Changes in B cell and T cell subsets in bovine leukaemia virus-infected cattle

Direct immunofluorescence and fluorescence-activated cell sorter techniques were used for the detection of surface immunoglobulin positive (SIg+) cells in peripheral blood lymphocytes (PBL's) of bovine leukaemia virus (BLV) infected cattle with or without persistent lymphocytosis (PL+, PL-) and in BLV-free cattle. The percentage of SIg+ cells was more than twice as high in BLV+PL+ cattle than in BLV-free and BLV+PL- cattle. Bovine T cells, and T cell subsets were identified indirectly by the same techniques using three monoclonal antibodies (MAb's) specific for all T cells (IL-A43), T helper (BoT4) cells (IL-A12) and T cytotoxic (BoT8) cells (IL-A17). The major histocompatibility complex (MHC) determinants of both class II (BoT4) and class I (BoT8) as well as all T cells were significantly reduced in BLV+PL+ compared to BLV-free cattle. The actual decrease in the BoT8 cell subset or the dilution effect that would change effector:target cell ratio suggests that a resultant decrease in cytotoxic activity in BLV+PL+ cattle may play an important role in the progress of BLV infection in cattle.     

Cattle Infected with Bovine Leukaemia Virus may not only Develop Persistent B‐cell Lymphocytosis but also Persistent B‐cell Lymphopenia

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non‐persistent lymphocytotic, PL^?) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL^? cattle and compared with six age‐matched animals with persistent lymphocytosis (PL⁺) and five non‐infected healthy controls (BLV^?). In the PL^? group, the percentage and number of surface immunoglobulin‐positive (sIg⁺) B cells were significantly reduced. Whereas in BLV^? cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg⁺ and 24% were sIgM⁺ B cells. In the PL^? group, less than 20% of the PBL were sIg⁺ and sIgM⁺ B cells. Only 5% of the PBL co‐expressed sIgM⁺ and CD5⁺ versus 16% in BLV^?. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM⁺ B cells co‐expressing CD5 and CD11b; and (iii) equally both λ‐ and κ‐type light chain B‐cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5^? and CD11b^? sIgM⁺ B cells and CD2⁺ T cells did not differ. In PL⁺ animals, about 75% of the PBL were sIgM⁺CD5⁺ B cells. These cells were of polyclonal origin, as light chains of the λ‐ and κ‐type were expressed in a ratio of 4:1 (57.7% of PBL λ⁺, 14% κ⁺) as in BLV^? animals (33.6% of PBL λ⁺, 8.7% κ⁺). In PL⁺ cattle the absolute number of B‐cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T‐cell numbers, the relative percentage of T‐cells could be lower than in normal controls. The cause for the observed B cell decrease in PL^? cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B‐cell lymphopenia.     

Experimental infection of calves with bovine leukemia virus (BLV): an applicable model of a retroviral infection

An experimental model of chronic infection with bovine leukemia virus (BLV) was established in young calves within a relatively short time. In the sera of all infected calves, precipitating antibodies were detected within 5 weeks after infection but upon disease progression pattern of cellular profiles varied. Three calves exhibited transient lymphocytosis 3-5 weeks after infection, two became persistent lymphocytotic (PL+) by that time and one stayed non-lymphocytotic (PL-) for 11 weeks and became PL+ after 4.5 months. Eventually all infected calves became PL+ by the end of the experiment, 6-12 months after infection. Increase of total counts of peripheral blood mononuclear cells (PBMC) related to polyclonal expansion of B-cells. The latter was assessed in all infected calves where the expansion of CD5-bearing cells (B+ CD5+) correlated with increase or decrease of total PBMC counts. Other cell populations such as CD4 and CD8 were also affected. Percentages decreased by 5 weeks after experimental infection to about half their original values though actual cell numbers stayed relatively stable. The experimental model we established compared well with field cases of naturally BLV-infected cattle and thus permitted the investigation of the disease at early stages of infection.     

