HLA-DRB1*15 association with multiple sclerosis is confirmed in a multigenerational Italian family.

Environmental and genetic factors seem to play a pathogenetic role in multiple sclerosis (MS). The genetic component is partly suggested by familial aggregation of cases; however, MS families with affected subjects over different generations have rarely been described. The aim of this study was to report clinical and genetic features of a multigenerational MS family and to perform a review of the literature on this topic. We describe a multigenerational Italian family with six individuals affected by MS, showing different clinical and neuroradiological findings. HLA-DRB1* typing revealed the presence of the DRB1*15:01 allele in all the MS cases and in 4/5 non-affected subjects. Reports on six multigenerational MS families have previously been published, giving similar results. The HLA-DRB1*15:01 allele was confirmed to be linked to MS disease in this family; moreover, its presence in non-affected subjects suggests the involvement of other susceptibility factors in the development and expression of the disease, in accordance with the complex disease model now attributed to MS.

[Development of an antigen detection dot blot assay for the diagnosis of human onchocerciasis based on the biotin-avidin binding system]. / Développement d'un test dot blot de détection d'antigène, basé sur le système de fixation biotine- avidine, pour le diagnostic de l'onchocercose humaine.

The development of a highly sensitive and specific diagnostic test is of urgent need for the field assessment of human onchocerciasis and for monitoring the success of control programs. We report here the development and evaluation of a Dot blot Immunobinding Assay (DIA-BA) based on the biotin-avidin binding system, for the detection of O. volvulus specific antigens in body fluids. Specific antibodies were produced by immunizing rabbits with the O. volvulus recombinant antigen Oncho-C71 and labelled with biotin. The biotinylated probes were then used to detect O. volvulus specific antigens initially blotted onto a nitrocellulose membrane. The smallest amount of blotted antigens detectable by the new test is 0.5ng, 1ng, 1ng and 2ng respectively in urine, dermal fluid, tears and serum samples. Out of 456 onchocerciasis endemic subjects examined, 98.4%, 96.5%, 90.8% and 75.0% were positive by the DIA-BA test on urine, dermal fluid, tears and serum respectively The test was most sensitive (100%) when used on urine and least (54.76%) when used on serum from skin snip positive subjects. The specificity of the test, determined amongst non-exposed individuals, was 100% on all but for dermal fluid samples (97.5%). Also, the color intensities on the blot were observed to positively correlate (r = 0.8 on urine) with the skin microfilaria loads on the individuals. We conclude that DIA-BA test could be very useful for mass diagnosis of prepatent, of low and high level infections due to O. volvulus.

Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast.

A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33. Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O. volvulus worms was screened. Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O. volvulus. These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S. cerevisiae. Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae. The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin. Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae. The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients. The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively. The combined sensitivity using both recombinant proteins was 94%.

The Kiddie Formal Thought Disorder Rating Scale: clinical assessment, reliability, and validity.

The Kiddie Formal Thought Disorder Story Game and the Kiddie Formal Thought Disorder Scale were administered to schizophrenic, schizotypal, and normal children, aged 5 to 13 years. The story game elicited more elaborate speech samples than did a structural clinical interview focused on psychotic symptomatology. The sum of illogical thinking and loose associations was a reliable kappa = 0.77), sensitive (79%), and specific (90%) indicator of schizophrenia in this sample. It also demonstrated significant developmental changes in the schizophrenic and normal subjects. Incoherence and poverty of content of speech were infrequently rated in both schizophrenic and normal subjects.

Homocysteine, vitamin B12, serum and erythrocyte folate in peritoneal dialysis patients.

