Increased mitochondrial DNA copy number in maternal peripheral blood is associated with low birth weight in Lombok, Indonesia.

Fetal development depends on maternal metabolic energy from mitochondria. We investigated the association of maternal mitochondrial function, represented by mitochondrial DNA copy number (mtDNA-CN) of venous blood, with child birth weight (BW) from 528 randomly selected mothers enrolled in the Supplementation with Multiple Micronutrients Intervention Trial (ISRCTN 34151616). Real-time quantitative PCR of archived blood specimens and regression analysis adjusting for other primary determinants of BW showed that loge mtDNA-CN was inversely associated with BW (ß = -204.6, p < 0.001), particularly in the third trimester (ß = -376.8, p<0.001). Maternal mtDNA-CN may be a marker for low BW and fetal growth restriction.

Stunting is associated with blood lead concentration among Bangladeshi children aged 2-3 years.

BACKGROUND: Lead toxicity is of particular public health concern given its near ubiquitous distribution in nature and established neurotoxicant properties. Similar in its ubiquity and ability to inhibit neurodevelopment, early childhood stunting affects an estimated 34 % of children under 5 in low- and middle-income countries. Both lead and stunting have been shown to be associated with decreased neurodevelopment, although the relationship between these childhood burdens is underexplored. The association between lead exposure and stunting has been previously established, yet limited data are available on susceptibility windows. METHODS: Whole blood lead samples were collected from rural Bangladeshi children at delivery (umbilical cord blood) and at age 20-40 months (fingerstick blood). Stunting was determined using the Child Growth Standards developed from the World Health Organization Multicentre Growth Reference Study. Children with height for age < -2 z-scores below the median of the WHO Child Growth Standards were classified as stunted in all analyses. RESULTS: Median (IQR) umbilical cord and fingerstick blood lead levels were 3.1 (1.6-6.3) µg/dl and 4.2 (1.7-7.6) µg/dl, respectively. In adjusted multivariable regression models, the odds of stunting at 20-40 months increased by 1.12 per µg/dl increase in blood lead level (OR = 1.12, 95 % CI: 1.02-1.22). No association was found between cord blood lead level and risk of stunting (OR = 0.97, 95 % CI: 0.94-1.00). CONCLUSIONS: There is a significant association between stunting and concurrent lead exposure at age 20-40 months. This association is slightly attenuated after controlling for study clinic site. Additional research including more precise timing of lead exposure during these critical 20-40 months is needed.

Modifying effect of maternal nutritional status on the impact of maternal multiple micronutrient supplementation on birthweight in Indonesia.

BACKGROUND/OBJECTIVE: Low birthweight (LBW) and intrauterine growth restriction are linked with maternal nutritional status during pregnancy, and maternal supplementation with multiple micronutrients (MMNs) is reported to increase birthweight. Responses to MMN, however, might be modified by maternal nutrition. SUBJECTS/METHODS: To examine the differential effects of maternal nutritional status on birthweight responses to prenatal MMN supplementation, data from the Supplementation with Multiple Micronutrient Intervention Trial, a cluster-randomized trial in Indonesia was analyzed. Birthweight outcomes of 7001 infants whose mothers received iron/folic acid were compared with 7292 infants whose mothers received MMN. The modifying effects of maternal short-term nutritional status (mid-upper arm circumference (MUAC) and long-term nutritional status (height) on the birthweight response to MMN supplementation were assessed. RESULTS: For women with higher MUAC (≥23.5 cm), MMN increased mean birthweight by 33 g (95% confidence interval (CI): -1 to 66, P=0.06) and significantly reduced LBW by 21% (relative risk: 0.79, 95% CI: 0.64-0.99, P=0.04). The modifying effect of MUAC on mean birthweight, LBW and small for gestational age was significant. There was no evidence of a modifying effect of maternal height on the response to MMN. CONCLUSIONS: Supplementation with MMN in pregnancy increased birthweight, but maternal nutritional status modified this response, with infants born to women with better short-term nutrition having greater birthweight response.