Monoclonal antibodies that distinguish bovine T and B lymphocytes

The specificities of three monoclonal antibodies (MAbs) were investigated using microcytotoxicity, fluorescence microscopy and laser flow cytometry (LFC) techniques. By microcytotoxicity, bovine thymocytes (n = 4) were estimated to be 85% B26A+, 4% TH21A+, and 1% H4+. Nylon wool enriched peripheral blood T lymphocytes (n = 3) were 90% B26A+, 10% TH21A+ and 10% H4+. Adherent B cell enriched fractions (n = 3) were 10% B26A+, 90% TH21A+ and 90% H4+. The two fluorochrome method was used to simultaneously identify lymphocytes that were sIg+ and MAb+. In these experiments, 92% of all sIg+ cells were H4+. An identical result was obtained for TH21A. 85% of all sIg- cells were B26A+. Using LFC, the mean percentages of sIg+, H4+ and TH21A+ PBL (n = 5) were not significantly different. B26A recognized a significantly greater population of cells, equivalent to the expected percentage of T lymphocytes. LFC also revealed two relatively discrete sizes of B26A+ PBL. The larger population overlapped the size range in which sIg+, H4+, TH21A+ PBL were found. The more numerous smaller B26A+ PBL were in a size range in which few sIg+, H4+ and TH21A+ PBL were found. In a study of MAb reactions with PBL of 185 cows, it was shown that in 92% of the animals H4 and TH21A were positively correlated (r = +.93), when H4 and TH21A were negatively correlated with B26A (r = -.94 and r = -.92, respectively). These correlation coefficients indicate a converse relationship between B26A and both H4 and TH21A. The remaining 8% of the animals were heterogeneous in their expression of the H4 and TH21A markers but not the B26A marker. These results provide strong evidence that: 1) B26A is a pan-T lymphocyte MAb in cattle, 2) a small but significant degree of heterogeneity exists in the expression of the epitopes recognized by H4 and TH21A. However, both MAbs recognize all B lymphocytes of most individuals, and 3) using a variety of immunological methods these three MAbs can now reliably be used to assay bovine T and B lymphocytes.     

Experimental infection of sheep with bovine leukemia virus: infectivity of blood, nasal and saliva secretions

This study was designed to determine the relative infectivity of lymphocytes and secretions from BLV-infected cattle with and without persistent lymphocytosis (BLV+PL+ and BLV+PL-). Ninety-seven sheep of mixed sex and age were assembled into 21 experimental groups. The recipient sheep were inoculated intravenously with serial dilutions of whole blood, saliva or nasal secretions from BLV+PL+ and BLV+PL- donor cows. Between 200 to 20,000 cells from single and mixed BLV+PL+ or single and mixed BLV+PL- donor cattle were used for inoculation. A very small number of BLV-infected lymphocytes (200 cells) was sufficient to induce BLV infection in sheep inoculated with diluted whole blood from BLV+PL+ cattle. The inoculation of whole blood (containing up to 20,000 lymphocyte cells) from BLV+PL- cattle did not induce BLV infection in recipient sheep. Saliva and nasal secretions also failed to bring about BLV transmission.     

Cellular immune response cytokine expression during the initial stage of bovine leukemia virus (BLV) infection determines the disease progression to persistent lymphocytosis

We have established experimental models of bovine leukemia virus (BLV) infection followed by progression to persistent lymphocytosis (PL) positive (BLV+PL+) or PL negative (BLV+PL-) stages of infection. Two out of six BLV infected animals developed PL+ 4 weeks after BLV infection. One other animal became PL+ late in the course of infection and three infected animals stayed PL-. These animals (PL-) exhibited transient lymphocytosis 3-4 weeks after infection and sustained PL- lymphocyte counts up to 24 weeks after infection. Competitive RT-PCR analysis of IFN-gamma mRNA expression revealed that peripheral blood mononuclear cells (PBMC) of animals with PL+ status developed by 4 weeks after infection had augmented IFN-gamma mRNA expression 3-4 weeks after BLV infection. However PBMC of animals that sustained a long-termed PL- lymphocyte count had elevated IFN-gamma mRNA expression 1-24 weeks after infection. Competitive RT-PCR analysis of IL-2 mRNA expression showed an increase in the levels of IL-2 mRNA in PL animals. Interleukin-10 (IL-10) mRNAs expression were elevated both in PL+ and PL- animals from 3 and 12 weeks after infection respectively. We suggest that early and extended expression of cellular response cytokines may delay the progression to PL+ in enzootic bovine leukemia.     

