Molecular analysis of two enzyme genes, HPRT1 and PRPS1, causing X-linked inborn errors of purine metabolism.

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. On the other hand, PRPS1 mutations cause PRPP synthetase superactivity associated with hyperuricemia and gout, sometimes including neurodevelopmental abnormalities. We have identified two mutations in two Lesch-Nyhan families after our last report. One of them, a new single nucleotide substitution (130G>T) resulting in a missense mutation D44Y was detected in exon 2 of HPRT1. RT-PCR amplification showed not only a cDNA fragment with normal size, but also a small amount of shorter fragment skipping exons 2 and 3. The other missense mutation F74L (222C > A) was detected in a Japanese patient but has been reported previously in European families. In four hyperuricemic patients with mild neurological abnormality, no mutations responsible for partial HPRT deficiency were identified in HPRT1. In these four patients, we also performed molecular analysis of PRPS1, but no mutations in PRPP synthetase were found.

Hippocampal volumes and diffusion-weighted image findings in children with prolonged febrile seizures.

OBJECTIVES: To assess hippocampal volumes (HV) and signal changes on diffusion-weighted imaging (DWI) within 5 days of prolonged febrile seizures (PFS) and compare them with the PFS duration and EEG. METHODS: We studied 12 children (mean age: 32 +/- 21 months, range 10 months-5 years) within 5 days of a first episode of PFS (a seizure or series of seizures lasting for 30 min or longer, without return of consciousness between the seizures). The HV measurements were carried out using high-resolution magnetic resonance imaging and signal intensity abnormalities were evaluated visually on DWI. HV in patients were compared with those of 13 neurologically normal controls (mean age 31 +/- 16 months, range 15 months-5 years). HV abnormalities correlated with PFS duration. HV and DWI abnormalities were compared with EEG abnormalities. RESULTS: Seizure duration ranged from 40 to 95 min. In seven out of twelve patients, seizures were refractory and lasted for 60 min or longer despite intravenous infusion of diazepam. In the patients with PFS for 60 min or longer, HV were significantly larger than that of controls. In all patients, there was a positive correlation between HV and seizure duration. DWI showed hyperintensity in unilateral hippocampus in three patients with intractable seizures, ipsilateral thalamus in two, and cingulate in one. EEG showed abnormalities in temporal areas ipsilateral to the DWI abnormalities in these patients. CONCLUSIONS: Large HV and hippocampal hyperintensity on DWI were seen in patients with refractory PFS. Our results suggest that medically refractory PFS lasting for 60 min or longer may cause structural changes in limbic structures that could promote later epileptogenesis.

Hippocampal volumes and diffusion-weighted image findings in children with prolonged febrile seizures.

OBJECTIVES: To assess hippocampal volumes (HV) and signal changes on diffusion-weighted imaging (DWI) within 5 days of prolonged febrile seizures (PFS) and compare them with the PFS duration and EEG. METHODS: We studied 12 children (mean age: 32 +/- 21 months, range 10 months-5 years) within 5 days of a first episode of PFS (a seizure or series of seizures lasting for 30 min or longer, without return of consciousness between the seizures). The HV measurements were carried out using high-resolution magnetic resonance imaging and signal intensity abnormalities were evaluated visually on DWI. HV in patients were compared with those of 13 neurologically normal controls (mean age 31 +/- 16 months, range 15 months-5 years). HV abnormalities correlated with PFS duration. HV and DWI abnormalities were compared with EEG abnormalities. RESULTS: Seizure duration ranged from 40 to 95 min. In seven out of twelve patients, seizures were refractory and lasted for 60 min or longer despite intravenous infusion of diazepam. In the patients with PFS for 60 min or longer, HV were significantly larger than that of controls. In all patients, there was a positive correlation between HV and seizure duration. DWI showed hyperintensity in unilateral hippocampus in three patients with intractable seizures, ipsilateral thalamus in two, and cingulate in one. EEG showed abnormalities in temporal areas ipsilateral to the DWI abnormalities in these patients. CONCLUSIONS: Large HV and hippocampal hyperintensity on DWI were seen in patients with refractory PFS. Our results suggest that medically refractory PFS lasting for 60 min or longer may cause structural changes in limbic structures that could promote later epileptogenesis.

