Fc gamma receptor IIa-H131R polymorphism and malaria susceptibility in sympatric ethnic groups, Fulani and Dogon of Mali.

It has been previously shown that there are some interethnic differences in susceptibility to malaria between two sympatric ethnic groups of Mali, the Fulani and the Dogon. The lower susceptibility to Plasmodium falciparum malaria seen in the Fulani has not been fully explained by genetic polymorphisms previously known to be associated with malaria resistance, including haemoglobin S (HbS), haemoglobin C (HbC), alpha-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Given the observed differences in the distribution of FcγRIIa allotypes among different ethnic groups and with malaria susceptibility that have been reported, we analysed the rs1801274-R131H polymorphism in the FcγRIIa gene in a study of Dogon and Fulani in Mali (n = 939). We confirm that the Fulani have less parasite densities, less parasite prevalence, more spleen enlargement and higher levels of total IgG antibodies (anti-CSP, anti-AMA1, anti-MSP1 and anti-MSP2) and more total IgE (P < 0.05) compared with the Dogon ethnic group. Furthermore, the Fulani exhibit higher frequencies of the blood group O (56.5%) compared with the Dogon (43.5%) (P < 0.001). With regard to the FcγRIIa polymorphism and allele frequency, the Fulani group have a higher frequency of the H allele (Fulani 0.474, Dogon 0.341, P < 0.0001), which was associated with greater total IgE production (P = 0.004). Our findings show that the FcγRIIa polymorphism might have an implication in the relative protection seen in the Fulani tribe, with confirmatory studies required in other malaria endemic settings.

Influence of infection on malaria-specific antibody dynamics in a cohort exposed to intense malaria transmission in northern Uganda.

The role of submicroscopic infections in modulating malaria antibody responses is poorly understood and requires longitudinal studies. A cohort of 249 children ≤5 years of age, 126 children between 6 and 10 years and 134 adults ≥20 years was recruited in an area of intense malaria transmission in Apac, Uganda and treated with artemether/lumefantrine at enrolment. Parasite carriage was determined at enrolment and after 6 and 16 weeks using microscopy and PCR. Antibody prevalence and titres to circumsporozoite protein, apical membrane antigen-1 (AMA-1), merozoite surface protein-1 (MSP-119 ), merozoite surface protein-2 (MSP-2) and Anopheles gambiae salivary gland protein 6 (gSG6) were determined by ELISA. Plasmodium falciparum infections were detected in 38·1% (194/509) of the individuals by microscopy and in 57·1% (284/493) of the individuals by PCR at enrolment. Antibody prevalence and titre against AMA-1, MSP-119 , MSP-2 and gSG6 were related to concurrent (sub-)microscopic parasitaemia. Responses were stable in children who were continuously infected with malaria parasites but declined in children who were never parasitaemic during the study or were not re-infected after treatment. These findings indicate that continued malaria infections are required to maintain antibody titres in an area of intense malaria transmission.

Interaction of malaria with a common form of severe thalassemia in an Asian population.

In many Asian populations, the commonest form of severe thalassemia results from the coinheritance of HbE and beta thalassemia. The management of this disease is particularly difficult because of its extreme clinical diversity; although some genetic and adaptive factors have been identified as phenotypic modifiers, the reasons remain unclear. Because the role of the environment in the course of severe thalassemia has been neglected completely and because malaria due to both Plasmodium falciparum and Plasmodium vivax has been prevalent in Sri Lanka, we carried out a pilot study of patients with HbE beta thalassemia that showed high frequencies of antibodies to both parasite species and that 28.6% of the children had DNA-based evidence of current infection with P. vivax. Malarial antibodies then were assessed in patients with HbE beta thalassemia compared with those in age-matched controls. There was a significant increase in the frequency of antibodies in the thalassemic patients, particularly against P. vivax and in young children. There was also a higher frequency in those who had been splenectomized compared with those with intact spleens, although in the latter it was still higher than that in the controls. The thalassemic patients showed significant correlations between malaria antibody status and phenotype. Patients with HbE beta thalassemia may be more prone to malaria, particularly P. vivax, which is reflected in their clinical severity. Because P. vivax malaria is widespread in Asia, further studies of its interaction with HbE beta thalassemia and related diseases are required urgently as a part of ongoing thalassemia control programs.

Estimating medium- and long-term trends in malaria transmission by using serological markers of malaria exposure.

