Histidinuria: A renal and intestinal histidine transport deficiency found in two mentally retarded children

Two siblings presenting slight mental retardation showed an abnormal elimination of histidine, their blood levels for the same amino acid being normal. The percentage of tubular resorption of histidine was calculated in both boys, and the values were 40.1 per cent (case 1) and 52.8 per cent (case 2). All other amino acids essayed were normal. After an oral overload test with histidine, a low intestinal absorption was found in the two boys, the values of this test in the parents being intermediate between those of the children and of the three normal controls and corresponding to heterozygosity. In view of the studies carried out on the two boys, it is possible to conclude that they are suffering from an impairment in their histidine membrane transport system which affects the kidney and intestines. Since they are siblings a genetically determined trait may be suspected.     

Lymphocyte activation: IV. The ultrastructural pattern of the response of mouse T and B cells to mitogenic stimulation in vitro

Thymocytes from cortisone-treated mice (`T' cells), `B' spleen cells (B lymphocytes from thymectomized, irradiated, marrow reconstituted mice) and normal spleen (T + B) cells were examined by electron microscopy after 60 hours stimulation by Concanavalin A (a T cell specific mitogen), endotoxin (B cell specific mitogen), and pokeweed mitogen (which stimulates both T and B cells). Stimulation of T cells by Con A or PWM induced the appearance of lymphoblasts (Type I) and only PWM or endotoxin stimulated B cells developed `plasmablast' features (dilated, vesicular rough endoplasmic reticulum; Type II). A few stimulated B cells also had lymphoblast morphology. Large cells from normal (T + B) spleen stimulated by PWM were heterogeneous consisting of 55–60 per cent plasmablasts and 40–45 per cent lymphoblasts. It was concluded that the ultrastructure of stimulated lymphocytes depended on whether T or B cells were stimulated and not primarily on the mitogen used. In general, the response evoked by mitogens paralleled at the ultrastructural level that induced by antigens. It was also found that multivesicular bodies and glycogen particles occurred predominantly in the cytoplasm of stimulated T cells (lymphoblasts).     

Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells

Human embryonic stem (ES) cells are pluripotent cells derived from blastocyst-stage embryos. It has been suggested that these cells should play a major role in transplantation medicine and be able to advance our knowledge in human embryology. We propose that these cells should also play a vital role in the creation of models of human disorders. This aspect would be most valuable where animal models failed to faithfully recapitulate the human phenotype. Lesch-Nyhan disease is caused by a mutation in the HPRT1 gene that triggers an overproduction of uric acid, causing gout-like symptoms and urinary stones, in addition to neurological disorders. Due to biochemical differences between humans and rodents, a mouse lacking the HPRT expression will fail to accumulate uric acid. In this research we demonstrate a model for Lesch-Nyhan disease by mutating the HPRT1 gene in human ES cells using homologous recombination. We have verified the mutation in the HPRT1 allele at the DNA and RNA levels. By using selection media, we show that HPRT1 activity is abolished in the mutant cells, and the HPRT1-cells show a higher rate of uric acid accumulation than the wild-type cells. Therefore, these cells recapitulate to some extent the characteristics of Lesch-Nyhan syndrome and can help researchers further investigate this genetic disease and analyze drugs that will prevent the onset of its symptoms. We therefore suggest that human diseases may be modeled using human ES cells.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Four cases of wheat-dependent exercise-induced anaphylaxis with negative gluten cap-rast score]

BACKGROUND: Either omega-5 gliadin or high molecular weight glutenin is known to be a major allergen in wheat-dependent exercise-induced anaphylaxis (WDEIA). It is generally considered that gluten specific IgE score is more reliable than that of wheat specific IgE score for the diagnosis of WDEIA. Our aim was to verify the significance of gluten specific IgE in the diagnosis of WDEIA. METHODS: We evaluated the result of gluten CAP-RAST score and omega-5 gliadin specific IgE score on four WDEIA patients who visited our hospital during the years 2004 and 2005, whose diagnosis were onfirmed by prick tests, immunoblot tests and provocation tests. RESULTS: Contrary to our expectations, all four patients showed negative gluten CAP-RAST scores, however all patient's omega-5 gliadin specific IgE scores were positive. CONCLUSION: Our results suggest that gluten specific CAP-RAST score is unreliable in the diagnosis of WDEIA. On the other hand omega-5 gliadin specific IgE score is possibly a better candidate as a diagnostic tool for WDEIA.     

