Epidemiological analysis of a large enterohaemorrhagic Escherichia coli O111 outbreak in Japan associated with haemolytic uraemic syndrome and acute encephalopathy.

A large outbreak of enterohaemorrhagic Escherichia coli (EHEC) O111 and O157 occurred in Japan in April 2011. We conducted an unmatched case-control study and trace-back investigation to determine the source of EHEC O111 infection and risk factors for severe complications. Pulsed-field gel electrophoresis was performed to help define cases. A total of 86 individuals met the case definition. Of these, 40% experienced haemolytic uraemic syndrome (HUS), 24% acute encephalopathy, and 6% died. Illness was significantly associated with eating the raw beef dish yukhoe (odds ratio 19·64, 95% confidence interval 7·03-54·83), the likely food vehicle. EHEC O111 and its closely related stx-negative variants were found in the beef. HUS occurred most frequently in individuals aged 5-9 years, and this age group was significantly associated with acute encephalopathy. The prevalence of HUS and acute encephalopathy was higher than in previous non-O157-related outbreaks, indicating a high risk of severe complications.

The effects of dexmedetomidine on inflammatory mediators after cardiopulmonary bypass.

Cardiac surgery with cardiopulmonary bypass is associated with the development of a systemic inflammatory response that can often lead to dysfunction of major organs. We hypothesised that the highly selective α2-adrenergic agonist, dexmedetomidine, attenuates the systemic inflammatory response. Forty-two patients were randomly assigned to receive dexmedetomidine or saline after aortic cross-clamping). The mean (SD) levels of the nuclear protein plasma high-mobility group box 1 increased significantly from 5.1 (2.2) ng ml(-1) during (16.6 (7.3) ng ml(-1) ) and after (14.3 (8.2) ng ml(-1) ) cardiopulmonary bypass in the saline group. In the dexmedetomidine group, the levels increased significantly only during cardiopulmonary bypass (4.0 (1.9) ng ml(-1) baseline vs. 10.8 (2.7) ng ml(-1) ) but not after (7.4 (3.8) ng ml(-1) ). Dexmedetomidine infusion also suppressed the rise in mean (SD) interleukin-6 levels after cardiopulmonary bypass (a rise of 124.5 (72.0) pg ml(-1) vs. 65.3 (30.9) pg ml(-1)). These suppressive effects of dexmedetomidine might be due to the inhibition of nuclear factor kappa B activation and suggest that intra-operative dexmedetomidine may beneficially inhibit inflammatory responses associated with ischaemia-reperfusion injury during cardiopulmonary bypass.

Predominant involvement of the cerebellum in guinea pigs infected with bovine spongiform encephalopathy (BSE).

This study reports the experimental transmission of bovine spongiform encephalopathy (BSE) to guinea pigs and describes the cerebellar lesions in these animals. Guinea pigs were inoculated intracerebrally with 10% brain homogenates from BSE-affected cattle. These animals were designated as the first passage. Second and third passages were subsequently performed. All guinea pigs developed infection at each passage. The mean incubation period of the first passage was 370 days post-infection (dpi) and this decreased to 307 dpi and 309 dpi for the second and third passages, respectively. Mild to severe spongiform degeneration and gliosis were observed in the cerebral cortex, thalamus and brainstem. In addition, the affected animals had marked pathological changes in the cerebellum characterized by severe cortical atrophy associated with Bergmann radial gliosis of the molecular layer and reduction in the width of the granular cell layer. Immunohistochemically, intense PrP(Sc) deposition and scattered plaque-like deposits were observed in the molecular and granular cell layers. Cerebellar lesions associated with severe atrophy of the cortex have not been reported in animal prion diseases, including in the experimental transmission of PrP(Sc) to small rodents. These lesions were similar to the lesions of human kuru or the VV2 variant of sporadic Creutzfeldt-Jakob disease, although typical kuru plaques or florid plaques were not observed in the affected animals.