Plasma homocysteine (tHcy) is an important risk factor for atherosclerosis in dialysis patients. Few data were reported on the prevalence and severity of hyperhomocysteinemia in peritoneal dialysis (PD) patients. In addition, little attention was paid to the search of factors possibly involved in the pathogenesis of hyperhomocysteinemia in these patients. A cross-sectional study was performed in 107 stable PD patients. None of them was given folate or vitamin B12 supplementation before or during the study. Plasma tHcy, serum vitamin B12, serum and erythrocyte folate were measured by immunoenzymatic methods. Genetic analysis of the methylentetrahydrofolate-reductase thermolabile mutation (tMTHFR) was performed in 61 patients. 97% of patients had tHcy levels higher than normal. tHcy was not different between men and women, patients with or without malnutrition, with or without clinically evident atherosclerotic vasculopathy, with or without anemia. tHcy levels were significantly higher in homozygotes for the tMTHFR mutation than in patients carrying the wild type form. Significant univariate correlation was found between hyperhomocysteinemia and time since the start of dialysis, serum and erythrocyte folate and vitamin B12. The best fitted model equation was log tHcy = 108.53 + 0.1606 (duration of dialysis) -1.1053 (s-F) -0.7980 (age) 0.0215 (vitamin B12). Our results agree with those reported by other authors in hemodialysis patients. Despite the large number of PD patients with normal serum vitamin B12 and folate status, the relation between tHcy and vitamin B12 or folate suggests that the supplementation of these vitamins could be useful irrespective of their serum levels, especially in younger patients or in those treated for a long period of time with peritoneal dialysis.

Effect of incremental doses of folate on homocysteine and metabolically related vitamin concentrations in nondiabetic patients on peritoneal dialysis.

The role of folate supplementation in reducing hyperhomocystinemia in patients on dialysis has been reported, but the optimal dose of folate is still unknown. The aim of the present study was to investigate whether greater than 5 mg/day folate supplementation provides any additional effect on plasma homocysteine (HCY) levels. The study was prospective, open, and had no control group. Of the 64 eligible nondiabetic patients on peritoneal dialysis with hyperhomocystinemia (>20 micromol/L), 56 were given oral folate (5 mg/day) for 3 months. When Hcy did not fall below 20 micromol/L, folate doses were increased by 5 mg every 3 months to up to 15 mg/day. With 5 mg/day supplementation, serum folate concentrations increased above the upper confidence limit in 23 patients and erythrocyte folate concentrations in 27 patients. Hcy levels decreased to less than 15 micromol/L in 6 cases and by more than 50% in 12 cases. Nineteen of the remaining patients were given 10 mg/day folate. After increasing the dose, serum and erythrocyte folate levels rose above the upper detection limit. In one patient, plasma Hcy concentrations decreased to less than 15 micromol/L. Ten patients were given 15 mg/day oral folate for an additional 3 months with no effect on homocystinemia. This study confirms that oral folate supplementation may improve hyperhomocystinemia even in patients on dialysis with normal serum or erythrocyte folate concentrations. In fact, serum and erythrocyte levels cannot predict the effect of supplementation on plasma Hcy levels. However, 5 mg/day folate supplementation normalized Hcy in 10% of cases and reduced Hcy levels in another 21%. Increasing the folate dose to greater than 5 mg/day had a minimal (10 mg/day) or no (15 mg/day) additional effect on Hcy concentrations. Despite the minimal effect of increasing folate doses, given the low cost, the absence of side effects, and the high cardiovascular risk for patients on peritoneal dialysis, a careful attempt to increase the dose of oral folate up to 10 mg/day might be suggested.

[Prognostic value of lactate determination in cerebral malaria in the Cameroonian child]. / Intérêt pronostic du dosage du lactate dans le paludisme cérébral chez l'enfant camerounais.

