Analysis of 66 patients definitive with transfusion-associated graft-versus-host disease and the effect of universal irradiation of blood.

BACKGROUND: Transfusion-associated graft-versus-host disease (TA-GVHD) is a potentially fatal adverse reaction to blood transfusion. Although TA-GVHD was formerly considered to be rare and to occur only in immunocompromised patients, it was confirmed to occur even in immunocompetent patients in Japan, based on a definitive diagnostic test for TA-GVHD using highly polymorphic microsatellite repeat sequences. We clarify the clinical picture of TA-GVHD via definitive diagnosed cases and argue the validity of blood irradiation for TA-GVHD prevention. PATIENTS AND METHODS: Two-hundred and ninety patients who were suspected of having TA-GVHD and referred to us for diagnostic testing from October 1992 to August 1999 were analysed for the associated clinical characteristics and risk factors. Effects of universal irradiation were followed up until 2010. RESULTS: Sixty-six of the 290 study patients were diagnosed as having definite TA-GVHD by microsatellite DNA analysis. Regarding the symptoms of patients with definite TA-GVHD, a fever of over 38 °C, erythema and leucocytopenia were found in virtually all of these patients. Among patients in whom human leucocyte antigen (HLA) typing was carried out, TA-GVHD almost always developed in HLA heterozygous patients following the transfusion of HLA homozygous blood. TA-GVHD was reported significantly more frequently in older patients. In this study, TA-GVHD was caused by the transfusion of HLA one-way match blood stored for 14 days. CONCLUSION: No cases of TA-GVHD development have been confirmed since 2000, when the supply of irradiated blood products became widespread. No major problems have been encountered since the start of universal irradiation, more than 10 years ago.

Consecutive national surveys of ABO-incompatible blood transfusion in Japan.

BACKGROUND AND OBJECTIVES: Morbidity and mortality from ABO-incompatible transfusion persist as consequences of human error. Even so, insufficient attention has been given to improving transfusion safety within the hospital. MATERIALS AND METHODS: National surveys of ABO-incompatible blood transfusions were conducted by the Japanese Society of Blood Transfusion, with support from the Ministry of Health, Labor and Welfare. Surveys concluded in 2000 and 2005 analysed ABO-incompatible transfusion data from the previous 5 years (January 1995 to December 1999 and January 2000 to December 2004, respectively). The first survey targeted 777 hospitals and the second, 1355 hospitals. Data were collected through anonymous questionnaires. RESULTS: The first survey achieved a 77.4% response rate (578 of 777 hospitals). The second survey collected data from 251 more hospitals, but with a lower response rate (61.2%, or 829 of 1355 hospitals). The first survey analysed 166 incidents from 578 hospitals, vs. 60 incidents from 829 hospitals in the second survey. The main cause of ABO-incompatible transfusion was identification error between patient and blood product: 55% (91 of 166) in the first survey and 45% (27 of 60) in the second. Patient outcomes included nine preventable deaths from 1995 to 1999, and eight preventable deaths from 2000 to 2004. CONCLUSION: Misidentification at the bedside persists as the main cause of ABO-incompatible transfusion.

Kudo type-5 total elbow arthroplasty in mutilating rheumatoid arthritis: a 5- to 11-year follow-up.

We studied 11 patients (14 elbows) with gross rheumatoid deformity of the elbow, treated by total arthroplasty using the Kudo type-5 unlinked prosthesis, and who were evaluated between five and 11 years after operation. Massive bone defects were augmented by autogenous bone grafts. There were no major complications such as infection, subluxation or loosening. In most elbows relief from pain and stability were achieved. The results, according to the Mayo Elbow Performance Score, were excellent in eight, good in five and fair in one. In most elbows there was minimal or no resorption of the grafted bone. There were no radiolucent lines around the stems of the cementless components. This study shows that even highly unstable rheumatoid elbows can be replaced successfully using an unlinked prosthesis, with augmentation by grafting for major defects of bone.

Human peripheral blood Valpha24+ Vbeta11+ NKT cells expand following administration of alpha-galactosylceramide-pulsed dendritic cells.

