Biomedical subjects
R Lucius
Publications and source records attributed to R Lucius.
Copper stained protein gels can be efficiently used for immunoblotting.
We have found that the previously described fast and sensitive copper staining of proteins resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis does not interfere with the subsequent electrotransfer of these proteins to a solid support and their detection by specific antibodies. After the gel is copper stained and photographed it is simply destained and then equilibrated in transfer buffer prior to immunoblotting. We find that this treatment has no significant effect on transfer efficiency or band sharpness and is compatible with all common detection methods for the blotted proteins. It thus permits the separation of proteins to be checked in a simple way before immunoblotting is performed.
Degradation of the neuropeptide somatostatin by cultivated neuronal and glial cells.
The enzymatic degradation of the neuropeptide somatostatin was investigated in cultivated cells and subcellular fractions from rat brain. Dissociated neurones, astrocytes, and oligodendrocytes obtained from rat cerebral cortex were of more than 85-98% purity as evidenced by immunostaining with antisera to cell specific markers. All of these cell types were able to cleave radiolabeled somatostatin to smaller fragments, especially cultivated astrocytes with the highest specific activity. The neuroblastoma cell line N1E-115 did not measureably cleave somatostatin. The somatostatin-degrading proteases of the cultivated brain cells could be differentiated by their sensitivity to protease inhibitors and by the fragments produced: astrocytes contain a metallo-endoprotease sensitive to phenanthroline which cleaves somatostatin at the Phe6-Phe7 and Thr10-Phe11 bonds, whereas the endoprotease(s) of neurones and oligodendrocytes was insensitive to chelating agents but strongly inhibited by the antibiotic bacitracin. In accordance with this, the bacitracin-sensitive activity was mainly recovered in the synaptic plasma membrane and myelin subcellular fractions obtained by differential centrifugation of rat cerebral cortex homogenate. However, the highest total and specific somatostatin-degrading activity was detected in the cytosolic fraction.
Acanthocheilonema viteae: vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens.
Jirds (Meriones unguiculatus) were immunized with irradiated (35 krad) stage-3 larvae (L3) of Acanthocheilonema viteae. The induced resistance against homologous challenge infection and the antibody response of the animals were studied. Immunization with 3, 2, or 1 dose of 50 irradiated L3 induced approximately 90% resistance. Immunization with a single dose of only 5 irradiated L3 resulted in 60.8% protection while immunization with a single dose of 25 L3 induced 94.1% protection. The protection induced with 3 doses of 50 irradiated L3 did not decrease significantly during a period of 6 months. Sera of a proportion, but not all resistant jirds, contained antibodies against the surface of vector derived L3 as defined by IFAT. No surface antigens of microfilariae or adult worms were recognized by the sera. Vaccinated animals had antibody responses against antigens in the inner organs of L3 and in the cuticle and reproductive organs of adult worms as shown by IFAT. Immunoblotting with SDS-PAGE-separated L3 antigens and L3-CSN revealed that all sera contained antibodies against two exported antigens of 205 and 68 kDa, and against a nonexported antigen of 18 kDa. The 205-kDa antigen easily degraded into fragments of 165, 140, 125, and 105 kDa which were recognized by resistant jird sera. Various antigens of adult worms, but relatively few antigens of microfilariae, were also recognized. To test the relevance of exported antigens of L3 to resistance, jirds were immunized with L3-CSN together with a mild adjuvant. This immunization induced 67.7% resistance against challenge infection and sera of the immunized animals recognized the 205- and 68-kDa antigens of L3.
Immunohistological and electron microscopic studies of microfilariae in skin and lymph nodes from onchocerciasis patients after ivermectin treatment.
Microfilariae were studied in skin and lymph node biopsies from Liberian patients with generalised onchocerciasis 12-78 hours after administration of a single dose of 150 micrograms/kg body weight using histology, transmission electron microscopy and immunocytological staining with antibodies against an immunodominant antigen of Onchocerca volvulus. Most microfilariae in the skin appeared morphologically intact and beginning signs of degeneration were seen only on the ultrastructural level. The densities of microfilariae in the lymph nodes were about thousandfold higher in ivermectin treated patients. More than 90% of the microfilariae in the lymph nodes showed distinct signs of degeneration. Early changes were seen in the muscle cells. The disintegrating microfilariae in the lymph nodes were always encircled by eosinophils or macrophages or both cells. Immunohistological staining with antifilarial antibodies increased the detection of small and disintegrating pieces of microfilariae considerably.
