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A total of 246 Japanese composed of 121 with benign prostatic hyperplasia (BPH) and 125 with prostatic cancer (PCa) were analyzed clinically to explain fusion gene anomalies between erythroblast transformation-specific family transcription factor-related gene (ERG) and transmembrane protease serine 2 (TMPRSS2) on chromosome 21q22. Among the 246 cases, 12 (10%) BPH and 18 (14%) PCa showed prostate-specific antigen (PSA) values of >20 ng/ml. It was shown that PSA was not only secreted from the prostates of BPH and PCa, but also actively from bone metastases, which occurred in the 18 PCa, although PCa bone metastases through lymphatic vessels were shown to be blocked by a cystic abscess formation or skin lipoma derived from lymphatic vessels. Among the 30 cases of >20 ng/ml PSA, lymphatic-related lung lesions were observed in 4 (33%) BPH and 12 (67%) PCa. Except for 1 case with pleural PCa metastasis, pleural, lung, and liver metastases were not observed in the 125 PCa. Instead, primary pleural squamous cell carcinoma was detected in two BPH and one PCa and primary lung adenocarcinoma/lung neuroendocrine cell carcinoma (LNCC) was detected in two BPH and five PCa in which adenocarcinoma changes to LNCC were shown. Besides early PCa bone metastases, hematopoietic normal stem cell suppression was observed in two BPH and two PCa, probably on the basis of periosteal stem cell activation. The correlation between insulin and blood sugar (BS) was examined in 66 BPH and 24 PCa. A negative correlation coefficient of −0.297 was calculated in the nine PCa with abnormal BS. Among 23 PCa and 52 BPH of the 90 cases measured insulin, 10 (43%) PCa and 7 (13%) BPH were diagnosed with diffuse fatty liver. Insulin-resistant diabetes mellitus was observed with a high spike of insulin. High levels of triglyceride and total cholesterol (TC) were well improved by prostatectomy. The TMPRSS2:ERG gene fusions/deletions had caused bone marrow and fat metabolism anomalies and other primary cancers not only in PCa, but also in BPH, which indicated a better prognosis of early prostatectomy. more...

A total of 101 cases of 81 infertile men and 20 men with testicular tumors were analyzed clinically for suspected X-chromosome-linked gene abnormalities. Among the 81 infertile men analyzed by testicular biopsy, 11 Klinefelter syndrome (KS) cases showed Leydig cell nodular hyperplasia with azoospermia. They had low levels of testosterone (131 ± 81 ng/dl) and high levels of luteinizing hormone (21 ± 7 mIU/ml) and follicle-stimulating hormone (36 ± 11 mIU/ml). From Leydig cell hyperplasia combined with atrophic tubules, X-linked androgen receptor dysfunctions in Sertoli and Leydig cells were judged in the KS cases. One azoospermia case of non-KS had primary yolk sac tumor of the mediastinum and liver. The removed yolk sac tumor showed complete necrosis after bleomycin, etoposide, and cisplatin (BEP) chemotherapy, which indicated cisplatin-sensitive apoptosis. X-linked inhibitor of apoptotic protein (XIAP) must exhibit an apoptosis-inducible mutation (XIAP-S87A) in the non-KS case. Subsequently, 20 testicular tumor cases were classified into ten with seminoma, eight with mixed germ cell tumors (MGCT), one with mature teratoma, and one with diffuse large B cell lymphoma. Although among the eight MGCT cases, six cases had high levels of α-fetoprotein (68–43647 ng/ml), their blood levels of human chorionic gonadotropin-β were more...

To investigate the increased expression of activation-induced cytidine deaminase (AID) in vascular diseases, 379 Japanese and 2 non-Japanese composed of 87 varix, 81 arteriosclerosis obliterans (ASO), 206 aortic aneurysm (AA), and 7 venous thrombosis (VT) patients, whose ages were ranged from 60.7 ± 13.3 to 74.5 ± 8.8 years old, were analyzed clinically. Their coagulation anomalies were described well using their levels of thrombin antithrombin III complex (TAT), α2-plasmin inhibitor-plasmin complex (PIC), and fibrin/fibrinogen degradation products-D dimer (FDP-DD) in all the 4 diseases. VT showed the most severe coagulopathy with the highest levels of TAT, PIC, and FDP-DD, followed by AA. Among the seven VT cases, a lympho-plasmacytic hypophysitis (IgG4-related hypophysitis) case and a retroperitoneal fibrosis case combined with malignant lymphoma (ML) were found. Another VT case involving portal vein thrombosis (PVT) demonstrated a low level (59%) of protein C antigen (Ag) together with ulcerative colitis. In the 81 ASO, two showed ML combined with gastric cancer (GC). Cancers were examined in 87 varix, 81 ASO, and 199 AA cases. The frequency of cancer was 9% (33/367), in which five cases showed double cancer. The most common cancer was gut cancers which were found in 5% cases (20/367). GC was found in 14 cases, and intestinal cancers were found in six cases. The AA onset of ten GC cases seemed to occur at the same time as the onset of GC, 60–70% of which showed postoperative serum amylase elevation. Ectopic splenosis aggravated the clinical symptoms in three AA patients who had undergone gastrectomy, cholecystectomy, or hepatectomy. Bleeding (hemolysis) and thrombosis were main trigger signs in the three AA patients. One of the three developed PVT. Existing high levels of activation-induced cytidine deaminase (AID) were judged from severe coagulopathy, combined B cell malignancy, accompanied autoimmunity, the highest frequency of GC, and aggravated splenosis. AID downregulation was concluded to be an important factor in order to stop vascular disease progressions. more...

