Anti-A and anti-B titers, age, gender, biochemical parameters, and body mass index in Japanese blood donors.

It has been reported that anti-A and anti-B (ABO antibody) titers decrease with age, but little is known about the association between ABO antibody titers and physiologic/biochemical parameters such as body mass index (BMI), gamma-glutamyl transpeptidase (GGT), and total cholesterol (T-Cho). We investigated the present situation of ABO antibody titers among healthy blood donors in Japan and the physiologic/biochemical factors that may be associated with changes in ABO antibody titers. Plasma from 7450 Japanese blood donors was tested for ABO antibody titers using ABO reverse typing reagents by an automated microplate system; donor samples were classified into low, middle, and high titers according to the agglutination results obtained with diluted plasma samples. Multivariate regression analysis was performed to analyze the association between ABO antibody titers and age, gender, biochemical parameters (alanine transaminase [ALT], GGT, globulin, T-Cho, and glycosylated albumin [GA]), and BMI according to the ABO blood groups. A significant correlation between ABO antibody titers and age/gender, except for gender in anti-A of blood group B donors, was observed. BMI showed significant but negative correlations with anti-A and anti-B (ß = -0.085 and -0.062, respectively; p < 0.01) in blood group O donors. In addition, significant but negative correlations between GGT and T-Cho with anti-B of blood group A donors (ß = -0.055 and -0.047, respectively; p < 0.05) were observed. Although differences existed among the ABO blood groups, ABO antibody titers seem to be associated with physiologic and biochemical parameters of healthy individuals.

Regulation of the lysophosphatidylserine and sphingosine 1-phosphate levels in autologous whole blood by the pre-storage leukocyte reduction.

BACKGROUND AND OBJECTIVES: The effect of leukoreduction and storage periods on the accumulation of bioactive lysophospholipids and substances in human autologous blood (AB units) has not been fully investigated. MATERIALS AND METHODS: The accumulation of bioactive lysophospholipids such as sphingosine 1-phosphate (S1P) and lysophosphatidylserine (LysoPS) in AB units during the storage was investigated. The time-dependent changes and the effect of the filtration in pre-storage leuckoreduction (LR) and unmodified samples derived from 46 AB units were analysed. Additionally, the changes of lysophospholipids and platelet releasate, namely ß-thromboglobulin (ß-TG), induced by exposure of whole blood (WB) or platelet-rich plasma (PRP) to the filter material were analysed. RESULTS: LysoPS, but not S1P levels, time-dependently and significantly increased in both unmodified and LR samples. LysoPS significantly decreased in LR compared with unmodified samples, whereas S1P increased in LR compared with unmodified samples. In addition, exposure of WB and/or PRP to the filter material in vitro resulted in increased levels of S1P, LysoPS and ß-TG. CONCLUSIONS: LR effectively reduced the accumulation of LysoPS in AB units. On the other hand, it increased concentrations of S1P due to platelet activation by exposure to the filter material. These suggest that increases of S1P levels in LR and LysoPS in the unmodified samples were mainly caused by the leukocytes and/or platelets and that LR was effective in inhibiting the accumulation of LysoPS.

Perinatal management of neonatal alloimmune thrombocytopenia associated with anti-group A antibody.

OBJECTIVE: To prevent neonatal alloimmune thrombocytopenia due to anti-group A antibody perinatal management was performed. BACKGROUND: We previously reported a case of severe intracranial haemorrhage associated with neonatal alloimmune thrombocytopenia due to anti-group A isoantibody. MATERIAL/METHODS: A 40-year-old Japanese woman, gravida 4 para 1, was pregnant with her second baby. The previous sibling developed severe thrombocytopenia and died 10 days after birth due to intracranial haemorrhage. He was diagnosed with neonatal alloimmune thrombocytopenia; the causative antibody was found to be the anti-group A antibody. Prednisone was started at 7 weeks' gestational age. Intravenous immunoglobulin 1 g kg(-1) week(-1) was started at 29 weeks' gestational age and continued to delivery. Serological studies and genotyping were performed. RESULTS: The second boy was delivered at 33 weeks' gestational age by caesarean section. He was discharged without intracranial haemorrhage or thrombocytopenia. The anti-group A antibody titre in the maternal serum was 2048-4096 (normal range: 4-64). The anti-group A antibody titre in the newborn's serum was 4. Cross-matching between the maternal serum and the paternal platelets was positive. CONCLUSION: Owing to the history of neonatal alloimmune thrombocytopenia causing intracranial haemorrhage and death of the previous sibling, strict follow-up of the subsequent pregnancy was conducted.

