Cyclosporine inhibition of calcineurin activity in human leukocytes in vivo is rapidly reversible.

Despite increasing information about the mechanism of action of cyclosporine A (CsA), little is known about the way lymphocytes recover from CsA. Recovery is central to understanding the pharmacodynamics of CsA in vivo. We studied the recovery of calcineurin phosphatase (CN) activity in CsA-treated cells. Single dose kinetics in renal transplant patients showed that inhibition of CN activity in PBL increased and fell concomitant with CsA blood vessels. In vitro, control PBL treated with CsA 100 micrograms/l, washed, and resuspended in CsA-free medium showed little recovery (0-20%) after 24 h. Erythrocytes or anti-CsA Ab added to the recovery medium increased recovery to 50% within 4 h. Similar recovery was seen in the ability of cells to produce IFN-gamma after OKT3 stimulation. Recovery of CN activity was associated with the efflux of [3H]CsA, was not blocked by cycloheximide and was temperature sensitive. A cell line with high expression of surface P glycoprotein (PGP), showed rapid recovery. However, PGP blockade did not prevent recovery in PBL, indicating a different PGP-independent mechanism. In PBL, recovery from CsA is slow and limited in vitro, but rapid in vivo, where CsA equilibrates among a complex set of extralymphocytic binding sites.

A new class of infectious agents detectable by the production of chorioallantoic membrane lesions by human lymphoblastoid cell lines and their culture supernatants.

Intravenous injection of cells and their tissue culture supernatants (CS) from human lymphoblastoid cell lines (LCL) induced the formation of lesions on the chorioallantoic membrane (CAM) of the chicken embryo. Injection of cells and CS from non-LCL and normal human lymphocytes induced few or no lesions. Irradiated chick embryos were more sensitive to lesion formation than were nonirradiated embryos. The log10 CAM lesions induced in irradiated (500 rads) embryos were a linear function of the log10 cells (from LCL) in the inoculum; the slope was 1.0, within experimental error. The formation of CAM lesions did not depend on the presence of Epstein-Barr virus (EBV) since lesions were also induced by cells and extracts derived from EBV genome-free LCL. Lesion-inducing activity associated with CS was filterable through 0.22-mu filters, sedimented at 78,000 x g, and sensitive to inactivation by heat (56 degrees C for 30 min), UV irradiation, chloroform, sera from chickens immunized against CS, and certain human sera. Lesion-inducing activity associated with cells and extracts was resistant to 5,000 rads of gamma-radiation. B2/B2 embryos (the B locus is the major histocompatibility locus of chickens) were more sensitive to lesion formation than were B15/B21 and outbred embryos; this suggested a genetic influence on lesion formation. Our data suggest that the irradiated chicken embryo may be a highly sensitive and useful means for the detection of an unidentified or unknown agent or agents that may play an important role in human oncogenic lymphocyte transformation or might interact with transforming viruses.

Monozygotic twins concordant for the narcoleptic syndrome.

We report the first documented monozygotic twins who both had the narcoleptic syndrome. We assessed monozygosity by HLA antigens and by blood groups. In contrast to virtually all other narcoleptics, they had HLA-DQ1 instead of HLA-DR2; this helped to localize the gene, and perhaps explains its greater expressivity in these than in other twins.

Calcineurin activity is only partially inhibited in leukocytes of cyclosporine-treated patients.

