Surface antigens of male worms and microfilariae of Onchocerca gibsoni.

Living adult males and microfilariae of the cattle filarial parasite Onchocerca gibsoni were externally labelled with radioactive iodine using the iodogen and Bolton-Hunter procedures. Characterization of labelled surface proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed clear cut differences in the two life cycle stages. In addition, the two radiolabelling procedures yielded some differences in the profiles of radiolabelled surface proteins for both adults and microfilariae. Immunoprecipitation analysis revealed a number of labelled antigens recognized by antibodies in human onchocerciasis serum pools, thereby demonstrating the usefulness of O. gibsoni as a model in Onchocerca volvulus vaccine studies. The reactivity of microfilarial antigens extended to antibodies from other human nematode infections, whereas male surface antigens, particularly those of low molecular weight, were Onchocerca specific. This indicates that O. gibsoni can provide a convenient source of specific diagnostic antigen.

[Hearing and language sequelae in survivors of a neonatal intensive care unit]. / Secuelas de audición y lenguaje en sobrevivientes de una Unidad de Cuidados Intensivos Neonatales.

INTRODUCTION: The purpose of this study is to record the hearing and language sequelae in a sample of children from the Intensive Care Unit between the ages of two and three. MATERIALS AND METHODS: Forty-one children were studied after being submitted to neuropsychological, hearing and language tests. Both normal and abnormal parameters for each test allowed the categorization of the children. RESULTS: In the general sample there were 24 full-term children versus 17 preterm children. The neurological testing showed a tendency towards normality. The same was seen in language testing since the proportion of normality corresponded to those children born after a full term. There were practically no differences in those suspected from both groups and a greater percentage of abnormal children were found among the pre-term infants. From an audiological standpoint there was a predominance of normal children; there was only one patient with severe bilateral hypoacusis who needed an electric auxiliary hearing device and two other patients with peripheral ear dysfunction classified as serous middle ear otitis. CONCLUSIONS: The incidence of hypoacusis in this sampling type is similar to that reported in the literature. Those patients with a history of assisted mechanical ventilation have been later found to have, as a sequelae, serous middle ear otitis, which corresponds to that reported by Paradise. The development of language stages were altered more so in preterm children. In some patients, the lack of stimulation associated with a low socioeconomic back group favors the delay in the development of language skills.

Specific and cross-reacting antibodies in human responses to Onchocerca volvulus and Dracunculus medinensis infections.

Immunoelectroblotting and enzyme-linked immunosorbent assay were used to identify non-cross-reacting antigenic components of Dracunculus medinensis and the filarial worms Onchocerca volvulus, Loa loa, Wuchereria bancrofti, Brugia malayi, and Mansonella ozzardi. Parasite specific serodiagnostic ELISA systems for onchocerciasis and dracunculiasis were devised based on these findings. Phosphate buffered saline extracts of adult worms were passed through a column of monoclonal antibodies to phosphorylcholine (PC). Crude and PC-depleted extracts were reacted on ELISA plates with individual sera from subjects infected with a range of nematodes. Binding of total antibody (Ig) or IgG class antibody and IgG4 subclass antibody was revealed using goat antihuman-Ig-phosphatase conjugate, or appropriate mouse monoclonal antihuman-Ig-type-specific reagents, followed by goat antimouse-Ig-phosphatase conjugate. Specificity of ELISA was improved by restricting reaction to the host's IgG4 antibody subclass, and/or by removing PC determinants from crude antigens. In parallel immunoelectroblots, crude and PC-depleted extracts probed with pooled sera showed potentially useful diagnostic antigens, including a 12 kDa protein from D. medinensis and 14, 18, and 27 kDa proteins from O. volvulus. Two Onchocerca specific ELISA systems non-reactive with antibodies to D. medinensis were devised.

Specific detection of human antibodies to Onchocerca volvulus.

