Search

The aim was to assess the therapeutic strategy of patients with chylothorax in a neonatal intensive care unit.Twenty-eight infants with chylothorax were included in this study. Their clinical characteristics and outcomes were reviewed retrospectively.The male-to-female ratio was 1:1. The mean gestational age and birthweight were 35.1 ± 3.5 weeks and 2,692 ± 791 g, respectively. Eighteen patients were diagnosed with congenital chylothorax; chylothorax occurred postoperatively in 10 patients. Chromosomal anomalies were diagnosed in 8 patients. Six patients received surgical therapy, such as pleurodesis, thoracic duct ligation, or lymphaticovenous anastomosis. Two patients required surgery due to resistance to pleurodesis. In surgically managed patients, the daily maximum amount of pleural effusion (mL)/bodyweight (kg) ratio was significantly larger than in non-surgically managed patients: 229.0 ± 180.5 versus 59.7 ± 49.2 mL/kg. In the receiver operating characteristic analysis of the daily maximum amount of pleural effusion/bodyweight ratio, the area under the curve was 0.889 when the cut-off value was 101 mL/kg, and the sensitivity was 0.8333 and the specificity was 0.8095 (P = 0.0059).Pleurodesis using OK432 could become a surgical first-line therapy for chylothorax even for neonates. It was important to initiate pleurodesis for refractory chylothorax at an earlier stage. A daily chylous effusion/bodyweight ratio of101 mL/kg was a good predictor and seemed to be a useful parameter for prompt surgical intervention.

The adipocyte is important not only for the storage of excess energy as fat, but also for the secretion of homeostatic factors. Gene expression profiles during adipocyte differentiation have been reported previously for mouse 3T3-L1 cells. However, the profiles of adipogenic gene expression in mice and cattle may be different because several metabolic pathways of the ruminant adipose tissue are different from those of non-ruminants. The gene expression profile in a clonal bovine intramuscular preadipocyte cell line during adipogenesis was examined using the polymerase chain reaction-subtraction method. Six hundred and twenty-one clones, which were expressed at an early stage of differentiation, from the preadipocyte to adipocyte, were isolated and characterized. Further detailed studies were carried out for 86 selected genes using northern blotting. Ten genes were found to be highly expressed after differentiation of bovine intramuscular preadipocyte cells. In particular, the expression profiles of genes for stearoyl CoA desaturase and FK506 binding protein were quite different from the time course of differentiation of that seen in the 3T3-L1 cells reported previously. In addition, these genes were assigned to bovine chromosomes using a bovine/hamster somatic cell hybrid panel and public database.

Prostaglandin (PG) D(2) is abundantly produced by mast cells in the sites of allergic inflammations and acts on various cell types through its receptors DP and CRTH2. Among human T cells, CRTH2 is preferentially expressed on Th2-type cells. However, distribution of DP among T cells and impacts of CRTH2- and DP-mediated signals on T cell functions are presently unclear. Here, we show that CD4(+) and CD8(+) T cells producing IFN-gamma and IL-2 were reduced by DP-mediated signals, while CRTH2-mediated signals enhanced IL-2, IL-4, IL-5, and IL-13 production by Th2 cells. CRTH2 signals also caused up-regulation of CD11b and CD40L in resting Th2 cells. RT-PCR analysis revealed distribution of DP among Th cell subsets. On CRTH2(+) Th2 cells, the CRTH2-mediated PGD(2) effects were dominantly observed. Thus, PGD(2) favors Th2 functions through CRTH2 while restraining Th1 functions via DP, which may contribute to development of Th2-dominated status in allergic inflammations.

Semenogelin I and II (Sg I and II) are the major components of human semen coagulum. The protein is rapidly cleaved after ejaculation by the chymotrypsin-like protease prostate-specific antigen (PSA), which results in the liquefaction of the semen coagulum and the progressive release of motile spermatozoa. One of the cleavage products of the protein, a 14-kDa protein, is a sperm motility inhibitor (seminal plasma motility inhibitor [SPMI]). We developed a monoclonal antibody (mAb) that is specific to the fragment of Sgs, SPMI, and a sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of Sgs using this mAb. Then, we measured SPMI/Sg levels in human seminal plasma from healthy male volunteers (n = 100, aged 18–24 years). The mean level of SPMI/Sg in seminal plasma was 19 ± 13 mg/mL (range, 4–68 mg/mL). Log-transformed SPMI/Sg levels were negatively correlated with the sperm motility (r = −0.229, P = .0220) and positively correlated with the total protein concentration (r = 0.793, P < .0001). This result supports that SPMI, one of the fragments of Sg, has its inhibitory effect on ejaculated spermatozoa in liquefied semen under physiological conditions.

