Successful cessation of transmitting healthcare -- associated infections due to Burkholderia cepacia complex in a neonatal intensive care unit in a Japanese children's hospital.

BACKGROUND: Burkholderia cepacia strains have been known to possess the capability to cause serious infections especially in neonatal intensive care units (NICUs), and their multi-drug resistances become a severe threat in hospital settings. The aim of this investigation was to evaluate the B. cepacia complex infections in the NICU in Nagano Children's Hospital, Azumino 399-8288, Japan, and to report the intervention leading to the successful cessation of the outbreak. METHODOLOGY: The incidence of isolation and antimicrobial susceptibilities of nosocomial Burkholderia cepacia complex strains during a four-year period were retrospectively examined by clinical microbiological records, and by pulsed-field gel electrophoresis analyses along with the bacteriological verification of disinfectant device itself and procedures for its maintenance routinely used in the NICU. RESULTS: During the period surveyed between 2007 and 2009, only an isolate per respective year of B. cepacia complex was recovered from each neonate in the NICU. However, in 2010, the successive 6 B. cepacia complex isolates were recovered from different hospitalized neonates. Among them, an isolate was originated from peripheral blood of a neonate, apparently giving rise to systemic infection. In addition, the hospitalized neonate with bacteremia due to B. cepacia complex also exhibited positive cultures from repeated catheterized urine samples together with tracheal aspirate secretions. However other 5 isolates were considered as the transients or contaminants having little to do with infections. Moreover, the 5 isolates between July and October in 2010 revealed completely the same electrophoresis patterns by means of pulsed-field gel electrophoresis analyses, strongly indicating that they were infected through the same medical practices, or by transmission of the same contaminant. CONCLUSIONS: A small outbreak due to B. cepacia comlex was brought about in the NICU in 2010, which appeared to be associated with the same genomovar of B. cepacia complex. The source or the rout of infection was unknown in spite of the repeated epidemiological investigation. It is noteworthy that no outbreak due to B. cepacia complex was noted in the NICU after extensive surveillance intervention.

[A case of hookworm infestation with dissociation values between FDP-E and FDP-D dimer].

We previously reported a five-year-old girl showing bleeding tendency and transient morphological and functional platelet abnormalities probably due to a hookworm, Necator Americanus, infestation. In this report, we describe the rarely accelerated fibrinogenolysis and/or fibrinolysis in this patient whose value of fibrinogen and/or fibrin degradation products(FDP) determined with an FDP-E assay was much higher than that determined with a D-dimer assay. Namely, on day-1 and day-13 of hospitalization, her D-dimer values were only 10 to 20% of the prospected values from FDP-E values. We speculated this phenomenon was induced by circulating protease(-like) agent(s) produced by hookworm, because the only slightly participation of plasmin and/or granulocyte elastase was evaluated by the determination of enzyme-inhibitor complexes. And the other possibility of fibrinogen degradation by blast- or tumor-associated protease was excluded by the clinical manifestations and primary disorders. In conclusion, we report a very rare case with the accelerated fibrinogenolysis and/or fibrinolysis in a patient with the hookworm infestation. We are interested in the mechanism that manifested the patient's bleeding tendency accompanied with morphological and functional platelet abnormalities.

Significance of the Grb2 and son of sevenless (Sos) proteins in human bladder cancer cell lines.

The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine-nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP. However, the significance of Grb2 and Sos proteins, which is related to EGF-triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU-7, UMUC-2, UMUC-6) and two kinds of cultured normal urothelial cells (HMKU-1, HMKU-2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c-H-ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H-ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer.

Sex-determining gene(s) on distal 9p: clinical and molecular studies in six cases.