The implementation and evaluation of malaria control programs would be greatly facilitated by new tools for the rapid assessment of malaria transmission intensity. Because acquisition and maintenance of antimalarial antibodies depend on exposure to malaria infection, such antibodies might be used as proxy measures of transmission intensity. We have compared the prevalence of IgG antibodies with three Plasmodium falciparum asexual stage antigens in individuals of all ages living at varying altitudes encompassing a range of transmission intensities from hyper- to hypoendemic in northeastern Tanzania, with alternative measures of transmission intensity. The prevalence of antibodies to merozoite surface protein-1(19) was significantly more closely correlated with altitude than either point-prevalence malaria parasitemia or single measures of hemoglobin concentration. Analysis of age-specific seroprevalence rates enabled differentiation of recent (seasonal) changes in transmission intensity from longer-term transmission trends and, using a mathematical model of the annual rate of seroconversion, estimation of the longevity of the antibody response. Thus, serological tools allow us to detect variations in malaria transmission over time. Such tools will be invaluable for monitoring trends in malaria endemicity and the effectiveness of malaria control programs.

Ammonia cleaves polypeptides at asparagine proline bonds.

Polypeptides that contain the sequence Asn-Pro undergo complete cleavage at this amide bond with ammonia. One cleavage product possesses Pro as the new amino terminus and the other Asn or isoAsn as the new C-terminus, the formation of the latter probably arising by way of a cyclic succinimide intermediate. Other Asn-X bonds where X = Tyr, Gln, Ile, Glu, Ala, Gly, Asn or Phe did not exhibit any peptide bond cleavage, whereas when X = Leu, Thr and Ser partial cleavage was observed. Asn residues not involved in chain-cleavage underwent deamidation to Asp as shown by MALDI-ToF mass spectrometry (MS) analysis. The partial conversion of in-chain Asp residues to isoAsp under the reaction conditions was inferred from RP-HPLC and MS analysis of reaction mixtures.

Application of an in vitro endopeptidase assay for detection of residual toxin activity in tetanus toxoids.

Tetanus vaccine is composed of chemically denatured tetanus toxin (TeNT), thus safety testing requires confirmation of freedom from residual and reversible toxicity. Currently, TeNT activity is estimated using in vivo assay models. Information that TeNT acts by selectively inactivating protein leading to the blocking of release of neurotransmitters has provided the opportunity to develop in vitro biochemical assay for toxin activity. In this study we describe development and use of an in vitro endopeptidase assay for detection of TeNT activity in toxoid vaccine formulations.

Differences between neonates and adults in tissue-type-plasminogen activator (t-PA)-catalyzed plasminogen activation with various effectors and in carbohydrate sequences of fibrinogen chains.

Our study investigates the effect of fetal and adult soluble fibrin (SF), fetal and adult fibrinogen Aalpha- and gamma-chains, as well as adult CNBr-fibrinogen fragments on tissue-type plasminogen activator (t-PA)-catalyzed plasminogen activation of both fetal and adult Glu-plasminogen types 1 and 2. In addition, we determined carbohydrate sequences of fetal and adult Bbeta- and gamma-chains by mass spectrometric analysis. In the absence of an effector, no substantial differences in the rate of plasmin formation could be seen between the fetal and adult plasminogen types. In the presence of an effector, both fetal Glu-plasminogen types revealed lower values for k(cat app) than the respective adult types. No differences could be seen in the values for K(m app). The resulting differences in catalytic efficiencies between the fetal and adult plasminogen types were much less than previously reported. No differences could be seen between fetal and adult effectors in stimulating t-PA-catalyzed plasminogen activation. Detailed analyses of the activation kinetics revealed a longer initial phase of slow plasmin formation of both fetal Glu-plasminogen types compared to their respective adult types, indicating a slower plasmin-induced modification of CNBr-fibrinogen fragments or SF by fetal plasmin. Mass spectrometric analysis of the N-glycans present on adult and fetal Bbeta- and gamma-fibrinogen chains showed the presence of a major monosialylated biantennary structure with lesser amounts of the disialylated form. In contrast to previous data, we conclude that catalytic efficiency of t-PA-catalyzed plasminogen activation in neonates is only slightly lower than in adults.

Multicenter collaborative study to calibrate salmon calcitonin by bioassay and high-performance liquid chromatography: establishment of the third international standard.

