Helminths, hygiene hypothesis and type 2 diabetes.

Worldwide, there is little overlap between the prevalence of soil-transmitted helminths and type 2 diabetes (T2D). Helminth-induced type 2 immune responses and immune regulatory network might modulate the obesity-induced activation of inflammatory pathways that are associated with the development of insulin resistance, a strong predictor of the development of T2D. However, other factors such as helminth-associated changes in adiposity and gut microbiome might also contribute to improved metabolic outcomes. In this review, we summarize epidemiological evidence for the link between helminths and T2D and discuss the potential mechanisms, based on findings from experimental studies as well as the limited number of studies in humans.

The effect of three-monthly albendazole treatment on Th2 responses: Differential effects on IgE and IL-5.

Helminth parasites induce a strong Th2 response, characterized by high levels of IgE and elevated signature cytokines such as IL-5. As many global deworming programmes are underway, there is concern that this might lead to emergence of Th1-mediated pathologies when the counterbalancing helminth-induced Th2 response is absent. Therefore, we assessed the effect of deworming on Th2-mediated responses in a household-clustered randomized controlled trial in Indonesia. Total plasma IgE and whole-blood IL-5 responses to mitogen phytohaemagglutinin (PHA) were measured in 1494 and 682 subjects, respectively, at baseline, 9 and 21 months after three-monthly single-dose treatment with albendazole or placebo. Anthelmintic treatment did not result in complete removal of helminth infections in the community. However, treatment significantly decreased IgE levels in albendazole- compared to placebo-treated subjects. IL-5 responses to PHA were not significantly affected by anthelmintic treatment and tended to increase in albendazole-treated subjects, indicating that intensive treatment of helminth parasites has different outcomes on B-cell (IgE levels) and T-cell (IL-5) responses. The data shows that 2 years of deworming can have differential effects on responses typified as Th2-mediated, which needs to be taken into account when examining the impact of helminths on noncommunicable diseases.

Maternal and child cytokine relationship in early life is not altered by cytokine gene polymorphisms.

The development of immune responses is influenced by the interaction between environmental and genetic factors. Our previous study showed a close association between maternal and young infant's cytokine responses. The question is how this association evolves over time and the contribution of genetic polymorphisms to this association. Five cytokines in mitogen-stimulated whole blood culture were measured from pregnant mothers and their children aged 2, 5, 12, 24 and 48 months. Cytokine gene polymorphisms were determined in both mothers and children. High production of maternal interleukin (IL)-10, tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was significantly associated with higher levels of the corresponding cytokines in their children at 2 months (T2), but the association decreased over time. Maternal single-nucleotide polymorphism (SNP) in IFN-γ gene, rs3181032, was found to be associated with child's IFN-γ levels at T2 only, whereas maternal IL-10 rs4579758 and child's TNF-α rs13215091 were associated with child's corresponding cytokines at later ages but not at T2. In the final models including the gene polymorphisms, maternal cytokines were still the strongest determinant of child cytokines. Maternal cytokine during pregnancy, which could be a proxy for child's environmental factors, showed its highest impact at early age, with no or little influence from genetic factors.

Allergic disorders and socio-economic status: a study of schoolchildren in an urban area of Makassar, Indonesia.

BACKGROUND: In urban centres of developing countries, there is great variation in socio-economic status (SES) and lifestyle; however, little information is available on allergic disorders in groups with high- or low-SES within the same urban area. OBJECTIVE: To determine the prevalence of allergic disorders and investigate risk factors related to them among high- and low-SES schoolchildren in Makassar, the capital city of South Sulawesi, Indonesia. METHOD: This cross-sectional study was performed in 623 children originating from high- (N = 349) and low-SES (N = 274) schools. Information on reported allergic symptoms and potential factors associated with allergic disorders was obtained by questionnaire. Specific IgE and skin prick test (SPT) reactivity were determined against aeroallergens [Dermatophagoides pteronyssinus (HDM) and cockroach]. Total IgE and helminth infections were also assessed. RESULT: The prevalence of SPT to any aeroallergens was significantly higher in high-SES than in low-SES school (25% vs. 8%, P < 0.001, respectively). However, specific IgE against cockroach and total IgE were significantly lower in high- than in low-SES children. Allergic symptoms were reported more often in low- compared to high-SES children. Specific IgE to aeroallergens significantly increased the risk of SPT positivity to the same aeroallergen in the high-, but not in the low-SES children. In the high- but not in low-SES, there was a significant positive association between SPT to HDM and wheeze. Similarly, cockroach skin reactivity and elevated BMI increased the risk of eczema in the high-SES children only. CONCLUSION AND CLINICAL RELEVANCE: Skin prick test is higher in high-SES, whereas IgE and allergic symptoms are higher in low-SES children. Specific IgE is a risk factor for being SPT-positive, and SPT positivity is a risk factor for allergic symptoms but only in children of high- and not low-SES school. Therefore, the socio-economic status of a child might affect the diagnosis of allergic disease in a developing country.

