Oxygen radical generation and expression of NADPH oxidase genes in bottlenose dolphin (Tursiops truncatus) neutrophils.

Oxygen radical generation by stimulation with phorbol myristate acetate (PMA) was evaluated in bottlenose dolphin neutrophils. A Cypridina luciferin analog-dependent chemiluminescent assay demonstrated that dolphin neutrophils generate superoxide by the addition of PMA, and that its superoxide-forming activity is completely suppressed by diphenylene iodonium, a specific inhibitor of NADPH oxidase. These results indicate that dolphin neutrophils possess NADPH oxidase activity. Furthermore, the NADPH oxidase activity (hydrogen peroxide production) in dolphin neutrophils, as well as in human neutrophils, was greater at 37 degrees C than at a lower temperature. RT-PCR with specific primers revealed that dolphin neutrophils expressed the mRNAs of the major NADPH oxidase components, which included membrane-associated flavocytochrome b (gp91(phox) and p22(phox)) and cytosolic factors (p40(phox), p47(phox), and p67(phox)), implying the existence of these protein homologues in dolphin neutrophils.

Development of monkey C-reactive protein (CRP) assay methods.

Monkey-specific C-reactive protein (CRP) assay methods (enzyme-linked immunosorbent assay (ELISA) and turbidimetric immunoassay (TIA)) were developed. The anti-monkey CRP serum was prepared by immunization of rabbits with the immune complex formed between the acute-phase serum from turpentine oil-inoculated monkeys and goat anti-human CRP serum. The specificity of the rabbit anti-monkey CRP serum was confirmed by immunoelectrophoresis and Western blotting. The purity of monkey CRP prepared by chromatography procedures was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The serum CRP levels in nine normal monkeys, as measured by sandwich ELISA were ranged from 0.26 to 1.42 microg/ml (mean 0.71+/-0.37). The CRP levels in five acute-phase sera of turpentine oil-inoculated monkeys were 248-451 microg/ml (mean 371.2+/-73.8). This monkey-specific CRP assay method was found more sensitive than the human-specific CRP assay method in detecting monkey CRP by TIA.

Possible causal association between ulcerative colitis and idiopathic thrombocytopenic purpura.

A 24-year-old Japanese male having ulcerative colitis and idiopathic thrombocytopenic purpura is described. When the symptoms of ulcerative colitis were manifest during his clinical course, the platelet life-span was shortened and the platelet associated IgG level was increased. Thus, causal association between the two diseases may be suggested.

Variation of serum alpha2-macroglobulin concentration in healthy rats and rats inoculated with Staphylococcus aureus or subjected to surgery.

BACKGROUND AND PURPOSE: The aim of the study reported here was to investigate variations in the serum concentration of alpha2-macroglobulin (alpha2M) from healthy rats and rats inoculated with Staphylococcus aureus or subjected to surgery. METHODS: Concentration of alpha2M was measured by use of an enzyme-linked immunosorbent assay. RESULTS: Serum alpha2M in healthy rats at intervals of 3 h, 1 day, and 1 week ranged from 17.5 to 38.0 (mean +/- SD, 27.2 +/- 6.6) microg/ml, 15.8 to 48.2 (27.9 +/- 8.7) microg/ml, and 17.2 to 39.6 (23.9 +/- 5.9) microg/ml, respectively. Concentrations of alpha2M did not undergo significant variations within individuals or between rats. Serum alpha2M concentration increased at one day and peaked two days after inoculation with S. aureus or being subjected to surgery. Peak concentration was eight to 33 times preinoculation values after inoculation with S. aureus, four to 25 times pre-surgical values when rats were subjected to castration, and seven to 28 times pre-surgical values when rats were subjected to oophorohysterectomy, respectively. CONCLUSIONS: Physiologic variation in the concentration of alpha2M in rats was not found. Induction of alpha2M in rats inoculated with S. aureus or subjected to surgery was documented.

Contrast-enhanced MRI with gadodiamide injection in rabbit carcinoma models.