Salmon calcitonin (sCT) is widely used therapeutically in the treatment of patients with postmenopausal osteoporosis, Paget's disease, and some forms of hypercalcemia. Preparations of synthetic calcitonin peptides of high purity and reproducibility are now routinely produced and physicochemical methods, particularly reverse-phase high-performance liquid chromatography (RP-HPLC), are replacing the in vivo biological assay for monitoring and calibration. Although the bioassay is no longer required for routine batch control in Europe, calcitonin bioassays are still required in some countries and in the development of new products. Stocks of the Second International Standard (IS) for salmon calcitonin are now depleted and, to replace it with a new calibrant, an international collaborative study was organized in which the aims were to: determine the activity of the candidate sCT by in vivo bioassay in terms of the second IS; assess the stability of the preparation after accelerated thermal degradation; estimate the purity of the ampouled candidate preparation; and determine the sCT content in gravimetric units by HPLC. The HPLC data in terms of ampoule content were in good agreement giving an estimate of 23.1 (coefficient of variation [CV] 3.8%) microg per ampoule. The HPLC chromatograms revealed a small, but detectable, degree of heterogeneity, which possibly occurred during the formulating or ampouling procedures, resulting in a reduction in monocomponent content (purity) from 96% to 92%. The biological activity of the ampoule contents in international units (IU) was calculated from the mass value and the internationally agreed-upon figure of 6000 IU/per mg for the specific activity of salmon calcitonin. This gave a value of 138 IU per ampoule, which was in good agreement with the biological assay estimate (140 IU per ampoule). The preparation of sCT was subsequently adopted as the Third International Standard by the World Health Organization with an assigned content of 138 IU per ampoule.

Recombinant human albumin as a stabilizer for biological materials and for the preparation of international reference reagents.

Recombinant human albumin expressed in Saccharomyces cerevisiae was compared with native human serum albumin in its physicochemical properties and in its use as a stabilizer in lyophilized preparations of thyroid-stimulating hormone (TSH), interleukin 15 (IL-15) and granulocyte colony-stimulating factor (G-CSF). Advantages of recombinant albumin include its lack of potential human contaminants and infectious agents. When used at concentrations of 0.1-0.2% (w/v), recombinant albumin was equivalent to native serum albumin in its capacity to protect immunological, biological and biochemical properties of TSH, IL-15 and G-CSF. Physicochemical characteristics of the two forms of albumin including their binding to fatty acids were also similar. The recombinant form of albumin used in this study should be considered as a suitable stabilizer in the preparation of lyophilized products and reference reagents.

Characterisation of the chains of human fibrinogen isolated by perfusion chromatography using fibrin specific monoclonal antibodies.

In order to study the epitopes on fibrin to which monoclonal antibodies are directed, we required pure individual polypeptide chains of human fibrinogen in milligram quantities. High purity chains of human fibrinogen were rapidly obtained, in under 3 minutes, by the novel procedure of reversed-phase perfusion chromatography and these chains were subjected to immunological characterisation using monoclonal antibodies specific to the individual chains. Cross-reactivity against these antibodies in both immunoblotting and enzyme linked immunospecific assay (ELISA) procedures showed that these isolated fibrinogen chains were of high purity and retained high immunoreactivity. These chains were employed to initiate studies to define the epitopes in fibrin to which four fibrin specific monoclonal antibodies, B10, A11, 5F3 and 1H10 are targeted. Two of these antibodies, B10 and A11, were shown to be directed to a linear sequence on the A alpha chain, although the binding profiles for the two antibodies suggested that different epitopes may be involved for each of these two antibodies. MAbs, 1H10 and 5F3, however, did not bind to any of the three fibrinogen chains, suggesting that conformational epitopes in fibrin are likely to be involved in the binding of these two antibodies to fibrin.

Lysine vasopressin undergoes rapid glycation in the presence of reducing sugars.

Lysine vasopressin (LVP) readily reacts with reducing saccharides both in lyophilized preparations and in aqueous solution. Incubation of LVP with, for example, lactose over a pH range of 3.0-8.5 in phosphate buffer or simply in water, gives rise to a number of reaction products, some of which form rapidly (in a matter of hours) even in the frozen state. Reaction mixtures were analysed by reversed-phase HPLC and the structures of the products were deduced from the amino-acid composition of isolated components, by comparison with product profiles obtained with analogues under similar conditions and by FAB mass-spectral analysis of derivatives isolated after reduction with cyanoborohydride. The primary products arise from the formation of Schiff's bases at one or both of the two amino functions. The alpha-amino group of the N-terminal cystine is considerably more reactive than is the epsilon-amino group of lysine and it is the N-terminal adduct which rapidly forms even at -20 degrees C. It is concluded that caution must be shown in using reducing sugars in formulations containing peptides and proteins, particularly the vasopressins and oxytocin.

Ultra-rapid preparation of milligram quantities of the purified polypeptide chains of human fibrinogen.

In order to study the epitopes in fibrin towards which monoclonal antibodies are directed we needed the pure individual polypeptide chains of human fibrinogen in reasonable quantity. We report here a simplified, rapid method of separation of high-purity human fibrinogen chains. Following reduction and S-carboxymethylation of human fibrinogen, the sample was injected directly onto a column of the polymeric reversed-phase perfusion packing POROS 20-R2, and the chains were completely resolved in less than 3 min at a flow-rate of 10 ml/min. The capacity was equivalent to that of a similar sized conventional silica-based column. However the throughput was approximately five to ten times as high. The column was durable and robust in day-to-day use.

