Nylon Wool Columns Research Articles

Deletion of Bid Impedes Cell Proliferation and Hepatic Carcinogenesis

Mechanisms that control the proliferation capability of the initiated cells during hepatocarcinogenesis are still largely unclear. We investigated the role of a pro-death Bcl-2 family protein, Bid, in liver tumor development using a neonatal diethylnitrosamine model. Diethylnitrosamine was administrated to 15-day-old wild-type and bid-null mice. The development of microfoci at the early stage and of gross tumors at the later stage was compared between the two groups of mice. Both microfoci and gross tumor development were significantly retarded in the bid-null mice, despite reduced cell death as measured by TUNEL staining. Further studies indicated that there were significantly less proliferating cells in diethylnitrosamine-treated bid-null livers. The regulation of cell proliferation by Bid was confirmed in two other systems not involving carcinogenesis. Hepatocyte proliferation following partial hepatectomy and T lymphocyte proliferation following anti-CD3 stimulation were both retarded in bid-null mice. Thus, these studies revealed a previously undisclosed function of Bid in regulating cell proliferation, which can be important to tumor development. Furthermore, the role of Bid in promoting hepatocarcinogenesis is in contrast to its reported role in suppressing myeloid leukemia and thus suggests an organ- and/or etiology-specific role of the Bcl-2 family proteins in regulating oncogenesis.

Activation of CD38 by Interleukin-8 Signaling Regulates Intracellular Ca2+ Level and Motility of Lymphokine-activated Killer Cells

CD38 is an ADP-ribosyl cyclase, producing a potent Ca(2+) mobilizer cyclic ADP-ribose (cADPR). In this study, we have investigated a role of CD38 and its regulation through interleukin-8 (IL8) signaling in lymphokine-activated killer (LAK) cells. Incubation of LAK cells with IL8 resulted in an increase of cellular cADPR level and a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)), which was sustained for a long period of time (>10 min). Preincubation of an antagonistic cADPR analog, 8-Br-cADPR (8-bromo-cyclic adenosine diphosphate ribose), abolished the sustained Ca(2+) signal only but not the initial Ca(2+) rise. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist blocked both Ca(2+) signals. Interestingly, the sustained Ca(2+) rise was not observed in the absence of extracellular Ca(2+). Functional CD38-null (CD38(-)) LAK cells showed the initial rapid increase of [Ca(2+)](i) but not the sustained Ca(2+) rise in response to IL8 treatment. An increase of cellular cADPR level by cGMP analog, 8-pCPT-cGMP (8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate), but not cAMP analog or phorbol 12-myristate 13-acetate was observed. IL8 treatment resulted in the increase of cGMP level that was inhibited by the IP(3) receptor blocker but not a protein kinase C inhibitor. cGMP-mediated Ca(2+) rise was blocked by 8-Br-cADPR. In addition, IL8-mediated LAK cell migration was inhibited by 8-Br-cADPR and a protein kinase G inhibitor. Consistent with these observations, IL8-induced migration of CD38(-) LAK cells was not observed. However, direct application of cADPR or 8-pCPT-cGMP stimulated migration of CD38(-) cells. These results demonstrate that CD38 is stimulated by sequential activation of IL8 receptor, IP(3)-mediated Ca(2+) rise, and cGMP/protein kinase G and that CD38 plays an essential role in IL8-induced migration of LAK cells.

Adrenergic Modulation of Erythropoiesis Following Severe Injury Is Mediated Through Bone Marrow Stroma

