Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.     

Clinicopathological and long-term prognostic features of membranous nephropathy with crescents: a Japanese single-center experience

Background

Three recent studies from the United States and China reported the clinicopathological features and short-term prognosis in patients with membranous nephropathy (MN) and crescents in the absence of secondary MN, anti-glomerular basement membrane (GBM) antibodies, and anti-neutrophil cytoplasmic antibodies (ANCA).

Methods

We compared clinicopathological and prognostic features in 16 MN patients with crescents (crescent group) and 38 MN patients without crescents (control group), in the absence of secondary MN, anti-GBM antibodies, and ANCA. Median follow-up periods in the crescent and control groups were 79 and 50 months, respectively.

Results

Decreased estimated glomerular filtration rates (<50 mL/min/1.73 m²), glomerulosclerosis, and moderate-to-severe interstitial fibrosis were more frequently observed in the crescent group than in the control group (P = 0.043, P = 0.004, and P = 0.035, respectively). Positive staining rates for glomerular IgG2 and IgG4 were significantly different between the 2 groups (P = 0.032, P = 0.006, respectively). Doubling of serum creatinine during follow-up was more frequently observed in the crescent group than in the control group (P = 0.002), although approximately two-thirds of patients in the crescent group were treated with immunosuppressive therapy. Crescent formation and interstitial fibrosis were risks for doubling of serum creatinine [hazard ratio (HR) = 10.506, P = 0.012; HR = 1.140, P = 0.009, respectively].

Conclusions

This is the first Japanese study demonstrating significant differences in clinicopathological and prognostic features between the 2 groups. Most patients in the crescent group may develop a long-term decline in renal function despite immunosuppressive therapy.

     

Intravital imaging of DSS-induced cecal mucosal damage in GFP-transgenic mice using two-photon microscopy

Background

Two-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging.

Materials and methods

An organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM.

Results and conclusions

We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin–GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.     

Assessment of coronary artery disease in hemodialysis patients with delayed systolic blood pressure response after exercise testing

BACKGROUND: We evaluated usefulness of the postexercise systolic blood pressure (SBP) response for detecting coronary artery disease (CAD) in hemodialysis patients. METHODS: A treadmill exercise testing was done, and the SBP response was measured in 44 hemodialysis patients (30 men, 14 women; age 41 to 81 years). The postexercise SBP response was defined as the ratio of SBP after 3 minutes of recovery to SBP at peak exercise. RESULTS: The SBP ratio of the 25 subjects with coronary artery stenosis (1.01+/- 0.13) was significantly greater (p<0.01) than 19 subjects without coronary artery stenosis (0.83+/- 0.10). An SBP ratio greater than 0.92 identified CAD with higher sensitivity, specificity, and accuracy than did the conventional ST-segment depression criterion (76 vs. 56%, 90 vs. 53%, and 82 vs. 55%, respectively). CONCLUSION: Determination of the SBP ratio is a clinically useful, noninvasive method for accurately detecting CAD in hemodialysis patients.     

Sensitivities to various epidermal growth factor receptor‐tyrosine kinase inhibitors of uncommon epidermal growth factor receptor mutations L861Q and S768I: What is the optimal epidermal growth factor receptor‐tyrosine kinase inhibitor?

