We tested the hypothesis that lymphocytes from swine with susceptibility to malignant hyperthermia (MH) had calcium extrusion activity higher than unaffected swine. Cytoplasmic concentration of ionized calcium was determined by use of dual emission spectrofluorometry and measurement of the ratio of free to calcium-bound form of the fluorescent calcium dye indo-1. Net calcium accumulation and unidirectional calcium extrusion rate were dependent on intracellular calcium concentration. Calcium extrusion from calcium-loaded lymphocytes was monitored while calcium influx was inhibited by suspending the cells in calcium-free medium with a calcium chelator. Net calcium accumulation of untreated lymphocytes was monitored in calcium-replete medium. A novel method of calculation of ionized calcium was used. This method confirmed our previous findings of lower ionized calcium concentration (86 +/- 40 and 370 +/- 216 nmol/L; P < 0.01) and slower rates of calcium accumulation (39 +/- 16 and 127 +/- 52 nmol/L/min) in untreated lymphocytes from MH-susceptible swine compared with controls. These changes were attributable to calcium extrusion activity two- to three-fold higher in lymphocytes of MH-susceptible swine (154 +/- 36 and 408 +/- 47 nmol/L/min at 175 nmol/L; 972 +/- 111 and 1,690 +/- 505 nmol/L/min at 425 nmol/L). These data were compatible with our model of higher calcium extrusion activity being a compensatory adaptation of MH-susceptible swine lymphocytes to their hypersensitivity to stimuli that increase cytoplasmic calcium concentration.

Peanut agglutinin (PNA) and surface immunoglobulin (SIg) were investigated as markers for T and B lymphocytes in blood and lymphoid tissues of dogs of various ages. In the blood study, 4 age groups (n = 8 dogs/group) were used. The mean (+/- SD) percentages of PNA-positive (PNA +) cells were 68.4 +/- 8.6% (group 1, < 1 year old), 70.3 +/- 9.2% (group 2, 1 to 2 years old), 72.0 +/- 3.7% (group 3, 5 to 6 years old), and 63.8 +/- 10.1% (group 4, 10 to 11 years old). The mean percentages of SIg-positive (SIg+) cells in blood were 32.1 +/- 10.6% (group 1), 43.2 +/- 7.0% (group 2), 34.3 +/- 4.8% (group 3), and 35.0 +/- 6.8% (group 4). The mean total percentages of PNA+ and SIg+ cells were 100 +/-6% (group 1), 113.5 +/- 4.9% (group 2), 106.3 +/- 5.3% (group 3), and 98.9 +/- 9.2% (group 4). The proportions of PNA+ and SIg+ cells in dogs of group 2 were significantly (P < 0.05) different from those in dogs of the other groups. Serial changes in PNA+ and SIg+ cells were investigated in blood of 6- to 29-week-old pups (n = 8). A significant (P < 0.05) transient decrease in PNA+ cells and a corresponding increase in SIg+ cells was observed in pups between 14 and 17 weeks old. Lymphoid tissue specimens and blood samples were obtained from 2- to 6-month-old dogs (n = 11) and from 6- to 12-month-old dogs (n = 10). Percentages reflected the combined data from both groups because there were no significant differences between the 2 age groups. The mean percentages of PNA+ cells were: blood, 68.4 +/- 8.6%; thymus, 86.6 +/- 16.3%; spleen, 29.5 +/-16.0%; lymph node, 48.5 +/- 16.0%; and bone marrow, 30.8 +/- 26.4%. The mean percentage of SIg+ cells were: blood, 32.1 +/- 10.6%; thymus, 3.1 +/- 5.5%; spleen, 69.3 +/-10.3%; lymph node, 55.4 +/- 15.2%; and bone marrow, 65.4 +/- 22.4%. The procedure to identify T lymphocytes in blood and lymphoid tissue was easy to perform, was reproducible, and could be performed on as few as 10(6) cells.