Effect of maternal multiple micronutrient supplementation on fetal loss and infant death in Indonesia: a double-blind cluster-randomised trial.

BACKGROUND: Maternal nutrient supplementation in developing countries is generally restricted to provision of iron and folic acid (IFA). Change in practice toward supplementation with multiple micronutrients (MMN) has been hindered by little evidence of the effects of MMN on fetal loss and infant death. We assessed the effect of maternal supplementation with MMN, compared with IFA, on fetal loss and infant death in the setting of routine prenatal care services. METHODS: In a double-blind cluster-randomised trial in Lombok, Indonesia, we randomly assigned 262 midwives to distribute IFA (n=15 ,86) or MMN (n=15,804) supplements to 31 290 pregnant women through government prenatal care services that were strengthened by training and community-based advocacy. Women obtained supplements, to be taken daily, every month from enrolment to 90 days post partum. The primary outcome was early infant mortality (deaths until 90 days post partum). Secondary outcomes were neonatal mortality, fetal loss (abortions and stillbirths), and low birthweight. Analysis was by intention to treat. The study is registered as an International Standard Randomised Controlled Trial, number ISRCTN34151616. FINDINGS: Infants of women consuming MMN supplements had an 18% reduction in early infant mortality compared with those of women given IFA (35.5 deaths per 1000 livebirths vs 43 per 1000; relative risk [RR] 0.82, 95% CI 0.70-0.95, p=0.010). Infants whose mothers were undernourished (mid upper arm circumference <23.5 cm) or anaemic (haemoglobin <110 g/L) at enrolment had a reduction in early infant mortality of 25% (RR 0.75, 0.62-0.90, p=0.0021) and 38% (RR 0.62, 0.49-0.78, p<0.0001), respectively. Combined fetal loss and neonatal deaths were reduced by 11% (RR 0.89, 0.81-1.00, p=0.045), with significant effects in undernourished (RR 0.85, 0.73-0.98, p=0.022) or anaemic (RR 0.71, 0.58-0.87, p=0.0010) women. A cohort of 11 101 infants weighed within 1 h of birth showed a 14% (RR 0.86, 0.73-1.01, p=0.060) decreased risk of low birthweight for those in the MMN group, with a 33% (RR 0.67, 0.51-0.89, p=0.0062) decrease for infants of women anaemic at enrolment. INTERPRETATION: Maternal MMN supplementation, as compared with IFA, can reduce early infant mortality, especially in undernourished and anaemic women. Maternal MMN supplementation might therefore be an important part of overall strengthening of prenatal-care programmes.

Validation of a rapid enzyme immunoassay for the quantitation of retinol-binding protein to assess vitamin A status within populations.

OBJECTIVE: To compare the prevalence of vitamin A deficiency (VAD) among Cambodian preschool children as determined by the retinol-binding protein-enzyme immunoassay (RBP-EIA) and direct measurement of serum retinol by high-performance liquid chromatography (HPLC). SETTING AND SUBJECTS: Sera from 359 children were randomly selected from archived specimens collected in a national VAD prevalence survey in Cambodia. METHODS: Sera were first analyzed for retinol content by HPLC and then subjected to analysis using RBP-EIA to determine serum RBP concentrations. National Institute of Standards and Technology and control sera were used to ensure quality and accuracy for each set of analyses. To classify VAD, the same cutoff point of <0.70 micromol/l was employed for each indicator. RESULTS: Overall, the prevalence of VAD based on serum retinol was 22.3% (95% confidence interval (CI): 18.0, 26.6), whereas the RBP-EIA indicated a VAD prevalence of 20.9% (95% CI: 16.7, 25.1). A simple linear regression model indicated an R2 of 0.79, and a receiver operating curve analysis revealed an area under the curve of 0.92. CONCLUSIONS: We found no significant difference between the results of RBP-EIA compared to retinol analyzed by HPLC in estimating the prevalence of VAD. Use of the test could enable public health authorities to assess the extent of VAD and track progress in control programs in resource-poor settings.