Surgical stress response after colorectal resection: a comparison of robotic, laparoscopic, and open surgery.

BACKGROUND: The perioperative immune status of colorectal robotic surgery (RS), laparoscopic surgery (LS), and open surgery (OS) patients has not been compared. Our aim was to evaluate perioperative stress and immune response after RS, LS and OS. METHODS: This prospective study included 46 colorectal surgery patients from the Department of Surgical Oncology of the University of Tokyo Hospital. Peripheral venous blood samples were obtained preoperatively and on postoperative days 1, 3, and 6. We evaluated expression of HLA-DR (marker of immune competence), C-reactive protein (CRP) levels, and lymphocyte subset counts (natural killers, cytotoxic T cells and helper T cells). RESULTS: Fifteen, 23, and 8 patients underwent RS, LS and OS, respectively. HLA-DR expression was the lowest on day 1 and gradually increased on days 3 and 6 in all the groups. There was no significant difference in postoperative HLA-DR expression between the RS and LS group. However, on day 3, HLA-DR expression in the RS group was significantly higher than in the OS group (p = 0.04). On day 1, CRP levels in the LS group were significantly lower than in the RS group (p = 0.038). There were no significant perioperative changes in the lymphocyte subset cell count between the three groups. CONCLUSIONS: Perioperative surgical stress, as evaluated by immunological parameters, was comparable between robotic and laparoscopic surgery and higher with open surgery. Robotic surgery may be an alternative to laparoscopic surgery, as a minimally invasive surgery option for colorectal cancer.

The frequencies of human neutrophil alloantigens among the Japanese population.

Human neutrophil antigens (HNAs) play an important role in a variety of clinical conditions including immune-mediated neutropenia, non-hemolytic transfusion reactions, and transfusion-related acute lung injury. The aim of this study was to investigate the frequency distribution of HNAs-1 to -5 among the Japanese population. We analyzed samples from 570 healthy Japanese by molecular and serologic techniques to estimate the gene frequencies of HNAs-1 to -5. DNA samples were obtained and typed for the HNA-1 (n = 523), -3 (n = 570), -4 (n = 570), and -5 (n = 508), by molecular techniques. The HNA-1 genotype was determined by using a commercial polymerase chain reaction-reverse sequence-specific oligonucleotide probes (PCR-rSSOP) kit. The HNA-3 to -5 genotypes were determined by the PCR-sequence specific primer (PCR-SSP), previously described, with a small modification. The HNA-2a phenotype was determined in 301 donors by granulocyte immunofluorescence test. In Japanese, the gene frequencies of HNA-1a, -1b, and -1c were 0.623, 0.377, and 0.000, respectively. The frequency of HNA-2a phenotype was 0.987, and the gene frequencies of HNA-3a and -3b were 0.654 and 0.346, respectively. HNA-4a and -4b were found at 1.000 and 0.000, respectively, and HNA-5a and -5b at 0.840 and 0.160, respectively. We describe, for the first time, the frequencies of all HNAs (HNA-1 to -5) among the Japanese population. This study will be helpful for the prediction of the risk of alloimmunization to HNA, especially to determine the risk of HNA alloantibody production by transfusion of HNA incompatible blood and feto-maternal incompatibility.

Three novel beta3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency beta3-HPA antibodies.

BACKGROUND: Antibodies against human platelet antigens (HPA) are clinically important in fetal-maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the beta3 subunit of the alphaIIb beta3 integrin. Antibody detection is generally based on platelet-derived alphaIIb beta3 from HPA-genotyped donors. Recombinant allelic beta3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging. OBJECTIVES: We aimed to generate three recombinant beta3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency beta3 alloantigens. METHODS: The removal of the specificity-determining loop from the betaA domain and fusion of truncated beta3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens. RESULTS: Reactivity of the three peptides with beta3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes. CONCLUSIONS: These data demonstrate that beta3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.