BACKGROUND AND OBJECTIVES: We have undertaken the first clinical trial involving the administration of alpha-GalactosylCeramine (alpha-GalCer)-pulsed dendritic cells (DCs) to human subjects, to determine safety, optimal dose, optimal administration route and immunological effects. MATERIALS AND METHODS: Subjects (n = 4) with metastatic malignancy received two infusions of alpha-GalCer-pulsed DCs intravenously, and two infusions intradermally. The percentages of Valpha24 Vbeta11 NKT cells in peripheral blood (PB) were determined by three-colour flow cytometry and the PB NKT cell numbers were calculated using the total number of PB lymphocytes/ml determined by automated full-blood counts. RESULTS: No serious treatment related adverse events were observed during the study period. Administration of alpha-GalCer-pulsed DCs in vivo can significantly (P < 0.03) increase PB Valpha24+ Vbeta11+ NKT cell numbers above pretreatment baseline levels after the transient fall in the NKT numbers within 48 h. CONCLUSIONS: Administration of alpha-GalCer-pulsed DCs is well tolerated, modulates PB Valpha24+ Vbeta11+ NKT cells and may have a role in the therapy of malignancies sensitive to activities of Valpha24+ Vbeta11+ NKT cells, or for autoimmune diseases.

Polymorphisms in TNFA and TNFR2 affect outcome of unrelated bone marrow transplantation.

Effects of polymorphisms in TNFA and TNFR2 on the outcome of 462 cases of unrelated bone marrow transplantation (uBMT) were studied retrospectively. Four alleles of TNFA (U01-U04) distinguished by polymorphism in the upstream region, -1031 (T/C), -863 (C/A) and -857 (C/T), and two alleles of TNFR2 (196M/196R) distinguished by polymorphism at codon 196 were determined. Transplantation involving TNFA-U02- and/or U03-positive donors and/or recipients resulted in a higher incidence of graft-versus-host disease (GVHD) of grade III-IV (P < 0.05 for donor type, P < 0.01 for recipient type) and a lower relapse rate than that involving TNFA-U01 homozygous recipients and/or donors (P < 0.025 for donor type, P < 0.01 for recipient type). These results include the HLA mismatching effect due to linkage disequilibirium of TNFA with HLA loci. However, the effects were also observed in HLA-A, -B and -DRB1 allele-matched transplantation. Transplantation from TNFR2-196R-positive donors exhibited a higher incidence of severe GVHD (P < 0.05) and tendency for a lower relapse rate than that from TNFR2-196M homozygous donors. TNFR2-196R of recipient origin had no effect on GVHD but increased the relapse rate (P < 0.025). These results suggest that TNFA and TNFR2 typings are helpful for predicting uBMT outcome and for preventing severe complications at an early stage.

Supernatants of stored polymorphonuclear neutrophils exhibit growth-promoting activity in several cell lines: perforin expression in polymorphonuclear neutrophils and its role in down-regulation of growth-promoting activity.

BACKGROUND AND OBJECTIVES: Polymorphonuclear neutrophils (PMNs) play important roles in the host immune defence. This study was performed to identify roles of PMNs other than those already known. MATERIALS AND METHODS: PMNs were separated from the peripheral blood of healthy individuals and stored in vitro for 24 h in the presence or absence of an anti-human Fc receptor (FcR) gamma III antibody, namely, anti-CD16 monoclonal antibody (mAb). Stored supernatants were harvested and incubated with several leukaemia and transformed cell lines for 48 h. The increase in growth rate was assessed by the increase in the amount of 3H-thymidine incorporated into these cells. Expression of perforin on PMNs, which is thought to decrease cell viability, was elucidated by flow cytometry (FCM) analysis. The presence of perforin in the stored supernatants was determined using an enzyme-linked immunosorbent assay (ELISA). A serine protease inhibitor, which is known to block the effect of perforin, was added to the cultures of several leukaemia and transformed cell lines to confirm the effect of perforin in reducing cell viability. RESULTS: Growth promotion of some cell lines cultured with the stored supernatants of PMNs was observed both in the presence and absence of anti-CD16 mAb, which was used as a trigger molecule of PMNs. This was particularly notable in the case of Raji and Daudi (both Burkitt lymphoma) cell lines and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (B-LCLs) derived from healthy individuals. Perforin expression was observed in both freshly prepared and stored PMNs, regardless of the presence or absence of anti-CD16 mAb. ELISA also detected perforin in the stored supernatants in both the presence and absence of anti-CD16 mAb. The increase in growth rate was induced in the presence of not only a serine protease inhibitor but also an anti-perforin mAb. CONCLUSIONS: Stored supernatants of PMNs exhibit up-regulation of cell growth in several cell lines; this up-regulation is particularly prominent in B-lineage cell lines. Furthermore, perforin appeared to be expressed on PMNs constitutively and secreted into the extracellular fluid. Results of this study strongly suggest that the growth-promoting activity in supernatants of stored PMNs is partially inhibited by perforin, which is thought to be produced by PMNs themselves.

