Host conditioning with total lymphoid irradiation and antithymocyte globulin prevents graft-versus-host disease: the role of CD1-reactive natural killer T cells.

Our previous studies in mice showed that the nonmyeloablative conditioning regimen of fractionated irradiation of the lymphoid tissues (total lymphoid irradiation; TLI) and depletive anti-T-cell antibodies (anti-thymocyte serum) markedly increased the percentage of regulatory DX5+ and natural killer 1.1+ T cells in the mouse spleen, and prevented acute lethal graft-versus-host disease (GVHD) in BALB/c mice (H-2(d)) following the transplantation of bone marrow (BM) and peripheral blood mononuclear cells (PBMC) from C57BL/6 (H-2(b)) donors. The object of the current study was to determine whether the TLI and anti-thymocyte serum regimen protected natural killer T-cell deficient CD1(-/-) BALB/c mice against GVHD after BM and PBMC transplantation from C57BL/6 donors, and whether a similar conditioning regimen of TLI and anti-thymocyte globulin (ATG) can prevent GVHD in Lewis rat (RT1(l)) hosts after BM and PBMC transplantation from ACI rat (RT1(a)) donors. The experimental results in mice showed that, although wild-type BALB/c hosts are protected in association with a marked increase in CD1- reactive T cells expressing the invariant TCR identified with a CD1 tetramer reagent; CD1(-/-) BALB/c hosts are not. Studies of chimeric donor cells in mice protected from GVHD showed donor T-cell polarization to a Th2 cytokine pattern. Results in rats showed that approximately 1000 fold more donor PBMC cells were required to induce a similar incidence of lethal GVHD in TLI and ATG conditioned hosts as compared with hosts conditioned with single-dose total-body irradiation or total-body irradiation and ATG. Surviving TLI and ATG conditioned rat hosts were complete chimeras. In conclusion, the TLI and ATG/anti-thymocyte serum conditioning regimen protects against GVHD in rats and mice, and regulatory natural killer T cells are required for protection.     

Differential dynamics of the peripheral and intrahepatic cytotoxic T lymphocyte response to hepatitis B surface antigen

The distribution and dynamics of the cytotoxic T lymphocyte (CTL) response to hepatitis B surface antigen (HBsAg) were studied in mice after intramuscular DNA immunization and after hepatic infection by a recombinant adenovirus that expresses the hepatitis B virus genome (Ad-HBV). CTLs specific for HBsAg accumulate preferentially in the spleen after DNA immunization but are primarily intrahepatic after Ad-HBV infection. The secondary CTL response to Ad-HBV in DNA-primed mice is characterized by rapid depletion of effector CTLs from the spleen, and their expansion in the liver where they cause hepatitis, secrete interferon gamma (IFNγ), and inhibit HBV gene expression. Suppression of HBsAg synthesis is accompanied by disappearance of intrahepatic IFNγ-producing CTLs and their reaccumulation in the spleen. The data suggest a possible explanation for the paucity and functional deficiency of HBV-specific CTLs in the periphery during chronic HBV infection, and that the severity of infection can be worsened by a preexisting CTL response if neutralizing antibody is not also present.     

Spontaneous age-associated amyloidosis in senescence-accelerated mouse (SAM)

Morphological studies on spontaneous systemic amyloidosis were conducted on 222 senescence-accelerated mice (SAM) (P) and on 150 mice in the senescence-resistant series (R).Among the pathologic findings, amyloidosis showed the highest incidence in both SAM (79.7%) and R (32.7%) Although an extensive deposition of amyloid was evident in some aged mice in the R series, a more severe amyloidosis occurred with a higher incidence in the P series. There was a statistical significance between the incidence of amyloidosis and age, in both the P and R series. There were no differences in organ distribution and mode of amyloid deposition between the P and R series or between the sexes. In about 60% of the amyloid-positive cases in the 28 killed SAM and 7 mice in the R series, there were no signs of inflammation or neoplasm.The morphological features in SAM more closely resembled those seen in cases of murine spontaneous senile amyloidosis than the features seen in cases of experimentally induced amyloidosis. This model is expected to be a valuable tool with which to assess the relationship between amyloid deposition and the aging process or senescence, perhaps even cases of human senile amyloidosis.     

