Cannabinoid receptors CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system.

Cannabinoid receptor (CB) expression was characterized in immunological cell and tissue preparations. Northern analysis revealed approximately 6-kb transcripts for CB1 (brain-type) in mouse spleen and brain and in rat cerebellum. CB1 was not detected in mouse thymus or rat spleen RNA by Northern analysis. CB2 (peripheral) was detected as a approximately 4-kb transcript in mouse spleen and thymus and as approximately 2.4-kb transcripts in rat spleen. Quantitation of CB2 transcripts in mouse spleen and thymus revealed approximately 4 x 10(3) and approximately 4 x 10(2) molecules/100 ng RNA, respectively, with no quantifiable CB2 in mouse brain. Conversely, CB1 was expressed in mouse brain (approximately 2 x 10(5) molecules/100 ng RNA) with lower expression in mouse spleen (approximately 2 x 10(2) molecules/100 ng RNA) and was not quantifiable in mouse thymus. Competition binding in intact mouse splenocytes demonstrated that nonradiolabeled cannabinoids CP-55940, Win-55212-2, CP-56667, delta 9-THC, and cannabinol all competed for receptor binding with 3H-CP-55940, a high-affinity nondiscriminating CB1 and CB2 receptor ligand. Based on previous findings which demonstrated a marked inhibition of T-cell-dependent immune responses by cannabinoids, primary T cells and several T-cell lines were characterized. Radioligand binding analysis identified 100-300 cannabinoid receptor binding sites/cell with an approximate Kd of 200-700 pM in purified splenic T cells which also exhibited cannabinoid-induced inhibition of adenylate cyclase. Northern analysis of human T-cell lines revealed approximately 2.4-kb CB2 mRNA transcripts but no CB1 in HPB-ALL cells, a cell line which also exhibited inhibition of adenylate cyclase by delta 9-THC. Conversely, Jurkat E6-1 cells expressed an unusual mRNA banding pattern for CB2 expressing three distinct transcript sizes, none of which were 2.4 kb, the size for human CB2. Jurkat also did not express CB1 mRNA and did not exhibit inhibition of adenylate cyclase when treated with delta 9-THC. Collectively, these results provide further evidence that CB2 is the predominant cannabinoid receptor within the immune system and that this form of the receptor is expressed on T cells.

Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

Systemic hemodynamic and hepatic microvascular responses to a 33% blood volume exchange with whole blood, stroma-free hemoglobin, and oxypolyhemoglobin solutions.

Little is known about the microvascular effects of blood replacement solutions. This study was undertaken to develop an animal model suitable for studies of the microcirculatory effects of such solutions and to investigate microvascular responses to isovolemic transfusion with stroma-free hemoglobin (SFH), whole donor blood, or a new potential blood substitute solution containing oxypolyhemoglobin (OPH) as an oxygen carrier. Hamster livers were exposed and the microcirculation studied using intravital epifluorescent video microscopy. 33% blood volume replacement with SFH elevated systemic blood pressure by 25 Torr. Accompanying this increase in pressure was a 36% decrease in sinusoidal blood flow velocity and a 10% decrease in terminal hepatic venular diameters. Terminal portal venular diameters did not change. Decrease in liver sinusoidal perfusion was not due to neutrophil mediated injury, as myeloperoxidase activity in jejunum, liver, kidney, and lung remained unchanged. The reduction in perfusion was likely due to systemic vasoconstriction produced by SFH. In contrast, transfusion with whole blood did not change any of the measured parameters showing the excellent stability of the model. OPH transfused animals exhibited only a small 10 Torr transient increase in MAP 15 min post-transfusion. By 30 min MAP returned to the pre-infusion value. No significant changes were observed in either venular diameters or sinusoidal velocities in this group of animals. These results demonstrate suitability of this model for studies of the microcirculatory and hemodynamic effects of blood replacement solutions. Furthermore, OPH solution produced only minor transient disturbances in microvascular and systemic parameters.

Examination of the role of the colloids hydroxyethylstarch, dextran, human albumin, and plasma proteins in a modified UW solution.

The delayed contralateral nephrectomy procedure (three-step) produced inconsistent results, indicating that the preserved autotransplanted kidney tends to remain unfunctional and to regenerate incompletely unless the demand for work is placed upon it. Omission of HES in UW (UW-plain) did not affect preservation success, but resulted in increased graft edema. Substitution of HES in UW with plasma (SGF-V) or albumin (MAlb) gave significantly worse results than UW-like solutions with or without synthetic colloids. Replacement of HES in UW with UMdex-70 or UMdex-500 gave nonsignificantly worse results than UW-like solutions with or without synthetic colloids. The use of UMdex-40 as the main colloid in UW cheapened the solution, equalled the preservation success of UW and UW-plain but surpassed UW-plain in edema prevention, and exceeded UW concerning recovery of graft microcirculation.

Renal preservation after warm ischemia using oxygen free radical scavengers to prevent reperfusion injury.