Comparative immunobiology of thymic DC mRNA in autoimmune-prone mice.

New Zealand Black (NZB) mice have multiple defects in both innate and acquired immunity. A fundamental defect, described more than 25 years ago, is premature thymic involution. Subsequent studies have disclosed multiple defects in thymic epithelial cells, and it has been proposed that thymic dendritic cells (DCs) play an important role not only in thymic involution but also in the appearance of immunopathology. However, the number of available thymic DCs makes this population extremely difficult to study. We have taken advantage of our ability to isolate pure populations of thymic DCs and have examined several key mRNA levels of enzymes involved in signal transduction. Our data on NZB mice was compared to that of NZB x NZW F1 (B/WF1), BXSB-Yaa, MRL/lpr, NOD and control mice. Importantly, we demonstrate herein that a common feature in autoimmune-prone mice is an increase of thymic DC c-met mRNA. Indeed, the increase in c-met mRNA levels appeared specific to the thymus and was not noted in the spleen. Additionally, we demonstrate that E-cadherin, a downstream molecule of c-met, is also reduced. Finally, we note that the levels of HGF mRNA are normal in the autoimmune strains examined herein, confirming that the abnormality of c-met mRNA is not due to primary defects in thymic stromal cells. We submit that these results highlight the possibility of a selective defect in thymic DCs which will be a pivotal step in loss of tolerance, and suggest that future studies focus on adoptive cell transfer involving this population.

B cells are selectively associated with thymic cortical but not medullary pathology in NZB mice.

Abnormal expansion of autoantibody-synthesizing B cells and self-reactive T cells, which most likely escape negative selection within the thymus, have both been characterized and reasoned to play a role in the pathogenesis of autoimmunity in NZB mice. Support for this thesis has been our observation that NZB mice have severe cortical and medullary thymic microarchitectural defects. As a means to dissect the roles of T and B cells in the induction of such abnormalities, B cell-deficient NZB mice were bred by backcrossing the Igh6(null)allele on to the NZB background (NZB-muMT mice). Such mice showed undetectable levels of autoantibodies. NZB-muMT mice, as compared to wild-type NZB mice, had lower absolute numbers of CD4(+)T cells. Furthermore, thymic abnormalities in NZB-muMT mice were restricted to the medulla. These data suggest that, while B cells may play a role in thymic cortical abnormalities, the medullary abnormalities are induced by other mechanisms.

Increased expression of mXBP-1 (TREB-5) in thymic B cells in New Zealand mice.

New Zealand Black mice as well as several other murine models of murine lupus are well known for premature degeneration of thymus and development of autoimmunity. To focus on molecular events unique to murine lupus, we performed differential display using arbitrary primer pairs to distinguish NZB versus BALB/c thymus at 5 weeks of age. Following an extensive analysis of DNA bands that were either consistently up or downregulated and from studies of expression pattern of thymic genes by in situ nucleic acid hybridization, we focused on one clone that was consistently differentially expressed between NZB and BALB/c thymus. This clone was isolated, sequenced, and identified as the murine homologue of the human X box binding protein (hXBP-1), also known as TREB 5. mXBP-1 was found to be consistently upregulated in B cells in the thymic cortex of NZB and (NZBxNZW)F1, but not BALB/c, C3H/HeJ or C57BL/6 mice. In addition, it was dramatically elevated in MRL/ lpr but not MRL/++ mice; similarly, it was increased in BXSB/ Yaa male but not BXSB female thymic cortex. Of particular interest was an absence of mXBP-1 expression in the thymus of NZB/ Bln- Igh6(null)homozygotes. mXBP-1 has several putative functions, including the regulation of MHC class II expression and by virtue of its ability to recognize CRE-like elements shown to be involved in HTLV-1 transcription.

