Protein-specific helper T-lymphocyte formation initiated by dendritic cells

Antibody responses to hapten-polypeptide conjugates require peptide-specific helper T cells. The latter can be primed in tissue culture by providing small numbers of dendritic cells. Primed, irradiated helper T cells then induce B-cell growth and differentiation in the apparent absence of dendritic cells. Both stages of the antibody response--the induction of helper T lymphoblasts by dendritic cells and the delivery of help from T to B cell--occur in discrete cell aggregates that can be isolated by velocity sedimentation. If helper T blasts revert to smaller "memory" lymphocytes, dendritic cells again are needed to initiate the antibody response.     

Detection of equine immunoglobulin-secreting cells by a plaque assay.

A protein A-hemolytic plaque assay was applied to detect immunoglobulin (Ig)-producing cells in horse peripheral blood, using pokeweed mitogen as a B lymphocyte activator. A maximum number of Ig-secreting cells was obtained when horse peripheral blood lymphocytes were cultured in a medium containing horse serum. The number of Ig-secreting cells in young horses (2 years old) was lower than that in adult horses (6 to 23 years old). In addition, the plaque formation was unchanged from blood samples kept at 4 degrees C for 24 hours, while blood samples kept for 72 hours did not yield plaques. These results indicate that the plaque assay is a reliable and useful method for detecting Ig-secreting cells in the peripheral blood of the horse.     

Characterization of Japanese isolates of Aujeszky's disease virus by restriction endonuclease cleavage patterns, virulence in mice and thymidine kinase activity.

Twenty four cloned isolates of Aujeszky's disease virus collected from outbreaks of Aujeszky's disease from 1981 through 1989 in Japan were characterized by their restriction endonuclease (RE) cleavage patterns, virulence for mice and thymidine kinase (TK) activity. All of the isolates belonged to Type II of the four types classified by Herrmann et al. (1984). Based on the number and migration rate of the restriction fragments, the isolates were divided into 7 groups with Bam HI, 9 groups with Kpn I, 3 groups with BstE II and 2 groups with Sal I. The results indicate that the RE analysis, especially with Bam HI and Kpn I, provides useful epidemiological information about field isolates of Aujeszky's disease virus. All of the isolates showed virulence for mice ranging from 6.9 to 63.0 (PFU/LD50). It was interesting that the Nagano S87 strain, which had the highest virulence for the mouse, showed unique RE cleavage patterns with four enzymes. On the other hand, ara-T-resistant, TK-negative strain, was avirulent for mice (greater than 10(6.4) PFU/LD50). All of the isolates investigated in this study showed TK activity by the thymidine plaque autoradiography.     

Avirulent ts and thymidine kinase-deficient mutant of Aujeszky's disease virus.

The temperature-sensitive (ts), thymidine kinase-deficient (TK-) mutant designated ZHtsTK- strain, of Aujeszky's disease virus (ADV) was isolated from a virulent strain with the treatments using 5-bromodeoxyuridine and arabinosylthymine. The ZHtsTK- strain was easily distinguished from the other virulent ADV strains by plaque size on HmLu-1 and chicken embryo fibroblast cells and by restriction endonuclease analyses using Bam HI, Sal I and Kpn I. The ZHtsTK- strain was avirulent for mice, guinea pigs and rabbits, and produced neutralizing antibodies to ADV in these animals. The rabbits inoculated with the ZHtsTK- strain did not shed detectable amounts of virus after dexamethasone treatment. The ZHtsTK- strain was also avirulent for 5-day-old piglets and did not cause disease. No virus was detected from the piglets inoculated intramuscularly in the nasal swabs or the tissues examined on postinoculation day 9. These findings presented here suggested that there is a significant correlation between pathogenicity and properties such as ts and TK-, and the combination of ts and TK- properties plays a much larger role in reducing virulence for animals.     

Changes in acute-phase proteins and cytokines in serum and milk whey from dairy cows with naturally occurring peracute mastitis caused by Klebsiella pneumoniae and the relationship to clinical outcome

This study was conducted to evaluate the changes in acute-phase proteins and cytokine concentrations in dairy cows with naturally occurring peracute Klebsiella pneumoniae (K. pneumoniae) mastitis and their association with the outcome of the disease. Seventeen Holstein cows with K. pneumoniae mastitis from 8 dairy farms were divided on the basis of outcome after local and systemic therapy into 2 groups comprising 8 euthanized cows and 9 that recovered. Changes in acute-phase proteins and cytokine concentrations in cows with K. pneumoniae mastitis were evaluated at the onset of the disease (day 0) and at days 3, 7 and 14 after therapy and compared with those of 13 healthy dairy cows. The concentrations of haptoglobin (Hp) and interleukin (IL)-6 in serum and α(1)-acid glycoprotein and IL-1β in serum and whey on day 0 were significantly (P<0.05) higher in the euthanized cows than in those that recovered and the healthy cows. A correlation (r=0.90, P<0.01, n=17) was found between IL-6 and Hp concentrations in sera from recovered and euthanized cows at day 0. This indicated that serum concentrations of Hp and IL-6 at the initial examination were prognostic factors for survival, and the cutoff values were 2,020 μg/ml and 32 ng/ml, respectively. These results suggest that IL-6 and Hp concentrations are involved in the manifestation of K. pneumoniae mastitis and may be possible indicators of the prognosis of peracute K. pneumoniae mastitis.     