Frequency of lymphocytes bearing Fc receptors and surface membrane immunoglobulins in normal, persistent lymphocytotic and leukemia cows.

Fluoresceinated, heat-aggregated bovine immunoglobulins (B-IgG) and human immunoglobulins (H-IgG) were used to detect a receptor for the crystallizable fragment (Fc) of the immunoglobulin molecule on peripheral blood lymphocytes (PBL) of cattle. The aggregated and B-IgG and H-IgG bound to the bovine PBL, but aggregated H-IgG was found to be more sensitive for the detection of Fc receptors. The specificity of aggregated H-IgG binding to the Fc receptors was established by demonstrating that antigen-antibody complexes inhibited this binding, and unaggregated H-IgG did not bind significantly to PBL. Double-labeling experiments suggested that all Fc+ cells have surface immunoglobulins (SIg), a marker for B lymphocytes. The percentage of Fc+ and SIg+ cells in normal animals was 9.5% (range 4-15%) and 16.2% (range 4.5-30.2%), respectively. Persistent lymphocytotic cows had 2.71 times more Fc+ and 3.85 times more SIg+ lymphocytes than did normal cows. Cows with lymphosarcoma had a lower percentage of Fc+ and SIg+ cells than did cows with persistent lymphocytosis. Cases with thymic lymphosarcoma and those with the skin form of leukemia had normal percentages of Fc+ and SIg+ cells.     

Serum immunoglobulin concentrations in cattle naturally infected with bovine leukemia virus

In order to elucidate whether natural infection of BLV in cattle might induce humoral immunological responses, changes in IgG1, IgG2, and IgM concentrations in the sera of infected cattle were determined. Twelve BLV-infected cattle were used. Cattle of different breeds were classified serologically and haematologically into BLV + PL+, BLV + PL- and BLV-free groups. Ig concentrations in the sera of the three groups were quantitated using a commercial single radial immunodiffusion assay. The findings were compared to those of BLV-free cattle. The serum IgM concentrations were significantly lower in cattle with PL (P less than 0.001) than in BVL + PL- and BLV-free cattle. The IgM concentrations tended to be lower in BLV+ PL- than those of BVL-free cattle. There were no significant differences in IgG1 and IgG2 serum concentrations between the three experimental cattle groups. IgG1 was the predominant subtype in the sera of all cattle groups.     

Development and serial passage of persistent lymphocytosis associated with bovine leukemia virus infection in cattle

Two calves each were inoculated with 1.5 x 10(8) or 5 x 10(9) lymphocytes collected from each one cow which had persistent lymphocytosis (PL) and antibodies to bovine leukemia virus (BLV). A sudden increase in the number of peripheral blood lymphocytes (PBL) was observed 14 and 23 days, respectively, after inoculation and the maximum number reached 29,000 and 52,000/microliters 72 and 57 days after inoculation. Although the degree of PL decreased gradually in these cattle, it continued until 14 and 44 months after inoculation when one animal was sacrificed and the other died of lymphosarcoma. The PL was passaged in cattle by inoculation of a large number of PBL obtained from cattle at the stage of PL (PLL). The degree of PL was severer in cattle inoculated with a larger number of PLL. PL was not caused by inoculation of PBL obtained from either BLV-infected non-PL cattle or cattle free of BLV. The PL was also caused by inoculation of PLL into BLV-infected non-PL cattle. On the other hand, it was not observed after inoculation of a large amount of cell-free virus obtained from short-term cultures of PLL. Antibodies to BLV developed earlier and to higher levels in cattle inoculated with PLL than in those inoculated with cell-free virus. These facts show that infection with BLV was established more effectively by PLL than by cell-free virus, the infection may occur by lymphocyte to lymphocyte interaction and the actual number of infected BLV may have an important role in development of PL.     