Bladder and upper urinary tract (UUT; pelvic and/or ureteral) cancers were studied in a total of 118 Japanese patients using serum amylase to clarify their prognosis and treatment. Among 114 patients with urothelial carcinoma (UC), 99, 10, and five had bladder UC, bladder-UUT-UC, and UUT-UC alone, respectively. Three had bladder squamous cell carcinoma, and one had undifferentiated bladder carcinoma. During the 3 years of this study (2007–2009), 35 (30%) patients received radical cystectomy at a mean age of 72 ± 8 years old, of which 23 (66%) demonstrated muscle invasion (pT2≤). Five (4%) patients showed post-operative hyperamylasemia with serum amylase levels of 1,353–2,986 IU/l. Except for one case of UUT-UC with mild operative bleeding, in four other bladder UC cases, severe fibrinolytic bleeding was observed, and their continuing bleeding was accompanied by a post-operative elevated serum amylase. One patient with a serum amylase level of 2,986 IU/l showed severe post-operative pelvic bleeding and severely necrotic UC recurrence involving prostate and urethral remnants and died of intestinal perforation about 1 year post-cystectomy. Serum amylase was secreted from bladder cancers in parallel with the amount of tissue-type plasminogen activator (t-PA), although serum amylase secreted maximally a little later than t-PA after serum Ca was returning to normal. Five patients showed brain metastases with a low level of serum amylase, near to normal serum Ca, and high levels of alkaline phosphatase at advanced cancer stages, and another four with hypercoagulation showed cerebral infarction with similar laboratory findings to those of the former five, of which three had severe chronic renal failure. The proteolytic reactions of the urokinase-type PA secreted from bladder cancer caused tissue necrosis, especially in the intestine, resulting in intestinal perforation and remote bone and brain metastases. In the CRF patients, recombinant erythropoietin was effective for treating hypoxia. more...

Japanese patients were selected from 875 patients who had undergone an orthopedic operation. Seventy-two cases of rheumatoid arthritis (RA) and 17 cases of suspected RA were analyzed. In parallel with high levels of rheumatoid factor (RF), Sjoegren syndrome, finger and toe deformans, and knee and hip arthritis deformans were observed at high frequencies. The arthritis deformans of 51 patients displayed a mean serum iron levels of 50 ± 33 (SD) μg/dl and transferrin saturation (TS) of 18 ± 11%. The hypothyroidisms diagnosed in the 12 patients were caused by autoimmune, hypopituitary, and papillary thyroid cancer (PTC). Two of the insulinoma cases had hypothyroidism due to down-regulated transcription of genes related to insulin. Two PTC, four breast ductal carcinomas, and three lung adenocarcinomas, which were observed in patients with bone diseases, were assumed to be caused by epidermal growth factor receptor (EGFR) gene mutations. One lung cancer with gefitinib-sensitive EGFR mutations, a breast cancer that expressed human epidermal growth factor receptor 2 (HER2), and a lung-metastasized popliteal epitheloid sarcoma showed disseminated bone and/or intra-lung metastases. When an HER2-positive breast cancer metastasized to the bones, TS was elevated to 73.4–81.4% with anemic attacks and high levels (12.4–16.8 ng/ml) of pyridinoline cross-linked carboxy-terminal telopeptide type I. In the other six cancer patients with noninvasive phenotypes, the metastases became cysts. One noninvasive lung cancer was confirmed to express thyroid transcription factor-1 (TTF-1). Polyps, varix, and multiple thromboses were found to be clinical signs of pre-EGFR cancers. more...