Molecular cloning, tissue expression, and subcellular localization of porcine peptidoglycan recognition proteins 3 and 4

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are present in most invertebrates and vertebrates. Mammals have four PGRPs, PGLYRP1-4. In the present study, we cloned the cDNAs encoding porcine PGLYRP3 and 4 from the esophagus of adult swine. The length of the complete open reading frames of porcine PGLYRP3 and 4 are identical and contain 1125bp encoding 374 amino acid residues. The amino acid sequences of these two proteins were more similar to their human orthologs (78.9% [PGLYRP3] and 73.9% [PGLYRP4]) than to their mouse orthologs (71.3% [PGLYRP3] and 67.9% [PGLYRP4]). Expression analysis revealed that both PGLYRP3 and 4 were more strongly expressed in digestive tract, especially the esophagus, than in immune organs such as spleen or mesenteric lymph nodes in both newborn and adult swine. To analyze the subcellular distribution of porcine PGLYRP1-4, we constructed transfectant cell lines. Western blot and flow cytometric analyses revealed that porcine PGLYRP3 and 4 are not only secreted, but also expressed on the cell surface, unlike PGLYRP1 and 2. These results should help contribute to the understanding of PGLYRP3- and 4-mediated immune responses via their recognition of intestinal microorganisms in newborn and adult swine.     

Parasitological survey on wild carnivora in north-western Tohoku, Japan.

In the winter of 1997-1998, we collected parasitological data from 60 wild carnivora in the north-western part of Tohoku region, Japan. These included 7 foxes (Vulpes vulpes japonica), 20 raccoon dogs (Nyctereutes procyonoides viverrinus), 29 martens (Martes melampus melampus), 3 weasels (two Mustela sibirica itatsi and one M. nivalis namiyei), and one Japanese badger (Meles meles anakuma). Roundworms (Toxocara canis in foxes and Toxocara tanuki in raccoon dogs), hookworms (Ancylostoma kusimaense and Arthrostoma miyazakiense) and Molineus sp. in the small intestine were the most prevalent in foxes and raccoon dogs. In martens, Aonchotheca putorii in the stomach, Concinnum ten in the pancreatic duct, Molineus sp. and Euryhelmis costaricensis in the small intestine were the most prevalent. Collected parasites include some new helminth species for this region or Japan; the strobilar stage of Taenia polyacantha from foxes, Pygidliopsis summa from a raccoon dog, Eucoleus aerophilus, A. putorii, and Soholiphyme baturini from martens.     

Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)

Adhesion tests are complex, time‐consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.     

Three cases of idiopathic sterile pyogranulomatous inflammation of epidural fat in miniature dachshunds

Progressive ataxia and paralysis in three Miniature Dachshunds were found to be caused by idiopathic sterile pyogranulomatous inflammation of epidural fat between T5 and L4. All dogs were managed by hemilaminectomy and removal of epidural compressive material. Surgical findings and histopathological evaluation were necessary to diagnose epidural pyogranulomatous inflammation. A dog did not regain motor and sensor function after the surgery. Two dogs had exhibited improved neurological function after the surgery, but they recurred. Oral cyclosporine treatment was useful for their long remission. Idiopathic sterile pyogranulomatous inflammation of epidural fat can be considered to be a cause of thoracolumbar myelopathy in dogs.     

Clathrin-mediated endocytosis of mammalian erythroid AE1 anion exchanger facilitated by a YXXΦ or a noncanonical YXXXΦ motif in the N-terminal stretch

To explore the roles of the conserved YXXΦ-type motif in the erythroid-specific N-terminal stretch of anion exchanger 1 (AE1), cell surface expression and internalization of various mutants derived from murine erythroid AE1 tagged with an N-terminal enhanced green fluorescent protein and an extracellular FLAG (EGFP-mAE1Flag) were explored in K562 and HEK293 cells. EGFP-mAE1Flag showed rapid internalization, in association with the internalizations of transferrin and the endogenous AE1 chaperone-like protein glycophorin A in K562 cells. Disruption of the conserved Y72VEL sequence markedly reduced the internalization and increased the relative abundance of cell-surface AE1, whereas substitution of the N-terminal region from bovine AE1 that lacks the relevant motif for the corresponding region had less of an effect on internalization. Deletion or substitution mutations of the Y7EDQL sequence in the bovine N-terminal stretch resulted in the decreased internalization of the AE1 proteins. Cell surface biotinylation and deglycosylation studies showed that approximately 30% of the cell-surface EGFP-mAE1Flag and several other mutants was sorted to the plasma membrane without N-glycan maturation in the Golgi apparatus. These findings indicate that the conserved YXXΦ sequence or a noncanonical YXXXΦ sequence in the N-terminal region facilitates the endocytic recycling of erythroid AE1 through a clathrin-mediated pathway.