Processing and major histocompatibility complex class II presentation of Legionella pneumophila antigens by infected macrophages

To better understand interactions between the intracellular pathogen Legionella pneumophila and macrophages (Mphis), host and bacterial determinants important for presentation of antigens on major histocompatibility complex class II molecules (MHC-II) were investigated. It was determined that immune CD4 T-cell responses to murine bone marrow-derived Mphis (BMphis) infected with wild-type L. pneumophila were higher than the responses to avirulent dotA mutant bacteria. Although this enhanced response by immune T cells required modulation of vacuole transport mediated by the Dot/Icm system, it did not require intracellular replication of L. pneumophila. Intracellular cytokine staining identified a population of immune CD4 T cells that produced gamma interferon upon incubation with BMphis infected with wild-type L. pneumophila that did not respond to Mphi infection with dotA mutant bacteria. Endocytic processing was required for presentation of L. pneumophila antigens on MHC-II as determined by a defect in CD4 T-cell responses when the pH of BMphi endosomes was neutralized with chloroquine. Investigation of MHC-II presentation of antigens by BMphis infected with L. pneumophila icmR, icmW, and icmS mutants indicated that these mutants have an intermediate presentation phenotype relative to those of wild-type and dotA mutant bacteria. In addition, it was found that antigens from dot and icm mutants are presented earlier than antigens from wild-type L. pneumophila. Although immune CD4 T-cell responses to proteins secreted by the L. pneumophila Lsp system were not detected, it was found that the Lsp system is important for priming L. pneumophila-specific T cells in vivo. These data indicate that optimal antigen processing and MHC-II presentation to immune CD4 T cells involves synthesis of L. pneumophila proteins in an endoplasmic reticulum-derived compartment followed by transport to lysosomes.     

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.     

Ultrastructural myeloperoxidase activity and expression of myeloid-associated antigens in acute lymphoblastic leukemia

Ultrastructural myeloperoxidase (MPO) activity and myeloid-associated antigen (MyAg) expression were investigated in 12 adult patients with acute lymphoblastic leukemia (ALL). Ultrastructural MPO was detected by 3 different methods. Immunophenotyping was performed by flow cytometry, using a series of monoclonal antibodies. Ultrastructural MPO-positive blast cells were detected in 6 patients (50%). In 5 of these 6 patients, the methods detecting both MPO and platelet peroxidase (PPO) activities found MPO-positive blast cells more frequently than those detecting MPO activity alone. In 2 patients (17%), at least one kind of MyAg was positive. Ultrastructural MPO activity was detected more frequently than MyAg expression in ALL patients. This method of detecting PPO and MPO is recommended for detection of ultrastructural MPO-positive ALL.     

The value of external morphology in the identification of larval anisakid nematodes: a scanning electron microscope study

We studied larval nematodes of four genera of the Anisakidae using a scanning electron microscope (SEM). The anterior and posterior extremities and cuticular structures of the 3rd-stage larvae (L3) ofAnisakis type I,Pseudoterranova decipiens, Contracaecum type B andHysterothylacium were examined. The 4th-stage larvae (L4) ofAnisakis type I,P. decipiens, recovered after infection into laboratory rats, and the L3 and L4 ofAnisakis type I larvae from human were also examined in the same way. There were generic differences in the shape and size of the lip bulges, external papillary structures, the appearance of the boring tooth, the width and depth of the grooves and ridges of the cuticle and the caudal structures of the L3. InAnisakis type I andP. decipiens L3, changes were seen in the anterior extremity, cuticle and posterior extremity after molting to the L4. Similar changes can be expected in larvae infecting man. The L4 ofAnisakis type I from rat and man were similar, while the L4 ofAnisakis type I andP. decipiens showed differences. These ultrastructural differences might be of value in the identification of fragments recovered during endoscopy in man.     

Resolving the composite trait of hypertension into its pharmacogenetic determinants by acute pharmacological modulation of blood pressure regulatory systems

Acute pharmacogenetic analysis was carried out in an intercross F2 population derived from Prague hypertensive-hypertriglyceridemic and Lewis rats. Quantitative trait loci (QTL) mapping was performed for baseline blood pressure (BP) and for BP after blockade of the renin-angiotensin system by losartan, of the sympathetic nervous system (SNS) by pentolinium, and of the nitric oxide system by N(G)-nitro- L-arginine methyl ester. Two significant loci for baseline BP were found on chromosome (Chr) 3 (logarithm of likelihood, LOD, 3.8) and Chr 5 (LOD 3.6), and one suggestive locus on Chr 1 (LOD 2.7). The QTL on Chr 3 persisted after treatment with the three agents while the QTL on Chr 5 and Chr 1 disappeared after pentolinium administration. This suggests independence of the locus on Chr 3 from each acute BP regulatory system examined, whereas the loci on Chr 5 and Chr 1 appeared to be controlled mainly by the SNS. Although not apparent at baseline, a significant locus appeared on Chr 8 (LOD 7.0) after blockade of the SNS, and NO system blockade led to the appearance of a new QTL on Chr 1 (LOD 3.6), indicating the contribution of the inhibited systems to these loci. Pharmacogenetic dissection of the BP trait is a powerful tool to unravel the underlying physiological mechanisms of QTL affecting baseline BP and to identify specific QTL for the response to drugs. This pharmocogenetic approach enabled us to determine the main causative acute BP regulatory systems and should lead to better selection of suitable antihypertensive drugs for individual patients.     