Measurement of the degree of immunosuppression induced clinically by drugs such as cyclosporine is an important but elusive goal. In lymphocytes in vitro, cyclosporine (CsA) blocks the phosphatase activity of the enzyme calcineurin, preventing cytokine induction. We sought to measure the degree of calcineurin blockade in patients on CsA. Calcineurin activity was measured in peripheral blood mononuclear cells (PBL) from stable CsA-treated renal transplant patients, compared with controls. Cytokine expression was assessed by challenging ex vivo PBL with calcium ionophore A23187 (5 microM) for 60 min and measuring interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) mRNA induction. In vitro, CsA inhibited both calcineurin activity and cytokine induction with an IC50 of 10-20 micrograms/L. In CsA-treated patients with therapeutic CsA levels (mean trough CsA blood level = 180 +/- 55 micrograms/L), calcineurin activity was detectable but reduced by 50% compared with controls (P < or = 0.001) and correlated with CsA trough levels (r = -0.390, P < or = 0.01). The induction of cytokine mRNA in such patients was not blocked, but was sensitive to CsA in vitro, suggesting that CsA is much less available in vivo in body fluids than it is for isolated cells in vitro. In lymphocytes of patients on CsA, calcineurin activity is reduced but 50% of the activity persists, permitting strong signals to trigger cytokine expression. Partial calcineurin inhibition may explain why the immune responsiveness of patients on CsA is reduced but still sufficient for host defense.

Cyclosporine inhibition of leukocyte calcineurin is much less in whole blood than in culture medium.

Recent reports have shown that cyclosporine (CsA)-treated patients have abundant calcineurin phosphatase (CN) activity in vivo despite CsA blood concentrations that would be completely inhibitory in vitro. We sought to determine whether this disparity was a result of altered distribution of CsA in culture medium (CM) compared with whole blood (WB). CN activity was measured in peripheral blood leukocytes (PBL) that had been exposed in vitro to CsA in either WB or CM. Cells from both groups were also stimulated with OKT3 to determine the effect of CsA on the induction of IFN-gamma synthesis. CsA accumulation in PBL was determined by liquid scintillation counting of PBL exposed to 3H-CsA. The IC50 for CsA inhibition of CN activity was significantly lower for PBL in CM (2 micrograms/L) compared with PBL in WB (102 micrograms/L, P < or = 0.005). Likewise, for CsA inhibition of IFN-gamma induction, the IC50 was 18 micrograms/L for PBL in CM compared with 690 micrograms/L for PBL in WB (P < or = 0.005). The shift in IC50 was accompanied by a 10-fold increase in 3H-CsA in PBL in CM compared with PBL in WB. We conclude that PBL exposed to CsA in CM accumulate significantly more CsA than PBL in WB. The result is that CsA inhibition of CN activity and cytokine induction appears at least an order of magnitude greater than its true effect in biologic fluids. This disparity is due to partitioning of CsA to irrelevant CsA binding sites, which are abundant in WB and in vivo, but absent in culture medium.

Pathologic features of acute renal allograft rejection associated with donor-specific antibody, Analysis using the Banff grading schema.

Alloantibody frequently appears during the immune response to alloantigens in renal transplant recipients. We studied whether the presence of antibody against donor class I antigens correlated with the clinical and pathologic features of acute rejection episodes. We identified patients who had (1) clinical evidence of acute rejection, (2) a renal biopsy showing pathologic features of acute rejection, defined by the Banff criteria, and (3) pre- and posttransplant sera screened against donor T cells. We divided these patients into those with or without donor-specific alloantibody reactive with donor T cells. Of 44 patients with biopsy-proven rejection, 20 were antibody negative (Ab-R) and 24 were antibody positive (Ab+R). The biopsies from Ab+R patients had a higher incidence of severe vasculitis (P=0.0009) and glomerulitis (P=0.01). Fibrin thrombi in the glomeruli and/or vessels, fibrinoid necrosis, and dilatation of peritubular capillaries were also more frequent in the Ab+R group. Infarction was present in biopsy specimens from 9/24 Ab+R patients versus none in the Ab-R group (P=0.002). The Ab+R biopsy specimens more often had polymorphonuclear leukocytes in the peritubular capillaries (P=0.003). In contrast, specimens of Ab-R patients showed tubulitis more often than the specimens of Ab+R patients: moderate and severe tubulitis was present in 19/20 (95%) Ab-R patients versus 12/24 (50%) Ab+R patients (P=0.002). Graft loss was increased in Ab+R patients, particularly in the first 3 months (12/24 compared with 3/20, P=0.025). Thus, during biopsy-proven acute rejection episodes, anti-class I antibody correlates with severe vascular lesions, glomerulitis, and infarction, whereas more severe tubulitis predominates in rejection episodes without antibody.