Antioxidant status and acute malaria in children in Kampala, Uganda.

Although antioxidant status has been implicated in the pathogenesis of malaria, these factors need further characterization. A longitudinal study was conducted involving 273 children 1-10 years of age with acute, uncomplicated malaria in Kampala, Uganda. Plasma vitamin A, carotenoids, and vitamin E were measured at enrollment and on day 7. Malaria parasitemia was measured at enrollment, on day 3, and on day 7. Malaria parasitemia had completely cleared in 57.1% and 85.3% of children by day 3 and day 7, respectively. Plasma vitamin A, alpha-carotene, beta-carotene, lycopene, lutein/zeaxanthin, and vitamin E were depressed at enrollment and increased by day 7. Multivariate analyses showed that higher plasma lycopene concentrations at enrollment were associated with clearance of parasitemia between enrollment and day 3 (odds ratio = 1.46, 95% confidence interval = 1.07-2.06, per 0.10 micromol/L of lycopene). This study suggests that children with acute malaria have depressed plasma concentrations of antioxidants, and that higher plasma lycopene is associated with more rapid clearance of malaria parasitemia.

Nutritional modulation of malaria morbidity and mortality.

This review critically examines the relationship between nutritional status and malaria. The data indicate that protein-energy malnutrition is associated with greater malaria morbidity and mortality in humans. In addition, controlled trials of either vitamin A or zinc supplementation show that these nutrients can substantially reduce clinical malaria attacks. Data for iron indicate that supplementation may minimally aggravate certain malariometric indices in some settings and also strongly improve hematologic status. Withholding of iron supplements from deficient population is, therefore, not currently indicated. Available evidence for other nutrients describe varied effects, with some deficiencies being exacerbative (e.g., thiamine), protective (e.g., vitamin E), or both exacerbative and protective in different settings (e.g., riboflavin, vitamin C). The roles of folate, other B vitamins, unsaturated fatty acids, amino acids, and selenium are also examined. Study of the interactions between nutrition and malaria may provide insight to protective mechanisms and result in nutrient-based interventions as low-cost and effective adjuncts to current methods of malaria prevention and treatment.

Relation of serum retinol to acute phase proteins and malarial morbidity in Papua New Guinea children.

BACKGROUND: Acute phase proteins (APPs) are associated with malaria-induced hyporetinemia (serum retinol <0.70 micromol/L); however, the degree of the association is not well documented. OBJECTIVE: The association between malaria-induced hyporetinemia and APPs was assessed. DESIGN: In a cross-sectional study, 90 children with serum retinol concentrations from <0.35 to >1.05 micromol/L were selected from children in a clinical trial of vitamin A supplementation. Serum was collected before treatment allocation. Retinol binding protein (RBP) concentrations were determined by radioimmunoassays, and transthyretin, alpha(1)-acid glycoprotein (AGP), alpha(1)-antichymotrypsin, C-reactive protein (CRP), haptoglobin, and albumin concentrations by radial immunodiffusion assays. RESULTS: Children in the subsample had high rates of splenomegaly and Plasmodium-positive blood-smear slides (P < 0.01); AGP (Pearson's r = -0.40, P < 0.001) and CRP (r = -0.21, P = 0.04) were inversely correlated with retinol. The negative APPs RBP, transthyretin, and albumin were positively and significantly associated with retinol. All APPs, except alpha(1)-antichymotrypsin, were significantly correlated with splenomegaly. Of the positive APPs, AGP correlated with CRP (r = 0.37, P < 0.001), indicating chronic inflammation. In a stepwise regression analysis, 73% of retinol's variability was explained by RBP and transthyretin. The model predicted that a 1-SD increase in RBP or transthyretin increases retinol by approximately 0.38 or 0.47 micromol/L, respectively, whereas an equivalent increase in AGP decreases retinol by 0.12 micromol/L. CONCLUSIONS: The RBP-transthyretin transport complex of retinol is not altered by inflammation. Positive APPs are useful markers of type and severity of inflammation; however, except for AGP, it is unlikely that they can correct for malaria-induced hyporetinemia.