We report on clinical and molecular findings in five karyotypic males (cases 1-5) and one karyotypic female (case 6) with distal 9p monosomy. Cases 1-3 and 6 had female external genitalia, case 4 showed ambiguous external genitalia, and case 5 exhibited male external genitalia with left cryptorchidism and right intrascrotal testis. Gonadal explorations at gonadectomy in cases 3 and 4 revealed that case 3 had left streak gonad and right agonadism, and case 4 had bilateral hypoplastic testes. Endocrine studies in cases 1-4 and 6 showed that cases 1, 3, and 6 had definite primary hypogonadism, with basal FSH levels of 54, 39, and 41 IU/L, respectively, whereas case 2 with severe malnutrition was unremarkable for the baseline values, and case 4 had fairly good testicular function. Fluorescence in situ hybridization and microsatellite analyses demonstrated that all cases had hemizygosity of the 9p sex-determining region distal to D9S1779, with loss of the candidate sex-determining genes DMRT1 and DMRT2 from the abnormal chromosome 9. Sequence analysis in cases 1-4 and 6 showed that they had normal sequences of each exon of DMRT1 and the DM domain of DMRT2 on the normal chromosome 9, and that cases 1-4 had normal SRY sequence. The results provide further support for the presence of a sex-determining gene(s) on distal 9p and favor the possibility of DMRT1 and/or DMRT2 being the sex-determining gene(s). Furthermore, as hemizygosity of the 9p sex-determining region was associated with a wide spectrum of gonadogenesis from agonadism to testis formation in karyotypic males and with primary hypogonadism regardless of karyotypic sex, it is inferred that haploinsufficiency of the 9p sex-determining gene(s) primarily hinders the formation of indifferent gonad, leading to various degrees of defective testis formation in karyotypic males and impaired ovary formation in karyotypic females.

A novel non-pathogenetic polymorphism of the APC gene in a patient with familial adenomatous polyposis coli.

DISORDER: Familial adenomatous polyposis coli. ETHNICITY OF PATIENT: Japanese. GENE: APC. GENBANK ACCESSION NUMBER: M 74088. CHROMOSOMAL ASSIGNMENT: 5q21. TYPE OF DNA VARIANT: A germline missense mutation. A germline nonsense mutation. MUTATION: CGG (Arg, wild type) to TGG (Trp) substitution at codon 88 in exon 3 of the APC gene. CGA (Arg, wild type) to TGA (term.) at codon 213 in exon 5 of the APC gene. ALLELIC FREQUENCY: <0.014 (missense mutation, TGG at codon 88). METHOD OF MUTATION DETECTION: PCR-SSCP/direct sequencing.

Proteasome inhibition circumvents solid tumor resistance to topoisomerase II-directed drugs.

Physiological cell conditions, such as glucose deprivation and hypoxia, play a role in developing drug resistance in solid tumors. These tumor-specific conditions cause decreased expression of DNA topoisomerase IIalpha (topo IIalpha), rendering cells resistant to topo II-targeted drugs, such as etoposide and doxorubicin. We show here that inhibition of proteasome attenuated drug resistance by inhibiting topo IIalpha depletion induced by glucose starvation and hypoxia. topo IIalpha restoration was seen only at the protein levels, indicating that the topo IIalpha protein depletion occurred through a proteasome-mediated degradation mechanism. The stress-induced etoposide resistance was effectively prevented in vitro by the proteasome inhibitor lactacystin in both intrinsically resistant and sensitive tumor cells (colon cancer HT-29 and ovarian cancer A2780 cells, respectively). Furthermore, lactacystin effectively enhanced the antitumor activity of etoposide in the refractory HT-29 xenograft. These results indicate that lactacystin could serve as a new therapeutic agent to circumvent resistance to topo II-targeted chemotherapy in solid tumors.

Suppressive effects of dominant negative ras mutant N116Y on transformed phenotypes of human bladder cancer cells.

To investigate the suppressive effect of dominant negative H-ras mutant N116Y on transformed phenotypes, we established two N116Y ras mutant stable transfectant clones (C5, C13) of human bladder cancer cell line, UMUC-2. These N116Y ras mutant transfectants, especially the C5 cells, showed a dramatic change of cellular morphology and significantly reduced growth in soft agar compared to their control. Furthermore, phosphorylation of the Jun NH2-terminal kinase (JNK) was significantly decreased in these transfectants compared to the control. These results suggest that the N116Y-induced suppression of transformed phenotypes in UMUC-2 cells is associated with inhibition of JNK phosphorylation.

An improved intravesical model using human bladder cancer cell lines to optimize gene and other therapies.

Orthotopic implantation of human bladder cancer cells into immunodeficient mice is an important tool for studying the biology and effects of therapy. Nevertheless, the incidence of tumor implantation and growth by transurethral instillation of the human bladder cancer cells into murine bladders has been low or not reproducible. However, using a modified intravesical technique and the human bladder cancer cell lines, KU-7 and UM-UC-2, we have been able to obtain a high and reproducible incidence of superficial bladder tumors. Furthermore, intravesical administration of the LacZ adenovirus vector resulted in significant beta-galactosidase expression in these bladder tumors as well as the normal urothelium, which was associated with the removal of the glycosoaminoglycan layer. Because this modified technique produces a high incidence of superficial human tumor growth and allows the efficacy of gene transfer to be evaluated, it should be a useful model for the study of intravesical gene therapy for human bladder cancer.