A comparison of ex vivo cytokine production in venous and capillary blood.

We performed a randomized study of the immunological effects of an early measles vaccine given at 4.5 months of age and aimed to obtain venous samples from the infants at baseline and 6 weeks later. If this was not feasible, a capillary sample was obtained. We analysed baseline samples from the first 50 children enrolled in the study to investigate the potential differences in ex vivo cytokine production between venous blood and capillary blood. We also obtained paired venous and capillary blood samples from 11 adult volunteers. Whole blood was stimulated with lipopolysaccharide (LPS) [a Toll-like receptor (TLR)-4 ligand], (S)-(2, 3-bis (palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH, trihydrochloride (PAM3Cys) (a TLR-2 ligand), phytohaemagglutinin (PHA) or purified protein derivative (PPD). Cytokine concentrations in the supernatants were assessed by a multiplexed assay and were compared between venous and capillary samples in both infants and adults. The production of both the pro- and the anti-inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10, was higher in cultures of capillary blood compared with venous blood. This was found in non-stimulated control samples as well as in blood stimulated with PAM3Cys and PPD. Adults produced more IL-5 in venous blood than in capillary blood upon PHA stimulation. We found no other difference in the levels of IL-5 or IFN-gamma between venous and capillary blood. In capillary blood we found sex differences in response to PHA but this was not the case in venous blood. We found significant differences in the production of cytokines between venous and capillary blood. Such differences should be taken into account when setting up immuno-epidemiological studies.

Campaigns with oral polio vaccine may lower mortality and create unexpected results.

Three studies from Guinea-Bissau found conflicting effects of OPV-at-birth (OPV0) on child survival. One study from 2004 suggested excess male mortality among children receiving OPV0 compared with children receiving NoOPV0 during a period of shortage of OPV. However, two subsequent studies showed beneficial effects of OPV0. In 2004, two national OPV-campaigns had been conducted in Guinea-Bissau. In a reanalysis of the 2004-study, in a survival analysis the age-adjusted mortality rate of study participants was 67% (95% CI=42-81%) lower after the OPV-campaigns than before the campaigns. In the OPV0 group only 22% (655/3031 person-years (pyrs)) of follow-up time was "after" the OPV-campaigns whereas 55% (473/859 pyrs) of the time in the NoOPV0 group was post-campaign (p<0.0001, Chi2). Censoring for OPV-campaigns in the original study removed excess male mortality and made the three studies more homogeneous. Overall, there is now considerable evidence that OPV, like other live vaccines, has important beneficial non-specific effects.

Effect of anthelmintic treatment on leptin, adiponectin and leptin to adiponectin ratio: a randomized-controlled trial.

Emerging evidence suggests that helminths might confer protection against the development of type 2 diabetes. We aimed to assess the role of adipokines in mediating the effect of helminths on insulin resistance. Serum samples were obtained from a randomized-controlled trial of anthelmintic treatment in an area endemic for soil-transmitted helminths (STH), Flores Island, Indonesia. In STH-infected subjects, anthelmintic treatment significantly increased the ratio of leptin to adiponectin (treatment effect factor (95% confidence interval (CI)), P-value for interaction: 1.20 (1.06-1.35), P=0.010), which largely stemmed from a significant reduction in adiponectin (0.91 (0.85-0.98), P=0.020) and a trend for an increase in leptin level (1.10 (1.00-1.21), P=0.119). No significant effect on resistin level was observed. This increase in leptin to adiponectin ratio seemed to contribute to the observed effect of deworming on increased insulin resistance (IR) as adjustment for leptin to adiponectin ratio attenuated the effect on IR from 1.07 (1.01-1.14, P=0.023) to 1.05 (0.99-1.11, P=0.075). Anthelmintic treatment in STH-infected subjects increases leptin to adiponectin ratio which may in small part contribute to the modest increase in IR. Further studies will be needed to assess the effect of the changes in adipokine levels on the host immune response and metabolism.