Granulysin is a cytolytic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) with a broad range of antimicrobial and tumoricidal activities. Two molecular forms of granulysin, the 15-kDa precursor and 9-kDa mature form, are produced in these cells. In this study, we developed monoclonal antibodies against granulysin and found that the 15-kDa granulysin is spontaneously secreted by peripheral blood NK and T cells via a non-granule exocytotic pathway. When NK cells killed the target cells, the released granulysin levels in culture supernatants significantly increased through the granule exocytosis. The granulysin protein was found in the sera of healthy individuals at an average concentration of 3.7 +/- 3.2 ng/ml (age 0-99 years, n=244). The serum levels of granulysin were transiently highly elevated among patients with acute viral infections. In addition, the serum granulysin levels in patients with severe immunodeficiency treated bycell therapy fluctuated proportionately to the improvement of other immunological parameters. Our results suggest that granulysin is well associated with diverse activities of NK cells and CTL in physiological and pathological settings and could be a useful novel serum marker to evaluate the overall status of host cellular immunity.

Prostaglandin D 2 is known to be the major prostanoid produced by allergen-activated mast cells, but its role in the formation of allergic diseases is not well established because of complexity of its receptor system and lack of appropriate inhibitors. We have recently identified a new-type prostaglandin D2 receptor, named CRTH2. Studies with normal subjects have shown that CRTH2 appears to be selectively expressed by T helper 2 cells but not T helper 1 cells among circulating CD4 + lymphocytes. The exact correlation between CRTH2 and T helper 2 cells in various disease settings and the impact of CRTH2-mediated prostaglandin D 2 activities on various T helper 2 responses in vivo still remain to be elucidated, however. In this study, we investigated the correlation between CRTH2 and T helper 2 cells among circulating CD4 + lymphocytes in normal adults and patients with atopic dermatitis, a T-helper-2-involving disease. The results showed that virtually all CRTH2 + CD4 + lymphocytes had a pure T helper 2 phenotype and formed not all but a large proportion of circulating T helper 2 cells for both normal and atopic dermatitis subjects. In chemotaxis assays, peripheral blood CRTH2 + CD4 + lymphocytes were significantly attracted by prostaglandin D2 as well as by a typical T-helper-2-attracting chemokine, thymus and activation regulated chemokine, whereas they showed little chemotactic migration toward typical T-helper-1-attracting chemokines, macrophage inflammatory protein 1β and interferon-γ-inducible protein 10. Furthermore, in atopic dermatitis patients, a preferential increase of CRTH2 + cells was noted within the disease-related cutaneous lymphocyte-associated antigen-positive, but not the cutaneous lymphocyte-associated antigen-negative, CD4 + lymphocyte compartment. Our results suggest the involvement of the prostaglandin D 2 /CRTH2 system in both normal and pathogenic T helper 2 responses.

Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3(-) CD16(+) cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression ( P

Among various seminal plasma proteins, semenogelin (Sg), produced in the seminal vesicle, has been considered a candidate for demonstrating the presence of semen. Sg consists of two proteins, one 52 kDa (Sg-I) in size, and the other a mixture of 71 and 76 kDa proteins (Sg-II). Recombinant Sg-I and Sg-II proteins were obtained using a baculovirus system and then injected into a rabbit to produce the respective antibodies [Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells, Int. J. Mol. Med. 2 (1998) 693]. When liquefied seminal plasma was immunoblotted with the anti-Sg-I and Sg-II antibodies, the anti-Sg-II antibody identified a wider range of the polypeptides originating from Sg than did the anti-Sg-I antibody. A dot-blot-immunoassay using anti-Sg-II antibody revealed a clear immunoreactive spot even when the semen was diluted 6400-fold. However, this assay showed that the Sg antigen was undetectable in saliva, urine, vaginal secretions, sweat, nasal secretions and serum. To determine the stability of Sg antigenic activity, filter paper with dried semen stains were kept at 37, 4 and 22 degrees C for 1, 6 and 18 months, respectively, and the Sg antigenic activity was examined. The activity was detectable in an area not less than 0.5 cm x 0.5 cm under all of the above environmental conditions during each period. Finally, semen was mixed with saliva or blood at various volumetric ratios, and used as a source of dried stains. The Sg antigenic activity was detectable in the stains until the ratio of semen to saliva or blood reached 1:8. These results suggest that Sg may be useful as a marker for semen identification.