Severe trauma leads to hematopoietic failure and bone marrow (BM) dysfunction that manifests clinically as a persistent anemia and leukopenia. The impact of severe trauma and its associated hyperadrenergic state on erythropoiesis has not been described. The aim of this study was to demonstrate the effects of adrenergic agonists and antagonists on erythropoiesis, both in normal bone marrow mononuclear cells (BMNC) and stroma-depleted BM. Urine epinephrine (EPI) and norepinephrine (NE) excretion from severely injured patients was assessed via enzyme-linked immunoadsorbent assay (ELISA). Erythropoiesis was assessed by the growth of erythroid progenitors-erythroid burst forming units and colony forming units (BFU-E and CFU-E)-in normal human BM in the presence of adrenergic agonists and antagonists at varying concentrations. Parallel cultures, depleted of BM stroma by passage through nylon wool columns, were compared. Urine NE excretion was elevated in all samples from days 1 to 10 following injury (average 139 +/- 59 mcg/day vs. control 35 +/- 9 mcg/day). In vitro doses of NE, EPI, and isoproterenol (ISO) exerted a stimulatory effect on BFU-E colony growth in BMNCs (expressed as percentage of control: 324 +/- 30, 272 +/- 16, 212 +/- 95, vs. 100%), but had no effect on stroma-depleted BM. There is a substantial and persistent hyperadrenergic state seen after severe injury that may last for up to a week. Adrenergic agonists have a clear stimulatory effect on the growth of primitive erythroid precursors in normal BM. The adrenergic stimulus appears to be mediated via BM stroma.

Isolation and tracking of a rare lymphoid progenitor cell which facilitates bone marrow transplantation in mice

Bone marrow cells are composed of pluripotent stem cells to terminally differentiated cells, with a wide variety of abundance of each cell type. In the past, many of the cell types within this heterogeneous population have been characterized either by expression of specific proteins or using functional markers. In spite of promising results obtained with the latter method, various cell types within bone marrow have not been well characterized due to the low abundance of a specific cell type. Considering the demand for a reliable technique to enrich cell types, a wide variety of approaches, ranging from simple nylon wool columns to high-speed cell sorting, have evolved. Only limited success has been obtained with approaches ranging from the detection of MHC antigen to positron emission tomography to track the ontogeny of specific bone marrow-derived cells in studies of syngeneic or allogeneic transplantation. The present study describes a relatively simple method to enrich and track a rare bone marrow cell (facilitating cell, FC), which can facilitate allogeneic bone marrow stem cell transplantation in mice. The isolation technique is comprised of enrichment of FC by magnetic activated cell sorting (MACS) system followed by purification through high-speed cell sorter. An initial inoculation of 30,000 FC obtained from male mice was detected in the thymus, spleen, and bone marrow of allogeneic female recipients, by using 32P-labeled dCTP in a specific PCR for Y-chromosome. This technique may improve the efficiency of isolation of other rare cells from the bone marrow.

In vitro detection of functional humoral immunocompetence in juvenile chinook salmon ( Oncorhynchus tshawytscha) using flow cytometry

A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of chinook salmon ( Oncorhynchus tshawytscha) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a fluorescein isothiocyanate labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM-FITC), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM). Through a series of calibration steps, it was confirmed that anti-RBT IgM-FITC was specific for B-lymphocyte SIgM in chinook salmon. Binding of anti-RBT IgM-FITC to chinook salmon SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM-FITC binding. Using anti-RBT IgM-FITC as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile chinook salmon was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (±2.82), 34.8 (±1.86) and 56.7% (±4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of cortisol in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.

Differential expression of gamma interferon mRNA induced by attenuated and virulent Mycobacterium tuberculosis in guinea pig cells after Mycobacterium bovis BCG vaccination.

To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-gamma) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-gamma from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-gamma, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a (32)P-labeled probe derived from the cDNA clone. Compared to the IFN-gamma mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-gamma mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN-gamma mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4(+) T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-gamma mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-gamma mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-gamma mRNA accumulation is currently under investigation.

Local and Systemic T Cell Responses to Purified Proteins Following Cow's Milk Feeding in Guinea Pigs

