Batmunkh Munkhbat

Quantification of serum-soluble HLA class I antigens in patients with gastric cancer

The amount of sHLA-I in serum was examined in 74 patients with gastric cancer and 15 normal healthy controls. For mAbs, W6/32 specific for HLA-A, -B, -C, and biotin IOT2 specific for HLA class I associated with beta 2 microglobulin, were used to determine the values of sHLA-I using an ELISA. The patients in stage-IV gastric cancer showed lower values of sHLA-I (445.4 +/- 247.1 ng/ml) than those in stage I (725.9 +/- 575.8 ng/ml), stage II (752.8 +/- 255.0 ng/ml), and normal controls (868.9 +/- 715.0 ng/ml) (P < 0.05). In analysis of the patients with HLA-A24, the allele that has been reported to secrete more sHLA-I than other alleles, the results were nearly the same. These results suggest that the secretion of sHLA-I is low in patients with very advanced cancer. However, there was no correlation between the sHLA-I level and the metastasis or prognosis in longitudinal studies in 11 patients.

Xenogeneic (pig to rat) fetal liver fragment transplantation using macrocapsules for immunoisolation

Acute liver failure caused by viral infection, surgical resection of a large part of the liver or by drug use has a high mortality. For its treatment, hepatocyte or liver tissue transplantation is useful. We report here the beneficial effects of xenogeneic fetal liver fragment (FLF) transplantation with an immunoisolation macrocapsule. The macrocapsules were made of a microporous polypropylene membrane. Pig FLFs (1 mL) was inserted into each capsule to serve as a graft in LEW rats. Acute liver failure was induced by 90% liver resection on day 0. Group 1: transplantation of encapsulated FLF into the omentum 2 days before liver resection (n = 17). Group 2: FLF transplantation into the omentum on day -2 (n = 11). Group 3: liver resection (control) (n = 19). The survival rate, the histology of the grafts and the biochemical parameters [blood sugar (BS), GPT, and GOT] were evaluated. The survival rates of groups 1, 2, and 3 on day 7 were 70.6, 0, and 11.1%, respectively. There were significant differences in BS, GPT, and GOT levels between groups 1 and 3 on day 1 (p < 0.05). On day 28, the histological analyses of the grafts of encapsulated FLFs revealed that the hepatocytes appeared viable, but that the haematopoietic cells had degenerated. Xenogeneic FLFs with macrocapsules survived more than 1 mo, and supported the hosts liver function.

HLA-A*24-B*07-DRB1*01 haplotype implicated with genetic disposition of peak bone mass in healthy young Japanese women.

HLA exhibits the most extensive polymorphism of any of the known human genes and is known as a genetic marker which allows genetic background of many diseases and physical phenomena. In this study, we, therefore, tried to investigate the regulation of HLA polymorphism and peak bone mass (PBM) in order to elucidate the genetic backgrounds of bone metabolism in young women. Subjects were 67 healthy young Japanese women (average age: 23.6 +/- 2.6 years, Body Mass Index (BMI): 19.9 +/- 2.0 who were randomly chosen. Allelic polymorphisms in HLA class I (HLA-A and -B) and HLA-class II (DRB1) were investigated by PCR-SSOP and PCR-SSP. Vitamin D Receptor (VDR) and Estrogen Receptor (ER) gene polymorphisms were also analyzed. Lifestyle factors, such as exercise and nutrition, were examined by questionnaire. Bone mineral density was examined using with Lunar DPX-L. Subjects who possessed HLA-B*07 had a significantly lower PBM than those without B*07 (p < 0.05). All subjects were divided into 3 groups according to HLA haplotypes linked with HLA-B*07, as follows: A*24(+/-)B*07(-)DRB1*01(+/-), A*24(+)B*07(+)DRB1*01(-), and A*24(+)B*07(+)DRB*01(+). There were no significant differences between these three groups in factors that affect bone metabolism, such as age, age at menarche, BMI, calcium intake, exercise habits, VDR or ER allele frequency. The HLA-A*24-B*07-DRB1*01 haplotype had a significantly lower Z score in the lumbar spine compared with subjects without this haplotype (p < 0.05). When the Z score was divided by values higher or lower than +1 or -1, all 3 subjects whose Z score was lower than -1.0 were found to have the HLA-A*24-B*07-DRB1*01 haptotype. A significant association between HLA-A*24-B*07-DRB1*01 and Z score < -1 was found (Yates correction chi(2) = 10.82, p = 0.001, RR = 204). In conclusion, the HLA-A*24-B*07-DRB*01 haplotype can be considered a new genetic marker implicated with low PBM in healthy young Japanese women.