Cytotoxic lymphocytes such as CTL and NK cells play principal roles in the host defense mechanisms. Monitoring these effector cells in vivo is helpful to understand the immune responses in disorders such as cancer and infectious diseases. In this study, we identified a novel secretory protein, killer-specific secretory protein of 37 kDa (Ksp37), as a Th1-specific protein by a subtractive cloning method between human Th1 and Th2 cells. In peripheral blood leukocytes, Ksp37 expression was limited to Th1-type CD4+ T cells, effector CD8+ T cells, γδ T cells, and CD16+ NK cells. Most of these Ksp37-expressing cells coexpressed perforin, indicating that Ksp37 is selectively and commonly expressed in the lymphocytes that have cytotoxic potential. Ksp37 was released at constant rate from both unstimulated and stimulated PBMCs in vitro and also detected in normal human sera. In healthy individuals, serum Ksp37 levels were significantly higher in children (mean ± SD; 984 ± 365 ng/ml for age 0–9) than in adults (441 ± 135 ng/ml for age 20–99), consistent with reported differences in the absolute counts of blood T and NK cells between children and adults. In patients with infectious mononucleosis, transient elevation of serum Ksp37 levels was observed during the early acute phase of primary EBV infection. These results suggest that Ksp37 may be involved in an essential process of cytotoxic lymphocyte-mediated immunity and that Ksp37 may also have clinical value as a new type of serum indicator for monitoring cytotoxic lymphocytes in vivo.

Prostaglandin (PG)D2, which has long been implicated in allergic diseases, is currently considered to elicit its biological actions through the DP receptor (DP). Involvement of DP in the formation of allergic asthma was recently demonstrated with DP-deficient mice. However, proinflammatory functions of PGD2 cannot be explained by DP alone. We show here that a seven-transmembrane receptor, CRTH2, which is preferentially expressed in T helper type 2 (Th2) cells, eosinophils, and basophils in humans, serves as the novel receptor for PGD2. In response to PGD2, CRTH2 induces intracellular Ca2+ mobilization and chemotaxis in Th2 cells in a Gαi-dependent manner. In addition, CRTH2, but not DP, mediates PGD2-dependent cell migration of blood eosinophils and basophils. Thus, PGD2 is likely involved in multiple aspects of allergic inflammation through its dual receptor systems, DP and CRTH2.

Several effector molecules, including cytokines, are differentially produced by Th1 and Th2 cells. We used a gene expression screen method to identify a gene encoding hematopoietic PG D synthase (hPGDS) which was preferentially expressed in human Th2 but not Th1 clones. Studies with anti-hPGDS mAbs confirmed the Th2-dominated expression of hPGDS protein. Upon stimulation with anti-CD3 plus anti-CD28 mAbs, coordinated cyclooxygenase-2 expression and PGD2 production were induced in Th2 lines. hPGDS expression was also observed in a small population (