Priming of a beta-galactosidase (beta-GAL)-specific type 1 response in BALB/c mice infected with beta-GAL-transfected Leishmania major.

To determine whether an ongoing response to Leishmania major would affect the response to a non-cross-reacting, non-leishmanial antigen, susceptible BALB/c mice and resistant C3H mice were infected with L. major parasites expressing Escherichia coli beta-galactosidase (beta-GAL); this parasite was designated L. major-betaGAL. BALB/c and C3H mice responded to infection with L. major-betaGAL by mounting a CD4 T-cell response to both parasite antigens and to the reporter antigen, beta-GAL. The phenotypes of these T cells were characterized after generating T-cell lines from infected mice. As expected, BALB/c mice responded to infection with L. major-betaGAL by producing interleukin 4 in response to the parasite and C3H mice produced gamma interferon (IFN-gamma) in response to the parasite and beta-GAL. Interestingly, however, BALB/c mice produced IFN-gamma in response to beta-GAL. Taken together, these results demonstrate that priming of IFN-gamma-producing cells can occur in BALB/c mice despite the fact the animals are simultaneously mounting a potent Th2 response to L. major.

The influence of zinc supplementation on morbidity due to Plasmodium falciparum: a randomized trial in preschool children in Papua New Guinea.

Zinc is crucial for normal immune function and can reduce morbidity from multiple infectious diseases. To determine the influence of zinc on malaria morbidity we conducted a randomized placebo-controlled trial of daily zinc supplementation in children residing in a malaria endemic region of Papua New Guinea. A total of 274 preschool children aged 6 to 60 months were given 10 mg elemental zinc (n = 136) or placebo (n = 138) for 6 days a week for 46 weeks. Slide-confirmed malaria episodes were detected by surveillance of cases self-reporting to a local health center. Cross-sectional surveys were conducted at the beginning, middle, and end of the study to assess infection rates, parasite density, spleen enlargement, and hemoglobin levels. Zinc supplementation resulted in a 38% (95% CI 3-60, P = 0.037) reduction in Plasmodium falciparum health center-based episodes, defined as parasitemia > or = 9200 parasites/microl with axial temperature > or = 37.5 degreesC or reported fever. Episodes accompanied by any parasitemia were also reduced by 38% (95% CI 5-60, P = 0.028), and episodes with parasitemia > or = 100,000/microl were reduced by 69% (95% CI 25-87, P = 0.009). There was no evidence of the effects of zinc on Plasmodium vivax morbidity or on health center attendance for causes other than P. falciparum. Zinc had no consistent effect on cross-sectional malariometric indices. Although P. falciparum prevalence tended to be lower at the end of the study in children given the placebo, such changes were absent at the mid-study survey. These results suggest that improved dietary zinc intake may reduce morbidity due to P. falciparum.

Effect of vitamin A supplementation on morbidity due to Plasmodium falciparum in young children in Papua New Guinea: a randomised trial.