Extensive and long-term ex vivo production of dendritic cells from CD34 positive umbilical cord blood or bone marrow cells by novel culture system using mouse stroma

We previously developed a system using murine strome (HESS-5), which could expand umbilical cord blood (UCB) stem and progenitor cells, especially CD34+/38- cells, in the presence of human recombinant cytokines. In this study, the ability of expanded UCB- or bone marrow (BM)-CD34+ cells to differentiate into dendritic cells (DCs) was examined. DCs could be induced either from short or long term cultured CD34+ cells after switching the cytokines from Flk-2/Flt-3 ligand, stem cell factor (SCF), thrombopoietin (TPO) to granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) (immature type) plus tumor necrosis factor (TNF)-alpha with stimulation by CD40L transfectant (mature type). Each immature or mature UCB-DCs showed a dextran uptake or a potent allo-T lymphocytes proliferative ability, respectively. Furthermore, those DCs from BM significantly stimulated auto-T lymphocytes in an antigen (varicella zoster virus) specific manner. In conclusion, a novel culture system using HESS-5 is useful to support a rapid and sustained generation of primitive myeloid cells which can develop into functional DCs.

Soluble HLA class I and class II in Japanese

Soluble human leukocyte antigens (sHLA) class I (sHLAI) and class II (sHLA-II) in sera or plasma can be quantitatively measured by enzyme-linked immunosorbent assay (ELISA) [1, 2]. Several reports state that individuals with particular HLA allotypes have a higher sHLA-I concentration than those without that particular allotype [1-5]. HLA-A9 especially is widely known to be accompanied by the highest sHLA-I concentration [2-5] . Meanwhile, there are only a few reports discussing sHLA-II quantification [6, 7] and no definite conclusion about the association with particular HLA allotypes has been reported. In the present study, sHLA-I and II (DR) in plasma were measured by ELISA and their association with particular HLA allotypes was examined in Japanese subjects. The frequency of HLA-A9-positive individuals in Japan is much higher (68%) than that in North America (22%) [8]; therefore, the Japanese population could provide the appropriate samples to investigate the relationship between sHLA-I and A9 allotype.

Association between HLA-DPB1 matching and 1-year rejection-free graft survival in high-risk corneal transplantation

We analyzed the effect of matching for HLA class II alleles on corneal graft outcome in a single-center, retrospective study from January 1991 through April 1996. The study involved 81 transplant recipients at high and low risk of corneal graft rejection, who were typed by the polymerase chain reaction-restriction fragment length polymorphism method and who completed at least 1-year of follow-up. The DRB1, DQB1, and DPB1 alleles were analyzed together and transplant recipients were subdivided into groups with matching (one to four alleles matched in the high risk or one to five alleles matched in the low risk) and without matching (no allele matched) for HLA class II. A significantly higher rate of 1-year rejection-free graft survival was revealed in high-risk transplant recipients with matching, compared with those without matching (P=0.0238). We have shown that matching for at least one HLA class II allele was actually beneficial in high-risk transplants. An analysis of matching for each allele separately, detected that only HLA-DPB1 matching was significantly associated with a higher rate of 1-year rejection-free graft survival in high-risk transplant recipients with matching (one or two alleles matched) compared with those without matching (no allele matched) (P=0.0139). In particular, matching for one DPB1 allele was significantly beneficial compared with no matching (P=0.0140). There was no significant effect of HLA-DRB1 and -DQB1 matching (P=0.3177 and P=0.2878, respectively). Furthermore, a strong association between DPB1 matching and 1-year rejection-free graft survival was observed in DRB1-incompatible high-risk transplant recipients (P=0.0308). Nevertheless, no significant effect of DPB1 matching was detected in DQB1-incompatible transplant recipients. Our findings indicate that HLA class II DNA typing is clinically relevant for corneal transplant recipients and that especially HLA-DPB1 matching has a beneficial effect in high-risk corneal transplantation.

Serum concentrations of soluble HLA-class I and CD8 forms in patients with viral hepatic disorders

Soluble HLA-class I and CD8 molecules were determined by sandwich ELISA in patients with viral-induced hepatic disorders. As a whole, the patients with hepatic disorders (acute hepatitis: AH; chronic hepatitis: CH; liver cirrhosis: LC; hepatocellular carcinoma: HCC) showed higher sHLA-class I and sCD8 levels than normal controls (P<0.001). AH patients had the highest sHLA-class I levels (mean, 3513±2112ng/ml), followed by CH (2896±1290ng/ml), LC (2293±1266ng/ml), and HCC (2221±1212ng/ml) sCD8 levels were highest in AH, followed by HCC, LC, and CH, in that order. Among histologically defined C virus-positive patients, sHLA-I levels were higher in those with chronic active hepatitis (CAH) 2A (3802±1124ng/ml) than in those with chronic persistent hepatitis (CPH; 2200±711ng/ml;P<0.01), the levels then decreased as the disease progressed (CAH2B, 3564±1783ng/ml, LC, 2376±1265ng/ml). In contrast, sCD8 values showed little difference among the disorders. sHLA-class I levels showed a positive correlation with sCD8 values both in whole patients and in patients with AH (P<0.01), but no correlation was shown, in any patients, with biochemical parameters such as GPT and GOT. These findings, taken together, suggest that hepatic destruction is not the only cause of sHLA-class I production, but that sHLA-class I levels, together with sCD8 levels, may reflect immunological activity in hepatic disorders.