Most patients with non‐small cell lung cancer (NSCLC) harboring common epidermal growth factor receptor (EGFR) mutations, such as deletions in exon 19 or the L858R mutation in exon 21, respond dramatically to EGFR tyrosine kinase inhibitors (EGFR‐TKI), and their sensitivities to various EGFR‐TKI have been well characterized. Our previous article showed the in vitro sensitivities of EGFR exon 18 mutations to EGFR‐TKI, but little information regarding the sensitivities of other uncommon EGFR mutations is available. First, stable transfectant Ba/F3 cell lines harboring EGFR L858R (Ba/F3‐L858R), L861Q (Ba/F3‐L861Q) or S768I (Ba/F3‐S768I) mutations were created and their drug sensitivities to various EGFR‐TKI were examined. Both the Ba/F3‐L861Q and Ba/F3‐S768I cell lines were less sensitive to erlotinib, compared with the Ba/F3‐L858R cell line, but their sensitivities to afatinib were similar to that of the Ba/F3‐L858R cell line. The Ba/F3‐L861Q cell line was similarly sensitive and the Ba/F3‐S768I cell line was less sensitive to osimertinib, compared with the Ba/F3‐L858R cell line. The results of western blot analyses were consistent with these sensitivities. Next, similar experiments were also performed using the KYSE270 (L861Q) and KYSE 450 (S768I) cell lines, and their results were compatible with those of the transfectant Ba/F3 cell lines. Our findings suggest that NSCLC harboring the EGFR L861Q mutation might be sensitive to afatinib or osimertinib and that NSCLC harboring the EGFR S768I mutation might be sensitive to afatinib. Overall, afatinib might be the optimal EGFR‐TKI against these uncommon EGFR mutations.     

Current Issues and Future Directions of Oncolytic Adenoviruses

Oncolytic adenoviruses (Ads) constitute a promising new class of anticancer agent. They are based on the well-studied adenoviral vector system, which lends itself to concept-driven design to generate oncolytic variants. The first oncolytic Ad was approved as a drug in China in 2005, although clinical efficacy observed in human trials has failed to reach the high expectations that were based on studies in animal models. Current obstacles to the full realization of efficacy of this class of anticancer agent include (i) limited efficiency of infection and specific replication in tumor cells, (ii) limited vector spread within the tumor, (iii) imperfect animal models and methods of in vivo imaging, and (iv) an incomplete understanding of the interaction of these agents with the host. In this review, we discuss recent advances in the field of oncolytic Ads and potential ways to overcome current obstacles to their clinical application and efficacy.     

Influences of exposure to cigarette smoke on concentration of nicorandil in plasma of rats.

Influences of acute exposure to cigarette smoke on plasma concentrations of nicorandil administered orally and parenterally were investigated in rats by HPLC. The animals were exposed to tobacco smoke of two kinds of cigarettes using a smoking machine (i.e., the cigarette smoke contained either low or high nicotine and tar). The plasma concentration of nicorandil administered orally at a dose of 10 mg/kg had a lower absorption phase in two cigarette smoke-exposed groups, particularly in the high nicotine and tar-containing cigarette smoke-exposed group, compared with the nonsmoking control group. The AUC and MRT values in a high nicotine and tar-containing cigarette smoke-exposed group were lower and higher, respectively, than in the nonsmoking control group. However, there was no marked difference in nicorandil plasma concentrations between the cigarette smoke-exposed group and the nonsmoking control group when nicorandil was administered ip or iv at a dose of 5 mg/kg. These results suggest that cigarette smoke exposure causes the suppression or delay of absorption of nicorandil from the gastrointestinal tract.     

Cellular Immune Response against Tropomyosin Isoform 5 in Ulcerative Colitis

We have reported an autoantibody response in ulcerative colitis (UC) against human tropomyosin isoform 5 (hTM5), the predominant colonic epithelial cell hTM isoform. In this report, we determined the number of IFN-gamma-secreting cells (spot-forming cells, SFC) against hTM5 by an enzyme-linked immunospot (ELISPOT) assay. Another cytoskeletal protein, caldesmon, CaD40, was used as a control antigen. Peripheral blood mononuclear cells were separated by a Ficoll density gradient from 28 patients with UC, 13 patients with Crohn's disease (CD), and 9 healthy subjects (HS). The mean (+/-SEM) SFC values against hTM5 in UC, CD, and HS were 48.8 +/- 8.1, 18.6 +/- 4.6, and 20.8 +/- 8.6, respectively. The value in UC was significantly higher than those in CD (P < 0.005) and HS (P < 0.025). SFC values in CD did not differ from those in HS. None of the 50 samples (except 1 UC) reacted to the CaD40 antigen. This study demonstrates, for the first time, a defined colon epithelial cell antigen, hTM5, that is capable of inducing a significant T cell response in UC but not in CD.