[A case positive for the inhibitor for coagulation factor V].

We report a 71-year-old man who exhibited hepatocellular carcinoma and the inhibitor for coagulation factor V (FV). The inhibitor was found when his coagulation screening tests revealed an abnormally prolonged prothrombin time (71.1 sec) and activated partial thromboplastin time (more than 120 sec) but normal values of fibrinogen (241 mg/dl), the thrombo test (84%) and hepaplastin test (71%). In addition, FV-coagulation activity of the patient's plasma showed less than 1% of the pooled normal plasma and inhibitory activity for FV of his plasma was 32 Bethesda units. This inhibitory activity was neutralized by the addition of anti-human immunoglobulin-gamma-chain serum. The patient was treated with a fibrin sealant including human thrombin when he underwent an partial hepatectomy (32 months before onset) and received 2 doses of thrombin orally (5 months and 2 weeks before onset) to stop bleeding from phlebeurysm. Several studies have reported that the inhibitor for FV was produced after treatment with bovine thrombin containing FV as a contaminant. These findings suggest that our patient may produce an immunoglobulin specific for FV after similar stimulation of human thrombin containing FV.

[A case of simple coarctation].

We report a successful surgical repair of the simple coarctation of a 80-day-old girl by extended end-to-end aortic arch reconstruction. She was admitted to our hospital at the age of 4 days because of poor pulsation of femoral arteries. The systolic blood pressure gradient between the arm and the leg was 30 mmHg. Echocardiography on admission revealed a simple coarctation and patent foramen ovale, with the mildly impaired left ventricular contraction (left ventricular fractional shortening was 23%). Although aortography demonstrated an isolated interrupted segment at the aortic isthmus with collaterals (type A classification of Celoria-Patton), the tubular connection between the distal arch and the descending aorta, of which intralumen was obstructed with abundant ductal tissues, was found at operation. The obstruction of the lumen of aortic isthmus in our case, which was originally patent, might be caused by ductal closure and present as a simple coarctation.

Glucose-regulated stresses cause degradation of DNA topoisomerase IIalpha by inducing nuclear proteasome during G1 cell cycle arrest in cancer cells.

The glucose-regulated stress response of cancer cells leads to a decreased expression of DNA topoisomerase IIalpha (topo IIalpha) and a cell cycle arrest at the G1 phase. In this study, we found that the topo IIalpha decrease occurred specifically during the G1 arrest in human colon adenocarcinoma HT-29 cells. The intracelluar level of topo IIalpha in HT-29 cells was relatively constant regardless of cell cycle position in the exponentially growing state, determined using a centrifugal elutriation technique and synchronizing the cells with a mitotic inhibitor nocodazole. Interestingly, when the cell cycle was arrested in the M phase by nocodazole, the topo IIalpha level remained high even in stressed cells. After the stressed cells were released from the M phase, topo IIalpha steeply decreased along with cell cycle progression followed by the next G1 arrest. This decrease in nuclear topo IIalpha protein was completely inhibited by selective inhibitors for proteasome. Furthermore, we found that proteasome activity was elevated three to fourfold in the nuclear extract of stressed cells over unstressed cells. Accordingly, there were increased amounts of nuclear proteasome subunits, although total intracellular content of the subunits did not change in stressed cells. These findings indicate that the expression of topo IIalpha in stressed cells is downregulated at the G1 phase by proteasome-mediated degradation and that the proteolysis of topo IIalpha can be facilitated by the nuclear accumulation of proteasome.

Glucose starvation and hypoxia induce nuclear accumulation of proteasome in cancer cells.

Solid tumors commonly contain regions with glucose-starved and hypoxic conditions. Tumor cells under the adverse conditions can survive through the stress response, such as cell cycle arrest. In this study, we found that the stress conditions stimulated nuclear accumulation of proteasomes, large multicatalytic protease complexes, in human colon cancer HT-29 cells. The nuclear proteasome levels both in amount and in activity were increased approximately 4 and 2 times by glucose starvation and hypoxia, respectively. No changes were detected in the total expression levels of proteasome. The nuclear proteasome accumulation was also observed in ovarian cancer A2780 cells under glucose starvation, suggesting that this response was regardless of the origin of cancer cells. Our results indicate that the nuclear proteasome distribution is enhanced by glucose starvation and hypoxia, and suggest that the proteolysis by proteasome in the nucleus may play roles in the stress response of solid tumor cells.