Abnormal thymic expression of epithelial cell adhesion molecule (EP-CAM) in New Zealand Black (NZB) mice.

New Zealand Black (NZB) mice have been well documented to have a variety of thymic epithelial cell microenvironmental abnormalities, including disruption of corticoepithelial cell networks and medullary cell clusters. These abnormalities of the thymic stromal network are particularly important because similar observations have been noted in other models of murine lupus. Thymic epithelial cells, a key component of the microenvironment, play an important role in selection of the mature T cell receptor repertoire. Recently, a homotypic calcium-independent human and murine epithelial cell adhesion molecule, Ep-CAM, has been described which is located at the thymocyto-cortical cell junction. The function of Ep-CAM is still unclear but its unique location within the thymus suggests that it is critical in the process of providing maturation signals. Consequently, we examined the thymic expression of Ep-CAM in a series of autoimmune prone mice by thymic distribution of Ep-CAM in NZB, NZW, NZB/W, BXSB-Yaa, MRL- lpr/lpr, C3H- gld/gld and the control strains BALB/c, C57BL6, C3H and MRL(+/+), by immunohistology and flow cytometry. Interestingly, NZB mice are similar to control mice from day 4 to 2 weeks of age, having a very low expression of Ep-CAM at the thymocyto-cortical junction. In control strains, there is a marked increased in expression of Ep-CAM beginning at 5 weeks of age. In contrast, NZB mice fail to show significant expression of Ep-CAM even well into adulthood. This abnormality of NZB mice was also noted in NZB/W F1 and BXSB mice, but not MRL- lpr/lpr or C3H- gld/gld mice. Given the potential importance of Ep-CAM in thymic selection, this study provides important evidence that a defective stromal microenvironment is likely to be of etiological significance in the susceptibility of NZB to autoimmune disease.

Apoptosis and the thymic microenvironment in murine lupus.

The thymus of New Zealand black (NZB) mice undergoes premature involution. In addition, cultured thymic epithelial cells from NZB mice undergo accelerated preprogrammed degeneration. NZB mice also have distinctive and well-defined abnormalities of thymic architecture involving stromal cells, defined by staining with monoclonal antibodies specific for the thymic microenvironment. We took advantage of these findings, as well as our large panel of monoclonal antibodies which recognize thymic stroma, to study the induction of apoptosis in the thymus of murine lupus and including changes of epithelial architecture. We studied NZB, MRL/lpr, BXSB/Yaa, C3H/gld mice and BALB/c and C57BL/6 as control mice. Apoptosis was studied both at basal levels and following induction with either dexamethasone or lipopolysaccharide (LPS). The apoptotic cells were primarily found in the thymic cortex, and the frequency of apoptosis in murine lupus was less than 20% of controls. Moreover, all strains of murine lupus had severe abnormalities of the cortical network. These changes were not accentuated by dexamethasone treatment in cultured thymocytes. However, the thymus in murine lupus was less susceptible to LPS-induced apoptosis than control mice. Finally we note that the number of thymic nurse cells (TNC) was lowest in NZB mice. Our findings demonstrate significant abnormalities in the induction of apoptosis and the formation of TNC-like epithelial cells in SLE mice, and suggest that the abnormalities of the thymic microenvironment have an important role in the pathogenesis of murine lupus.

Effect of genetic background on establishment of mouse embryonic stem cells.

We established 13 embryonic stem (ES) cell lines from 542 embryos crossed between various strains of mice: 10 lines from 129/Sv-ter embryos (10/48, 20.8%) and 3 lines out of other 9 combinations of intra- or inter-strain matings (1 from intracross of C57BL/6CrSlc, 1 from B6D2F1 x C57BL/6CrSlc, 1 from Yok:ddY x Slc:ICR). No ES cell line from 129/Sv-ter x Slc:ICR embryos suggests that ICR strain might have inhibitory genetic factor(s) for the ES cell formation. Some ES cell lines could be obtained from hybrids even if none or few lines from their parental strains, suggesting a heterosis effect can be expected for establishing ES cell lines in mice.

