Iron transport across the human placenta is regulated by hepcidin.

BACKGROUND: Transport of iron across the placenta is critical for appropriate development of the fetus. Iron deficiency during pregnancy remains a major public health concern, particularly in low- and middle-income countries, often exacerbated by infectious diseases leading to altered iron trafficking via inflammatory responses. Herein, we investigate the role of hepcidin, a master regulator of iron homeostasis, on regulation of iron transport across trophoblast cells. METHODS: We utilized the Jeg-3 choriocarcinoma cell line for analysis of the expression of transferrin receptor, ferritin, and ferroportin as well as the export of 59Fe in the presence of hepcidin. Placental tissue from human term pregnancies was utilized for immunohistochemistry. RESULTS: Hepcidin treatment of Jeg-3 cells decreased the expression of ferroportin and transferrin receptor (TfR) and reduced the cellular export of iron. Lower expression of TfR on the syncytiotrophoblast was associated with the highest levels of hepcidin in maternal circulation, and ferroportin expression was positively associated with placental TfR. Placentas from small-for-gestational-age newborns had significantly lower levels of ferroportin and ferritin gene expression at delivery. CONCLUSIONS: Our data suggest that hepcidin plays an important role in the regulation of iron transport across the placenta, making it a critical link in movement of iron into fetal circulation. IMPACT: Hepcidin has a direct impact on iron transport across the human placenta. This study provides the first evidence of direct regulation of iron efflux from human trophoblast cells by hepcidin. These data extend our understanding of iron transport across the maternal-fetal interface, a process critical for fetal health and development.

Developing a novel risk prediction model for severe malarial anemia.

As a pilot study to investigate whether personalized medicine approaches could have value for the reduction of malaria-related mortality in young children, we evaluated questionnaire and biomarker data collected from the Mother Offspring Malaria Study Project birth cohort (Muheza, Tanzania, 2002-2006) at the time of delivery as potential prognostic markers for pediatric severe malarial anemia. Severe malarial anemia, defined here as a Plasmodium falciparum infection accompanied by hemoglobin levels below 50 g/L, is a key manifestation of life-threatening malaria in high transmission regions. For this study sample, a prediction model incorporating cord blood levels of interleukin-1ß provided the strongest discrimination of severe malarial anemia risk with a C-index of 0.77 (95% CI 0.70-0.84), whereas a pragmatic model based on sex, gravidity, transmission season at delivery, and bed net possession yielded a more modest C-index of 0.63 (95% CI 0.54-0.71). Although additional studies, ideally incorporating larger sample sizes and higher event per predictor ratios, are needed to externally validate these prediction models, the findings provide proof of concept that risk score-based screening programs could be developed to avert severe malaria cases in early childhood.

The effect of weekly iron and vitamin A supplementation on hemoglobin levels and iron status in adolescent schoolgirls in western Kenya.

BACKGROUND/OBJECTIVES: Iron deficiency anemia is a major public health problem in developing countries and may affect school performance and physical work capacity in nonpregnant adolescents, and may increase the risk of anemia during subsequent teenage pregnancies. We assessed the effect of weekly iron (120 mg elemental iron) and vitamin A (25 000 IU) supplementation on hemoglobin, iron status and malaria and nonmalaria morbidity in adolescent schoolgirls. SUBJECTS/METHODS: A total of 279 schoolgirls aged 12-18 years from public primary schools in Kisumu, western Kenya. Double-blind randomized placebo-controlled trial using a factorial design. RESULTS: Five months of iron supplementation was associated with a 0.52 g dl(-1) (0.21, 0.82) greater increase in hemoglobin relative to iron placebo. The effect was only observed in girls with iron deficiency on enrollment (1.34 g dl(-1) (0.79, 1.88)), but not in iron-replete girls (-0.20 g dl(-1) (-0.59, 0.18)). Similar differences in treatment effect were seen between menstruating and nonmenstruating girls. The effect of iron was independent of vitamin A. The baseline prevalence of vitamin A deficiency was low (6.7%) and no sustained increase in hemoglobin was seen with weekly vitamin A (-0.07 g dl(-1) (-0.38, 0.25)). Incidence of malaria parasitemia was higher in the iron than iron-placebo groups (Rate ratio 1.33 (0.94, 1.88)). CONCLUSIONS: Weekly iron supplementation results in substantial increases in hemoglobin concentration in adolescent schoolgirls in western Kenya, which may outweigh possible risks caused by malaria, but only in iron-deficient or menstruating girls and not in iron-replete and nonmenstruating girls.