Antigen retrieval using sodium hydroxide for prion immunohistochemistry in bovine spongiform encephalopathy and scrapie.

Formalin-fixed and paraffin wax-embedded (FFPE) tissue sections are usually used for histopathological and immunohistochemical analyses in prion diseases in animals and man. However, formalin fixation cross-links proteins, reducing disease-associated prion protein (PrP(Sc)) immunolabelling. To detect PrP(Sc) in animals naturally affected with bovine spongiform encephalopathy (BSE) and scrapie, we applied minimal pretreatment with sodium hydroxide (NaOH). This simple pretreatment, combined with enzymatic digestion using proteinase K (PK), was equally effective in the detection of PrP(Sc) in FFPE tissue, and superior in terms of speed, compared with the usual autoclaving method. The most effective results, without any section loss, were obtained with 10 µg/ml PK in phosphate buffered saline containing 0.1% Triton-X at room temperature for 10 min and 150 mM NaOH at 60 °C for 10 min. By this simple procedure, PrP(Sc) was visualized in the brain of animals with BSE and scrapie using a range of anti-PrP primary antibodies.

Evaluation of telomerase activity in non-genital Bowen's disease.

We investigated the level of telomerase activity (TA) in 17 specimens of non-genital Bowen's disease (BD) and in 14 specimens of skin without sun exposure (non-exposed skin) using a non-isotopic PCR-based telomeric repeat amplification protocol (TRAP) assay. Expression of human telomerase reverse transcriptase (hTERT; the catalytic subunit of telomerase) was also evaluated by immunochemistry in the non-genital BD tissues. Moderate to high levels of TA were detected in 41.2% of 17 non-genital BD specimens (P = 0.001). In contrast, TA was not evident in non-exposed skin. Recently, nucleolin was reported to be associated with hTERT, so we used this antibody instead of hTERT antibody. Immunohistochemistry showed that nucleolin expression was associated with high TA levels in non-genital BD. Our results also revealed differences of TA levels among non-genital BD specimens. High levels of TA in those specimens were not age related. Five out of 7 specimens (71.4%) with moderate to high TA levels were from sun-exposed sites, while the remaining 10 specimens with low levels of TA were from non-exposed sites. These results suggested that cellular DNA damage caused by ultraviolet irradiation might be associated with an increase of TA in non-genital BD. Among non-genital BD specimens, 4 out of 17 (23.5%) showed high levels of TA (median relative TA value: 79.8%; P = 0.003), which might be associated with immortalization or transformation to invasive squamous cell carcinoma.

Mechanism of NKT cell activation by intranasal coadministration of alpha-galactosylceramide, which can induce cross-protection against influenza viruses.

In a nasal vaccine against influenza, the activation of natural killer T (NKT) cells by intranasal coadministration of alpha-galactosylceramide (alpha-GalCer) can potently enhance protective immune responses. The results of this study show that the NKT cell-activated nasal vaccine can induce an effective cross-protection against different strains of influenza virus, including H5 type. To analyze the mechanism of NKT cell activation by this nasal vaccine, we prepared fluorescence-labeled alpha-GalCer by which we detect a direct interaction between NKT cells and alpha-GalCer-stored dendritic cells in nasal mucosa-associated tissues. Accordingly, although very few NKT cells exist at mucosa, the nasal vaccination induced a localized increase in NKT cell population, which is partly dependent on CXCL16/CXCR6. Furthermore, we found that NKT cell activation stimulates mucosal IgA production by a mechanism that is dependent on interleukin (IL)-4 production. These results strengthen the basis of nasal vaccination via NKT cell activation, which can induce immune cross-protection.

Tissue distribution of cynomolgus adeno-associated viruses AAV10, AAV11, and AAVcy.7 in naturally infected monkeys.

