[A multicenter epidemiological study of acute bacterial meningitis in children].

Objective: To analyze the clinical epidemiological characteristics including composition of pathogens , clinical characteristics, and disease prognosis acute bacterial meningitis (ABM) in Chinese children. Methods: A retrospective analysis was performed on the clinical and laboratory data of 1 610 children <15 years of age with ABM in 33 tertiary hospitals in China from January 2019 to December 2020. Patients were divided into different groups according to age,<28 days group, 28 days to <3 months group, 3 months to <1 year group, 1-<5 years of age group, 5-<15 years of age group; etiology confirmed group and clinically diagnosed group according to etiology diagnosis. Non-numeric variables were analyzed with the Chi-square test or Fisher's exact test, while non-normal distrituction numeric variables were compared with nonparametric test. Results: Among 1 610 children with ABM, 955 were male and 650 were female (5 cases were not provided with gender information), and the age of onset was 1.5 (0.5, 5.5) months. There were 588 cases age from <28 days, 462 cases age from 28 days to <3 months, 302 cases age from 3 months to <1 year of age group, 156 cases in the 1-<5 years of age and 101 cases in the 5-<15 years of age. The detection rates were 38.8% (95/245) and 31.5% (70/222) of Escherichia coli and 27.8% (68/245) and 35.1% (78/222) of Streptococcus agalactiae in infants younger than 28 days of age and 28 days to 3 months of age; the detection rates of Streptococcus pneumonia, Escherichia coli, and Streptococcus agalactiae were 34.3% (61/178), 14.0% (25/178) and 13.5% (24/178) in the 3 months of age to <1 year of age group; the dominant pathogens were Streptococcus pneumoniae and the detection rate were 67.9% (74/109) and 44.4% (16/36) in the 1-<5 years of age and 5-<15 years of age . There were 9.7% (19/195) strains of Escherichia coli producing ultra-broad-spectrum ß-lactamases. The positive rates of cerebrospinal fluid (CSF) culture and blood culture were 32.2% (515/1 598) and 25.0% (400/1 598), while 38.2% (126/330)and 25.3% (21/83) in CSF metagenomics next generation sequencing and Streptococcus pneumoniae antigen detection. There were 4.3% (32/790) cases of which CSF white blood cell counts were normal in etiology confirmed group. Among 1 610 children with ABM, main intracranial imaging complications were subdural effusion and (or) empyema in 349 cases (21.7%), hydrocephalus in 233 cases (14.5%), brain abscess in 178 cases (11.1%), and other cerebrovascular diseases, including encephalomalacia, cerebral infarction, and encephalatrophy, in 174 cases (10.8%). Among the 166 cases (10.3%) with unfavorable outcome, 32 cases (2.0%) died among whom 24 cases died before 1 year of age, and 37 cases (2.3%) had recurrence among whom 25 cases had recurrence within 3 weeks. The incidences of subdural effusion and (or) empyema, brain abscess and ependymitis in the etiology confirmed group were significantly higher than those in the clinically diagnosed group (26.2% (207/790) vs. 17.3% (142/820), 13.0% (103/790) vs. 9.1% (75/820), 4.6% (36/790) vs. 2.7% (22/820), χ2=18.71, 6.20, 4.07, all P<0.05), but there was no significant difference in the unfavorable outcomes, mortility, and recurrence between these 2 groups (all P>0.05). Conclusions: The onset age of ABM in children is usually within 1 year of age, especially <3 months. The common pathogens in infants <3 months of age are Escherichia coli and Streptococcus agalactiae, and the dominant pathogen in infant ≥3 months is Streptococcus pneumoniae. Subdural effusion and (or) empyema and hydrocephalus are common complications. ABM should not be excluded even if CSF white blood cell counts is within normal range. Standardized bacteriological examination should be paid more attention to increase the pathogenic detection rate. Non-culture CSF detection methods may facilitate the pathogenic diagnosis.

[On-pump total arterial revascularization in coronary artery disease patients with left ventricular dysfunction: a multi-center retrospective study].