Elevated natural killer cytotoxicity in HLA-B8 and HLA-DR3-positive individuals.

Increased natural killer cytotoxicity in persons positive for HLA-B8 and HLA-DR3 has been found. It is suggested that linkage disequilibrium between the genes coding for HLA-B8 and HLA-DR3 may have evolved as a result of strengthened immunological surveillance in individuals bearing them, as decrease in the frequency of both these antigens has recently been reported in patients with tumours.

The functional consequences of partial calcineurin inhibition in human peripheral blood mononuclear leucocytes.

Calcium-dependent signal transduction is essential to the induction of cytokine expression by stimuli acting through the T cell receptor. In vitro, the immunosuppressant cyclosporine (CyA) blocks this pathway by inhibition of calcineurin (CN) phosphatase activity. But in vivo, patients on CyA have only 50% inhibition of CN and can mount cytokine responses. To simulate this state of partial inhibition, we studied the responses of human peripheral blood mononuclear leucocytes (PBL) in vitro at low CyA concentrations. PBL were challenged in vitro with calcium ionophores or anti-CD3 monoclonal antibody. The induction of IFN-gamma (interferon-gamma) and IL-2 (interleukin 2) steady-state mRNA was studied by Northern blotting and reverse transcriptase-polymerase chain reaction. IFN-gamma was assessed in a radiolabelled antibody binding assay or by ELISA (enzyme-linked immunosorbent assay). CN was assessed by dephosphorylation of a 32P-serine labelled 19 amino acid substrate. CyA inhibited CN with an IC50 (concentration giving 50% inhibition) of 10 ng/ml (95% confidence interval, CI = 8-13 ng/ml). Likewise, the induction of IFN-gamma and IL-2 mRNA by calcium ionophore A23187 was inhibited with IC50 of 14 ng/ml (95% CI = 8-27 ng/ml) and 32 ng/ml (95% CI = 5-178 ng/ml), respectively, while the IC50 for inhibition of IFN-gamma protein secretion was 8 ng/ml (95% CI = 9-18 ng/ml). Partial inhibition of CN also altered the threshold for IFN-gamma induction. CyA 10 ng/ml inhibited IFN-gamma induction by anti-CD3 monoclonal antibody OKT3 significantly more at low OKT3 concentrations (10 ng/ml, mean +/- SEM = 72 +/- 9% inhibition) compared to high OKT3 concentrations (1000 ng/ml, 47 +/- 6%, p < 0.01). Similar results were seen using high and low concentrations of A23187. Finally, cells pretreated with CyA recovered the ability to respond to high concentrations of A23187 (5 microM) faster than low concentrations (0.5 microM). We conclude that the principal defect in lymphocytes with partial CN inhibition is a reduction in maximum cytokine output which is closely related to the degree of CN inhibition. In addition, there is significantly greater inhibition of weak stimuli compared to maximal stimuli. These defects may explain why patients on CyA can have a reduction in immune responsiveness but still retain protection from infection.

Role of the major histocompatibility complex in resistance to Marek's disease: restriction of the growth of JMV-MD tumor cells in genetically resistant birds.

B21 is associated with resistance to Marek's disease (MD). Forty populations of chickens from all over the world were examined for the presence of the B21 allele. B21 was found in twelve of these populations and it's presence was confirmed by GVH testing in all ten populations which were tested. The populations in which B21 was detected represent the extreme production types of the species and include the progenitor of the species, the Red Jungle Fowl. Our studies suggest that B21 may have strong survival value for the species. An allogeneic transplantable lymphoma of MD, the JMV tumor cell line, grows more slowly in MD resistant (B21/B21) chicks than in MD susceptible (B2/B2) chicks. This is the first direct evidence that genetic resistance to MD may involve an active (immunological?) restriction of tumor cell growth. JMV cells were further characterized as a transplant of B1 carrying lymphoblastoid cells, an allele which may be associated with susceptibility to MD.