Vaccina virus (VV) infection induces specific antibodies and cytotoxic T cells in various animal species. Therefore, helper T cells also should be induced that stimulate the humoral and cellular immune responses. We determined such helper T-cell activity in 2 species after VV infection. Rabbits and rhesus macaques were infected with the Copenhagen strain of VV or with recombinant VV expressing retroviral proteins. Animals of both species developed antibodies and specific proliferative T-cell response. This reactivity could be enhanced by booster infection with VV. The proliferating macaque cells were CD4+ and major histocompatability complex class II-restricted. These data confirm the broad immunogenicity of VV. Expression of additional polypeptides expressed from a recombinant VV does not lead to altered immune response to VV antigens. However, strength of the helper T-cell response, as well as clinical reactions, differed between macaques and rabbits. Infection with recombinant VV as delivery vectors offers the opportunity for combined vaccination against recombinant proteins and does not diminish cellular and humoral immune responses to VV itself.

Addition of alanine and taurine blocked killing of lymphocytes caused by culture at 45 C. The optimal concentration for thermoprotection was achieved at 12.5 mM for L-alanine and 5 mM for taurine. Both D and L forms of alanine provided thermoprotection. The effect of these agents was not simply to increase osmolarity of the culture medium, because NaCl did not provide thermoprotection at comparable concentrations. Alanine and taurine were each tested at concentration of 50 mM for ability to block heat shock-induced killing and developmental retardation of 8- to 16-cell mouse embryos. Both agents enhanced embryo development after exposure to high temperature, though development remained less than that for embryos not exposed to high temperature. In one experiment, for example, 81% of embryos cultured at 38 C advanced in development during culture vs 0% at 42 C, 15% at 42 C with alanine, and 32% at 42 C with taurine. The beneficial effect of alanine at high temperature may have been partly attributable to effects independent of thermoprotection, because development of embryos cultured at 38 C was also improved by alanine.

Cows naturally infected with Brucella abortus developed antibody (Ab) responses to a nonlipopolysaccharide antigen (NLA) purified from B abortus strain 1119-3. Sera from strain 19-vaccinated cows did not have detectable amounts of Ab. Weak lymphoproliferative responses to NLA were observed in blood mononuclear cell suspensions obtained from infected cows. There was no evidence of NLA-specific lymphoproliferation in cell suspensions from healthy cows. Nonlipopolysaccharide antigen binding to bovine blood mononuclear cells was observed by antigen-consumption assays and direct binding of radiolabeled antigen. Cells from infected cows bound less NLA than did cells from healthy cows when assays were conducted with intact blood mononuclear cell preparations (monocytes plus lymphocytes). Monocytes obtained from any group did not bind NLA. Purified B lymphocytes from infected and healthy vaccinated cows bound about 3 times more NLA than did T lymphocytes, but there were no apparent differences between the 2 groups in extent of binding. Results of the study indicate that bovine lymphocytes have binding sites for a NLA purified from B abortus strain 1119-3.

Cytologic examination of bronchoalveolar lavage fluid (BALF), including phenotypic analysis of lymphocytes, was performed on 32 Standardbreds with poor race performance and endoscopic examination findings characteristic of inflammatory airway disease (IAD). Nucleated cell counts in BALF from IAD-affected horses were higher than those in control horses; the cytologic profile of BALF in affected horses included mixed inflammation, characterized by mild neutrophilia, lymphocytosis, and monocytosis. Eosinophil and mast cell counts were not higher in the IAD-affected group, compared with those in the control group; however, 4 IAD-affected horses had marked eosinophilia (24.7 +/- 4.8% SEM) in BALF. Phenotypic analysis of lymphocytes in BALF obtained from IAD-affected horses revealed a low proportion of CD4-positive cells and B cells, compared with those in the control group; these findings may have been representative of a greater proportion of non-B, non-T cells (null cells) in horses with IAD. The cytologic profile of BALF obtained from horses with IAD differed from that in horses affected with chronic obstructive pulmonary disease, suggesting that the pathogenesis of inflammation in horses with IAD may differ from that of chronic obstructive pulmonary disease.