Adeno-associated virus (AAV) is used in gene-therapy studies, but its tissue distribution is unknown in natural infection. We examined cynomolgus AAVs (previously isolated AAV10 and AAV11 and novel AAVcy.7) for their tissue distribution in 14 cynomolgi by type-specific PCR. We found AAV10, AAV11, and AAVcy.7 in 6, 10, and 14 monkeys, respectively, and two or three types in 11 monkeys, showing that these AAVs are widespread in the monkeys. We detected AAV at a higher level mainly in the lymphatic tissues and ileum, which suggests that AAV may invade the host through Peyer's patches in the ileum and infect immune cells.

Species-specificity of a panel of prion protein antibodies for the immunohistochemical study of animal and human prion diseases.

Monoclonal antibodies to the prion protein (PrP) have been of critical importance in the neuropathological characterization of PrP-related disease in men and animals. To determine the influence of species-specific amino-acid substitutions recognized by monoclonal antibodies, and to investigate the immunohistochemical reactivity of the latter, analyses were carried out on brain sections of cattle with bovine spongiform encephalopathy, sheep with scrapie, mice infected with scrapie, and human beings with Creutzfeldt-Jakob disease (CJD) or Gerstmann-Sträussler-Sheinker disease (GSS). Immunoreactivity varied between the antibodies, probably as the result of differences in the amino-acid sequence of the prion protein in the various species. Some monoclonal antibodies against mouse recombinant PrP gave strong signals with bovine, ovine and human PrP(Sc), in addition to murine PrP(Sc), even though the amino-acid sequences determined by the antibody epitope are not fully identical with the amino-acid sequences proper to the species. On the other hand, in certain regions of the PrP sequence, when the species-specificity of the antibodies is defined by one amino-acid substitution, the antibodies revealed no reactivity with other animal species. In the region corresponding to positions 134-159 of murine PrP, immunohistochemical reactivity or species-specificity recognized by the antibodies may be determined by one amino acid corresponding to position 144 of murine PrP. Not all epitopes recognized by a monoclonal antibody play an important role in antigen-antibody reactions in immunohistochemistry. The presence of the core epitope is therefore vital in understanding antibody binding ability.

Effects of epidural anaesthesia on surgical stress-induced immunosuppression during upper abdominal surgery.

BACKGROUND: Previously, we have demonstrated that surgical stress rapidly induced transient hyporesponsiveness of blood cells to endotoxin and that monocyte mCD14 and HLA-DR expression decreased soon after the start of surgery under general anaesthesia. This study was designed to investigate the effects of epidural anaesthesia on surgical stress-induced immunosuppression in patients undergoing upper abdominal surgery. METHODS: After having obtained informed consent, patients were randomly allocated to receive general anaesthesia (Group G) or general anaesthesia with epidural anaesthesia (Group E). Perioperative changes in neutrophil phagocytic activity, neutrophil respiratory burst activity, monocyte mCD14 and HLA-DR expression, plasma IL-10 concentration, and the LPS-induced TNF-alpha production in whole blood were measured. RESULTS: Surgical stress rapidly depressed neutrophil phagocytic activity, monocyte mCD14 and HLA-DR expression, and LPS-induced TNF-alpha production ex vivo (P < 0.05 vs preoperation) in both Group G and Group E. In contrast, the plasma IL-10 concentration increased significantly 2 h after the start of surgery (P < 0.05) in both groups. There were no significant differences between the two groups. The neutrophil respiratory burst activity did not change during the operation in either group. CONCLUSION: This study showed that the innate immune system is suppressed from the early period of upper abdominal surgery. Subgroup analysis suggested that epidural anaesthesia to T4 dermatome as well as general anaesthesia may not protect patients from this immunosuppression. These results in part explain the impairment of host-defense mechanisms seen in the perioperative period.

