Relationship between Epidermal Growth Factor Receptor Mutations and Adverse Events in Non-Small Cell Lung Cancer Patients treated with Afatinib.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors such as afatinib are used for non-small cell lung cancer (NSCLC) and show varying efficacy depending on EGFR gene mutation. Few studies have examined the relationship between EGFR gene mutations and the adverse events of afatinib in NSCLC. This retrospective study included 32 Japanese patients with NSCLC with EGFR gene mutation who were treated with afatinib between May 2014 and August 2018 at Kagawa University Hospital. Among the 32 Japanese patients with NSCLC treated with afatinib, 19 patients were positive for exon 19 deletion mutation (Del 19) and 13 patients were negative for Del 19. The incidence of grade ≥ 2 skin rash was slightly higher in patients positive for Del 19 (42.1% vs. 7.7%, P = 0.050). No significant differences were detected in other adverse events between the two patient groups. Patients positive for Del 19 also showed significantly longer median progression-free survival (288 vs. 84 days, P = 0.049). Our study indicates a higher incidence of skin rash associated with afatinib treatment in Japanese patients with NSCLC positive for Del 19 compared with patients without Del 19. The Del 19 positive patient group also showed better progression-free survival. J. Med. Invest. 68 : 125-128, February, 2021.

Uterine sarcoma with posterior reversible encephalopathy syndrome associated with pazopanib.

WHAT IS KNOWN AND OBJECTIVE: Our objective is to report on a case of posterior reversible encephalopathy syndrome associated with pazopanib. CASE DESCRIPTION: A 64-year-old patient with uterine sarcoma developed PRES 3 days after pazopanib was initiated. After the discontinuation of pazopanib, the symptoms of PRES improved. WHAT IS NEW AND CONCLUSION: The first report worldwide to describe a patient with uterine sarcoma experiencing PRES caused by pazopanib. Patients with uterine sarcoma may experience PRES, even in the early phase of pazopanib therapy.

Relationship between epidermal growth factor receptor mutations and skin rash in non-small cell lung cancer patients.

Several reports have investigated relationships between epidermal growth factor receptor (EGFR) mutations and the efficacy of EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer; however, there have been insufficient analyses of relationships between EGFR mutations and adverse reactions. This study investigated the relationships between EGFR mutations and skin rash. We first compared skin rash grades between different mutations, then tested factors possibly affecting skin rash by multivariate analysis. The main outcome measure was the significant difference in incidence of skin rash between each group with different mutations. Our study suggested that the risk of skin rash is low in patients with exon 19 deletion mutations who are taking EGFR-TKIs, whereas it is high in those with exon 21 point mutations. These results will be useful indicators for instructions regarding daily examinations, skin care, and use of oral antibiotics or topical steroids in patients taking EGFR-TKIs with skin rash.

Hypertension and Proteinuria as Predictive Factors of Effects of Bevacizumab on Advanced Breast Cancer in Japan.

Bevacizumab (BV), an inhibitor of vascular endothelial growth factor, is used in combination with paclitaxel (PTX) to treat advanced breast cancer. Hypertension and proteinuria are characteristic adverse events of BV therapy. We assessed the potential of these adverse events as predictors of BV treatment responses. Our results revealed that groups that developed hypertension and proteinuria early (by day 56) had a stronger antitumor response (Fisher's exact test p<0.05). However, no significant difference was observed in progression-free survival (the Kaplan-Meier method and Log-rank test). As a reference, age, the treatment line, subtypes, liver and renal function, diabetes mellitus and hyperlipidemia history, body mass index, influencing concomitant medicine, average relative dose intensity and hematotoxicity did not significantly differ between groups with or without hypertension and with or without proteinuria. These results indicate the potential of the development of hypertension and proteinuria as predictors of improved outcomes with PTX plus BV therapy in patients with breast cancer. However, since both adverse events may preclude the continuation of treatment, their earlier management may be required.

Indication of metronomic chemotherapy for metastatic breast cancer: Clinical outcomes and responsive subtypes.