Effectiveness of washed platelet concentrate and red cell transfusions for a patient with anhaptoglobinemia with antihaptoglobin antibody.

A 71-year-old Japanese male with myelodysplastic syndrome progressing to overt leukaemia and hepatocellular carcinoma developed dyspnea and urticaria immediately after infusion of platelet concentrate (PC). He exhibited an identical reaction following blood transfusion. Serum haptoglobin was undetectable. The patient was determined to be homozygous for Hp(del) by polymerase chain reaction (PCR). Antibody to haptoglobin was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. No antibodies against human leucocyte antigen (HLA) or platelet-specific antigens were detected. Washed PC and washed red blood cells were effective in preventing the transfusion-related anaphylactoid reactions.

Usefulness of absorbable screws in the Sauvé-Kapandji procedure for rheumatoid wrist reconstruction.

Abstract In the Sauvé-Kapandji (S-K) procedure for rheumatoid wrist reconstruction, the distal end of the ulna is fixed to the radius with screws. Recently, absorbable screws have increasingly been used instead of metal ones. However, the clinical usefulness of absorbable screws in S-K procedures for rheumatoid patients is still unknown. The purpose of this article is to evaluate the effect of absorbable screws in this procedure by comparing their clinical results with those of metal screws. Poly-l-lactic acid (PLLA) absorbable screws were used in 23 wrists, and metal screws were used in 20 wrists. We evaluated the presence of general or local reactions to PLLA, the stability of the ulnar head, the time to bone union, changes in the shape of the distal ulna, and the presence of bone resorption around the screws. There were no complications with the use of PLLA screws, and their fixation stability was adequate to form sufficient bone union. In five cases in the metal screw group, bone resorption around the screws occurred between 1 and 2 years after surgery. Bone resorption around the PLLA screws was not observed. We conclude that absorbable screws may be more useful than metal screws in the S-K procedure for rheumatoid wrist reconstruction.

E variants found in Japanese and c antigenicity alteration without substitution in the second extracellular loop.

BACKGROUND: The molecular basis of E variants in the Japanese population is poorly understood. In this study, molecular analysis of E variants detected in Japanese by serologic methods was carried out. STUDY DESIGN AND METHODS: E variants from healthy Japanese blood donors were screened by serologic analysis using E MoAbs. Fifteen E variant samples were divided into three types--EFM, EKH, and EKK-on the basis of patterns of reactivity with five distinct E antibodies. The entire coding region of the Rh cDNAs from the E variant samples was analyzed by sequencing. RESULTS: Although the Rh cDNA sequences of the three types were different from each other, those of the EFM-type variants (RHEFM) had a partial DNA exchange in exon 5 between the RHCE and RHD genes, generating an RHcE variant (Gln233Glu, Met238Val). The cDNA of EKH-type variants (RHEKH) exhibited a point mutation (G461C) in exon 3 of the RHcE allele that resulted in an Arg154Thr substitution in the third external loop of the RhcE peptide. The EKK-type variant (RHEKK) carried a hybrid gene structure characterized by replacement of exons 1-3 (or 2-3) of the RHCE gene with those of the RHD gene. The RHD gene of a person possessing an E variant of the EKK type was also a hybrid gene, D-cE(2-3)-D or cE(1-3)-D (RHDKK). The E variants of types EKH and EKK showed weak c antigenicity. CONCLUSION: In serologic screening of 140,723 Japanese blood donors, 15 were found to possess E variants (0.011%). A new RHCE variant, RHEKH, was identified. On the basis of the variants found in this study, the c antigenicity seemed to be determined not only by Pro-103 but also by the structure of the third extracellular loop or the amino acids contained in it.

