Impact of prior bevacizumab therapy on the incidence of ramucirumab-induced proteinuria in colorectal cancer: a multi-institutional cohort study.

BACKGROUND: The association between prior bevacizumab (BEV) therapy and ramucirumab (RAM)-induced proteinuria is not known. We aimed to investigate this association in patients with metastatic colorectal cancer (mCRC). METHODS: mCRC patients who received folinic acid, fluorouracil, and irinotecan (FOLFIRI) plus RAM were divided into with and without prior BEV treatment groups. The cumulative incidence of grade 2-3 proteinuria and rate of RAM discontinuation within 6 months (6M) after RAM initiation were compared between the two groups. RESULTS: We evaluated 245 patients. In the Fine-Gray subdistribution hazard model including prior BEV, age, sex, comorbidities, eGFR, proteinuria ≥ 2 + at baseline, and later line of RAM, prior BEV treatment contributed to proteinuria onset (P < 0.01). A shorter interval between final BEV and initial RAM increased the proteinuria risk; the adjusted odds ratios (95% confidence intervals) for the intervals of < 28 days, 28-55 days, and > 55 days (referring to prior BEV absence) were 2.60 (1.23-5.51), 1.51 (1.01-2.27), and 1.04 (0.76-1.44), respectively. The rate of RAM discontinuation for ≤ 6M due to anti-VEGF toxicities was significantly higher in the prior BEV treatment group compared with that in the no prior BEV treatment group (18% vs. 6%, P = 0.02). Second-line RAM discontinuation for ≤ 6M without progression resulted in shorter overall survival of 132 patients with prior BEV treatment (P < 0.01). CONCLUSION: Sequential FOLFIRI plus RAM after BEV failure, especially within 55 days, may exacerbate proteinuria. Its escalated anti-VEGF toxicity may negatively impact the overall survival.

Virulence of representative Japanese Francisella tularensis and immunologic consequences of infection in mice.

Francisella tularensis, which causes tularemia, is widely distributed in the Northern hemisphere. F. tularensis strains isolated in Japan are genetically unique from non-Japanese strains; however, their phenotypic properties have not been well studied. Thus, mice were infected with representative Japanese strains of F. tularensis and their virulence and mouse immune responses to them assessed. Of four representative Japanese strains, the Ebina, Jap and Tsuchiya strains were susceptible to H2 O2 and did not grow well intracellularly. Only Yama strain grew intracellularly and was lethal to mice. Infection with Yama strain resulted in drastic increases in IFN-γ, CD4 and CD8 double-positive T cells and Th1 cells (CD3, CD4 and Tim3-positive cells), and a decrease in the ratio of CD8-positive CD4-negative T cells in mice. C57BL/6J mice that survived infection produced IgM antibodies to LPS and IgG2c antibodies to 43, 19 and 17 kDa proteinase K-sensitive components. These data are valuable for understanding the phenotypic properties of F. tularensis in Japan.

Tensin2-deficient mice on FVB/N background develop severe glomerular disease.

Tensin2 (Tns2) is an essential component for the maintenance of glomerular basement membrane (GBM) structures. Tns2-deficient mice were previously shown to develop mild glomerular injury on a DBA/2 background, but not on a C57BL/6J or a 129/SvJ background, suggesting that glomerular injury by the deletion of Tns2 was strongly dependent on the genetic background. To further understand the mechanisms for the onset and the progression of glomerular injury by the deletion of Tns2, we generated Tns2-deficient mice on an FVB/N (FVB) strain, which is highly sensitive to glomerular disease. Tns2-deficient mice on FVB (FVBGN) developed severe nephrotic syndrome, and female FVBGN mice died within 8 weeks. Ultrastructural analysis revealed that FVBGN mice exhibited severe glomerular defects with mesangial process invasion of glomerular capillary tufts, lamination and thickening of the GBM and subsequent podocyte foot process effacement soon after birth. Aberrant laminin components containing α1, α2 and ß1 chains, which are normally expressed in the mesangium, accumulated in the GBM of FVBGN, suggesting that these components originated from mesangial cells that invaded glomerular capillary tufts. Compared to Tns2-deficient mice on the other backgrounds in previous reports, FVBGN mice developed earlier onset of glomerular defects and rapid progression of renal failure. Thus, this study further extended our understanding of the possible genetic background effect on the deterioration of nephrotic syndrome by Tns2 deficiency.

Survey of Francisella tularensis in Wild Animals in Japan in Areas Where Tularemia is Endemic.

Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.

Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.

In vitro antibiotic susceptibility of Francisella tularensis isolates from Japan.

The antibiotic susceptibilities of 36 isolates of Japanese Francisella tularensis, an etiological agent of the zoonotic disease tularemia, were analyzed using the E test. All the isolates were susceptible to ciprofloxacin, doxycycline, erythromycin, and gentamicin but resistant to benzylpenicillin and cephalothin. The susceptibility to seven other ß-lactams (aztreonam, cefotaxime, cefoxitin, ceftriaxone, cefuroxime, imipenem, and meropenem) varied among the isolates. These findings suggest that the guidelines for the antibiotic treatment of tularemia issued by the World Health Organization are appropriate for Japanese tularemia patients.

Identification of the source of Francisella tularensis infection by multiple-locus variable-number tandem repeat analysis.

Tularemia is a zoonotic disease caused by Francisella tularensis. Most patients in Japan have reportedly acquired such infections through direct contact with infected Japanese hares. We recently encountered a patient who contracted tularemia after skinning and butchering a dead hare. Because the remains of the hare were available, we attempted to determine whether the patient actually contracted infection by handling the carcass. F. tularensis-specific sequences were successfully amplified by PCR from the patient specimens as well as from the remnants of discarded hare carcass. PCR amplification of the ISFtu2 and RD1 regions indicated infection by F. tularensis subsp. holarctica, which was considered as a prevalent strain in Japan. Furthermore, high-resolution multiple-locus variable-number tandem repeat analysis (MLVA) showed that the combination of repeat numbers in sequences from the patient and hare were indistinguishable, thus indicating that the patient had been infected with F. tularensis strain that had also infected the hare. These findings demonstrated that MLVA is a useful epidemiological investigational tool to identify possible sources of certain zoonotic diseases such as tularemia.

Tenc1-deficient mice develop glomerular disease in a strain-specific manner.

BACKGROUND/AIMS: Tenc1 (also known as tensin2) is an integrin-associated focal adhesion molecule that is broadly expressed in mouse tissues including the liver, muscle, heart and kidney. A mouse strain carrying mutated Tenc1, the ICR-derived glomerulonephritis (ICGN) strain, develops severe nephrotic syndrome. METHODS: To elucidate the function of Tenc1 in the kidney, Tenc1(ICGN) was introduced into 2 genetic backgrounds, i.e. DBA/2J (D2) and C57BL/6J (B6), strains that are respectively susceptible and resistant to chronic kidney disease. RESULTS: Biochemical and histological analysis revealed that homozygous Tenc1(ICGN) mice develop nephrotic syndrome on the D2 background (D2GN) but not on the B6 background (B6GN). Initially, abnormal assembly and maturation of glomerular basement membrane (GBM) were observed, and subsequently effacement of podocyte foot processes was noted in the kidneys of D2GN but not B6GN mice. These defects are likely to be involved in the integrin signaling pathway. CONCLUSION: This study suggests that Tenc1 contributes to the maintenance of GBM structures and that the genetic background influences the severity of nephrotic syndrome.

Dependence of psychophysical threshold on rate of applied force to the upper first molar in humans.

OBJECTIVE: The study aims to investigate the dynamic perception of a force applied to the upper first molar for different rates of force increase. DESIGN: Six volunteers (four male and two female; mean age, 27.2±2.4 years) with full natural dentition (except for the third molars) participated in this study. The psychophysical threshold for a force applied to the right maxillary first molar and the reaction time corresponding to each threshold were measured for rate of force increase of 103.74, 236.23, 354.58, 478.22 and 584.63 mNs(-1). The physical impulse, which is the integral of force over time, was calculated for each threshold. RESULTS: Psychophysical thresholds in the upper first molar increased with the rate of force increase. The reaction time corresponding to each threshold decreased with increasing force rate. Impulses corresponding to each threshold were independent of force rate. CONCLUSIONS: In the present study, the psychophysical threshold for a force applied to a molar tooth was shown to change depending on the rate of increase of the exerted force. From the viewpoint of the impulse, the dissipated energy necessary to reach the psychophysical sensation threshold was almost constant, regardless of the rate of force increase.

Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form.

BACKGROUND: In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009-2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs). FINDINGS: Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. DISCUSSION: Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly advantageous for the rapid development of molecules to detect target antigens. However, our results also indicated that this strategy might not have been effective for selecting H1N1pdm-specific antibodies during the 2009 pandemic, where the co-circulating sH1N1 virus shared similar antigenic properties. This suggests that it might be advisable to use a synthetic scFv phage display library by strategically considering the characteristics of target antigens and the potential situations.

