Nutritional anaemia and malaria in preschool and school age children.

BACKGROUND: The most common cause of anemia is a deficiency of iron; but it may also be caused by deficiencies of folates, vitamin B12 and protein. Some anemias are not caused by nutritional factors, but by congenital factors and parasitic diseases such as malaria. This study attempted to estimate the prevalence of anemia among pre-school and school- aged children in two rural areas of Odogbolu Local government area, and to determine whether its cause was nutritional or could be attributed to malaria. METHODS: A total of 177 children between the ages of 2 and 11 years were included in the study. Children were examined for malaria parasites by microscopy. The World Health Organization (WHO) age-adjusted cut-off for hemoglobin and hematocrit were used to classify anemia. An enzyme linked immunosorbent assay for serum ferritin was compared with standard methods of determining iron deficiency. Under-nutrition (stunting, wasting and underweight) was classified according to the National Centre for Health Statistics standards. Values below-2SD were defined as mild-moderate under-nutrition, and those below-3SD as severe malnutrition. RESULTS: Most of the children were anemic, 87.1%, having PCV values below the 32% cut-off and 95% with hemoglobin levels lower than the 11 g/dl, although parasite prevalence and density were low. Malnutrition was patent; 36% of the children were stunted, 18.3% wasted and 44.2% underweight. Serum ferritin was more sensitive than PCV in detecting anemic children. Although anemia was higher in boys and preschoolers compared to girls and school aged children, the difference was significant only in preschoolers (P? = ?.004). Anaemia was also significantly higher in Irawo village school than in Iloti (P? = ?.0001) CONCLUSION: The anemia detected in this population may be due more to under-nutrition than to malaria.

Variation in the relationship between anti-MSP-1(19) antibody response and age in children infected with Plasmodium falciparum during the dry and rainy seasons.

Malaria remains a major parasitic disease in Africa, with 300-500 million new infections each year. There is therefore an urgent need for the development of new effective measures, including vaccines. Plasmodium falciparum merozoite surface protein-1(19) (MSP-1(19)) is a prime candidate for a blood-stage malaria vaccine. Blood samples were collected from children aged 10 days to 15 years in the months of January-March (N = 351) and October-November (N = 369) corresponding to the dry and rainy seasons, respectively. P. falciparum infection was determined by microscopy and enzyme linked immunosorbent assay (ELISA) was used to determine the total IgG and IgG subclasses. There was a significant increase in the mean anti-MSP-1(19) antibody titre in the dry season (p < 0.05), compared to the rainy season. A significantly positive correlation between the anti-MSP-1(19) antibody titre and parasite density (p < 0.01, r = 0.138) was observed. In the rainy season, unlike in the dry season, P. falciparum positive children had higher anti-MSP-1(19) antibody titres than P. falciparum negative children and this difference was significant (p < 0.05). When all individuals were grouped together, the anti-MSP-1(19) antibody titre increased with age in both seasons (r = 0.186 and 0.002), this increase was more apparent in the dry season. However, when the study population was divided into P. falciparum positive and negative groups, it was observed that in the rainy season, there was a negative correlation between anti-MSP-1(19) titre and age in P. falciparum positive individuals, while those who were P. falciparum negative had a positive correlation between anti-MSP-1(19) titre and age. Analysis of anti-MSP-1(19) IgG subclass showed that IgG1 and IgG3 mean titres were highest in both the dry and rainy seasons with an increase in the mean antibody titres for IgG1, IgG2 and IgG3 in the rainy season. In the dry season there was a positive correlation between IgG1, IgG2, and IgG3 titres with age, while IgG4 was negative, whereas in the rainy season there was a positive correlation between IgG2 and IgG4 (non-cytophilic antibodies) with age and a negative correlation for IgG1 and IgG3 (cytophilic antibodies) with age. Seasonal differences in the level of MSP-1(19) IgG subclass titres were observed for P. falciparum negative and positive individuals. Only samples, which were positive for IgG2 and IgG4, showed positive correlation between parasitemia and total IgG. The incidence of P. falciparum infection, which increases during the rainy season, might be an important determinant of anti-MSP-1(19) antibody levels in children living in Igbo-Ora and the results point to the fact that non-cytophilic antibodies to MSP-1(19) in children might be associated with an increase in total IgG and parasitemia.