In this study, we investigated the disturbances of glucose metabolism in 61 Cameroonian children aged 1 to 15 years old, infected with cerebral malaria and severe malaria cases admitted in hospitals in Yaoundé. Parasitaemia in the two groups was respectively 8.10 +/- 0.35% in cerebral malaria group and 4.47 +/- 0.84% in those admitted for severe malaria. The majority of our patients (n = 42) had normal glycaemia, while 15 cases were hyperglycemic and only 4 subjects all belonging to the cerebral malaria group were hypoglycemic. No case of hyperinsulinaemia was recorded. The level of lactate was higher in 74% of patients (19 over 26) admitted for cerebral malaria virsus 37% patients (13 over 35) admitted for severe cases. We found a positive correlation between the level of parasite and that of lactate (P < 0.01 r = 0.47). Whereas no positive correlation was found in one hand between the level of parasite and glycaemia, the level of parasites and insulin, and in the other hand between glycaemia and the level of lactate, glycaemia and insulin.

Serum lipids and atherogenic risk in sickle-cell trait carriers.

This work studies the relationship between a common genetic marker in our population, sickle-cell trait, and atherogenic risk factors. One hundred and twenty-two healthy subjects were studied. Forty-six subjects had normal haemoglobin (HB-AA) while 76 had sickle-cell trait Hb-AS. Mean cholesterol level was higher in subjects with sickle-cell trait (2.82 +/- 0.21 g/I) than in subjects with normal haemoglobin (2.08 +/- 0.40 g/I) used as controls. The difference was statistically significant (p < 0.05). HDL cholesterol levels remained in the normal range in controls (Hb-AA) (0.560 +/- 0.10 g/I) while subjects with Hb-AS had high HDL-c levels (0.618 +/- 0.30 g/I). Mean LDL cholesterol levels in Hb-AS subjects were about 57% higher than those in controls (1.84 +/- 0.10 g/I and 1.17 +/- 0.30 g/I, respectively) (p < 0.05). Apolipoprotein A1 levels of Hb-AS subjects were lower (1.37 +/- 0.10 g) than those of Hb-AA subjects (1.98 +/- 0.15 g/I), (p < 0.05) while apolipoprotein B concentrations of Hb-AS subjects were higher (1.70 +/- 0.27 g/I) than in controls (1.30 +/- 0.10 g/I) (p < 0.05). The atherogenicity index given by the Apo-B/Apo-AI ratio was higher in Hb-AS subjects (1.24) than in those with normal haemoglobin (0.65). We conclude that Hb-AS subjects are at high atherogenic risk if subsequent diet management is not undertaken.

[Proteolytic activity of adult worm extracts of Onchocerca volvulus]. / Activité protéolytique de l'extrait de vers adultes de l'Onchocerca volvulus.

Studying proteolytic activity of Onchocerca volvulus (nematode causing "river blindness") shows that it is able to digest a variety of substrates such as: azoalbumine, azocoll and elastin-orcein with specific activity of 0.28, 0.57 and 1.48 mg/hour/mg of extract respectively. These enzymes are active at various pH such as pH 5.0, 8.0 and 10.0 with highest activity at pH 8.0. The effect of specific inhibitors and activators indicates that the extract might contain serine, metallo and thyoproteases. The electrophoresis of the extract on a polyacrylamide gel copolymerized with gelatin shows many proteins with enzymatic activities with molecular weight of 16.6, 43.6, 45.7, 56.2, 60.2, 61.6 and 63.1 KD respectively. The Onchocerca volvulus worm contains proteases of various enzymatic activities: a non specific activity on protein such as on azoalbumin and specific activities on collagen and elastin. These enzymes could play an important role in the survival of parasites in human hosts.

[Variations under genetic control of onchocerca infection as a function of clinical profile in the endemic center of Cameroon]. / Variations sous contrôle génétique de l'infection onchocerquienne en fonction du profil clinique dans les foyers endémiques du Cameroun.