Objective: To evaluate the clinical outcomes of on-pump total arterial revascularization with bilateral radial artery (BRA) and left internal mammary artery (LIMA) as conduits in coronary artery bypass grafting (CABG) patients with left ventricular dysfunction (LVD). Methods: All the perioperative medical records and follow-up results of coronary artery disease patients with left ventricular ejection fraction (LVEF) ≤ 40% undergoing CABG from 24 heart centers of 15 provinces and autonomous regions in China between July 2015 and December 2019 were retrospectively analyzed. Results: A total of 87 consecutive patients (55 males and 32 females) underwent on-pump CABG with BRA and LIMA, with a mean age of (57.5±9.1) years old. There were 22 patients complicated with primary hypertension, 12 with diabetes mellitus, 8 with peripheral vascular disease, 7 with chronic obstructive lung disease, 12 with mild renal injury and 3 with partial aortic calcification. There were 43 cases with in-stent stenosis, and 21 had left main disease. The mean LVEF and left ventricular end-diastolic diameter (LVEDD) was (35.5±7.3)% and (65.5±2.6) mm, respectively. The mean graft number, aortic cross-clamp time and cardiopulmonary bypass duration was 3.2±0.9, (90.5±22.7) min and (113.4±19.2) min, respectively. There were 32 mitral and 9 aortic valve replacements, and 5 tricuspid annuloplasties. Prophylactic intra-aortic balloon pumps were implanted in 27 patients. There were 2 operative deaths from acute heart failure. After surgery, there were 15 cases of atrial fibrillation, 1 case of acute kidney injury, 1 case of acute myocardial infarction, and 1 cases of stroke. All the patients fulfilled the follow-up, with a mean time of (39.5±7.7) months. At 3 months after surgery, LVEDD was decreased and LVEF was improved significantly compared with pre-operative indicators [(53.0±1.5) mm vs (65.5±2.6) mm, t=9.51 P=0.02; (45.2±3.3)% vs (35.5±7.3)%, t=13.79, P=0.001]. No major cardiac events were reported during the follow-up. At (30.5±7.4) months after surgery, 62.4% of patients (53/85) underwent coronary CT angiography examination, and the results indicated that the graft patency was 98.8%, with only one case of RA occlusion occurred. Conclusion: In selected patients of LVD, on-pump total arterial revascularization with BRA and LIMA conduits was proved to be safe and effective.

[Mid-term multi-center outcomes of bilateral radial artery as conduits in coronary artery bypass grafting].

Objective: To evaluate the mid-term outcomes of bilateral radial artery (BRA) grafts in coronary artery bypass grafting (CABG). Methods: All perioperative medical records and follow-up results of CABG with BRA grafts in multi-centers of China were analyzed retrospectively. Results: A total of 211 patients (170 males and 41 females) underwent CABG grafting with BRA conduits between August 2013 and September 2018, with a mean age of (56.5±9.7) years old (rang 41 to 73 years). There were 161 cases of triple-vessel disease and 50 cases of two-vessel disease. Ninety patients had diabetes mellitus (DM), 35 patients with peripheral vascular disease, 4 patients with chronic obstructive pulmonary disease and 11 with heart valve disease. Two patients underwent off-pump CABG and 209 patients accepted on-pump CABG with commitment valve surgery. There were 210 cases of total arterial revascularization and 161 cases using left thoracic artery conduits, with a graft number of 2-4 (2.7±0.9). No operation-related death occurred, atrial fibrillation happened in 12 patients, hemothorax in 7 cases, and forearm hematoma in one case, hypoxemia in 13 cases and pneumonia in one case. The duration of mechanical ventilation was (8.3±4.7) hours and the mean hospital length of stay was (7.1±2.9) days. Follow-up was completed in 191 patients (90.52%) with a duration of 3-59 (35.5±9.3) months. The mean left ventricular ejection fraction at 3 months after operation was significantly improved, compared to that of the pre-operation (61.0%±7.2% vs 47.1%±5.3%, P=0.017). All patients survived, except that one died from brain injury. No major cardiac events occurred, with a cumulative survival rate of 100% at 1 year and 99.53% at 3 year after operation, respectively. It was showed in coronary CT angiography (CTA) examination that all grafts in 132 patients were patent at the mean follow-up duration of (21.5±6.4) months. Conclusions: BRA grafts as arterial conduit in CABG are proved to be safe, easy for total arterial revascularization and have good mid-term clinical results.