Proline-specific proteases in cultivated neuronal and glial cells.
The activities of several proline-specific endo- and exopeptidases were determined in homogenates of immunocytochemically defined cultures of astrocytes, oligodendrocytes and neurones obtained from rat cerebral cortex. Astrocytes are significantly enriched in post-proline cleaving dipeptidyl peptidase II, prolidase and aminopeptidase P activities; neurones and astrocytes contain prolyl endopeptidase. Dipeptidyl peptidase IV and prolyl carboxypeptidase activities are low or absent in the cultures. The physiological significance of these findings is discussed.
Molecular cloning of an immunodominant antigen of Onchocerca volvulus.
We have cloned and characterized the gene for an immunodominant antigen of O. volvulus that is recognized by the sera of 96% of patients with onchocerciasis. Its 1.2-kb mRNA constitutes 0.3% of adult worm poly(A)+ RNA and its cDNA sequence reveals that it is not a highly conserved structural protein such as actin or tubulin. Similar but not identical genes occur in the genomes of related filarie, Brugia malayi and Dirofilaria immitis. The recombinant antigen has both immunodiagnostic and immunoprophylactic significance.
Characterization of an immunodominant Onchocerca volvulus antigen with patient sera and a monoclonal antibody.
Adult Onchocerca voluvlus and infective larvae, but not microfilariae contain an immunodominant antigen (33,000 and 21,000 Mr in females, 39,000, 33,000, and 21,000 Mr in males, 133,000 Mr in infective larvae) which is recognized by an Onchocerca-specific mAb. The component is part of the reproductive organs and muscles. 96.2% of onchocerciasis sera contained antibodies detectable by immunoblotting against it. Antigen purified by immunoaffinity chromatography was specifically recognized in immunoblots by onchocerciasis sera, but not by sera from other filarial infections. The high immunogenicity, the specificity, and the occurrence in infective larvae of this antigen indicate an immunodiagnostic potential and a possible role in the immunobiology of the parasite.
Construction of Onchocerca volvulus cDNA libraries and partial characterization of the cDNA for a major antigen.
Adult Onchocerca volvulus were isolated from nodules removed from onchocerciasis patients at four locations--two in the West African Sudan-savanna region (near Bamako, Mali, and Touboro, Cameroon), one in a West African forest region (Kumba, Cameroon) and one near Guatemala City, Guatemala. Four different cDNA expression libraries were constructed in bacteriophage lambda gt11 using poly(A)+ RNA from the adult female worms. Individual cDNA clones of single copy genes were used to compare the genomes of parasites from the different locales and to show that the haploid genome of O. volvulus is 1.5 x 10(8) base pairs. About 1 in 700 recombinant clones in each of the four amplified cDNA libraries produces a fusion protein recognized by pooled human anti-O. volvulus antisera. Partial sequence determination of a 2.0 kb cDNA clone for an O. volvulus protein that induces an immunodominant response in rabbits revealed that this antigen has sequence similarities with Caenorhabditis elegans myosin and with schistosome paramyosin (which confers partial protection against schistosome infection). The four cDNA libraries have been deposited with American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, U.S.A., for general distribution under ATCC Number 37509.
Dipetalonema viteae infection in three species of rodents: species specific patterns of the antibody response.
Groups of jirds (Meriones unguiculatus), multimammate rats (Mastomys natalensis) and golden hamsters (Mesocricetus auratus) were infected with third stage larvae (L3) of Dipetaloneam viteae and the course of infection was followed until 20 weeks post infection. Worm growth was best and microfilaraemia was high and long lasting in jirds and in multimammate rats, whereas golden hamsters were poor hosts as measured by these parameters. The IgG and IgM antibody responses of the species were compared by immunoblotting and ELISA using proteins of D. viteae, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The levels of antibodies against most proteins of high molecular weight declined during infection in jirds and in multimammate rats, whereas an increase was observed in golden hamsters. In contrast, several antigens of low molecular weight induced increasing antibody levels in all species. Species specific antigen recognition was observed for a number of protein bands of L3, microfilariae and female worms. The data suggest that susceptibility to immunesuppression and the species specific pattern of antigen recognition might determine the qualities of a rodent species as host for D. viteae.