The human colorectal tumor cell line LoVo has a homozygous deletion in the hMSH2 gene from exon 3 to exon 8, is deficient in mismatch repair (MMR) activity, and exhibits microsatellite instability. To determine whether the introduction of a wild type hMSH2 into LoVo restores MMR activity and stabilizes microsatellite loci, we transferred a chromosome 2 fragment containing hMSH2 into a homologous recombination–proficient chicken DT40/human hybrid (DT40 2C) and a human chromosome 2 in a mouse A9 hybrid to LoVo. Transfers of these chromosomes into LoVo resulted in LoVo both with and without a wild-type hMSH2. Complete correlation was found between the presence of the wild-type hMSH2 and hMSH2 expression, an increased stability in microsatellite loci, and competency in MMR. The hMSH2-positive LoVo hybrids also showed an increased sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine. This enhanced toxicity is associated with G2 cell-cycle arrest followed by premature mitosis and cell death. These results suggest that hMSH2 may be responsible for complementing mutator and drug-resistant phenotypes in chromosome 2-transferred LoVo cells. To test whether the hMSH2 in DT40 2C cells can be modified by homologous recombination, we transfected DT40 2C with a targeting vector containing an hMSH2 exon 4 disrupted by the zeocin-resistant gene. The results showed that the hMSH2 locus in DT40 2C was efficiently targeted by an exogeneously transfected homologous sequence, suggesting that transfer of a modified hMSH2 from DT40 2C to LoVo via chromosome transfer could be used to determine the function of hMSH2. Mol. Carcinog. 29:37–49, 2000. Published 2000 Wiley-Liss, Inc.

Stanniocalcin (STC) acts as a regulator of calcium and phosphate homeostasis in an endocrine manner in bony fish. Recently, complementary DNAs encoding human and mouse STC have been characterized, and the messenger RNA (mRNA) expression was identified in various tissues, such as kidney, small intestine, prostate, thyroid, and ovary. Because previous studies concerning the effects of fish STC on mammalian bone have been discussed, there is a good possibility that mammalian STC is a local factor in bone. Here, we demonstrated STC mRNA expression in neonatal mouse calvaria, the primary cultured mouse osteoblast-rich fractions, and human and mouse osteoblastic cell lines. We also mapped the cellular distribution of the STC mRNA in femur and calvaria in developing mice. Several transcripts with a major 4-kb band were detected in all samples. The cellular distribution of the mRNA expression corresponded closely to osteoblasts in both femur and calvaria. Significant labeling of the STC mRNA was also identified in chondrocytes but not in osteoclasts and other bone marrow elements. These results are the first evidence that hormone may be actually expressed in osteoblasts and chondrocytes, and they strongly implicate the involvement of local STC in both endochondral and membrane bone as an autocrine/paracrine factor.

Stanniocalcin (STC) is a glycoprotein hormone that was first identified in fish, where it regulates the calcium level in the body fluid. The cDNA which encodes human STC has recently been reported but the function has not been completely elucidated. We have prepared a monoclonal antibody against human STC using an analogous peptide of the putative antigenic domain in human STC; it was conjugated with keyhole limpet hemocyanin (KLH). The monoclonal antibody specifically stained the distal convoluted tubules in human kidney which is a putative target organ of STC. The ELISA was established using the monoclonal antibody and recombinant human STC as a standard antigen. The monoclonal antibody prepared in this study provides a useful tool for clinical studies of STC in human.

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood gammadelta T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 10(7)) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN-alpha and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood gammadelta T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 10(9) BRM-activated killer (BAK) cells composed of CD56+ gammadelta T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90-100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated gammadelta T cells producing IFN-gamma were assayed as an indication marker of BAK therapy. The normal range of IFN-gamma producing gammadelta T cells comprised 6.9 +/- 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN-gamma producing gammadelta T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood gammadelta T cells of Patients Nos. 1-7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN-gamma producing gammadelta T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial gammadelta T cell counts, a low frequency of these cells may contraindicate BAK therapy.