Identification of a subpopulation of immune Nigerian adult volunteers by antibodies to the circumsporozoite protein of Plasmodium falciparum.

Collections of human sera from malaria-endemic areas would be valuable for identifying and characterizing antigens as malaria vaccine candidates if the contributing serum donors' ability to resist infection were fully characterized. We prepared such a serum collection from 26 apparently immune Nigerian adults who failed to develop patent parasitemia for at least 20 weeks following a documented increase in antibodies to the circumsporozoite protein (CSP) from Plasmodium falciparum. Volunteers were evaluated five times per week for malaria symptoms and bimonthly for parasites by examining thick blood smears. The incidence rate over 13 months for the cohort was 42% (47 malaria-confirmed volunteers) and the risk of infection was 1.3 infections/year. Responses to CSP did not correlate with protection. Because antibody responses to antigens other than CSP may be associated with protection, the sera from these immune individuals may be useful for identifying and characterizing other potential malaria vaccine candidates.

Neurogenic vasodilation in rabbit basilar isolated artery: involvement of calcitonin-gene related peptide.

Neurogenic vasodilation in cranial arteries may be an important mechanism in the pathogenesis of migraine headache. We describe a novel, in vitro assay to characterise neurogenic vasodilator responses in endothelium-denuded segments of rabbit isolated basilar artery, with particular focus on calcitonin-gene related peptide (CGRP). In arterial segments precontracted with prostaglandin F(2alpha), relaxations evoked by exogenously applied alphaCGRP (EC(50)=2.9 nM) were inhibited by alphaCGRP-(8-37) (pA(2)=6.49) or by desensitisation resulting from prior exposure to alphaCGRP. Relaxations evoked by exogenously applied vasoactive intestinal polypeptide (VIP) (EC(50)=2.5 nM) were inhibited by VIP-(7-28) 1 microM. The 5-HT(1) receptor agonists L-771,331 ((3S)-3[N-(S)-alpha-methylbenzyl]aminomethyl-(S)-1-[2-(5-(2-oxo-1, 3-oxazolidin-4-ylmethyl)-1H-indol-3-yl)ethyl]pyrrolidine) and sumatriptan exerted contractile effects (EC(50)=293 and 95 nM, respectively). In neurogenic experiments, vasodilation evoked by electrical field stimulation was markedly attenuated by pre-treatment with capsaicin (10 microM) or by prior CGRP receptor desensitisation and to a lesser extent by pre-treatment with VIP-(7-28) 1 microM. L-771,331 (100 nM) exerted a weak inhibitory effect, marked only by a short reduction in the recovery time (post-electrical stimulation) and sumatriptan (30 nM) had no effect. The neurogenic response was potentiated by alphaCGRP-(8-37) 1 microM (reversible on wash-out). Short application (5-10 min) of capsaicin (10 microM) produced vasodilation that was inhibited by alphaCGRP-(8-37) 1 microM. These data suggest that electrically evoked neurogenic vasodilation in rabbit basilar artery has a large component resulting from the release of sensory neuropeptides in particular CGRP and a smaller component involving the release of VIP.

Trypanosoma (Nannomonas) congolense: properties of hexokinase and phosphofructokinase from cultured procyclic trypomastigotes and bloodstream forms.

The distribution and kinetics of two key glycolytic enzymes hexokinase (HK) and phosphofructokinase (PFK) were studied in animal-infective bloodstream forms (haematozoic trypomastigotes) and uninfective procyclic forms (insect trypomastigotes) of Trypanosoma congolense. The results show that in both forms of T. congolense HK and PFK are particulate and are probably localized in a membrane-delimited organelle, the glycosome. Hexokinases of bloodstream and procyclic forms of T. congolense are kinetically similar with respect to their affinity for glucose and ATP, the apparent Km for glucose being within the range, of 91 microM to 100 microM and that for ATP, 65 microM to 91 microM. Phosphofructokinase of both forms responds to its substrate in a complex manner: a plot of initial velocity versus substrate concentration displays intermediary plateau regions.

Regulation of glycolysis in Trypanosoma brucei: hexokinase and phosphofructokinase activity.

