Association of tumor necrosis factor receptor type II polymorphism 196R with Systemic lupus erythematosus in the Japanese: molecular and functional analysis.

OBJECTIVE: To investigate whether a polymorphism(s) or mutation(s) in the tumor necrosis factor receptor II (TNFRII) gene is involved in the pathogenesis of systemic lupus erythematosus (SLE). METHODS: All 10 exons of the TNFRII gene were analyzed by exon-specific polymerase chain reaction-single-strand conformation polymorphism, followed by nucleotide sequencing of exons that displayed aberrant bands. To analyze the function of the TNFRII polymorphisms, the full-length TNFRII complementary DNA of each allele was transfected in HeLa cells and then studied for specific binding of 125I-TNFalpha, as well as interleukin-6 (IL-6) production and cytotoxic activity after treatment with recombinant human TNFalpha. RESULTS: We identified 4 polymorphisms, at codons 56, 181, 196, and 232. The latter 2 had amino acid substitutions M196R and E232K, respectively. Only the 196R allele was significantly associated with SLE in our 105 Japanese SLE patients, with an allele frequency of 20.5%, compared with 12.6% in 99 healthy controls (P = 0.0335). More importantly, using TNFRII-transfected HeLa cells, we demonstrated significantly increased IL-6 production by 196R TNFRII compared with 196M TNFRII. The cytotoxic activity induced by 196R TNFRII was also increased compared with that of 196M TNFRII. This increase was achieved without affecting the binding affinity of TNFalpha to TNF-RII, as demonstrated by the finding that specific TNFalpha binding to the HeLa transfectants of 196R and 196M TNFRII was similar, with Kd values of 3.12 x 10(-10)M and 4.34 x 10(-10)M, respectively. CONCLUSION: These results suggest that 196R TNFRII, which transduces the signals of TNFalpha more effectively than does 196M TNFRII, is involved in the pathogenesis of SLE.

Severe thrombocytopenia caused by digitoxin intoxication in a patient with heart failure associated with Sjögren's syndrome.

Congestive heart failure (CHF) related to Sjögren's syndrome is extremely rare. This report concerns a patient who presented with CHF and severe thrombocytopenia (5,000/microl). Serum concentrations of K, Mg and digitoxin were 3.2mmol/L, 1.4mg/L and 57.2ng/ml, respectively. Digitoxin intoxication was evident, seemingly evoked by hypokalemia, hypomagnesemia, hepatorenal dysfunction and hypothyroidism. The severe thrombocytopenia was considered to have been caused by this intoxication, as it disappeared soon after the digitoxin was discontinued and potassium was supplemented.

[Pneumatosis cystoides intestinalis associated with intravenous pulse cyclophosphamide treatment for systemic lupus erythematosus].

Pneumatosis cystoides intestinalis (PCI) is an uncommon disease manifestation characterized by the presence of air in the bowel wall. PCI is sometimes observed in patients with progressive systemic sclerosis or mixed connective tissue disease but extremely rare in patients with systemic lupus erythematosus (SLE). We here report a patient with SLE who developed PCI after the treatment with intravenous cyclophosphamide (IVCY). This is the first case that association between IVCY and PCI was suggested. A 51-year-old woman with a 24-year history of SLE was admitted to our hospital because of skin ulcers in the lower legs. She had been receiving prednisolone orally. Laboratory findings on the present admission showed a elevated titer of anti-double stranded DNA antibody and positive LE test. She was successfully treated with three pulses of methylprednisolone followed by two IVCY together with vasodilators for her disease activity of SLE including skin manifestation. Just after the second IVCY, abdominal distention was gradually developed without any other abdominal symptoms, including abdominal pain. Abdominal radiography and computed tomography revealed pneumoperitoneum and multiple intramural air collections which involved the ascending colon primarily. Gastrointestinal series, however, showed no evidence of intestinal perforation. The diagnosis of PCI was made radiologically. After she was treated with a combined therapy with intravenous hyperalimentation and breathing with high concentration of oxygen for three weeks, PCI and pneumoperitoneum disappeared. It would be necessary that IVCY is carefully administrated, especially for the patients under the risk of PCI, such as collagen diseases.

Growth inhibition of leukemic cells by (-)-epigallocatechin gallate, the main constituent of green tea.