This study was designed to demonstrate the broad utility of contrast-enhanced magnetic resonance imaging (MRI) for diagnosis of carcinoma. Twenty-six New Zealand White rabbits of either sex (1.7-3.4 kg) were used for the investigation. VX2 carcinoma (squamous cell carcinoma) was implanted in either the brain, lung, ovary, bone or muscle of rabbits. Contrast agent, Gadodiamide Injection, was administered intravenously at a dose of 0.1 mmol/kg. MR images were obtained by a 1.5 T, or a 2.0 T magnetic field strength super-conductive MRI unit. The intensity of the signal for the carcinoma was increased after administration of contrast agent, and the detectability or diagnostic information of post-contrast images was superior to that of pre-contrast images in all models. In addition, no significant side effects were observed during the MRI examination. After diagnosis using MRI, morphological damage in each model was assessed by gross and histopathological examinations. In contrast-enhanced MRI, though there were variations between the models employed, contrast effects in brain and muscle carcinoma models were generally related to differences in capillary permeability, while imaging in lung, ovary and bone carcinoma models was dependent on differences in blood flow rate and the size of interstitial spaces. Overall, our results demonstrate that contrast-enhanced MRI is a useful and safe method for diagnosing tumors.

Serum alpha2-macroglobulin and cytokine measurements in an acute inflammation model in rats.

An enzyme-linked immunosorbent assay (ELISA) for rat alpha2-macroglobulin (alpha2M) using a monoclonal antibody was developed, and used to measure alpha2M in sera from rats injected intramuscularly with turpentine oil as an inflammatory agent. The mean concentration of alpha2M gradually increased and peaked 2 days after the turpentine oil injection. The peak alpha2M concentration ranged from 2362-8472 microg/ml (mean 4531 microg/ml), which was 50-290 times higher than the pre-dosing levels of 23-61 microg/ml. In addition, interleukins (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, and interferon (IFN)-gamma were measured using commercial ELISA reagent kits. IL-6 and IL-8 increased and peaked 12 h after turpentine oil injection, the levels being 5-51 times and 2-38 times the pre-dosing ones, respectively. The concentrations of IL-1, IL-2, IL-4, IL-10 and IFN-gamma in rats injected with turpentine oil did not change.

A comparison of the concentrations of C-reactive protein and alpha1-acid glycoprotein in the serum of young and adult dogs with acute inflammation.

The concentrations of C-reactive protein (CRP) and alpha1-acid glycoprotein (AAG) were evaluated in 1-, 3- and 18-month-old dogs (four of each age) that had been inoculated with turpentine oil. The CRP and AAG in 3-month-old and younger dogs subjected to surgery or inoculated with either Staphylococcus aureus or a viral vaccine were also evaluated. The average CRP concentration in the sera peaked 2 days after inoculation of turpentine oil. The peak CRP concentrations in 3- and 18-month-old dogs were significantly (p < 0.05) greater than those in 1-month-old dogs. The average AAG concentration in the sera peaked 4 days after inoculation of turpentine oil. No significant difference was found in AAG concentrations between any of the age groups. When experimentally inoculated with S. aureus or subjected to oophorohysterectomy, the CRP and AAG concentrations increased in 3-month-old dogs, but they increased little in 1-month-old dogs. The CRP and AAG in dogs inoculated with the viral vaccine did not increase. In dogs with fractures or subjected to percutaneous gastrostomy, the CRP and AAG concentrations correlated with the condition of dogs.

Immunological determination of faecal haemoglobin concentrations in dogs.