Specific diagnosis of antibodies to Onchocerca was achieved through (1) the construction of direct and indirect ELISA systems, and (2) restricting ELISA assays to the IgG4 class. The direct ELISA was based on the isolation of a surface derived, low molecular weight surface antigen preparation containing two main antigens (M. wt. 16.2 and 12.8 kDA) as defined by Western blot analysis. The direct ELISA system detected antibodies in children of six years old, and may therefore be applicable to detecting reinvasion in OCP areas of Onchocerca volvulus control. The indirect ELISA system was a competitive binding ELISA-based assay using a monoclonal antibody recognising two Onchocerca components (M. wts. 15.6 and 25.9) on a Western blot. The direct and indirect ELISA systems were similarly specific and sensitive when evaluated in a preliminary survey. The direct ELISA system yielded a specificity and sensitivity of: 100% and 100% respectively, using Mexican endemic and Mexican intestinal nematode infection sera as positive and negative controls respectively: 91% and 96% respectively, using Venezuelan endemic and Venezuelan Mansonella ozzardi infection sera as positive and negative controls, respectively: 87% and 93% respectively, using African endemic and Papuan (New Guinea) Wuchereria bancrofti infection sera as positive and negative controls respectively: 93% and 93% respectively, using African endemic and Indian W. bancrofti infection sera as positive and negative controls respectively. Similar specificity and sensitivity levels were obtained when the same comparisons were made using the indirect (inhibition) ELISA assay. These values may be contrasted with the currently used PBS extract of O. volvulus which yielded specificities of less than 10% in all the above comparisons.(ABSTRACT TRUNCATED AT 250 WORDS)

[The prone position as an indicator of deviated motor development during the first year of life]. / La posición prona como indicador de un desarrollo motor desviado en el primer año de la vida.

The adequate control and alignment of a baby in the prone position during the first year of life is a basic necessary milestone for a normal, neuromotor development. In this study the prone position was registered in 246 babies with a previous pediatric evaluation, three options could be made: normal retarded or altered. The Milani Comparetti neuromotor evaluation was also realized for each baby at the same time with the same classification options. Both results were compared. A sensitivity and specificity = 1.0 were found for the prone position registered. This test is fast and simple, so it is recommended at a first attention level in high risk population for neuromotor disability.

[The Apgar score as a predictor of neurologic sequellae]. / El puntaje de Apgar como predictor de secuelas neurológicas.

65 newborns with Apgar score less than 7 were studied. Initially 2 groups were formed. Group 1 with severe asphyxia Apgar 0-3 and group 2 with moderate asphyxia Apgar 4-7. Each group was divided according to Apgar score at 5 minutes; with good recuperation Apgar greater than or equal to 6 and bad recuperation Apgar less than 6. So four subgroups were formed. All patients were evaluated with Amiel Tison neurological examination at 12 months old. Of the 65 newborns, 52 (80%) had a normal neurologic examination, and only 12 (20%) were not normal. There were not significant statistic differences neither between the moderate and severely asphyxiated groups nor between the four subgroups at 5 minutes and their neurological examination. We conclude that the Apgar score at 5 minutes is not a good predictive of neurological sequelae, because there is a myriad of factors difficult to investigate in relation to the etiology of neurologic sequelae. We stress the importance of reviewing the original idea about the Apgar score as a useful method (instrument) to evaluate the hemodynamic and homeostatic conditions of the newborns.

[Neurologic follow-up of polycythemic newborn infants]. / Seguimiento neurológico de neonatos policitémicos.

Seventy five newborn infants with neonatal polycythemia diagnosis (venous hematocrit levels greater than 65% were studied). They were born in the Instituto Nacional de Perinatologia and follow through their first year of life searching for neurologic findings. The more frequent signs related to polycythemia in this group were: presence of plethora, respiratory distress, cyanosis and tremors; the associated findings were hypoglycemia, hyperbilirubinemia, necrotizing enterocolitis and meconium aspiratum. Sixty eight of the seventy five neonates developed symptomatology and seven didn't. The eight neonates who developed neurologic transient impairments belonged to the symptomatic group in a 0.11 proportion. We concluded that the number of babies with neurologic findings in our sample was small in comparison with other reference studies; we also found that the exchange transfusion is not a preventive measure for neurologic sequel, but it is useful to diminish the symptomatology caused by polycythemia; besides we don't recommend this procedure in asymptomatic babies because the complication risks as the enterocolitis. We propose a simple treatment scheme.

Unique recognition of a low molecular weight Onchocerca volvulus antigen by IgG3 antibodies in chronic hyper-reactive oncho-dermatitis (Sowda).