A novel action of stargazin as an enhancer of AMPA receptor activity

Stargazin (γ-2) is disrupted in the ataxic and epileptic mutant mouse, stargazer (stg). The striking defect in the stg cerebellum is the lack of functional AMPA receptors on granule cells. Recently, it has been reported that γ-2 and its related molecules are crucial for the surface expression, synaptic targeting and recycling of AMPA receptors, being termed collectively as the transmembrane AMPA receptor regulatory proteins (TARPs). However, it is still unclear whether TARPs directly modulate AMPA receptor activity. Here we report that coexpression of GluR1 (GluR1) with γ-2 using HEK293 cells and Xenopus oocytes markedly enhanced glutamate-induced currents. This effect was far beyond the increase of AMPA receptor surface expression and accompanied by increased glutamate affinity and subunit cooperativity. Other member of TARPs (γ-3, γ-4, and γ-8) also enhanced the current response through the AMPA receptors. The enhancing effect by γ-2 coexpression was further observed for homomeric GluR2 (GluR2) channels, which, when expressed alone, are known to produce only a small or negligible current response. These results suggest that γ-2 not only promotes AMPA receptor surface expression but also directly modulates AMPA receptor activity.     

Expression of the endothelial markers PECAM-1, vWf,and CD34 in vivo and in vitro

EC culture models are essential to study pathological alterations of endothelial cells (ECs) in pulmonary vascular diseases under standardized conditions. Nevertheless, little is known about the spectrum of alterations of vessel-specific endothelial phenotypes in monolayer cultures. For the comparative study of endothelial markers in vivo and in vitro we investigated immunohistochemically the expression of PECAM-1, vWf, and CD34 by pulmonary ECs in vivo and in stimulated/unstimulated human umbilical vein endothelial cells (HU-VEC) and human pulmonary microvascular endothelial cells (HPMEC). In vivo, vessel type-specific expression patterns were found for vWf and CD34, while PECAM-1 was homogeneously and strongly expressed. While all HUVEC showed a marked vWf staining, about two-thirds of HPMEC exhibited a strong and the rest a moderate vWf staining. In both in vitro models all ECs were clearly PECAM-1-positive. However, only about 20% of the HUVEC and HPMEC were CD34-positive. Our results demonstrate the reduced expression of vessel type-specific endothelial phenotypes by endothelial monolayer cultures, stressing the need to improve culture conditions as well as develop cocultures and three-dimensional culture models. Moreover, the need for endothelial markers specific for single microvascular type ECs becomes obvious in order to establish cultures consisting of only one microvascular ECs subpopulation.     

Molecular cloning and expression analysis of a macrophage-colony stimulating factor receptor-like gene from rainbow trout, Oncorhynchus mykiss

The M-CSF and its receptor (M-CSFR, CSF-1R or c-fms proto-oncogene) system were initially implicated as essential in mammals for normal monocyte development as well as for pregnancy. To allow a comparison with the M-CSF and M-CSFR system of an oviparous animal, we cloned a M-CSFR-like gene from rainbow trout (Oncorhynchus mykiss). The gene was cloned from a cDNA library of head kidney. It contained an open reading frame encoding 967 amino acids with a predicted size of 109 kDa. The putative amino acid sequence of rainbow trout M-CSFR showed 54% amino acid identity to fugu (Takifugu rubripes) M-CSFR, 52% to zebrafish (Danio rerio) M-CSFR and 40% to mouse (Mus musculus) and human (Homo sapiens) M-CSFR. The M-CSFR-like gene was constitutively expressed in head kidney, kidney, intestine, spleen and blood. The gene was detected especially in the ovary of immature female rainbow trout. These results suggest that a M-CSFR-like receptor may be involved in female reproductive tracts even in an oviparous animal like fish.     

Feline coronavirus serotypes 1 and 2: seroprevalence and association with disease in Switzerland

To determine the prevalence of antibodies to feline coronavirus (FCoV) serotypes 1 and 2 in Switzerland and their association with different disease manifestations, a serological study based on immunofluorescence tests was conducted with Swiss field cats using transmissible gastroenteritis virus (TGEV), FCoV type 1 and FCoV type 2 as antigens. A total of 639 serum samples collected in the context of different studies from naturally infected cats were tested. The current study revealed that, with an apparent prevalence of 83%, FCoV serotype 1 is the most prevalent serotype in Switzerland. FCoV type 1 viruses induced higher antibody titers than FCoV type 2, and were more frequently associated with clinical signs and/or feline infectious peritonitis. The antibody development in seven cats experimentally infected with FCoV type 1 revealed that, with progressing duration of infection, antibodies to FCoV type 1 significantly increased over those to FCoV type 2. There was a significant relationship between antibody titers against TGEV, FCoV 1, and FCoV 2 and TGEV antigen detected the highest proportion of seropositive cats. We conclude that a vaccine against FCoV should be based on FCoV type 1-related antigens and that for serodiagnosis of FCoV infection TGEV should be used to attain the highest diagnostic efficiency. When serology is used in addition to clinical signs, hematology, and clinical chemistry results as an aid to diagnose clinical FIP, TGEV shows a diagnostic efficiency equal to that of a FCoV antigen.