The survival of patients with metastatic breast cancer (MBC) has not improved, despite recent advances in therapeutic strategies. This is mainly due to the fact that cytotoxic agents cannot be administered over a long period, even if they exhibit favorable activity, due to treatment-related side effects or acquisition of tumor resistance to the administered agents. Thus, the development of therapeutic strategies that may be used over a long time period is required to improve survival. We assessed the availability and clinical outcomes of metronomic chemotherapy, which is defined as continuous or frequent treatment with low doses of cytotoxic drugs. A total of 80 patients with MBC received chemotherapy in the metastatic setting, and the clinicopathological factors and clinical outcomes were retrospectively compared between 52 patients who received metronomic regimens and 28 patients who received other cytotoxic regimens. As regards clinical outcomes, the median time-to-treatment failure (TTF) and overall survival (OS) were significantly longer in the metronomic group compared with those in the non-metronomic group (TTF, 15 vs. 4 months, P=0.0001; and OS, 53 vs. 28 months P=0.0012, respectively). In the metronomic group, none of the 18 patients who responded to the regimen had triple-negative (TN) cancer (17 had luminal-type tumors and 1 had a human epidermal factor receptor 2-type tumor). Furthermore, TTF and OS were significantly longer in patients with non-TN cancer compared with those in patients with TN cancer in the metronomic group (TTF, 16 vs. 7 months, P=0.0014; and OS, 108 vs. 20 months, P=0.000007, respectively). The proportion of patients who experienced treatment-related adverse events was significantly lower in the metronomic group compared with that in the non-metronomic group (36.5 vs. 61.5%, respectively; P=0.038). In conclusion, metronomic chemotherapy is a viable option for luminal-type MBC in terms of effectiveness and minimal toxicity, regardless of metastatic sites or prior treatment. However, an alternative treatment is required for TN cancer.

Effect of endurance for adverse drug reactions on the preference for aggressive treatments in cancer patients.

PURPOSE: Cancer patients receiving chemotherapy will sometimes conceal their discomfort, but an excessive endurance for adverse drug reactions (ADRs) can lead to a poorer prognosis. The aim of this study was to clarify the association between ADR endurance and a preference of cancer patients for aggressive treatments. METHODS: A cross-sectional study was undertaken of inpatients under 75 years of age receiving injectable systemic chemotherapy or oral chronic medications at hospitals in Japan. Subjects were asked to respond to a validated questionnaire to assess the extent of their ADR endurance and whether they would choose a novel, more aggressive therapy if their life expectancy was estimated at 2 years. RESULTS: Study participants were separated into the chemotherapy group (n = 36) and the non-chemotherapy group (n = 78). In the chemotherapy group, patients who had moderate ADR endurance scores were more likely to choose the new therapy (0-33, 34-67, and 68-100 points: 0.0, 54.5, and 27.3 %; χ (2) test, p = 0.15). Additionally, every patient on long-term chemotherapy (≥3 years) had high ADR endurance scores but did not choose the new, riskier treatment. In the non-chemotherapy group, the proportion of those choosing the new therapy was linearly associated with higher ADR endurance scores (25.9, 38.2, and 64.7 %; p = 0.04). CONCLUSION: Cancer patients may prefer aggressive therapies, even when self-estimations of ADR endurance are not very high, especially if they have been receiving chemotherapy for a short period of time. These patients should be observed with great caution.

Factors responsible for long-term survival in metastatic breast cancer.

BACKGROUND: Although survival of patients with metastatic breast cancer (MBC) has been significantly prolonged over the past decade due to improvement of anti-cancer therapeutics, only a few patients survive for more than 10 years. It has not been determined which patients can have long-term survival with treatment. METHODS: To determine prognostic factors responsible for long-term survival, we retrospectively compared clinicopathologic factors of patients with MBC who survived for 50 months or more after diagnosis with patients who did not. Of 70 patients with MBC who received chemotherapy between November 2005 and September 2011, 23 patients who survived for 50 months or more after diagnosis and 28 patients who died within 50 months after diagnosis were assessed for their clinicopathologic factors and outcomes. RESULTS: The proportion of patients with hormone receptor-positive (HR+) tumors was significantly higher and the proportion of patients with triple negative tumors (TN) was lower in long-term survivors than in non-long-term survivors (HR+: 87% versus 28.6%, P=0.000037; TN: 13.1% versus 53.6%, P=0.0028). Metastatic site, number of disease sites, prior chemotherapeutic regimens and human epidermal growth factor receptor-2 (HER2) status did not differ between the two groups. The proportion of patients who received metronomic regimens was significantly higher in long-term survivors than in non-long-term survivors (65.2% versus 35.7%, P=0.034) when the most effective regimen among regimens that were received in metastatic settings was compared between the two groups. Overall response rate was significantly higher (82.6% versus 17.9%, P<0.00001) and time to treatment failure after receiving the most effective regimen was longer in long-term survivors than in non-long-term survivors (26 versus 5 months, P=0.0001). The number of chemotherapeutic regimens for breast cancer and that for MBC did not differ between the two groups. CONCLUSIONS: Patients with luminal-type MBC who benefit at least once from chemotherapy including metronomic regimens, or patients who continued to receive the most effective regimen for more than two years can be expected to have long-term survival after diagnosis of MBC, regardless of the number of chemotherapeutic regimens they had received.