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.     

Cellular interactions regulating the in vitro response of bovine lymphocytes to ovalbumin.

We examined the contribution of MHC class II-restricted T cells (CD4+), MHC class I-restricted T cells (CD8+), gamma/delta T cell receptor (TCR)+ T cells, B cells and macrophages to the development and control of in vitro proliferative responses of bovine lymphocytes to ovalbumin (OA). Cell populations for in vitro assay were obtained from peripheral blood (peripheral blood leukocytes, PBL) of OA-primed cattle. Specific cell populations were depleted or purified from PBL by staining with monoclonal antibodies (MAbs) against the appropriate differentiation antigens and sorting on a Fluorescence Activated Cell Sorter (FACS). OA-specific in vitro responses of in vivo primed PBL were dependent on the presence of CD4+ T cells. Their presence could not be replaced by the inclusion of T cell growth factor (TCGF) in the culture system, indicating that CD4+ T cells probably actively proliferate in response to antigenic stimulation. Bovine CD8+ T cells and gamma/delta TCR+ T cells appeared to exert a suppressive effect on proliferative responses. No proliferation was observed in PBL after the depletion of MHC class II+ cells. In this case, the response could be restored by the addition of macrophages or LPS-activated B cells to the MHC class II- population.     

Binding of soluble immune complexes to fractionated Atlantic salmon (Salmo salar L.) leucocytes.

Binding of a fluorescent-labelled soluble immune complex to different types of Atlantic salmon leucocytes was investigated using flow cytometry. Peripheral blood leucocytes (PBL) were separated into sIg+ and sIg enriched populations by magnetic activated cell sorting, blood neutrophils were identified by electronic gating, and kidney macrophages were selected by plastic-adherence. About 60% of both sIg+ and sIg- enriched PBL, 44% of neutrophils and 34% of macrophages bound the soluble immune complexes.     

Influence of betulinic acid on lymphocyte subsets and humoral immune response in mice

Betulinic acid is a pentacyclic triterpene found in many plant species, among others, in the bark of white birch Betula alba. Betulinic acid was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial, anticancer and anti-inflammatory activities. The effects of betulinic acid (50, 5, 0.5 mg/kg) administered orally five times at 24 hours intervals to non-immunized and red blood cells (SRBC)-immunized mice were determined. The present study examined the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes, and the percentage of subsets of T cells (CD4+CD8+, CD4CD8, CD4+, CD8+) in thymus,T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes, as well as white blood cell (WBC) and differential leukocyte counts in non-immunized mice, and humoral immune response in SRBC-immunized mice. SRBC was injected 24 hours after administration of the last dose of betulinic acid. It was found that betulinic acid administered orally five times at the dose of 0.5 mg/kg increased the total number of thymocytes, splenocytes, lymphocytes of mesenteric lymph node cells, and the weight ratio of the spleen and mesenteric lymph nodes in non-immunized mice. Betulinic acid also changed the percentage of T cell subsets in the thymus and T and B lymphocytes in peripheral lymphatic organs. The effects of betulinic acid on T and B cell subpopulations depended on the dose applied. The strongest stimulating effect of betulinic acid was observed when the drug was administered at the dose of 0.5 mg/kg. Five exposures to betulinic acid (0.5 mg/kg) decreased the percentage of immature CD4+CD8+ thymic cells with corresponding increases in the percentage and absolute count of mature, single-positive CD4+ thymocytes and decreased the percentage and total count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Multiple administration of betulinic acid at the investigated doses augmented the percentage and absolute count of CD19+ cells in the peripheral lymphatic organs. Moreover, betulinic acid at the dose of 5 mg/kg administered prior to SRBC immunization increased the number of plaque forming cells (PFC) but decreased the production of anti-SRBC antibodies on day 4 after priming. Thus, betulinic acid is a potential biological response modifier and may strengthen the immune response of its host.     