Faecal haemoglobin (Hb) concentrations in apparently healthy experimental Beagle dogs and in dogs of various breeds kept in private households or at breeders were measured by reversed passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA) in an effort to define the physiological concentrations of faecal Hb in the dog. In 88% (53) of 60 experimental Beagle dogs (30 males and 30 females), the RPLA titres were 1:2 and 1:8 and the faecal Hb concentrations ranged from 40.0 to 431.5 (mean 184.1 +/- 92.6) micrograms/g faeces by ELISA. No significant difference was found in Hb levels or RPLA titres between males and females. Seven dogs (12%) had significantly greater RPLA titres and Hb concentrations by ELISA than the remaining dogs. In 84% (45) of the 53 dogs kept in private households or at breeders, the RPLA titres were < 1:1 to 1:8 and the faecal Hb concentrations ranged from 7.1 to 456.7 (mean 137.5 +/- 128.7) micrograms/g faeces in ELISA. Eight of these dogs (15.1% of 53 dogs) had significantly greater RPLA titres and Hb concentrations by ELISA than the remaining dogs. There were no significant differences between the Beagles and dogs kept in private households or at breeders. In conclusion, in 98 (86.7% of 113) dogs the physiological concentrations of RPLA titres were < 1:1 to 1:8 and the faecal Hb concentrations were 143.5-185.1 micrograms/g (95% confidence level). Approximately 13.3% of apparently healthy dogs had higher faecal Hb concentrations, suggesting the presence of subclinical haemorrhages. Four dogs suffering from colorectal cancer also had high faecal Hb concentrations.

Physiological levels of C-reactive protein in normal canine sera.

This study was undertaken to investigate whether the level of C-reactive protein (CRP) in the serum of dogs undergoes physiological variation, using 10 normal Beagle dogs (5 males and 5 females), 1-2 years old, maintained in a healthy condition in a controlled environment. The CRP concentration in the sera collected seven times each day at intervals of approximately 3 h ranged from 0.8 to 16.4 micrograms/ml (mean 5.06 +/- 3.60) in one experiment and from 0.8 to 14.0 micrograms/ml (mean 4.50 +/- 2.80) in a second experiment. On examining the 24-h variations in the concentration of CRP in serum, neither consistent changes nor a definite pattern of circadian rhythm was detected. During 28 days observation, only very slight changes, which seemed attributable to analytical errors, were seen in any of the dogs, except one. The concentration of CRP in the serum during the 28 days ranged from 0.8 to 22.6 micrograms/ml (mean 3.65 +/- 1.40). The concentrations underwent no significant variations in individual dogs, but significant differences were found between the dogs (p < 0.01).

Concentrations of C-reactive protein in normal monkeys (Macaca irus) and in monkeys inoculated with Bordetella bronchiseptica R-5 and measles virus.

The concentrations of C-reactive protein (CRP) in serum from normal crab-eating monkeys (Macaca irus) were measured by means of a monkey-specific turbidimetric immunoassay (TIA), and the changes in the serum CRP concentrations in crab-eating monkeys inoculated with Bordetella bronchiseptica R-5 and measles virus (Ichinose or NK 3 strain) were also examined. The CRP concentrations in sera from 54 normal crab-eating monkeys ranged from 0 to 8.3 microg/ml (mean 2.2 +/- 1.9). No significant difference was found in the CRP concentrations between males and females (p > 0.05). The concentrations of CRP in the sera from four crab-eating monkeys inoculated intrabronchially with 10(9) live B. bronchiseptica increased gradually to a peak at 2 days after inoculation. The peak concentrations of CRP were from 102.4 to 313.2 microg/ml, 54-96 times the preinoculative values of 1.9-5.6 microg/ml. When the same four crab-eating monkeys were inoculated intrabronchially with measles virus 34 days after inoculation of B. bronchiseptica, the serum CRP concentrations did not increase. Monitoring of CRP is useful for assessing monkeys with acute B. bronchiseptica infection and will probably be of value in the diagnosis of other bacterial infections.

Monitoring C-reactive protein in beagle dogs experimentally inoculated with Ehrlichia canis.

The concentrations of C-reactive proteins (CRP) in the plasma of five beagle dogs experimentally inoculated with Ehrlichia canis increased markedly. The concentrations began to increase between 4 and 16 days and peaked between 15 and 42 days after inoculation of E. canis. The peak concentrations ranged from 217.8 to 788.8 microg/ml (452.6 +/- 228.1 SD). After the peak, the concentrations of CRP decreased rapidly. The PCR product of 16S rRNA of E. canis became detectable in the five dogs between 18 and 27 days after inoculation of E canis. Antibodies to E canis were detected in plasma from the dogs between 5 and 15 days after inoculation of E. canis. The timings of seroconversion and of the start of the increase in CRP were approximately similar and the high concentrations of CRP in the plasma of the dogs tended to become apparent when the PCR product of 16 S rRNA of E. canis became detectable.