Onchocerciasis, also known as "river blindness", presents a plenum of clinical manifestations which vary from one individual to another, and from one area to another. This large spectrum of clinical manifestations of the disease is an indication of the complexity of the pathogenesis of onchocerciasis and suggests that many interacting factors might influence the clinical features of the disease. The present study has focused on the heterogenicity of the host immune response as a plausible explanation for differences in clinical manifestations of the infection. Host genetic factors, namely HLA genes, might play an important role in determining the nature of the immune response mounted against the parasite Onchocerca volvulus, and thus the development of different manifestations of the infection. Genetic diversity of onchocerciasis was assessed in different endemic foci in Cameroon. In order to investigate the possibility that the Major Histocompatibility Complex (MHC) genes might be associated with the different clinical types of onchocerciasis, 146 subjects living in three endemic areas of Cameroon were studied. They were classified in four groups: A (asymptomatic subjects), P (putatively immune subjects) L (patients with localised disease) and G (patients with generalised disease). The four groups differed in the distribution of HLA class II alleles as determined by Direct Heteroduplex Analysis. On the one hand, allele HLA-DQA1*0501 appeared to be associated with protection against severe onchocerciasis; on the other, allele HLA-DQB1*0201 might play an important role in the severe form of the disease.

Glycolipid antigens of adult O. volvulus reactive with patients sera from onchocerciasis endemic areas in Cameroon.

Lipid extraction of adult O. volvulus worms using a chloroform/methanol/water mixture yielded 10 lipid fractions of which 8 were demonstrated by the orcinol reagent to be glycolipids. In TLC, two of these lipid fractions had mobilities similar to cholesterol and cholesterol ester (Rf.: 0.95, 0.86) whereas two others migrated as sphingomyelin and lecithin (Rf.: 0.40, 0.35) respectively. Other components migrated at intermediate positions. The glycolipids were immunologically active and reacted with sera from onchocerciasis patients. The highest reaction was obtained with the IgG antibody class, followed by IgM while no appreciable reactivity was observed with IgE. Sera from patients infected with other filariae such as Loa-loa and Dipetalonema perstans did not show any significant reaction with these antigens. The significance of these results is discussed.

[Intravenous folinic acid and vitamin B12 supplementation and homocysteine concentration in hemodialysis patients]. / Supplementazione e.v. di acido folinico e vitamina B12 e concentrazioni di omocisteina nei pazienti emodializzati.

INTRODUCTION: Hyperhomocysteinemia is one of the causes of the increased incidence of cardiovascular disease in uremia. Since homocysteine (Hcy) metabolism depends on the availability of folate and vitamin B12, we have measured the effects of chronic i.v. supplementation of folinic acid and vitamin B12 in a group of patients on maintenance hemodialysis. METHODS: We compared the blood concentration of total Hcy (tHcy), vitamin B12 and folate and the intraerythrocyte concentration of folate in a group of 27 hemodialysis patients (Treated group), given an i.v supplementation with folinic acid (0.9 mg) and Vitamin B12 (cyanocobalamine 1.5 mg and hydroxycobalamine 1.5 mg) three times per week at the end of each dialysis session with those measured in a similar group of 28 hemodialysis patients without supplementation (No Treatment group). The patients were also characterized for the thermolabile variant (mutation C667-->T) of the enzyme methylene-tetrahydrofolate reductase (tMTHFR). RESULTS: High plasma levels (< 11.7 micromol/L) of tHcy were observed in 54/55 patients. T patients had Hcy values significantly lower than NT ones (31.7+/-3.6 vs. 1.1+/-8.3micromol/L, p < 0.05). Serum vitamin B12 (1200 73.6 vs. 762+/-72.2 pmol/L, p < 0.001) and intraerythrocyte folate levels were also significantly higher in the T group (2176+/-127 vs. 1511+/-156, p < 0.005), while no significant difference was observed for serum folate. The distribution of tMTHFR genotypes was similar in the two groups. Homozygous patients showed higher levels of Hcy in comparison with wild type patients both in the whole population (62.32+/-15.9 vs.30.43+/-3.2, p < 0.05) and in the NT group (87.8+/-25.3 vs.36.8+/-13.1., p < 0.05), while no significant difference was observed among genotypes in the T group. CONCLUSIONS: Uremic patients on hemodialysis, when supplemented with even low i.v. dose of folinic acid and vitamin B12, show significantly lower plasma levels of tHcy than non-supplemented patients.

