Yoshito Kusuhara

Reemerging murine typhus, Japan.

To the Editor: Murine typhus is an arthropod-borne infectious disease caused by Rickettsia typhi, which is distributed widely around the world (1–4). In Japan, tsutsugamushi disease occurs most frequently in persons infected with rickettsioses (5). Spotted fever caused by R. japonica also occurs in the southwestern part of Japan (6,7). In the 1940s and 1950s, many murine typhus cases were reported in Japan. These diagnoses were made according to the clinical features of the illness and the reactivity of the serum samples to OX19 in Weil-Felix tests. A few cases were diagnosed on the basis of symptoms exhibited by animals infected with isolated rickettsiae and complement fixation tests, in addition to results of the Weil-Felix tests. The Weil-Felix test is useful for preliminary screening of rickettsiosis; however, the reaction could indicate epidemic typhus or spotted fever in some cases. Since 1958, only three murine typhus cases have been reported in Japan (8). In these cases, no serologic tests for epidemic typhus were conducted. Serum sample from patients with epidemic typhus and murine typhus frequently possess serologic cross-reactivity to R. typhi and R. prowazekii, respectively (9). Thus, the possibility of epidemic typhus could not be excluded definitively in these cases. On May 4, 2003, a 56-year-old man living in Tokushima, Japan, sought medical care; he had a temperature of 39.1°C and exanthema on the trunk and the upper limbs. No surface lymph nodes were palpable. He was treated with lincomycin and cefditoren pivoxil with no improvement. On day 3, the patient informed caregivers that he had been in a bamboo grove on days 1 and 11 before the onset of symptoms. C-reactive protein of the serum sample collected on day 3 was positive (= 7.6 mg/dL). From this finding, spotted fever was suspected; the disease is endemic in Tokushima. On day 4, the exanthema had spread systemically, and treatment with minocycline was started, which led to a gradual decrease in fever and rashes. The patient was admitted to the Tokushima University Hospital on day 6 of the illness for diagnosis and further treatment. Serum samples were collected from the patient on days 5, 6, 9, 20, and 34. Indirect immunoperoxidase tests on the serum samples for tsutsugamushi disease, spotted fever, murine typhus, and Q fever on day 5 of the illness were negative for immunoglobulin (Ig) G and IgM antibodies (<1:40). Weil-Felix tests on the serum samples on days 5 and 9 of the illness were negative for OX2, OX19, and OXK. Indirect immunofluorescence of the serum samples on days 6, 9, 20, and 34 of the illness was conducted by using strains 18 and Wilmington of R. typhi, and the strain Breinl of R. prowazekii as typhus group rickettsiae; and the strain YH of R. japonica, the strain Malish 7 of R. conorii, and the strain Tick of R. montanensis as the spotted fever group rickettsiae. All serum samples tested for the rickettsiae showed an IgM titer of 1:200. On the other hand, the IgM titers of these serum samples, to the Orientia tsutsugamushi were <1:20. For the IgG antibodies of these serum, spotted fever group rickettsiae were negative (<1:20). However, the typhus group rickettsiae were positive for IgG antibodies. Among the typhus group rickettsiae, strain 18 of R. typhi had the highest elevated titers. The titers to the sera on days 6, 9, 20, and 34 of illness were 1:80, 1:160, 1:160, and 1:80, respectively. Another strain of R. typhi, the strain Wilmington, had lower titers of 1:40, 1:80, 1:80, and 1:40, on days 6, 9, 20, and 34 of the illness, respectively. This could have occurred because strain 18 may be a closer antigenic relation of the causative agent than is the strain Wilmington. R. prowazekii demonstrated the lowest IgG titers among typhus group rickettsiae for these serum samples, <1:20, 1:20, 1:40, and 1:20, on days 6, 9, 20, and 34 of the illness, respectively. These results suggested that the disease was murine typhus. To demonstrate more detailed antigenic reactivity, Western immunoblotting of rickettsiae was conducted by using a serum specimen from day 20. All of the rickettsiae were reactive to the serum to various extents. The serum reacted to the ladderlike lipopolysaccharide of R. typhi and R. prowazekii; the antigenicity of rickettsial lipopolysaccharide is group-specific. As expected from the immunofluorescence data, no reaction was demonstrated to the lipopolysaccharide of spotted fever group rickettsiae, R. japonica and R. montanensis, although trace cross-reactivity, mainly to rOmpB, was shown. Thus, typhus group rickettsiosis was suspected for this case on the basis of these data. Compared to the trace reaction of spotted fever group rickettsiae to rOmpB, a stronger, but still weak, reaction was detected to the heat-labile state of rOmpB of R. prowazekii, and an extremely strong reaction was demonstrated to the heat-labile and heat-stable states of rOmpB of R. typhi. These results strongly suggested that the disease was murine typhus. To confirm this diagnosis, we conducted absorption tests as described previously (10). The patient serum collected on day 20 showed complete absorption by the homologous antigen, the purified R. typhi strain 18, demonstrating no reaction to R. typhi or to R. prowazekii by immunofluorescence. However, the serum showed incomplete absorption by the heterologous antigen, the purified R. prowazekii, demonstrating no reactivity to R. prowazekii but some reactivity to R. typhi. These tests confirmed the diagnosis of murine typhus. Murine typhus has never been reported in Japan after the 1950s, except for the three suspected cases and this case. Although other undiagnosed cases may have occurred, they appear to be few; many febrile cases of exanthema have been examined for various rickettsioses, especially after spotted fever was diagnosed in Japan in 1984. Murine typhus may have reemerged because of the recent increase of black rats, Rattus rattus, in Japan. This patient mentioned that he had captured a rat and disposed of the carcass about a week before the onset of symptoms. Infection could have resulted at that time from contamination with feces of infected fleas such as the oriental rat flea, Xenopsylla cheopis. Historical review indicates that this is the first complete serologic diagnosis of a murine typhus case in Japan.