HLA and elephantiasis in lymphatic filariasis.

Lymphatic filariasis presents a spectrum of manifestations with infection-free asymptomatics at one end and elephantiasis at the other. In order to determine if any HLA antigens are associated with the development of elephantiasis, we compared the HLA frequencies in 55 elephantiasis patients with those in 40 controls consisting of individuals older than 45 years of age without any signs of elephantiasis. The only significant difference in class I antigen frequencies was observed for B27, which was present in 11% of the patients and absent in the controls. More differences were observed in HLA class II antigen frequencies. Both DR3 and the 2B3 epitope (on DQ6, DQ8, and DQ9 molecules) were significantly decreased in patients with elephantiasis whereas the DQ5 frequency was significantly higher in patients than in controls. Analysis of specific antibody isotype profiles revealed that DQ5-positive individuals had increased levels of antifilarial IgG3, an isotype known to be involved in tissue damage. These data suggest that HLA class II genes may control the course of Brugian filariasis by influencing the T-cell-dependent antibody repertoire.

Long-term follow-up of treatment with diethylcarbamazine on anti-filarial IgG4: dosage, compliance, and differential patterns in adults and children.

We have followed a population in an area endemic for Brugia malayi for three years after intensive treatment with diethylcarbamazine (DEC). Microfilariae were cleared from the circulation within four months in all eligible study participants (n = 60). There appeared to be a strong correlation between the maximum reduction in specific IgG4 and the number of days drug was taken under supervision (p = 0.41, P < 0.001), indicating that high total dosage of DEC is necessary for optimal reduction of active infection. In individuals with good compliance (at least 180 mg/kg of body weight, n = 34), we observed variable IgG4 patterns. All pre-treatment IgG4+ children (9-14 years old) and 40% of the IgG4+ adult population (> or = 15 years old) showed a gradual decrease in anti-filarial IgG4; 53% of these showed complete clearance of worm burden by the end of the study. In contrast, another group of male IgG4+ adults showed IgG4 patterns that started to increase between nine months and two years after treatment, indicating either a partial efficacy of DEC that allowed recovery of resident adult worms or reinfection.

Detection of filaria-specific IgG4 antibodies using Brugia Rapid test in individuals from an area highly endemic for Brugia timori.

The filarial parasite Brugia timori is of great public health importance in some islands of Eastern Indonesia. To establish a simple serological test for the identification and post-treatment monitoring of areas endemic for B. timori, a rapid immunochromatographic dipstick test (Brugia Rapid, BR) was evaluated on microfilaraemic and amicrofilaraemic individuals. This test is based on the detection of anti-filarial IgG4 antibodies that react with a recombinant Brugia malayi antigen (BmR1). In our study area on Alor island the prevalence of microfilaraemia was 26%. With the BR test, 100% of 196 sera from microfilaraemic persons and 76% of 563 sera from amicrofilaraemic persons, either symptomatic or asymptomatic, reacted positive. All 50 control sera from areas non-endemic for lymphatic filariasis gave negative BR test results. This study showed that the BR test can be also used to detect antibodies against B. timori. Due to the high prevalence of IgG4 antibodies as detected by the BR test (81%), no significant correlation with the prevalence of microfilaraemia could be detected within the endemic village. The BR test also shows great promise to be employed as a monitoring tool for B. timori in the framework of the Global Program to Eliminate Lymphatic Filariasis (GPELF).

Ivermectin and diethylcarbamazine trials in leaf monkeys (Presbytis cristatus) infected with Wuchereria kalimantani.

Clinical trials of Ivermectin in single oral doses of 200, 400, and 1,000 mg/kg body weight or in multiple doses of 200 mg/kg body weight for 5 consecutive days were performed in leaf monkeys (Presbytis cristatus) infected with Wuchereria kalimantani. Optimal microfilaricidal effect occurred at 200 mg/kg body weight. The drug was less effective than diethylcarbamazine in this animal model for human filariasis but had no adverse effects.

In Th2-biased lymphatic filarial patients, responses to purified protein derivative of Mycobacterium tuberculosis remain Th1.