Abstract Cell-mediated immune mechanisms may contribute to hypersensitivity reactions following ingestion of cow's milk. We assessed in vitro lymphoproliferation to purified cow's milk proteins in sensitized lymphocytes from the spleen, mesenteric lymph nodes and peripheral blood of guinea pigs consuming cow's milk. Blood and lymph node cells were quite responsive but splenocytes responded poorly. Orally sensitized guinea pigs responded best to a-casein, β-casein and β-lactoglobulin and minimally to a-lactalbumin. Even two months after oral exposure, guinea pig mesenteric lymph node lymphocytes still proliferated when cultured with a-casein and β-casein. Refeeding cow's milk to guinea pigs after a milk-free interval following the initial exposure did not boost the proliferative response and may have induced suppression. The effect of nylon wool column enrichment of T cells prior to culture demonstrated the essential role of antigen-presenting cells in T cell activation by cow's milk proteins. Our results suggest the optimum length of time required for oral sensitization to a-casein, β-casein and β-lactoglobulin is 2 weeks. However, prolonged feeding for 4 weeks with cow's milk down-regulated the antigenic response and upregulated the proliferative response to a polyclonal T cell mitogen (ConA). These results suggest that feeding cow's milk can induce antigen specific T cell responses which are both local and systemic. The lymphocyte proliferation assay with highly purified antigens could be useful in elucidating the fundamental cellular mechanisms of cow's milk allergy.

Two-step negative enrichment of CD4 + and CD8 + T cells from murine spleen via nylon wool adherence and an optimized antibody cocktail

We developed a method to highly purify CD4 + and CD8 + T cells from murine spleen by negative enrichment strategy. Single-cell suspensions of spleen cells were depleted from erythrocytes by ammonium chloride-mediated lysis. The obtained cell suspension contained approximately 28% CD4 + cells and 14% CD8 + cells. Passing of these cells over a nylon wool column removed up to 75% of all cells, leading to a suspension containing approximately 50% CD4 + and 23% CD8 + cells. These cells were further purified by a single immunomagnetic depletion step using a panel of eight antibodies in combination with MACS magnetic beads and an autoMACS machine. After purification, cells were viable and mostly non-activated based on the expression of activation markers and did not or only minimally respond to polyclonal stimuli such as soluble anti-CD3 antibodies or Concanavalin A. With this method, 19–38% of all CD4 cells and 10–29% of all CD8 cells in a spleen cell suspension were recovered at the mentioned purity. The whole procedure is fast (<4 h of preparation), simple and cost effective, as all antibodies and the magnetic beads have been titrated to the minimal concentration needed for purification. The method is highly reproducible, routinely leading to CD4 + cells with >97% purity (range 97.4–99%) and CD8 + cells with >96% purity (range 95.6–96.7%). The described protocol should facilitate studies aiming at the physiology of “untouched” murine T cells.

Sepsis-Induced Alteration in T-Cell Ca2+ Signaling in Neonatal Rats

Sepsis-induced suppression in T-cell proliferation follows deranged Ca²⁺ signaling in adult rats. In preliminary studies, we observed suppression in T-cell proliferation in septic neonatal rats as well. In this study, we assessed splenic T-cell cytosolic Ca²⁺ concentration, [Ca²⁺]_i, as its elevation plays an important role in T-cell proliferation. Also, we investigated the role of PGE₂ in sepsis-related changes in T-cell [Ca²⁺]_i in animals pretreated with cyclooxygenase-1 (COX-1) inhibitor (resveratrol) and cyclooxygenase-2 (COX-2) inhibitor (NS-398). Sepsis was induced in 15-day-old rat pups by intraperitoneal implantation of fecal pellets containing Escherichia coli and Bacteroides fragilis. The sham group consisted of pups implanted with sterile fecal pellets. Septic and sham pups were sacrificed 24 h after implantation and their spleens were removed. The spleens from sham and septic pups, along with spleens from unoperated control pups, were processed for single cell suspensions, and T cells were isolated using nylon wool columns. Fura-2 fluorophotometry was employed for the measurement of [Ca²⁺]_i (in nM units) in T cells stimulated with concanavalin A (ConA). Our results show that ConA-mediated T-cell [Ca²⁺]_i response is significantly suppressed in septic neonatal rats. Pretreatment of pups with COX-2, but not COX-1 inhibitor, prevented the decrease in the [Ca²⁺]_i response. These findings suggest that PGE₂ might induce the attenuation in T-cell Ca²⁺ signaling during sepsis in neonatal rats.

