In vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes from ponies.

The in vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes (PBL) from ponies were studied. Prednisolone inhibited lymphocyte stimulation by phytohemagglutin (PHA) in a dose-dependent manner, without inducing lysis even at large doses. The PBL from horses heterozygous for the combined immunodeficiency trait responded to corticosteroid treatment the same as did PBL from normal ponies. Removal of the corticosteroid after incubation with PBL from normal ponies partially restored responsiveness of these cells to PHA. Chronic in vivo treatment of ponies with corticosteroids caused a marked decrease in the absolute numbers of circulating lymphocytes. Most remaining lymphocytes had detectable surface immunoglobulin and C3 receptors, suggesting a greater decrease in the T-lymphocyte population. In spite of this, there was little change in the in vitro PHA- or keyhole limpet hemocyanin-sensitized ponies. In general, the corticosteroid effects of lysis, as well as the mitogenic and antigenic responses of PBL from ponies, were similar to those previously reported for human lymphocytes.     

In vitro stimulation of bovine peripheral blood lymphocytes: suppression of phytomitogen and specific antigen lymphocyte responses by aflatoxin.

The effects of aflatoxin B1 on responses of the peripheral blood lymphocytes (PBL) of 2 normal animals to phytohemagglutinin, concanavalin-A, and pokeweed mitogen and of the PBL of 2 Mycobacterium bovis-infected animals to phytohemagglutinin and purified protein derivative of M bovis (PPD) were studied. Aflatoxin concentrations of greater than or equal to 10 microgram/ml significantly suppressed the lymphocyte response of normal animals to the phytomitogens. Lymphocyte response of M bovis-infected animals to specific antigen PPD was significantly suppressed at aflatoxin concentration of 0.5 microgram/ml. Fifty- to 100-fold higher concentrations of aflatoxin were required to produce 50% suppression of lymphocyte response to phytomitogens, as compared with that produced to PPD.     

Optimal macroculture method for studying mitogenic stimulation of turkey lymphocytes.

This report describes a method for culturing turkey lymphocytes in disposable, unwashed glass test tubes with Morton closures and for recovering lymphocytes on fiber glass filters with a cell harvester made of common laboratory equipment for assay of mitogenic stimulation. Optimal conditions for culture were established.     

In vitro stimulation of bovine peripheral blood lymphocytes: effect of short-term storage of blood prior to lymphocyte culture

Storage of peripheral blood from Mycobacterium bovis-sensitized cattle from 1 to 48 hours at 4, 22, and 37 C was shown not to alter markedly the lymphocyte blastogenic response to M bovis-purified protein derivative. Concanavalin A-induced lymphocyte mitogenic responses were unaffected by storage of blood for 1, 24, or 48 hours at 22 C and 37 C; however, storage of blood for 48 hours at 4 C significantly lowered (P less than 0.05) mitogenic responses to concanavalin A, as compared with responses to blood kept at 22 C. Mononuclear cell recovery from stored blood at all temperatures was markedly less than that from freshly drawn blood samples. Cell recoveries were most affected by storage of blood at 4 C and 37 C.     

In vitro response of purified ovine peripheral blood lymphocytes to phytohemagglutinin-M.

Peripheral blood was collected from three normal yearling castrated male lambs. Lymphocytes were isolated by Ficoll-Hypaque gradient centrifugation. Lymphocyte response to phytohemagglutinin was evaluated in an isotopic uptake stimulation test under varying culture conditions in order to standardize the assay. Results were analyzed as log10 counts per minute and stimulation indices. Culture conditions consisting of 2 microL (20 micrograms) phytohemagglutinin-M/culture, 1 X 10(6) cells/mL and a 72 hour incubation period were selected for use in further studies.     

Optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes

A microculture technique was developed for the in vitro blastogenesis of feline lymphocytes. Blastogenesis of ficoll-diatriazoate gradient separated mononuclear cell, washed blood and whole blood were compared. In general the whole blood cultures yielded higher stimulation indices (SI) than the washed blood or separated mononuclear cell cultures.The effect of several variables on the stimulation of lymphocyte cultures was examined. A cell concentration of 3 × 10⁵ cells/well and a 1:20 dilution of washed and unwashed whole blood gave optimal stimulation with concanavalin A (Con A). Phytohaemagglutinin-P (PHA-P) did not give significant levels of stimulation. Inactivated fetal calf serum (FCS) at levels of 2.5% (for washed blood) and 5% (for separated mononuclear cell and whole blood) gave highest SI. Supplementation with FCS was preferable to autologous, homologous or horse sera for all cultures. Optimal SI was obtained in all cultures incubated for 3 days and labelled with 1 μCi tritiated thymidine (³H-TdR) for the last 16 hours. The highest SI were in the range of 70 to 105 (18,764 to 42,681 counts per minute (CPM) for separated mononuclear cell culture, 100 to 165 (28,403 to 45,334 CPM) for washed blood culture and 105 to 186 (41,076 to 69,999 CPM) for whole blood culture.     