Pheochromocytoma arising from an accessory adrenal gland in a patient with multiple endocrine neoplasia type 2A: transient development of clinical manifestations after hemorrhagic necrosis.

A case of pheochromocytoma arising from an accessory adrenal gland in a patient with multiple endocrine neoplasia type 2A (MEN 2A) is reported. This tumor resulted in autonecrosis which caused transient expression of clinical symptoms. Scintigraphy of the abdomen identified the existence of an additional accessory adrenal gland because of which the patient did not require a supplement of hydrocortisone after bilateral total adrenalectomy. Pheochromocytoma arising from an accessory adrenal gland is rarely reported, and spontaneous remission of clinical symptoms due to necrosis of the pheochromocytoma without a clinical emergency is also unusual. Accessory adrenal glands can be the cellular basis for pheochromocytoma, and the importance of continual follow up for pheochromocytoma in subjects with MEN 2A should be emphasized.

Suppression of Erk activation and in vivo growth in esophageal cancer cells by the dominant negative Ras mutant, N116Y.

Our previous studies demonstrated that introduction of a dominant negative H-ras mutant, N116Y, inhibits the growth of various types of cancer cells in vitro. In this study, we tested the efficacy of N116Y in blocking the growth of esophageal cancer cells using an adenoviral vector. Infection with N116Y adenovirus, (AdCMV-N116Y), in which N116Y expression is driven by the cytomegalovirus promoter, significantly reduced the in vitro growth of all esophageal cancer cell lines studied. Esophageal cancer cells that contained wild-type K-ras and H-ras (TE8, SGF3, SGF7) were more sensitive to AdCMV-N116Y than HEC46 cells that expressed mutant K-ras protein. Most importantly, direct injection of AdCMV-N116Y into TE8- or SGF3-induced tumors in nude mice suppressed their growth significantly. To examine the suppressive mechanism of N116Y, cell cycle profile and the activation of extracellular signal-regulated kinase 2 (Erk2) were examined by flow cytometry and Western blot analysis, respectively. In TE8 cells, progression into S phase was clearly blocked after infection with AdCMV-N116Y. Infection with AdCMV-N116Y did not strongly suppress the activation of Erk2 after EGF stimulation in serum-starved HEC46 cells, whereas it completely suppressed activation in TE8, SGF3 and SGF7 cells. Our observations suggest that N116Y reduces growth of human esophageal cancer cells and suppresses the activation of Erk2; they also indicate that N116Y is a potential candidate gene for human esophageal cancer gene therapy.

A comprehensive system to explore p53 mutations.

To establish an effective and reliable system for the detection of p53 mutations, we evaluated the detection efficiencies of nonisotopic polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), fluorescence in situ hybridization (FISH), and immunohistochemistry. Ten cell lines (AsPc1, BxPc3, Miapaca2, Panc1, Colo320-011, Lovo, MCF7, LNCaP, HL-60, and Daudi), a peripheral blood sample from a patient with a p53 germline mutation (p53GML), and a normal peripheral blood sample were used for examination. Direct nucleotide sequencing identified p53 mutations in 7 of 12 samples (AsPc1, BxPc3, Miapaca2, Panc1, Colo320-011, HL-60, and p53GML). The nonisotopic PCR-SSCP detected anomalies of the PCR fragments in 5 cell lines. In the FISH analysis, 2 cell lines exhibited loss of heterozygosity of the p53 locus. Immunohistochemistry detected an accumulation of the abnormal p53 in 4 cell lines. The combination of these 3 methods produced no false-negative or false-positive results. This combination may be an excellent and beneficial system for the clinical diagnosis of the various human cancers.

[Clinical evaluation of RT-PCR method for detection of HCV-RNA: second report availability of RT-PCR system using PCR internal control for detection of HCV-RNA and the influence of interfering substances on the detection].

