Neil Lempert

In vitro immunosuppressive properties of cyclosporine metabolites

The in vitro biological activity of cyclosporine (CsA) and four of its metabolites (M1, M8, M17, and M21) was determined. M1, M17, and M21 are primary metabolites, while M8 is a secondary metabolite derived from either M1 or M17. The order of inhibitory activity in production assays was phytohemagglutinin (PHA), con-canavalin A (ConA), mixed lymphocyte culture (MLC), and interleukin-2 (IL-2) CsA > M17 > M1 > M21 “ M8. In the PHA assay, CsA was significantly more inhibitory than M17, but in Con A and MLC assays, the inhibitory activity of M17 approached that of CsA. More importantly, M17 and Ml inhibited the production of IL-2 in the MLC to the same extent as CsA. M21 was significantly less inhibitory than either M17 or M1, and M8 appeared to be largely devoid of biological activity. These experiments demonstrate that single hydroxylations of amino acids 1 (M17) and 9 (M1) do not significantly affect the ability of the molecule to block IL-2 production, but hydroxylation of both amino acids renders the molecule virtually inactive. In addition, the presence of the N-methyl group on amino acid 4 appears to be very important, since removal of this group (M21) greatly diminishes the immunosuppressive activity.

Immunosuppressive metabolites of cyclosporine in the blood of renal allograft recipients.

Cyclosporine levels by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) were monitored in serial blood samples (n = 177) from 11 renal allograft recipients. HPLC analysis revealed three primary metabolites of CsA (M17, M1, and M21) in peak and trough blood samples; M17 was the preponderant metabolite. In 4 patients on whom serial metabolite assays were performed, M17 was found in the blood at 86-2004 ng/ml; M1 and M21 were found at up to 100 ng/ml. The immunosuppressive properties of purified metabolites M1, M17, M21, and M8 (which was not detected in the blood) were compared with CsA. M17--and, to a lesser extent, M1 and M21--were found to inhibit the in vitro response of human mononuclear cells in the mixed leukocyte culture and in mitogen (phytohemagglutinin [PHA], concanavalin A [Con A], and pokeweed mitogen [PWM]) assays at 1000 ng/ml. M8 exhibited no in vitro inhibitory activity. M17 was further tested at 10-1000 ng/ml in PHA and mixed lymphocyte culture (MLC) assays. M17 had considerably less inhibitory activity (12-43%) than CsA (18-70%) in the PHA assay. However, in MLC experiments M17 blocked the proliferative response by 39-72% at 100-800 ng/ml, which approached the degree of inhibition exhibited by CsA (63-87%). In 34 of 37 (92%) patient blood samples, the level of metabolite M17 was found to exceed the parent drug level and could not be measured accurately by RIA. The observed in vitro immunosuppressive activity of metabolites (particularly M17) and their presence in the blood of renal allograft recipients suggest a possible role for these metabolites in the immunopharmacology of CsA.

Complicated diverticulitis following renal transplantation

PURPOSE: Colonic perforations in renal transplant recipients have historically been associated with mortality rates as high as 50 to 100 percent. However, these previous series generally predate the use of cyclosporine-based immuno-suppressive protocols. METHODS: We retrospectively reviewed all patients who had undergone renal transplant from our institution and who developed complicated diverticulitis. Complicated diverticulitis was defined as diverticulitis involving free perforation, abscess, phlegmon, or fistula. Factors analyzed included the time interval since transplantation, use of cyclosporine, living-relatedvs. cadaveric donor, cause of renal failure, and presenting signs and symptoms. RESULTS: Between August 1969 and September 1996, 1,211 kidney transplants were performed in 1,137 patients. The first 388 patients (1969–1984) received prednisone and azathioprine, with cyclosporine added to the immunosuppressive regimen for the subsequent 823 recipients (1984–1996). Thirteen (1.1 percent) patients had episodes of complicated diverticulitis, occurring from 25 days to 14 years after transplant; all required surgical therapy. Clinical presentation was highly variable, ranging from asymptomatic pneumoperitoneum (2 patients) to generalized peritonitis. There was one perioperative mortality (7.7 percent). Patients with polycystic kidney disease as the cause of renal failure had a significantly higher rate of complicated diverticulitis. Specifically, patients with polycystic kidney disease (9 percent of the total transplant population) accounted for 46 percent of the cases of diverticulitis (P < 0.001, Fishers exact probability test). Neither treatment with cyclosporine nor donor source had a significant effect on the rate of diverticular complications (P=0.36 andP=0.99, respectively, Fishers exact probability test). CONCLUSION: Complicated diverticulitis following renal transplantation is rare, and the clinical presentation may be atypical in the immunosuppressed transplant recipient. Patients with polycystic kidney disease experience a significantly higher rate of complicated diverticulitis than do other transplant patients and, therefore, warrant aggressive diagnostic evaluation of even vague abdominal symptoms. In addition, pretransplant screening and prophylactic sigmoid resection in this high-risk population deserve consideration and further study.