Depletion of Accessory Cells by Adherence to Sephadex G‐10

Accessory cells such as macrophages and other antigen-presenting cells (APC) in suspensions of mouse spleen and lymph node are removed by means of their preferential adherence to Sephadex G-10 polymers. Unlike the use of nylon wool columns to remove B and accessory cells (UNIT 3.2), Sephadex G-10 columns are employed when the main purpose is to yield functional populations of T cells and B cells (by depleting mostly macrophages and other APC). The following technique is simple, fast, and reproducible; however, it results in some retention of B cells on the resin. The basic protocol in this unit involves assembling the column, loading and equilibrating the sterile Sephadex slurry, and passing the cells through the column followed by collection and analysis of eluate. Two support protocols describe preparation of the Sephadex G-10 slurry for use in the column by fining and sterilization, plus recycling resin from used columns.

Purification of functional T lymphocytes from splenocytes of the beluga whales ( Delphinapterus leucas)

In an effort to gain knowledge on immune functions in beluga whales, Delphinapterus leucas, we have used two physical methods for the purification of T lymphocytes of spleen cells. Isolation by sheep red blood cells (SRBC) rosetting and by adherence on nylon wool columns were tested. SRBC-rosetting gave unreliable results in obtaining purified T cells. Therefore, the purification of T cells was done using nylon wool columns. Less than 3% of the IgM + B cells remained in effluent populations. In the later population, 45% gave positive staining with mouse anti-human CD4 allowing us to verify functionality of the cells. The study of calcium mobilization and tyrosine kinase activation, mediated by CD4 cross-linking permitted verification of the functionality of cells. We also showed that upon activation with mitogens, beluga T cells upregulate the density of MHC class II molecules on their surfaces. CD4 cross-linking with a specific antibody inhibited the proliferation response. Overall, the activation of beluga whales lymphocytes did not differ markedly from what is known in other species. This study can help in the groundwork for functional investigation of the beluga whale’s immune system.

Purification and characterisation of bovine WC1+ gammadelta T lymphocytes from peripheral blood.

In order to isolate and characterise resting WC1+ gammadelta T cells from cattle, we developed a protocol for purifying these cells by negative selection from peripheral blood. The purification method included five steps: separation of mononuclear cells on lymphoprep, depletion of monocytes by adherence to plasma-coated gelatin, enriching T cells on a nylon wool column, depleting CD2+ T cells by sheep red blood cells (SRBC), and finally depleting CD4+ and CD8+ T cells by the magnetic cell sorting technique (MACS). This procedure proved efficient and reproducible, and the purity of the isolated WC1+ gammadelta T cells was more than 97% as analysed by flow cytometry (FACS). Cytokines and costimulatory molecules mRNA expression was assessed by the reverse transcriptase polymerase chain reaction (RT-PCR) technique in freshly isolated resting WC1+ T cells. We found that purified uncultured WC1+ T cells express TNF-alpha, CD28, CTLA-4 and IL-2R alpha mRNA transcripts but do not express those for IL-2, IL-4, IL-6, IL-10 and IFN-gamma. The expression of CD28 and CTLA-4 transcripts on bovine WC1+ T cells indicates that these genes are evolutionarily conserved.

Resistance of established porcine islet xenografts to humoral rejection by hyperimmune sera.

Although preformed natural antibodies cause hyperacute rejection of primarily vascularized xenografts, tissue grafts such as skin or islets are revascularized by in-growth of host capillaries and therefore might be resistant to circulating antibodies. We examined the effect of hyperimmune serum and primed T cells on the survival of long-term porcine islet xenografts in diabetic nude mice. Porcine islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic BALB/c athymic mice. Hyperimmune serum and sensitized splenocytes were prepared by repeated immunization of BALB/c mice with porcine lymph node cells. Splenic T cells were enriched by nylon wool column separation. Tissues were examined by immunohistology using murine- and porcine-specific monoclonal antibodies. Porcine islets survived in nude mice for > 100 days with high levels of circulating porcine C-peptide and maintenance of normoglycemia. Injection of the hyperimmune sera (IgG) into normoglycemic nude mice bearing porcine islets for > 70 days failed to induce rejection despite the continued presence of circulating anti-porcine cytotoxic antibody. Injection of sensitized T cells caused acute rejection of long-term (>140 days) porcine islets, whereas injection of naive T cells had no effect. Histologically, porcine islets removed from mice treated with hyperimmune serum showed no staining for IgG. Long-surviving porcine islet grafts showed strong staining for interleukin (IL)-10 and a lesser amount of IL-4 but no staining for IL-2 or interferon-gamma. Although fresh porcine islets were positive for swine leukocyte antigen class 1 antigen and intercellular adhesion molecule (ICAM)-1 but negative for mouse platelet endothelial cell adhesion molecule and ICAM-2, long-surviving porcine islets showed positive endothelial staining for mouse platelet endothelial cell adhesion molecule and ICAM-2. Established islet xenografts are resistant to hyperimmune serum as a result of a lack of target endothelial antigens, whereas they remain susceptible to rejection caused by primed T cells. Local production of Th2 cytokines may explain the inability of long-surviving islet xenografts to activate injected naive T cells.