Concentrations of C-reactive protein in normal monkeys (Macaca irus) and in monkeys inoculated with Bordetella bronchiseptica R-5 and measles virus.

The concentrations of C-reactive protein (CRP) in serum from normal crab-eating monkeys (Macaca irus) were measured by means of a monkey-specific turbidimetric immunoassay (TIA), and the changes in the serum CRP concentrations in crab-eating monkeys inoculated with Bordetella bronchiseptica R-5 and measles virus (Ichinose or NK 3 strain) were also examined. The CRP concentrations in sera from 54 normal crab-eating monkeys ranged from 0 to 8.3 microg/ml (mean 2.2 +/- 1.9). No significant difference was found in the CRP concentrations between males and females (p > 0.05). The concentrations of CRP in the sera from four crab-eating monkeys inoculated intrabronchially with 10(9) live B. bronchiseptica increased gradually to a peak at 2 days after inoculation. The peak concentrations of CRP were from 102.4 to 313.2 microg/ml, 54-96 times the preinoculative values of 1.9-5.6 microg/ml. When the same four crab-eating monkeys were inoculated intrabronchially with measles virus 34 days after inoculation of B. bronchiseptica, the serum CRP concentrations did not increase. Monitoring of CRP is useful for assessing monkeys with acute B. bronchiseptica infection and will probably be of value in the diagnosis of other bacterial infections.

Thymic microenvironment and NZB mice: the abnormal thymic microenvironment of New Zealand mice correlates with immunopathology.

There are distinct microenvironmental abnormalities of thymic architecture in several murine models of SLE defined using immunohistochemistry and a panel of mAb dissected at thymic epithelial markers. To address the issue of the relationship between the thymic microenvironment and autoimmunity, we studied backcross (NZB x NZW) F1 x NZW mice in which 50% of offspring develop nephritis associated with proteinuria and anti-DNA antibodies. We reasoned that if thymic abnormalities are associated with development of disease, the correlation of abnormalities with lupus-like disease in individual backcross mice will form the foundation for identification of the mechanisms involved. In parallel, we directed a genetic linkage analysis, using markers previously shown to be linked to nephritis and IgG autoantibody production, to determine if such loci were similarly associated with microenvironmental changes. Our data demonstrate that all (NZB x NZW) F1 x NZW backcross mice with disease have microenvironmental defects. Although the microenvironmental defects are not sufficient for development of autoimmune disease, the severity of thymic abnormalities correlates with titers of IgG autoantibodies to DNA and with proteinuria. Consistent with past studies of (NZB x NZW) F1 x NZW mice, genetic markers on proximal chromosome 17 (near MHC) and distal chromosome 4 showed trends for linkage with nephritis. Although the markers chosen only covered about 10-15% of the genome, the results demonstrated trends for linkage with thymic medullary abnormalities for loci on distal chromosome 4 and distal chromosome 1. We believe it will be important to define the biochemical nature of the molecules recognized by these mAbs to understand the relationships between thymic architecture and immunopathology.

Development of monkey C-reactive protein (CRP) assay methods.

Monkey-specific C-reactive protein (CRP) assay methods (enzyme-linked immunosorbent assay (ELISA) and turbidimetric immunoassay (TIA)) were developed. The anti-monkey CRP serum was prepared by immunization of rabbits with the immune complex formed between the acute-phase serum from turpentine oil-inoculated monkeys and goat anti-human CRP serum. The specificity of the rabbit anti-monkey CRP serum was confirmed by immunoelectrophoresis and Western blotting. The purity of monkey CRP prepared by chromatography procedures was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The serum CRP levels in nine normal monkeys, as measured by sandwich ELISA were ranged from 0.26 to 1.42 microg/ml (mean 0.71+/-0.37). The CRP levels in five acute-phase sera of turpentine oil-inoculated monkeys were 248-451 microg/ml (mean 371.2+/-73.8). This monkey-specific CRP assay method was found more sensitive than the human-specific CRP assay method in detecting monkey CRP by TIA.