Seventy-five parathyroidectomy (PTE) and 69 adrenalectomy (AE) patients were analyzed. The 75 PTE included 56 of secondary parathyroid hyperplasia (SPTH) with chronic renal failure (CRF) and 19 of primary parathyroid hyperplasia (PPTH) without CRF. The 69 AE included 23 with Cushing’s syndrome, 30 with primary aldosteronism, four with cortical carcinoma (three virilizing and one feminizing carcinomas), seven with pheochromocytoma (two multiple endocrine neoplasia 2A), two with paraganglioma, one with schwannoma, one with non-functional adenoma, and one with hepatic cancer metastasis. The 56 SPTH showed higher levels of intact (I)-parathyroid hormone (PT) of 1,359 ± 1,036 pg/ml than the 292 ± 372 pg/ml of the 19 PPTH. Measured vitamin D of 1, 25(OH)2D was higher in the nine PPTH (88.0 ± 34.7 pg/ml) than in the 46 SPTH (15.5 ± 13.9). High serum amylase of >350 IU/l and cyst formations were found in ten (18%) and 16 (29%) of the SPTH but none of the PPTH. At least ten (18%) of the SPTH had polycystic kidney disease (PKD). Saliva-type amylase of the SPTH resolved the high I-PTH. High levels (>6.0%) of Hb A1C (8.1%) were measured only in one SPTH case with mediastinal ectopic SPTH and PKD. High urine 17 ketosteroid (17 KS) levels of 161.5 ± 72.9 mg/l were measured in three virilizing carcinomas with metastases to the bone, liver, and/or lung. Low-grade feminizing carcinoma was observed in a 6-year-old boy. Cysts of the kidney and/or liver were found in 17 (74%) with Cushing’s syndrome, 12 (40%) with primary aldosteronism, and three (43%) with pheochromocytoma. One paraganglioma had liver and pancreatic cysts, and one schwannoma had polycystic ovary. High levels of Hb A1C were detected in six (26%) with Cushing’s syndrome, five (17%) with primary aldosteronism showing insulinoma, and two (29%) with pheochromocytoma. In all 13 patients with diabetes mellitus (DM), AE was an effective DM treatment. more...

The common causes and clinical features of gastric and colon (GC) carcinomas were investigated in 218 Japanese patients who underwent gastrectomy and 138 Japanese patients who underwent colectomy over a period of 2.9 years. One characteristic feature of common GC carcinoma was high levels of saliva-type serum amylase (S-Amy). High serum amylase of >400 IU/l, which was secreted from GC carcinoma, was identified in 9% to 12% of the patients immediately after surgery. Besides the high S-Amy, liver (bile duct) and/or kidney cysts and diabetes mellitus (DM) or borderline DM were found in 61–70% and 36–37%, respectively, of these GC carcinoma patients. The liver (bile duct) or kidney cysts may have been induced by a gene anomaly related to GC carcinoma. DM with low levels of insulin could also be connected to GC carcinoma. The pancreatic carcinoma markers, carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA), increased when GC carcinoma had metastasized to the lungs and/or liver. Seven (3%) patients with gastric carcinoma had liver metastasis, but none had lung metastasis. Colon carcinomas, which had better pathological findings than gastric carcinomas, metastasized more frequently with higher CEA levels. Both lung and liver metastases were found in four (3%) patients, lung metastasis in three (2%) patients, and only liver metastasis in five (4%) patients. Early stage metastases via lymph-vascular or large vascular vessels, but not small vein vessels, with a good prognosis of 3–4% mortality rates, were also characteristic findings of GC carcinomas. Some nodules of the metastases fell into necrosis in the metastatic organs. It was concluded that, among the GC carcinoma patients admitted to our hospital, at least 70% of GC carcinomas may be common GC carcinomas, which must have abnormal GC salivary AMY gene promoters. more...

Although hemophagocytic syndrome (HPS) signs are more severe in aortic aneurysm cases than in angina pectoris cases, HPS-associated postoperative death should be considered more in both cases. This study analyzed a total of 183 Japanese patients, with 101 aneurysms, 79 anginas, and 3 with both, who underwent aortic replacement (AR) or/and aortocoronary bypass grafting (ACBG) during a period of 1.92 years. In seven aneurysm and eight angina cases with 7.6–12.1 g/dl of hemoglobin (Hb), iron metabolism was studied. As clinical outcomes, diabetes, chronic renal failure (CRF), and thrombosis were more prevalent in the angina cases than in the aneurysm cases. In six cases of aneurysm (6:101) who died after AR, five who died of acute myocardial infarction (AMI) or pulmonary embolism (PE) during postoperative 3–101 days developed HPS, and no detail data was available for one case. Among the five diabetic cases of angina (5:79) who died 7–74 days after ACBG, four cases with cerebral infarction (CI) had infection-associated HPS, and one case had severe kidney atrophy without CI and HPS. Postoperative HPS signs in the aneurysm cases were also generally more severe than those of the angina cases. The aneurysm case with no CRF showed mostly typical HPS and died of AMI. She had pancytopenia, anemia with anisocytosis and nucleated red cells, leukopenia with immature myeloid cells, thrombocytopenia with coagulopathy, elevated lactate dehydrogenase, hyperbilirubinemia, liver dysfunction, and numerous phagocytes containing hemosiderin in the specimen of the aorta examined before the operation. Preoperatively, she also showed high levels of serum ferritin (SF) and low levels of both serum iron (S–Fe) and total iron-binding capacity (TIBC). All the 15 patients in whom iron metabolism was evaluated had low levels of S–Fe. Low levels of TIBC and high levels of SF were observed in 11:15 patients. Cardiac hemophagocytosis with no CRF was a suggestive of HPS. AR and ACBG operations became a direct trigger of HPS. Postoperative AMI, sepsis, and PE became a HPS trigger, but CI alone and CRF did not. For postoperative survival, it was important to protect the patients from infections. more...