Individual human Ig class responses to Onchocerca volvulus antigens have been evaluated by Western blotting using sera from cases of generalized onchocerciasis and chronic hyper-reactive onchocerciasis (Sowda). in all cases except IgG3 the patterns of recognition by human antibody classes were similar in Sowda and generalized onchocerciasis. Weak or undetectable responses were seen with IgG1, IgG2 and IgM. The total profiles of antigens recognized by the other Ig classes were different, although in some cases certain bands were commonly identified. The result with IgG3, however, was striking. Here, two major antigens (9 kD and 72kD) were recognized by IgG3 antibodies in Sowda sera but not generalized onchocerciasis sera. Furthermore, these two antigens were not recognised by any other Ig class, either in generalized or Sowda onchocerciasis, nor were they detected by antibodies of any class present in a collection of sera representative of other nematode infections. This difference in the IgG3 response was so pronounced that Sowda sera could be distinguished from generalized onchocerciasis sera by an IgG3-specific ELISA assay with a PBS parasite extract as the antigen. Thus, a correlation has been established between one particular clinical condition of onchocerciasis (Sowda) and a serological response, defined in terms of both the parasite antigens and an immunoglobulin class restricted antibody response.

Nematode antigens in protection, diagnosis and pathology.

A thorough study of parasite antigens is a prerequisite for control programmes based on protection by vaccination, accurate serodiagnosis and perhaps immune modulation to diminish pathological sequelae. Stage specific surface secreted and somatic antigens may be of particular value in proceeding towards these goals. The design of vaccines is most appropriately focused on surface antigens. With respect to pathology, certain antigens must stimulate humoral and, or cellular immune responses which are responsible for the undesirable immunopathologic consequences of the disease. The ultimate objective, therefore, is identification of those particular antigens followed by appropriate down regulation of the immune system in order to delete such potentially harmful immunological reactions. The relevant illustration presented in this context is an interesting correlation between one particular clinical condition of onchocerciasis ("sowda") and the serological response, defined both in terms of the parasite antigen and an immunoglobulin class restricted antibody response. Current parasitological methods of diagnosis consistently underestimate parasite prevalence. Failure to detect low level patent infections incurs the risk of having a reservoir capable of perpetuating infections. There is, then, an urgent requirement for accurate serodiagnosis, to be used in association with, and for the evaluation of, drug treatment and vector elimination in parasite control programmes. Given the high sensitivity of current immunoassay technology, the only bar to establishing the necessary immunological tests is the choice of suitably specific antibody-antigen systems. Once these are identified, a combination of recombinant nucleic acid biochemistry and hybridoma technology should provide the necessary reagents for inexpensive, robust and specific diagnostic tests. In addition, it may not be many years before the ubiquitous RIA and ELISA technology gives way to the newly developing biosensor systems. Finally, given the sensitivity and specificity of today's nucleic acid hybridization techniques, we may soon expect to see specific identification of infective larvae in their vectors of this, a cloned DNA probe specific for Onchocerca volvulus, and with potential for the detection of infective larvae in blackflies is described.

Isolation of an antigenic fraction for diagnosis of onchocerciasis.

A surface enriched fraction was prepared from adults of Onchocerca volvulus by brief extraction of entire worms with detergent. This was then gel filtered to yield a low molecular weight fraction which functioned specifically in ELISA analysis. An identical result was also obtained when the related cattle parasite, O. gibsoni, was similarly fractionated and tested. The low molecular weight fraction contained at least four antigenic components when examined by coprecipitation and immunoblotting studies. One ml of packed worms yielded sufficient low molecular weight antigen to examine about 2,000 human sera by the ELISA procedure, and the test was sensitive at human serum dilutions down to 1/400. A preliminary study with individual sera from Onchocerciasis endemic and non-endemic areas of Southern Mexico yielded 0/24 false positives, 3/24 false negatives and a significant ELISA value in 21/24 sera from proven cases of Onchocerciasis.

Onchocerca antigens in protection, diagnosis and pathology.

Characterization of the immune response to Onchocerca volvulus is important for the diagnosis, control and understanding of the disease it causes. The antibody response to surface, secreted and somatic antigens of the worm has therefore been examined at an individual immunoglobulin (Ig) class level, by using a panel of different human sera. Onchocerca-specific antigens tend to be of low molecular mass and preferentially recognized by IgG4 and IgE. There is considerable cross-reaction between O. volvulus and O. gibsoni, so that the latter may be an alternative source of material for use in diagnosis. A surface-enriched fraction of low molecular mass appears to be a most promising diagnostic tool. Amongst somatic antigens, two were uniquely recognized by IgG3 antibodies in sera from sowda patients, thereby providing a molecular correlate for a recognized pathological condition. Improved diagnosis is needed for detecting infection in both humans and the vector. Our target for detection in humans is a continuously released, nonimmunogenic product, which is ideally stage and parasite specific. The excretions of adult worms do contain components not recognized by antibodies in infected serum, but we cannot rule out that these are of host, rather than parasite origin. Excretions of Litomosoides carinii contain both host and parasite molecules and, in addition, stage-specific and sex-specific components. Unfortunately, however, the rate of production of excretions varies during the life of L. carinii. This finding may be relevant to the detection of Onchocerca excretions if they are produced at a similarly uneven rate. Finally, for detecting infective larvae in the vectors, we are currently screening a genomic library of O. volvulus for an appropriate probe. To date, one DNA sequence has been cloned that shows promising specificity.