Developing population pharmacokinetic parameters for high-dose methotrexate therapy: implication of correlations among developed parameters for individual parameter estimation using the Bayesian least-squares method.

Bayesian estimation enables the individual pharmacokinetic parameters of the medication administrated to be estimated using only a few blood concentrations. Due to wide inter-individual variability in the pharmacokinetics of methotrexate (MTX), the concentration of MTX needs to be frequently determined during high-dose MTX therapy in order to prevent toxic adverse events. To apply the benefits of Bayesian estimation to cases treated with this therapy, we attempted to develop an estimation method using the Bayesian least-squares method, which is commonly used for therapeutic monitoring in a clinical setting. Because this method hypothesizes independency among population pharmacokinetic parameters, we focused on correlations among population pharmacokinetic parameters used to estimate individual parameters. A two-compartment model adequately described the observed concentration of MTX. The individual pharmacokinetic parameters of MTX were estimated in 57 cases using the maximum likelihood method. Among the available parameters accounting for a 2-compartment model, V1, k10, k12, and k21 were found to be the combination showing the weakest correlations, which indicated that this combination was best suited to the Bayesian least-squares method. Using this combination of population pharmacokinetic parameters, Bayesian estimation provided an accurate estimation of individual parameters. In addition, we demonstrated that the degree of correlation among population pharmacokinetic parameters used in the estimation affected the precision of the estimates. This result highlights the necessity of assessing correlations among the population pharmacokinetic parameters used in the Bayesian least-squares method.

Effects of angiotensin II AT1-receptor blockade on high fat diet-induced vascular oxidative stress and endothelial dysfunction in Dahl salt-sensitive rats.

We examined the effects of angiotensin II AT1-receptor blockade with olmesartan on high fat (HF) diet-induced vascular oxidative stress and endothelial dysfunction in normal salt (NS) diet-fed Dahl salt-sensitive (DSS) rats. Treatment with NS + HF diet (32% crude fat, 0.3% NaCl) for 20 weeks significantly increased blood pressure in DSS rats. NS + HF diet-fed DSS rats also showed higher plasma levels of thiobarbituric acid-reactive substances, aortic superoxide production, and mRNA levels of p22(phox) and gp91(phox) in aortic tissues than NS diet-fed DSS rats. Furthermore, acetylcholine-induced vasorelaxation of aorta from NS + HF diet-fed DSS rats was significantly reduced. In NS + HF diet-fed DSS rats, treatment with olmesartan medoxomil (10 mg/kg per day, p.o.) and hydralazine (25 mg/kg per day, p.o.) similarly decreased blood pressure. However, in these animals, only olmesartan normalized plasma levels of thiobarbituric acid-reactive substances, vascular superoxide in aortic tissues, and acetylcholine-induced vasorelaxation. These data indicate that HF diet-induced hypertension is associated with vascular oxidative stress and endothelial dysfunction in NS diet-treated DSS rats. Inhibition of angiotensin II AT1 receptors may elicit beneficial effects on HF-induced hypertension and vascular injury in subjects that have genetically enhanced sodium-sensitive blood pressure.

Clinical usefulness of human epidermal growth factor receptor-2 extracellular domain as a biomarker for monitoring cancer status and predicting the therapeutic efficacy in breast cancer.

