Immunochemical analysis of baboon (Papio cynocephalus) IgG subclasses

Few reports are presently available on the existence of IgG subclasses in nonhuman primates. Papain and trypsin digestion of baboon (Papio cynocephalus cynocephalus and P. cynocephalus hamadryas) IgG proteins, in the absence of cysteine, revealed the occurrence of two different protein populations, one being protease-resistant, the other being protease-sensitive. The papain-resistant population is rendered sensitive to digestion upon addition of cysteine. Ion exchange chromatography of the papain-resistant IgG population subsequently demonstrated that it is composed of two different subpopulations varying in their ionic binding affinities. Peptide maps of the Fc fragments of the papain sensitive population and of the Fc fragments of the two papain-resistant subpopulations differing in their binding affinities for ion exchange resins, were different from each other. The biochemical identification of the baboon IgG proteins presented here strongly suggests that they are composed of at least three different subclasses.     

Potentiation of lymphocyte mitogenic responses to concanavalin A by antigen-activated peripheral blood monocytes.

Purified protein derivative (PPD)-stimulated monocytes derived from Mycobacterium bovis-sensitized cattle significantly potentiated lymphocyte mitogenic responses to concanavalin A (conA), as measured by incorporation of [3H] thymidine into cellular DNA. Monocytes were cultured for 24 hours in the presence of PPD, washed thoroughly, and mixed with purified lymphocytes; various doses of conA were added to these cultures, and the cultures were incubated for 4 days and assayed for DNA synthesis. The lymphocyte mitogenic responses to suboptimal, buy not optimal, doses of conA were significantly enhanced by the presence of PPD-activated monocytes from M bovis-sensitized cattle. Treatment nonsensitized cattle with PPD did not result in any significant enhancement of conA-induced lymphocyte mitogenic responses at any dose of conA tested.     

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.     

Isolation of equine peripheral blood mononuclear cells using Percoll.

The concentration of Percoll required for isolating equine peripheral blood mononuclear cells has been reinvestigated. A poor cell yield was obtained at the 60 per cent concentration already reported. It is recommended that workers specifically interested in high yields of mononuclear cells, for investigation of lymphocyte and monocyte functions, use a concentration of 65 per cent Percoll. However, workers wishing to isolate pure populations of equine neutrophils might consider a concentration of 70 per cent in the upper layer of Percoll used to retain the mononuclear cells.     

Generation of canine dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.     

Intestinal parasites of baboons (Papio cynocephalus anubis) and chimpanzees (Pan troglodytes) in Gombe National Park.

A survey of gastrointestinal parasites of nonhuman primates in Gombe National Park, Tanzania, was conducted during July and August 1989. Fresh fecal samples collected from 35 baboons (Papio cynocephalus anubis) and 20 chimpanzees (Pan troglodytes) were analyzed for intestinal parasites. Parasite identifications were based upon the size and appearance of eggs and larvae on both direct and fecal flotation examinations. Seven species of helminths and three species of protozoa were noted in the chimpanzees, and seven species of helminths, two species of trematodes, and one species of protozoa were noted in baboons. An unidentified parasite, with a thick egg shell similar to that of ascarids, was noted in both the chimpanzee and baboon population. Unfortunately a definitive identification of this parasite could not be made based upon egg size and morphology alone and insufficient numbers of larva were present to aid identification. One previously unreported parasite, Schistosoma mansoni, was identified in the baboon population. This is the first report of a schistosome in Gombe's nonhuman primate population.     

Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.     

Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro

The presence of PCV2 DNA or spliced capsid mRNA (Cap mRNA) for viral replication was assessed following addition of PCV2 to resting or concanavalin A (ConA) stimulated peripheral blood mononuclear cells (PBMCs). Real-time PCR or real-time RT-PCR assays were used to measure viral DNA or Cap mRNA, respectively. The study demonstrated that PCV2 replication increased in infected PBMCs over time. Replication within infected PBMCs was significantly (P<0.05) increased when PBMCs were stimulated with ConA, compared to unstimulated PBMCs. The data showed a strong correlation between the level of PCV2 Cap mRNA and the level of viral DNA in the ConA stimulated PBMCs. Replication of PCV2 was also assessed in T lymphocyte- and monocyte/macrophage-enriched or monocyte/macrophage-depleted PBMC populations which had been stimulated with ConA for 3 days. It was demonstrated that the enriched T lymphocytes and the monocyte/macrophage-depleted PBMCs had significantly higher Cap mRNA and viral DNA levels (P<0.05) compared to the monocyte/macrophage-enriched population, indicating that in addition to monocytes/macrophages, PCV2 replicates in lymphocytes, particularly T lymphocytes following stimulation. These results suggest that the presence of activated T lymphocytes may play an important role in PCV2 replication and potentially the development of clinical disease.     

Streptococcus equi but not Streptococcus zooepidemicus produces potent mitogenic responses from equine peripheral blood mononuclear cells

Streptococcus equi causes equine strangles. The acute disease has many of the hallmarks of an acute response including high fever, elevated plasma fibrinogen and neutrophilia, affects known to be mediated by proinflammatory cytokines. The objective of this study was to screen-culture supernatants from equine clinical isolates of S. equi and S. zooepidemicus for stimulation of mitogenic responses by horse peripheral blood mononuclear cells. Mitogenicity comparable to that of concanavalin A was detected in culture supernatants of S. equi strains but not in those of S. zooepidemicus. Mitogenicity was neutralised by Proteinase K and a post-strangles convalescent serum, and evidence for the presence of both thermo-resistant and thermo-labile mitogenic factors was obtained. Release of proteinaceous immunogenic mitogens in combination with the antiphagocytic protein SeM unique to S. equi may therefore contribute to some of the severe clinical manifestations of acute strangles in the horse.     

Reactions of peripheral blood mononuclear cells (PBMC) of camels with monoclonal antibodies against ruminant leukocytes

The particular immune system of the camel has been but little investigated. In this work circulating camel peripheral blood mononuclear cells (PBMC) were studied by flow cytometry. Monoclonal antibodies (mAbs) raised against ruminant leukocytes were used for the detection of cell surface antigens. Monoclonals to T-cell markers, CD4 (CACT138A) and CD8 (CACT80C), exhibited no reactivity towards camel PBMC in contrast to their reactivity to PBMC of other ruminant species and those of cattle in particular. A relatively high percentage (29.1+/-8.9%) of camel PBMC reacted with a non-immunoglobulin cell surface marker, B-B2, comparable to the reactivity of bovine PBMC. The B-B7 cell marker revealed 22.4+/-10.0% of reactive camel PBMC while the CD45 leukocyte common antigen was identified only on 19.4+/-3.1% of camel PBMC as compared to 74.7+/-4.9% for bovine PBMC. IgM (PIg45A) was detected on 9.1+/-1.4% of camel PBMC and on 46.6+/-19.5% of the bovine PBMC. Double fluorescent labeling with two B-cell markers and an anti-ruminant lambda light-chain mAb revealed 7-9% of cells bearing both B and lambda L-chain markers. Light chain reactivity was also assessed using an anti-goat F(ab')(2) antiserum. The values obtained, 14.3+/-5.8% for the camel and 47.8+/-2.7% for the cattle, are close to the values observed for surface IgM. These data suggest that camels, like other ruminants, possess L-chain bearing cells of the B-cell lineage. However, in the camel, Igs are different in that in addition to regular four chain Igs, about 65% of them possess two heavy chain Igs devoid of light chains. Because different sets of V(H) gene segments are used by four and two chain Igs, it is possible that there might be two lineages of B-cells each secreting a different form of antibodies.     

Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides

Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N=10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

Increased apoptosis of peripheral blood mononuclear cells (PBMC) during general and epidural anaesthesia in dogs

The objective of the study was to investigate the hypothesis that perioperative lymphocytopenia was due to apoptosis of these cells induced by either halothane or epidural anaesthesia in dogs. The relationship between apoptosis induction and plasma concentrations of the stress hormone cortisol and the cytokines TNF-alpha and IL-10 was examined as well. The study was performed on 22 healthy mongrel dogs, equal numbers from both genders, weighing 18.3 ± 2.9 kg, and aged between 3–5 years. Dogs were divided in three groups. Eight of the animals were anaesthetized with halothane, another eight received epidural anaesthesia using lidocaine, and six served as controls. Venous blood samples were obtained immediately before (0 minute) anaesthesia, during deep anaesthesia (120 minute), and on the next day (24 hour) in order to determine the following parameters: the total lymphocyte counts, the percentage of apoptotic peripheral blood mononuclear cells (PBMC) by flow cytometry, plasma concentrations of the cytokines tumor necrosis factor – alpha (TNF-alpha) and interleukin – 10 (IL-10) by enzyme-linked immunosorbent assay (ЕLISA), and plasma cortisol levels by radioimmune assay. Both halothane and epidural anaesthesia in dogs induces apoptosis of PBMC with slight decrease in total lymphocyte counts. These immunomodulatory effects were transient and faded till the 24th hour. Concerning the mechanism of inducing lymphocyte apoptosis by general or epidural anaesthesia, it seemed that neither cortisol, nor the tested cytokines TNF-alpha and IL-10 were implicated in this process. Further investigations are necessary to confirm this assumption.     

Response of bovine and porcine peripheral blood mononuclear cells to human recombinant interleukin 2(125)

Bovine and porcine peripheral blood mononuclear cells (PBMC) were tested for their response to human recombinant interleukin 2(125) (rIL 2(125)). The rIL 2(125) used in these experiments was purified to homogeneity from Escherichia coli, contained a site-specific modification at amino acid #125 replacing a cysteine with a serine residue and had a specific activity of 4 X 10(6) units/mg. Human rIL 2(125) was shown to be directly mitogenic for bovine and porcine PBMC and was able to maintain the long-term growth of mitogen-activated PBMC of both species. Long-term cultures were highly sensitive to low levels of rIL 2(125) and showed dose-dependent responses when used in short-term IL 2 assays. Bovine and porcine PBMC preincubated with human rIL 2(125) for 1 and 5 days demonstrated enhanced levels of cell-mediated cytotoxicity against both allogeneic and xenogeneic cell lines.     

2-Mercaptoethanol influences the in vitro function of bovine peripheral blood mononuclear cells.

The effects of 2-mercaptoethanol (2-ME) on some in vitro functions of bovine peripheral blood mononuclear cells (PBMC) were examined. It was shown that 2-ME enhanced, in a dose-dependent manner, the production of antibodies to bovine coronavirus. In this test the optimal concentration of 2-ME was 50 microM. This molarity of 2-ME was also optimal for the pokeweed mitogen (PWM)-induced proliferation of PBMC obtained from the 7 cattle tested. Similarly, the spontaneous proliferation of PBMC from 4 out of these cattle was enhanced. Thus, 2-ME evoked an increase (up to 2.5 times) or a decrease (at most 10 times) of the quota between the PWM-induced and the spontaneous proliferation (stimulation index). In general, the presence of 50 microM 2-ME enhanced the in vitro production of interferon by bovine PBMC. On the contrary, the highest proliferative response of PBMC to stimulation with bovine virus diarrhoea virus was achieved in cultures without 2-ME or in cultures with 0.5 or 5 microM 2-ME. Since the effects of 2-ME varied, for different tests as well as for cattle tested, attention should be paid to the use of 2-ME in cultures of bovine PBMC.     

Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells.

The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.     

Detection of bovine herpesvirus-1 in peripheral blood mononuclear cells eight months postinfection.