Increased expression of hypoxia-inducible factor-1α and metallothionein in varicocele and varicose veins.

OBJECTIVE: The increased blood stasis and venous volume pressure causing tissue hypoxia are observed in both varicocele and varicose veins. Metallothionein (MT), a metal-binding protein, protects against cell apoptosis under hypoxic stress. It also plays an important role in collateral flow recovery and angiogenesis. We studied the distribution of hypoxia-inducible factor-1α (HIF-1α) and MT in varicocele and varicose veins. METHODS: The study specimens consisted of 1 cm venous segments that were obtained from 12 male patients during vascular stripping surgery for varicose veins and 1 cm of internal spermatic vein (ISV) obtained from 12 patients during left varicocele repair. The control samples of 1 cm ISV were obtained from 10 male patients who underwent left inguinal herniorrhaphy. All vascular specimens were analysed for HIF-1α and MT expression by immunoblotting, immunohistochemical (IHC) staining and confocal microscopy. Data were analysed using one-way analysis of variance with Tukey's comparison test. RESULTS: In both venous diseases, the increased expression of HIF-1α and MT compared with the control group (P < 0.05) and most of the proteins distributed over smooth muscle layers were detected by IHC staining; HIF-1α and MT in the muscle layer with co-localization, and MT overexpression especially located in the endothelium of both venous diseases under confocal microscopy. CONCLUSIONS: Our results revealed the higher expression of HIF-1α and MT in varicocele and varicose veins than in the control group; MT overexpression in the muscle layer of both diseased vessels and especially located in the endothelium under confocal microscopy. MT has the function to protect vascular cells from apoptosis under hypoxia. Thus, this MT function may cause a decreased vascular cell apoptosis and then contribute to the dilated and thickened walls of varicocele and varicose veins.

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.

Mycobacterium avium complex-associated hemophagocytic syndrome in systemic lupus erythematosus patient: report of one case.

Hemophagocytic syndrome (HPS) in systemic lupus erythematosus(SLE) patients has not commonly been reported. In this case study, we report the first case of Mycobacterium avium complex (MAC)-associated hemophagocytic syndrome in a patient with systemic lupus erythematosus (SLE). This SLE patient, a 15-year-old girl, had been on a high dose of prednisolone (> 0.5mg/kg/day) for more than 3 years. She presented with a spiking fever, hepatosplenomegaly, pancytopenia, hyperferritinemia and adult respiratory distress syndrome. Bone marrow examination revealed hemophagocytosis as well as non-caseating granulomatosis. There was no indication of SLE fare-up. She responded poorly to initial treatment with methyl-prednisolone, intravenous immumoglobulin, etoposide, and drugs for Mycobacterium tuberculosis including rifampin, ethambutol, isoniazid and pyramide. However, gastric lavage culture revealed MAC. Following treatment with clarithromycin, ciprofloxacin and amikacin, her condition gradually improved and she was discharged 3 months after admission. In SLE patients with pancytopenia and hyperferritinemia, MAC-associated HPS should be considered in the differential diagnosis.

Efficacy of Re-188-labelled sulphur colloid on prolongation of survival time in melanoma-bearing animals.

