Vector potential of cockroaches for Helicobacter pylori infection

OBJECTIVES: The routes of human infection with Helicobacter pylori remain unclear. In the present study, we examined cockroaches as possible vectors for transmission of H. pylori. METHODS: We used a common species of cockroach (Periplaneta fuliginosa). After a 3-day fast, cockroaches were placed on agar plates containing freshly grown H. pylori (Sydney strain) (challenge group) or on sterile agar plates without H. pylori (control group). After 24 h of challenge, cockroaches were moved to disinfected containers, and sterile food and water were provided. The external surfaces (legs and body) and excreta of the cockroaches were sampled for culture, rapid urease test, and polymerase chain reaction (PCR). RESULTS: H. pylori were culturable from the excreta of the challenge group for 24 h postchallenge. Positive rapid urease test results were obtained up to day 3, and PCR analysis was positive for H. pylori DNA up to day 7 from the excreta. In contrast, H. pylori were not culturable from the external surfaces of the cockroaches. The rapid urease test was positive for only 8 h, and PCR analysis was positive for H. pylori DNA for 1 day from the external surface. CONCLUSIONS: Cockroaches usually live in unsanitary environments and may contaminate foods and food containment areas such as pantries. Transmission of H. pylori infection could be achieved via inadvertent ingestion of foods contaminated with cockroach excreta containing viable H. pylori.     

Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Transgenic mice expressing a pH and Cl- sensing yellow-fluorescent protein under the control of a potassium channel promoter

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl-, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl- sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl- concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.     

Growth Control of C6 glioma in vivo by Nerve Growth Factor

Treatment with nerve growth factor (NGF) causes differentiation of rat C6 glioma cells and strongly inhibits their proliferation in vitro. This suggests that induction of NGF-mediated differentiation may provide a novel therapeutic approach to growth control of glial tumors. We examined the effects of NGF treatment on the growth potential of C6 glioma, which expressed NGF receptor in vivo. C6 glioma cells (1 × 10⁶ cells/10 l) were transplanted into the rat striatum. After 4 days, the animals were given successive injections of 100 ng NGF into the growing tumor at every 4 days (n = 10 rats). Controls were subjected to identical procedures with vehicle which did not contain NGF (n = 10 rats). At 14 days after transplantation, we evaluated the tumor volume, proliferative cell index (PCI) based on the MIB-1 immunoreactivity and enzyme activities related to energy metabolism by enzyme histochemistry. We found that the NGF treatment markedly reduced the tumor volume of the C6 glioma (34.00 ± 8.47 mm³ to 7.22 ± 4.92 mm³, p < 0.01). NGF treatment also decreased the PCI (33.34 ± 9.57% to 3.85 ± 3.56%, p < 0.01) with a negative correlation with tumor volume (r = 0.972, p < 0.01), and the hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) activities (p < 0.01 and p < 0.01, respectively) which reflect the demand for nucleic acid synthesis for proliferation through the glycolytic and pentose phosphate pathways. The present results demonstrate for the first time that inhibition of tumor cell proliferation of C6 glioma by NGF occurs in vivo, probably through the NGF-mediated differentiation of C6 glioma cells which has been observed in in vitro studies.     

Quantitative analysis of cell-free Epstein-Barr virus genome copy number in patients with EBV-associated hemophagocytic lymphohistiocytosis

To determine whether the EBV genome content in serum or plasma reflects clinical features and outcome in EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), we quantified the cell-free EBV genome copy number by real-time PCR in 38 patients with EBV-HLH, and compared this to the values from 15 patients with infectious mononucleosis (IM). The median (range) cell-free EBV genome copy number at diagnosis was 3.0 x 10(3) (undetectable -5.5 x 10(7)) copies/ml in EBV-HLH, which was significantly higher than the 6.6 x 10(1) (undetectable -1.0 x 10(3)) copies/ml in IM (P = 0.0008). We serially analyzed cell-free EBV genome copy number in 10 cases of EBV-HLH up to 4 months from diagnosis. In four patients who achieved remission, the EBV genome became undetectable soon after starting therapy. In the remaining six patients who responded poorly to therapy, the EBV genome copy number in the serum or plasma remained at high levels except for one case. In addition, we confirmed that the EBV genome became undetectable after hematopoietic stem cell transplantation in 4 EBV-HLH cases. These results suggest that the quantitative analysis of cell-free EBV genome copy number is useful for evaluating disease activity and for predicting the response to therapy in EBV-HLH.     

Induction of Specific Cytotoxic T Lymphocytes against Autologous Brain Tumor by Crossreactive Allo-tumor Cell Stimulation

Cytotoxic T lymphocytes (CTL) against autologous malignant brain tumor were generated in peripheral blood lymphoid cells (PBL) prepared from a patient with a malignant brain tumor by stimulation of the cultured PBL for 7 days with attenuated Crossreactive malignant melanoma (MM2) cells pretreated with mitomycin C. The Crossreactive MM2 cells were effective for antigen stimulation for CTL induction in place of autologous glioblastoma cells, which are difficult to expand in culture. The optimal ratio between nylon wool-passed T lymphocytes and nylon wool-adherent accessory cells to induce CTL in the patient's PBL was found to be 25 to 1. In vitro -activated CTLs induced by MM2 were cytotoxic not only to MM2, but also to the autologous tumor cells in an HLA class I-restricted manner, and their surface phenotype was found to be CD3⁺ and CD8⁺. CTL therapy using cross-reactive allogeneic tumor cells as the stimulator could be clinically valuable to treat malignant brain tumors.     