The Involvement of Hepatocyte Growth Factor-MET-Matrix Metalloproteinase 1 Signaling in Bladder Cancer Invasiveness and Proliferation. Effect of the MET Inhibitor, Cabozantinib (XL184), on Bladder Cancer Cells

OBJECTIVE To clarify the invasive mechanisms of muscle-invasive bladder cancer (BCa) would be useful for the determination of appropriate treatment strategies. We previously showed that hepatocyte growth factor (HGF)-MET signaling is correlated with invasiveness of BCa cells. Here, we investigated the effects of the MET inhibitor, cabozantinib (XL184), on BCa cells. METHODS We first conducted Western blot analysis to investigate MET expression in BCa cell lines. Next, we examined the effect of cabozantinib on their proliferation and invasive abilities using MTT and Matrigel invasion assays, respectively. Invasion assays were performed using the xCELLigence system. Additionally, to investigate the biological function of HGF-MET signaling, we analyzed gene expression profiles and performed real-time polymerase chain reaction analyses of 5637 cells that were cultivated with or without HGF stimulation, with or without cabozantinib. RESULTS MET was highly expressed in 4 of 5 BCa cell lines, and 5637 and T24 cells showed especially high protein expression of MET. Cabozantinib suppressed cell proliferation and invasion (cell index; mock, 1.49 vs HGF, 2.26 vs HGF + XL184, 1.47, P < .05). Gene expression profile analysis indicated that matrix metalloproteinase 1 (MMP1) was significantly elevated at the mRNA level with addition of HGF. Moreover, cabozantinib suppressed HGF-induced MMP1 expression in 5637 T24 cells. CONCLUSIONS These data indicate that cabozantinib suppressed MMP1 expression by blocking HGF-MET signaling and that HGF-MET-MMP1 signaling is involved in the invasiveness and proliferation of BCa cells. These results suggest that cabozantinib might prove useful for future treatment of muscle-invasive BCa.

Complications of Flexible Ureteroscopic Treatment for Renal and Ureteral Calculi during the Learning Curve

Background: The flexible ureterorenoscope (URS) and associated devices have developed rapidly. However, despite its therapeutic benefits, URS may be associated with some complications. To the best of our knowledge, there are no studies discussing the complications of flexURS during the learning curve. Methods: A retrospective review of the records of patients who underwent flexURS from January 2005 to June 2013 was performed. To compare the complications after the introduction of flexURS, patients were divided into four groups based on the surgeons training experience, that is, based on the number of cases performed by the surgeon. A total of 219 cases underwent flexURS. Groups 1, 2, 3, and 4 included 35, 50, 50, and 84 cases, respectively. The complications were classified using the Clavien system (I-IV). Results: The mean operation time and stone-free rate were significantly different (p < 0.001, p = 0.013, respectively). The total complication rates were 13.6, 10, 8.3, and 3.2%, respectively (p = 0.068). The more the surgeons experience, the less was the complication rate. Despite our best efforts, the incidence of urosepsis was not reduced (p = 0.902). Conclusions: To reduce severe complications, it is necessary to have performed about 100 cases. Increased surgeon experience tended to decrease the risk of severe complications, but the incidence of urosepsis was not reduced.

Clinical Significance of Neoadjuvant Combined Androgen Blockade for More Than Six Months in Patients with Localized Prostate Cancer Treated with Prostate Brachytherapy.

Introduction: The aim of this study is to clarify the clinical significance of neoadjuvant combined androgen blockade (CAB) for ≥6 months in patients with localized prostate cancer. Patients and Methods: A total of 431 patients with localized prostate cancer who underwent prostate brachytherapy (BT) with or without neoadjuvant CAB for ≥6 months with mean follow-up time of 64.6 months (range 24-108 months) were evaluated retrospectively. Of those 431, 232 patients received BT in combination with neoadjuvant CAB for ≥6 months. Biochemical recurrence-free rates (BRFRs) in 364 patients with at least 3 years of follow-up were evaluated by log-rank test. Results: BRFR in patients with low-, intermediate- and high-risk prostate cancer were 98.1, 94.2 and 89.1%, respectively. In patients with intermediate-risk prostate cancer only, neoadjuvant CAB was significantly associated with BRFR (p = 0.0468). Especially in patients with intermediate-risk prostate cancer with radiation dose received by 90% of the prostate (D90) <180 Gy, neoadjuvant CAB exerted a favorable impact on BRFR (p = 0.0429). On multivariate analyses, neoadjuvant CAB and D90 were independent predictors of BRFR (p = 0.0061 and p < 0.0001, respectively). Conclusions: Neoadjuvant CAB for ≥6 months has a favorable impact on BRFR in patients with intermediate-risk prostate cancer, particularly in patients with relatively low radiation doses of D90.