An improved method for recovery of F-defective Sendai virus expressing foreign genes from cloned cDNA

An improved system is described to recover non-transmissible Sendai virus that lack the envelope fusion (F) gene from cloned cDNA. The system (1) used plasmids that expressed the F and the HN viral envelope proteins, as well as the plasmids that expressed the viral NP, P, and L proteins as helper plasmids for recovery, and (2) overlaid packaging cells that expressed the F protein. With this improved system, we have succeeded in recovery of F-defective Sendai virus expressing two foreign proteins, and expression vectors that do not contain the EGFP reporter gene. This system may provide the basis for constructing recombinant F-defective Sendai virus for preventing and treating human diseases in the form of vaccines and vectors for gene therapy.     

Association of Pasteurella multocida toxin with vimentin

To help understand the molecular mechanisms of Pasteurella multocida toxin (PMT) action, we searched for a cellular protein interacting with PMT. The ligand overlay assay revealed a 60-kDa cellular protein that binds to a region from the 840th to 985th amino acids of the toxin. This protein was identified as vimentin by peptide mass fingerprinting. The N-terminal head domain of vimentin was further found to be responsible for the binding to the toxin.     

P16-immunostaining pattern as a predictive marker of lymph node metastasis and recurrence in early uterine cervical cancer.

OBJECTIVES: We investigated the relationship between P16-immunostaining patterns and clinicopathological factors in early uterine cervix cancers and assessed whether P16-immunostaining patterns predict the prognosis of the patients with early uterine cervix cancers. METHODS: Twenty-nine early squamous cell carcinoma (SCC) specimens of the uterus were examined using immunohistochemistry for P16 expression. The P16-immunostaining pattern was classified into two groups: the homogeneous type and the heterogeneous type. P16-immunostaining patterns were evaluated in different parts of the carcinoma in situ (CIS): the center of the tumor and the front interface of the infiltrating tumor. RESULTS: All specimens were of the homogeneous type in CIS. The P16-immunostaining pattern was significantly of the heterogeneous type in the front interface of the infiltrating tumor with lymphatic invasion, vascular invasion, lymph node metastasis, and recurrence. Regarding the P16-immunostaining patterns in the front interface of the infiltrating tumor, the patients with the heterogeneous type showed a significantly worse prognosis than the patients with the homogeneous type. CONCLUSIONS: The prognosis of patients with early uterine cervical SCC may be predicted by evaluating the P16-immunostaining pattern in the front interface of the infiltrating tumor.     

cAMP-independent decrease of ATP-sensitive K+ channel activity by GLP-1 in rat pancreatic β-cells

Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic beta-cells induced by glucagon-like peptide 1 (7-36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2-100 nM). Exendin (9-39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from -90 to -50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K+ channel (KATP). This GLP-1 effect was prevented by exendin (9-39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through KATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on KATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of KATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced KATP activity. The half-maximal concentrations (ki) of ATP were 11.6 microM without and 5.6 microM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces KATP activity by elevating the sensitivity of KATP to ATP, resulting in depolarization of pancreatic beta-cells. This GLP-1 action is independent of the cAMP signalling pathway.     

Significance of an insular component in follicular thyroid carcinoma with distant metastasis at initial presentation

Risk factors for distant metastasis were studied in 82 patients with follicular thyroid carcinoma (FTC). Metastases to either the lung or bone existing at the time of presentation were confirmed by I-131 radio-iodine uptake in 10 patients. FTC with an insular component was found in eight patients. Univariate analysis of 14 possible risk factors showed 7 to be statistically significant: insular component, poorly differentiated carcinoma, trabecular component, serum thyroglobulin level before surgery, patient age at the time of presentation, solid component, and vascular invasion (ranked by p values). After further analysis of the interrelation of the factors and of the logistic regression curves, we concluded that presence of an insular component and patient age were the only independent risk factors. Distant metastasis was not detected in any of the 27 patients ≤49 yr old. Among the 55 older patients (≥50 yr old), 5 of the 49 (10%) without an insular component and 5 of the 6 (83%) with an insular component had distant metastasis. The remaining older patient with an insular component but without distant metastasis showed a gradual increase in thyroglobulin levels after total thyroidectomy.