H-2 allele specificity of the NK cell C-type lectin-like MHC class I receptor Ly49A visualized by soluble Ly49A tetramer

Ly49A is a C-type lectin-like receptor on NK cells that recognizes MHC class I ligands, H-2D(d) and D(k). The engagement of Ly49A with the ligands inhibits activation of NK cells and protects target cells from lysis by NK cells. Here we express the extracellular region of Ly49A with an N-terminal biotinylation tag in Escherichia coli to obtain soluble Ly49A (sLy49A) after refolding. sLy49A is indistinguishable from native Ly49A expressed on NK cells serologically and in the ability to specifically bind H-2D(d) after tetramerization with R-phycoerythrin-coupled streptavidin. The fluorescently labeled tetramer of sLy49A is applied to explore MHC class I haplotype specificity of Ly49A. We demonstrate the hierarchical reactivity of Ly49A with H-2 of various alleles in the order of d > k, r > p > v > q > s > z. Reactivity of sLy49A tetramer to spleen lymphocytes from B10.QBR mice (H-2K(b), I(b), D(q), Qa-1/Tla(b)) but not from C57BL/10 mice (H-2(b)) identifies H-2D(q) and L(q) as candidates for a Ly49A ligand. Binding of sLy49A tetramer to H-2D(q)- or L(q)-transfected cell lines demonstrates that the two highly related MHC class I molecules, H-2D(q) and L(q), are ligands for Ly49A. sLy49A tetramer staining also demonstrates preferential expression of Ly49A ligand on a subset of B cells in P/J mice. These results provide the basis to examine the molecular mechanism by which Ly49A discriminates polymorphic MHC class I molecules.     

Genotyping of Mycobacterium leprae on the basis of the polymorphism of TTC repeats for analysis of leprosy transmission

The polymorphism of TTC repeats in Mycobacterium leprae was examined using the bacilli obtained from residents in villages at North Maluku where M. leprae infections are highly endemic (as well as from patients at North Sulawesi of Indonesia) to elucidate the possible mode of leprosy transmission. TTC genotypes are stable for several generations of passages in nude mice footpads and, hence, are feasible for the genotyping of isolates and epidemiological analysis of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among residents at the same dwelling in villages in which leprosy is endemic and that some household contacts harbored bacilli with a different genotype from that harbored by the patient. Investigations of a father-and-son pair of patients indicated that infections of bacilli with 10 and 18 copies, respectively, had occurred. Genotypes of TTC repeats were found to differ between a son under treatment and two brothers. These results reveal the possibility that in addition to exposure via the presence of a leprosy patient with a multibacillary infection who was living with family members, there might have been some infectious sources to which the residents had been commonly exposed outside the dwellings. A limited discriminative capacity of the TTC polymorphism in the epidemiological analysis implies the need of searching other useful polymorphic loci for detailed subdivision of clinical isolates.     

Histochemical, immunohistochemical and ultrastructural investigations of giant cell tumors of bone

The origin and characteristics of so-called stromal cells (stromal cell) and the osteoclast-like giant cell series of 19 cases of giant cell tumor (G.C.T.) of bone were studied. Immunohistochemically, two interesting cases were found. The stromal cells of one case were alpha-1-antitrypsin positive and those of the other case were alpha-1-antichymotrypsin positive. The histiocytic stromal cells of the latter case seemed to be surely neoplastic since they showed mild to moderate cell atypism. There were foci consisting of fibroblastic cells or osteoid and osteoblasts within the tumor. Those cells in the foci were apparently continuous with the surrounding stromal cells, and they were, therefore, also considered to be neoplastic. These findings strongly indicate that the stromal cells originate from the undifferentiated mesenchymal cells in the bone marrow and may differentiate to osteoblastic, fibroblastic, and histiocytic cells. All cells of these three series were not stained for a high stable form of acid phosphatase (SAPhase). SAPhase activity was demonstrated only in osteoclast-like giant cells and some mononuclear cells, which are recently believed to be non-neoplastic. Therefore, the cell atypia of SAPhase negative stromal cells is considered to have a prognostic value.     