Antibody responses in forest and savanna onchocerciasis in Ivory Coast.
Three foci of onchocerciasis transmission (two forest and one savanna, respectively) in the Republic of Ivory Coast were chosen for a comparative analysis of Onchocerca volvulus antigens and of patients' antibody responses. Clear differences between frequency and intensity of ocular pathology existed between the forest foci and the savanna focus. We found heterogeneity of SDS-PAGE-separated components of female worms with respect to the mobility of three protein bands of the 100 KD region and to the ability of an 80 KD band to bind horseradish peroxidase. These variations were clearly not associated with the degree of ocular pathology. No differences in the composition of worm antigens were detected by immunoblotting. A variable with a possible relation to ocular pathology was the antibody response of patients: individuals from the savanna focus, where a high degree of ocular pathology is observed, tended to have a stronger and more differentiated IgG antibody response against O. volvulus antigens than patients from the forest foci. However, no antigens specifically recognized by patients from either forest or savanna were detected.
Dipetalonema viteae: resistance in Meriones unguiculatus with multiple infections of stage-3 larvae.
The jird, Meriones unguiculatus, infected with 80 normal infective larvae of Dipetalonema viteae, revealed a recovery rate of 27.9% 12 weeks after infection. A pretreatment by three injections of 50 normal larvae each and challenge by 80 larvae resulted in a recovery rate of 10.7%. The recovered worms were longer than those from the challenge control animals. When three times 50 irradiated larvae (35 krad) were inoculated, the recovery rate of the challenge decreased to 2.6%, representing a protection of 90.7%. The surviving adult worms were stunted and derived exclusively from the 80 normal larvae given for challenge, since absolutely no adult worms were recovered in eight animals inoculated three times with 50 irradiated larvae only. Sera of all pretreated jirds contained IgG and IgM antibodies which bound in immunoblotting experiments bound predominantly to three proteins of larvae with molecular masses of 68,140, and 165 kDa, respectively. Enzymatic surface iodination revealed that the three antigens were exposed on the larval surface. The coincidence of a partial resistance to a challenge infection and of an antibody response against surface proteins of infective larvae suggests an importance of these antigens for the rejection of D. viteae mediated by an acquired immunological resistance of M. unguiculatus.
A study on antigen recognition by onchocerciasis patients with different clinical forms of disease.
Sera from 40 onchocerciasis patients from the Yemen Arab Republic with either mild localized forms of onchocerciasis, intermediate or severe localized forms of the disease or generalized forms of infection were studied with respect to their IgG and IgM response against Onchocerca volvulus antigens. Immunoblotting, performed with SDS-PAGE-separated proteins of female O. volvulus and quantified by densitometric scanning, revealed IgG and IgM antibodies against worm components in sera of all patients. Persons with intermediate or severe localized forms of onchocerciasis had a stronger IgG response against more proteins than individuals of the other groups. However, some antigens (Mr 21, 23, 30, 33 kDa) induced comparable quantities of IgG in all groups. The IgM response of patients with mild localized forms of onchocerciasis was more intensive and directed against more antigens than in the other groups. No antigens were detected that were recognized only by individuals with low levels of microfilaridermia. In all groups, varying concentrations of antibodies against cuticle, muscle/hypodermis layer and/or uterus of female O. volvulus were detected by the indirect immunofluorescence test using frozen worm sections as antigen. The highest mean antibody titres were found in patients with intermediate and severe localized forms of disease.
Behavioural changes in adult rats produced by early postnatal maternal deprivation and treatment with choline chloride.