The kinetic properties of the glycosomal hexokinase (HG)2 and phosphofructokinase (PFK) from Trypanosoma brucei bloodstream forms were investigated. Hexokinase has a very high affinity for glucose (Km = 17 microM) and exhibits a broad pH optimum with a maximum at pH 7.8. No indications have been found for regulation of HK activity. Phosphofructokinase behaves as an allosteric protein with respect to its substrate, fructose-6-phosphate. 5'-AMP acts as a positive allosteric effector. The apparent Km for 5'-AMP is extremely low (7 microM). The other substrate for PFK is Mg2+-ATP chelate which activates the enzyme in a hyperbolic manner. Excess of ATP over Mg2+ is inhibitory. The enzyme needs Mg2+ for full activity. Compounds known to be positive or negative heterotrophic modifiers of PFK in other organisms are without effect. It is concluded that PFK and HK probably do not play a regulatory role in glycolysis in T. brucei.

Impaired growth and increased glucocorticoid-sensitive enzyme activities in tissues of rat fetuses exposed to maternal low protein diets.

Epidemiological evidence that hypertension and coronary heart disease are programmed by exposure to poor diet during intrauterine life, is supported by animal experiments. In the rat, fetal exposure to a maternal low protein diet is associated with abnormal fetal growth and later elevation of blood pressure. Fetal exposure to glucocorticoids of maternal origin are proposed to underlie this association. Pregnant female rats were fed control (18% casein) or moderately low protein diets (9% casein). Feeding of low protein was either throughout gestation (d0-22), or for specific periods (d0-7, d8-14, d15-22). Fetal and placental weight were determined at d14, 20 and 22. Low protein feeding in the periods d0-7, d8-14, d0-14 stimulated fetal growth to d14. At d20 gestation low protein exposed fetuses tended to be smaller than control fetuses, although low protein d8-14 fetuses were significantly larger than controls. Animals exposed to low protein diets were of lower weight at birth and had higher blood pressure at 4 weeks postnatal age. The activities of glucocorticoid-inducible enzymes in brain (fetal and neonatal) and liver (neonatal) were specifically elevated relative to control animals, by low protein exposure. The data suggest that low protein exposure, particularly in late gestation is associated with increased fetal glucocorticoid-exposure. This may both retard fetal growth and programme later increases in systolic blood pressure.

Platelet-aggregating activity of released factor(s) from Trypanosoma brucei brucei.

The effect of factors derived from Trypanosoma brucei brucei on rat platelets was studied. T. brucei at a concentration of 4 X 10(9) trypanosomes/ml phosphate saline glucose (PSG) was stored at -20 degrees C for 18 h, thawed, and a supernatant fraction, trypanosome-derived supernatant (TDS) was obtained by spinning the sample at 3000 g for 10 min at 20 degrees C. Normal rat platelets, prepared as platelet-rich plasma (PRP), were then incubated with TDS in the absence or presence of ADP (0.05-0.1 microM). The results showed that approximately 83% platelet aggregation was induced by addition of TDS (50 microliters; 113 micrograms protein) to 100 microliters PRP with a platelet count of 10(6). simultaneous addition of ADP and TDS to PRP produced a synergistic effect. It was also shown that a supernatant fraction, obtained by incubating live T. brucei (4 X 10(9)/microliters PSG) at 0 degrees C 1 h and spinning down the trypanosomes (3000 g for 10 min), also induced platelet aggregation. The nature of the factor(s) derived from, or released by, T. brucei inducing platelet aggregation is being investigated but it has been shown not to be ADP.

Evaluation of a rapid immunochromatographic card test for Plasmodium falciparum in Ibadan, Nigeria.

This short report describes the results of a rapid, simple and cost effective immunodiagnostic test for malaria in Ibadan, Nigeria. A total of 77% patients presenting at the children outpatient clinic, University College Hospital with malaria symptoms were screened for malaria parasites by microscopy using Giemsa stain and by the immunochromatographic card test. The immunodiagnostic test had a sensitivity of 93.1% and a specificity of 95.8%, making a good alternative for malaria diagnosis especially in rural areas without electricity, where microscopy is not possible, and a decision is to be made on when to start treatment.