Th2 cytokines are associated with persistent hepatic fibrosis in human Schistosoma japonicum infection.

We conducted a prospective cohort study in Leyte, the Philippines, among 611 Schistosoma japonicum-infected participants 7-30 years old, all of whom were treated with praziquantel at baseline. To detect hepatic fibrosis, abdominal ultrasound was performed at baseline and 12 months after treatment. Stool for assessment of S. japonicum infection was collected at baseline and at 3, 6, 9, and 12 months after treatment. Cytokines (interleukin [IL]-4, IL-5, IL-10, IL-13, tumor necrosis factor- alpha , and interferon- gamma ) produced by peripheral-blood mononuclear cells in response to soluble worm antigen preparation (SWAP), soluble egg antigen (SEA), and control medium were measured once 4 weeks after treatment. IL-4 to SWAP and IL-10 to both SWAP and SEA were associated with the presence of baseline fibrosis after adjustment for potential confounding variables (P<.03, for all). In participants with fibrosis at baseline, IL-4 to SWAP and IL-5 and IL-13 to both SWAP and SEA were associated with persistent fibrosis at 12 months after treatment (P<.05, for all). Males showed consistently stronger T helper 2 (Th2) cytokine responses to both SWAP and SEA than did females (P<.02, for all). These results suggest an independent role for Th2-biased cytokine responses to S. japonicum antigens in persistent hepatic fibrosis and indicate that Th2 cytokines may contribute to the male-biased prevalence of fibrosis.

Protective immunity induced by phage displayed mitochondrial related peptides of Schistosoma japonicum.

New antigens and strategies are necessary for vaccine development against schistosomiasis japonica. Using a pool of 43 high titred anti-SWA sera from individuals residing in an Schistosoma japonicum endemic area of China, we have cloned a S. japonicum gene by cDNA library screening. The recombinant Sj338 protein has 44-46% identity to a mitochondrial precursor receptor protein of humans and rats. Immunization of mice with the recombinant Sj338 conferred 27-32% (p<0.01) reduction in worm burdens following cercarial challenge. In an effort to identify protective epitopes in Sj338 and increase the level of protection, we screened a random 12-mer peptide library constructed in M13 using a polyspecific anti-Sj338 rabbit serum. After five rounds of biopanning, we identified 30 reactive clones consisting of 11 distinct peptide sequences. These clones shared limited primary sequence homology with the recombinant Sj338 protein. Anti-sera raised against these phage clones recognized recombinant Sj338 and SWAP by Western blot. In murine vaccination experiments using whole recombinant phage without adjuvant, four of these clones demonstrated worm reductions of 11.6-25.1% (p=ns - 0.05) compared to M13 vaccinated animals. Animals vaccinated with all four of these phage demonstrated 34.2% (p<0.01) worm reduction compared to controls vaccinated with M13 clone. These data suggest that mimotope peptides are potential vaccine candidates for S. japonicum.

Prevalence and severity of anemia and iron deficiency: cross-sectional studies in adolescent schoolgirls in western Kenya.

OBJECTIVE: Anemia is a major public health concern in preschool children and pregnant women in the developing world. While many studies have examined these two at-risk groups, there is a paucity of data on anemia in adolescents living in developing countries in the complex ecologic context of poverty, parasitism, and malnutrition. We evaluated the prevalence, severity, and risk factors of anemia in adolescent schoolgirls in an area with intense malaria transmission in western Kenya. DESIGN: Two cross-sectional surveys were conducted, using a multistage random sample design. SETTING: Public primary schools in an area with intense malaria transmission in western Kenya. SUBJECTS: A total of 648 randomly selected adolescent schoolgirls aged 12-18 y. RESULTS: The prevalence of anemia (Hb <120 g/l) was 21.1%; only one girl had an Hb less than 70 g/l. Ferritin levels were available from a subsample of 206 girls. The prevalence of iron deficiency (ferritin <12 microg/l) was 19.8, and 30.4% of anemic girls were iron deficient. Malaria and schistosomiasis were the main risk factors for anemia in younger girls (12-13 y), while menstruation was the principal risk factor in older girls (14-18 y). CONCLUSIONS: Iron deficiency and anemia in school-attending girls in western Kenya were more prevalent than in developed countries, but considerably less prevalent than in preschool children and pregnant women from the same study area. Our findings are consistent with other recent school-based surveys from western Kenya, but not with recent community- and school-based cross-sectional surveys from other parts of sub-Saharan Africa. It deserves further study to determine if adolescent girls not attending school are at higher risk of anemia.