The important roles of host liver metabotropic glutamate receptor 5 (mGluR5) and Fas ligand (FasL) genes in bone marrow (BM)/liver graft survival were shown using rat models. The bad effects of BM co-graft and granulocyte-macrophage colony-stimulating factor (GM-CSF) on liver graft survival were also assessed. Thirty-one Lewis rat livers were grafted a piece of Lewis or dark agouti (DA) BM alone or together with a section of liver, 9 of these were injected with GM-CSF. At 4–5 months post-transplantation, mGluR5 mRNA of host livers was measured quantitatively by reverse transcriptase (RT)-polymerase chain reaction (PCR). Eighteen experimental and 5 control rats were measured for FasL mRNA of the host liver. CD2+CD90− cellular density was measured by flow cytometer (FCM) in all rats. Eight of 31 grafted and 3 of 7 control host livers showed absence or 50% reduction of mGluR5 mRNA with normal mGluR5 DNA (29%). Among 7 females grafted with Lewis male BM alone, 5 host livers showed low amounts of mGluR5 mRNA with signs of early graft acceptance. Among 8 males grafted with Lewis BM/livers and 7 females grafted with DA BM/livers, 2 males and 1 female had low levels of liver mGluR5 mRNA, in which the shrinking liver graft showed characteristically only a partial fusion to the host liver. Two male livers with better graft survival and 2 control male livers showed large amounts of the FasL gene products. In the 2 males with the activated FasL mRNA, the liver grafts were examined with an electron microscope. The liver grafted onto a host with no mGluR5 showed apoptotic changes such as massively developed rough endoplasmic reticulum and small mitochondria with a normal nucleus. The graft on the liver with activated mGluR5 had almost normal structures. Nine experiment rats injected with GM-CSF, which resulted in liver and spleen dendritic cell proliferation, showed aggravated graft rejection. FCM results of the females grafted BM alone showed a similar suppression of spleen CD2+CD90− cells to those of the males injected with GM-CSF. more...

Objective To investigate the cause of thymoma connected with renal amyloid β (Aβ) overproduction. Design and methods Pathologic, immune-fluorescence analyses, flow cytometry (FCM) analyses, polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR were applied for tailless and tailed Lewis/Sea rat siblings at the age of 6 months. Results A homozygous defect of the winged helix DNA-binding domain of the fork head (FH) gene was detected in the RT-PCR of the tailless rat kidney and liver. Lymphocyte-rich thymoma was found in both rats, but was more prominent in the tailless rat. FCM showed that CD4+ and CD8a+ cells constituted of more than 90% of the thymus lymphocytes in both rats. Aβ deposits in the renal tubules of both rats were shown by positive staining with antihuman Aβ (11–28) antibody (Ab) together with the activation of the kidney presenilin (PSEN) genes. The mRNA level of transthyretin (TTR) was suppressed moderately with age by the homozygous FH1 gene defect. Conclusion Both homozygous and heterozygous defects of the DNA-binding domain of the FH gene caused lymphocyte-rich thymoma and renal Aβ overproduction. more...

