Association of a single nucleotide polymorphism in the SH2D1A intronic region with systemic lupus erythematosus.

SH2D1A, also known as signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), is an adaptor protein. Recently, it was reported that SAP deficient mice were protected from systemic lupus erythematosus (SLE). In this study, we postulated SH2D1A gene to be a candidate susceptibility gene for SLE and analyzed its association with SLE. A case-control association study was conducted on 5 tag single nucleotide polymorphisms (SNPs) in SH2D1A region in 506 Japanese female SLE patients and 330 healthy female controls. The luciferase assay was performed to determine the functional role of the SNP associated with SLE. One SNP in the intron 2, rs2049995, showed association with SLE (p=0.0110, odds ratio (OR) 1.97, 95% confidence interval (CI) 1.16-3.34, under the dominant model). The association of rs2049995 seemed to be stronger in the subset with the age of onset less than 20 years (p=0.0067, OR 2.65, 95% CI 1.28-5.46). Functional evaluation of rs2049995 showed that reporter gene activity was increased 1.9-fold for the susceptible allele compared with the resistant allele. An intronic SNP of SH2D1A is associated with SLE.

Systemic alkalinization inhibits the ability of flavone acetic acid to augment natural killer activity, induce cytokine gene expression, and synergize with interleukin 2 for the treatment of murine renal cancer.

Flavone acetic acid (FAA) is an investigational drug that augments natural killer activity, induces the genes for alpha- and gamma-interferon (IFN) and tumor necrosis factor alpha, and synergizes with recombinant interleukin 2 for the successful treatment of murine renal cancer. However, in most clinical studies of FAA only minimal immunomodulatory effects have been reported. Most of the patients in these studies have also been given sodium bicarbonate to prevent possible nephrotoxicity. The current study was performed to determine whether alkalinization had any effects on FAA-induced immune modulation and therapeutic activity in mice. The results showed that alkalinization inhibited the treatment of murine renal cancer by FAA plus recombinant interleukin 2 such that the survival rate of 84% in nonalkalinized mice was reduced to 0 in mice that were alkalinized during treatment. Alkalinization also significantly inhibited the ability of FAA to augment both splenic and hepatic natural killer activity in a dose-dependent manner. In contrast, alkalinization did not inhibit the ability of polyinosinic:polycytidylic acid and poly-L-lysine stabilized in carboxymethyl cellulose, maleic anhydride divinyl ether, or Propionibacterium acnes to augment liver-associated natural killer activity. By Northern blot analysis, it was shown that the induction of mRNA for IFN-alpha, IFN-gamma, and tumor necrosis factor alpha by FAA in the spleen cells of mice was significantly reduced in alkalinized mice. Consistent with a reduction in the FAA-induced expression of the cytokine genes, alkalinization also resulted in a significant decrease in both the peak serum concentration and duration of detectable IFN activity following FAA treatment. Increasing the dose of FAA in alkalinized mice to 300 mg/kg overcame the deleterious effects of alkalinization for treatment of murine renal cancer by FAA plus recombinant interleukin 2. These results demonstrate that the process of alkalinization inhibits the immunomodulatory and immunotherapeutic effects of FAA in mice and suggest that alkalinization might have similar deleterious effects on FAA-induced immune stimulation in human clinical trials.

Prevention of acute chemotherapy-induced death in mice by recombinant human interleukin 1: protection from hematological and nonhematological toxicities.

Previous studies have demonstrated that interleukin 1 (IL-1) can protect most mice from the acute lethal toxicity mediated by high doses of radiation and/or some chemotherapeutic drugs. The results presented herein demonstrate that the pretreatment of mice with recombinant human interleukin 1 alpha (rhIL-1 alpha) protects mice from the lethal effects of several myelotoxic chemotherapeutic drugs, including 5-fluorouracil (5FUra), cyclophosphamide, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II), and 1,3-bis-(2-chloroethyl)-1-nitrosourea. However, pretreatment with rhIL-1 alpha was not effective against the acute lethal toxicity generated by doxorubicin and cisplatin. The chemoprotective effects appear to be at least partially due to myeloprotection/restoration, since the recovery of myeloid colony-forming units and the total cellularity in the bone marrow and spleen were accelerated in the rhIL-1 alpha-pretreated mice. However, the chemoprotective effects of rhIL-1 alpha are apparently not limited to myeloprotection, since pretreatment with rhIL-1 alpha protects mice against the lethal toxicity of both 5FUra and cyclophosphamide, yet bone marrow transplants rescue mice treated with 5FUra but not those treated with cyclophosphamide. The chemoprotective effects of rhIL-1 alpha may be at least partially indirect, since the efficacy of chemoprotection by rhIL-1 alpha is reduced in athymic mice, and interleukin 6, but not tumor necrosis factor alpha, can substitute for rhIL-1 alpha in chemoprotection from 5FUra.