Inhibitory effect of gabapentin on N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

BACKGROUND: Gabapentin (GBP) is a prescription drug used for the treatment of neuropathic and post-operative pain. However, the mechanism by which it exerts its analgesic action is not well understood. Because intrathecal administration of GBP has been shown to exert antinociceptive effects in animal studies, we hypothesized that the spinal cord may be a plausible action site. METHODS: We examined the effects of GBP on neurotransmitter-gated ion channels and G protein-coupled inwardly rectifying potassium (GIRK) channels distributed in the spinal cord and involved in pain modulation. Recombinant human NR1/NR2A N-methyl-D-aspartate (NMDA), alpha(1)beta(2)gamma(2S)gamma-aminobutyric acid type A (GABA(A)) or alpha(1) glycine receptors, or GIRK1/GIRK2 channels were expressed in Xenopus laevis oocytes and the effects of GBP (0.1-1000 microM) on them were assessed using a two-electrode, voltage-clamp system. RESULTS: GABA(A) and glycine receptors and GIRK channels were not affected by GBP, even at the highest concentrations. Conversely, NMDA receptors were inhibited by GBP in a concentration-dependent manner, with significant inhibition observed at 10 microM. At 30 microM, GBP inhibited the glutamate-concentration response curve without changing the half-maximal effective concentration or the Hill coefficient, indicating a non-competitive inhibition. Glycine decreased the inhibitory effect in a concentration-dependent manner. CONCLUSIONS: These findings suggest that the inhibitory effect of GBP on NMDA receptors may play an important role in the antinociceptive property of GBP; however, it does not appear that GABA(A) and glycine receptors or GIRK channels contribute to the pharmacological properties of GBP.

HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination.

An ATM-dependent cellular signal, a DNA-damage response, has been shown to be involved during infection of human immunodeficiency virus type-1 (HIV-1), and a high incidence of malignant tumor development has been observed in HIV-1-positive patients. Vpr, an accessory gene product of HIV-1, delays the progression of the cell cycle at the G2/M phase, and ATR-Chk1-Wee-1, another DNA-damage signal, is a proposed cellular pathway responsible for the Vpr-induced cell cycle arrest. In this study, we present evidence that Vpr also activates ATM, and induces expression of gamma-H2AX and phosphorylation of Chk2. Strikingly, Vpr was found to stimulate the focus formation of Rad51 and BRCA1, which are involved in repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), and biochemical analysis revealed that Vpr dissociates the interaction of p53 and Rad51 in the chromatin fraction, as observed under irradiation-induced DSBs. Vpr was consistently found to increase the rate of HR in the locus of I-SceI, a rare cutting-enzyme site that had been introduced into the genome. An increase of the HR rate enhanced by Vpr was attenuated by an ATM inhibitor, KU55933, suggesting that Vpr-induced DSBs activate ATM-dependent cellular signal that enhances the intracellular recombination potential. In context with a recent report that KU55933 attenuated the integration of HIV-1 into host genomes, we discuss the possible role of Vpr-induced DSBs in viral integration and also in HIV-1 associated malignancy.

Novel real-time monitoring system for human cytomegalovirus-infected cells in vitro that uses a green fluorescent protein-PML-expressing cell line.

Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

Effusion and solid lymphomas have distinctive gene and protein expression profiles in an animal model of primary effusion lymphoma.

Lymphoma usually forms solid tumours in patients, and high expression levels of adhesion molecules are observed in these tumours. However, Kaposi's sarcoma-associated herpesvirus (KSHV)-related primary effusion lymphoma (PEL) does not form solid tumours and adhesion molecule expression is suppressed in the cells. Inoculation of a KSHV-associated PEL cell line into the peritoneal cavity of severe combined immunodeficiency mice resulted in the formation of effusion and solid lymphomas in the peritoneal cavity. Proteomics using two-dimensional difference gel electrophoresis and DNA microarray analyses identified 14 proteins and 105 genes, respectively, whose expression differed significantly between effusion and solid lymphomas. Five genes were identified as having similar expression profiles to that of lymphocyte function-associated antigen 1, an important adhesion molecule in leukocytes. Among these, coronin 1A, an actin-binding protein, was identified as a molecule showing high expression in solid lymphoma by both DNA microarray and proteomics analyses. Western and northern blotting showed that coronin 1A was predominantly expressed in solid lymphomas. Moreover, KSHV-encoded lytic proteins, including viral interleukin-6, were highly expressed in effusion lymphoma compared with solid lymphoma. These data demonstrate that effusion and solid lymphomas possess distinctive gene and protein expression profiles in our mouse model, and suggest that differences in gene and protein expression between effusion and solid lymphomas may be associated with the formation of effusion lymphoma or invasive features of solid lymphoma. Furthermore, the results obtained using this combination of proteomics and DNA microarray analyses indicate that protein synthesis partly reflects, but does not correlate strictly with, mRNA production.