The non-specific stimulation of avian peripheral blood lymphocytes from uninfected chickens by paramyxoviruses and influenza viruses

Avian paramyxovirus-3 was mitogenic to peripheral blood lymphocytes (PBL) from about half the normal birds sampled from 3 inbred flocks. Eight other myxoviruses including Newcastle disease virus, Sendai virus and influenza virus were also irregularly mitogenic. This could complicate in vitro assays for specific immunity.     

猪树突状细胞的体外培养及其主要免疫生物学特性研究

为建立体外诱导、培养猪外周血单核细胞来源树突状细胞(DC)的方法,并对其进行免疫生物学特性鉴定,本实验从猪外周血无菌分离单核细胞—DC前体细胞,以猪源重组GM-CSF和IL-4联合诱导培养,从形态学、表型及功能方面对其进行检测。结果证实,经体外诱导培养的猪DC具有典型的树突状形态;培养6d的未成熟DC表面SLA-II-DR、CD1、CD172a表达的阳性率分别为61.50%、83.10%和24.90%;培养8d的成熟DC表面SLA-II-DR、CD1、CD172a表达的阳性率分别为71.70%、50.10%和19.30%;培养的DC具有吞噬异物的功能,以及刺激同种异体淋巴细胞增殖的能力。本文首次在国内成功地建立了体外培养猪外周血单核细胞来源DC的方法,为进一步研究DC在猪传染性疾病致病机制中的作用奠定了基础。     

Sequential mitogen stimulation of peripheral blood lymphocytes from chickens inoculated with infectious bursal disease virus

Chickens were inoculated wih the pathogenic Edgar strain of infectious bursal disease virus at 1 week, 2 weeks, or 1 day of age. In the 3 experiments, phytohemagglutinin stimulation of peripheral blood lymphocytes was significantly decreased on day 3 or 4 after inoculation. Subsequently, on days 7 through 21, stimulations were similar between lymphocytes from inoculated birds and those from control birds. Pokeweed mitogen stimulation was affected minimally in virus-inoculated chickens. In each experiment, on day 7, the spontaneous [3H]thymidine uptake was greater in nonstimulated lymphocyte cultures from inoculated chickens than in such cultures from control chickens. In an additional experiment, chickens 1 week of age were exposed to a pathogenic vaccinal virus given in their water. The vaccinal virus exposure resulted in significant decrease of phytohemagglutinin stimulation of lymphocytes on days 3 and 7 of the experiment. A significant decrease in pokeweed mitogen stimulation was observed on day 10 after inoculation.     

Non-specific natural cytotoxic factor released from bovine peripheral blood lymphocytes.

Natural cytotoxicity against bovine leukemia cells (PC-3 cells) was found in bovine peripheral blood lymphocytes (PBL), and in non-adherent cells but not in adherent cells to nylon-wool column. Natural cytotoxic cells (NCC), which have natural cytotoxic activity, are found in T cell-rich fraction. When NCC were cocultured with PC-3 cells, natural cytotoxic factor (NCF) was released rapidly from NCC, and dose-response curve for NCF was almost linear induction. Cytotoxicity against PC-3 cells by NCC or NCF was increased with an increment of incubation period. Cytotoxicity against K562 cells, CL-1 cells, M1 cells or EL-4 cells by NCF was almost the same level as that against PC-3 cells, but that against those cell lines by NCC was not found. NCF activity in culture fluid from NCC cocultured with K562 cells or CL-1 cells was lower than that from NCC cocultured with PC-3 cells.     

Comparative studies on in vitro mitogen-induced proliferation of peripheral blood lymphocytes in dog and breeding fox.

In vitro blastogenesis of dog and fox lymphocytes was compared by a microculture technique. The highest 3H-thymidine incorporation in cultures of dog lymphocytes was observed at day 3, while in those of fox at day 2, incubated either at 37 degrees C or at 39 degrees C. Lymphocytes cultured at 39 degrees C incorporated more tritiated thymidine than did cells cultured at 37 degrees C. The stimulation index (SI) of dog peripheral blood lymphocytes to both mitogens concanavalin A (Con A) and leucoagglutinin (LA) was in a similar range, while pokeweed mitogen (PWM) showed a weaker but significant stimulatory action. The blastogenesis of fox lymphocytes was the greatest in Con A stimulated cultures. The mitogenic potency of LA and PWM was about half of that of Con A, with no essential difference between them. Maximum lymphocyte proliferation of dog and fox was observed when culture media were supplemented with 10% fetal calf serum (FCS).     

Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin A.

A microculture system in conjunction with a semiautomatic multiple sample harvester (SAMSH) was used to study the in vitro properties of chicken peripheral lymphocytes. This new procedure enabled doing rapid multiple tests, using relatively few cells, and was highly reproducible. Data were presented to show many variables that are involved in studying the concanavalin A (Con A) response of chicken lymphocytes in a microculture system. Analysis indicated that the conditions for optimal Con A stimulation as measured by incorporation of 3H-TdR include: (a) use of 2 x 10(6) cells per culture in RPMI 1640 culture medium in the absence of any serum, (b) use of 0.4 mug of Con A per culture, (c) incubation at 37 degrees C for 72 hours, and (d) addition of 1 muCi of 3H-TdR to each culture 12 to 24 hours prior to termination. This technique could be used to monitor immunocompetence of the chicken.     

A rapid microtechnique for in vitro stimulation of canine lymphocytes using whole blood

A simple, rapid microtechnique utilizing whole blood has been developed for evaluating the in vitro stimulation of canine lymphocytes. The test uses 5 ul. of heparinized whole blood per culture in a microtiter plate and requires no serum supplement. Cultures are harvested by an automated multiple sample cell harvester. This technique permits large numbers of replicate samples to be tested on individual animals with minimal amounts of blood and minimal technician time.     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

A recombinant feline immunodeficiency virus envelope fusion protein stimulates peripheral blood lymphocytes from naive cats to proliferate in vitro.

A region of feline immunodeficiency virus (FIV)/Glasgow-8 external envelope glycoprotein (env) incorporating the third and fourth variable regions (V3/V4) was cloned, inserted into the pGEX vector and expressed in Escherichia coli to yield milligram quantities of the recombinant polypeptide as a fusion protein with glutathione S-transferase. The fusion protein V3/V4GST was used in lymphocyte proliferation assays, where it consistently caused peripheral blood lymphocytes from naive cats to proliferate in a dose-dependent manner. Other FIV fusion proteins produced under identical conditions (V5GST and p24GST) and glutathione S-transferase alone did not cause proliferation in this system. The monoclonal antibody vpg15, which has been shown to block infection of susceptible cells in vitro, did not decrease the response to V3/V4GST. Human peripheral blood lymphocytes did not proliferate in response to V3/V4GST.     

Stimulation by phytohaemagglutinin of peripheral blood lymphocytes from horse,pig, sheep and man

Optimal conditions for stimulation by phytohaemagglutinin (PHA) were established for equine, porcine, ovine and human lymphocytes in MEMS medium. Optimal thymidine concentration was determined for assay of cell transformation. With all species tested horse serum gave highest thymidine incorporation. Homologous serum was not more appropriate for lymphocytes of man, pig and sheep. Optimal stimulation was achieved at 20, 0.5–5, 5, and 10–40 μg PHA per 10⁶ cells for human, equine, porcine and ovine lymphocytes, respectively.     

Optimal conditions for in vitro mitogen-induced proliferation of peripheral blood lymphocytes in breeding foxes

The proliferative response of fox peripheral blood lymphocytes to nonspecific mitogens: leucoagglutinin (LA), concanavalin A (Con A) and pokeweed mitogen (PWM) was studied. Microcultures were kept at 39 degrees C in a humidified atmosphere containing 5% CO2. The highest 3H-thymidine incorporation was observed, when Con A was used, while LA and PWM showed weaker but significant stimulatory action. Optimal doses of mitogens were: 5 micrograms/ml for Con A, 5 micrograms/ml for LA and a dilution of 1:100 for PWM. The maximal stimulation index for Con A was about 240 and up to 100 for LA or PWM. The maximal lymphocyte proliferation was observed when culture media were supplemented with 10% serum. When proliferation kinetics were studied, the peak response was observed on Day 2.     

Flow cytometric analysis of ovine peripheral blood lymphocytes.

Leukocyte suspensions were prepared from the peripheral blood of 12 sheep three times at two month intervals beginning at 12 months of age. Monoclonal antibodies and flow cytometric analysis were used to characterize the cells. There were no significant differences over time therefore the data from the three time intervals were pooled. The mean percentages and ranges (minimum to maximum) of the major lymphocyte subtypes were: B-cells (29.6%, 11-50), gamma delta T-cells (36.6%, 22-68), CD4+ T-cells (14.1%, 8-22) and CD8+ T-cells (12.0%, 4-22). Lymphocyte subtype percentages appeared less variable within than between individuals. Two populations of B-cells were noted; one population had more cytoplasm and light scatter characteristics similar to monocytes while a second population of B-cells was more typical of small lymphocytes. The nature of the large B-cells requires further study.