In this report, we presented the results that EGCG, the main constituent of the polyphenols present in Japanese green tea inhibited growth of leukemic cell lines of both human and mice. The proliferation of human leukemic cell lines and mouse NFS60 cell line was inhibited by EGCG. Sensitivity of each line to EGCG was different, and more than 50% of DNA synthesis was reduced in all the cell lines in the presence of 50 microM EGCG. On the other hand, normal hematopoietic progenitor cells retained their natural function of supplying mature cells of various lineages in the presence of less than 10 microM EGCG in vitro. Even in the presence of 100 microM EGCG, half the colonies containing all the lineages of cells were developed. All the dead cells of each line showed characteristics of apoptosis, which might be due to inhibition by EGCG of growth factors' signaling. Besides anticarcinogenic activity, EGCG is expected to have a new function for leukemia therapy without side effects.

Pharmacological studies on Puerariae flos III: protective effects of kakkalide on ethanol-induced lethality and acute hepatic injury in mice.

Kakkalide, one of the major isoflavonoid components of Puerariae flos, has been investigated for its effect on ethanol-induced intoxication and on hepatic injury, including hyperglycaemia, in mice. Kakkalide reduced mortality associated with administration of ethanol. At doses of 100 and 200 mg kg-1 the effect of kakkalide was significant. The same dose of kakkalide prevented increased serum glutamic oxaloacetic transaminase and glutamic pyruvic transaminase activity. At a dose of 200 mg kg-1 it also counteracted ethanol-induced elevation of glucose levels. These results suggest that kakkalide might be useful for counteracting the effects of alcohol and might be effective for treating hepatic injury.

Effect of (-)-epigallocatechin gallate on leukemic blast cells from patients with acute myeloblastic leukemia.

Information on the anti-carcinogenic effect of EGCG, the main constituent of the polyphenols present in Japanese green tea leaves, has recently been accumulating. In this report, we evaluate the effect of EGCG on leukemic blast cells from AML patients. The results showed that EGCG inhibited the proliferation of AML cells in all cases examined. Since AML cells might proliferate by autocrine or paracrine growth mechanisms, we also examined the effect of EGCG on the production of GM-CSF from AML cells. Although EGCG did not directly inhibit the production of GM-CSF, it did inhibit the effect of TNF-alpha or TPA, both of which stimulated AML cells to produce GM-CSF. On the other hand, the modulation of receptors for growth factors might play a role in the proliferation or carcinogenesis of AML cells. We also found that EGCG inhibited the modulation of c-kit, a receptor for stem cell factor, on leukemic cells. These findings suggested that EGCG might be available as a new therapeutic tool for AML patients.

Protective effect of granulocyte colony-stimulating factor against T-cell-meditated lethal shock triggered by superantigens.

The bacterial superantigens (SAg), toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB), are powerful T-cell stimulators, triggering systemic release of lymphokines causing lethal shock in D-galactosamine (D-Gal)-sensitized mice. We show that pretreatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) protects mice against T-cell-mediated SAg-shock. In mice challenged with D-Gal/TSST-1, lethal shock was caused within 30 hours. In contrast, animals pretreated with two consecutive subcutaneous injections of 2 micrograms rhG-CSF with a 12-hour time interval showed only marginal signs of illness and no lethality after challenge with D-Gal/TSST-1. Mice treated with 5 micrograms rhG-CSF either 12 or 6 hours in advance also survived otherwise lethal doses of D-Gal/TSST-1. The protective effects of rhG-CSF pretreatment was also evident against lethal doses of D-Gal/SEB challenge and this protection was accompanied by suppression of systemic interleukin-2. However, rhG-CSF affected neither the proliferative responses of SAg-reactive T cells in vivo or in vitro nor their interleukin-2 production in vitro, implying that rhG-CSF may indirectly interfere with cytokine synthesis in T cells but not with T-cell-SAg binding itself. These results represent another beneficial effect of rhG-CSF as an anti-inflammatory agent against T-cell-mediated toxicity triggered by SAg.

Successful second autologous blood stem cell transplantation after G-CSF-combined conditioning for the treatment of high-risk acute myelogenous leukemia.