[Effect of Onchocerca volvulus infestation on plasma vitamin A concentration in school children in a rural region of Cameroon]. / Influence de l'infestation par Onchocerca volvulus sur le taux sérique de vitamine A des enfants scolariss en zone rurale du Cameroun.

Vitamin A deficiency is known to be associated with immune dysfunction and common childhood infections. However, little is known about the relationship between vitamin A deficiency and onchocerciasis in children. The aim of this study was to determine the prevalence of vitamin A deficiency and to investigate the relationship between vitamin A status and onchocerciasis. A total of 231 children, aged 6 to 15 years, were randomly selected between March 1995 and April 1996 at Yambassa and Balamba (central province of Cameroon). They were examined: we determined their vitamin A status and whether they had onchocerciasis. We diagnosed onchocerciasis by skin biopsy and the detection of antibodies against Onchocerca volvulus in the blood. We found that 101 of the 231 children examined (43.73%) had palpable nodules and/or microfilariae and the remaining 130 (56.27%) had been exposed to the parasite but had no clinical signs of infestation. Some children tested negative for skin microfilariae but positive by ELISA. Thus, 197 (85.28%) children were found to be infested with O. volvulus (group A) and the remaining 34 (14.72%) were found to have been exposed to the parasite but to have no clinical signs of onchocerciasis (group B). Plasma vitamin A concentrations were marginal, with concentrations below 0.7 mumol/l (20 mug/dl) recorded for 82.25% of the subjects. Children with onchocerciasis were more likely to have low vitamin A status. The mean plasma vitamin A concentration of infested children (0.52 +/- 0.14 mumol/l) was significantly lower (p < 0.05) than that of the children exposed but not infested. The parasite, O. volvulus, uses the vitamin A present in host tissues during its development, leading to a decrease in plasma retinol concentration.

Development and evaluation of an antigen detection dipstick assay for the diagnosis of human onchocerciasis.

To improve on the diagnosis of onchocerciasis, especially light infections, we developed and evaluated an oncho-dipstick test based on the detection of Onchocerca volvulus specific antigens in urine and tears. The test was able to detect as little as 25 ng/ml of parasite specific antigens in samples and took as little as 3 h. Evaluation of the assay on 456 residents of an onchocerciasis hyperendermic area in Cameroon resulted in 408 (89.5%) positives in urine and 374 (82%) positives in tears. The prevalence of onchocerciasis in the study area, as determined by Rapid Epidemiological Mapping of Onchocerciasis (REMO) and skin snip methods, was 52 and 36.8%, respectively. The sensitivity of the oncho-dipstick assay was 100% in urine and 92% in tears; its specificity was 100% in both. Concordance between urine and tear test results from the same individuals was 87%. The test strips were sufficiently reactive when left at room temperature for up to 8 months. The test would be useful for laboratory diagnosis of onchocerciasis in low transmission zones and to ascertain successful treatment of patients in experimental drug studies.

Antigenic structure of sperm whale myoglobin. II. Characterization of antibodies preferentially reactive with peptides arising in response to immunization with the native protein.

The antigenicity of peptide fragments derived from CNBr-cleaved sperm whale Mb has been tested in competitive inhibition assays. These peptides appeared to be very poor inhibitors in a reaction involving radiolabeled sperm whale Mb with antipeptide antibodies, requiring about 10(4)-fold excess of peptides to achieve the same level of inhibition as cold native Mb. However, cold peptides competed much better with radiolabeled peptides for antibody binding than the native protein. These antibodies, preferentially reactive with peptides, originated from sera of animals immunized with the native molecule. This indicates heterogeneity of the antibodies produced against Mb and suggests the presence of diverse antibody subpopulations reactive with different antigenic forms of the protein. Results presented in this paper illustrate that competitive radioimmunoassays preferentially measure antibodies to the radiolabeled species, and this phenomenon can be used to demonstrate distinct populations among antibodies isolated to a single peptide.