Five male and 7 female Lewis rats unilaterally nephrectomized at the ages of 44-46 days were transplanted with the mesenteric lymph nodes (MLN) obtained from syngeneic rats 4 months after the nephrectomy. In addition, 6 males received unilateral nephrectomy plus splenectomy at the age of 38 days. All of these rats were observed for 8.5 months after the nephrectomy. Flow cytometry analyses indicated that 17-32% of MLN lymphocytes and 4-20% of peripheral blood (PB) lymphocytes lost CD2 antigen in the unilaterally nephrectomized rats. Approximately 20% of PB and MLN lymphocytes had lost CD2 antigen in the males with combined splenectomy and nephrectomy, one of which died of severe infection. The survival rates of the MLN graft after 4.5 months were 100% in the 5 nephrectomized males and 14% in the 7 nephrectomized females. The CD2 antigen on immunologically activated T cells was up-regulated. Twenty-eight% lymphocytes of the survived MLN graft and 51% PB T cells of the 7 females with MLN graft rejection showed the CD2 up-regulation. It was shown immunohistochemically that not only CD2-positive (+) cells but also CD4+ cells were increased in the cortex of the survived MLN graft. The hepatocyte growth factor receptor gene, c-met gene, was shown to be present in lymphocytes. The c-met was considered to cooperate with the CD2. Interstitial mononuclear cell infiltrations with glomerulosclerosis were found in the remaining kidneys of the males with unilateral nephrectomy plus MLN grafts. Glomerular angiogenesis and accompanying endothelial cell apoptosis were demonstrated without mesangial cell proliferation in the males with unilateral nephrectomy plus splenectomy. more...

One LEW/Sea (Lewis) strain rat, Rat P-1, had a homozygous defect of the serine proteinase inhibitor 2 (Spi-2) gene coding for serpin contrapsin. A sibling, Rat P-2, and a rat of the same strain, Rat P-3, had a heterozygous Spi-2 gene defect. The homozygous Spi-2 gene defect of Rat P-1 was diagnosed by the polymerase chain reaction (PCR). The presence of an alpha 1-proteinase inhibitor (PI) gene was confirmed by PCR in all 3 rats. Rat P-1 and the sibling Rat P-2 were sacrificed at 7 days of age because of the severe weakness of Rat P-1, although Rat P-2 appeared healthy. Rat P-3 was sacrificed at 40 days of age because of the severe weakness. Rat P-1's hepatocytes with the homozygous Spi-2 gene defect showed massive accumulation of polymerized alpha 1-antitrypsin (AT) in the rough endoplasmic reticulum (RER). Large, defined accumulations of alpha 1-AT were not found in the heterozygous rats with the Spi-2 gene defects. Nephrogenesis was retarded in all 3 rats, especially in Rat P-1. Extramedullary hematopoiesis was absent in the liver of Rat P-1 and suppressed in the spleen of Rat P-3, where activated alpha 1-AT synthesis was found. Atelectasis mixed with hyperinflated lung regions and thymic apoptosis were observed in the 2 weak rats. As Spi-2 gene defect had some similar clinical points to platelet-derived growth factor (PDGF) deficiency, it was suggested that increased vasoconstrictor peptide hormones competed with PDGF for binding to the PDGF-receptors. more...

Thymectomy is an inevitable therapy for thymoma. Therefore, determining hemato-immune changes post-thymectomy is important. Twenty-six normal LEW/Sea rats thymectomized (Tx) at the ages of 38 +/- 5 days were followed without any treatment for 4 months (experiments [Exp] A and B). In addition, 16 LEW/Sea rats Tx at the age of 42 days (Exp C and D) and 10 non-Tx control LEW/Sea rats (Exp E) were immunized with syngeneic male liver cells 2 to 3 times and followed 2.7 months after the first immunization. Flow cytometric (FCM) analysis of mesenteric lymph nodes (MLN) and peripheral blood (PB) showed as follows: among the 26 Tx rats (Exp A and B), MLN lymphocyte population at 4 months postthymectomy was characterized by decreased numbers of CD4+ cells (22%-36%) or alphabeta T-cell receptor (TCR)-positive cells (16%-54%) and increased numbers of interleukin 2 receptors (IL-2R) (>90%). In the 16 Tx-immunized rats (Exp C and D), both alphabeta TCR (MLN) and CD45R (PB) expression on lymphocytes was suppressed with rather high numbers of CD4. Bone marrow (BM) and PB hematological studies of the partially and totally Tx rats indicated the following: 8 BM of the 10 totally Tx males (Exp A) and the 7/10 male spleen showed erythroid hyperplasia with maturation arrest at the stage of basophilic erythroblasts and reticulocytopenia in the PB. One of the 10 males, which had both BM myeloid to erythroid (M/E) ratio of 4.3 and spleen myeopoiesis, was in a more advanced stage, a prestage of pure red cell aplasia (PRCA) with 22% of CD4+ MLN cells. Syngeneic liver cell immunization resulted in the Tx rats as follows: hepatotoxicity based on the immunization was weaker in the 16 Tx rats (Exp C and D) than in the 10 non-Tx rats (Exp E). Polymerase chain reaction (PCR) of PB and MLN showed compensatory activated leptin receptor (LR) and T-bet DNA in 19 Tx rats (Exp A and B) with abnormal FCM findings. The ineffective erythropoiesis at 4 months after thymectomy was explained by the erythroblast LR/erythropoietin receptor (EPOR) dysfunction. The low grade hepatotoxicity in the Tx rats (Exp C and D) was explained by the disturbed Th1 reactions (or the disturbed T-bet gene transcription) at postthymectomy. more...