Gene expression analysis in human monocytes, monocyte-derived dendritic cells, and alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha-GalCer (alpha-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha-GalCer, suggesting a novel immunomodulating effect of alpha-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days.

Polymorphisms in the coding region of mtDNA and effects on clinical outcome of unrelated bone marrow transplantation.

The entire protein-coding region was divided into 45 fragments, separately amplified and analyzed for polymorphism by the PCR-SSCP (single-strand conformation polymorphism) method. The effect of polymorphism mismatching on the clinical outcome of unrelated bone marrow transplantation was studied to clarify whether products from mtDNA become minor antigens. Variability in PCR-SSCP pattern combinations of the 45 fragments suggests that each individual has a different polymorphism combination in the protein-coding region if all the coding regions were compared at the nucleotide sequence level. Nonsynonymous polymorphisms were found at relatively high frequency in MTATP8 and MTND3. Both the polymorphisms with and without substitution matched the peptide-binding motifs of HLA-A*0201. The effects of the polymorphism matching were retrospectively analyzed in 340 recipients transplanted with HLA-A, -B, -DRB1 allele-matched bone marrow from unrelated donors. There were no effects of polymorphism matching on the incidence of acute GVHD and cumulative disease-free survival. These results suggest that polymorphisms which generate peptides, with and without substitutions, that bind the same HLA molecule hardly influence GVHD because the difference between the HLA-peptide complexes is minute.

In vitro anti-tumour activity of alpha-galactosylceramide-stimulated human invariant Valpha24+NKT cells against melanoma.

alpha-galactosylceramide (KRN 7000, alpha-GalCer) has shown potent in vivo anti-tumour activity in mice, including against melanoma and the highly specific effect of inducing proliferation and activation of human Valpha24+NKT-cells. We hypothesized that human Valpha24+NKT-cells activated by alpha-GalCer might exhibit anti-tumour activity against human melanoma. To investigate this, Valpha24+NKT-cells were generated from the peripheral blood of patients with melanoma after stimulation with alpha-GalCer pulsed monocyte-derived dendritic cells (Mo-DCs). Valpha24+NKT-cells did not exhibit cytolytic activity against the primary autologous or allogeneic melanoma cell lines tested. However, proliferation of the melanoma cell lines was markedly suppressed by co-culture with activated Valpha24+NKT-cells (mean +/- SD inhibition of proliferation 63.9 +/- 1.3%). Culture supernatants of activated Valpha24+NKT-cell cultures stimulated with alpha-GalCer pulsed Mo-DCs exhibited similar antiproliferative activities against melanoma cells, indicating that the majority of the inhibitory effects were due to soluble mediators rather than direct cell-to-cell interactions. This effect was predominantly due to release of IFN-gamma, and to a lesser extent IL-12. Other cytokines, including IL-4 and IL-10, were released but these cytokines had less antiproliferative effects. These in vitro results show that Valpha24+NKT-cells stimulated by alpha-GalCer-pulsed Mo-DCs have anti-tumour activities against human melanoma through antiproliferative effects exerted by soluble mediators rather than cytolytic effects as observed against some other tumours. Induction of local cytokine release by activated Valpha24+NKT-cells may contribute to clinical anti-tumour effects of alpha-GalCer.

Expression of renin-angiotensin system genes in immature and mature dendritic cells identified using human cDNA microarray.

Using a cDNA glass microarray, the expression of 1081 genes in immature and mature dendritic cells (DCs) of two different individuals has been studied. The upregulation of mRNA transcripts of genes encoding the transcription factor ZFM1, Mos proto-oncogene serine/threonine-protein kinase, B-cell-specific transcription factor, preB-cell growth stimulating factor, ets translocation variant 6, and epidermal growth-factor-like CRIPTO was for the first time detected in DCs. Using semiquantitative RT-PCR analysis the upregulation of the transforming growth factor-alpha, integrin alpha 6 and ZFM 1 transcription factor in mature DCs was confirmed in samples from four different individuals. On the other hand, the downregulation of renin-binding protein transcript was detected in mature DCs using a cDNA microarray. For the first time, the expression of renin-angiotensin system genes was evaluated during maturation of DCs in samples from four donors by semiquantitative RT-PCR. A possible role of the renin-angiotensin system in DCs is discussed.