We describe a patient with acute myelogenous leukemia (AML) who received his second autologous blood stem cell transplantation (ABSCT) following a G-CSF-combined pre-transplant conditioning regimen. The patient underwent ABSCT during first remission but suffered a relapse 8 months later. After achieving second remission, he was prepared for his second ABSCT; recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered in combination with Ara C, in addition to the same conditioning regimen as that used before the first ABSCT. There was no increase in regimen-related toxicity after this second G-CSF-combined conditioning regimen when compared with that observed after the first ABSCT. To date, the patient's second remission following the second ABSCT has lasted 26 months, which has exceeded that following the first ABSCT. The G-CSF-combined pretransplant conditioning regimen for ABSCT may be effective in the treatment of high-risk AML by increasing the chemosensitivity of the residual leukemic cells.

[Successful treatment of recurrent multiple lung metastasis from colon cancer with combination chemotherapy using methotrexate, 5-fluorouracil, and high-dose leucovorin: a case report].

A 66-year-old man, who had received sigmoidectomy for sigmoid cancer in 1985, was diagnosed as having multiple lung and liver tumors in September 1988. When celiac-angiography was performed, recurrent liver metastases from sigmoid cancer were suspected and he received a transarterial embolism with ADM 30 mg and MMC 20 mg. In addition, he was treated with a sequential chemotherapy with methotrexate (MTX), 1,200 mg intravenously (6 h-infusion) followed by 5-fluorouracil (5-FU), 600 mg/m2/day and leucovorin, 300 mg/body/day in continuous infusion for 5 days from day 2 with concomitant oral administration of dipyridamole (300 mg/day) over 14 days. Treatment was repeated every 28 days for two courses. For the third course, administration of only 5-FU, leucovorin and dipyridamole was performed. As a result, the size of pulmonary lesions was prominently reduced on computed tomography. Although mucositis, anal erosion, diarrhea and thrombocytopenia were noted, no severe side effects were observed. This sequential chemotherapy appears useful for metastatic lesions from colon cancer.

[A patient with liver cirrhosis manifesting various symptoms including cerebellar ataxia due to germanium intoxication].

A patient with hepatitis B virus-associated cirrhosis manifested various symptoms such as anemia, renal damage and neurological signs including cerebellar ataxia due to long-term administration of germanium-containing food. The patient was a 40-year-old male who had taken germanium containing mineral cheese for 26 months after he was diagnosed as having cirrhosis. Twenty four months after beginning to take the mineral cheese, he began manifesting paresthesia of the extremities, dysarthria and gait ataxia. Laboratory findings revealed anemia and renal damage. Biopsy of the peripheral nerve revealed loss of the large sheathed nerve, a characteristic feature of germanium intoxication. A high concentration of germanium (GeO2) was detected in patient's hair and urine. Cerebellar ataxia was characteristic in this patient, which was not reported in the previous papers.

[Treatment of iron overload with combination of recombinant human erythropoietin and phlebotomy].

A patient of Coombs negative autoimmune hemolytic anemia was massively transfused of 162 units concentrated red blood cells in 3 months and developed iron overload disease which was confirmed by liver biopsy. Hemolysis was successfully treated with high-dose methyl-prednisolone therapy and splenectomy. To treat iron overload, we administered recombinant human erythropoietin (Epo) in combination with phlebotomy. Total iron removed for 5 months was about 4 g. Thus, combination of Epo and phlebotomy was effective for the treatment of iron overload disease. Furthermore, we compared a degree of clinical effect of subcutaneous administration of Epo with that of intravenous administration in the clinical course and found the former more effective.

A traditional Chinese herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-yoei-to) augments the production of granulocyte-macrophage colony-stimulating factor from human peripheral blood mononuclear cells in vitro.

Ren-shen-yang-rong-tang (Japanese name: Ninjin-yoei-to, NYT) is a traditional Chinese herbal medicine. Leukocytosis and elevated levels of colony-stimulating factor (CSF) in peripheral blood were found previously after the administration of this compound to mice. In this study, human peripheral blood mononuclear cells (PBMC) were cultured in the presence of NYT in vitro, and the levels of granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF) in the supernatant of cultured PBMC were measured using a sensitive enzyme-linked immunosorbent assays. NYT significantly (P less than 0.01) augmented GM-CSF production but not G-CSF production by PBMC in vitro.

Production of interleukin 6 from human liver cell lines: production of interleukin 6 is not concurrent with the production of alpha-fetoprotein.