Clinical manifestations and immune response to MSP 1(19) in severe paediatric malaria in Adeoyo state maternity hospital, Ibadan.

A 10-week cross-sectional study was carried out at the Adeoyo State Maternity Hospital (Beere, Ibadan), Southwestern Nigeria in order to determine (a) the prevalence of severe malaria, (b) identify the predominant clinical presentations that characterise the disease in children below 5 years and the pattern of antibody responses to MSP 19 elicited in severe malaria complications. Three thousand, one hundred and thirty-one cases reported to the Out Patients' Department; of these, 372 (11.8%) subjects were recruited on the basis of doctors' diagnosis of severe malaria, malaria and other complications. Six per cent (188/3131) ofthe patients were admitted. Serum samples for 320 ofthe 372 subjects were analysed for antibodies specific to MSP 1(19) by ELISA. The highest antibody responses occurred in the age group 2-5 years. Parasite prevalence was 77.9% (290 of 372 subjects) and parasite density ranged from 80 to >100000 parasites/microL blood. Fever (an average temperature of 38.6 +/- 0.4 degrees C and peak at 41 degrees C) and severe malaria were the major clinical manifestations of malaria amongst the study population. Severe malaria was found to be associated with other features such as cough, vomiting and diarrhoea.

Total immunoglobulin G and IgG1 subclass levels specific for the MSP-1(19) of Plasmodium falciparum are different in individuals with either processing-inhibitory, blocking or neutral antibodies.

BACKGROUND: Some MSP-1(19) specific antibodies that inhibit merozoite invasion also inhibit the secondary processing of MSP-1. However the binding of these inhibitory antibodies can be blocked by another group of antibodies, called blocking antibodies, which recognize adjacent or overlapping epitopes, but themselves have no effect on either MSP-1 processing or merozoite invasion. These antibodies have been reported to be present in individuals living in a malaria endemic area. METHODS: Blood samples were obtained from children shown to have processing inhibitory, blocking, and neutral antibodies in a previous study. Enzyme linked immunosorbent assay (ELISA), was used to determine the total IgG, IgM and IgG subtypes. RESULTS: There was a significant difference in anti-MSP-1(19) IgG, while there was no significant difference in the anti-MSP-1(19) IgM. Only anti MSP-1(19) IgG1, amongst the IgG subtypes was significantly different between the groups. CONCLUSION: This study shows that antibodies against MSP-1 are different not only in specificity and function but also in the amount of total IgG and IgG subtype produced.

Antibody specificities of children living in a malaria endemic area to inhibitory and blocking epitopes on MSP-1 19 of Plasmodium falciparum.

Merozoite surface protein-1(19) (MSP-1(19)) specific antibodies which include processing inhibitory, blocking and neutral antibodies have been identified in individuals exposed to Plasmodium falciparum. Here we intend to look at the effect of single and multiple amino acid substitutions of MSP-1(19) on the recognition by polyclonal antibodies from children living in Igbo-Ora, Nigeria. This would provide us with information on the possibility of eliciting mainly processing inhibitory antibodies with a recombinant MSP-1(19) vaccine. Blood was collected from children in the rainy season and binding of anti-MSP-1(19) antibodies to modified mutants of MSP-1(19) was analysed by ELISA. The MSP-1(19) mutant proteins with single substitutions at positions 22 (Leu-->Arg), 43 (Glu-->Leu) and 53 (Asn-->Arg) and the MSP-1(19) mutant protein with multiple substitutions at positions 27+31+34+43 (Glu-->Tyr, Leu-->Arg, Tyr-->Ser, Glu-->Leu); which had inhibitory epitopes; had the highest recognition. Children recognised both sets of mutants with different age groups having different recognition levels. The percentage of malaria positive individuals (32-80%) with antibodies that bound to the mutants MSP-1(19) containing epitopes that recognise only processing inhibitory and not blocking antibodies, were significantly different from those with antibodies that did not bind to these mutants (21-28%). The amino acid substitutions that abolished the binding of blocking antibodies without affecting the binding of inhibitory antibodies are of particular interest in the design of MSP-1(19) based malaria vaccines. Although these MSP-1(19) mutants have not been found in natural population, their recognition by polyclonal antibodies from humans naturally infected with malaria is very promising for the future use of MSP-1(19) mutants in the design of a malaria vaccine.