Effects of isoproterenol on IL-2 and cAMP production in peripheral T cells from asthmatic and non-asthmatic subjects sensitive to Candida

Immunity to Candida albicans (Candida) is characterized by a Th-1 type pattern of reactivity. Candida is rarely a cause antigen for bronchial asthma. Beta agonists have been found to inhibit secretion of IL-2 from T cells through intracellular cAMP elevation. We examined effects of isoproterenol (ISO) on Candida-stimulated T cells. Peripheral T cells obtained from six Candida-sensitive asthmatics, six Candida-sensitive non-asthmatic subjects, and six normal donors by Ficoll-Hypaque gradient centrifugation and nylon-wool column chromatography were incubated with Candida antigen or concanavalin A (Con A) in the absence or presence of ISO. Secretion of IL-2 and intracellular accumulation of cAMP were assayed by ELISA. Con A induced secretion of IL-2 in each of the three groups. Candida antigen induced IL-2 secretion in the normal and the non-asthmatic subjects, but not in the asthmatics. ISO, which reduced Con A-induced secretion of IL-2 in a dose-dependent manner, had no effect on Candida-induced secretion of IL-2. Although ISO increased the intracellular concentration of cAMP in untreated and Con A-treated T cells from all donors, cells from the normal and the non-asthmatic subjects, but not from the asthmatics, that were co-incubated with ISO and Candida had lower levels of cAMP than those treated with ISO alone. It is suggested that Candida antigen induces secretion of IL-2 and reduces ISO-inducible accumulation of cAMP in Candida-responsive IL-2 secreting cells, which may make Candida-induced secretion of IL-2 insensitive to ISO.

Effect of genetic selection and MHC haplotypes on lymphocyte proliferation and Interleukin-2 like activity in chicken lines selected for high and low antibody production against sheep red blood cells

Chickens from third generation matings of lines of chickens selected for high (HA) and low (LA) antibody production to sheep red blood cells (SRBC) and typed for MHC genotypes B 13/13, B 13/21, and B 21/21 were used in this study. Chickens from both lines carried all the three genotypes B 13/13, B 13/21, and B 21/21. To study T- and B-lymphocytes mitogenic activity, 12-week-old female chickens were injected intravenously with 0.2 ml of 9% SRBC and spleens were collected at 0, 6 h, and 6 day post-antigen injection (pAg). Isolated lymphocytes were incubated with either Concanavalin-A (Con-A) for T-cell activity, or Pokeweed mitogen (PWM) for B-cell activity and thymidine 3 H uptakes were measured. To study the Interleukin-2 (IL-2)-like activity in the same lines and genotypes, splenic lymphocytes from 12-week-old chickens were passed through nylon wool columns to enrich the T-cell population. After a 24 h incubation with Con-A, the conditioned media (CM) were collected. The CM were tested for IL-2 like activity by determining whether they altered the proliferation of Con-A stimulated T cells. This proliferation effect was then compared to that of a reference conditioned media (RCM) prepared from K-strain birds and that were used as the standard for the assay. There was no significant difference ( p > 0.05) in IL-2 like activity between HA and LA lines, however, the LA was significantly higher than HA ( p < 0.05) in T- and B-cell mitogenic activity. The genotype B 13/13 had significantly higher ( p < 0.05) IL-2 like activity than the B 21/21. The genotype B 13/13 was also significantly higher ( p < 0.05) in T- and B-cell mitogenic activity than the B 21/21. At 0 h, pAg T- and B-mitogenic activity was significantly higher ( p < 0.05) than 6 h. In summary, our results indicate that although the birds were selected for high antibody production to SRBC, their lymphocyte mitogenic activity was lower than those selected for low antibody production. Hence, humoral and cell-mediated immune responses appear to be under different genetic controls, and that selection for greater humoral response may be at the expense of cellular responses. Our results also suggest differences in IL-2 like activity production between chickens carrying different MHC B-haplotypes, and that genetic control of such activity is possibly linked to the MHC genes.