Profiling of genes expressed in human monocytes and monocyte-derived dendritic cells using cDNA expression array.

Using a human cDNA expression array, we obtained expression profiles of 588 genes in CD14+ monocytes and monocyte-derived dendritic cells (DCs). Overall, 22 genes were upregulated, and nine genes were downregulated in DCs of both samples from two different individuals. Many of the genes that were upregulated in DCs encode proteins that are related to differentiation, cell structure, migration, termination of cell cycle as well as proliferation, e.g. tumour necrosis factor-alpha (TNF-alpha), tumour necrosis factor receptor II (TNFRII), thymosin beta-10, epithelial discoidin domain receptor 1, replication factor C, putative transcription factor DB1, alpha catenin, transforming growth factor-beta 1, prohibitin, p53-regulating protein and neu differentiation factor. Among the downregulated genes in DCs were genes that encode proteins of cell cycle regulation: mitotic growth and transcription activator, platelet-derived growth factor receptor-beta subunit, interleukin 2 receptor (IL-2R)-gamma subunit, IL-7R-alpha subunit, leucocyte interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR). Semi-quantitative reverse transcription-polymerase chain reaction method confirmed the upregulated expression levels in DCs for TNFRII, TNF-alpha, alpha catenin and downregulation of IFN-gamma, GM-CSFR on four different donor samples of DCs and monocytes. Moreover, our data show the presence of a 'switch-on' step for the TNF-alpha and TNFRII gene expression in immature DCs for further differentiation into mature DCs.

Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4+ Valpha24 natural killer T cells expressing CD40L.

Human Valpha24 natural killer T (Valpha24NKT) cells are activated by alpha-glycosylceramide-pulsed dendritic cells (DCs) in a CD1d-dependent and T-cell receptor-mediated manner. There are two major subpopulations of Valpha24NKT cells, CD4- CD8- Valpha24NKT and CD4+ Valpha24NKT cells. We have recently shown that activated CD4- CD8- Valpha24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of Valpha24NKT cells is currently limited. We aimed to investigate whether CD4+ Valpha24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4+ Valpha24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4+ Valpha24NKT cells, but not with resting CD4+ Valpha24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb. Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40-CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Valpha24NKT cells. The apoptosis of DCs from normal donors, triggered by the CD40-CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4+ Valpha24NKT cells by virtue of apoptosis of DCs.

TRAIL expression by activated human CD4(+)V alpha 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukemia cells.

Human Valpha24NKT cells are activated by alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells in a CD1d-dependent and a T-cell receptor-mediated manner. Here, we demonstrate that CD4(+)V alpha 24NKT cells derived from a patient with acute myeloid leukemia (AML) M4 are phenotypically similar to those of healthy donors and, in common with those derived from healthy donors, express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) when the cells are activated by alpha-GalCer-pulsed dendritic cells but not prior to activation. We also show that myeloid leukemia cells from patients with AML M4, but not from patients with AML M0 or M1, undergo apoptosis following culture with TRAIL-expressing autologous or allogeneic healthy donor V alpha 24NKT cells. Apoptosis of AML M4 leukemia cells from patient peripheral blood was almost completely blocked by a neutralizing monoclonal antibody against TRAIL, indicating that TRAIL on V alpha 24NKT cells is essential for the induction of apoptosis in AML M4 leukemia cells. A nonobese diabetic-severe combined immunodeficient human leukemia (AML M4) model showed that human activated CD4(+)V alpha 24NKT cells induced apoptosis of human leukemia cells in vivo. This is the first evidence that activated V alpha 24NKT cells express TRAIL and that TRAIL causes apoptosis of monocytic leukemia cells from patients with AML M4 in vitro and in vivo. Adoptive immune therapy with activated V alpha 24NKT cells, or other strategies to increase activated V alpha 24NKT cells in vivo, may be of benefit to patients with AML M4. (Blood. 2001;97:2067-2074)

Identification of novel single nucleotide substitutions in the NKp30 gene expressed in human natural killer cells.