Chronic granulocytic leukemia following successful renal transplantation

The use of immunosuppressive therapy has markedly increased over the past several years, and concomitant with its use has been an increased frequency of associated neoplasia. The patient presented is a 22‐year‐old white male who, following two renal transplants and prolonged immunosuppressive therapy with azathioprine and methylprednisolone, developed chronic granulocytic leukemia. Chromosome karyotyping demonstrated the somewhat unusual development of a Philadelphia chromosome with translocation to the # 7 of the C group. A review of transplantation centers revealed that five cases of chronic granulocytic leukemia have occurred in a population of 25,000 renal transplant patients, a 5‐fold increased incidence over the general population. Possible etiologies that may be responsible for the development of chronic granulocytic leukemia in patients on immunosuppressive therapy are discussed. It is our hope that by the introduction of these reports of chronic granulocytic leukemia into the medical literature, the need for caution in the use of immunosuppressive drugs in nonmalignant disease will again be emphasized.

THE EFFECTS OF IMMUNOSUPPRESSIVE AGENTS ON IN VITRO PRODUCTION OF HUMAN IMMUNOGLOBULINS

We have evaluated the effects of CsA, methylprednisolone (MP), 6-mercaptopurine (6-MP), and FK506 on T cell-dependent and T cell-independent immunoglobulin production. FK506 and 6-MP were potent inhibitors of IgG and IgM production by PWM-stimulated peripheral blood mononuclear cells, which depend on the presence of T cells. CsA was less effective in this system and MP actually enhanced IgG and IgM production. In order to assess the direct effects of these various immunosuppressive agents on B cells, we utilized human B cell lines representing different stages of B cell differentiation. The B cell lines CESS and SKW6.4 exhibit increased production of IgG and IgM, respectively, in response to interleukin-6. These cells represent activated, but not fully differentiated, B cells. CsA inhibited IL-6-induced IgG production by CESS cells by 64% at 100 ng/ml and 6-MP inhibited this response by 82% at 250 ng/ml. Neither CsA nor 6-MP effectively inhibited IL-6-induced IgM production by SKW6.4 cells. MP at 250 ng/ml inhibited IL-6-induced IgG production by 89%, but enhanced IL-6-induced IgM production more than twofold. FK506 did not inhibit IL-6-induced IgG or IgM production, suggesting that it has no direct effect on the ability of B cells to respond to this differentiation factor. These experiments clearly demonstrate that CsA, MP, and 6-MP have direct inhibitory effects on the response of human B cells to IL-6. In contrast, FK506 has no direct effect on these B cells lines, but is more potent than the other agents at inhibiting T cell-dependent immunoglobulin production.

The inhibitory effects of N-ethylmaleimide, colchicine and cytochalasins on human T-cell functions

The thiol-alkylating agent, N-ethylmaleimide (NEM), was found to inhibit the response of human peripheral blood mononuclear cells to the T-cell mitogen, concanavalin A (Con A). NEM (10 microM) blocked Con A-induced agglutination, production of interleukin-2 (IL-2) and expression of the IL-2 receptors (Tac) without toxicity. In order to determine whether the effects of NEM on lymphokine production were related to inhibition of agglutination, we compared the immunosuppressive effects of NEM with those of cytochalasin A, cytochalasin B and colchicine. NEM did not inhibit E-rosette (ER) formation, suggesting that it does not interfere with actin filaments. Low concentrations of NEM (4 microM) inhibited IL-2 production and Tac expression without inhibiting agglutination, while 6-10 microM NEM blocked agglutination and DNA synthesis as well. In contrast, 5-10 microM cytochalasin B (CB) inhibited ER formation, agglutination, Tac expression and DNA synthesis, but augmented IL-2 production by three- to ten-fold. Colchicine (0.1-10 microM) had no effect on ER formation or agglutination and augmented IL-2 production by as much as 18-fold. However, colchicine blocked Tac expression by greater than 40% and DNA synthesis by greater than 80%. Cytochalasin A (CA), which has the thiol-reactive properties of NEM, the actin filament-disrupting properties of CB, and the microtubule-disrupting properties of colchicine, exhibited the immunosuppressive effects of all three compounds. These studies suggest that the inhibitory effects of NEM on IL-2 production do not appear to be due to reactivity with the cytoskeleton, but are probably due to effects on signal transduction pathways leading to IL-2 production and expression of IL-2 receptors.

