Induction and separation of mouse helper T cells by lectins.

The capability of Lens culinaris agglutinin (LcA) to induce selectively the helper T cell activity affecting primary antibody response was demonstrated. In the presence of mouse spleen cells, activated with LcA at a concentration of 12 . 5 microgram/ml, optimal augmentation of the humoral immune response to sheep erythrocytes (SRBC) was observed. It was also demonstrated that Limulus polyphemus agglutinin (LPA), which was shown to possess carbohydrate-binding specificity directed to sialic acid residues, preferentially agglutinated helper T cells. Conversely, peanut agglutinin (PNA), which binds preferentially to the sugar sequence beta-D-Gal-(1 leads to 3)-D-GalNAc, did not agglutinate the helper cells. Furthermore, the stimulatory effect of LPA-agglutinated cells on the antibody response was abolished by treatment of the cells with anti-Thy-1 . 2 and complement. These results suggested that the helper cells induced by LcA were T cells and that they have abundant sialic acid residues exposed on the cell surface.     

Soluble intercellular adhesion molecule-1 (ICAM-1) in sera and bronchoalveolar lavage (BAL) fluids of extrinsic allergic alveolitis.

ICAM-1 plays an important role in inflammatory diseases. We analysed ICAM-1 expression on BAL fluid cells and measured soluble ICAM-1 (sICAM-1) concentrations in sera and BAL fluids from patients with extrinsic allergic alveolitis (EAA). We found significantly increased cellular ICAM-1 expression on BAL fluid lymphocytes and alveolar macrophages, and significantly increased values of circulating and BAL fluid sICAM-1 in EAA patients compared with controls. Successive measurement showed prompt decrease of both sICAM-1 values in EAA patients during periods when antigen exposure was prevented. In BAL fluids from EAA patients, sICAM-1 values significantly correlated to neutrophil and ICAM-1+ lymphocyte counts. In EAA patients, circulating and BAL fluid sICAM-1 values has significant negative correlations to values of carbon monoxide diffusing capacity and to time intervals between last episode and sample collection. However, these values had significant positive correlation to values of alveolar-arterial oxygen pressure difference. In EAA, antigen exposure appears to induce cellular ICAM-1 expression on BAL fluid cells, and also appears to up-regulate shedding of ICAM-1 in the alveolar lining fluid and in the circulation. The sICAM values appear to reflect disease activity of EAA.     

The origin of molecular disease: functional abnormalities and consequent clinical symptoms caused by single amino acid substitutions

The discovery of sickle cell hemoglobin in 1949 lead to the concept of "molecular disease". Since then, the total number of abnormal hemoglobins has been increasing, reaching 750 in April, 1998. Of these, 91% are variants with single amino acid substitutions. A "scratch" made in the protein moiety can cause alterations in physicochemical properties such as oxygen affinity, stability and autooxidation of 50% of all variants, further causing hematological or clinical disorders with significant frequencies. Generally, these alterations in properties and consequent hematological or clinical disorders are closely related to the mode and intramolecular location of the mutation. Conventional laboratory methods for mass screening of abnormal hemoglobins such as electrophoresis, isoelectric focusing and ion-exchange HPLC may overlook clinically important variants which possess amino acid substitutions without change in the charge. Oxygen affinity measurement may be a useful method to detect silent variants. Modern apparatuses can quickly and conveniently construct a continuous oxygen dissociation curve from one drop of whole blood. The dissociation curve becomes biphasic when the red cell contains abnormal hemoglobin with abnormal oxygen affinity. The continuous curve recording is advantageous because the proportion of the abnormal hemoglobin component can be estimated and the abnormal chain, either alpha or beta, can be predicted based on the inflection point of the biphasic curve.     

Granzyme B-positive primary gastric T-cell lymphoma: gastric T-cell lymphoma with the possibility of extrathymic T cell origin

A case of primary gastric T-cell lymphoma, which was positive for granzyme B, is reported. The patient was a 47-year-old Japanese female who complained of a dull upper abdominal pain. Radiographic and endoscopic examinations revealed an ulcerative infiltrative lesion in her stomach. Following the confirmation of a high-grade malignant lymphoma, a distal gastrectomy with regional lymph nodal dissection was performed. The histology of the gastric lesion revealed a malignant lymphoma of the diffuse pleomorphic type without lymph nodal involvement. Immunohistochemistry revealed that the tumor cells were positive for LCA, CD3, TIA-1 and granzyme B, but were negative for CD4, CD8, CD56, CD30, L-26, EMA, TCR alpha/beta and TCR gamma/delta. Because the tumor cells showed T cell nature with cytotoxic activity proved by TIA-1 and granzyme B, and without evidence of further maturation of T cell, a malignant lymphoma originating from extrathymic-derived T cells was suggested.     

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.     