The suppressive effect of sphingosine 1-phosphate on monocyte-endothelium adhesion may be mediated by the rearrangement of the endothelial integrins alpha(5)beta(1) and alpha(v)beta(3).

BACKGROUND: Sphingosine 1-phosphate (S1P), known to play important roles in vascular biology, is a bioactive lysophospholipid mediator that maintains endothelial integrity via its cell-surface receptors (S1Ps). In this in vitro study, we aimed to examine the role of S1P in monocyte-endothelium adhesion, which is an important event in the pathophysiology of atherosclerosis. METHODS AND RESULTS: S1P pretreatment of human umbilical vein endothelial cells (ECs), but not U937 cells, effectively suppressed U937-EC adhesion independently from the expression of adhesion molecules, namely ICAM-1, VCAM-1, and E-selectin. This S1P-induced suppressive effect was inhibited by the blockage of S1P(1) and S1P(3) receptors and the specific inhibitors of G(i) protein, Src family proteins, phosphatidylinositol 3-kinase, and Rac1, indicating involvement of these key downstream pathways. Moreover, the RGD peptide and antibodies, which neutralize adhesion via alpha(5)beta(1) and alpha(v)beta(3), effectively inhibited U937-EC adhesion with a degree similar to S1P pretreatment. Both an adhesion assay and flow-cytometric analysis demonstrated that U937 cells adhered through integrins alpha(5)beta(1) and alpha(v)beta(3) expressed on the apical surface of monolayer ECs, and S1P shifted the localization of these integrins from the apical surface to the basal surface. CONCLUSIONS: From the present results, we propose that S1P may contribute to the maintenance of vascular integrity and the regulation of atherogenesis through the rearrangement of endothelial integrins.

A study of dendritic and endothelial cell interactions in colon cancer in a cell line and small mammal model.

AIM: Historically, cancer therapy directly targeting tumor cells have yielded suboptimal clinical results, and therefore anti-angiogenic therapy that targets tumor cells indirectly through impairing tumor vasculature is now considered to be one of the novel approaches potentially effective against various types of cancer. In this study, we evaluated whether lysates of endothelium could be effectively pulsed in dendritic cells (DCs), to enhance their anti-tumor effects. METHODS: For this purpose, we prepared DCs of BALB/c mouse, incubated them with lysates of autologous or xenogeneic endothelium, and tested their anti-tumor effects in two syngeneic models of colon cancer. RESULTS: DCs pulsed with the respective endothelium lysates significantly inhibited the growth of subcutaneous tumors as well as pulmonary metastases in mice, and their anti-tumor effect was superior to that of unpulsed DCs. Immunohistopathological analysis showed significant decrease in the mean vascular density of tumors, correlating well with the extent of tumor inhibition. In vitro analysis of splenocytes isolated from immunized mice revealed an induction of cytotoxic T lymphocytes and activation of natural killer cells, with a lytic activity against activated endothelium but not tumor cells. In addition, antibodies reacting with activated endothelium, but not tumor cells, were detected in murine sera by ELISA, and their function was confirmed by complement-dependent cytotoxicity assay. CONCLUSIONS: Our present results suggest that lysates of endothelium can be effectively pulsed in DCs and enhance their anti-tumor effects through induction of anti-angiogenesis, and therefore should have important clinical implications for adjuvant cancer therapy.

Vaccines targeting tumour angiogenesis--a novel strategy for cancer immunotherapy.