PURPOSE: Four DNA mismatch repair genes have been identified as being susceptible genes for hereditary nonpolyposis colorectal cancer. Deficiency of one of the mismatch repair genes causes the replication error phenotype in more than 80 percent of patients with hereditary non-polyposis colorectal cancer and in 10 to 30 percent of patients with sporadic colorectal cancer. To determine which mismatch repair gene is lacking the function in patients with replication error-positive colorectal cancer, several approaches have been used at the nucleic acid and protein levels. We studied replication error in 40 samples of randomly selected colorectal cancers and expression of hMSH2 and hMLH1 proteins analyzed by immunoblot in the tumor and normal tissues of the replication error-positive and replication error-negative samples. MATERIALS AND METHODS: Frozen tumor and normal tissues were obtained from 40 Japanese patients who had colorectal cancer. According to the Amsterdam criteria, those patients were classified as having 39 sporadic and 1 unknown colorectal cancers. Genomic DNA was extracted from tumor and normal tissues for determining replication error with eight microsatellite markers. Expression of hMSH2 and hMLH1 proteins in cell lysates of tumor and normal tissues of 16 patients was analyzed by immunoblot. RESULTS: The replication error phenotype was found in 6 (15 percent) of the 39 sporadic cases. hMLH1 protein was not detected in two of the six replication error-positive tumor tissues and not in the normal tissues, indicating that the tumor cells of the two patients had severe mutations in both alleles of the hMLH1 gene. Another four replication error-positive and ten replication error-negative tumors and normal tissues expressed hMLH1 protein. hMSH2 protein was detected in all samples. CONCLUSION: hMLH1 protein was undetectable in the two tumor tissues of the six replication error-positive samples of sporadic colorectal cancer. The detection procedure used here may have potential use for determining a dysfunctional mismatch repair gene product.

Malignant rhabdoid tumor of the kidney (RTK) is a rare renal sarcoma of childhood. Its histogenesis is unclear, and it is highly resistant to multimodality therapy. To elucidate the origin and the oncogenetic potential of RTK, we investigated the characteristics of 2 newly established RTK cell lines, SWT-1 and SWT-2. Both cell lines were verified to be RTK, since they did not exhibit contact inhibition and exhibited intermediate filaments, a specific marker for RTK. These cells possess the characteristics of mesenchymal cells based on their positive reactions with anti-vimentin and anti-laminin antibodies and their negative reactions with anti-keratin and anti-desmin antibodies. The karyotype of SWT-1 was 46,XX and that of SWT-2 was 46,XX,del(II)(pter-p13::p12-qter). Since 11p13 is the location of the WT-1 tumor-suppressor gene, and del(11p13) is associated with the aniridia-Wilms'-tumor syndrome, these findings link RTK with Wilms' tumor. While SWT-1 was negative for the tumor markers examined, SWT-2 released tissue polypeptide antigen into the culture supernatant. No rearrangement or amplification of the myc and ras oncogenes or of the p53 tumor-suppressor gene were detected. Wild-type RB protein and cyclin A were expressed in both cells. Our data suggest that these 2 cell lines may be useful in identifying the oncogenetic pattern of RTK. © 1996 Wiley-Liss, Inc.

To precisely evaluate a protein-related mechanism of epileptogenesis, we quantitatively analyzed the 70-kDa protein (namely, P70), a specific protein found in the cobalt-induced epileptic focus, and examined its effect on the electrocorticogram (ECoG) and cortical neurons in cerebral slices and its immunocytochemical localization in rats. Cobalt-induced cortical epileptogenic cortex exhibited a marked induction of P70. Its initiation time was ahead of the generation of epileptogenic activities. The anticonvulsant phenytoin (PHT) attenuated the cobalt-induced epileptogenic activities, but failed to suppress protein induction. Injection of this protein into the motor region of normal rat cerebral cortex elicited an epileptic ECoG and behavioral seizures. It also caused epileptiform activity with paroxysmal depolarization shifts in cortical neurons. These epileptogenic phenomena elicited by P70 were abolished by prior treatment with PHT or phenobarbital. Immunocytochemical analysis with an antiserum against P70 revealed that the reactivity was confined to pyramidal cells only in the region of the focus and was mainly localized on somatic, dendritic, and nuclear membranes and microtubles. These findings suggest that P70 may be linked to epileptogenesis.

Plasma glutathione peroxidase (pGPx) is an anti-oxidative enzyme. Using the polymerase chain reaction subtraction method, we have previously identified pGPx as a large part of the genes that are expressed following adipocyte differentiation in a bovine intramuscular preadipocyte (BIP) line. Therefore, we have analyzed the mechanism of production of pGPx in adipocytes. The expression of pGPx and C/EBPdelta increases during adipogenesis, with dexamethasone being the main effector of these genes. The expression of pGPx gene has been clearly detected in BIP cells and human adipocytes, but hardly in 3T3-L1 cells. The production of pGPx in bovine tissues is greatest in kidney and in intraperitoneal fat. We consider that the transcriptional control of pGPx in cattle might be carried out by C/EBPdelta and that the expression of pGPx might be a characteristic phenomenon of bovine adipogenesis.