We assessed the clinical usefulness of human epidermal growth factor receptor-2 extracellular domain (HER2ECD) as a biomarker for detecting cancer and monitoring disease status and for predicting the efficacy of anticancer treatment in breast cancer. Five-hundred and eighty serum samples from 252 patients with breast cancer were examined for the concentration of HER2ECD to compare with conventional tumor markers (CEA, CA15-3, NCC-ST439 and BCA225). Also, in 19 patients with HER2-overexpressed advanced or recurrent breast cancer who were treated with trastuzumab, clinical outcomes were evaluated retrospectively to determine whether their serum HER2ECD levels predict clinical responses. The proportion of patients with elevated HER2ECD levels was 15.1%, which was compatible with those with elevated conventional marker levels. In patients with HER2-overexpressed breast cancer, the positive rate of HER2ECD was significantly higher (24.1%) than those of conventional markers (7.4-12.9%), suggesting the usefulness of HER2ECD for detecting cancer in this population. HER2-overexpressed patients responding to trastuzumab (12 of 19 patients) showed significantly higher serum HER2ECD level (p = 0.033) and longer time to progression (TTP) (p = 0.039) and overall survival (OS) (p = 0.031) than did patients not responding (seven patients). Furthermore, higher response rates were observed in patients with elevated HER2ECD levels than in patients without elevated HER2ECD levels (91.3% vs. 14.3%, p = 0.032), whereas there was no difference in survival between the two groups. The results suggest that HER2ECD is a useful biomarker not only for detecting breast cancer recurrence but also for predicting tumor responses to trastuzumab.

Metronomic chemotherapy for metastatic breast cancer to prolong time to treatment failure to 12 months or more.

The objective of treatment for metastatic breast cancer (MBC) is to control the disease or disease-related symptoms. Prolonged survival has also often been achieved by chemotherapeutic regimens in this setting. Long-term administration of one therapeutic regimen is essential for prolonging survival as well as for maintaining quality of life in these patients. In this study, we focused on time to treatment failure (TTF) as a parameter that predicts patient survival and we retrospectively compared clinical outcomes of patients with MBC who showed TTF of ≥12 months (26 patients) and <12 months (29 patients). The proportion of hormone receptor-positive tumors and the number of prior chemotherapy regimens for MBC were significantly higher and tumor grade was lower in patients with TTF ≥12 months compared to those with TTF <12 months. With regard to clinical outcomes, the objective response rate (ORR) in patients with TTF ≥12 months was significantly higher and median time to progression (TTP) and overall survival (OS) were longer compared to those with TTF <12 months. Of note, the proportion of patients who received metronomic regimens was significantly higher in patients with TTF ≥12 months compared to those with TTF <12 months (80.8 vs. 24.1%, P=0.00003). To assess the clinical benefit of metronomic regimens, the efficacy in patients receiving metronomic and those receiving non-metronomic regimens was compared. Although there was no difference in ORR between the two groups, median TTP and OS were significantly longer in the metronomic compared to the non-metronomic group (TTP: 30 vs. 4 months, P=0.0017; OS: 68 vs. 28 months, P=0.0005). The results suggested that metronomic chemotherapy is useful for palliative care and also improved clinical outcomes as a regimen for which long-term administration may be expected.

Erythroid recovery affects tacrolimus levels after engraftment during stem cell transplantation.

Tacrolimus is commonly used in stem-cell transplants (SCT) for prophylaxis of graft-versus-host disease and is continuously administered throughout transplantation. The dose of tacrolimus is frequently decreased to maintain a desired concentration during the recovery of hemocytes after engraftment. If parameters which affect tacrolimus clearance are identified, it is of clinical use to estimate concentrations and aid dosing. The objective of this study was to identify which hematologic parameters affect tacrolimus clearance. Seventeen consecutive Japanese patients with hematological malignancies who received allogeneic SCT between March 2004 and January 2007 were enrolled in this study. Their steady-state concentrations were routinely measured and standardized as the concentration/dose (C/D) ratio ((ng/mL)/(mg/kg/d)). Multivariate analysis was performed to identify which hemocyte parameters affected the C/D ratio. Of the 13 patients, gradual dose reduction was required to combat elevated tacrolimus concentrations. The mean post-engraftment C/D ratio was higher than the pre-engraftment C/D ratio in each patient. The mean C/D ratio for all patients after engraftment was 1.56-fold higher (p=0.00004, range: 1.04-3.03) than that before engraftment. The variation ratio was calculated by dividing the C/D ratio by that on the engraftment day. Multivariate analysis revealed that the reticulocyte (RET) level (×10(3) count/µL) was the sole parameter influencing this ratio, and both parameters were expressed as: Variation ratio=0.004×RET+1.0. RET recovery of patients could influence the C/D ratio and tacrolimus clearance was affected by recipient original red blood cells, but not that of transfused red blood cells.