MP72-06 CORRELATION OF THE INSULIN RECEPTOR EXPRESSION AND THE RESISTANCE TO VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR TYROSINE KINASE INHIBITORS IN ADVANCED CLEAR CELL RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) demonstrate the significant efficacy for advanced clear cell renal cell carcinoma (ccRCC), however, it eventually becomes resistant to VEGFR-TKIs during the treatment. So far, the mechanisms for the resistance to VEGFR-TKIs remain to be fully elucidated. Previously we have identified the gene set which could predict poor prognosis of ccRCC patients (Takahashi M et.al., Proc Natl Acad Sci U S A., 98: 9754, 2001). We examined whether the insulin receptor (INSR) expression in the gene set may correlate with the resistance to VEGFR-TKIs. METHODS: The INSR expression was examined immunohistochemically in the nephrectomy specimens of the RCC patients (n1⁄433) who then received axitinib as the VEGFR-TKI and correlated with their survival outcome. We compared the INSR expression of the nephrectomy or metastasectomy specimens before and after the administration of VEGFR-TKIs in 5 cases. The INSR expression of the human renal glomerular endothelial cells (HGEC) was compared before and after the administration of axitinib by Western blotting. In addition, we established patient derived Xenograft model (PDX) of ccRCC. Tumors of PDX were resected when it regrew and showed the resistance for axitinib and the INSR expression was compared before and after the treatment by Western blotting. RESULTS: The INSR was expressed at the vessels surrounding tumor cells. Progression-free survival (PFS) was significantly shorter in the INSR-negative group than in the INSR-positive group. Multivariate analysis revealed that the INSR expression was the significantly independent predictor of PFS. In the specimens resected after VEGFR-TKI, the INSR expression was more frequently decreased. As the concentration of axitinib increased, the INSR expression in the HGEC was decreased. The tumors of PDX that were resected after demonstrating the resistance to axitinib had the decreased INSR expression. CONCLUSIONS: The decreased INSR expression could be correlated with the resistance to VEGFR-TKI and its expression may be useful in selecting appropriate drugs for advanced ccRCC patients. Source of Funding: None

MP45-17 GALECTIN-3 PLAYS CRITICAL ROLES FOR THE GROWTH OF BENIGN PROSTATIC HYPERPLASIA

INTRODUCTION AND OBJECTIVES: Galectin-3, a multifunctional oncogenic protein, has been reported to play important roles of progression in a variety of cancer including prostate cancer through the regulation of cancer cell proliferation, apoptosis, invasion and metastasis. However, we also identified the frequent up-regulation of Galectin-3 in benign prostatic hyperplasia (BPH), yet its pathophysiological roles in BPH. Here, we report the involvement of Galectin-3 in the growth of BPH. METHODS: To investigate the association of Galectin-3 expression and prostate volume, we examined serum Galectin-3 (pg/ ml) with ELISA method in non-cancer cohort and analyzed the correlation between Galectin-3 expression and prostate volume with Speamans correlation coefficient. Next, to analyzed proliferation abilities and the effect of Galectin-3 on smooth muscle, we examined knockdown of Galectin-3 expression by siRNA in BPH1 cells (benign prostatic hyperplasia cell line) and a co-culture experiment of BPH1 and PrSMC (Normal Human Prostate Smooth Muscle. Cells). Moreover, to investigate biological function of Galectin-3, we examined the gene expression profiles in Galectin-3-depleted BPH1 cells with microarray and bioinformatics analyses. RESULTS: Correlation analysis revealed that serum Galectin-3 (ng/ml) were correlated with prostate volume (R1⁄40.643 p1⁄40.023) (Figure 1). Next, depletion of Galectin-3 suppressed cell proliferation in BPH1 cells. Moreover, depletion of Galectin-3 in BPH1 cells suppressed cell proliferation of PrSMC cells in a co-culture method, suggesting Galectin-3 enhanced proliferation of PrSMC cells. Bioinformatics analysis with GSEA revealed that depletion of Galectin-3 was involved in interferon a response and interferon a response and interferon ? response (FDR q value < 0.001) (Figure 2), suggesting Galectin-3 regulates the proliferation of PrSMC through interferon response. CONCLUSIONS: Our findings suggest that Galectin-3 is significantly involved in the growth of BPH and a promising therapeutic target for patients with BPH. Source of Funding: GSK Japan Research Grant 2016