Generation and characterization of monoclonal antibodies to equine NKp46

The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population.     

Distribution of T and B lymphocytes in mammary dry secretions, colostrum and blood of adult dairy cattle

The distribution of lymphocyte subpopulations from dry secretions, colostrum and blood from 10 healthy adult Hostein-Fresian cows was studied using the TH21A and B26A mouse monoclonal antibodies (MAb) to adult bovine B and T lymphocytes, respectively. The mammary gland lymphocytes (MGL) were isolated from composite sample of all four quarters by density centrifugation over discontinuous gradient of ficoll-diatrizoate. The peripheral blood lymphocytes (PBL) were purified using the ficoll-thrombin method. Isolated PBL and MGL were analyzed using the two fluorochromes method (TFM) and laser flow cytometry (LFC). The mean viability of isolated PBL and MGL from dry secretions and colostrum after the TFM and LFC were 92.4% +/- 3.2%, 91.4% +/- 6.0% and 87.1% +/- 6.1%, respectively. There was a good correlation between the two MAbs and the percentage of surface immunoglobulin (SIg) positive cells in the peripheral blood using the TFM. The PBL yielded a mean percentage of 21.2% B cells, 66.4% T cells and 9.4% "Null cells" (TH21A+; SIg-). The TFM on MGL from dry secretions and colostrum indicated two distinct patterns (group I and II) of SIg and reactivity to MAb markers (p less than 0.001). The MGL data included in group I and group II were gathered from both colostral and dry secretions. In comparison to the distribution of lymphocyte subsets within peripheral blood the mean percentages of B cells, T cells and "Null cells" in the mammary gland were respectively, 2.8%, 88.1% and 5.4% for group I and 3.5%, 89.0% and 15.1% for group II. In the mammary secretions, the use of SIg alone was not considered to be a good marker for B cells; in four animals a mean percentage of 15.6% (13.9/89.0 X 100) of the mammary gland T lymphocytes were also SIg+. Of the TH21A+ MGL, only 18.8% were SIg+ in group II compared with 34.1% for MGL from group I and 69.3% for the PBL. Marked differences in cell size distribution and cell surface antigen density were found when PBL and MGL from dry secretions were compared by LFC using the B26A MAb. The results of this study demonstrate a difference in the percentages of peripheral blood and mammary gland B and T lymphocytes and confirm previous findings in which the T lymphocytes were found to represent the major subpopulation of lymphocytes in bovine mammary secretions. This may represent an essential event in the adoptive transfer of cellular immunity through the colostrum in cattle.     

Use of alpha-naphthyl acetate esterase staining to identify T lymphocytes in cattle

Peripheral blood lymphocytes (PBL), lymph nodes, and/or spleens from clinically normal cattle were examined for cytochemical staining of alpha-naphthyl acetate esterase (ANAE). Two types of positive-staining patterns in ANAE staining resulted. By a combination of ANAE staining and latex-ingesting test, diffuse ANAE-positive cells were considered as mononuclear phagocytic cells. Using erythrocyte rosettes, erythrocyte antibody complement rosettes, nylon-wool column technique, surface immunoglobulin (SIg) staining, and the ANAE staining technique, granular ANAE-positive lymphocytes were shown to be T lymphocytes. The frequency of T and B lymphocytes in PBL, spleens, and lymph nodes of clinically normal cattle was measured, using ANAE staining and SIg staining. In PBL, 47.7% were ANAE-positive and 26.9% were SIg-positive; in spleens, 22.4% were ANAE-positive and 53.7% were SIg-positive; and in lymph nodes, 38.5% were ANAE-positive and 28.3% were SIg-positive. The frequencies of T and B lymphocytes in PBL, spleens, and/or lymph nodes from cattle with enzootic bovine leukosis (EBL) and cattle with persistent lymphocytosis and in tumor cells from cattle with EBL were measured. When compared with those of clinically normal cattle, PBL, spleens, and lymph nodes of cattle with EBL and the PBL of cattle with persistent lymphocytosis contained numerous SIg-positive cells and few ANAE-positive cells. Tumor cells from cattle with EBL contained 7.3% ANAE-positive and 78.0% SIg-positive cells.     

