Laila N. Islam

Association of respiratory complications and elevated serum immunoglobulins with drinking water arsenic toxicity in human.

We assessed the relationship between chronic arsenic exposure through drinking water with respiratory complications and humoral immune response by measuring serum immunoglobulin profiles in the affected subjects (arsenicosis patients) living in the arsenic endemic rural villages of Bangladesh. The duration of exposure was determined through detailed history of the patients (n = 125) and the levels of arsenic in the drinking water and urine samples were determined. The mean duration of exposure in the patients was 7.4 ± 5.3 y, and the levels of arsenic in the drinking water and urine samples were 216 ± 211 and 223 ± 302 μ g/L, respectively, compared to 11 ± 20 and 29 ± 19 μ g/L, respectively, in the unexposed subjects. There was high prevalence of respiratory complications like breathing problems including chest sound, asthma, bronchitis and cough associated with drinking water arsenic toxicity. Arsenicosis patients had significantly elevated levels of IgG (P < 0.001) and IgE (P < 0.001) while the levels of IgA were also significantly higher (P < 0.005) but IgM were similar to that of the control subjects. Analysis of the clinical symptoms based on skin manifestations showed the levels of both IgG and IgE were significantly elevated during the initial stages while IgE were further elevated with the duration of arsenic exposure. Arsenicosis patients with respiratory complications had mean serum IgE levels of 706 ± 211 IU/mL compared to 542 ± 241 IU/mL in patients without apparent involvement with the respiratory system (P < 0.01). The eosinophil counts in the patients did not differ significantly from the unexposed subjects indicating that elevated levels of serum IgE might not be due to allergic diseases, rather it could be due to direct effects of arsenic. We found significant linear relationships between the levels of serum IgE and inorganic phosphorus (P < 0.05), and serum IgA levels with urinary excretion of arsenic (P < 0.001). These observations suggested that arsenic toxicity caused respiratory complications, induced changes in the humoral as well as mucosal immune responses.

The use of collagen or fibrin gels for the assay of human neutrophil chemotaxis

Neutrophil leucocytes are known to migrate actively into 3-dimensional gels of collagen or fibrin. In this paper, we have used such gels to study chemotaxis of human blood neutrophils towards gradient sources of formyl-methionyl-leucyl-phenylalanine (FMLP) using 2 assay systems. The first resembled the micropore filter assay in that neutrophils on the upper surface of collagen gels were allowed to invade in the presence of either an isotropic concentration or a gradient of FMLP. Neutrophils invaded the gel vigorously in both cases. The effect of the gradient was assessed by determining the population distribution at different levels in the gel. Cells moving randomly should be distributed normally, and directional locomotion should cause deviation from normal distribution. Such a deviation was seen, but was of marginal significance. A more direct demonstration of chemotaxis was achieved by the second assay in which an agarose slab containing FMLP was incorporated into a gel, and the paths of nearby neutrophils were filmed. These cells showed an unequivocal directional response to the FMLP gradient. Protein gels can thus be used in the same way as both the presently used filter assays and visual assays using plane substrata, but with the advantage of providing a more physiological environment for the study of chemotaxis than either.

Evaluation of methods for isolating human peripheral blood monocytes: Studies on chemotactic locomotion and other functional characteristics

We have compared six different methods of purifying human blood monocytes for their usefulness in relation to assays of polarization, locomotion and chemotaxis. For polarization assays it is essential to prepare an unstimulated, spherical, cell population in suspension. The techniques compared were based either on density differences between monocytes and lymphocytes using Percoll or Nycodenz, or on the separation of adherent monocytes from non-adherent cells on protein-coated surfaces, i.e., foetal calf serum (FCS); gelatin-FCS; gelatin-plasma; baby hamster kidney (BHK) microexudate coats. The BHK microexudate technique (Ackerman and Douglas, 1978) gave the best yield and purity of monocytes. These were spherical and had not been activated by the separation procedure. This technique provided monocytes in suspension that were functionally normal in locomotion and chemotaxis assays, phagocytosis, chemiluminescence and Fc receptor expression. To achieve a good yield of spherical cells, it was necessary to use tubes to which monocytes did not adhere. Siliconized glass was superior to tissue culture plastic for this purpose.

