Modulation of host immune responses by protozoal DNA

The pathology caused by acute Babesia bovis infection is similar to that seen in severe human malaria caused by Plasmodium falciparum infection, which is related to dysregulated production of inflammatory cytokines and nitric oxide (NO). We have observed induction of NO, inducible nitric oxide synthase (iNOS) and inflammatory cytokines in macrophages by B. bovis. Furthermore, proliferation of lymphocytes from individuals never exposed to certain protozoal pathogens can be induced by crude protozoal parasite extracts. We have repeatedly observed stimulation of naive PBMC from cattle to antigenic extracts of Babesia bovis. Based on recent studies demonstrating the mitogenicity of bacterial and other non-vertebrate DNAs for murine B cells and macrophages, the mitogenic properties of B. bovis DNA were examined. B. bovis and E. coli DNAs induced proliferation of PBMC and purified B cells from non-exposed cattle. Stimulatory activity was reduced by DNase treatment and methylation with CpG methylase, indicating the presence of stimulatory non-methylated CpG motifs in the B. bovis genome. B. bovis and E. coli DNAs enhanced IgG secretion by cultured B cells, stimulating IgG1 and more strongly, IgG2. Several hexameric CpG immunostimulatory sequences (ISS) active for murine B cells were identified in an 11 kb fragment of B. bovis DNA. An oligodeoxyribonucleotide containing one of these (AACGTT), located in the rhoptry associated protein-1 (rap-1) open reading frame, stimulated B cell proliferation. These studies identify a potential mechanism by which protozoal parasites may modulate host immune responses, leading to consequences such as hypergammaglobulinemia and splenomegaly. These results also support the use of ISS as vaccine adjuvants to enhance Type 1 immune responses in cattle.     

Pathogenicity of Rhodococcus equi expressing a virulence-associated 20 kDa protein (VapB) in foals

Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79-100kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10(6) and 10(9) cells of R. equi IMV strain A5, respectively. The foal infected with 10(9) cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10(6) cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10(5) or 10(6) of virulent R. equi ATCC 33701 having 15-17kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10(9) bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.     

Rapid and simple mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of deoxyribonucleic acid from various forms of canine whole-blood specimens.

This report describes a rapid and simple method for mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine beta-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of G(M1) gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.     

Decolorization of kraft pulp bleaching effluent by a newly isolated fungus, Geotrichum candidum Dec 1

Kraft pulp bleaching effluent supplemented with glucose was decolorized by a newly isolated fungusGeotrichum candidum Dec 1 (Dec 1) that showed a wide decolorizing spectrum to synthetic dyes. When the glucose concentration in the effluent was 30 g/l, the color removal and the reduction of absorbable organic halogens were 78% and 43% after 7 days culture, respectively. The average molecular weight of colored substances measured by gel filtration chromatography was lowered to less than 3000 from 5600 after 7 days culture. As the contribution of extracellular enzymes such as peroxidase (DyP), manganese peroxidase, and laccase to the decolorization of the kraft pulp bleaching effluent was small, Dec 1 appears to have a different mechanism of decolorizing kraft pulp bleaching effluent when compared with enzymes used to decolorize synthetic dyes.     

Retrospective diagnosis of feline GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japan: possible spread of the mutant allele in the Japanese domestic cat population

GM2 gangliosidosis variant 0 (human Sandhoff disease) is a lysosomal storage disease caused by simultaneous deficiencies of acid beta-hexosaminidase (Hex) A and Hex B due to an abnormality of beta-subunit, a common component in these enzyme molecules, which is coded by the HEXB gene. In the present study, a retrospective diagnosis was performed in 2 previous suspected cases of feline Sandhoff-like disease using a DNA test to detect the causative mutation identified previously in 4 cats in 2 other families of Japanese domestic cats. Enzymic analysis was also performed using stored leukocytes and plasma collected from the subject families in order to investigate the usefulness of enzymic diagnosis and genotyping of carriers. The DNA test suggested that the 2 cases were homozygous recessive for the mutation. Consequently, 6 cats homozygous for the same mutation have been found in 4 separate locations of Japan, suggesting that this mutant allele may be spread widely in the Japanese domestic cat populations. In enzymic analysis, Hex A and Hex B activities in leukocytes and plasma measured using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate were negligible in affected cats, compared with those in normal and carrier cats. However, there was a wide overlap in enzyme activity between normal and carrier cats. Therefore, it was concluded that enzymic analysis is useful for diagnosis of affected cats, but is not acceptable for genotyping of carriers.     