AIMS: To review the concept of tumour angiogenesis and anti-angiogenic therapy, limitations of recently used anti-angiogenic therapeutics; provide an up-to-date overview of the growing number of reports on vaccines targeting tumour angiogenesis; and finally discuss potential complications and future directions in the development of more potent and specific vaccines. METHODS: A literature search was carried out from PubMed for indexed articles. The most important articles were analysed and discussed. FINDINGS: The search yielded a large number of important indexed published articles that were reviewed, screened and tracked for other relevant publications. The most relevant articles, including those previously published by authors, were analysed and discussed. CONCLUSIONS: Recently, different vaccine strategies have been reported to inhibit tumour growth and metastasis by induction of specific cellular and/or humoral immunity against angiogenesis-associated antigens in pre-clinical models, suggesting effective combination of anti-angiogenesis and cancer immunotherapy. Evaluation of tumour endothelial cells and clinical phase I study of the vaccines are recently ongoing, and should give us better insight into the possibilities of this novel strategy for cancer immunotherapy.

Quantification of minimal residual disease in patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia using a real-time RT-PCR assay.

Using a real-time RT-PCR method, we analyzed the expression of e1a2 BCR-ABL mRNA in bone marrow samples from 13 patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia (ALL) at different time points during chemotherapy and after bone marrow transplantation (BMT). The detection limit of the method, assessed using serial dilutions of ALL/MIK cells, was found to be 1:10(5), similar to what is observed for the conventional RT-nested PCR method. The e1a2 BCR-ABL values were normalized with respect to those of the housekeeping gene GAPDH. The decrease in the e1a2 BCR-ABL/GAPDH ratio after remission induction chemotherapy reflects well the response to chemotherapy and consequently correlates with the prognosis. Although molecular remission was achieved by chemotherapy alone, some patients relapsed, and the e1a2 BCR-ABL/GAPDH ratios in these cases progressively increased to the levels seen prior to hematological relapse. Long-term hematological complete remission (more than 30 months) could be achieved in cases in which a more than 4.0 log decrease in the e1a2 BCR-ABL/GAPDH ratio was obtained by chemotherapy alone, and BMT was then performed. In conclusion, real-time RT-PCR allows for an evaluation of the kinetics of e1a2 BCR-ABL/GAPDH expression during the various phases of chemotherapy or after BMT and may be effective for the indication and control of disease relapse in Ph-positive ALL patients.

Peripheral blood dendritic cells, but not monocyte-derived dendritic cells, can augment human NK cell function.

Dendritic cells (DCs) are essential antigen-presenting cells with a wide variety of functions relating to both adaptive and innate immunity. Recently, interactions of DCs with natural killer (NK) cells and NK1.1-positive T cells have been reported in mice. However, in humans, this interaction is not well understood. Here we report the use of a coculture method to analyze the modulation of NK cell function in antitumor immunity by DCs. We found that peripheral blood DCs (PDCs) enhanced NK cell activity in cytotoxicity assay, even without direct contact between DC and NK cells. In contrast, neither monocyte-derived DCs (MoDCs), nor TNF-alpha-treated MoDCs, stimulated NK lytic activity. Secretion of IL-12 and TNF-alpha into the PDC-NK coculture supernatant was increased. However, blocking antibodies against these cytokines could not completely abolish the upregulation of NK activity, suggesting the presence of other soluble factor(s) that affect DC-NK cell interaction. To summarize, this study demonstrates for the first time the direct activation of human NK cells by DC-NK cell interaction in vitro, suggesting that DCs may have a central role linking the innate and adaptive immune responses. Moreover, in stimulating NK cell function, PDCs appear to have a different potential from MoDCs.

Dendritic cells activate antitumor immunity for malignant intracranial germ cell tumor: a case report.

We report a 22-year-old male patient with a history of intracranial malignant germ cell tumor (GCT) who had undergone tumor resection twice, followed by radiation and chemotherapy. The tumor had rapidly recurred along the entire ventricular wall with extensive invasion into the brain parenchyma. The serum level of human beta-chorionic gonadotropin (beta-hCG) was 232.3 ng/ml on admission. Although tissue samples of the recurrent tumor could not be obtained, the previous histological diagnosis of germinoma and elevated serum beta-hCG levels suggested recurrence of malignant GCT. The patient declined chemotherapy but accepted dendritic cell (DC)-based immunotherapy. DC inoculation five times resulted in rapid tumor shrinkage and a significant decrease in the serum level of beta-hCG. Here we discuss the effectiveness of immunotherapy using DCs for recurrent intracranial malignant GCTs.