Thirty-two normal LEW/Sea rats were transplanted a piece of syngeneic pancreas between the peritoneum and abdominal muscle. Among them, 17 (68%) of the 25 rats that received pancreatic transplantation at 41-50 days of age had a surviving beta-cell mass at 5.5-7.1 months after transplantation. Among the 25 rats, 12 rats injected with interleukin-1 receptor (IL-1R) and IL-2Rbeta peptides at post-transplantation showed better surviving grafts at 5.5 months' observation. Only 2 (25%) of the other 7 young rats that received a pancreatic graft at 20 days of age had a small mass at 21 days post-transplantation. Flow cytometer (FCM) analyses showed that thymus OX40(+) (CD134(+)) T-cells were increased up to 37+/-4% at the graft rejection in the 13 old rats without the IL-R peptide injections. The 7 young rats had 99% of thymus OX40(+) T-cells. However, the 12 old rats injected with the IL-R peptides showed suppressed numbers of thymus OX40(+) T-cells (8-13+/-3%). The long-term surviving, but apoptotic, grafted beta-cells were stained positively both with anti-insulin monoclonal antibody (mAb) and with anti-c-erbB-2/human epidermal growth factor receptor (HER)-2/neu mAb. Expression of a c-erb family oncogene was shown on the pancreatic graft surviving for 7.1 months. Electron microscopic analysis of the grafted beta-cells showed abnormally large beta granules and loss of functioning mitochondria in the cytoplasm. In 18 (56%) of the 32 rats, the 220-bp and 380-bp specific products of insulin-degrading enzyme (IDE) gene were amplified using the polymerase chain reaction (PCR) of the liver DNA. Among the 18 rats, 6 rats expressed 2 extra hands of 280-bp and 700-bp in a correlation with the high levels of the transforming growth factor-alpha (TGF-alpha) cDNA of 120-bp which was amplified in the quantitative reverse-transcriptase (RT)-PCR of the liver cDNA. Among the selected 11 rats, 5 rats showed large amounts of the 120-bp TGF-alpha cDNA. Host pancreatic RT-PCR showed 235-bp or 250-bp bcl-2 and 181-bp bcl-xS gene products. The bcl-2 cDNA of the host pancreas was amplified actively when the pancreatic graft was being rejected. Exceptionally, the one female injected with the IL-R peptides showed a low level of the liver TGF-alpha cDNA together with the pancreatic expressions of Bax (140-bp), bcl-2 and like interleukin converting enzyme (LICE) (318-bp) cDNA. Insulin secretion from the grafted beta-cells and IL-1beta-induced Fas-mediated apoptosis of the beta-cells were suspected to be present at the same time in the female with the best graft survival. more...

The transduction of exogenous hepatocyte growth factor (HGF) genes to spleen T lymphocytes and the immune effects of syngeneic spleen graft on spleen lymphoma cells were studied in LEW/Sea rats. Three different systems were designed. (1) Six female rats and six male rats received irradiated spleen graft and were followed for 7 months. (2) Five female rats and six male rats received intra-peritoneal (i.p.) injections of the Lewis red cells incorporated 20-bp HGF genes and anti-interleukin-6 (IL-6) antibody (Ab) with spleen graft and were followed for 5.5 months. (3) Four females and five males received i.p. injections of the Lewis red cells incorporated 20-bp HGF genes and anti-IL-6 Ab without spleen graft and were followed for 5.5 months. Hemato-pathological analyses, flow cytometer analyses of gamma-delta (gammadelta) T-cell receptor (TCR)-positive lymphocytes and reverse-transcription-polymerase chain reaction (RT-PCR) of TCRgamma gene, TCRValpha3 gene and apoptotic genes were performed. Results showed that one of the six females received irradiated spleen graft developed nodal gamma-delta (gammadelta) T-cell pre-lymphoma with 100% of gammadelta TCR+ lymphocytes in the mesenteric lymph nodes. One female injected with the HGF genes and anti-IL-6 Ab and grafted spleen died of advanced hepatosplenic gammadelta T-cell lymphoma at 3.5 month observation. All the five males injected with the HGF genes and anti-IL-6 Ab without spleen graft developed early hepatosplenic gammadelta T-cell lymphoma at 5.5 month observation. In two rats with spleen graft and the two rats without spleen graft, which were injected with the HGF genes and anti-IL-6 Ab, the lymphoma was suspected uncertainly with activated TCRValpha3 mRNA. The i.p. injections of HGF genes, which were incorporated in the red blood cells, triggered hapatosplenic gammadelta T-cell lymphoma. Surviving spleen graft rejected the lymphoma cells, but not in rats rejected the graft. Spleen graft was a good organ transplantation to expect graft versus lymphoma effects. more...