Differential inhibition of human T-lymphocyte activation by maleimide probes

Cellular thiols are known to be involved in lymphocyte activation, differentiation, and growth. In theory, alkylation of selective cellular thiols could be used to regulate specific processes in the activation sequence by inactivating particular enzymes or structural proteins, although to date specific alkylating probes have not been reported. N-Ethylmaleimide (NEM) is a lipophilic sulfhydryl-alkylating agent that is known to block the in vitro proliferative response of T lymphocytes. NEM (10 microM) was found to be fully inhibitory in PHA, Con A, and MLC assays only when added prior to or simultaneously with the mitogens or allogeneic cells; the addition of NEM only 15 sec after stimulating the cells with PHA resulted in a loss of greater than 50% of the inhibitory activity. The addition of 50 microM 2-ME 10 min after treating the cells with NEM failed to block the inhibitory effect. NEM (10-20 microM) had no adverse effect on lymphocyte viability, but completely blocked lymphocyte agglutination in response to mitogens or allogeneic cells. The lymphocytes overcame the inhibitory effects of NEM after 48 hr in both the PHA and MLC experiments. Resumption of the proliferative response was associated with the onset of agglutination in the PHA assay. In experiments using various analogs of NEM, we noted that the presence of a nonpolar N-linked side group was necessary for inhibitory activity. Pretreatment of PBMC with NEM decreased the total cellular thiols by 50% and blocked proliferation by 99%, whereas N-hydroxymaleimide decreased the total cellular thiols by 38% but had no effect on the proliferative response. The additional 12% of the cellular thiols that react with NEM, but not NHM, account for the inhibitory effect of NEM on lymphocyte proliferation. These findings suggest that selective cellular thiols are critical for T-cell activation.

Vascular access for cancer chemotherapy.

Eleven bovine heterografts were utilized in the repetitive administration of chemotherapy in 10 patients with insufficient vascular access. Six grafts remained patent until the time of death and five grafts clotted from 71 to 1110 days postoperatively. Three patients are alive and well. All grafts were initially patent and no wound infections resulted. The bovine heterograft appears to be a useful adjunct for securing vascular access for patients requiring cancer chemotherapy.

The pathology of post-catheterization brachial artery occlusion

Between 1971 and October 1975, of 1084 diagnostic cardiac catheterizations via the brachial artery, 61 patients suffered from immediate loss of pulses at the wrist with signs of ischemia of the hand. In the majority of instances, the ischemic signs were not of a limb threatening nature and in only one of these patients were there immediate indications of impending tissue loss. However, for reasons which have been well described by Machelder et al. [5] and because of the probability of future catheterizations in some of these patients it has been the established policy of the Cardiology Unit to request vascular consultation with a view to reinstatement of pulsatile flow in all of these patients. Three patients declined this type of surgery and 58 patients were explored in an attempt to restore brachial artery patency. In each of these patients, the operation was so conducted that an attempt was made to define precisely the cause of loss of pulsatile flow in the brachial arteries (Karmody and Lempert [4]). In no patient was spasm of the brachial artery found to be a causative factor in the loss of wrist pulses. In each instance an occlusive thrombus was found within the lumen of the artery. In 33 of the patients the apparent cause of this thrombosis was related either to an unsatisfactory closure of the original arteriotomy and/or to the “wipe-

Inhibition of interleukin-2 production in the human T cell line JURKAT by nonpolar maleimides☆

The immunosuppressive properties of polar and nonpolar maleimides were studied by measuring their ability to inhibit mitogen-induced interleukin-2 (IL-2) production by JURKAT T cells. The nonpolar maleimides N-ethylmaleimide (NEM) and N-phenylmaleimide (NPM) inhibited IL-2 production by 85-99%, but only when added to JURKAT cells prior to the mitogen. The polar maleimides N-hydroxymaleimide (NHM) and 4-maleimidosalicylic acid (M84) did not suppress IL-2 production significantly, even though NHM reacted with more cellular thiols (12%) than did NPM (8%). Both NEM and NPM suppressed IL-2 production at doses that did not affect proliferation. NEM inhibited IL-2 production induced by PHA, anti-CD3 (alpha CD3) monoclonal antibodies or PMA, and A23187, but did not interfere with the binding of alpha CD3 to the cells. NEM inhibited IL-2 production at concentrations that did not interfere with the PHA-induced increase in intracellular free calcium [( Ca]i). Neither NPM nor NHM inhibited the rise in [Ca]i, even at the highest concentrations tested. Although JURKAT T cells require both PMA and A23187 to induce IL-2 production, we found that cells pretreated with PMA could respond to A23187 added 18 hr later. PMA-treated cells were not resistant to the immunosuppressive effects of NEM or NPM. However, PMA-pretreated cells became resistant to the inhibitory effects of NEM upon the addition of A23187, suggesting that nonpolar maleimides inhibit activation events induced by the rise in [Ca]i.