Peripheral blood mononuclear cells (PBMCs) from 5 calves (3 controls and 2 vaccinates) used in a bovine herpesvirus 1 (BHV-1) vaccine study with a BHV-1 Cooper strain challenge were collected 6 months after challenge. The PBMCs from the control animals were positive by immunofluorescence for the BHV-1 glycoprotein D (gD) while the vaccinates were negative. The PBMC samples from 4 of the 5 animals were examined for BHV-1 DNA by polymerase chain reaction (PCR) and for gD immunofluorescence at 8 months after challenge. The BHV-1 DNA and viral antigen were detected in PBMC samples at 8 months postinfection, but no virus was isolated.     

Mitogenic reactivity of mononuclear cells isolated from thymus, spleen and umbilical cord blood of pig foetuses

The in vitro mitogenic reactivity of mononuclear cells from the thymus, spleen and umbilical cord blood of Danish Landrace pig foetuses ranging in gestational age (GA) from 48 to 112 days was monitored by means of a microculture lymphocyte transformation test (LTT). Dose-response profiles for concanavalin A (Con A), pokeweed mitogen (PWM) and leucoagglutinin (LAG) were set up for the various age-groups and the results showed that the onset and development of mitogenic reactivity in the pig foetus is age-related. The results indicate the occurrence of mitogen responsive cells in the thymus and cord blood at 48 days GA and in the spleen at 54 days but statistically significant reactivity (p less than 0.01) for the various tissues could only be demonstrated at later stages of gestation. Thymus cells from all foetuses ranging in GA from 54 to 112 days exhibited significant reactivity to Con A, PWM and LAG. While the first detectable definite response of spleen cells was seen at 60 days GA when 50% of the foetuses exhibited significant reactivity to the 3 mitogens, spleen cells from all foetuses beyond that age responded significantly. Cord blood cells from only 50% of the foetuses of 60 and 70 days GA responded significantly to Con A and PWM but after this stage, cord blood cells from all foetuses did. The first significant response of cord blood cells to LAG was seen at 70 days GA but only in 50% of the foetuses and it was not until 100 days GA that significant reactivity to LAG was detected in all foetuses.     

Equine herpesvirus-1-specific interferon gamma (IFNgamma) synthesis by peripheral blood mononuclear cells in thoroughbred horses

REASONS FOR PERFORMING STUDY: An assay has been developed that measures EHV-1 specific interferon gamma synthesis (IFNgamma), a cytokine produced following the activation of memory T lymphocytes and therefore a measure of cell mediated immunity. The method requires validation in the field. OBJECTIVES: To measure the frequency of EHV-1 specific, IFNgamma synthesising peripheral blood mononuclear cells (PBMC) in a population of Thoroughbred horses, and examine its relationship with age, gender, premises and history of vaccination or field infection with EHV-1. METHODS: Lymphocytes from 200 Thoroughbred horses were stimulated with EHV-1 in vitro, and IFNgamma detected using a monoclonal antibody and indirect immunofluorescence. Percent positive cells were enumerated by flow cytometric analysis and the results described and compared statistically between groups. RESULTS: The frequency of IFNgamma+ PBMC was significantly higher in animals age >5 years compared with 2-4 years, in females vs. males, on stud farms vs. training yards and following vaccination of 2-year-olds with inactivated virus compared with nonvaccinates. Age strongly confounded all these associations and care must therefore be taken interpreting these results. Mares exposed to a field infection with EHV-1 also had higher frequencies of IFNgamma+ PBMC than other vaccinated horses. CONCLUSIONS: The frequency of EHV-1 specific, IFNgama+ PBMC among the sample Thoroughbred population was diverse but lowest in young, unvaccinated horses-in-training. POTENTIAL RELEVANCE: The frequency of EHV-1 specific lymphocytes synthesising IFNgamma in this population may be associated with its susceptibility to infection with this virus. This easy technique may be applied to monitor the antigenicity of vaccines and their effectiveness at stimulating cellular immunity.