A method is proposed for the determination of heat-stable alkaline phosphatase (HSAP) in serum. It comprises the following steps. The serum is heat-inactivated at 65°C and the residual alkaline phosphatase is allowed to react with Naphthol AS-MX phosphate. After stopping the reaction by acetone, the liberated Naphthol AS-MX is measured fluorometrically using high-performance liquid chromatography (HPLC). This method can be applied to cancer-screening in clinical examination. It is sensitive and rapid as compared with existing methods. Linearity, accuracy and detection limit have been examined (linearity 0–2190 ng Naphthol AS-MX; coefficient of variation ≈7%; recovery ≈80%). more...

Syngeneic liver grafts were implanted in the livers of 22 LEW/Sea strain rats. To prolong the graft survival, anti-CD2 monoclonal antibody (MAb) or anti beta type platelet-derived growth factor receptor (PDGFR-beta) antibody (Ab) was injected, or splenectomy was performed in the rats which were then followed until 10 to 11 weeks posttransplantation. The 22 rats with chronic graft rejection showed increased CD8a-like antigen (probably Fas ligand) on the peripheral blood T cells. All the liver grafts had both necrosis and apoptosis. The liver graft apoptosis was indicated by histopathological abnormalities, and by DNA strand breaks and hemosiderin depositions in the cytoplasm. PDGFR-beta expression in the apoptotic liver graft was demonstrated immunohistochemically. Among the 17 rats injected with anti-CD2 MAb, CD2 signaling on host T cells was effectively suppressed by the injection of anti-CD2 MAb in 4 rats with better-surviving liver grafts. In these 4 rats, CD28 antigen on thymic lymphocytes was down-modulated and high numbers (136-233-positive cells per lobe) of the epithelial reticular cells with apoptotic lymphocytes were counted. Anti-PDGFR-beta Ab caused high pulmonary secretions of growth factors and reticular fibrosis in the lungs of 5 rats injected with the Ab. Anti-PDGFR-beta Ab injection reduced the host cell apoptosis in the lung and thymus, but did not prolong the survival of liver grafts. In the 9 rats with both splenectomy and anti-CD2 MAb injection, pulmonary apoptosis was induced with the 6-16% reductions of CD4+ lymphocytes. Prolonged graft survival was observed in only one of the 9 rats. Anti-CD2 MAb was effective for prolonging the liver graft survival with suppressed CD28 antigen, but anti-PDGFR-beta Ab and splenectomy were not. more...

Heparin effects were studied on Lewis rats with alpha 1 antitrypsin (AT) defect. Among 8 rats that were born at the same birth, three rats were shown to have mild defect of alpha 1 AT. Heparin was injected repeatedly into all the 8 rats. Interstitial pneumonia and localized periodic acid-Schiff (PAS) stain of hepatocytes were found in alpha 1 AT defective male. One of the three alpha 1 AT defective rats had about a half of normal alpha 1 AT level. Antithrombin (AT) III level was slightly low in the alpha 1 AT defective female with splenomegaly. Lung electron micrograph of the other alpha 1 AT defective female showed edematous changes of capillaries and alveolar basement membranes and also proliferated collagen fibers. In the lung of alpha 1 AT defective male, many thrombocytes adhered to alveolar degenerated smooth muscles that were recognized as Masson bodies. Extracted platelet-activating factor (PAF) in the plasma of the alpha 1 AT defective male was shown to trigger T lymphocyte chemotaxis. Five normal Lewis rats were immunized with bovine serum albumin (BSA). IgG1 antibody to BSA was produced in all the rats. The rats with high titers of IgG1 anti BSA antibody showed more strongly atrophic changes of glomerulus than those of the mild alpha 1 AT defective rats treated with heparin. more...

After 8 months of storage of normal red cells in liquid nitrogen, red cell enzyme activities were essentially unchanged, except for triose phosphate isomerase which was reduced by about half. Mutant pyruvate kinase (PK), PK Tokyo I, showed no changes in kinetic or electrophoretic properties after 8 months of storage. These findings indicate that red cells stored for a long term in liquid nitrogen can still be used for the study of mutant PK. more...