[Case of encephalopathy seemingly caused by tacrolimus at blood concentration near the upper limit of therapeutic range].

We report a case of encephalopathy seemingly caused by tacrolimus (FK506) in spite of blood concentration near the upper limit of therapeutic range. A 26-year-old man received FK506 to prevent acute graft-versus-host disease after hematopoietic stem cell transplantation. He underwent an intravenous injection of FK506 the day before transplantation (day -1). He developed headache, hypertension, nausea and vomiting from day 2 to day 3. A computed tomography scan showed a low density area with unclear border in the bilateral cerebellar hemispheres. Thereafter, these symptoms improved with discontinuation of FK506, which was strongly suggestive of encephalopathy caused by FK506. The blood concentration of FK506 at the onset of encephalopathy was 21.7 ng/mL. Although this value was slightly higher than the standard therapeutic range (10-20 ng/mL), it was within clinically acceptable range in the early stage after stem cell transplantation. This indicates that even if the blood concentration of FK506 is within the therapeutic range, encephalopathy may develop. In summary, although the blood concentration of FK506 is useful as an indicator for prevention of encephalopathy, we propose careful monitoring not only of the blood concentration but also clinical status for the detection of initial symptoms and prevention of aggravation.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²âº independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A1 (PLA1) activity was dominant over the PLA2 activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens.

Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl-L-arginine p-nitroanilide and acyl-L-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO.

Effects of exposure to a time-varying 1.5 T magnetic field on the neurotransmitter-activated increase in intracellular Ca(2+) in relation to actin fiber and mitochondrial functions in bovine adrenal chromaffin cells.

BACKGROUND: It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca(2+) mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca(2+) release from Ca(2+) stores in adrenal chromaffin cells. METHODS: We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca(2+) in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2h. RESULTS: Exposure to the magnetic field significantly reduced the increase in [Ca(2+)]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca(2+) release by the exposure was unaffected. CONCLUSIONS AND GENERAL SIGNIFICANCE: These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca(2+) release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca(2+)]i.

Peritoneal pharmacokinetics of cefepime in laparotomy patients with inflammatory bowel disease, and dosage considerations for surgical intra-abdominal infections based on pharmacodynamic assessment.

This study aimed to examine the peritoneal pharmacokinetics of cefepime and to assess its pharmacodynamic exposure in peritoneal fluid (PF). Cefepime (1 g) was administered to eight patients with inflammatory bowel disease before abdominal surgery. Venous blood and PF samples were obtained at the end of the 0.5-h infusion and at 1, 2, 3, 4, 5, and 6 h thereafter. Drug concentrations in plasma and PF were determined, analyzed pharmacokinetically, and used for a Monte Carlo simulation with minimum inhibitory concentration (MIC) data. Cefepime penetrated well into PF, with a maximum drug concentration in a PF/plasma ratio of 0.59 +/- 0.15 (mean +/- SD, n = 8), and an area under the concentration-time curve ratio of 0.90 +/- 0.10. The probabilities of attaining the bacteriostatic and bactericidal targets (40% and 70% of the time above the MIC, respectively) in PF were > or =85% against Escherichia coli, Klebsiella species, and Enterobacter cloacae with 0.5 g every 12 h, 1 g every 12 h, 1 g every 8 h, and 2 g every 12 h. However, 1 g every 8 h or 2 g every 12 h was required for bacteriostatic and bactericidal probabilities > or =85% against Pseudomonas aeruginosa. These conventional regimens did not achieve a high probability against Bacteroides species. These results should help to give us a better understanding of the peritoneal pharmacokinetics of cefepime while also helping to choose the appropriate dosage to prevent surgical intra-abdominal infections on the basis of the pharmacodynamic assessment.