Establishment of the second international standards for porcine and human calcitonins: report of the international collaborative study.

The biological potency of calcitonins in clinical use in long-term treatment of Paget's disease of bone and, increasingly, in osteoporosis is usually expressed international units defined by the relevant World Health Organization international reference preparation. The international reference preparations for porcine and human calcitonins were ampouled in 1970 and stocks are now exhausted. Replacement standards were ampouled in 1989 and have been evaluated and calibrated by an international collaborative study comprising 16 laboratories in 12 countries. Evaluations included high-performance liquid chromatography and in vitro bioassay; calibration of each new ampouled preparation in terms of its international reference preparation was by in vivo rat hypocalcaemia bioassay. On the basis of the results of the study and with the agreement of the participants, replacement standards were established by the Expert Committee on Biological Standardization of the World Health Organization in 1991: the international standard for porcine calcitonin (ampoule code 89/540), with an assigned potency of 0.8 international units per ampoule, and the international standard for human calcitonin, with an assigned potency of 17.5 international units per ampoule. Both international standards appeared to be sufficiently stable to serve as the international standards for in vivo biological assays. Comparison of the two species of calcitonin in the same hypocalcaemia assay showed that they were approximately equipotent when the doses were given intravenously but that the human peptide was four- to sixfold more potent than porcine calcitonin when doses were given subcutaneously, emphasizing the need to compare "like with like".

Identification of gonadorelin (LHRH) derivatives: comparison of reversed-phase high-performance liquid chromatography and micellar electrokinetic chromatography.

A number of reversed-phase (RP) HPLC systems for the separation of gonadorelin (gonadoliberin, LHRH) and five therapeutically important analogues have been systematically examined. The selectivity of RP-HPLC has been compared with several micellar electrokinetic chromatographic (MEKC) systems and free solution capillary electrophoresis. RP-HPLC exhibits greater selectivity towards structural differences, but complete separation of the peptides in one isocratic analytical run is tedious due to the large differences in retention. Gradient elution gives satisfactory separation in an acceptable time span. Of the micellar systems examined (sodium dodecyl sulphate, cetrimide, 3-[(cholamidopropyl)dimethylamino]-1-propanesulphonate and Triton X-100) only MEKC with cetrimide micelles gave a complete separation showing selectivity similar, but not identical, to RP-HPLC, and providing a complete separation of all six compounds as rapidly as gradient RP-HPLC.

Comparison of selectivities of reversed-phase high-performance liquid chromatography, capillary zone electrophoresis and micellar capillary electrophoresis in the separation of neurohypophyseal peptides and analogues.

A number of RP-HPLC systems, including those prescribed in several official monographs, have been evaluated for separating oxytocin, the vasopressins, some clinically important analogues and two additional neurohypophyseal nonapeptides. The separation has been compared with capillary zone electrophoresis and micellar electrophoresis in four micellar systems: cationic, anionic, zwitterionic and neutral. Complete separation was achieved by both RP-HPLC and micellar CE but the importance of charge as a major parameter of separation in CE confers a distinct and useful selectivity to micellar CE based separations.

International Standards for salmon calcitonin, eel calcitonin, and the Asu1-7 analogue of eel calcitonin: calibration by international collaborative study.

Regulatory specifications in most countries require that the potency of salmon calcitonin (sCT) clinical products be expressed in International Units (IU) defined by the World Health Organization (WHO) International Standard. The first ampouled standard was prepared in 1972 and has been distributed world-wide since then. A batch of ampoules to serve as the replacement standard is now required. Other piscine calcitonins, eel calcitonin (eCT) and an amino-suberic acid analogue of eCT (Asu1-7 eCT) are now clinical products in some countries and international standards are required for these peptides which are similar to, but not identical with, sCT. This paper describes the preparation of three new ampouled standards and their biological calibration by international collaborative study comprising 17 participants from 10 countries. Following the recommendations in the final report of the collaborative study, the 2nd International Standard (IS) for sCT, the 1st IS for eCT and the 1st IS for Asu1-7 eCT were recently established by WHO, each with an assigned potency in IU, and are now available for issue.

Determination of L-thyroxine in reference serum preparations as the o-phthalaldehyde-N-acetylcysteine derivative by reversed-phase liquid chromatography with electrochemical detection.

A simple procedure for the assay of L-thyroxine in serum preparations with D-thyroxine as internal standard is described. The L-thyroxine is extracted with acetonitrile, fractionated on a reversed-phase silica cartridge and analysed by reversed-phase high-performance liquid chromatography of the o-phthalaldehyde-N-acetyl-L-cysteine derivative. This derivative is not fluorescent, but may be detected with suitable sensitivity and selectivity with an electrochemical detector.