Natural infection with filarial nematode parasites results in immune responses skewed towards T helper (Th)2, while infection with mycobacteria shows many characteristics of a Th1-dominated response. Cytokines typifying Th1, interferon (IFN)-gamma, and Th2, interleukin (IL)-4, were measured following stimulation of peripheral blood mononuclear cells from filarial patients with Brugia malayi adult worm antigen (BmA) and purified protein derivative of Mycobacterium tuberculosis (PPD). In response to PPD, only 1 out of 81 patients produced IL-4, and this at an amount (4.4 pg/ml) just above the detection limit, whereas 59% of patients responded to BmA by releasing IL-4. Conversely, substantial quantities of IFN-gamma were released in response to PPD (geometric mean 37.43 U/ml) compared to low BmA-stimulated IFN-gamma production in the same patients (geometric mean 5.02 U/ml). These results demonstrate that the strong skewing of the cytokine environment towards Th2 in filarial patients in vivo does not influence the predominance of a Th1 type immune response to PPD.

T-cell activation and the balance of antibody isotypes in human lymphatic filariasis.

Human filarial infection presents a spectrum of clinical states with two major poles: asymptomatic microfilaraemia and amicrofilaraemic chronic disease. Microfilaremia is associated with a Th1-type tolerance, and maximal IgG4 antibodies, while elephantiasis patients react across a broad range of immune parameters. In this review, Rick Maizels and his colleagues discuss recent advances in the immunology of human filariasis and present a summary of their latest studies in an endemic area of Indonesia.

Clustering of filarial infection in an age-graded study: genetic, household and environmental influences.

A statistical method that analyses correlation structures in families to delineate the contribution of genetic, household and environmental factors on clustering of infection, has been applied to data collected in an area endemic for brugian filariasis in South Sulawesi, Indonesia. Infection was assessed both by microfilaraemia and by anti-filarial IgG4. The results confirmed earlier findings that genetic factors play an important role in clustering of infection. When clustering of infection was analysed in children (< 10 years of age) and adults (> 20 years of age) separately, it was found that the genetic factors influence clustering of infection in children more profoundly than environmental or household effects. In contrast, genetic factors could not fully explain the clustering of infection seen in adults, which seemed to be mainly determined by household and environmental effects. The data have implications for genotyping studies in brugian filariasis; they indicate that it may be important to concentrate on the younger age groups where individual environmental effects have not yet overruled the genetic influences on gain/loss of infection.

Differential decline in filaria-specific IgG1, IgG4, and IgE antibodies in Brugia malayi-infected patients after diethylcarbamazine chemotherapy.

In human filariasis, the predominant serum antibody is IgG4, accompanied by significant IgE production. The ratio of IgG4 to IgE is highest in asymptomatic microfilaremic carriers, while chronic disease is associated with elevated IgG1-3. The changes in isotypes following chemotherapy with diethylcarbamazine (DEC) were studied in 2 groups of Brugia malayi-infected patients from Sumatra and South Kalimantan, Indonesia. Similar results were obtained from each group. IgG4 levels decreased sharply (65%-78%) within 12 months. IgG1 levels declined in a less consistent and extreme manner, and levels of IgG2 and IgG3 declined only in patients with elephantiasis, who also had the highest initial levels of these antibodies. IgE responses were relatively stable to therapy in microfilaremic patients (7%-28% reduction) and showed only moderate decline (56% over 2 years) in elephantiasis patients. Active filarial infection is thus associated with specific IgG4 antibodies, but there is independent expression of the IgE and IgG4 isotypes in filariasis.

Specific T cell unresponsiveness in human filariasis: diversity in underlying mechanisms.

In an attempt to overcome T cell unresponsiveness to filarial antigens, 65 individuals belonging to the three clinical groups of elephantiasis patients, microfilaraemics, and asymptomatic amicrofilaraemics who exhibited unresponsiveness to Brugia malayi adult worm antigen (BmA) were studied. Peripheral blood mononuclear cells were cocultured with antigen and one of the following reagents that have been reported to be effective in reconstituting T cell proliferation: interleukin-2 (IL-2), interleukin-7 (IL-7), anti-interleukin-4, anti-interleukin-10, anti-CD2, anti-CD27, anti-CD28, indomethacin, phorbol myristate acetate (PMA), or calcium ionophore (A23187). We were able to overcome antigen-specific unresponsiveness in only a minority of the individuals studied. Co-culture with IL-2, IL-7, indomethacin and PMA were the only conditions which resulted in enhanced proliferation to BmA in these individuals. In general, unresponsiveness in elephantiasis patients was easier to reverse than in other clinical groups: in 50% of elephantiasis patients, in 12.5% of microfilaraemics and in 20% of asymptomatic amicrofilaraemics. The results indicate that more than one distinct immunological mechanism may account for the antigen-specific unresponsiveness in individuals exposed to and infected with brugian filariasis.

