ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.     

Protection of rabbits against HTLV-II infection with a synthetic peptide corresponding to HTLV-II neutralization region

Summary Rabbit immune sera raised against synthetic peptides of the HTLV-II envelope gp46 region were examined for HTLV-II neutralization ability by HTLV-vesicular stomatitis virus (VSV) pseudotype assay and syncytium inhibition assay. HTLV-II neutralization activity was detected in the sera against HTLV-II Env gp46, 80–103 but not in those to HTLV-II Env gp46, 171–196. Three rabbits immunized with the synthetic peptide of HTLV-II Env gp46, 80–103 and three non-immunized rabbits were challenged with intravenous inoculation of an HTLV-II-producing human cell line (MOT, 1×10⁷ cells). The non-immunized rabbits showed seroconversion for HTLV-II after 2 weeks and maintained persistent infection but the immunized rabbits were protected from HTLV-II infection. Nested or repeated polymerase chain reaction revealed the presence of HTLV-II provirus sequences in the non-immunized rabbits but not in the immunized rabbits. These results suggest that peptide vaccination with a synthetic peptide corresponding to the HTLV-II neutralization region is useful for preventing HTLV-II infection.     

Cold-adaptation of human rotavirus

A human rotavirus strain was cold-adapted for possible future use as a live vaccine. The original strain was isolated in 1980 in primary cynomolgus monkey kidney cells and has a serotype I and subgroup II antigenicity. The virus was serially passaged in African green monkey kidney cells; it was cultivated at 37 degrees C at the first stage of passages, and the cultivation temperature was then shifted down stepwise by 3 degrees C per each 10 passages. Finally the virus was passaged 10 times at 25 degrees C (total passage number of 55). The virus formed small-size plaques with irregular shaped borders at 31 degrees C. Growth at 25 degrees C of the cold-adapted virus was higher than that of the original virus. There was no difference between the migration patterns of 11 dsRNA segments in polyacrylamide gel electrophoresis of the original and the cold-adapted viruses.     

Lamivudine and IFN-beta sequential therapy in HBe antigen-positive patients with chronic hepatitis B virus genotype C infection.

Sequential treatment with lamivudine and interferon (IFN) has induced sustained biochemical and virologic responses in the majority of patients with chronic hepatitis B in France. However, the efficacy of sequential treatment in patients with chronic hepatitis B virus (HBV) genotype C infection has not been evaluated. Twenty-four HBe antigen-positive patients were treated with 100 mg lamivudine alone for 16-32 weeks, then with both 6 MU IFN-beta and lamivudine for 4 weeks, and lastly with IFN-beta alone for 20 weeks. Sustained response was achieved in 7 (29%) patients 24 weeks after the end of therapy. No lamivudine-resistant variants emerged in any patient. Hepatitis flare occurred in 3 patients after the withdrawal of lamivudine, but none had decompensation. The patients with sustained response were significantly younger at baseline (p = 0.033) and had a significantly lower HBV DNA level at the start of IFN (p = 0.020) than those without sustained response. In conclusion, the rate of response to sequential therapy with lamivudine and IFN in HBe antigen-positive patients with HBV genotype C infection was lower than the rate reported previously. Patients who were young or who had a favorable virologic response to lamivudine were more likely to have a sustained response.     

Pineal malignant rhabdoid tumor with chondroid formation in an adult

A pineal tumour in a 27-year-old male is presented with the characteristic histological features of a pineal malignant rhabdoid tumor (MRT) with chondroid formation. Occasionally, tumor cells contained a single well-demarcated hyaline globular inclusion within the cytoplasm adjacent to the nucleus. The stroma of these tumors tends to be densely hyalinized and become chondroid. Immunohistochemical staining was positive for vimentin, epithelial membrane antigen, chromogranin A, synaptophysin, neuron-specific enolase, S-100 protein, and muscle actin. Despite surgery and radiochemotherapy, the tumor recurred in the pineal region and metastasized to the lower lobe of right lung. The patient died 2 years after the initial diagnosis. This is the second published case of central nervous system-MRT appearing in an adult. The clinical and pathological features of pineal MRT in this patient are presented.     