Adult onset cardiac dilatation in a transgenic mouse line with Galß1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes: a new model for dilated cardiomyopathy.

Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases. Here, we report an adult onset cardiac dilatation in a transgenic mouse line with Galß1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes. The transgenic hearts at the end-stage, at around 7 months old, were enlarged, with enlarged cavities and thin, low-tensile walls, typical of dilated cardiomyopathy. Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered. Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed. Significant increases in calreticulin and calnexin were observed in hearts of the transgenic mice. These results suggest that expression of ST3Gal-II transgenes induces abnormal protein glycosylation, which disorganizes the endoplasmic/sarcoplasmic reticulum quality control system and elevates the calreticulin/calnexin level, resulting in suppression of cardiac function. The transgenic mice showed 100% incidence of adult onset cardiac dilatation, suggesting great potential as a new model for dilated cardiomyopathy.

Type 2 diabetes mellitus in a non-obese mouse model induced by Meg1/Grb10 overexpression.

We assessed the possibility of C57BL/6-Tg (Meg1/Grb10)isn(Meg1 Tg) mice as a non-obese type 2 diabetes (2DM) animal model. Meg1 Tg mice were born normal, but their weight did not increase as much as normal after weaning and showed about 85% of normal size at 20 weeks of age. Body mass index of Meg1 Tg mice was also smaller than that of control mice. The glucose tolerance test and insulin tolerance test showed that Meg1 Tg mice had reduced ability to normalize the blood glucose level. Blood urea nitrogen (BUN) in Meg1 Tg mice (19.6 +/- 1.2 mg/dl) was significantly lower than in controls (22.0 +/- 0.8 mg/dl), while plasma triglyceride, insulin, adiponectin, and resistin levels were significantly higher (202.0 +/- 23.4 mg/dl vs 146.3 +/- 23.4 mg/dl, 152.4 +/- 16.3 pg/ml vs 88.1 +/- 16.9 pg/ml, 74.4 +/- 10.9 microg/ml vs 48.3 +/- 7.0 microg/ml, and 4.0 +/- 0.2 ng/ml vs 3.6 +/- 0.2 ng/ml, respectively). Body, visceral fat weight and liver weights were significantly lower (19.6 +/- 0.4 g vs 24.3 +/- 0.3 g, 376.7 +/- 29.6 mg to 507.5 +/- 23.0 mg, and 906.0 +/- 41.8 mg to 1,001.0 +/- 15.1 mg, respectively). Thus, hyperinsulinemia observed in Meg1 Tg mice indicates that their insulin signaling pathway is somehow inhibited. With high fat diet, the diabetes onset rate of Meg1 Tg mice increased up to 60%. These results suggest that Meg1 Tg mice resemble human 2DM.

Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray.

The genetic diagnosis of pathogenic agents using microarrays has the advantage of high-throughput detection, but a relatively large amount of DNA sample is required. To obtain a sufficient amount of DNA for molecular diagnoses, several whole genome amplification (WGA) methods have been proposed. In this study, using Francisella tularensis and Escherichia coli as models, we compared four WGA methods in terms of their efficiency of amplification of whole genomic DNA in order to identify the most suitable method for preparing DNA to be used for microarray analysis. It was possible to obtain more than 1.5 microg of products from 10 ng of F. tularensis and E. coli genomic DNA using four methods, but biases in the amplification of bacterial genes were least prominent in the multiple displacement amplification (MDA) or OmniPlex WGA. When the amplified DNAs were applied to microarray slides consisting of 32 different genes probes, DNAs amplified by Phi29 v2 of MDA and OmniPlex WGA showed high signal intensity as well as a high signal-to-noise ratio for all 32 genes. These results indicate that Phi29 v2 and OmniPlex WGA are useful methods for obtaining sufficient DNA from a limited amount of samples for the detection of microbes using microarrays.

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I.

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4(-) and CD8(-), but CD44(+), CD25(+) and cytoplasmic CD3(+). This phenotype is indicative of a thymus-derived pre-T-cell phenotype, and disease development was associated with the constitutive activation of NF-kappaB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.

Transgene insertion pattern analysis using genomic walking in a transgenic mouse line.