We have examined the alpha globin gene complex for 49 individuals with alpha-thalassemia-2 (-alpha 3.7). Crossovers resulting in alpha-thalassemia-2 (type I) were observed in all 57 chromosomes with the -alpha 3.7 defect. Except for one alpha-thalassemia-2 chromosome, all were linked to the absence of an Rsa I restriction site located 0.7 kb 5' to the alpha 2-globin gene; this polymorphic site was observed for 10 of 38 non-alpha-thalassemia chromosomes from Black Americans. In four Black families with a heterozygous alpha-thalassemia-2 [-alpha 3.7 (I)], an Apa I restriction site has been identified in the IVS-2 of the alpha 2 gene of the normal chromosome (labeled the alpha *2 gene). The alpha *2 gene of one Black subject was cloned and a segment located 5' to the Cap site as well as the IVS-2, exon 3, and a 3' segment were sequenced. The data show that the alpha *2 gene is an alpha 2 gene except for a segment between nucleotides (nts) 580-81 and nt 509 (Cap site = nt 1), and perhaps as far upstream as nt -634, which has an alpha 1 sequence. This alpha *2 hybrid gene probably originated through a double crossover; the structural identity of its IVS-2 with that of the alpha 1 gene adequately explains the presence of the Apa I restriction site. more...

A new improved electrophoretic method of glucose-6-phosphate dehydrogenase (G6PD) was devised by using a slab disc electrophoresis with a discontinuous gel of 1-mm thickness. This was suitable for electrophoresis of thermolabile G6PD variants with low activities. Using this system, sharp G6PD bands and acceptable separations of G6PD Konan, G6PD Tokyo, and G6PD Yokohama, were obtained and compared with those of starch gel electrophoresis. more...

A total of 3000 men living in Yamaguchi were screened for glucose-6-phosphate dehydrogenase (G6PD) deficiency using Beutler's spot test and three types of starch gel electrophoresis. These electrophoresis used a phosphate buffer system at pH 7.0, a TRIS-EDTA-borate buffer system at pH 8.6, and a TRIS-hydrochloride buffer system at pH 8.8. Fifteen G6PD-deficient variants were found at the rate of 0.5% and classified into four groups. As new variants, G6PD Konan, Kamiube, and Kiwa were identified. These three variants had a mild to moderate G6PD deficiency and were not associated with any clinical signs. G6PD Konan had fast electrophoretic mobility as compared with normal levels, G6PD Kiwa had slightly elevated electrophoretic mobility, and G6PD Kamiube had normal electrophoretic mobility. These three variants had normal levels of Km G6P, Km NADP, and Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NAPD, normal heat stability, and a normal pH curve. The other variant was G6PD Ube, which we had previously found in Yamaguchi (Nakashima et al., 1977). One boy with G6PD Ube was Korean. more...

A case of red cell adenosine deaminase (ADA) overproduction associated with hereditary hemolytic anemia is reported here. This appears to be the second report. Proband is a 38-year-old Japanese male who had hemoglobin, 15.8 g/100 ml; reticulocyte count, 4.5%; serum indirect bilirubin, 4.9 mg/100 ml; 51Cr-labeled red cell half-life, 12 days; red cells showed moderate stomatocytosis. His red cell ADA activity showed 40-fold increase while that of the mother showed 4-fold increase. The mother was hematologically normal. The father had a normal enzyme activity. The proband and the mother showed slightly high serum uric acid levels. The proband's red cell showed: ATP, 628 nmoles/ml (normal, 1,010--1,550); adenine nucleotide pool, 46% of the normal mean; 2,3-diphosphoglycerate content, 3,782 nmoles/ml (normal 4,170--5,300); increased oxygen affinity of hemoglobin, P50 of intact erythrocytes being 21.8 mmHg (normal, 24.1--26.1). Red cell glycolytic intermediates in the proband were low in general, and the rate of lactate production was low. Kinetic studies using crude hemolysate revealed a normal Km for adenosine, normal electrophoretic mobility but slightly abnormal pH curve and slightly low utilization of 2-deoxyadenosine. The ADA activity of lymphocytes was nearly normal. more...

Four new glucose 6-phosphate dehydrogenase (G6PD) variants which had electrophoretically slow-moving characteristics associated with congenital nonspherocytic hemolytic anemia were discovered in Japan. Gd(-) Kurume was found in a 17-year-old male whose red cells contained only 0.8% of normal enzyme activity. Gd(-) Kurume had a normal Km G6P, normal Km NADP, low Ki NADPH, normal utilization of 2-deoxy-G6P and deamino-NADP, very low heat stability and a biphasic pH curve. The hemolytic anemia was mild. Gd(-) Fukushima was characterized from a 33-year-old male and had 2.8% of normal enzyme activity, normal Km G6P, normal Km NADP, low Ki NADPH, normal utilization of 2-dcoxy-G6P, low utilization of deamino-NADP, low heat stability and a normal pH curve. The hemolytic anemia was mild. Gd(-) Yamaguchi was characterized from an 8-year-old boy and had 3.5% of normal enzyme. more...