Reversal of enzymic phenotype of thymidine metabolism in induced differentiation of U-937 cells.

Exposure of U-937 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in specific alterations in thymidine metabolism. Within 24 h after treatment with 1.62 x 10(-9) M TPA, the reciprocal alteration in the activities of opposing enzymes of thymidine metabolism observed during normal cell culture growth was reversed. In TPA-treated cells, the activities of anabolic enzymes thymidine kinase (EC 2.7.1.75)and thymidylate synthase (EC 2.1.1.45) declined with time linearly to 20 and 16% of those of untreated cells by 72 h. Incorporation of [3H]thymidine and [3H]deoxyuridine into acid-insoluble fractions also decreased in parallel with the decline in enzyme activities. In contrast, the activities of catabolic enzymes thymidine phosphorylase (EC 2.4.2.4) and dihydrothymine dehydrogenase (EC 1.3.1.2) increased. The rise in thymidine phosphorylase activity peaked at 48 h with 406% elevation over the control. The activity of dihydrothymine dehydrogenase was not altered for the first 24 h, but it increased up to 338% by 96 h. Immunotitration of dihydrothymine dehydrogenase with monoclonal antibody against this enzyme showed that the rise in activity in the differentiated cells was due to the increase in the amount of enzyme protein. No significant difference was observed in the Km values for the substrate of each enzyme between untreated and TPA-treated cells. These metabolic alterations during induced differentiation were in line with the changes in cell morphology and accompanied by an accumulation of the cells in G1 at the expense of S phase. These observations indicate that induced differentiation of U-937 cells results in a reversal of the enzymic phenotype of thymidine metabolism and suggest that emergence of thymidine metabolic imbalance may serve as an early marker of differentiation of these cells.

Flavone acetic acid directly induces expression of cytokine genes in mouse splenic leukocytes but not in human peripheral blood leukocytes.

Flavone-8-acetic acid (FAA) is a flavonoid drug that augments mouse natural killer activity, induces cytokine gene expression, and synergizes with recombinant interleukin 2 for the treatment of murine renal cancer. However, FAA has been largely inactive in human clinical trials. In the present study we investigated the ability of FAA treatment to directly induce cytokine mRNA expression in total mouse splenic leukocytes and selected leukocyte subsets, as well as in total human peripheral blood leukocytes. Analysis of RNA isolated from FAA-treated mouse splenic leukocytes demonstrated that treatment with greater than or equal to 100 micrograms/ml of FAA induced expression of tumor necrosis factor alpha (TNF-alpha) mRNA by 1 h and induced maximal expression of TNF-alpha, alpha-interferon, and gamma-interferon mRNA within 3 h. The expression of all cytokine genes was diminished by 6 h. Interferon biological activity was detected in the supernatants of mouse splenic or peripheral blood leukocytes after treatment with FAA. These results correlate well with the previously reported induction of cytokine mRNA genes and biological activity by FAA in vivo. In contrast, FAA did not induce detectable mRNA expression or cytokine protein secretion by human peripheral blood leukocytes under similar conditions. These results demonstrate that FAA can directly stimulate cytokine gene expression in mouse but not in human leukocytes. Further studies performed with highly purified positively selected mouse CD4+ or CD8+ splenic T-lymphocytes, as well as purified B-cells, demonstrated that the FAA-induced expression of gamma-interferon mRNA was mainly induced in the CD8+ lymphocyte subset. alpha-Interferon mRNA was expressed largely in the B-cell population, while TNF-alpha mRNA was induced in all leukocyte subsets tested. Therefore, these results suggest that the immunomodulatory effects of FAA in mice are direct, but different cytokines are induced from different leukocyte subsets. Further, the data suggest that flavonoid compounds or analogues that stimulate cytokine gene expression in human cells might be therapeutically active in cancer patients.