A case of malignant fibrous histiocytoma of the ilium,evaluation by blood-pool scintigraphy

A rare case of malignant fibrous histiocytoma (MFH) of the ilium was presented and blood-pool scintigraphic images and angiographic images were correlated. Hypervascular tumor and laking of contrast material with arteriovenous shunts were shown by angiography. Intensive tracer accumulation was shown by blood-pool scintigraphy. After radiation therapy and transcatheter arterial embolization of the tumor, tracer accumulation was noticeably reduced. These findings suggested a vascular tumor such as angiosarcoma, but surgery revealed MFH of the bone. Blood-pool scintigraphy was useful in the evaluation of the vascular characteristics of the tumor.     

Neurocutaneous melanosis associated with Hirschsprung's disease in a male neonate

Hirschsprung's disease is an inherited disorder characterized by the absence of ganglion cells in the distal bowel. Neurocutaneous melanosis is a rare congenital syndrome characterized by proliferation of melanin-producing cells in the skin and leptomeninges. The authors described a newborn patient with neurocutaneous melanosis associated with Hirschsprung's disease. This male baby had congenital hydrocephalus, large and multiple pigmented skin nevi, and severe abdominal distension. He showed marked hydrocephalus at birth and underwent a ventriculo-peritoneal shunt at the age of 5 days. Investigations for gut motility disorders revealed typical findings consistent with Hirschsprung's disease involving the rectosigmoid colon. He was surgically treated for Hirschsprung's disease after transanal endorectal pull-through at the age of 7 months. After settlement of the ventriculo-peritoneal shunt, the transanal approach was of significant value for keeping the intraperitoneal catheter clean. The association of developmental disorders of melanocytes and enteric ganglia, both of which originated from the neural crest, suggested the presence of mutual pathogenetic factors in the patient.     

Impaired expression of myogenic regulatory molecules in the pelvic floor muscles of murine embryos with anorectal malformations

Background/Purpose

Recent biological studies have elucidated the molecular mechanism of muscle development, in which various regulatory factors (myogenic regulatory factors [MRFs]) play key roles during embryogenesis. To investigate the development of anorectal malformations (ARMs), we studied MRF expressions in myogenic cells in the pelvic floor using murine embryos affected with ARM.

Methods

Anorectal malformation embryos were obtained from the 10.5th embryonal day (E10.5) to the 7.0th postnatal day (D7.0) in a natural mutant strain (Sd/+, RSV/Le). Serial frozen sections were prepared for immunohistochemistry using specific antibodies to M-cadherin, myoD, Myogenin, myosin heavy chain, and alfa-actin molecule.

Results

In normal mice, embryonal caudal somites differentiated into myogenic stem cells and migrated to the pelvic floor between E11.0 and E14.0. In the ARM mice, however, caudal somites were irregularly arranged and MRF expressions in myogenic cells were markedly decreased in the dorsocaudal region at E11.5 to E13.0, leading to hypoplastic pelvic floor muscles.

Conclusions

The maldevelopment of pelvic floor muscles in ARM is derived from a deficient supply of myogenic stem cells, with impaired MRF expression. These results suggest that myogenic stem cells, available from bone marrow contents, may be used for postnatal muscle regeneration to reinforce the pelvic floor muscle function in children with ARM.     

Developmental study of tethered spinal cord in murine embryos with anorectal malformations

Background/Purpose

Tethered spinal cord is frequently associated with anorectal malformations (ARMs). However, it remains unknown how the tethered spinal cord develops and relates to the severity of ARM. We studied the development of the spinal cord in ARM mouse embryos induced by all-trans retinoic acid (ATRA).

Methods

Pregnant ICR-Slc mice were administered 100 mg/kg of ATRA on the ninth embryonic day (E9.0). Embryonic specimens were obtained from the uteri between E11.0 and E18.5. Midsagittal histologic sections focusing on the spinal cord and pelvis were prepared for immuonhistochemistry specific for neurofilament and Protein Gene Product 9.5 molecules.

Results

More than 98% of ATRA-treated embryos demonstrated ARM with rectourethral or rectocloacal fistula. Normal embryos exhibited progressive ascent of the spinal cord from E14.5. However, in ARM embryos, the distal spinal cord ended with meningomyelocelelike or atypical hamartomatous lesions at E11.5 to E13.5, which later caused stretch force that damaged the spinal cord, resulting in tethered cord between E16.0 and E16.5.

Conclusions

In ATRA-induced ARM mouse embryos, tethered spinal cord was mostly established, accompanied by caudal neural maldevelopment, during early fetal development. This experimental model may be useful for researching detailed neuropathologic conditions in ARM children accompanied with tethered spinal cord.