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine.

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.

Comparison of self-reported and observed water contact in an S. mansoni endemic village in Brazil.

Estimates of exposure are critical for immuno-epidemiologic and intervention studies in human schistosomiasis. Direct observation of human water contact patterns is both costly and time consuming. To address these issues, we determined whether individuals residing in a Schistosoma mansoni endemic village in Brazil could accurately self-report their water contact patterns. We compared the results of a water contact questionnaire to the present gold standard, direct observation of water contact in 86 volunteers, aged 8--29. We administered a survey to estimate volunteers' frequency and type of water contact and directly measured each volunteers' water contact patterns during 5 weeks of detailed water contact observations. We found a poor correlation between self reported frequency of contact and directly observed exposure (rho=0.119, P=NS). The questionnaire data was supplemented by information about average body surface area of exposure and duration of contact for specific activities derived from observations of this cohort. This 'supplemented questionnaire' data was significantly correlated with their exposure index (rho=0.227, P=0.05). It provides a starting point from which questionnaires may develop to provide a more cost-effective and less labor intensive method of assessing water contact exposure at the level of the individual.

Human resistance to Plasmodium falciparum increases during puberty and is predicted by dehydroepiandrosterone sulfate levels.

Immunity to Plasmodium falciparum develops slowly in areas of endemicity, and this is often ascribed to poorly immunogenic or highly variant parasite antigens. However, among populations newly exposed to malaria, adults acquire immunity more rapidly than children. We examined the relationship between pubertal development and resistance to P. falciparum. During two transmission seasons in western Kenya, we treated the same cohort of young males to eradicate P. falciparum and then obtained blood smears each week for 4 months. We determined pubertal development by Tanner staging and by levels of dehydroepiandrosterone sulfate (DHEAS) and testosterone in plasma. In multivariate and age-stratified analyses, we examined the effect of pubertal development on resistance to malaria. In both seasons (n = 248 and 144 volunteers, respectively), older males were less susceptible than younger males. Age-related decreases in the frequency and density of parasitemia were greatest during puberty (15- to 20-year-olds). DHEAS and testosterone were significant independent predictors of resistance to P. falciparum parasitemia, even after accounting for the effect of age. Fifteen- to 20-year-old males with high DHEAS levels had a 72% lower mean parasite density (P<0.01) than individuals with low DHEAS levels. Similarly, 21- to 35-year-old males with high DHEAS levels had a 92% lower mean parasite density (P<0.001) and 48% lower frequency of parasitemia (P<0.05) than individuals with low DHEAS levels. These data suggest that the long period needed to attain full immunity could be explained as a consequence of host development rather than as the requirement to recognize variant or poorly immunogenic parasite antigens.

Evaluation of mild-to-moderate dysplasia on cervical-endocervical (Pap) smear: a subgroup of patients who bridge LSIL and HSIL.

The Bethesda System recommends Pap smear diagnosis to be based on the most abnormal cells present, regardless of number. Our reporting system includes a diagnostic category of mild-to-moderate dysplasia (D1-2), defined as cases with only a few moderately dysplastic cells. We evaluated the validity of a D1-2 diagnostic category by reevaluation of 58 cases with subsequent follow-up (up to 24 months). On biopsy and/or Pap smear follow-up, 24 cases (41%) showed LSIL and 34 cases (59%) showed HSIL. Index smears from these cases were examined by two cytopathologists blinded to patient follow-up for the following morphologic features: volumes (scale 1-4) of LSIL, HSIL, and dyskeratosis, HSIL as single cells or syncytial fragments, and acute inflammation. None of the morphologic features evaluated were significantly different between the LSIL and HSIL follow-up groups based on univariate and multivariate logistic regression analysis. The diagnosis of D1-2 on Pap smear is a valid diagnostic category that defines a subgroup of patients with both LSIL and HSIL follow-up, which cannot be reliably predicted based on morphology alone.

Interleukin-10 responses to liver-stage antigen 1 predict human resistance to Plasmodium falciparum.