BACKGROUND: Many individuals at risk of malaria also have micronutrient deficiencies that may hamper protective immunity. Vitamin A is central to normal immune function, and supplementation has been shown to lower the morbidity of some infectious diseases. We investigated the effect of vitamin A supplementation on malaria morbidity. METHODS: This randomised double-blind placebo-controlled trial of vitamin A supplementation took place in a P. falciparum endemic area of Papua New Guinea. Of 520 potentially eligible children aged 6-60 months, 480 were randomly assigned high-dose vitamin A (n=239) or placebo (n=241), every 3 months for 13 months. Malaria morbidity was assessed through weekly community-based case detection and surveillance of patients who self-reported to the health centre. Cross-sectional surveys were also done at the beginning, middle, and end of the study to assess malariometric indicators. Analyses were by intention to treat. FINDINGS: The number of P. falciparum febrile episodes (temperature > or = 37.5 degrees C with a parasite count of at least 8000/microL) was 30% lower in the vitamin A group than in the placebo group (178 vs 249 episodes; relative risk 0.70 [95% CI 0.57-0.87], p=0.0013). At the end of the study P. falciparum geometric mean density was lower in the vitamin A than the placebo group (1300 [907-1863] vs 2039 [1408-2951]) as was the proportion with spleen enlargement (125/196 [64%] vs 148/207 [71%]); neither difference was significant (p=0.093 and p=0.075). Children aged 12-36 months benefited most, having 35% fewer febrile episodes (89 vs 141; relative risk 0.65 [14-50], p=0.0023), 26% fewer enlarged spleens (46/79 [58%] vs 67/90 [74%], p=0.0045), and a 68% lower parasite density (1160 [95% CI 665-2022] vs 3569 [2080-6124], p=0.0054). Vitamin A had no consistent effect on cross-sectional indices of proportion infected or with anaemia. INTERPRETATION: Vitamin A supplementation may be an effective low-cost strategy to lower morbidity due to P. falciparum in young children. The findings suggest that clinical episodes, spleen enlargement, and parasite density are influenced by different immunological mechanisms from infection and anaemia.

PIP: A randomized double-blind placebo-controlled trial was conducted to assess the efficacy of vitamin A supplementation on morbidity due to Plasmodium falciparum among 520 children aged 6-60 months in a malaria-endemic area of Papua New Guinea. Malaria morbidity was assessed through weekly community-based case detection and surveillance of patients who self-reported to the health center. Cross-sectional surveys were also conducted at the beginning, middle, and end of the study to assess malariometric indicators. Laboratory tests were also analyzed for species-specific density. Findings showed that the number of episodes of falciparum malaria among young children in Papua New Guinea was 30% lower in those who received vitamin A than in the placebo recipients. The children, mostly aged 12-36 months, had fewer febrile episodes, fewer enlarged spleens and lower parasite density. No significant differences were observed for hemoglobin concentration or prevalence of anemia for any age group. The findings suggest that clinical episodes, spleen enlargement, and parasite density were influenced by immunological mechanisms that were different from infection and anemia. It also suggests that vitamin A is effective, inexpensive, and a programmatically practical tool in controlling P. falciparum malaria.

Zinc and immune function: the biological basis of altered resistance to infection.

Zinc is known to play a central role in the immune system, and zinc-deficient persons experience increased susceptibility to a variety of pathogens. The immunologic mechanisms whereby zinc modulates increased susceptibility to infection have been studied for several decades. It is clear that zinc affects multiple aspects of the immune system, from the barrier of the skin to gene regulation within lymphocytes. Zinc is crucial for normal development and function of cells mediating nonspecific immunity such as neutrophils and natural killer cells. Zinc deficiency also affects development of acquired immunity by preventing both the outgrowth and certain functions of T lymphocytes such as activation, Th1 cytokine production, and B lymphocyte help. Likewise, B lymphocyte development and antibody production, particularly immunoglobulin G, is compromised. The macrophage, a pivotal cell in many immunologic functions, is adversely affected by zinc deficiency, which can dysregulate intracellular killing, cytokine production, and phagocytosis. The effects of zinc on these key immunologic mediators is rooted in the myriad roles for zinc in basic cellular functions such as DNA replication, RNA transcription, cell division, and cell activation. Apoptosis is potentiated by zinc deficiency. Zinc also functions as an antioxidant and can stabilize membranes. This review explores these aspects of zinc biology of the immune system and attempts to provide a biological basis for the altered host resistance to infections observed during zinc deficiency and supplementation.

Potential contribution of maternal zinc supplementation during pregnancy to maternal and child survival.