The production of interleukin (IL) 6 from six human liver cell lines, including Chang liver, HLF, HLE, HepG2, PLC/PRF/5, and HuH-7, was investigated using enzyme-linked immunosorbent assay and Northern blot analysis. When cells were cultured in the presence of 12-O-tetradecanoylphorbol-13-acetate, significant amounts of IL6 were detected in the culture supernatants of Chang liver cells, HLF cells, and HLE cells. However, IL6 was not detected in the culture supernatants from HepG2 cells, PLC/PRF/5 cells, or HuH-7 cells which had been treated similarly. To further investigate the production of IL6, expression of the IL6 gene was studied. Results of Northern blot analysis using IL6 complementary DNA as a probe showed that the induction was initiated at the mRNA level. Moreover, IL6 mRNA was also induced by IL1 beta and tumor necrosis factor but not by a calcium ionophore (A23187) or IL6 itself in Chang liver cells. This is the first study to demonstrate the production of human IL6 in liver cells. Furthermore, when the production of alpha-fetoprotein (AFP) from the liver cell lines was examined, the three that were able to produce IL6 failed to produce AFP, whereas the other three cell lines succeeded in producing AFP. These observations may indicate the heterogeneous origin of the liver cell lines.

[Successful treatment of advanced gastric cancer with multiple liver metastasis by combination chemotherapy using mitomycin C, 5-fluorouracil, and high-dose leucovorin: a case report].

A 68-year-old woman complaining of severe iron deficiency anemia was found to have an advanced gastric cancer (poorly differentiated adenocarcinoma) with multiple liver metastases. The patients was treated three times with combination chemotherapy using a monthly schedule consisting of bolus infusion of mitomycin C (10 mg/m2) on day 1, continuous infusion of 5-fluorouracil (600 mg/m2) on day 1 to 6, and continuous infusion of high-dose leucovorin (300 mg/body) on day 1 to 6, with concomitant oral administration of dipyridamole (300 mg/day) over 14 days. Endoscopically, cancerous ulcer in the primary gastric lesion improved like a healed peptic ulcer. Metastatic lesions in the liver almost disappeared on computed tomography. The most prominent side effect was oral mucositis which was tolerable and healed in a week. This regimen appears potentially useful in the treatment of gastric cancer.

Milk of calcium renal stone in a patient with acute promyelocytic leukemia.

A case of milk of calcium renal stone is reported. This is a rare disease in which a suspension of calcium salts is formed within a renal cyst. The pathognomonic sign is a fluid level seen in a standing position and an oval density seen in a supine position. In the present case, the milk of calcium was found to develop in a hydronephrotic kidney during the course of acute promyelocytic leukemia and this condition was suggested to be a complication of infection.

Stimulatory effect of forphenicinol on normal human bone marrow granulocyte-macrophage progenitor cells mediated by T-lymphocytes.

Forphenicinol, L-2-(4-formyl-3-hydroxymethylphenyl) glycine, is a newly discovered low molecular weight immunomodifier. Its effects on normal human bone marrow granulocyte-macrophage progenitor cells (CFU-C) were studied in vitro. Addition of forphenicinol to cell cultures resulted in a significant (p less than 0.05) increase in the number of, and preservation of the ability to form CFU-C colonies per fixed number of human non-adherent bone marrow cells. This effect was not observed in T-lymphocyte depleted fractions of human non-adherent bone marrow cells. Furthermore, colony stimulating factor was released when T-lymphocytes were incubated with forphenicinol. These data suggested that T-lymphocytes mediated the stimulatory effect of forphenicol on human bone marrow CFU-C.

Evaluation of cefoperazone in the treatment of bacterial infections in patients with hematological diseases.

This study was conducted as a multicenter trial in 12 institutions in the Kyushu region of Japan. The clinical effects of cefoperazone were evaluated in 101 patients with 112 infections complicated by hematological disease. In about half of the patients, the baseline neutrophil count was below 1,000/mm3. Results were excellent in 23 of 101 cases and good in 48, with an efficacy rate of 70.3%. Cefoperazone alone or in combination with other antibiotics was effective in 60.7% of patients who had not responded to previous antibiotic therapy. No serious side effects attributable to cefoperazone were noted. The results indicate that cefoperazone is a useful antibiotic for the treatment of severe infections in patients with hematological diseases.