Intrathymic inoculation of donor B cells prolongs cardiac allograft survival by non-deletional mechanisms

Resting B (rB) cells and splenic dendritic cells (DCs) were purified from splenocytes as described. A typical preparation of rB cells as analysed by flow cytometry was found to be 95.7% major histocompatibility class (MHC) II, 1.2% CD80, 2.5% CD86, and 92.3% sIg. A typical preparation of splenic DCs was 92.2% MHC II, 68.8% CD80, 88.2% CD86, 68% N418, and 9.4% sIg. In functional assays, DCs but not rB cells were able to stimulate proliferation of allogeneic T cell and generate cytotoxic T cells. T cells were made from spleen using a nylon wool column as described and were sorted negatively by FACS Vantage with turbo sort option (Becton Dickinson, Mountain View, Calif). Typical preparations as analysed by FACS was found to be .90% CD3, ,0.5% IA, and ,1% Ig.

Method of lymphocytotoxic crossmatch test for feline renal transplantation.

The optimal condition for methods of lymphocytotoxic crossmatch test for feline renal transplantation was investigated. On separation of viable lymphocytes from whole blood, the best results were obtained when Ficoll-diatrizoate with 1.078 of a specific gravity at 20 degrees C was centrifuged with 800 x g for 30 min at 4 degrees C. A nylon wool column was used to separate T and B cells from lymphocyte fraction. The ratio of T cells in nylon wool effluent cells was 95%, while the ratio of B cells in adherent cells was 41%. Lymphocytotoxic crossmatch tests were performed by using the effluent cells as T cells and the adherent cells as B cells, at 37 degrees C (warm) and 4 degrees C (cold). The ratio of B cells in adherent cells was low, however, the result was utilized as a matching test before transplantation by combining with the T cell result. The trypan blue stain method made it easier than the eosin stain method to distinguish living and dead cells. The lymphocytotoxic crossmatch tests were performed on 15 pairs of healthy cats, and only one pair showed doubtful positive against anti-B cell cold antibodies. During acute rejection after renal transplantation in two pairs which were negative on any anti-lymphocyte antibodies before the transplantation, the anti-T cell warm antibodies became positive in both pairs, and the anti-T cell cold antibodies became positive on one of the two pairs.