Cytokine induction and therapeutic synergy with interleukin-2 against murine renal and colon cancers by xanthenone-4-acetic acid derivatives.

Derivatives of xanthenone-4-acetic acid (XAA) have been found to have similar activity to flavone-8-acetic acid against transplantable solid tumors. Some of these compounds were compared to flavone acetic acid (FAA) in their ability to induce cytokines as well as to mediate antitumor effects against murine renal cancer (Renca) and a mouse colon cancer (MCA-38). 5-Methyl-XAA and 5-chloro-XAA proved to be more potent than FAA on a mg/kg basis for induction of the genes for IFN alpha, IFN gamma, and TNF alpha, and for IFN and TNF activities in the sera of treated mice. These effects were sharply dose dependent. On the other hand, 7-methyl-XAA, which has no antitumor activity, did not induce these genes. In addition, 5-methyl-XAA and 5-chloro-XAA but not 7-methyl-XAA synergized with recombinant human interleukin-2 (rhIL-2) for the treatment of Renca and MCA-38. Doses of the active derivatives that failed to induce cytokines also exhibited no therapeutic synergy with rhIL-2. These results suggest that at least some of the antitumor effects of these XAA derivatives are related to their ability to induce cytokines.

Reversal of enzymic phenotype of thymidine metabolism in induced differentiation of HL-60 cells.

Exposure of HL-60 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in specific alterations in thymidine (TdR) metabolism. Within 12 h after treatment with 1.62 nM TPA, the reciprocal alteration in the activities of opposing pathways of TdR metabolism observed during normal culture cell growth was reversed. In TPA-treated cells, the activities of anabolic enzymes, TdR kinase (TK; EC 2.7.1.21) and thymidylate synthase (TS; EC 2.1.1.45), declined to 15% and 18% of those of untreated cells by 96 h. Incorporation of 3H-TdR and 3H-deoxyuridine also decreased in parallel with decline in enzyme activities. In contrast, the activities of catabolic enzymes, TdR phosphorylase (TP; EC 2.4.2.4) and dihydrothymine dehydrogenase (DHT DH; EC 1.3.1.2), increased to 399% and 318% by 96 h. Immunotitration of DHT DH with monoclonal antibody showed that the rise in activity in the differentiated cells was due to the increase in protein amount. Kinetic properties of the enzymes were not altered during differentiation. These metabolic alterations were accompanied by an accumulation of the cells in G1 at the expense of S-phase. Present data indicate that induced differentiation of HL-60 cells results in a reversal of enzymic phenotype of TdR metabolism due to a consequence of decreased proliferation and suggest that emergence of TdR metabolic imbalance may serve as early markers of differentiation of these cells.

Suppression of natural killer activity in patients treated with cisplatin and methylprednisolone.

The achievable concentrations of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl-3-nitrosourea (ACNU), adriamycin, mitomycin C, vindesine, and epipodophyllotoxin suppressed the natural killer (NK) activity of human peripheral blood lymphocytes in vitro, whereas NK activity was not decreased 24 h after incubation with the maximum achievable concentration of cisplatin (cDDP). NK activity of patients treated with 80 mg per square meter of cDDP alone was not decreased for 3 weeks after cDDP administration. In contrast, NK activity of patients treated with the same dose of cDDP and methylprednisolone was significantly decreased 1 week after cDDP administration. It was concluded that methylprednisolone, used as an antiemetic agent, had an immunosuppressive effect.

Pyoderma gangrenosum complicating ulcerative colitis: Successful treatment with methylprednisolone pulse therapy and cyclosporine.