Serum soluble HLA-DR antigens in autoimmune hepatitis.

To investigate the significance of HLA-class II, especially DR antigens, in autoimmune hepatitis (AIH), the serum concentrations of soluble HLA-DR antigen (sDR) were measured in 16 patients with AIH. The expression of HLA-DR antigens in the liver tissues of AIH patients was also studied by immunohistochemistry. AIH at diagnosis showed markedly higher serum sDR levels than controls, in which the liver tissues exhibited positive staining of HLA-DR antigens. Seven patients received corticosteroid therapy, in whom the serum sHLA-DR concentration was reduced dramatically from activated to remission stage. In sequentially follow-up cases, sDR correlated well with the disease activity, and also with the change of surface DR expression in the liver. A single major band with a molecular size of 60 kDa was detected, both in patients sera and in normal control sera, by Western blotting. In conclusions, serum sHLA-DR level could be a marker reflecting immunological activity of the disease.

Effects of iso and xeno fetal liver fragments transplantation on acute and chronic liver failure in rats.

Isogeneic (rat) and xenogeneic (swine) fetal liver fragments (FLF) transplantation into the omentum was performed for d-galactosamine (d-Gal)-induced acute and carbon tetrachloride (CCI4)-induced chronic hepatic failure in rats. The recipients that had iso or xeno FLF showed higher survival rates than the nontransplanted controls on a lethal dose (2.6 g/kg body weight) of d-Gal (survival rates: Iso 70%, Xeno 80%, and control 9.1%). On a sublethal dose (1.0 or 1.2 g/kg) of d-Gal, iso, or xeno FLF caused marked improvement of the values of GPT, GOT, and total bilirubin (T. Bil); at 72 h after d-Gal injection they went significantly lower than those of controls (Iso vs. control; p < 0.01, Xeno vs. control; p < 0.05). Histological examination of the livers revealed severe damage in controls, however, only a slight damage was found in iso or xeno FLF transplanted rats. Iso grafts were fairly well preserved in the omentum at 72 h posttransplants, however, xeno graft had almost changed into a necrotic tissue. CCI4 was administered subcutaneously for 14 wk to induce chronic hepatic failure and then iso FLF were transplanted 3 days after the last CCI4 injection. Iso FLF transplanted rats showed higher improvement of GPT and GOT values at 12 days posttransplants compared with controls (GPT p < 0.01, GOT p < 0.05), although histological improvement was not so remarkable in both group. Iso grafts formed nodules with many hepatocytes in the omentum 12 days posttransplant. The results indicate that iso or xeno FLF transplantation could be an alternative approach for incurable liver insufficiencies.

The Generation of Immunocompetent Dendritic Cells From CD34^+ Acute Myeloid or Lymphoid Leukemia Cells

The ability of CD34+ leukemic cells to differentiate to dendritic cells (DCs) was investigated in 18 acute myeloid leukemia (AML) and 4 lymphoid leukemia (ALL) patients. The generation of DCs was determined by the expression of DC-associated CD1a or CD83 (more than 30%) with costimulatory molecules, by CD80 antigens (>20%), and by the exhibition of allostim-ulatory activity. In the AML patients, allostimulatory mature DCs were generated from 3 of 9 M0 or M1, 2 of 5 M2, 2 of 4 M4 or M5, and 3 of 4 ALL (L2) cases. In total, DCs were more efficiently induced from cases expressing over 75% of CD34+ among whole bone marrow mononuclear cells (8 of 12), compared with those under 75% (2 of 10; P < .05). B-cell (CD19), natural killer (NK)—cell (CD56), or T-cell (CD7) lineage markers, which were aberrantly expressed on the blasts, were rarely found on leukemic DCs at the end of the culture period, and myeloid (CD13, CD33), not lymphoid (CD10), markers were shown on ALL-derived DCs. In Philadelphia chromosome—positive ALL or AML patients with t (8;21), DCs were confirmed to be of leukemic origin by fluorescence in situ hybridization analysis.