Specific cellular immune responses to pancreatic antigen in chronic pancreatitis and Sjögren's syndrome

The specific cellular immune response to the partially purified pancreatic antigen was studied by the peripheral blood lymphocyte proliferation assay in patients with chronic pancreatitis, Sjögren's syndrome, and primary biliary cirrhosis. A significant positive result (stimulation index >2.0) was observed in 7 of 21 patients with idiopathic chronic pancreatitis (33%;P<0.05), 6 of 7 patients with Sjögren's syndrome-associated chronic pancreatitis (86%;P<0.0005), and 6 of 11 patients with Sjögren's syndrome (55%;P<0.01), compared to normal controls whose stimulation index was 0.94±0.28 (mean ± SD;n=14; range, 0.56–1.60). On the other hand, patients with alcoholic chronic pancreatitis (17%;n=12), stone-related chronic pancreatitis (0%;n=7), primary biliary cirrhosis-associated chronic pancreatitis (33%;n=3), primary biliary cirrhosis (0%;n=4), systemic lupus erythematosus (17%;n=6), and autoimmune thyroiditis (0%;n=6) showed no significant difference from normal controls. Furthermore, in patients with idiopathic chronic pancreatitis who had positive results, a lymphocyte proliferative response to the pancreatic antigen was observed in T cells, especially in the CD4⁺ T cell subpopulation. These results suggest that the pancreatic antigen plays a role in the pathogenesis of a part of idiopathic chronic pancreatitis and Sjögren's syndrome in association with T cell responses and, also, suggest that autoimmunity may be a possible etiological factor in chronic pancreatitis.     

Expression enhancement of the Tn5 neomycin-resistance gene by removal of upstream ATG sequences and its use for probing heterologous upstream activating sequences in yeast

We have constructed a series of promoter or upstream activating sequence (UAS)-probe plasmids carrying the Tn5-derived neomycin resistance gene whose seven additional ATG codons in the 5-untranslated region were completely or partially removed. When the deleted version of the neo sequence retaining only one additional ATG (NeoD) was expressed under the control of a TDH3 promoter whose UAS was deleted, the transformed cells were unable to grow at a low concentration of the antibiotic G418. In contrast with this, yeast cells expressing the NeoC sequence and having no additional ATG exhibited a high level of G418-resistance. Moreover, the UAS-probe system using NeoD has been successfully applied for the identification of several E. coli DNA sequences that clearly function as UASs in yeast cells. Two of these prokaryotic sequences with UAS activity were identified as a part of the coding region of the tgt and the hydG gene, respectively.     

Relative frequencies of G (VP7) and P (VP4) serotypes determined by polymerase chain reaction assays among Japanese bovine rotaviruses isolated in cell culture.

The relative frequencies of both the G (VP7) and P (VP4) serotypes of 40 bovine rotaviruses isolated in cell culture from diarrheic calves in Japan between January 1983 and February 1991 were determined by recently developed polymerase chain reaction assays. Isolates with G serotype 6 and P serotype 5 (UK-like strains) were most frequently found (42.5%) followed by isolates with G6P11 (17.5%), G6P1 (10%), or G10P5 (10%). Isolates with G10P11 (B223-like strains) were least frequently found (7.5%). The presence of various combinations of G and P serotypes suggests frequent reassortment in nature among bovine rotaviruses.