Cytotoxicity of natural killer (NK) cells is regulated by a balance of signals from two kinds of NK receptors, activating receptors and inhibitory receptors. Natural cytotoxicity receptors (NCR) family, which consists of NKp30, NKp44 and NKp46, is a major human activating NK receptor. NKp30 has been mapped to the HLA class III region near tumor necrosis factor (TNF) family loci. We have analyzed the NKp30 gene of healthy Japanese and found two synonymous substitutions in the coding region, c.111G>A and c.156C>T, and also identified two single-nucleotide polymorphisms (SNPs) in the promotor region, -201G>A and -163G>C. Furthermore, it was confirmed that these polymorphisms of the NKp30 gene show strong linkage disequilibria with each other and with HLA-DRB1 or TNFA polymorphisms. Since susceptibilities to certain diseases were mapped near this region, the NKp30 polymorphisms could be useful genetic markers.

Identification of a telomeric boundary of the HLA region with potential for predisposition to human narcolepsy.

We report on a study performed to determine a boundary of the region with the potential to contribute to the predisposition to human narcolepsy (the susceptibility region) in the human leukocyte antigen (HLA) region. We investigated a Japanese narcolepsy family, in which a de novo chromosomal recombination occurred between the HLA-DRB1 and HLA-B genes in the proband. The recombinant chromosome carrying HLA-DRB1*1501 was transmitted to the affected child and grandchild, suggesting that a strong genetic factor(s) predisposing to the disorder was (were) present on the chromosome, and that the recombination breakpoint could be regarded as a boundary to the susceptibility region. To search for the breakpoint, we carried out allele typing at various polymorphic sites, e.g., microsatellite repeat polymorphisms, restriction fragment length polymorphisms, and single-nucleotide polymorphisms in the HLA region, and examined haplotypes with the polymorphic sites in the family members. Haplotype analyses revealed that the recombination breakpoint was present approximately 50 kb to the telomeric side of the palmitoyl-protein thioesterase-2 (PPT2) gene in the HLA class III region. From the gene map of the HLA region, the cyclic AMP response element-binding protein-related protein gene (CREB-RP) appeared to be located at the telomeric end in the 50-kb region. Therefore, the data presented here suggest that the susceptibility region for the disorder in the family is present on the centromeric side of the CREB-RP gene in the recombinant Chromosome 6 carrying HLA-DRB1*1501.

Population-based mapping of pulmonary adenoma susceptibility 1 locus.

Pulmonary adenoma susceptibility 1 (Pas1), the major locus affecting inherited predisposition to lung tumor development in mice, maps near the Kras2 gene. We previously reported a significant association between a KRAS2/RsaI polymorphism and the risk and prognosis of lung adenocarcinoma (ADCA) in the Italian population. In the present case-control study, we examined 269 lung ADCA patients, 121 squamous cell lung carcinoma patients, and 632 healthy individuals (general population controls) in the Japanese population with genetic markers spanning approximately 1200 kb in the KRAS2 region. Allele-specific oligonucleotide hybridization revealed the same KRAS2/RsaI polymorphism associated with risk and prognosis as in Italian lung ADCA patients; the polymorphism was significantly associated with clinical stage (P < 0.001) and survival rate (log rank = 0.0014), confirming the mapping of PAS1 and pointing to the role of this locus in human lung cancer.

Alpha-glycosylceramides enhance the antitumor cytotoxicity of hepatic lymphocytes obtained from cancer patients by activating CD3-CD56+ NK cells in vitro.

Alpha-glycosylceramides, such as alpha-galactosylceramide and alpha-glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, V alpha 14 TCR+NK1.1+ T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by alpha-glycosylceramides. We recently reported that V alpha 24 TCR+ NKT cells, the human homologues of murine V alpha 14 TCR+NK1.1+ cells, are rarely seen among freshly isolated human hepatic lymphocytes. Therefore, it is important to examine whether alpha-glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of alpha-glycosylceramides in cancer immunotherapy in humans. Here, we show that alpha-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro. The direct effector cells of the elicited cytotoxicity were CD3-CD56+ NK cells. Even though V alpha 24 TCR+NKT cells proliferated remarkably in response to alpha-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of alpha-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3-CD56+ NK cells in the liver.