Antibody responses to filarial infective larvae are not dominated by the IgG4 isotype.

The humoral immune response in humans to filarial parasites is generally dominated by the IgG4 isotype, when measured by ELISA against somatic adult worm extract. In contrast, as we report here, antibodies reactive to somatic extracts of infective larvae are more equally represented by IgG1 and IgG4. Moreover, binding to surface exposed epitopes in immunofluorescence on larval stages is mediated foremost by IgG1 and IgM, secondarily by IgG2 and IgG3, and very little by IgG4. Both anti-L3 surface and somatic antibodies are strongest in elephantiasis patients, and tend to increase with age. Antibody to the L3 surface is also present in most microfilaraemic individuals who bear no detectable antibodies to the surface of the microfilarial stage. These results demonstrate that a stage- and isotype-specific response is mounted to the L3 surface which should be considered as a possible mediator of concomitant immunity in filariasis.

Specificity of predominant IgG4 antibodies to adult and microfilarial stages of Brugia malayi.

Human infections with filarial nematodes such as Brugia malayi are accompanied by unusually high titres of parasite-specific IgG4 antibodies. We have compared the profile of antigens recognised by filarial-specific IgG1 and IgG4 isotypes by Western blotting. Serum samples were collected from 120 subjects exposed to Brugia malayi, divided into three groups of asymptomatic amicrofilaraemic (endemic normal), microfilaraemic, and elephantiasis patients. Antigen preparations were tested from both adult B. malayi parasites, and from microfilariae; 24 distinct bands were analysed from the former, and 19 from the latter. Both qualitative scoring for band reactivity, and densitometric scanning of major bands, were employed. The consistent result was one of high and preferential IgG4 reactivity to a set of low molecular weight bands, of 15, 17, 20, 31 and 33 kDa; most of the 19 other bands showed higher reactivity with IgG4. Analysis of Western blot patterns showed an overall tendency for stronger IgG4 responses in microfilaraemic cases, and higher IgG1 responses in elephantiasis patients, consistent with published studies using ELISA on unfractionated parasite extracts. This study has defined an array of filarial antigens from each stage, and relative levels of IgG4 recognition, which will be important in unravelling distinct immune responses to this complex parasite.

Depression of antigen-specific interleukin-5 and interferon-gamma responses in human lymphatic filariasis as a function of clinical status and age.

In an area in which brugian filariasis is endemic, when cytokine levels were analyzed as a function of clinical status comparing those who were asymptomatic and amicrofilaremic with those who were microfilaremic, it was found that both interferon (IFN)-gamma and interleukin (IL)-5 were suppressed in microfilariae carriers (P < .01 and P < .001, respectively), but IL-4 was unabated. Age had a significant effect on cytokine production in both groups. In asymptomatic amicrofilaremic subjects, IL-4 production was high in young persons and decreased with age, whereas in microfilaremic subjects, IL-4 increased significantly with age. Conversely, IFN-gamma showed a tendency to increase with age in asymptomatic amicrofilaremic subjects but not in microfilaremic subjects. IL-5 decreased significantly with increasing age in both asymptomatic amicrofilaremic and microfilaremic groups. These results indicate that the length of exposure to and infection with filarial parasites can each exert a substantial effect on the cytokine response profiles of host T cell populations.

Adults acquire filarial infection more rapidly than children: a study in Indonesian transmigrants.

To dissociate the influence of host age from length of exposure on acquisition of filarial infection, we examined the development of microfilaraemia and anti-filarial IgG4 in all ages of a naive population that became suddenly exposed to Brugia malayi as a result of transmigration. Responses in 247 transmigrants, who had settled for periods of several months up to 6 years in their new homesteads, were compared with those of 133 life-long residents. As shown in earlier studies, anti-filarial IgG4 increased with age in the indigenous populations, whose age is equivalent to length of exposure. However, by examining transmigrants, it became clear that development of specific IgG4 was influenced by age, since levels of this antibody were consistently higher in transmigrant adults than in transmigrant children, despite an equal length of exposure to filarial infection. Examining microfilaraemia, it was confirmed that infection establishes more rapidly in adults than in children.