proliferation in culture following exposure to potassium ion.

Following brain injury astrocytes swell acutely and proliferate thereafter. To investigate the direct relationship between swelling and proliferation, cultured astroglial cells were exposed to 60 mM K+ ion or hypo-osmolar (205 mOsm) electrolyte solution for 1 h to cause transient swelling. Proliferation was assessed by bromodeoxyuridine (BrdU) uptake 24 and 96 h later. The cells were double-labelled with antibodies to glial fibrillary acidic protein (GFAP) and BrdU. At 96 h the GFAP immunoreactive cells exposed to K+ exhibited 44.9% uptake versus 27.0% and 21.1% for control and hypo-osmolar exposed cells, respectively. We conclude that swelling of astroglia in vitro does not induce cell proliferation but transient depolarization following exposure to high K+ does.

Hepatotoxicity associated with dietary iron overload in mice.

1. Weanling male CD-1 mice were fed 120 (control), 5000 and 8000 mg of iron kg-1 for seven weeks. The haematocrit (P = 0.265), water consumption (P = 0.170) and percentage body weight ratios of kidney, spleen and heart were not affected by iron supplementation. 2. Iron supplementation reduced weight gain (P = 0.023), increased weight of liver (P = 0.0001), the iron deposition index and concentration of iron in the liver (P < 0.01). A strong correlation between liver iron concentration and level of iron in the diet (r = 0.989) was observed. Histologically, the deposition of iron was restricted to the hepatocytes, Kupffer cells and splenic macrophages. 3. Consumption of 5000 and 8000 mg of iron kg-1 resulted in hepatic damage, as judged by elevated serum alkaline phosphatase and alanine aminotransferase activities (P < 0.05). 4. This study indicates that prolonged feeding of excess dietary iron has the potential to cause hepatic accumulation of iron with resultant liver toxicity, and that mice may be a suitable model to study the mechanisms of dietary iron overload.

Vitamin E is protective against iron toxicity and iron-induced hepatic vitamin E depletion in mice.

This study examined the effect of excess dietary iron on liver function, iron and vitamin E status and the protective activity of vitamin E. Consumption of excess dietary iron (3000, 5000, 8000 mg iron/kg/diet) compared with consumption of the control diet (120 mg iron/kg diet) by weanling male CD-1 mice for 7 wk resulted in accumulation of iron in liver, increased relative liver weights and a reduction in hepatic vitamin E stores. The concentration of vitamin E in the liver was negatively correlated with dietary iron concentration (r = 0.998). Weekly administration of vitamin E (20 mg/kg, subcutaneously) prevented iron-induced liver damage without altering hepatic iron stores. Pretreatment of adult male CD-1 mice with a single subcutaneous dose of vitamin E (20 mg/kg body wt) 24 h prior to a lethal dose of iron (60 mg/kg, intraperitoneally) resulted in 100% protection. A similar dose of vitamin E given 5, 30 or 60 min (intravenously) after iron intoxication enhanced survival to 90, 70 and 80%, respectively, compared with the untreated control group. Vitamin E treatment 30 min after iron intoxication reduced mortality by 75% compared with intravenous treatment with 10 mg/kg of deferoxamine (Desferal). Data in this study indicate that vitamin E may be a useful antidote for iron toxicoses and that iron-induced depletion of vitamin E may play a role in the pathogenesis of iron toxicity.

Influence of low dietary iron and iron overload on urethan-induced lung tumors in mice.

Weanling male CD-1 mice were fed low iron (7 ppm), control (120 ppm) and iron loaded diets (3000 or 5000 ppm) for 19 weeks. After seven weeks, the mice received 1.5 mg urethan/g ip, and tumor development was evaluated 12 weeks later. The low iron diet increased the incidence of lung adenomas by 86%. The iron loaded diets did not influence adenoma development. Tumor size was unaffected by iron status (p = 0.297). These results indicate that low iron body status promotes tumor development and are inconsistent with the hypothesis that excess iron promotes cancer growth and that low iron protects against tumor growth.

Evaluation of potential antidotes for sodium fluoroacetate in mice.

Pathogenesis in fluoroacetate poisoning is multifactorial. Biochemically it is characterized by lethal synthesis of fluorocitrate, causing hypocalcemia, and energy deficiency through blockade of the TCA cycle. Calcium gluconate (CaG) was chosen to antagonize hypocalcemia, while sodium alpha kelogluterate (NaKG) and sodium succinate (NaSuc) were selected as potential antidotes to revive the TCA cycle. Effectiveness of each of these antidotes individually and in certain combinations was tested in mice exposed to lethal doses (15 mg/kg ip) of sodium fluoroacetate (NaFAC). Antidotal treatments were administered at 15 min, 4 h, 10 h, 24 h, and 36 h after NaFAC. All 3 of the antidotes alone, as well as a combination of CaG with NaKG, were ineffective in reducing mortality in mice after NaFAC. On the other hand, a combination of CaG (130 mg/kg) with NaSuc (240 mg/kg) was effective if the 2 solutions were either injected at separate sites or mixed in the same syringe just prior to injection. Similar solutions, if mixed for 24 h or longer before administrations, were ineffective. Increasing the dose of NaSuc to 360 or 480 mg/kg with CaG (130 mg/kg) was unrewarding. These results indicate that CaG in combination with 240 mg NaSuc/kg offer a promising therapy modality in NaFAC intoxication. Additional studies involving biochemical parameters and other species are needed to confirm the efficacy and mechanism(s) of action of this combination.

Record linkage study of hypokalaemia in hospitalized patients.

Retrospective analysis of biochemical data from 58,167 hospital inpatients revealed that 21% developed hypokalaemia during hospitalization-in 5.2% the serum potassium was less than 3.0 mmol/l. Subsequent evaluation showed a positive correlation between hypokalaemia and both female sex and hospital mortality. Patients with leukaemia and lymphoid tumours, especially when receiving antibiotic or cytotoxic therapy, and patients with gastro-intestinal malignancy were amongst those most frequently experiencing hypokalaemia. There was no significant association with cardiovascular disease. Drug and intravenous fluid administration accounted for the hypokalaemia in 56% of patients. While drug-related hypokalaemia was most commonly seen with diuretics, it was also apparent following use of steroids, insulin and haematinics.

Changes in glycosylated haemoglobin after oral glucose load.

To study the relation between hyperglycaemia and a change in the concentration of glycosylated haemoglobin (HbA1) blood glucose and HbA1 concentrations were measured during an oral glucose tolerance test and for 120 days afterwards in 20 normal subjects. These measurements showed that a minor degree of hyperglycaemia led to a significant increase in lycosylated haemoglobin concentrations. The increase appeared 10 days after the test, and values remained raised until 30 days and returned to normal 60 days after the test. If such a minor fluctuation of blood glucose can lead to a significant increase in HbA1 concentrations the test may be too sensitive as an index of long-term blood glucose control in diabetics.