Enhanced engraftment of human peripheral blood lymphocytes into anti-murine interferon-gamma monoclonal antibody-treated C.B.-17-scid mice.

To improve the engraftment of human peripheral blood lymphocytes (PBL) to severe combined immunodeficient (SCID) mice, and to elucidate the factors which prevent the PBL's survival, we treated SCID mice with mAb, which neutralizes murine IFN-gamma's ability to activate cell-mediated immunity. Compared with untreated mice, mAb-treated mice retained significantly higher numbers and ratios of human PBL in the peritoneal cavity and spleen, as well as significantly higher serum titers of human IgG and IgM. Histologically, host versus graft reaction (HVGR) was less severe in the mAb-treated mice. Moreover, these phenomena were completely abrogated when mice were also treated with murine recombinant IFN-gamma. These results suggest that murine IFN-gamma plays an important role in the rejection of human cells in SCID mice and that its depletion by means of mAb treatment can significantly reduce HVGR and improve the engraftment of human PBL.

Beta-galactosidase-deficient mouse as an animal model for GM1-gangliosidosis.

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid beta-galactosidase, which hydrolyses the terminal beta-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted beta-galactosidase gene showed beta-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7-10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis.

TCR beta-independent development of CD4+ CD8+ thymocytes observed in a strain of scid mice.

Mice homozygous for severe combined immunodeficiency (scid) mutation usually halt their thymocyte development at the CD4-CD8- double negative (DN) stage due to their inability of TCR gene rearrangement. In this study, we report that SCID-bg mice, which were originally generated by mating CB-17-scid mice with KSN-bg mice, spontaneously develop dominant CD4+ CD8+ double positive (DP) thymocytes. Their thymi were mainly composed of DN, CD4-CD8+ and DP cells, and the majority of them did not present CD3. Similarly, they lacked TCR beta expression both on cell surface and in cytoplasm, which suggests that the thymocyte development to the DP stage observed in SCID-bg mice, was independent of CD3 and TCR beta expression. In spite of significant DP thymocytes in SCID-bg mice, the histology of their thymi was not so different from those of CB-17-scid mice. Analysis of bone marrow cells in SCID-bg mice showed that the development of B lineage cells was not altered when compared with CB-17-scid mice. These findings point out the fact that thymocytes in SCID-bg mice have a peculiar characteristic compared to CB-17-scid mice, and provide evidence of TCR beta-independent development of thymocytes to the DP stage.

Report on a patient with paroxysmal cold hemoglobinuria.

Antibodies against the human blood group P antigen (anti-P alloantibodies) agglutinate phenotype P1 and P2 erythrocytes treated with papain at 4 degrees C but not phenotype PK and p erythrocytes. This condition is referred to as an autoimmune disease of paroxysmal cold hemoglobinuria (PCH), and the anti-P specificity is found in the cold hemagglutinins. Serum from a patient suspected to be suffering from PCH by the cold auto-agglutination properties was tested for anti-P specificity, using papain-treated O blood group erythrocytes. The serum-mediated hemagglutination and the serum and complement-mediated immunohemolysis were inhibited by globoside (P antigen GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide) and Forssman glycosphingolipid (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide). Therefore, we concluded that she had PCH. She was completely cured 6 months later.

Neurological manifestations of knockout mice with beta-galactosidase deficiency.

We succeeded in producing the beta-galactosidase-deficient knockout mouse by gene targeting in embryonic stem cells. The mutant mice developed progressive spastic diplegia within a few months after birth, and died of emaciation at 7-10 months of age. This is an authentic murine model of human GMI-gangliosidosis, and is useful for studies of its pathogenesis and treatment.

A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride membrane after thin-layer chromatography blotting and in situ mass spectrometric analysis of the ligands.