A transgene mapping technique (Noguchi et al., Exp. Anim. 53:103-111, 2004) is described that can be used to analyze transgene integration patterns in transgenic mice. The technique was used to reveal that a transgenic mouse line (GM1-sy#116) harbored inverted and direct tandem repeats of both intact and partial pCAGGS-based transgenes in the G2 region of chromosome 1. This complicated concatenation of transgenes may have been caused by simple end-joining of DNA constructs fragmented by exposure to UV transillumination during gel-purification, and by nuclease digestion inside zygote pronuclei. The results suggest that care should be taken to avoid unwanted fragmentation during the preparation of vector constructs.

Erythropoietin-producing cells in the liver of ICR-derived glomerulonephritis (ICGN) mice.

The ICR-derived glomerulonephritis (ICGN) mouse is an appropriate model for anemia associated with chronic renal disorder (CRD). Insufficient renal production of erythropoietin (EPO) induces the anemia associated with CRD. EPO mRNA is expressed in both kidneys and liver of progressing-stage ICGN mice. Hypoxic stimulation induced the EPO mRNA expression in the liver as well as in the kidneys of ICGN mice. The localization of EPO-producing cells in the liver remains controversial. Present study using an amplified in situ hybridization technique identified that nonparenchymal cells were the source of hepatic EPO production in ICGN mice under both normoxia and hypoxia.

Dysfunction of erythropoietin-producing interstitial cells in the kidneys of ICR-derived glomerulonephritis (ICGN) mice.

Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O2) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD.

Effect of oral glucose administration on serum potassium concentration in hemodialysis patients.

BACKGROUND: Extrarenal potassium disposal is particularly critical in patients with end-stage renal disease. Exogenous insulin stimulates this disposal by enhancing potassium uptake into cells in hemodialysis (HD) patients and healthy subjects. However, the effect of physiological levels of endogenous insulin on this disposal in these patients or healthy subjects is unknown. METHODS: Effects of an oral glucose tolerance test (37.5, 75, and 150 g) on serum potassium levels were determined in 13 HD patients and 7 healthy controls. Serum potassium and insulin levels and plasma aldosterone and epinephrine levels were measured before and after glucose loads. RESULTS: In HD patients and controls, serum insulin levels increased to a similar magnitude in parallel with increased serum glucose levels, but serum potassium levels decreased significantly only in HD patients. In HD patients, plasma aldosterone or epinephrine levels were not changed significantly after a glucose load. In HD patients, the decrease in serum potassium levels was dependent on the increase in serum insulin levels and was more prominent when 150 g of glucose was administered. In HD patients, the decrease in serum potassium levels correlated negatively (r = -0.45; P < 0.001) with the increase in serum insulin levels, and maximal decrease in serum potassium levels correlated negatively (r = -0.54; P < 0.001) with maximal increase in serum insulin levels. CONCLUSION: Endogenous production of physiological concentrations of insulin in response to exogenous glucose administration decreases serum potassium levels only in HD patients, independently of plasma aldosterone and epinephrine levels.

Transforming growth factor-beta1 mediated up-regulation of lysyl oxidase in the kidneys of hereditary nephrotic mouse with chronic renal fibrosis.

Lysyl oxidase (LOX), an extracellular enzyme, plays a key role in the post-translational modification of collagens and elastin, catalyzing inter- and intra-crosslinking reactions. Because the crosslinked extracellular matrices (ECMs) are highly resistant to degradative enzymes, it is considered that the over-expression of LOX may cause severe fibrotic degeneration. In the present study, we addressed the role of LOX-mediated crosslinking in chronic renal tubulointerstitial fibrosis using an animal model of hereditary nephrotic syndrome, the Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse. Ribonuclease protection assay (RPA) revealed that LOX mRNA expression was up-regulated in the kidneys of ICGN mice as compared with control ICR mice. High-level expression of LOX and transforming growth factor (TGF)-beta1 (an up-regulator of LOX) mRNA was detected in tubular epithelial cells of ICGN mouse kidneys by in situ hybridization. Type-I and -III collagens, major substrates for LOX, were accumulated in tubulointerstitium of ICGN mouse kidneys. The present findings imply that TGF-beta1 up-regulates the production of LOX in tubular epithelial cells of ICGN mouse kidneys, and the excessive LOX acts on interstitial collagens and catalyzes crosslinking reactions. As a result, the highly crosslinked collagens induce an irreversible progression of chronic renal tubulointerstitial fibrosis in the kidneys of ICGN mice.