Polyclonal rabbit antiserum to human T-cell CD3 was used to study its reactivity in lymphoid tissues (lymph nodes, spleen, aggregated lymphoid follicles [Peyer's patches], thymus) of several animal species (cattle, sheep, goats, rats, and mice). Using a peroxidase-antiperoxidase technique on formalin-fixed and paraffin-embedded tissues, immunoreactive cells were detected in T cell-dependent areas of the lymphoid tissues. Reactivity was high in all species tested, but mouse tissues had reduced reactivity, compared with the other species. To obtain a reaction, it was necessary to digest tissues with pronase before application of the immunocytochemical technique. Our results indicate that CD3 antiserum may specifically recognize T-lymphoid cells as it does in human lymphoid tissues and can be used as a marker to study physiologic and pathologic conditions of the lymphoid system of these species.

We used monoclonal antibodies and immunohistologic examination of lymph nodes, to elucidate the pathogenesis of lymphosarcoma induced by, infection with bovine leukemia virus (BLV). The superficial cervical lymph nodes from 3 BLV-infected but apparently healthy sheep and 5 sheep with full-blown lymphosarcoma were examined. We also investigated the integration of bovine leukemia provirus by use of Southern blotting. In lymph nodes from sheep lacking clinical signs of infection, in which the provirus had been integrated at multiple sites in the genome, many large hypertrophic follicles were observed in the cortex. These follicles had germinal centers consisting of CD4+T cells and B cells that expressed surface IgM (sIgM) and major histocompatibility complex (MHC) class-II antigens, but not B cell-specific B2 molecule. The percentage of CD4+T cells in the cortex was significantly (P < 0.05) higher than that of the controls and sheep with lymphosarcoma. In all sheep with lymphosarcoma, the lymph nodes were completely destroyed by proliferating neoplastic cells, and in addition, small atrophic follicles, which consisted of normal B-cell marker-positive cells, were seen near the trabecula and the subcapsule. In these instances, neoplastic cells appeared to be a monoclonal population derived from a single CD5- B-cell lineage and to be classified as 2 types, CD5-CD4-CD8-B2+MHC class-II+sIgM+ and CD5-CD4-CD8-B2+MHC class-II+sIgM-. Moreover, CD8+T cells infiltrated diffusely throughout the tumorous lymph nodes apart from the atrophic follicles, and CD4+T cells were observed around atrophic follicles. Both types of T cells were small-size, normal lymphocytes with round and noncleaved nuclei, and were apparently non-neoplastic cells. In fact, after separation by use of a panning method, it seems that, in blood mononuclear cells from BLV-infected sheep without clinical signs of infection, but in lymphosarcomatous stages, the proviral genome was integrated only in B cells.

In these studies, the effects of recombinant human interleukin 2 (rHuIL-2) were examined on in vitro tumor cytotoxicity by canine blood lymphocytes obtained from peripheral vessels through use of a chromium release microcytotoxicity assay. Cytotoxic activity by lymphokine-activated killer cells was significantly increased, compared with that by untreated lymphocytes in a dose-dependent manner. The maximal effect was attained with 300,000 IU of rHuIL-2/ml. Lymphokine-activated killing also was dependent on the duration of incubation with rHuIL-2. After 1 day of rHuIL-2 incubation, cytotoxicity was significantly increased, compared with that of untreated lymphocytes. Of the 3 times examined, cytotoxicity peaked after 3 days of rHuIL-2 incubation. High levels of cytotoxic activity were still present at 7 days of incubation. Numbers of granular lymphocytes increased over the times examined. These results demonstrate functional and morphologic changes in canine peripheral blood lymphocytes obtained from peripheral vessels after incubation with rHuIL-2 in a dose- and time-dependent manner.