Oxygen free radicals damage kidneys and accumulate during the period of preservation prior to transplantation. We hypothesized that a perfusate containing either an oxygen free radical scavenger such as ceruloplasmin, or an iron-chelating agent such as deferoxamine, would improve kidney preservation. Thirty-eight mongrel dogs underwent autotransplantation of the left kidney after 30 min of warm ischemia and 48 hr of machine perfusion (MOX-100, Water Instruments, Rochester, MN) at 5 degrees C and pH of 7.4. The right kidney was removed at the time of autotransplantation. Four blind code-labeled preservation solutions were tested. SGF-I was used for the control group (Group 1, n = 13), and the remaining animals were transplanted with kidneys preserved with one of three solutions modified from the basic SGF-I solution: Group 2, SGF-I plus deferoxamine (656 mg/liter), n = 8; Group 3, SGF-I ceruloplasmin enriched (72 mg/dl), n = 8; and Group 4, SGF-I ceruloplasmin reduced (3.4 mg/dl), n = 9. Serum creatinine levels were measured daily for 2 weeks and survival curves for each of the four groups were estimated by the Kaplan-Meier method. Peak mean serum creatinine levels +/- standard errors in Groups 1 through 4 were 12.6 +/- 1.97, 7.8 +/- 0.90, 7.1 +/- 1.26, and 8.2 +/- 1.09, respectively. Repeated measures analysis of variance showed statistically significant differences between the groups with respect to their serum creatinine profiles (Wald's test x2 with 3 df = 22.39, P value less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and characterization of an intravenous solution of IgG from human immunodeficiency virus-seropositive donors.

An intravenous solution of 99% pure globulin (hyperimmune IgG, HIVIG) was obtained from pooled plasma of selected human immunodeficiency virus (HIV-1)-seropositive asymptomatic donors with greater than 400 CD4+/microliters cells per microliter and a high titer of antibody to HIV-1 p24 protein. HIVIG had high titers of antibody to p24, glycoprotein 41 (gp41), and gp120, group-specific neutralizing activity, and binding to the gp120 hypervariable loop region. It inhibited syncytia formation. At low concentration, it enhanced viral production of HIV-1 in infected peripheral blood monocytes but was inhibitory at higher concentration. HIVIG directed group-specific antibody-dependent cellular cytotoxicity against HIV-infected targets. For a period of 6 to 28 months, plasma donors kept stable antibody titers and had a 1.0% decrease in CD4+ cells per month. One gram per kilogram HIVIG injected in two juvenile chimpanzees was well tolerated and did not transmit HIV, as measured by negative cell culture, IgM immune response to HIV proteins, and polymerase chain reaction. The mean half-life of HIV-1 p24 antibody was 15 days. These preliminary data suggest that HIVIG is a safe product suitable for clinical trial in HIV-1-infected individuals.

Beneficial and detrimental effects of RBC-adsorbed antilymphocyte globulin and prednisone on purified canine islet autograft and allograft function.

We have examined the effects of prednisone, cyclosporine, azathioprine, and RBC-adsorbed goat antidog antilymphocyte globulin on islet graft function in totally pancreatectomized canines with purified, quantitatively defined, autologous, or allogeneic islets transplanted to the liver. The objectives were twofold: (1) to determine the potential detrimental effects to islet autograft function of the aforementioned agents, and (2) to determine the relative efficacy of the "nontoxic" agents in prolonging purified islet allograft function administered in doses that would be considered tolerable in human. The islet autograft studies demonstrated that prednisone given in doses of 1-2 mg/kg/day had a detrimental effect on islet autograft function, and that the combinations of immunosuppression involving CsA, azathioprine, and ALG were not detrimental to islet autograft function to the extent that hyperglycemia would ensue. In the subsequent allograft studies, three groups of canines received islet transplants: (1) controls (n = 5; 7860 +/- 750 islets/kg/weight), (2) canines given CsA and azathioprine (n = 6; 6810 +/- 890 islets/kg/body weight), and (3) canines given CsA, azathioprine, and RBC-adsorbed goat antidog ALG (n = 8; 6540 +/- 710 islets/kg/body weight). The mean (+/- SE) day of rejection (serum glucose greater than or equal to 200 mg/dl) in the group of canine islet allograft recipients receiving CsA, azathioprine, and ALG was 11.8 +/- 1.4 days--significantly prolonged versus islet allograft recipients receiving no immunosuppression (mean survival 4.8 +/- 1.1 days, P less than 0.03), and versus allograft recipients receiving CsA/azathioprine without ALG (mean survival 4.4 +/- 1.4 days, P less than 0.05). Prednisone appears to be detrimental to islet graft function, even at low doses. ALG was not toxic, and significantly extended the survival of canine islet allografts. The inclusion of steroids as part of maintenance immunosuppression, or as treatment for acute rejection of islets, in human islet transplants should be reconsidered, whereas ALG or other antilymphocyte agents should continue to be used.

Antenatal diagnosis of molybdenum cofactor deficiency.

Analysis of uncultured chorionic villus material from a woman at risk of fetus with sulfite oxidase deficiency revealed a deficiency of sulfite oxidase. This was confirmed on termination of the pregnancy.