Antigenic structure of sperm whale myoglobin. I. Partition of specificities between antibodies reactive with peptides and native protein.

We have resolved anti-sperm whale Mb antibodies into two distinct populations: One population is reactive only with the native molecule, and the second is reaction with both Mb and peptide fragments. CNBr cleavage of sperm whale Mb yields three peptides: peptide I (1-55), peptide II (56-131), and peptide III (132-153). Immunoadsorbent columns made with these three peptides were used to fractionate antibody to sperm whale Mb. These columns cumulatively bind 60 to 70% of the total antibody present in the four sera studied (the amount of antibody bound by each peptide was proportional to its size). The remaining 30 to 40% of antibody could only bind specifically to an immunoadsorbent column of native Mb. This result indicates that not all antigenic determinants on the native Mb molecule are represented on peptide fragments.

Serum antibodies against human brain myelin proteins in Gambian trypanosomiasis.

Human brain myelin was obtained by flotation centrifugation on 0.88 M sucrose. The myelin proteins were extracted with 0.1% SDS-0.01 M Tris, pH 7.4 from the myelin floating on 0.88 M sucrose following centrifugation. Using ELISA and immunoblotting, we have shown that antibodies against the myelin proteins exist in sera collected from Gambian trypanosomiasis patients. The antibodies were more prevalent in patients with CNS involvement since of 21 sera from patients with more than 4 cells/ml cerebrospinal fluid (CSF), 15 (71.4%) had antimyelin antibodies, as compared to 3 of 13 (23.1%) from patients with less than 4 cells/ml CSF. Thus, of 18 sera that had antimyelin antibodies, 15 (83.3%) were from patients with CNS involvement. Using the immunofluorescence test, selected sera detected antigens in cryocuts of human brain.

Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite.

Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop O. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologically most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difficulty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate oligonucleotides. These oligonucleotides have been shown to support the amplification of the 150-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microfilariae. In addition, the method is capable of detecting O. volvulus infective larvae directly in extracts of blackfly vectors.

Immunity to Onchocerca volvulus. Serum mediated leucocyte adherence to infective larvae in vitro.

Leucocyte adherence to infective larvae of Onchocerca volvulus in the presence of serum was evaluated using sera from four clinically distinct groups of patients with Onchocerciasis from an area hyperendemic for the disease. Significant cellular adherence to infective larvae occurred for the most part in the presence of sera obtained from subjects with either no microfilaridemia and few or no palpable nodules. These patients had, as well, the highest serum titres of specific anti-O. volvulus IgG antibodies. In contrast, sera from subjects with many palpable nodules and heavy skin infiltration with microfilariae (generalised disease) did not mediate significant adherence of leucocytes to infective larvae targets. Further, this group had the lowest serum levels of specific anti-O. volvulus antibodies of the IgG isotype. The findings are in keeping with the hypothesis that specific protective immunity may occur in O. volvulus infections.

Comparative studies of clinical groups of patients in an onchocerciasis endemic area for evidence of immune-mediated protection.

Four clinical groups of persons from an area endemic for onchocerciasis were compared using certain immunological parameters. The groups were: generalised onchocerciasis, patients with restricted distribution of onchocercal skin lesions, microfilaredermia patients with no clinical manifestations, and a group which clinically, had successfully resisted the infection. Specific serum antibodies to O. volvulus antigens were found in all groups. The IgG specific antibodies were highest in patients with generalised onchocerciasis and lowest in the group who had apparently contained the infection. The sera of persons from the latter group mediated leukocyte adherence to and immobilised microfilariae of O. volvulus. Using the Western blot technique, there were no onchocercal protein antigens that reacted exclusively with sera from the "protected group". However, the staining of the reaction bands was most intense when sera from this patient group reacted with low molecular weight specific onchocercal antigens (M. W. 10-57 KD).