A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride (PVDF) membrane has been developed. Glycosphingolipids were separated on a high-performance thin-layer chromatography (HPTLC) plate and transferred onto a PVDF membrane by TLC blotting [Taki, T., Handa, S., and Ishikawa, D. (1994) Anal. Biochem. 221, 312-316]. The PVDF membrane was blocked with phosphate-buffered saline containing 4% casein and 0.1% methionine and overlaid with 35S-labeled Escherichia coli possessing K99 fimbriae (E. coli K99) at 37 degrees C for more than 2 h. Binding of the 35S-labeled E. coli K99 was detected with a bioimaging analyzer. Radioactivities were located on the bands corresponding to N-glycolylneuraminic acid containing glycosphingolipids such as sialylparagloboside, GM2, and GM3 with hydroxy fatty acid in ceramide moiety, and a weak binding was detected on the band of N-acetylneuraminylparagloboside. Furthermore, an in situ mass spectrometric analysis of the ligand glycosphingolipids on the membrane was demonstrated. The present method has several advantages compared with the overlay binding assay on the HPTLC plate as follows: (i) the method is simple and rapid; (ii) the membrane is easy to handle; (iii) binding is clear with low background; (iv) a small amount of [35S]methionine is required; and (v) sensitive ligand characterization can be done by in situ mass spectrometric analysis.

Failure of human immunoresponse to N-glycolylneuraminic acid epitope contained in recombinant human erythropoietin.

Recombinant human erythropoietin (rHUEPO) was produced by Chinese hamster ovary cells and commercially distributed to hospitals by two pharmaceutical companies in Japan ('ESPO' by Kirin Brewery Co. Ltd., and Sankyo Co. Ltd., and 'EPOGIN' by Chugai Pharmaceutical Co. Ltd.) These products contained about 1% N-glycolylneuraminic acid (Neu5Gc) in total sialic acid content. Since humans do not synthesize Neu5Gc, successive injection of Neu5Gc-containing products was feared to lead to allergic-like symptoms. Therefore, serum levels of antibodies of Neu5Gc epitope in 90 patients who received repeated i.v. injections of ESPO or EPOGIN were determined by an enzyme immunoassay using Neu5Gc alpha 2-3Gal beta 1-4Glc-Cer, GM3(Neu5Gc), as an antigen and compared with those in 100 healthy persons. Either no or low antibody levels were detected in both groups with no significant difference. In 40 patients who received s.c. injections of ESPO or EPOGIN, serum HD antibody levels were determined before and after weekly therapeutic injections carried out for one to several months, but no significant elevations were detected in all patients. The above results indicated that therapeutic administration of rHuEPO to patients to patients with chronic renal failure is safe from allergic-like side effects associated with the production of Neu5Gc-specific antibodies, and it was concluded that Neu5Gc epitope of rHuEPO is minimally antigenic in humans.

Neurotropin inhibits accumulation of eosinophils induced by allergen through the suppression of sensitized T-cells.

A non-protein extract isolated from the inflamed dermis of rabbits inoculated with vaccinia virus (Neurotropin) has been clinically used in Japan as an analgesic and anti-allergic drug. To clarify its anti-allergic mechanism, the effect of Neurotropin on eosinophil accumulation induced by ragweed pollen extract or platelet-activating factor (PAF) was examined in BALB/c mice. Neurotropin inhibited the T-cell-dependent accumulation of eosinophils induced by allergen in a dose-dependent manner when administered during sensitization. However, Neurotropin was unable to inhibit the T-cell-independent accumulation of eosinophils induced by PAF. A T-cell transfer experiment was performed to address the inhibitory mechanism. A marked accumulation of eosinophils was observed when recipients were injected i.p. with allergen and Nylon wool column-passed splenic T-cells from the sensitized donor mice. However, significant accumulation of eosinophils was not observed when sensitized donor mice were administered with Neurotropin but not saline. Taken together, these results suggest that Neurotropin inhibits the accumulation of eosinophils induced by allergen via the suppression of sensitized T-cell induction, or alternatively by interfering with T-cell function.