Experimental detection of canine haemoglobin (occult blood) in canine faeces by reversed passive latex agglutination.

A reversed passive latex agglutination test (RPLA) using anti-canine haemoglobin (Hb) antibody was developed for detecting bleeding in the lower digestive organs in dogs, and its applicability as a simple test for faecal occult blood was assessed. In Ouchterlony's gel immunodiffusion test, the anti-canine Hb antibody used to sensitize the latex reacted with canine Hb but not with Hbs, plasmas or meat extracts from pigs, goats, sheep, cattle, horses or chickens, or with fish extracts. Using latex sensitized with 50 micrograms/mg of anti-canine Hb IgG antibody, the lowest limit of detection for canine Hb was 21 micrograms/ml, and the latex reacted negatively with all test specimens other than canine Hb. In an in vitro experiment with a mixture of canine faeces and erythrocytes, the antigenicity of the Hb was found to undergo only very slight changes even when the specimens were allowed to stand for 12 h at room temperature. Hb could not be detected by RPLA in any of four successive faecal samples from three experimental dogs after infusion of autologous blood (5, 3 or 1 ml) into the stomach. In 3 other experimental dogs given an infusion of autologous blood (5, 3 or 1 ml) into the ascending colon, the presence of Hb was confirmed by RPLA in all four successive faecal samples obtained from those which received 5 or 3 ml of blood and in all except that obtained following the first defecation from the animal which had received 1 ml of blood.

[Complete remission of acute promyelocytic leukemia accompanied by DIC in an elderly patient treated with all-trans retinoic acid].

In this paper we report on a 74-year-old female patient who was suffering from acute promyelocytic leukemia (APL) and who, upon admission to our hospital on February 20, 1992, was also found to be stricken with disseminated intravascular coagulation (DIC). The DIC, however, was quickly arrested by administration of heparin and there was no exacerbation. Also, on admission her peripheral blood leukocyte count was 700/ul, but after oral administration of all-trans retinoic acid (ATRA) (45 mg/m/day) was begun on February 22, this count gradually increased and peaked at 35,200/ul on March 7. Some of these matured leukocytes revealed dysplastic features; some had Auer bodies. At this time cytogenetic analysis of bone marrow cells showed 46XX, t (15;17). The leukocyte count gradually decreased to 1,500/ul, and the dysplastic features disappeared. On March 21 her thrombocytes and reticulocytes began to increase, and she achieved complete remission when her abnormal karyotype disappeared on March 24. She suffered no severe complications such as infection or hemorrhage during treatment. We therefore suggest that ATRA is very effective for APL in elderly patients. It neither exacerbates DIC nor increases the risk of infection. In fact, when ATRA treatment is compared to the standard cytotoxic chemotherapy there is a reduced risk of infection.

[The role of protein kinases and phosphatases in erythroid colony formation of elderly].

Using H-7, HA1001, FK506, cyclosporin A (CsA) and okadaic acid (OA), which are protein kinase and phosphatase inhibitors, we examined qualitative changes in hematopoietic precursor cells due to aging from the viewpoint of the role of protein kinases and phosphatases. Though H-7 and OA suppressed erythroid colony formation both in the elderly (age: 72-92, median: 86) and the young (age: 22-39, median: 29), no change due to aging was noted. HA1001 did not affect erythroid colony formation either in the elderly or the young. Erythroid colony formation was enhanced by FK506 and CsA in the young, however, erythroid colony formation was suppressed in the elderly. Similar examinations using cell fractions of non-T, non-macrophage, non-T + T, and CD34 positive cells were performed in both groups. Enhancement of erythroid colony formation in the young and suppression in the elderly by FK506 using unseparated MNC disappeared after removal of T cells. Enhancement of colony formation in the young and suppression of colony formation in the elderly were recovered when T cells were added again. The effects of FK506 and CsA on erythroid colony formation were thought to be the results of T cell inactivation, and the different sensitivity to FK506 and CsA in the elderly and young seemed to be the result of changes in the control mechanisms of hematopoiesis, such as the regulation of cytokine production by T cells, caused by aging.

[Autoimmune hemolytic anemia with eosinophilia in elderly patient].