Clinical and clinico-pathologic characteristics of Shiba dogs with a deficiency of lysosomal acid beta-galactosidase: a canine model of human GM1 gangliosidosis

The present study was conducted to determine the clinical and clinico-pathologic characteristics of Shiba dogs with GM1 gangliosidosis, which is due to an autosomal recessively inherited deficiency of lysosomal acid beta-galactosidase activity. Clinical and clinico-pathological features were investigated in 10 homozygous Shiba dogs with GM1 gangliosidosis. The age at onset was 5 to 6 months and the dogs manifested progressive neurologic signs including loss of balance, intermittent lameness, ataxia, dysmetria and intention tremor of the head. The dogs were unable to stand by 10 months of age due to a progression of ataxia and spasticity in all limbs. Corneal clouding, a visual defect, generalized muscle rigospasticity, emotional disorder and a tendency to be lethargic were observed at 9 to 12 months. The dogs became lethargic from 13 months of age. The survival period seemed to be 14 to 15 months. As a clinico-pathologic feature, lymphocytes with abnormally large vacuoles were observed in peripheral blood (30 to 50% of total lymphocytes) through the lifetime of the dogs. The clinical and clinico-pathologic characteristics of this animal model are useful for not only the development and testing of potential methods of therapy, but also the diagnosis of affected homozygous Shiba dogs in veterinary clinics.     

Comparison of polymerase chain reaction-restriction fragment length polymorphism assay and enzyme assay for diagnosis of G(M1)-gangliosidosis in Shiba dogs.

In the present study, diagnostic methods for canine G(M1)-gangliosidosis were examined by comparing a DNA mutation assay with an enzyme assay. Sixty-two Shiba dogs of a pedigree with G(M1)-gangliosidosis were differentiated into 3 genotypes, i.e., normal, heterozygous, and homozygous affected dogs, using a DNA mutation assay, which consists of polymerase chain reaction amplification and the determination of restriction fragment length polymorphisms. The beta-galactosidase activity in leukocytes, umbilical cords, and plasma was measured using 4-methylumbelliferyl beta-D-galactoside and p-nitrophenyl beta-D-galactoside as artificial substrates and compared among the 3 genotypes. The results showed that it was possible to identify homozygous dogs with the enzyme assay using leukocytes and umbilical cords. When using leukocytes, heterozygous carriers could be differentiated from normal dogs in many cases. However, the use of the DNA mutation assay is essential for a complete determination of heterozygous carriers because of the overlap in the distribution of enzyme activity between these 2 groups. When umbilical cords were used, heterozygous carriers could not be differentiated from normal dogs because of no significant difference in enzyme activity between these 2 groups. The beta-galactosidase activity in plasma was not applicable to the diagnosis and genotyping of G(M1)-gangliosidosis in Shiba dogs.     

GM2-gangliosidosis variant 0 (Sandhoff-like disease) in a family of Japanese domestic cats

A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.     

Delay of gratification in children

To function effectively, individuals must voluntarily postpone immediate gratification and persist in goal-directed behavior for the sake of later outcomes. The present research program analyzed the nature of this type of future-oriented self-control and the psychological processes that underlie it. Enduring individual differences in self-control were found as early as the preschool years. Those 4-year-old children who delayed gratification longer in certain laboratory situations developed into more cognitively and socially competent adolescents, achieving higher scholastic performance and coping better with frustration and stress. Experiments in the same research program also identified specific cognitive and attentional processes that allow effective self-regulation early in the course of development. The experimental results, in turn, specified the particular types of preschool delay situations diagnostic for predicting aspects of cognitive and social competence later in life.     

High Performance Size Exclusion Chromatography of Water Extract from Sewage Sludge-Soil Mixture

In order to investigate the degradation or synthetic process of organic matter in soil, some instrumental analyses on the water extract from soil have been recently performed. SCHAUMBERG et al. (1) who observed the changes in the chemical nature of water-soluble components of sewage sludge-soil mixtures by using infrared spectroscopy, supplied valuable information on the fate of sludge organic components in soil. The authors reported (2, 3) that the stabilization process of sewage sludge in soil can be monitored by the conventional gel chromatography method with Sephadex G-15 in water extracts from sewage sludge-soil mixtures.

The previous works (2, 3) showed that macromolecular compounds which cannot be separated by Sephadex G-15 gel were present in the water extracts from sewage sludge-soil mixtures. High performance size exclusion chromatography (HPSEC) offers the possibility to estimate the molecular size distribution of such macromolecules, because HPSEC allows rapid size separation and high resolution. In the size separation of water-soluble compounds by HPSEC, however, the charge exclusion and the hydrophobic interaction between solutes and the stationary phase of the column caused accelerated or retarded transportation of water-soluble compounds through the column, leading to the erroneous estimation of the molecular size (4).

In this study, the authors applied HPSEC to water extracts from sewage sludgesoil mixtures during their process of decomposition in soil to observe the change of the molecular size distribution of the components.