A 32-year-old woman with ulcerative colitis had a relapsed of pyoderma gangrenosum during puerperium. Both the pyoderma gangrenosum and ulcerative colitis had been well controlled with oral prednisolone, but ulcerative colitis relapsed in pregnancy, and pyoderma gangrenosum relapsed in the puerperium. The pyoderma gangrenosum responded to methylprednisolone pulse therapy initially, but relapsed when prednisolone was tapered. A second trial of pulse therapy combined with cyclosporine resulted in complete remission of the pyoderma gangrenosum, and no recurrence was recognized after prednisolone was tapered. This is a very rare case of successful treatment with methylprednisolone pulse therapy combined with cyclosporine for pyoderma gangrenosum complicating ulcerative colitis.

Adenoma of the common human bile duct in Gardner's syndrome may cause relapsing acute pancreatitis.

Familial adenomatous polyposis of the colon, or Gardner's syndrome, is often accompanied by adenomas of the stomach and duodenum. We experienced a rare case of Gardner's syndrome, with adenomas of the common bile duct, in a patient who presented with relapsing acute pancreatitis. Our findings indicate that adenoma in the common bile duct or pancreatic duct should be considered as a possible etiology when patients with familial polyposis or Gardner's syndrome present with pancreatitis, particularly relapsing acute pancreatitis.

Relation between interleukin-1beta messenger RNA in gastric fundic mucosa and gastric juice pH in patients infected with Helicobacter pylori.

The effects of Helicobacter pylori infection on gastric acid secretion has not been clarified. The aim of this study was to elucidate the effects of H. pylori infection on gastric juice pH in relation to gene expression of interleukin-1beta (IL-1beta), which is reported to inhibit gastric acid secretion. Gastric juice pH and serum gastrin levels were measured in patients with peptic ulcer disease. The amount of IL-1beta mRNA in gastric fundic gland mucosa was also measured by a competitive reverse transcription-polymerase chain reaction method. These parameters were determined before and after treatment with lansoprazole and amoxicillin. Before treatment a significant positive relation was observed between the amount of IL-1beta mRNA in gastric fundic gland mucosa and gastric juice pH. After treatment significant decreases in the amount of IL-1beta mRNA, gastric juice pH, and serum gastrin levels were observed in patients with eradication of H. pylori, whereas no significant changes were observed in patients without eradication. These results suggest that H. pylori infection induces IL-1beta and suppresses acid secretion, resulting in increases in gastric juice pH and serum gastrin levels. Eradication of H. pylori decreases IL-1beta induction, resulting in an increase in gastric juice acidity and normalization of serum gastrin levels.

A case of AFP-producing early gastric carcinoma with rapid growth liver metastasis.

An 84-year-old man presented with complaints of epigastric discomfort. Upper gastrointestinal series and endoscopy showed an elevated lesion at the posterior wall of greater curvature on the gastric fundus. Diagnosed as moderately differentiated tubular adenocarcinoma by biopsy, wedge resection and 4sa regional lymph node dissection were carried out. The tumor morphology showed type I with slight elevation, 2.5 x 1.7 cm in size; histological showed papillary, tubular, and solid formations having clear cytoplasm and large bizarre nuclei invading the deep submucosal layer (sm2). This case was evaluated as T1(sm) N0 M0 stage Ia early gastric cancer. In the 5th month after operation, multiple liver metastases were detected. He died of liver failure by rapid growth of metastatic tumors in the 6th month after operation. The serum alpha-fetoprotein level at recurrence was 1,900 ng/mL, and alpha-fetoprotein-positive cells were immunohistochemically detected in operative and liver biopsy specimens.

[Peripheral neuropathy caused by cisplatin in patients with lung cancer].

The correlation between peripheral neuropathy and cisplatin (CDDP) was elucidated in 27 patients with primary and metastatic lung cancer, who were treated with Adriamycin 30 mg/m2 day 1, CDDP 80 mg/m2 day 1, and VP-16 70 mg/m2 day 1-5 every 4 weeks. The incidence of peripheral neuropathy was 33% (9 of 27 patients) and it increased to 60% in the patients who received over 320 mg/m2 of CDDP, demonstrating a positive correlation between the incidence of this toxicity and the total dose of CDDP. However, no significant relation was observed between the grade of neuropathy and CDDP. The neuropathy was manifested in the sensory system of the distal extremities and was developed into proximal portions. The peripheral neuropathy with grade 3 was irreversible, resulting in the dose-limiting factor of this regimen.