Molecular evidence for polyphyletic origin of human immunodeficiency virus type 1 subtype C in Bangladesh

HIV-1 positive blood samples were collected between 1999 and 2005 from population groups most at risk of HIV infection in Bangladesh through the national surveillance, from clients of the Voluntary Counseling and Testing (VCT) Unit for HIV at ICDDR,B and a survey of HIV in patients with tuberculosis. Partial sequences of the gag gene were used for subtyping the HIV strains by nested PCR using selective primers. Of the 198 HIV strains tested, subtype C (41.4%) was the commonest strain identified. Phylogenetic analysis of Bangladeshi subtype C strains showed that they clustered in polyphyletic branches representing HIV strains from different parts of the world. Most of the strains from injecting drug users (IDU) clustered together and were similar to Indian strains. The VCT strains however were very heterogeneous and clustered with strains from India, Myanmar, Ethiopia and Zimbabwe. Data suggest that there have been few introductions into the IDU population where the epidemic is driven by indigenous transmission. On the other hand there have been many and regular introductions of subtype C viruses through migrant workers in the VCT group. Very little overlap was observed in the strains obtained from IDU and those from other population groups.

Renin gene polymorphisms in bangladeshi hypertensive population.

Objective: Linkages of renin gene polymorphisms with hypertension have been implicated in several populations with contrasting results. Present study aims to assess the pattern of renin gene polymorphisms in Bangladeshi hypertensive individuals. Methodology: Introns 1, 9 of renin gene and 4063 bases upstream of promoter sequence of renin gene were amplified from the genomic DNA of the total 124 (hypertensive and normotensive) subjects using respective primers. Polymerase chain reaction-based restriction fragment length polymorphisms were performed using BglI, MboI and TaqI restriction enzymes. Results: Homozygosity was common in renin gene regarding BglI (bb=48.4%, Bb=37.9%, BB=13.7%, χ2 =1.91, P>0.05), TaqI (TT=81.5%, Tt=14.5%, tt=4.0%, χ2 =7.50, P<0.01) and MboI (mm=63.7%, Mm=32.3%, MM=4.0%, χ2=0.00, P>0.05) polymorphisms among total study population. For BglI and TaqI genotype distribution, hypertensive subjects (BglI: χ2 =6.66, P<0.05; TaqI: χ2 = 10.28, P<0.005) significantly deviate from Hardy-Weinberg Equilibrium law compared to normotensive subjects (BglI: χ2=0.51, P>0.05; TaqI: χ2=0.20, P>0.05). On the other hand, with respect to MboI polymorphisms of renin gene, only normotensive subjects deviate from the law (patients: χ2=1.28, P>0.05; vs controls: χ2=6.81, P<0.01). In the context of allelic frequency, common T allele was clearly prevalent (T frequency=0.86, t frequency = 0.14) for TaqI, but rare alleles b and m were more frequent for both BglI (b frequency=0.69, B frequency=0.31) and MboI (m frequency=0.80 M frequency=0.20) polymorphisms, respectively. Conclusion: Thus, we report that Bangladeshi hypertensive subjects did not show any distinct pattern of renin gene polymorphisms compared to their healthy control subjects with regard to their genotypic and allelic frequencies.

Function of Serum Complement in Drinking Water Arsenic Toxicity

Serum complement function was evaluated in 125 affected subjects suffering from drinking water arsenic toxicity. Their mean duration of exposure was 7.4 ± 5.3 yrs, and the levels of arsenic in drinking water and urine samples were 216 ± 211 and 223 ± 302 μg/L, respectively. The mean bactericidal activity of complement from the arsenic patients was 92% and that in the unexposed controls was 99% (P < 0.01), but heat-inactivated serum showed slightly elevated activity than in controls. In patients, the mean complement C3 was 1.56 g/L, and C4 was 0.29 g/L compared to 1.68 g/L and 0.25 g/L, respectively, in the controls. The mean IgG in the arsenic patients was 24.3 g/L that was highly significantly elevated (P < 0.001). Arsenic patients showed a significant direct correlation between C3 and bactericidal activity (P = 0.014). Elevated levels of C4 indicated underutilization and possibly impaired activity of the classical complement pathway. We conclude reduced function of serum complement in drinking water arsenic toxicity.

Immune response of Bangladeshi children with acute diarrhea who subsequently have persistent diarrhea.

Background Because altered immune responses may be a risk factor for persistent diarrhea, various aspects of the immune response were examined to elucidate the underlying immune mechanisms that may be involved in the development of persistent diarrhea. Methods Children (7–12 months of age) with watery diarrhea for 6 to 8 days from the Dhaka Hospital of the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), were enrolled. Children were classified as having acute diarrhea (AD) or persistent diarrhea (PD) if diarrhea resolved within 14 days or persisted for more than 14 days, respectively. Uninfected control children (n = 13), from the Nutrition Follow-Up Unit of ICDDR,B were also enrolled. Of the 123 children with diarrhea who were enrolled, 85 had AD and 38 had PD. Comparisons were performed for clinical features, nutritional status (weight for age, plasma transferrin, and serum albumin levels), and immune responses: neutrophil function; peripheral blood mononuclear cell function, delayed-type hypersensitivity (DTH) responses, plasma levels of immunoglobulins, tumor necrosis factor-&agr;, and interferon-&ggr;. Univariate analyses were conducted to assess differences among the three groups of children and between children with AD and PD. Logistic regression was performed to determine risk factors for PD. Results There were no differences in clinical features and nutritional status among the groups of children studied. More children in whom PD developed had a negative DTH response to tuberculin than those with AD (P = 0.021). Also, a negative DTH response to tuberculin was a significant risk factor for PD (odds ratio [OR] = 3.8, 95% confidence interval [CI] = 1.5–9.9). Conclusions Children with acute diarrhea with a negative DTH response to tuberculin are more likely to have development of persistent diarrhea.