The design of an effective vaccine against Plasmodium falciparum, the most deadly malaria parasite of humans, requires a careful definition of the epitopes and the immune responses involved in protection. Liver-stage antigen 1 (LSA-1) is specifically expressed during the hepatic stage of P. falciparum and elicits cellular and humoral immune responses in naturally exposed individuals. We report here that interleukin-10 (IL-10) production in response to LSA-1 predicts resistance to P. falciparum after eradication therapy. Resistance was not related to gamma interferon or tumor necrosis factor alpha production. This is the first report that human IL-10 responses are associated with resistance after eradication therapy, and our findings support the inclusion of LSA-1 in a vaccine against malaria.

Identification of the Schistosoma japonicum 22.6-kDa antigen as a major target of the human IgE response: similarity of IgE-binding epitopes to allergen peptides.

Human resistance to reinfection with Schistosoma mansoni and Schistosoma haematobium correlates with elevated IgE titers against worm antigens (soluble worm antigen preparation, SWAP). In S. mansoni infection, low levels of reinfection following chemotherapy are associated with the recognition of a cloned tegumental protein Sm22.6. Because of potential species-specific differences in resistance to schistosomes, we attempted to identify Schistosoma japonicum antigens recognized by human IgE. Following a survey of 176 infected individuals in Leyte, Philippines, we show that IgE antibodies from the majority of older, high-IgE/SWAP responders recognize antigens in the 22 (Sj22)-, 45-, 78- and 97-kDa range in SWAP. Limited IgE cross-reactivity between Sj22 and Sm22 was observed following a comparison of Filipino IgE responses to these antigens. The antigen was cloned from an adult S. japonicum lambda-ZAP cDNA library (Mindoro strain) by immunoscreening with pooled high-titer IgE antisera and a rabbit anti-Sj22 polyclonal antibody. The deduced amino acid sequence of the identified cDNA clone, MJ-1, showed significant homology to Sm22.6 (74%) and Sj22.6 (99%). Although the molecular sequence of Sj22.6 has already been reported, this is the first demonstration of its recognition by human IgE, thereby strengthening its potential as a vaccine candidate. Using an overlapping peptide approach, four IgE-binding epitopes were identified in Sj22.6, two of which exhibited similarities to known IgE-binding epitopes from codfish (Gad c 1) and beta-lactoglobulin-related allergens. These findings suggest that allergy and protective immunity to helminth infection may be linked by the structural similarities of epitopes recognized by human IgE.

Identification and molecular cloning of a 67-kilodalton protein in Schistosoma japonicum homologous to a family of actin-binding proteins.

A monoclonal antibody to Schistosoma japonicum which conferred significant protection against cercarial challenge in mice was produced. The predicted translation product of the cDNA corresponding to the antigen recognized by this antibody was homologous to a newly identified family of actin-binding proteins. The expressed protein bound polymerized actin and was recognized by serum from patients infected with S. japonicum.

Paramyosin: a candidate vaccine antigen against Schistosoma japonicum.

Paramyosin, a 97 kDa myofibrillar protein, is a candidate vaccine antigen for prevention of infection with the human parasite Schistosoma mansoni. To determine if paramyosin would also induce protection against Schistosoma japonicum, paramyosin was biochemically purified from S. japonicum adult worms. SDS-PAGE demonstrated a single protein with a molecular weight of 97 kDa. In four separate experiments, vaccination of mice with S. japonicum paramyosin without adjuvant induced significant resistance (62%-86%, P < 0.001) against cercarial challenge as compared to controls. These data suggest that S. japonicum paramyosin may represent a candidate vaccine for immunization against schistosomiasis japonica.

Gene cloning and complete nucleotide sequence of philippine Schistosoma japonicum paramyosin.

The development of an effective vaccine is recognised as a necessary adjunct to the control of schistosomiasis japonica, a disease affecting several million people in China and the Philippines. Currently, recombinant Schistosoma japonicum molecules are considered most suitable for large scale vaccine production and a number of genes encoding vaccine candidate polypeptides have been cloned and expressed (see Waine et al., 1993a). One of the molecules providing most promise as a vaccine target is paramyosin (Butterworth, 1992), a major structural protein of thick filaments in the muscle of most invertebrates; paramyosin genes have now been cloned from a range of parasitic helminths, including schistosomes (Limberger and McReynolds, 1990; Laclette et al., 1991; Dahmen et al., 1993; Landa et al., 1993; Mühlschlegel et al., 1993, Nara et al., 1994). The cloning and nucleotide sequence of S. Japonicum paramyosin is described.