Mild-to-moderate zinc deficiency may be relatively common worldwide, but the public health importance of this degree of zinc deficiency is not well defined. The purpose of this review was to provide a conceptual framework for evaluating the public health importance of maternal zinc deficiency as it relates to fetal growth and development, complications of pregnancy, labor and delivery, and maternal and infant health. The mechanisms through which zinc deficiency could influence health outcomes are well described. The results of experimental studies conducted in animal models have motivated concern about the potential health effects of mild-to-moderate maternal zinc deficiency. Observational studies in human populations have produced strong associations between poor maternal zinc status and various indicators of poor pregnancy outcome, but supplementation trials have not produced strong, or even consistent results. Supplementation trials are needed to define the public health importance of maternal zinc deficiency worldwide.

The influence of antigen-presenting cell type and interferon-gamma on priming and cytokine secretion of Leishmania major-specific T cells.

A Leishmania major-specific primary in vitro system that mimics the immune response of infected mice was used to determine the role that dendritic cells, B cells, and macrophages play in L. major T cell priming. Their relative priming potential (in order) was dendritic cells, B cells, and macrophages. Initiating primary in vitro responses with cell populations depleted of either B or dendritic cells modestly enhanced interferon (IFN)-gamma production; deleting both cells markedly enhanced IFN-gamma production. Thus, macrophages were the most effective cell for eliciting L. major Th1 cells. The effects of exogenously added IFN-gamma or neutralizing anti-IFN-gamma were also studied. With cells from genetically susceptible BALB/c mice, IFN-gamma inhibited proliferation and interleukin-4 secretion by T cells, whereas with resistant C57BL/6 cells, IFN-gamma enhanced IFN-gamma secretion. These results could not be explained by differences in IFN-gamma receptor expression.

Leishmania major: differential resistance to infection in C57BL/6 (high interferon-alpha/beta) and congenic B6.C-H-28c (low interferon-alpha/beta) mice.

In murine cutaneous leishmaniasis caused by Leishmania major (Lm), resistance often associates with the outgrowth of Lm-specific Th1 cells. Parasites are eliminated by Th1-mediated activation of infected macrophages (M phi) which destroy Lm by producing toxic nitrogen and oxygen radicals. The cytokine IFN-alpha activates microbicidal functions of M phis and facilitates outgrowth of Th1 cells. Therefore, we compared the course of infection with Lm in resistant C57BL/6 mice, bearing the If-1h high expression allele for IFN-alpha/beta, with the congenic B6.C-H-28c mouse, bearing the If-1I low expression allele from the Lm-susceptible BALB/c strain. We observed that B6.C-H-28c animals developed up to 70% larger footpad lesions and harbored up to 1000-fold more parasites than C57BL/6 mice. Furthermore, peak Lm-specific IFN-gamma production in the B6.C-H-28c animals was lower and delayed by approximately 2 weeks, whereas IL-4 production was higher and persisted approximately 2 weeks longer. Since these results suggested that IFN-alpha/beta plays a protective role in mice infected with Lm, we determined whether infusing B6.C-H-28c mice with IFN-alpha would influence the course of infection with Lm. Unfortunately, the mice developed severe peritoneal hemorrhaging in response to injection with IFN-alpha. Therefore, we examined the ability of IFN-alpha to activate M phis to destroy Lm in vitro. We observed that rIFN-alpha could synergize with subactivating doses of LPS to activate both C57BL/6 and BALB/c peritoneal M phis to produce NO and to kill intracellular Lm. Taken as a whole, these results suggest that type I interferons may play a protective role in cutaneous leishmaniasis.

T cell and non-T cell compartments can independently determine resistance to Leishmania major.

In experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm), the cellular determinants governing development of protective or exacerbative T cells are not well understood. We, therefore, attempted to determine the influence of T cell and non-T cell compartments on disease outcome. To this end, T cell chimeric mice were constructed using adult thymectomized lethally irradiated, bone marrow-reconstituted (ATXBM) animals of genetically resistant, C57BL/6, or susceptible, BALB/c, backgrounds. These hosts were engrafted with naive T cell populations from H-2-congenic susceptible, BALB.B6-H-2b, or resistant, C57BL/6.C-H-2d, animals, respectively. Chimeric mice were then infected with Lm, and disease outcome was monitored. BALB/c T cell chimeric mice, BALB/c ATXBM hosts given naive C57BL/6.C-H-2d T cells, resolved their infections as indicated by reductions in both lesion size and parasite numbers. Furthermore, the mice developed typical Th1 (interferon[IFN]-gamma hiinterleukin[IL]-4lo) cytokine patterns. In contrast, both sham chimeric, BALB/c ATXBM hosts given naive BALB/c T cells, and control irradiated euthymic mice succumbed to infection, producing Th2 profiles (IFN-gamma loIL-4hiIL-10hi). C57BL/6 T cell chimeras, C57BL/6 ATXBM hosts given naive BALB.B6-H-2b T cells, resolved their infections as did C57BL/6 sham chimeras and euthymic controls. Interestingly, whereas C57BL/6 control animals produced Th1 cytokines, chimeric animals progressed from Th0 (IFN-gamma hiIL-4hiIL-10hi) to Th2 (IFN-gamma loIL-4hiIL-10hi) cytokine profiles as cure ensued. Both reconstitution and chimeric status of all mice were confirmed by flow cytometry. In addition, T cell receptor V beta usage of Lm-specific blasts was determined. In all cases, V beta use was multiclonal, involving primarily V beta 2, 4, 6, 8.1, 8.2, 8.3, 10, and 14, with relative V beta frequencies differing between H-2b and H-2d animals. Most importantly, however, these differences did not segregate between cure and noncure outcomes. These findings indicate that: (a) genetic traits determining cure in Lm infection can direct disease outcome from both T cell and non-T cell compartments; (b) the presence of the curing genotype in only one compartment is sufficient to confer cure; (c) curing genotype T cells autonomously assume a Th1 cytokine profile-mediating cure; (d) noncuring genotype T cells can mediate cure in a curing environment, despite the onset of Th2 cytokine production; and lastly, (e) antigen specificity of responding T cells, as assessed by V beta T cell receptor diversity, is not a critical determinant of disease outcome.

Leishmania major-specific, CD4+, major histocompatibility complex class II-restricted T cells derived in vitro from lymphoid tissues of naive mice.

Several studies indicate that the outcome of experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm) is determined by immunological events occurring shortly after infection. These events lead to outgrowth of either protective CD4+ T cells in the C57BL/6 mouse, which cures, or exacerbative cells in the BALB/c mouse, which succumbs to disease. Potential factors influencing the outgrowth of protective or exacerbative T cells include antigen-presenting cells (APC), cytokines, and parasite antigens. An in vitro system, in which one could precisely control the factors shaping early events in the T cell response to Lm, would be very useful. To this end, we have examined the in vitro response of naive lymphocytes to Lm promastigotes. The data presented here show that Lm-specific CD4+ T cell receptor alpha/beta + T cells can be generated in vitro from spleen and lymph node cell populations of naive mice. Furthermore, they can be obtained from the CD44low (unprimed) population of T lymphocytes, indicating that in vitro priming occurs. The ability to generate these T cells is dependent on the presence of live parasites and is not due to a parasite-derived nonspecific T cell mitogen. Restimulation, as assayed by proliferation, requires APC bearing syngeneic I-A. Optimal restimulation of the in vitro derived T cells is achieved only when live promastigotes are used. The T cells do not proliferate in response to a frozen-and-thawed lysate of promastigotes, yet they exhibit mild reactivity to lysates prepared from heat-shocked promastigotes. Furthermore, they do not recognize two predominant antigens on the promastigote surface, lipophosphoglycan and gp63. T cells derived in vitro with Lm show crossreactivity with live L. donovani, less crossreactivity with live L. mexicana, and no crossreactivity with live Bacillus-Calmette-Guerin or live Brugia malayi microfilariae. Finally, these early T cells, whether derived from healing C57BL/6 or nonhealing BALB/c mice, produce interleukin 2 (IL-2), IL-4, and interferon gamma.