UNLABELLED: In this study, the effectiveness of a 188Re labeled sulfur colloid with two particle size ranges was used to evaluate the effectiveness of this agent on melanoma tumors in mice in terms of animal lifespan. METHODS: Two separate group of animals were used for investigating biodistribution and survival time. A total of 188 B16F10-melanoma-bearing BDF(1) mice were injected intraperitoneally with 3.7 MBq (0.1mCi)/2mL of radiolabeled sulfur colloid ten days after intraperitoneal inoculation of 5x10(5) B16F10 melanoma cells/2ml. For group 1, 30 mice were sacrificed at 1, 4, 24, 48 and 72 hours for biodistribution studies. In group 2, 158 mice were divided into 9 groups (n=16 approximately 18/groups)each receiving respectively tumor alone, tumor with normal saline, cold colloid or hot colloid with 16, 23, 31, 46, 62, or 124 MBq activity. Each of these colloid groups was further divided into two groups, one receiving smaller particle sizes (<3 microm:80.4 +/-7.2%, colloid 1) and the other receiving larger particle sizes (<3 microm:12.3+/-1.0%, colloid 2). The animals were checked daily until death and their survival recorded. RESULTS: Colloid 2 showed higher accumulation in almost all tissues, the highest accumulation organ was tumor ( approximately 40%), then spleen ( approximately 20%), stomach ( approximately 15%), diaphragm ( approximately 3%), and liver ( approximately 2%). There was a significant increase in survival time with increasing amount of the larger-particle-size colloid. Administered levels of 16-31 MBq/mouse were most efficacious and with higher amounts the survival times decreased significantly below that of the controls. There was a significant difference in the dose-response curves for the two preparations. Protection factors (1/Relative-risk) of nearly 5 were achieved using the larger colloid size, and nearly 30 using the smaller colloid size. An amount of 16-31 MBq of the colloid 2 was the optimal activity in these studies. On the one hand, the survival data agreed well with the biodistribution data, where higher accumulation was found in tumor with colloid 2. CONCLUSION: Rhenium-188 offers on-site availability, medium half-life, higher beta-particle energy of 2.12 MeV for therapy and emission of 155keV gamma photon suitable for imaging. The present study demonstrated that 188Re-sulfur colloid is an effective agent in controlling tumor cells in the abdominal cavity in animals.

Tumor invasiveness and liver metastasis of colon cancer cells correlated with cyclooxygenase-2 (COX-2) expression and inhibited by a COX-2-selective inhibitor, etodolac.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to reduce the risk and mortality of colorectal cancer (CRC). Although the exact mechanisms remain unclear, the inhibition of cyclooxygenase (COX) by NSAIDs appears to abort, if not prevent, CRC carcinogenesis or metastatic tumor progression. The aim of our study was to investigate the association between COX-2 expression and CRC tumor cell invasiveness. The differences in immunoblot-detectable COX-2 protein contents in primary CRCs, metastatic hepatic lesions and corresponding normal mucosa from the same individual were evaluated in 17 patients. Three different colon cancer cell lines, SW620, Lovo, HT-29 and a metastatic variant of HT-29, HT-29/Inv3, were employed to evaluate COX-2 expression and prostaglandin E(2) (PGE2) production in relation to their invasive abilities in vitro. The effects of a COX-2-selective inhibitor, etodolac, on cell proliferation and invasive activity were also determined. The results showed that 15 of 17 (88%) metastatic CRC cells from the liver and 14 of 17 (82%) primary CRC tissue exhibited much higher levels of COX-2 than corresponding adjacent normal mucosa from the same patient. Among those patients with relatively high COX-2 expression in the primary tumors, almost all exhibited even higher levels of COX-2 in their hepatic metastases. Among the 4 colon cancer cell lines, HT-29/Inv3 manifested the highest COX-2 expression, PGE2 production and in vitro invasive activity. The selective COX-2 inhibitor, etodolac, could especially exert cytotoxicity and markedly suppress the invasive property and PGE(2) production, although not the COX-2 protein level, in HT-29/Inv3 cells. Our results imply that COX-2 expression may be associated with the invasive and metastatic properties of CRC tumor cells.

An analysis of cytokine status in the serum and effusions of patients with tuberculous and lung cancer.