Genomic characterization of HBV genotype F in Bolivia: genotype F subgenotypes correlate with geographic distribution and T(1858) variant.

Hepatitis B virus (HBV) strains were classified into eight genotypes from A to H. Genotype F, an indigenous genotype in Central and South America, has been classified into subgenotypes. An in-depth phylogenetic analysis was performed using two full-length Bolivian HBV sequences and other genotype F strains from the database. A novel nomenclature of subgenotypes of genotype F was proposed, in which Bolivia strains belonged to subgenotype F4. This subgenotype had both Leu(45) and Ile(110) in the S gene, and linked to the T(1858) in the precore. This novel nomenclature demonstrated the relation between variability of the HBV genome and the restricted geographical distribution of the virus in some parts of Central and South America.

Bowen's carcinoma of the scrotal skin associated with human papillomavirus type 82.

We have previously cloned human papillomavirus type 82 (HPV-82) from a vaginal intraepithelial neoplasia, but it is not known whether HPV-82 can induce a cutaneous lesion. A large erosive nodule developed on the scrotum of a 50-year-old Japanese patient. Histopathologically, the lesion was composed of two distinct parts; one part showing changes characteristic of Bowen's disease in the epidermis, and the other showing elongated rete ridges and proliferation of atypical basaloid cells in the dermis. These parts were partially connected, giving the diagnosis of Bowen's carcinoma. Immunohistochemically, HPV capsid antigen was detected only in the nuclei of a few cells on the upper part of the epidermis. HPV-82 was identified in the lesion by blot hybridization and viral DNA was demonstrated in the lesion by in situ hybridization. HPV-82 has tropism for both the skin and the genital regions.

Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid.

We assessed the requirement of the host cytoskeleton for the intracytosolic transport of incoming human cytomegalovirus (HCMV) capsids. Treatments with microtubule (MT)-depolymerizing drugs nocodazole and colchicine led to a drastic decrease in levels of IE1 antigen, whereas cytochalasin B had no effect on the level of IE1 as determined by Western blot analyses. Sequential treatment including nocodazole washout and removal of cell surface virion revealed that HCMV entry into the cells occurred normally in the absence of the MT network. This finding was also supported by data obtained by monitoring pUL83 signals with an immunofluorescent assay (IFA). Furthermore, we demonstrated a close association of incoming HCMV capsids with MTs by IFA and ultrastructural analyses. In the absence of the MT network, the capsids which had entered the cytoplasm did not move to close proximity of the nucleus. These data suggest that HCMV capsids associate with the MT network to facilitate their own movement to the nucleus before the onset of immediate-early (IE) gene expression and that this association is required to start efficient IE gene expression.

Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice.

The present study demonstrates that a clonal derivative (HF10) of HSV-1 strain HF effectively treated disseminated peritoneal neoplasm in an immunocompetent animal model and that all of survived mice acquired resistance to rechallenge with tumor cells. The survival time of mice treated with HF10 was longer than that of mice treated with hrR3, indicating that the oncolytic effect of HF10 was more potent than that of hrR3 in this animal model. HF10 induces syncytia formation in vitro, whereas hrR3 forms rounded CPE. The sequential administration of HF10 gave a long term survival of more than 90 days after tumor injection, with no signs of disease, in 8 of the 9 treated mice. The results suggest that treatment of disseminated peritoneal tumor with HF10 induces a specific antitumor immune response. Genomic structure determination showed that HF10 has a deletion of 3.9-kilobase pair (kbp) in the right end of UL and UL/IRL junction, resulting in the loss of UL 56 expression. A 2.3 kbp deletion and extensive rearrangement were also observed in the left end of the genome.