Kinetics of antibody-secreting cell and fecal IgA responses after oral cholera vaccination in different age groups in a cholera endemic country

Immune responses to oral enteric vaccines in children and infants may be influenced by factors such as age, previous priming with related microorganisms and breast feeding. In this study, we aimed to determine optimal time points to assess immune responses to oral enteric vaccines in different clinical specimens. This was done by investigating antibody secreting cell (ASC) and fecal antibody responses on different days after vaccination using the licensed oral cholera vaccine Dukoral, containing cholera toxin B-subunit (rCTB) and inactivated Vibrio cholerae bacteria, as a model vaccine. Two vaccine doses were given 2weeks apart to infants (6-11months), young children (12-18months), toddlers (19months-5years) and adults in a cholera endemic country (Bangladesh). IgA ASC responses, as determined by the antibodies in lymphocyte supernatant (ALS) assay, plasma IgA and IgG responses and secretory IgA (SIgA) responses in extracts of fecal samples were evaluated 4/5 and 7days after each vaccination. After the first vaccine dose, anti-CTB ALS IgA responses in adults and toddlers were high and comparable on day 5 and 7, while responses were low and infrequent in young children. After the second dose, highest ALS responses were detected on day 5 among the time points studied in all age groups and the responses declined until day 7. In contrast, plasma IgA and IgG anti-CTB responses were high both on day 5 and 7 after the second dose. Fecal SIgA responses in young children and infants were highest on day 7 after the second dose. Our results suggest that ASC/ALS responses to two doses of the oral cholera vaccine Dukoral and related oral vaccines should be analyzed earlier than previously recommended (day 7) at all ages. Fecal antibody responses should preferably be analyzed later than ASC/ALS responses to detect the highest antibody responses.

Analyses of Genetic Variations of Glutathione S-Transferase Mu1 and Theta1 Genes in Bangladeshi Tannery Workers and Healthy Controls

Glutathione S-transferases (GSTs) belong to a group of multigene detoxification enzymes, which defend cells against oxidative stress. Tannery workers are at risk of oxidative damage that is usually detoxified by GSTs. This study investigated the genotypic frequencies of GST Mu1 (GSTM1) and GST Theta1 (GSTT1) in Bangladeshi tannery workers and healthy controls followed by their status of oxidative stress and total GST activity. Of the 188 individuals, 50.0% had both GSTM1 and GSTT1 (+/+), 12.2% had GSTM1 (+/−), 31.4% had GSTT1 (−/+) alleles, and 6.4% had null genotypes (−/−) with respect to both GSTM1 and GSTT1 alleles. Among 109 healthy controls, 54.1% were double positive, 9.2% had GSTM1 allele, 32.1% had GSTT1 allele, and 4.6% had null genotypes. Out of 79 tannery workers, 44.3% were +/+, 16.8% were +/−, 30.5% were −/+, and 8.4% were −/−. Though the polymorphic genotypes or allelic variants of GSTM1 and GSTT1 were distributed among the study subjects with different frequencies, the differences between the study groups were not statistically significant. GST activity did not vary significantly between the two groups and also among different genotypes while level of lipid peroxidation was significantly higher in tannery workers compared to controls irrespective of their GST genotypes.

Immunotoxic Effects of Arsenic Exposure

Endemic arsenic exposure emerged as a global public health problem affecting millions of people worldwide because of its association with various cancers and numerous other pathological effects. Increasing lines of research indicate that arsenic may adversely affect the immune system, but its specific target to impair immune function is poorly understood. While inorganic arsenicals arsenite and arsenate showed strong cytotoxicity to both macrophages and lymphocytes, arsenic trioxide has important antitumor properties. Arsenic can have significant effects on blood leukocytes and many aspects of the immune system including impaired T-cell activation, altered expression of cytokines such as IL-2, IL-4, IL-5, IL-10, IFN-γ, TNF-α, and GM-CSF, suppression of contact hypersensitivity responses, loss of adhesion, impairment of function, morphologic changes in human macrophages, and the humoral immune response. Arsenic induces overexpression of keratinocyte-derived growth factors, which are likely to have a significant role in arsenic-induced skin hyperkeratoses and cancer. The immunotoxic effects of arsenic exposure have been evaluated in this chapter.