Directed differentiation of telencephalic precursors from embryonic stem cells

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.     

Selection by phage display of llama conventional V(H) fragments with heavy chain antibody V(H)H properties

A llama single domain antibody (dAb) library designed and constructed to contain only heavy chain antibody variable domains (V(H)Hs) also contained a substantial number of typical conventional antibody heavy chain variable sequences (V(H)s). Panning the library against two carbohydrate-specific antibodies yielded anti-idiotypic dAbs and enriched solely for sequences from the V(H) subpopulation of the library. The conventional antibody origin of these V(H)s was confirmed by using oligonucleotide probes, specific for the enriched V(H)s, to identify the parental sequences in the message employed in library construction. Surprisingly, these V(H) dAbs, which are produced in high yield in Escherichia coli, are highly soluble, have excellent temperature stability profiles and do not display any aggregation tendencies. The very close similarity of these molecules to human V(H)s makes them potentially very useful as therapeutic dAbs.     

Cytomegalovirus infections following umbilical cord blood transplantation using reduced intensity conditioning regimens for adult patients.

Cytomegalovirus (CMV) infection is a major complication after allogeneic hematopoietic stem cell transplantation (Allo-HSCT); however, we have little information on the clinical features of CMV reactivation after cord blood transplantation using reduced-intensity regimens (RI-CBT) for adults. We reviewed medical records of 140 patients who underwent RI-CBT at Toranomon Hospital between January 2002 and March 2005. All the patients were monitored for CMV-antigenemia weekly, and, if turned positive, received preemptive foscarnet or ganciclovir. Seventy-seven patients developed positive antigenemia at a median onset of day 35 (range, 4-92) after transplant. Median of the maximal number of CMV pp65-positive cells per 50,000 cells was 22 (range, 1-1806). CMV disease developed in 22 patients on a median of day 35 (range, 15-106); 21 had enterocolitis and 1 had adrenalitis. CMV antigenemia had not been detected in 2 patients, when CMV disease was diagnosed. CMV disease was successfully treated using ganciclovir or foscarnet in 14 patients. The other 8 patients died without improvement of CMV disease. In multivariate analysis, grade II-IV acute graft-versus-host disease was a risk factor of CMV disease (relative risk 3.48, 95% confidential interval 1.47-8.23). CMV reactivation and disease develop early after RI-CBT. CMV enterocolitis may be a common complication after RI-CBT.     

Monocarboxylate transporter 1 (MCT1) plays a direct role in short-chain fatty acids absorption in caprine rumen

Despite the importance of short-chain fatty acids (SCFA) in maintaining the ruminant physiology, the mechanism of SCFA absorption is still not fully studied. The goal of this study was to elucidate the possible involvement of monocarboxylate transporter 1 (MCT1) in the mechanism of SCFA transport in the caprine rumen, and to delineate the precise cellular localization and the level of MCT1 protein along the entire caprine gastrointestinal tract. RT-PCR revealed the presence of mRNA encoding for MCT1 in all regions of the caprine gastrointestinal tract. Quantitative Western blot analysis showed that the level of MCT1 protein was in the order of rumen ≥ reticulum > omasum > caecum > proximal colon > distal colon > abomasum > small intestine. Immunohistochemistry and immunofluorescence confocal analyses revealed widespread immunoreactive positivities for MCT1 in the caprine stomach and large intestine. Amongst the stratified squamous epithelial cells of the forestomach, MCT1 was predominantly expressed on the cell boundaries of the stratum basale and stratum spinosum. Double-immunofluorescence confocal laser-scanning microscopy confirmed the co-localization of MCT1 with its ancillary protein, CD147 in the caprine gastrointestinal tract. In vivo and in vitro functional studies, under the influence of the MCT1 inhibitors, p -chloromercuribenzoate (pCMB) and p -chloromercuribenzoic acid (pCMBA), demonstrated significant inhibitory effect on acetate and propionate transport in the rumen. This study provides evidence, for the first time in ruminants, that MCT1 has a direct role in the transepithelial transport and efflux of the SCFA across the stratum spinosum and stratum basale of the forestomach toward the blood side.