Protean manifestations of human papillomavirus type 60 infection on the extremities.

BACKGROUND: Human papillomavirus type 60 (HPV-60) induces a ridged wart or an epidermal cyst on the sole of the foot, exhibiting identical pathological changes, with a single refractile eosinophilic intracytoplasmic inclusion body in infected cells. However, there is no information on the role of HPV-60 in the development of cutaneous lesions on other anatomical sites. OBJECTIVES: To perform the clinicopathological analysis of various cutaneous lesions of a patient in relation to HPV genotype. PATIENT: A 50-year-old male patient developed multiple papules, plaques and nodules on his hand, arm and legs. RESULTS: Clinicopathologically, the lesions were classified into three categories. A common wart on the finger showed papillomatosis and acanthosis characterized by numerous keratohyalin granules. Plane warts on the arm showed perinuclear vacuolization of the cells in the upper Malpighian layer. On the other hand, a pigmented papillomatous nodule on the finger, and the other lesions on the hands and legs exhibited similar histological features with a unique cytoplasmic eosinophilic inclusion body. All the three categorized lesions were equally positive for HPV capsid antigen by immunohistochemistry. By blot hybridization analysis for HPV sequences, it was revealed that a common wart on the finger and plane warts on the arm harboured HPV-27 and HPV-3, respectively, while all the other lesions harboured HPV-60. The histological localization of each viral DNA was confirmed in the corresponding lesions by in situ hybridization. CONCLUSIONS: HPV-60 is able to induce papular and nodular lesions on the extremities.

Positive pressure ventilation during fibreoptic intubation: comparison of the laryngeal mask airway, intubating laryngeal mask and endoscopy mask techniques.

BACKGROUND: The efficacy of delivery of mechanical ventilation through different airway devices during fibreoptic intubation is not known. METHODS: We compared the laryngeal mask airway (LMA), intubating laryngeal mask (ILM) and endoscopy mask for positive pressure ventilation (PPV) during fibreoptic intubation. In 80 adult paralysed patients, fibreoptic intubation was performed during PPV using a combination of a size 3 or 4 LMA with a 6.0 mm nasal RAE tracheal tube (LMA3/4 group; n=22), a size 5 LMA with a 7.0 mm nasal RAE tube (LMA5 group; n=18), an ILM with an 8.0 mm special reinforced tracheal tube (ILM group; n=20) or an endoscopy mask (Patil mask) with a 7.5 mm standard tracheal tube (Patil group; n=20). The inspiratory and expiratory tidal volumes (VI and VE) with a ventilation pressure of 20 cm H2O were measured using a pneumotachograph. RESULTS: Mean VE values during fibreoptic intubation in the LMA5 [5.3 (SD 1.5) ml kg(-1)] and ILM [7.1 (2.3) ml kg(-1)] groups were greater than in the LMA3/4 group [2.6 (1.0) ml kg(-1), P<0.0001]. The mean VE was greater in the Patil group [20.6 (4.9) ml kg(-1)] than in the other three groups (P<0.0001). Gastric insufflation during intubation was more frequent in the Patil group (30%) than in the other three groups (4.5-5.6%) (P<0.05). CONCLUSION: PPV is possible with the LMA, ILM or endoscopy mask during fibreoptic intubation. With an airway pressure of 20 cm H2O, ventilation during intubation using a size 3 or 4 LMA was almost insufficient, while ventilation using a size 5 LMA or an ILM was almost acceptable. Ventilation during intubation with the endoscopy mask was greater than that with the LMA or ILM, but gastric insufflation was more frequent.