Analysis of class switch recombination and somatic hypermutation in patients affected with autosomal dominant hyper-IgM syndrome type 2

Autosomal recessive form of hyper-IgM syndrome type 2 (AR-HIGM2) is secondary to mutations affecting both alleles of AICDA gene encoding activation-induced cytidine deaminase, characterized by defects of immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in most of the patients. We herein report the immunological phenotype of seven patients carrying a single heterozygous R190X mutation in AICDA. Variable defect in in vivo CSR inherited as an autosomal dominant (AD) trait strongly suggests that this heterozygous AICDA mutation causes HIGM (AD-HIGM2). In AD-HIGM2 B cells, CSR was consistently found impaired in vitro. However, in contrast to AR-HIGM2, the CSR-induced double-stranded DNA breaks in the switch region of IgM heavy chain gene were detected. The SHM frequency in V regions of IgM heavy chain gene in B cells was normal in all (but one patient). The characteristics of the AD-HIGM2 phenotype indicate that the AID C-terminal region may be involved in DNA repair machinery required for CSR.     

Proventricular glands in fowl.

Some researchers have already described the fowl proventriculus. However, we believed there was a need for detailed carbohydrate histochemical investigations on the same glands. Moreover, some researchers had erred about the lamina muscularis mucosae. The results of these investigations are as follows. 1. The proventricular glands consist of both superficial and profound gastric glands. 2. The superficial glands are distributed in the lamina propria mucosae while the profound glands exist in the tela submucosa. 3. The superficial glands are simple, branched tubular glands. The columnar glandular cells are arranged in a simple layer and react strongly to PAS, AB (pH 2.5 and 0.5). These appear to be dark purple when they are stained with PAS-AB (pH 2.5). Some other methods have also been tried. 4. Judging from the data 3), the superficial gastric glands contain neutral, weak and strong acids, sulfuric and acid mucopolysaccharides, sialomucin, and II and III neutral mucus type. 5. Glandular cells in the body and basal portions of the superficial gastric glands contain a large number of fine pepsinogen granules. 6. Judging from the data of 3)-5), we believe that the superficial gastric glands are undifferentiated gastric glands and that they are same kinds of glands that are found in mammals. 7. A large number of profound gastric glands fill the tela submucosa. They are compound tubular glands, and are composed of many glandular alveoli. Their columnar glandular cells are arranged in a simple layer. 8. These glandular cells react moderately to PAS, negatively to AB (pH 2.5 and 0.5) and PAS-AB (pH 2.5). Moreover, we observed some other reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The vomeronasal chemosensory system as a route of neuroinvasion by herpes simplex virus

We have investigated the potential of neurotropic microbes to invade the central nervous system (CNS) via the peripheral nervous system. Herpes simplex virus type 1 (HSV-1) strain KH6 and herpes simplex virus type 2 (HSV-2) strain 186 were found to infect chemosensory neurons in the vomeronasal organ (the pheromone detector) following intranasal inoculation of mice. HSV-1 strain KH6 infection was further transmitted to the accessory olfactory bulb (first relay), the medial amygdala (second relay), and the bed nucleus of the stria terminalis and the ventromedial hypothalamus (third relay). HSV-1 strain KH6 also targeted the olfactory and trigeminal systems. HSV-2 strain 186 predominantly attacked the brainstem including the trigeminal system. While both viruses did not induce apoptosis in infected chemosensory neurons, they did in infected brain tissue. These results suggest that neurotropic viruses can invade the brain by infecting vomeronasal chemosensory neurons and that the restrained induction of apoptosis in the infected neurons may facilitate viral transmission to the CNS.     

Effects of vasopressin on water and NaCl transport across the in vitro perfused medullary thick ascending limb of Henle's loop of mouse,rat, and rabbit kidneys

Direct tubular effects of arginine vasopressin (AVP) on water and NaCl transport across the medullary thick ascending limb of Henle (MAL) were examined by the in vitro perfusion of isolated nephron fragments of mice, rats, and rabbits. Osmotic water permeability of the MAL of mice and rats was low and remained unchanged with 2 mU/ml AVP added to the bath. A dose-dependent increase in transepithelial electrical potential difference (PD) with AVP was observed in the mouse MAL when the ambient medium was isotonic. A similar result was also obtained when 2×10^–4 mol/l dibutyryl adenosine 3,5-cyclic-monophosphate was added to the bath. In this preparation, AVP also caused an increase in the unidirectional Cl efflux from 323±45 to 398±61 pmoles·mm^–1 ·min^–1 (n=6,P<0.05). In contrast, under similar condition, we could not demonstrate any effect of AVP on PD, Cl efflux, or net Na flux in the rat MAL and on PD and Cl efflux in the rabbit MAL. Both PD and Cl efflux in the rat MAL were unaffected by AVP when the perfusate was made hypotonic. However, when the ambient medium was made hypertonic by adding NaCl and urea, a significant increase in PD was observed. In addition, we confirmed that AVP stimulated adenylate cyclase activity in the MAL as well as in the collecting tubule of mice and rats. We conclude that AVP stimulates Cl transport across the MAL of mice and rats by activating adenylate cyclase-cyclic AMP system. However, this effect of AVP may quantitatively vary among species.