Atypical development of Sertoli cells and impairment of spermatogenesis in the hypogonadal (hpg) mouse.

Testes of hypogonadal (hpg) mice show arrested postnatal development due to congenital deficiencies of gonadotrophin-releasing hormone (GnRH) and gonadotrophin synthesis and secretion. Follicle-stimulating hormone (FSH), androgen or oestrogen treatment restore qualitatively normal spermatogenesis in hpg testes. Understanding the cellular and molecular changes accompanying hormone-induced spermatogenesis in hpg mice requires detailed morphological analyses of the germ cells and Sertoli cells in the untreated hpg testis. We compared seminiferous epithelial cytology in adult hpg, immature and adult wild-type mice using unbiased optical disector-based stereology, immunolocalization of Sertoli cell microtubules (MT), espin (a component of the blood-testis barrier), markers of Sertoli cell maturity (p27(kip1) and WT-1), and electron microscopy. Hpg testes had marked reductions in weight, seminiferous cord volume and length, and severe spermatogenic impairment with germ cells per testis < 1% of adult wild-type testes. Sertoli cell nuclei expressed WT-1 in hpg testes, but often were centrally located, similar to 9-14-day-old wild-type testes, and they expressed p27(kip1), indicating that hpg Sertoli cells were post-mitotic. Hpg testes had significantly (P < 0.05) reduced Sertoli cells per testis (0.56 million) compared with 10-day wild-type (1.15 million) and adult wild-type testes (2.06 million). Immunofluorescence labelling of normal adult Sertoli cells showed supranuclear MT columns and basally located espin, but these features were absent in 10-day-old and hpg Sertoli cells. Hpg Sertoli cells showed pleomorphic nuclear ultrastructure with mature-type nucleoli, similar to normal adult-type Sertoli cells, but hpg Sertoli cells exhibited incomplete tight junctions that lacked ectoplasmic specializations. We conclude that in hpg mice, chronic gonadotrophin insufficiency restrains Sertoli cell proliferation and maturation, forming pseudo-adult-type Sertoli cells that are incapable of supporting germ cell proliferation and maturation.

Preparation of polyribosome aminoacyl-transfer ribonucleic acid from the muscle of chick embryos.

A procedure for preparing polyribosome aminoacyl-tRNA free from contamination by supernatant aminoacyl-tRNA and free amino acids is described. Important features of the procedure are the use of acidic buffers to help protect the amino acid-tRNA linkage and the inclusion of sodium dodecyl sulphate, to inhibit ribonuclease activity. The specific radioactivity of polyribosome aminoacyl-tRNA is high within 30s and reaches a maximum in 2 1/2 min, well ahead of polyribosome peptides which, as described by Herrmann et al. (1971), attain maximum specific radioactivity in about 10 min.

Activity of polyribosomes from the muscle of normal and dystrophic mice in cell-free amino-acid incorporation.

Polyribosomes sedimenting in the manner characteristic of those from embryonic chick muscle, as described by Heywood et al. in 1967 (Proc. Natl Acad. Sci. U.S.A. 57,1002--1009) were reproducibly obtained from normal mouse muscle by homogenization of the muscle with a Dounce homogenizer. The polyribosome profiles of dystrophic muscle were qualitatively similar to those of normal muscle except that the relative amount of ribosomes in polyribosome complexes was smaller (44% +/- 3S.E.) in dystrophic muscle than in normal muscle (67% +/- 4S.E.). In spite of this difference, polyribosomes from dystrophic muscle incorporated amino acids in vitro at a faster rate and produced a larger amount of polypeptide at the end of the reaction than polyribosomes from normal muscle.

Transfer of serine into polypeptides and myosin by chromatographic species of seryl-transfer ribonucleic acid.

The efficiencies of two chromatographic species of [3-H]seryl-tRNA, namely peaks I and II, in cell-free amino acid incorporation were investigated. The maximum yield of polypeptide seems to be the same for the reaction mixtures containing either peak I or peak II, suggesting that the efficiency of both peaks in total protein synthesis is the same. The efficiency of transfer of serine into myosin heavy subunit (myosin H) by peaks I and II was also investigated. Peak II of [3-H]seryl-tRNA transfers three times as much serine into myosin H as peak I.