The present study was designed to ascertain whether or not the pleural effusion and serum cytokine levels (granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-10 [IL-10], and interferon-gamma [IFN gamma]) in lung cancer patients differ from tuberculous (TB) pleural effusion, in which a strong cellular immune reaction is found; and, whether cytokine levels are a prognostic factor in lung cancer patients with malignant effusion. A total of 202 lung cancer patients with malignant pleural effusion and 26 patients with TB pleural effusion were studied consecutively between 1995 and 1998. Serum and effusion cytokine levels were analyzed with ELISA assays. The results showed that pleural effusion GM-CSF and IL-10 levels were significantly higher than serum levels in both cancer and TB patients. Pleural effusion IFN gamma levels were significantly higher than serum levels in TB patients. IFN gamma levels in both pleural effusion and serum were significantly higher in TB patients than in those with cancer. No significant difference was found, between TB and cancer patients, in the serum or pleural effusion levels of either IL-10 or GM-CSF. The ratio of pleural effusion IFN gamma to serum IFN gamma, effusion IFN gamma to effusion IL-10, and effusion IL-10 to serum IL-10, were all significantly higher in TB than in cancer patients, suggesting a higher cellular activity and T-helper 1 (Th1) reaction in TB pleural effusion than in malignant effusions, which were predominantly Th2 type. Survival analysis showed no significant difference in lung cancer patients with different levels of these cytokines. It was concluded that lung cancer patients with malignant pleural effusion had poorer immune profiles than those with TB pleurisy, both locally and systemically; and the cytokine profiles were not prognostic factors for lung cancer patients with malignant pleural effusion.

PIK3CA as an oncogene in cervical cancer.

Amplification of chromosome arm 3q is the most consistent aberration in cervical cancer, and is implicated in the progression of dysplastic uterine cervical cells into invasive cancer. The present study employed the 'positional candidate gene' strategy to determine the contribution of PIK3CA, which is located in 3q26.3, in cervical tumorigenesis. PIK3CA is known to be involved in the PI 3-kinase/AKT signaling pathway, which plays an important role in regulating cell growth and apoptosis. The results of comparative genomic hybridization show that the 3q26.3 amplification was the most consistent chromosomal aberration in primary tissues of cervical carcinoma, and a positive correlation between an increased copy number of PIK3CA (detected by competitive PCR) and 3q26.3 amplification was found in tumor tissues and in cervical cancer cell lines. In cervical cancer cell lines harboring amplified PIK3CA, the expression of gene product (p110alpha) of PIK3CA was increased, and was subsequently associated with high kinase activity. In addition, transformation phenotypes in these lines, including increased cell growth and decreased apoptosis, were found to be significantly affected by the treatment of specific PI 3-kinase inhibitor, suggesting that increased expression of PIK3CA in cervical cancer may result in promoting cell proliferation and reducing apoptosis. These evidences support that PIK3CA is an oncogene in cervical cancer and PIK3CA amplification may be linked to cervical tumorigenesis. Oncogene (2000).

Restoration of cytotoxic T lymphocyte function in malignant pleural effusion: interleukin-15 vs. interleukin-2.

The present study attempts to define the role of interleukin-15 (IL-15), as compared with IL-2, in generating cytotoxic T lymphocytes (CTL) from the malignant effusions of cancer patients. Effusion-associated lymphocytes (EAL) from malignant effusion were incubated with IL-15 or IL-2 with or without alphaCD3. Proliferation and cytotoxicity assays were performed. IL-15 was found to have at least an equivalent, if not higher, activity to IL-2 in terms of lymphocyte proliferation and generation of CTL from EAL. The proliferative response of EAL, cocultured with IL-15, with or without alphaCD3, was partly inhibited by pretreatment with an anti-IL2 receptor beta chain monoclonal antibody (mAb). The proliferative response of EAL, cocultured with alphaCD3, IL-2, or both, was partly inhibited by pretreatment with an anti-IL-2 receptor alpha chain mAb. Overnight [5lCr] release assays against K562, Daudi, and the patients' autologous tumor cells were done to evaluate EAL's cytolytic activity. MHC class I Ab blocked the stimulated cytolytic activity of EAL against autologous tumors. An mAb depletion assay showed that the phenotype of the restored EAL was CD16-CD4-CD8+; thus, the restored activity of EAL was CTL activity. The results suggest that both IL-15 and IL-2 can restore CTL activity from EAL in the presence of T cell receptor (TCR)-CD3 engagement, but the effect of IL-15 was superior.

Gene therapy with tumor vaccine increases the survival of hepatoma-bearing mice.