The effects of florfenicol on lymphocyte subsets and humoral immune response in mice

Florfenicol is a broad-spectrum bacteriostatic antibiotic used in domestic animals. The aim of the study was to determine the effect of florfenicol on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the peripheral lymphatic organs in non-immunized mice and humoral immune response in sheep red blood cells (SRBC)-immunized mice. Florfenicol was administered orally at a dose of 30 mg/kg six times at 24 h intervals to non-immunized mice and four or seven times at 24 h intervals to SRBC-immunized mice. SRBC was injected 2 hours prior to the first dose of the drug. Florfenicol increased the percentage of CD4CD8- thymocytes and the absolute number of CD4+ and CD8+ thymocytes on day 7. The increased percentage and absolute number of CD3+, CD4+ and CD8+ lymphocytes in mesenteric lymph nodes and decreased percentage of lymphocytes B were also observed 24 hours from the last administration of florfenicol. Florfenicol administered after SRBC immunization reduced the number of plaque forming cells (PFC) and the production of anti-SRBC antibodies on days 4 and 7 after priming.     

Age-related changes in leukocytes and T cell subsets in peripheral blood of Japanese Black cattle

It is well known that the immune system changes with age during development and maturation in Holstein cattle. But age-related changes in leukocytes and T cell subsets in peripheral blood of Japanese Black cattle still remain unclear. The aim of the present study was to investigate comparative changes of leukocytes (granulocytes, monocytes, B cells and T cells) and T cell subsets (CD4⁺, CD8⁺, γδ, CD8⁺γδ and WC1⁺γδ T cells) in Japanese Black cattle aged 0.5, 1, 2, 6, 18 and 36–41 (adult) months on flow cytometry using specific monoclonal antibodies for the cell surface markers. T cell proportion was approximately 40% in 2-month-old cattle and decreased to 20.6% in adults. In contrast, B cell proportion significantly increased from 7.4% to 28.2% with age. In T cell subsets the percentage of CD4⁺ T cells significantly increased from 40.5% to 60%, but that of WC1⁺γδ T cell subset significantly decreased with age. The percentages of CD8⁺ and CD8⁺γδ T cells did not change. The present study details the proportional changes in leukocyte and T cell subsets with age in the peripheral blood of Japanese Black cattle and these findings are similar to those described for Holstein cattle.     

Influence of bestatin, an inhibitor of aminopeptidases, on T and B lymphocyte subsets in mice

Bestatin, a low-molecular weight dipeptide, is a potent inhibitor of aminopeptidase N which has been demonstrated to have antitumor and immunomodulatory effects. The effects of bestatin (10, 1 and 0.1 mg/kg) administered intraperitoneally once, five or ten times to mice on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes were studied. It has been found that bestatin administered ten times at doses of 10, 1 and 0.1 mg/kg increased the total number of thymocytes, splenocytes and lymphocytes of mesenteric lymph nodes. Bestatin also changed the percentage and the absolute number of T cell subsets in the thymus and T and B lymphocytes in the peripheral lymphatic organs. Five and ten exposures to bestatin (10, 1 and 0.1 mg/kg) increased the absolute count of both immature CD4+CD8+ and CD4-CD8- thymic cells. Moreover, both a single and multiple administration of bestatin (1 and 0.1 mg/kg) decreased the percentage and absolute count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Both a single and multiple administration of bestatin at all the doses under investigation augmented the percentage and the absolute count of CD19+ (B lymphocytes) in the peripheral lymphatic organs. The results of the study show that there is a relationship between the effect induced by bestatin and the dose of the drug as well as the number of doses applied. The strongest effect on the T and B lymphocyte subsets was noted after five injections of bestatin at doses of 1 and 0.1 mg/kg.