[A case of second-degree atrioventricular block caused by high dose cisplatin treatment].

A 56-year-old man was admitted to our hospital because of stage IV squamous cell carcinoma of the lung. He was treated with two courses of high dose cisplatin (40 mg/m2/day, day 1-5). On day 4 of each course, a second-degree atrioventricular block was observed, which disappeared after cisplatin treatment. This case suggests that, although the cardiotoxicity caused by cisplatin is believed to be rare, one should take special care in high dose usage.

[Adriamycin, cisplatin and etoposide combination chemotherapy in non-small cell lung cancer].

Nineteen patients with non-small cell lung cancer (eight patients with adenocarcinoma, nine patients with squamous cell carcinoma, one patient with large cell carcinoma and one patient with sarcoma) who had not received previous chemotherapy were treated with a combination of adriamycin (30 mg/m2, i.v., on day 1), cisplatin (80 mg/m2, i.v., on day 1) and etoposide (70 mg/m2, i.v., on day 1-5). This chemotherapy regimen was repeated as long as possible for patients in whom PR was induced. Among all patients, CR was induced in none and 6 showed a PR (response rate 32%). However, 4 (56%) squamous cell carcinoma patients also showed PR. The median response duration in 6 PR patients was 28 weeks, and the median survival time in all patients was also 28 weeks. Mild to severe hematologic toxicities were induced and one patient died during myelosuppression. However almost all cases were reversible. Other toxicities included alopecia, nausea/vomiting, diarrhea, stomatitis, peripheral neuropathy and myocardial infarction which were reversible and manageable. The APVp therapy may be a valuable regimen for non-small cell lung cancer, especially squamous cell carcinoma.

[Fundamental study and clinical testing of human tumor clonogenic assay (HTCA)].

Through our retrospective analysis of HTCA and clinical effects as well as fundamental studies, the following results were obtained: In this retrospective study, HTCA for lung cancer showed a predictive accuracy of 71%, a true positive rate of 50% and a true negative rate of 77%. To obtain good predictive accuracy, HTCA should be modified to provide conditions comparable to those in vivo with regard to drug concentration and drug exposure time. More precise analysis of the pharmacokinetics of anticancer agents might yield methodological improvement. A decrease in the chemosensitivity spectrum in vitro was observed after chemotherapy. This might be related to evidence that patients with prior chemotherapy exhibited a poor response rate to chemotherapy. There were no active anticancer agents against specimens with aplating efficiency of more than 0.04%. More extensive prospective trials will be necessary to determine the clinical value of HTCA. HTCA could be a superior assay for detecting the anti-tumor activity of new agents and a useful method for in vitro phase II study.

[Adriamycin, cisplatin, and etoposide combination chemotherapy for small cell lung cancer].

Twenty-three patients with small cell lung cancer (11 with limited disease and 12 with extensive disease) who had not received previous chemotherapy were treated with a combination of adriamycin (30 mg/m2, i.v., on day 1), cisplatin (80 mg/m2, i.v., on day 1) and etoposide (70 mg/m2, i.v., on day 1-5). This chemotherapy regimen was repeated at 3- or 4-week intervals for 3 to 5 treatment cycles. Among 22 evaluable patients, 5 showed complete response and 17 had a partial response (response rate 100%). The median response duration of 12 extensive disease patients was 21 months. There were 5 survivors for more than 2 years. Toxicity included moderate to severe hematologic toxicity, alopecia, nausea and vomiting. This combination chemotherapy appears to be optimal for the treatment of small cell lung cancer.

[Randomized control study of high-dose metoclopramide in the prevention of CDDP-induced emesis].

The effect of high-dose metoclopramide (2 mg/kg, 4 times every 2 hours) on the emesis of patients treated with CDDP (80 mg/m2) was examined by randomized control trial. The above metoclopramide regimen significantly suppressed the frequency of vomiting on the day of CDDP administration. The duration of nausea and anorexia after CDDP treatment was also shortened by high-dose metoclopramide administration.