RESISTANCE OF ISLET XENOGRAFTS TO HUMORAL REJECTION

122 Hyperacute rejection of primarily vascularized xenografts is caused by performed natural antibodies to the gal-α1,3-gal epitope expressed on the vascular endothelium. However, islet xenografts are revascularized by in-growth of host capillaries and therefore might be resistant to circulating antibodies. We examined the effect of hyperimmune serum and primed T cells on the survival of long-term islet xenografts in nude mice. Materials and Methods: Islets (104 IE) isolated from 6 month old Yorkshire pigs were transplanted beneath the kidney capsule of streptozocin-induced Balb/c nu/nu (nude) mice. Hyperimmune serum and sensitized splenocytes were prepared by repeated immunization of Balb/c mice with porcine lymph node cells. Splenic T cells were enriched by a nylon-wool column separation. Tissues were examined by immunohistology using specific monoclonal antibodies. Results: Porcine islets (PI) survived in nude mice for >100 days with high levels of circulating porcine C-peptide. When hyperimmune serum (IgG) which was cytotoxic to porcine lymphocytes at a 1/2048 dilution was injected into normoglycemic nude mice bearing PI for >70 days (n=6), no rejection was induced despite continued presence of circulating anti-porcine cytotoxic antibody up to 14 days after injection. In contrast, 5×106 primed T cells injected into mice bearing long-term PI (>140 days) caused acute rejection. Injection of 5×106 naive Balb/c T cells had no effect on the survival of longstanding PI. When nude mice were treated early (12 days) after islet transplantation, naive T cells, but not hyperimmune serum, caused acute rejection. Histologically, PI removed from mice treated with hyperimmune serum showed no staining for IgG whereas incubation of freshly isolated PI with hyperimmune serum resulted in heavy deposition of IgG within the islets. PI removed after rejection caused by injected T cells showed heavy cellular infiltration. Long-surviving xenografts showed strong staining for IL-10 and a lesser amount of IL-4, but no staining for IL-2 nor γIFN. While fresh PI were positive for SLA class I antigen and ICAM-1 but negative for mouse PECAM and ICAM-1, long surviving PI showed positive endothelial staining for mouse PECAM, ICAM-1 and ICAM-2. Conclusion: Islet xenografts are resistant to hyperimmune serum and probably to natural antibodies due to lack of target endothelium antigens in the grafts as a result of in-growth of host vessels while they remain susceptible to rejection caused by primed T cells. Local production of Th2 cytokines may explain the inability of long-surviving islet xenografts to activate transferred naive T cells.

Role of the liver in T cell differentiation--generation of CD3-CD4+/CD8+TCRbeta- cells and CD3-4-8-TCRbeta+ cells from CD4-8-TCRbeta- athymic nude bone marrow cells by culture with parenchymal liver cells.

To investigate the influence of the liver on differentiation of hematopoietic stem cells/pro-T cells, TN-NWP-BMC (athymic nude bone marrow cells that were treated with anti-TCRbeta, anti-CD4, and anti-CD8 Abs plus complement and then passed through a nylon wool column) were cultured on parenchymal liver cells. After culture for 2.5 days, CD3-4-8-TCRbeta+ cells and CD3-CD4+/CD8+TCRbeta- cells were developed from TN-NWP-BMC. TCRVbeta8+ cells comprised 19.9% of CD3-4-8-TCRbeta+ cells, and Vbeta8 mRNA was detected in the CD3-4-8-TCRbeta+ cells by reverse transcriptase-polymerase chain reaction. The CD3-CD4+/CD8+TCRbeta- cells contained not only single-positive cells but also CD4+8+ double-positive cells. The CD8 protein consisted of 88.9% CD8alpha+beta-, 10.1% CD8alpha+beta+, and 1% CD8alpha-beta+ molecules. From these results and the finding of co-expressed antigens, CD3-4-8-TCRbeta+ cells and CD3-CD4+/CD8+TCRbeta- cells appear to be immature cells not committed to a certain cell lineage.

Isolation of rainbow trout (Oncorhynchus mykiss) intestinal intraepithelial lymphocytes (IEL) and measurement of their cytotoxic activity

Established methods for isolating intraepithelial lymphocytes (IEL) from mouse small intestine were modified to facilitate the isolation of IEL from rainbow trout gut epithelium. A 1h incubation in 1mMDTT/EDTA was required to maximise the separation of the epithelial compartment from the underlying lamina propria. Epithelial cell content was reduced by filtration on nylon wool columns followed by centrifugation on a 40% Percoll cushion. Most IEL pelleted through the 40% Percoll [high density (HD) IEL] although significant numbers banded out at the medium/40% Percoll interface [low density (LD) IEL]. The majority (∼90%) of HD IEL were<12·5μm in size, had a pleiomorphic nucleus and obvious mitochondria. LD IEL were larger cells, containing more cytoplasm with abundant ribosomes. Neither HD nor LD IEL appeared to contain ‘cytotoxic’ granules. Despite this, in a non-radioactive cytotoxicity assay (LDH release) with EL4 mouse thymoma cells as targets, significant killing was observed by both the HD and LD IEL.