BACKGROUND: Tumor vaccines combined with cytokine gene therapy and Bacillus Calmette-Guérin (BCG) were tested for prevention and therapeutic effects in the H6 mouse hepatoma model. METHODS: Plasmid DNA of expression vectors carrying cDNA of mouse interleukin (IL)-2 and mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) were used for transfection to obtain H6 mouse hepatoma cells that secreted IL-2 (H6/IL-2) or GM-CSF (H6/GM-CSF). For tumor prevention, groups of mice were immunized twice with irradiated tumor cells with untransduced H6, H6/IL-2, H6/GM-CSF, or an equal mixture of H6/IL-2 and H6/GM-CSF. Three weeks later, these mice were inoculated subcutaneously with live H6 hepatoma cells, and tumor growth was measured. For therapeutic studies, mice first inoculated with live H6 cells were treated three days later with various irradiated tumor cell vaccines alone or in combination with BCG. Subsequent tumor growth was measured. RESULTS: In tumor prevention studies, significant protection from tumor growth has been observed in animals vaccinated with irradiated cytokine-secreting H6 cells compared with those immunized with irradiated parental H6 cells. In tumor therapy studies, subsequent administration of irradiated H6/GM-CSF cells in combination with BCG impeded the tumorigenicity of preinoculated live H6 hepatoma cells. CONCLUSIONS: These results suggest that cytokine-secreting tumor vaccines have a prophylactic effect and BCG, in combination with irradiated H6/GM-CSF cells, shows a synergistic effect on delaying the growth of H6 mouse hepatomas.

Comparative tyrosine-kinase profiles in colorectal cancers: enhanced arg expression in carcinoma as compared with adenoma and normal mucosa.

There is strong evidence that tyrosine kinases are involved in the regulation of cellular growth and tumor progression. Over-expressions of tyrosine kinases have been documented in a number of neoplasms. To study the roles of tyrosine kinases in colon cancer, we developed a tyrosine-kinase-expression profile for each of the four different stages of colon carcinogenesis, using normal colon mucosa, adenomatous polyps, primary carcinoma and hepatic metastases collected from the same patient. We identified 30 tyrosine kinases expressed in these tissues: they include 10 non-receptor tyrosine kinases (yes, fyn, lyn, brk, abl, arg, jak1, jak3, tyk2 and itk), 17 receptor tyrosine kinases (erbB2, PDGF-Ralpha, PDGF-Rbeta, kit, c-fms, met, ron, FGF-R1, FGF-R2, FGF-R3, FGF-R4, cek5, tie-1, tkt, axl, sky and Ins-R), 2 dual kinases (mek and sek) and one possible novel kinase. Among these kinases, arg kinase appears to be expressed at a higher level in primary carcinoma and metastatic tumor than in adjacent normal mucosa or adenomatous polyp. This result was confirmed by extensive analysis of 50 additional matched sets of normal colon and colon-tumor specimens, using arg-specific primers and RT-PCR reactions. This study identifies a possible role for arg tyrosine kinase in colon carcinogenesis, especially in the transition from adenoma to carcinoma.

Involvement of Fas (CD95/APO-1) and Fas ligand in apoptosis induced by ganciclovir treatment of tumor cells transduced with herpes simplex virus thymidine kinase.

Transduction of cancer cells with herpes simplex virus thymidine kinase gene (HSVtk) followed by prodrug ganciclovir (GCV) treatment has been shown to induce apoptosis. In this study, four murine tumors including B16F10 melanoma, NG4TL4 sarcoma, H6 hepatoma and 1MEA 7R.1 hepatoma were found to vary in sensitivity to this gene therapy strategy in vitro but, at effective doses of GCV, the HSVtk-transduced cells of all four tumors showed similar kinetics of early rise in p53 protein levels, then cell cycle S-/G2-phase arrest and finally signs of apoptosis. Immunoblot analyses revealed that Fas (CD95/APO-1), Fas ligand (FasL) and two downstream mediators, RIP and caspase-3, (CPP32, YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tumor cells the cell cycle arrest and before apoptosis. Increased expression of FasL could also be observed in vivo in HSVtk-transduced tumors induced to regress by GCV treatment. Enzyme measurements using specific substrate showed that the caspase-3 activation followed kinetically the FasL expression. More than half of the HSVtk/GCV-induced cell death could be abrogated by addition to the cell culture medium of a specific antisense oligonucleotide to block FasL synthesis, a recombinant Fas/Fc chimeric protein to compete with Fas receptor for FasL binding, or cell-permeable specific tetrapeptide inhibitors of caspase-3 or caspase-8.