[Infection treatment caused by multiple-drug-resistant Pseudomonas aeruginosa in a patient undergoing allogeneic hematopoietic stem cell transplantation].

Infections caused by multiple-drug-resistant Pseudomonas aeruginosa (MDRP) are a clinically significant problem. We reported here the effective use of combination therapy in a patient with infection caused by MDRP according to an interventional treatment strategy suggested by a pharmacist. The patient was a 70-year-old male who underwent allogeneic hematopoietic stem cell transplantation. On day 45 after transplant, MDRP was newly isolated from urine, but the diagnosis at that time was colonization. On day 61, the patient developed a fever (> or =38.0 degrees C). In addition, laboratory data showed that C-reactive protein (CRP) was also increased. At the medical team conference, the pharmacist proposed the following treatment strategy for this infection. Aztreonam and amikacin were intravenously administered at doses of 2 g/day and 800 mg/day, respectively. The subsequent clinical course was well controlled, but the infection recurred and was aggravated. Aztreonam and ciprofloxacin were then intravenously administered at doses of 4 g/day and 600 mg/day, respectively, resulting in the alleviation of fever in the patient as well as a decrease in CRP and disappearance of MDRP isolates from urine on day 67; that is, MDRP infection was consequently well controlled. In conclusion, the combination therapy between aztreonam and amikacin, or ciprofloxacin may be clinically useful for severe infections of MDRP in compromised hosts.

Construction and characterization of a clostripain-like protease-deficient mutant of Clostridium perfringens as a strain for clostridial gene expression.

The inherent difficulty of expressing clostridial AT-rich genes in a heterologous host has limited their biotechnological application. We previously reported a plasmid for high-level expression of clostridial genes in Clostridium perfringens (Takamizawa et al., Protein Expr Purif 36:70-75, 2004). In this study, we examined the extracellular proteases of C. perfringens strain 13. Zymographic analysis and caseinase assaying of a culture supernatant showed that it contained a protease activated by dithiothreitol and Ca(2+), suggesting that clostripain-like protease (Clp) is the most likely candidate for the major extracellular protease. Disruption of the clp gene by homologous recombination markedly decreased the level of caseinase activity in the culture supernatant. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Clp(-) mutant but not the wild type strain increased the levels of many polypeptides in the culture supernatant after the late exponential growth phase. Such polypeptides included both cytoplasmic and secretory proteins, suggesting proteins secreted or released into the medium were degraded by Clp. To assess the effects of Clp on the productivity and stability of recombinant proteins, 74-kDa NanI sialidase was expressed in the two strains. The mutant strain produced a higher level of NanI activity than the wild type strain. Furthermore, under the conditions where Clp was activated, NanI was degraded easily in the latter culture but not in the former one. These results indicate that the Clp(-) mutant could serve as a useful strain for efficiently expressing and preparing protease-free clostridial proteins.

[Preventive effects of newquinolones for endogenous infection in patients receiving allogeneic hematopoietic stem cell transplantation--comparison between bone marrow transplantation, peripheral blood stem cell transplantation, and cord blood transplantation].

We performed a retrospective study to examine the preventive effects of newquinolones for endogenous infection in patients receiving various allogeneic hematopoietic stem cell transplantation including bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT), and cord blood transplantation (CBT). Forty-nine patients were enrolled. Ciprofloxacin or norfloxacin was orally administered for intestinal sterilization from day -14 until engraftment. As a result, the period from transplantation until engraftment was significantly longer in CBT group than in BMT group. The febrile index (the ratio of the febrile (> or =38.0 degrees C) period during neutropenia (< or =500 cells/mm(3)) and C-reactive protein (CRP)-positive index (the ratio of CRP-positive (> or =2.0 mg/dl) period during neutropenia) were comparable among the three groups. In addition, no gram-negative bacteria in stool was isolated in the three groups; that is, an endogenous infection of gram-negative bacteria, a potential pathogen, was well controlled by newquinolones. We should be careful when interpreting the results of this small study; however, newquinolones are clinically effective for endogenous infection of gram-negative bacteria in patients receiving not only BMT, but also PBSCT and CBT.