A 70-year-old woman was admitted to our hospital in November 1992 for evaluation of anemia. Physical examination revealed anemia, jaundice, swelling of axial and inguinal lymph nodes, and splenomegaly. Abnormal hematological findings were as follows: Hb of 3.9 g/dl, reticulocyte count of 58.2% (61.7 x 10(4)/microliters), hyperplasia of normal erythroblasts in bone marrow, and eosinophilia (21.0%, 2352/microliters) in peripheral blood. Routine laboratory examinations revealed polycolonal hypergammaglobulinemia 3.0 g/dl, a high level of serum LDH (797 IU/I) and a total bilirubin of 2.4 mg/dl (indirect, 1.6 mg/dl). The serum haptoglobin level was very low (< 5 mg/dl). Results of serological examinations were as follows: IgG of 3366 mg/dl, CH50 of 16.0 U/ml, positive Coombs test 2+, and positive tests for antinuclear antibody, rheumatoid factor, and cold agglutinin. CRP was negative. PHA-stimulated lymphocyte blast formation, NK activity, and ADCC activity were found to be suppressed, and the percentage of CD4-positive lymphocytes in peripheral blood was also low. An axillary lymph node biopsy revealed reactive lymphadenitis. No signs or history suggested allergy, collagen disease, or parasitic infection. Autoimmune hemolytic anemia (AIHA) complicated by immunologic abnormalities and eosinophilia was diagnosed. Oral prednisolone markedly reduced the hemolytic anemia, eosinophilia, lymph node swelling, and splenomegaly, but NK activity remained low.

Coamplification of the L-myc and N-myc oncogenes in a neuroblastoma cell line.

The L-myc, N-myc and c-myc genes are members of the myc oncogene family. In particular, L-myc is novel, and amplification of L-myc is still unknown except in small cell lung carcinoma. We examined L-myc amplification in 30 human neuroblastomas using Southern blot hybridization, and found that the L-myc gene was amplified approximately 5-fold in GOTO, a human neuroblastoma cell line. The N-myc gene was also amplified approximately 60-fold and furthermore, over-expression of L-myc and N-myc genes was observed in this cell line. In this report, we describe the coamplification of the myc gene family in the GOTO neuroblastoma cell line.

Spontaneous regression in B cell, diffuse large cell type non-Hodgkin's lymphoma.

A rare case of non-Hodgkin's lymphoma (NHL) that showed transient spontaneous regression (SR) is described. After 6 months of remission, recurrence was noted in the lymph nodes, pleura and the spleen. Although transient improvement was observed following combination chemotherapy, the pleural effusion became refractory to chemotherapy and the patient died 9 months after the relapse.

Suppressed natural killer cell activity in ulcerative colitis.

We describe a 31-year-old patient with ulcerative colitis who was successfully treated with prednisolone (PSL). Immunologic analyses were performed during the treatment course. Suppressed natural killer (NK) cell activity and increased levels of the CD4/CD8 ratio were observed on admission. Transient restoration of NK cell activity was obtained by PSL treatment. However, it returned to the initial low level in spite of the improvement of clinical symptoms. Possible mechanisms are discussed for involvement of the immune system in the pathogenesis of ulcerative colitis (UC).

[Results of radiotherapy for stage III uterine cervical carcinomas and preliminary study of combined radiotherapy and transarterial infusion chemotherapy].

This is a retrospective analysis of 55 patients with stage III carcinoma of the uterine cervix treated with radiation from November 1984 through December 1991. Eleven of the patients were treated with radiation and transarterial infusion chemotherapy (TAI), using cis-platinum and pepleomycin. The 3- and 5-year cumulative survival rates for all patients were 61% and 58%, respectively, and the 3-year cumulative survival rate for the group with combined radiation and TAI was 47%. According to initial failure site, the locoregional recurrence rate was 36.8%, and that for para-aortic lymph node metastasis and distant metastasis was 31.6%. The failure pattern was similar between the irradiation only group and the group with combined radiation and TAI. The incidence of intestinal complications of grades 1 and 2 was 20%. Irradiation combined with TAI did not increase the incidence of complications.