Acute fatal poisoning cases due to furathiocarb ingestion.

Seven cases involving acute fatalities due to ingestion of furathiocarb, a carbamate insecticide, are presented. Furathiocarb was detected in the gastric contents using thin layer chromatography (TLC) and gas chromatography/mass spectrophotometry (GC/MS), and quantified in the blood using a gas chromatograph equipped with a nitrogen-phosphorus detector (NPD). The fatal levels of furathiocarb in the blood ranged from 0.1 to 21.6 micrograms/ml.

Depressed cytolytic activity of peripheral blood mononuclear cells in unusually high paclitaxel concentrations: reversal by IL-2 and IL-12.

BACKGROUND: Human lymphocyte function was inhibited by high concentrations of paclitaxel and the effect was reversed by interleukin (IL)-2. However, there was no parallel study determining the relationship between paclitaxel concentrations in the lymphocyte cultures and pharmacokinetic analysis in human patients, nor was there any study on the reversal by cytokines, other than IL-2, of the paclitaxel-induced suppression of lymphocyte cytotoxicity. METHODS: We tested the effect of different doses of paclitaxel with various incubation times on the cytolytic activity of peripheral blood mononuclear cells (PBMNCs) against K-562 target cells. RESULTS: Our results showed that using a schedule similar to that for treating patients with tolerable doses of paclitaxel, no inhibition of cytolytic activity of PBMNCs was seen. When the paclitaxel concentration was increased 10-fold, the cytolytic activity of PBMNCs was significantly reduced. This suppression was reversed by the simultaneous addition of a low dose (10 U/ml) of IL-2 or IL-12. Addition of granulocyte macrophage-colony stimulating factor (10 U/ml) did not affect the cytolytic activity of PBMNCs, whereas addition of IL-4 reduced it. Time kinetic studies revealed that, with the addition of IL-2 or IL-12, most of the mononuclear cellular cytolytic activity recovered within 48 to 72 hours. CONCLUSIONS: These findings suggested that, to reduce the toxicity on mononuclear cellular function when high-dose paclitaxel treatment is elected in clinical practice, paclitaxel should be infused over a longer duration of time, or the treatment should be combined with the administration of a low dose of IL-2 or IL-12.

Establishment and characterization of a strial marginal cell line maintaining vectorial electrolyte transport.

E6/E7 genes of human papilloma virus type 16 were used to immortalize a primary culture of marginal cells (MC) from gerbils. One of the cloned lines was selected which demonstrated preservation of the main characteristics of the MC, both morphologically and physiologically. Electron microscopic examination showed well-developed junctional complexes and apical microvilli which suggested its epithelial origin. Polymerase chain reaction (PCR) demonstrated the incorporation of E6/E7 genes with the genome. Reverse transcription PCR revealed the existence of mRNA of the IsK channel, a unique marker of MC among the inner ear cells, in this clone. Flow cytometric analysis of this cell line's DNA content was diploid. Numerous large domes formed after confluence of the cell monolayer. Electrophysiologic studies displayed evidence of apical K+ and Na+ channels which were blocked by Ba2+ (2 mM) and amiloride (10(-5) M), respectively. Existence of basolateral Na,K-ATPase and Na+/Cl-/K+ cotransporter was shown by blockage by ouabain (10(-3) M) and bumetanide (50 microM), individually. Injection of the cell line to nude mice failed to induce growth of tumors. This cell line was serum-, density- and anchorage-dependent when cultured in plastic dishes. In conclusion, this cell line shows characteristics of well-differentiated MC maintaining the major ionic transport processes, and provides us a good model to study the possible mechanisms and regulating factors of endolymph production.

Protein-tyrosine kinase and protein-serine/threonine kinase expression in human gastric cancer cell lines.