Exogenously added granulysin was reported to kill mammalian target cells. The sites of actions and molecular mechanisms of granulysin in target cell killing, however, are presently unclear. We here examine the effects of granulysin with the target HeLa cells transiently expressed with GFP-fused 9 kDa granulysin. Endogenously expressed GFP-fused granulysin was preferentially localized in the nucleus and induced apoptotic cell death accompanying with phosphatidylserine translocation and nuclear condensation in a caspase-independent manner. These results suggest that granulysin enters the nucleus of target cells and induces apoptosis.

In order to isolate candidate genes involved in bovine adipocyte differentiation, we have constructed a subtraction library from a clonal bovine intra-muscular pre-adipocyte (BIP) cell line using the suppression subtractive hybridization method. We have isolated a set of subtracted cDNA fragments whose respective mRNA levels are up-regulated during the adipogenic differentiation of BIP cells, and cloned cDNAs from a differentiated BIP-lambda ZAP II cDNA library. Two cDNA clones were highly homologous to the sequence of mouse and human type XII collagen alpha-1, determined by a BLAST homology search. As type XII collagen has been reported to have four types of splicing isoform, two clones were determined to be XII-1 and XII-2 splicing isoforms, respectively, because of a difference in the C-terminal NC1 domain. From the expression analysis of type XII collagen, the XIIA-2 isoform was mainly expressed in differentiated BIP cells and adipose tissues. Although the function of type XII collagen has not been established as yet, these results suggest that type XII collagen may be associated with adipocyte differentiation and adipose formation in cattle and is a potentially useful marker for adipogenesis.

Genetic testings are commonly employed in various fields of clinical medicine and the test items performed at clinical laboratories are increasing rapidly in number. They are utilized to make early and/or definite diagnoses of infectious diseases, leukemia, cancers and molecular inherited diseases and also to monitor the progress of the diseases. However, these genetic testings except for infectious diseases have been developed independently at each clinical laboratory and the test results obtained at each laboratory are not always compatible each other. Under these situations it is widely expected to construct advanced genetic testing systems that can supply standardized data at any of domestic and international clinical laboratories. For the period from April, 1999 to March, 2002 three major clinical laboratories, SRL, Inc., BML, Inc. and MBC, Inc., were consigned by JBA (Japan Bioindustry Association) to collaborate in standardizing the evaluation methods for genetic testing systems among the clinical laboratories. The aim of the study is to develop the standardized genetic testing systems and to propose them as international standard operational procedures to the ISO/TC212 working group. Although one of the most important issues for standardization is the external quality assessment, they have not been carried out in reality. In this study we evaluated the difference of the genetic testing results obtained during the year of 2000 and 2001 among the clinical laboratories. The genetic testings for hematopoietic tumor, CML were selected to be evaluated since they are widely accepted as clinically useful tests.

Indomethacin is a widely used nonsteroidal anti-inflammatory drug and is generally known to exhibit its multiple biological functions by inhibiting cyclooxygenases or activating peroxisome proliferator-activated receptors. In this study, we present evidence demonstrating that the novel PGD2 receptor chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is another functional target for indomethacin. Indomethacin induced Ca2+ mobilization in CRTH2-transfected K562 cells at submicromolar concentrations (approximate EC50, 50 nM) in a Gαi-dependent manner as PGD2 did. Other nonsteroidal anti-inflammatory drugs (aspirin, sulindac, diclofenac, and acemetacin) had no such effect even at micromolar concentrations. In chemotaxis assay, three CRTH2-expressing cell types, Th2 cells, eosinophils, and basophils, were all significantly attracted by indomethacin (EC50, 50–500 nM) as well as by PGD2 (EC50, 2–20 nM), and the effects of indomethacin were blocked by anti-CRTH2 mAb. These results suggest the involvement of CRTH2 in mediating some of therapeutic and/or unwanted side effects of indomethacin, independently of cyclooxygenases and peroxisome proliferator-activated receptors.