Ribonucleic acid synthesis in embryonic chick muscle, rates of synthesis and half-lives of transfer and ribosomal RNA species.

In this study the term 'rates of synthesis' does not refer to rates of transcription but to rates of accumulation of newly synthesized RNA. The rates of synthesis and half-lives of tRNA, 18S and 28S rRNA of 14-day and 17-day embryonic chick muscle were determined by following the accumulation of radioactive AMP in RNA after administration of [3H]8-adenosine to chick embryos and correcting for the average specific activity of the precursor ATP pool Transfer RNA of 14-day embryonic chick muscle is synthesized at a rate of 2.4 x 10(5) molecules per min per 2N DNA content and decays with a half-life of 50h. Transfer RNA of 17-day embryonic chick muscle is synthesized, and decays, at similar rates. Ribosomal RNA, 18S and 28S, are synthesized at a rate of 9.94 x 10(3) and 8.48 x 10(3) molecules per min per 2N DNA, respectively, in 14-day embryonic chick muscle. The rates of rRNA syntheis in 17-day embryonic muscle are also similar. In both 14-day and 17-day embryonic muscle, 18S and 28S rRNA each decays with a half-life of 65h. We conclude that the constant level of tRNA and rRNA in embryonic chick muscle from 14 to 17 days (Nwagwu & Nana, 1974) is maintained also by a constant rate of synthesis and turnover.

Collagen synthesis in the muscle of developing chick embryos.

Radioactive protein was prepared from the leg muscle of chick embryos, 11, 14, 16 and 17 days old, each injected with radioactive proline and incubated for 30, 60 or 90 min afterwards. The radioactive protein was incubated with collagenase purified by chromatography on a Sephadex G-100 column. Under this condition, only collagen is digested into products soluble in trichloroacetic acid. The relative rate of collagen synthesis was determined by comparing the amount of radioactivity released into the supernatant fraction and that in the residue, by the method of Diegelmann & Peterkofsky [(1972) Dev. Biol. 28, 443--453]. The results show that the rate of collagen synthesis remains at approx. 10% of the rate of synthesis of other non-collagenous proteins during the development of chick embryonic muscle from 11 to 17 days. This suggests that the synthesis of collagen and that of other proteins are co-ordinately regulated at these stages of development.

Evidence of progressive deterioration of renal function in rats exposed to a maternal low-protein diet in utero.

Intrauterine growth retardation associated with maternal undernutrition is proposed to play a significant role in the aetiology of hypertension and CHD. Animal experiments suggest that the kidney, which is extremely vulnerable to the adverse effects of growth-retarding factors, may play an important role in the prenatal programming of hypertension. Maintenance of renal haemodynamic functions following structural impairment in fetal life is proposed to require adaptations which raise systemic blood pressure and promote a more rapid progression to renal failure. Rats were fed on diets containing 180 g casein/kg (control) or 90 g casein/kg (low protein) during pregnancy. The offspring were studied in terms of blood pressure, creatinine clearance, blood urea N, plasma and urinary albumin, renal morphometry and metabolic activity at 4, 12 and 20 weeks of age. Blood pressure was elevated at all ages in the low-protein-exposed offspring, relative to control rats. Rats (4 weeks old) exposed to the low-protein diet had smaller kidneys which were shorter and wider than those of control animals. Creatinine clearance was significantly reduced in 4-week-old rats exposed to the low-protein diet. Renal morphometry and creatinine clearance at older ages were not influenced by prenatal diet. Blood urea N, urinary output and urinary albumin excretion were, however, significantly greater in low-protein-exposed rats than in control rats at 20 weeks of age. These findings are suggestive of a progressive deterioration of renal function in hypertensive rats exposed to mild maternal protein restriction during fetal life. This is consistent with the hypothesis that adaptations to maintain renal haemodynamic functions following impairment of fetal nephrogenesis result in an accelerated progression towards glomerulosclerosis and increased intrarenal pressures mediated by rising vascular resistance.