Thoracic empyema associated with recurrent colon cancer: report of a case and review of the literature.

Many types of infections associated with colorectal cancer have been reported. Here, we describe a rare case of thoracic empyema that was observed during immunotherapy for recurrent colon cancer. Culture of the pleural fluid yielded Streptococcus bovis, which is known to be associated with gastrointestinal lesions, especially colorectal malignancies. The possible correlation between these two clinical entities-empyema and colon cancer-is discussed.

Prognostic significance of glutamine synthetase expression in unifocal advanced hepatocellular carcinoma.

BACKGROUND/AIMS: Glutamine synthetase (GS) catalyzes the synthesis of glutamine, a major energy source of cells, and is upregulated in a subset of human hepatocellular carcinomas (HCCs). GS expression may be related to tumor recurrence, since GS-expressing tumors have a growth advantage in that they are independent of the extracellular glutamine supply. However, there are no studies concerning the prognostic value of GS expression in patients with HCC. METHODS: Seventy-three patients with a single advanced HCC nodule who underwent curative hepatectomy were included in the study. GS expression in the HCC nodules was analyzed immunohistochemically and was compared with clinicopathologic features and the behavior of the tumors. Survival curves were assessed according to the Kaplan-Meier product-limit method and multivariate analysis based on the Cox regression model was performed. RESULTS: GS expression was strong in 26 cases (35.6%, high-GS group) and weak or absent in 47 cases (64.4%, low-GS group). Univariate analysis showed that the high-GS group had a significantly shorter disease-free survival time than the low-GS group (p=0.042). Multivariate analysis revealed that GS expression (p=0.021), as well as Child's classification (p=0.005) and portal invasion (p=0.039), was a significant and independent prognostic parameter that affected tumor recurrence. CONCLUSION: The results of this study indicate that GS expression may enhance the metastatic potential in HCC, and GS immunostaining may be helpful in identifying HCC patients at high risk for disease recurrence.

Cyclooxygenase-2 overexpression correlates with tumour recurrence, especially haematogenous metastasis, of colorectal cancer.

Epidemiological studies have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), known to inhibit cyclooxygenase (COX), reduce the risk of colorectal cancer. COX is a key enzyme in prostaglandin biosynthesis, and two isoforms of COX, COX-1 and COX-2, have been identified. Recently COX-2 has been reported to frequently overexpress in colorectal neoplasms and to play a role in colorectal tumorigenesis and tumour progression. In this study, using immunohistochemistry, we examined COX-2 expression in advanced human colorectal cancer and its correlation with clinicopathological features. COX-2 expression was observed mainly in the cytoplasm of cancer cells in all the specimens examined, but some stromal cells and endothelial cells were also stained. According to the grade of COX-2 expression of the cancer cells, patients were divided into high- and low-COX-2 expression groups. High-COX-2 expression significantly correlated with tumour recurrence, especially haematogenous metastasis. These results suggest that a selective COX-2 inhibitor can be a novel class of therapeutic agents not only for tumorigenesis but also for haematogenous metastasis of colorectal cancer. To our knowledge, this is the first report on the correlation between COX-2 overexpression and recurrence of colorectal cancer.

Effects of exposure to repetitive pulsed magnetic stimulation on cell proliferation and expression of heat shock protein 70 in normal and malignant cells.

This study focused on the effects of repetitive pulsed magnetic stimulation (RPMS) on normal and malignant cells of humans. We used three human cell lines, HBL-100 (human normal breast epithelium), MCF-7 (human breast cancer), and HeLa (human cervical cancer). Cell proliferation at 37 degrees C and 40 degrees C and the expression of heat shock protein (HSP) 70 at 37 degrees C, 40 degrees C, and 42 degrees C, before and after the exposure to RPMS, was investigated. Cell proliferation showed no effects of exposure to RPMS in both normal and malignant cells; however, HSP70 expression was increased by RPMS exposure under thermal stress at 40 degrees C and 42 degrees C in HBL-100 and HeLa. We concluded that RPMS exposure potentiates the effect of thermal stress on both normal and malignant cells, and malignant cells derived from different organs respond differently to RPMS exposure.