A clonal bovine intramuscular preadipocyte (BIP) line has been established from the intramuscular white adipose tissue of the M. longissimus thoracis in each of three Japanese Black cattle. Exponentially growing BIP cells exhibited a fibroblastic appearance. Adipocyte differentiation was initiated by treating confluent BIP cells with differentiation medium containing insulin and dexamethasone. Small lipid droplets appeared 5-6 days after stimulation and occupied a large fraction of the cell volume at 10 days and beyond. During the adipose conversion, the incorporation of acetate to the cells gradually increased by 10-fold and reached a maximum at day 5. However, incorporation of glucose increased only S-folds prior to this conversion, even though GLUT-1 level increased by 13-fold at day 7. GLUT-4 on the other hand, was not detected during the course of differentiation. These results suggested that adipose tissue metabolisms in ruminants were different from that of non-ruminants.

The low molecular weight fraction (mol wt less than 1,000) of Ehrlich cancer ascitic fluid has been known to enhance Listeria infection in mice. Chemical characterization of the entities in this fraction revealed four purine and pyrimidine analogues, i.e. uric acid, uracil, pseudouridine and 1-methyladenosine (m1Ado). When the effect of each of these components was studied on Listeria infection in mice, only m1Ado markedly enhanced the infection and killed the mice within a short period. The optimal enhancement was obtained when m1Ado was given intravenously to mice 3-6 days before the infection at a concentration of between 1 and 100 micrograms/mouse. On the other hand, uric acid, uracil and pseudouridine failed to show such an enhancing effect. m1Ado inhibited macrophage accumulation in the peritoneal cavity of mice after an intraperitoneal injection of phytohemagglutinin. Although m1Ado did not show any inhibitory effect on the phagocytic and bactericidal activities of macrophages in vitro, peritoneal macrophages obtained from mice which received m1Ado 3 days ahead revealed impaired bactericidal activity, suggesting the migration of different cell populations from the bone marrow of m1Ado-receiving mice. The results may suggest that m1Ado is a major factor in tumor ascites causing, in small doses, an impairment of macrophage functioning as can be detected in tumor-bearing hosts.

A factor which impairs the capacity of normal recipient mice to resist Pseudomonas infection has been isolated from the ascitic fluids of tumor-bearing mice. This factor was dialyzable (molecular weight between 4000 and 10,000), showed potent suppressive activity when as little as 10 μg was given ip and was resistant to heating at 56 for °30 min. The factor in the ascitic fluids was detectable rather late after tumor implantation and the production of the factor in the ascitic fluids was closely related to the tumor growth.

A definite increase in two low molecular weight factors, G10-2 and G10-3 was found in Ehrlich ascitic fluids, parallel to tumor growth. The isolation and identification of the two factors were attempted through gel filtration and reversed phase column chromatography, using ascitic fluids obtained 13 days after intraperitoneal implantation of Ehrlich tumor cells. As a result, two highly purified factors were observed upon examination by high performance liquid chromatography. Additional analytical data, collected by UV spectrum, NMR spectrum and mass analysis, allowed us to identify G10-2 as uric acid and G10-3 as uracil. Detailed immunological analysis of uric acid and uracil revealed that the augmenting activities of mouse and human NK cells by mouse IFN alpha/beta or human rIFN alpha A/D were impaired in the presence of either compound at concentrations of 0.07 mM, the concentration detectable in the ascitic fluid of tumor bearing mice.

The isolation of Erwiniae from the vagina became positive just before the occurrence of erwiniuria. It is proposed that the appearance of Erwiniae in the vagina is an important indicator in the prediction of urinary tract infection by Erwiniae.

AmB/NCS combination therapy against cancer was evaluated. Six gastric cancer patients were treated in this manner, and 7 gastric cancer patients treated with NCS alone to serve as the control. AmB sirup was administered orally for 4 days before surgical operation. NCS was given intravenously at the onset of gastric surgery. Lesion tissues and healthy tissues were collected from each patient and the NCS titers were measured by bioassay. It was shown in the majority of the gastric cancer cases that the NCS levels in the lesion tissues were substantially higher than in the surrounding normal tissues, whereas in the gastric cancer patients who received NCS alone, no significant differences were found between the tissues.