Protein kinases play key roles in cellular functions. They are involved in many cellular functions including; signal transduction, cell cycle regulation, cell division, and cell differentiation. Alterations of protein kinase by gene amplification, mutation or viral factors often induce tumor formation and tumor progression toward malignancy. The identification and cloning of kinase genes can provide a better understanding of the mechanisms of tumorigenesis as well as diagnostic tools for tumor staging. In this study, we have used degenerated polymerase-chain-reaction primers according to the consensus catalytic domain motifs to amplify protein kinase genes (protein-tyrosine kinase, PTK, and protein-serine/threonine kinase, PSK) from human stomach cancer cells. Following amplification, the protein kinase molecules expressed in the gastric cancer cells were cloned into plasmid vectors for cloning and sequencing. Sequence analysis of polymerase-chain-reaction products resulted in the identification of 25 protein kinases, including two novel ones. Expression of several relevant PTK/PSK genes in gastric cancer cells and tissues was further substantiated by RT-PCR using gene-specific primers. The identification of protein kinases expressed or activated in the gastric cancer cells provide the framework to understand the oncogenic process of stomach cancer.

S- and G2-phase cell cycle arrests and apoptosis induced by ganciclovir in murine melanoma cells transduced with herpes simplex virus thymidine kinase.

Mechanism of cell killing by transfer of Herpes simplex virus type-1 thymidine kinase (HSVtk) and subsequent ganciclovir (GCV) treatment was examined in B16F10 murine melanoma model. While parental B16F10 melanoma cells were resistant to GCV at 100 microM or higher, HSVtk-transduced B16F10 melanoma cell clones became susceptible to GCV with IC50 of 0.1 to 0.3 microM. By means of various parameters including characteristic morphological changes, in situ DNA end-labeling, DNA ladder pattern, flow cytometric detection of sub-G1 DNA content, and annexin V binding of inverted cell surface phosphatidylserine, apoptosis was shown to be associated with the cell killing of ganciclovir on HSVtk-transduced melanoma B16F10 cells. Kinetic analysis showed that the signs of apoptosis were observed not until 60 h of continued GCV treatment and preceded first by a rise in p53 protein level in 12 h and then by S-phase/G2-phase cell cycle arrest associated with corresponding increases in the level of cyclin B1 protein but no apparent change in protein level of Bax or Cdc2. These results suggest that apoptosis occurred as a result of ganciclovir-induced cell cycle arrests rather than direct chemical effect on HSVtk-transduced B16F10 melanoma cells.

Overexpression of cyclin A but not Skp 2 correlates with the tumor relapse of human hepatocellular carcinoma.

Cyclin A is an S- and G2-M-phase regulatory protein, and its abnormal expression has been implicated in cellular transformation. This work was undertaken to investigate the frequency of cyclin A overexpression and the correlated clinical outcome in human hepatocellular carcinoma (HCC). Herein, 12 of 31 (39%) patients exhibited cyclin A overexpression in their tumorous tissues, resulting from gene amplification in 6 of 12 patients, (post)transcription in 4 of 12 patients, and (post)translation in 2 of 12 patients. Patients who overexpressed cyclin A had significantly more tumor cells in the S and G2-M phases compared with those expressing a normal cyclin A level (P = 0.007 and 0.039, respectively). Increased levels of Skp 2, a cyclin A-interacting protein, were also found in 17 of 31 (55%) of HCC patients who showed a trend to have more S-phase tumor cells (P = 0.07). By an unpaired Student's t test and a Fisher's exact or chi2 analysis, overexpression of cyclin A had a strong correlation with elevated Skp 2 expression and increased alpha-fetoprotein levels (P = 0.001 and 0.009, respectively), but it was not associated with patients' age, tumor size, cirrhosis, or the positive detection of hepatitis B virus surface antigen. In the disease-free survival analysis, patients whose tumors overexpressed cyclin A had a median disease-free survival of 6 months, whereas patients who lacked cyclin A overexpression exhibited a longer median period of 29 months (P = 0.046). The overall survival analysis revealed the same trend, i.e., cyclin A-overexpressing patients had shorter overall survival periods (median, 12 versus 50 months; P = 0.09). By multivariate analysis, the correlation of cyclin A overexpression with shorter disease-free periods remained significant after adjustment for Skp 2 overexpression and alpha-fetoprotein induction (P = 0.019). These data suggest that overexpression of cyclin A can be an independent prognostic factor for the tumor relapse of human HCC.