Immunization of hamsters with TLCK-killed parasites induces protection against Leishmania infection.

Hamsters immunized with N-p-tosyl-L-lysine-chloromethyl ketone TLCK-treated L. brasiliensis brasiliensis (LB) from culture, infected with LB amastigotes presented: a gradual increase in T and B cell responsiveness to mitogens by lymph node lymphocytes, and an increased response to concanavalin A with no changes for dextran sulphate and pokeweed mitogen in splenocytes. Absence of parasites in lymph nodes after 6 weeks post-infection and a nodule 4 times smaller than that of infected control animals. The nodule was undetectable after 70 days of infection. Hamsters preimmunized with TLCK-treated L. donovani (LD) from culture did not show suppression of the blastogenic response to mitogens of spleen and lymph node cells after infection with LD amastigotes and survived for more than one year, whereas infected, unimmunized animals died five months after infection. Animals preimmunized with culture parasites (LB or LD) treated with phenyl-methyl-sulphonyl-fluoride (PMSF) and infected with LB or LD amastigotes did not show any protective effect.

Identification of antigens of Onchocerca volvulus and Onchocerca gibsoni for diagnostic use.

Adults of Onchocerca volvulus and Onchocerca gibsoni were identically fractionated into a surface-enriched fraction, a phosphate buffered saline (PBS) extract and a PBS insoluble-detergent soluble fraction. Glycoproteins were prepared from these extracts and all fractions were examined by the Western blot technique using sera from individuals infected with a variety of filarial and non-filarial nematode worms. Using antisera to O. volvulus, a number of antigens were demonstrated in all of the extracts, with some antigens of each extract being unique. Many antigens were glycoproteins, and a high cross-reactivity was observed between O. volvulus and O. gibsoni. The different fractions of both species were also analysed using a panel of different sera in order to identify Onchocerca-specific antigens. The studies revealed that the lower molecular weight antigens showed greater Onchocerca specificity in all of the extracts examined. The surface-enriched fraction, however, clearly contained less widely cross-reacting components than the somatic and glycoprotein fractions. Finally, using surface labelling and coprecipitation techniques, O. gibsoni was shown to possess a 20 kDa Onchocerca-specific antigen, previously described for O. volvulus. The findings indicate a number of Onchocerca-specific antigens which may have potential in diagnosis of human onchocerciasis. They also show that the related bovine parasite O. gibsoni, may be an alternative source of material.

Differential recognition patterns of human immunoglobulin classes to antigens of Onchocerca gibsoni.

Adult Onchocerca gibsoni worms were fractionated into a surface-enriched fraction, a saline extract, a saline insoluble-detergent soluble fraction and a total glycoproteins extract. The antigens in each fractions were separated by electrophoresis in polyacrylamide gels and examined with an immuno-blot technique for reactive antibodies in sera from individuals infected with a variety of filarial and non-filarial nematode worms. Radiolabelled monoclonal antibodies were used to determine the Ig heavy chain isotypes. A number of antigens were demonstrated in all of the extracts, with many antigens of each extract being unique. Although some Onchocerca antigens stimulated antibodies of all human immunoglobulin classes, the panel of antigens recognized by each Ig isotype was different. The IgE response was restricted and directed at antigens not recognized by antibodies to other nematode parasites. IgM and IgA responses tended to recognize many antigens, whilst IgG responses were directed at intermediate numbers of antigens. The control of isotype balance to individual parasite antigens is thus independently regulated. This survey provides a rational basis for the exploration of Onchocerca antigen-human antibody class systems with relevance for diagnosis, protection and pathology.

Serum protein from Leishmania brasiliensis infected hamster that suppress lymphocyte response of normal hamster lymphocytes.

Immunosuppression in Leishmania brasiliensis (LB) or L. donovani (LD) infected hamsters is correlated with the appearance of two serum protein bands found at 21, 60, 68 and 76 days post LB-infection and with eight bands at 21 days post-LD-infection probably of host origin. A protein band from LB-infected hamster serum isolated by electrofocusing, suppressed the blastogenic response of normal lymphocytes to T and B cell mitogens.