The COBAS Amplicor system is an automated diagnostic PCR system which contains a PCR internal control (P. I. C.) template to monitor the amplification. The applicability of COBAS Amplicor HCV was examined using sera of patients with hepatitis. Furthermore, the effects of possible interfering substance (total protein, triglyceride, hemoglobin, glucose, total bilirubin, heparin, lysis reagent including guanidium) on HCV-RNA detection were investigated. The sensitivity of COBAS Amplicor HCV was equivalent to the manual method of Amplicor HCV, moreover all of the results in 54 clinical samples analyzed on both COBAS Amplicor HCV and Amplicor HCV were in agreement. Detection sensitivity of HCV-RNA decreased in the presence of total bilirubin and heparin. Ten and 25mg/dl of total bilirubin affected HCV-RNA detection but did not affect P. I. C. This result suggested that total bilirubin interfered with the protein denature caused by the lysis reagent. Fifteen U/ml of heparin in the sample completely inhibited amplification both of the HCV-RNA and P. I. C. One U/ml of heparin did not affect amplification, but heparinized blood samples should not be used for the detection of HCV-RNA. To examine the effect of possible carry over contamination on the lysis reagent which contains guanidium, various concentrations of lysis reagent in P. I. C. were tested. RT-PCR was inhibited by 1/500 volume contamination of lysis reagent in specimen diluent. Other substances did not affect the sensitivity. Our results indicate that the carryover contamination of lysis reagent cause more "false negative" results than interfering substances in sera. In conclusion, HCV-RNA detection system containing P. I. C., such as COBAS Amplicor HCV, will become a very useful to differentiate "false negative" and "true negative" result.

The significance of Ras guanine nucleotide exchange factor, son of sevenless protein, in renal cell carcinoma cell lines.

PURPOSE: The aim of the present study is to clarify the significance of the Ras guanine-nucleotide exchange reaction in the proliferation of human renal cell carcinoma cell lines. MATERIALS AND METHODS: We examined the expression of human son of sevenless-1 (hSos-1) protein and the epidermal growth factor (EGF) receptor in human renal cell carcinoma cell lines by Western blot analysis. Additionally, a dominant negative H-ras mutant, N116Y, which is known to inhibit the Ras guanine-nucleotide exchange reaction, was transfected into these cell lines by lipofection. RESULTS: Human renal cell carcinoma cell lines expressed much higher amounts of the EGF receptor and hSos-1 protein than normal kidney tissue. Moreover, the N116Y ras mutant could strongly suppress cellular proliferation in these cell lines. CONCLUSIONS: Augmentation of the Ras guanine-nucleotide exchange reaction might be essential to the proliferation of human renal cell carcinoma cells.

[Clinical evaluation of RT-PCR method for detection of HCV-RNA--with special reference to Amplicor HCV].

Detection of the viral genome in serum is the most reliable way to analyze HCV viremia. In the present study, we evaluated the availability of a new detection kit for HCV-RNA, Amplicor HCV, which is based on the RT-PCR microplate hybridization protocol. The procedure of Amplicor HCV is simple, unlike the conventional RT-nested-PCR method. Although Amplicor HCV assay exhibited the lower sensitivity than the the conventional RT-nested-PCR method (10 x for HCV type 1a, 1b and 2b; 10(3) x for type 2a), Amplicor HCV assay could detect the HCV-RNA in all HCV-RNA positive cases by conventional RT-nested PCR method, except for one case who contained low concentration of HCV-RNA (10 copies/ml). The coincidence rate was 99.2% in 120 clinical samples between two assays. Amplicor HCV assay, moreover, could efficiently evaluate the viremia regardless of anti-HCV-2 antibody titer. This assay was useful for monitoring the effect of interferon therapy during and after administration. These results suggest that Amplicor HCV has an excellent availability for the clinical laboratories.

Involvement of Ras in the expression of glycolipid sulfotransferase in human renal cancer cells.

Glycolipid sulfotransferase activity in a human renal cell carcinoma cell line, SMKT-R3, is enhanced by epidermal growth factor (EGF); tyrosine kinase inhibitors suppress this enhancement. To investigate the involvement of Ras in the signal transduction pathway from the EGF receptor to the expression of glycolipid sulfotransferase, we introduced v-H-ras into SMKT-R3 cells. In a quiescent state, the percent GTP bound to Ras in v-H-ras-expressing cells increased about 2.5-fold compared with control cells, suggesting that v-Ras introduced into the renal cancer cells is in an active form without EGF stimulation. Glycolipid sulfotransferase activity in v-H-ras-expressing cells was higher than in control cells. The sulfotransferase activity was affected neither by EGF nor by genistein, a tyrosine kinase inhibitor, in v-H-ras-expressing cells, whereas it was enhanced by EGF and reduced by genistein in control cells. Our observations suggest that Ras mediates the regulation pathway of glycolipid sulfotransferase activity in SMKT-R3 cells.