Rats deprived temporarily of maternal contact and nutrition from the 3rd to 14th day of life showed decreased emotionality and poor memory capacity when tested in adulthood. Simultaneous administration of choline chloride, an acetylcholine precursor, in neonatal life permanently increased the emotionality and restored memory capacity to levels of control animals. However, no effect was demonstrated when choline chloride administration occurred after the deprivation period. The results of this study reveal that the effects on later behaviour appear to be dependent on the timing of choline chloride exposure in early life. Profound and long-lasting abnormalities in brain function produced by early postnatal maternal deprivation can be attenuated by correcting abnormal acetylcholine levels during brain development. Hence, acetylcholine can be considered as an important, environment-dependent local organizer of the brain.
Schistosoma mansoni: immunoblot analysis of adult worm proteins.
Proteins of adult Schistosoma mansoni were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed in immunoblots for reactions with individual mouse sera. Four weeks after a heavy infection with a few hundred cercariae, IgG antibodies directed predominantly against a protein of 31 kDa were detected. The protein was only weakly recognized by antibodies of mice harboring a 4-week-old light infection with about 60 cercariae. After 6 weeks or more, mice infected with either dose formed antibodies, not only against the 31-kDa protein and a 67-kDa protein, but also against a number of other components. While reactions with the 31- and 67-kDa proteins occurred with sera of all individual mice of four different strains, the reactions with other components were less consistently observed. Mice vaccinated with a heavy or light dose of 20,000-rad-irradiated cercariae did not form antibodies detectable in the blotting system. However, in immunofluorescence assays with living skin schistosomula, but not lung schistosomula, antibodies against the larval surface were detected with all sera obtained 4 weeks after infection or vaccination. In addition, immunofluorescence studies using the same sera and sectioned adult parasites demonstrated the presence of antibodies against the parasite surface in all sera except those obtained from mice exposed to a light infection with normal cercariae. Mice infected in this latter way were the only animals that did not develop a significant resistance against a challenge infection 4 weeks after exposure to normal or irradiated cercariae. The presence of an immunofluorescent reaction against the schistosome gut always coincided with a reaction of the sera with the 31-kDa protein in the immunoblots. Although a role in immune resistance could not be ascribed to any of the proteins reacting in the immunoblots, the data demonstrate important differences in the antibody specificities induced by various infection schemes.
Studies on the protein and antigen composition of individual female Onchocerca volvulus after collagenase digestion.
The composition of proteins and antigens of female Onchocerca volvulus from one focus of transmission was studied by SDS-PAGE and immunoblotting. Worms that had been exposed to collagenase digestion of onchocercomata for different periods of time and parasites of different age and status of reproduction were tested. Some O. volvulus antigens were found to be sensible to prolonged digestion (molecular weights: 18 KD, 21 KD, 24 KD) but the majority of the antigens was stable up to 64 hours of incubation at 34 degrees C. The composition of proteins and antigens varied with the age and the status of reproduction of the worms. Slight differences between individual filariae were found, even when worms of comparable age and status of reproduction were tested that had been exposed to nodule digestion for comparable time.
Identification of immunogenic proteins of Dipetalonema viteae (Filarioidea) by the "Western Blotting" technique.
Sodiumdodecylsulphate (SDS)-solubilized proteins of several developmental stages of Dipetalonema viteae (adults, uterus content, microfilariae, infective larvae) were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter sheets. Their antigenicity in natural infections of Meriones unguiculatus was investigated by immuno-recognition with pooled sera of infected hosts. The antibodies were labelled by radioiodinated protein A and visualized by autoradiography. Immunorecognized protein bands were demonstrable in all stages, some of them being stage specific, others common to several or all stages. The highest number of labelled protein bands was found in extracts of adult worms, the lowest in preparations of microfilariae and infective larvae.
Camponotus compressiscapus André (Hymenoptera, Formicidae) an experimental second intermediate host of Dicrocoelium hospes Looss, 1907 (Trematodes, Dicrocoeliidae).
Infection experiments showed that the African ant Camponotus compressiscapus can serve as a second intermediate host of Dicrocoelium hospes. Most of the cercariae penetrated the crop-wall and encysted in the hemocoel, whereas one or more larvae invaded the ant's central nervous system. These larvae cause behavioral changes, which increase their chance of being ingested by final hosts. Experimental infections of definitive hosts were obtained in golden hamsters, Meriones unguiculatus, guinea-pigs, and sheep.