A retrospective study of the Framingham cardiovascular risk scores in a liver transplant population.

OBJECTIVE: Evaluate the relationship between Framingham cardiovascular risk scores (FRS) and transplant-related factors, particularly renal function, in a stable liver transplant population. METHODS: Single-center retrospective study of 54 post-liver transplant patients followed in one outpatient clinic. Demographics and laboratory data were assessed using the modified FRS (2009). Standard statistical analyses were performed between FRS and transplant-related factors; patient demographics, new-onset diabetes after transplant (NODAT), immunosuppressives, and estimated glomerular filtration rate (eGFR) measured by isotope dilution mass spectrometry (IDMS) and Cockcroft-Gault (CG) equations. RESULTS: Forty percent of patients were classified as low FRS, 29.6% as moderate FRS, and 29.6% as high FRS (of whom 50% had NODAT). Immunosuppressant use was similar between the high- and low-risk groups. FRS inversely correlated with eGFR (P = .0001) measured by either equation. eGFR measured by IDMS in the high-risk group (60.4 ± 22.1 mL/min/1.73 m(2)) was significantly lower than that in the low-risk group (97.1 ± 54 mL/min/1.73 m(2); P = .0001). In the multivariate analysis, age, eGFR and NODAT were significantly different between the low- and high-risk FRS groups. Receiving operational characteristic (ROC) analysis identified eGFR measured by IDMS at 42.7 mL/min/1.73 m(2) with a sensitivity of 92%, specificity of 19%, and positive predictive value of 72% to identify high-risk patients. Box-plot analysis of variance between eGFRs in the three risk groups showed a P value of .001. CONCLUSIONS: In this study one-third of liver transplant patients had a high FRS, and 14.8% had an eGFR below 40 mL/min/1.73 m(2). Low eGFR predicts those with high FRS. Liver transplant patients particularly those with NODAT, with low eGFR should undergo close management of cardiovascular risk factors.

Prevention of CAN graft loss in the medium term without HLA disparity reduction.

INTRODUCTION: This study compares the incidence of cadaveric graft failure from chronic allograft nephropathy in the medium term (1 to 5 years) using older and newer immunosuppressive regimens. The older regimen was established triple therapy and the newer regimen, almost universal replacement of azathioprine by mycophenolate. MATERIALS AND METHODS: In the older series, 76 (71 after death censoring) cadaveric renal grafts done from 1990 to mid-1996 in patients who survived for more than 1 year were analyzed. In the newer series, 49 (45 after death censoring) cadaveric grafts done 5 or more years ago in patients surviving 1 year were analyzed. In the older series, immunosuppression was combined steroids, cyclosporine, and azathioprine. In the newer series, mycophenolate replaced azathioprine in 85%, historically conventional immunosuppression was used in 7.5%, and miscellaneous in 7.5%. RESULTS: Cumulative deaths in years 1 to 5 with renal graft function were as follows: older series, 6.6% (5/76), newer 8.2% (4/49) (P = NS). In the older series, death-censored 1- to 5-year cumulative graft failure was 35.2% (24/71), newer series 4.4% (2/45) (chi-square 13.5, relative risk reduction 0.87 [0.51 to 0.97], P =.00021). ACE-inhibitor antihypertensive therapy was used in 25% (18/71) of the patients in the older series and in 53% (24/45) of patients in the newer series (chi-square 6.1, relative risk 1.8 [1.1 to 2.9], P =.01). CONCLUSION: Replacement of azathioprine with mainly myocophenolate in triple immunosuppression and enhanced use of ACE inhibitors are associated with near complete prevention (87%) of medium-term CAN graft failure, making death with graft function now the major cause of graft loss in this time.

Analysis of the CD40/CD40L role in the sustenance of alloreactive antibody production.

CD40 ligand (CD40L) is important for T/B lymphocyte interaction. To understand the cellular basis of humoral allosensitization we, therefore: (1) measured CD40L protein and gene expression in sensitized and non-sensitized uremic unactivated peripheral CD4+ T lymphocytes; (2) studied the impact of blocking the CD40/CD40L pathway on alloreactive antibody (allo-Ab) production by engrafted sensitized PBLs into severe combined immunodeficient (SCID) mice after in vitro preactivation with IL2/LPs/HLA class II allopeptides and adjuvants as a potent stimulus to produce allo-Ab (Shoker et al. Transplantation 1999;68;1188); and (3) studied the modifying effect of CD40/CD40L blockade on T helper type I and II cytokine gene expression in the respective mice spleen. The CD40L protein was measured by flow cytometry and the gene expression was measured by quantitative RT-PCR. Alloreactive antibodies (alo-Abs) produced by sensitized PBLs engrafted into SCID mice with and without blockade of the CD40 receptor were measured by the PRA-STAT ELISA method. The modifying effects of CD40 blocking on allo-Ab production and cytokine gene expression by the engrafted cells measured by RT-PCR were then compared. There was no detectable CD40L protein expression in either the uremic or the control groups. The CD40L gene expression of 0.04 +/- 0.02 attomoles (aM) in the sensitized group was significantly higher than in the non-sensitized patients (0.009 +/- 0.007 aM, P < 0.0001) or the control CD4+ T cells (0.016 +/- 0.004 aM, P < 0.001). Blockade of the CD40 receptor abrogated the production of allo-Ab antibodies by the engrafted sensitized cells in 60% of the tested mice (n = 10); decreased the mean +/- S.D. optic density of allo-Ab to 0.1 +/- 0.13 and the mean +/- S.D. PRA to 12 + 16). In the presence of the control Ab, allo-Ab production in SCID sera was present in 100% of the 10 SCID mice tested; the mean +/- S.D. PRA was 75 +/- 20, and the mean + S.D. OD activity was 0.412 +/- 0.17. All cytokine genes were, otherwise, expressed in the presence or absence of CD40 blockade. The results suggest a potential role of an enhanced CD40/CD40L interaction in the sustenance of alloreactive antibody production without significant deviation to T helper-like I or II responses. Blocking the CD40/CD40L pathway may have a potential therapeutic benefit to treat sensitized uremic patients.

Analysis of the relationship between chimerism and the allgeneic humoral response.

BACKGROUND: Persistence of antigens has been suggested to play a role in two opposing immunological phenomena: tolerance and memory. Therefore, we studied the impact of chimerism on alloreactive antibody (allo-Ab) production in kidney transplant patients. METHODS: Thirty-five female renal transplant recipients of male donor organs were classified into the following groups: group 1, 13 sensitized uremic patients on dialysis; group 2, 5 nonsensitized uremic patients on dialysis; group 3, six sensitized patients experiencing graft rejection (3 acute vascular, 1 acute cellular, and 2 chronic); and group 4, 11 nonsensitized with functioning allografts (9 with good function, 1 with acute cellular rejection, and 1 with chronic rejection). Mean duration of dialysis after graft failure was similar in groups 1 (56+/-29.7 months) and 2 (41.8+/-42.4 months), as was dialysis efficiency. Chimerism was measured indirectly in the peripheral blood lymphocytes by polymerase chain reaction amplification of a specific Y chromosome DNA gene sequence with a detection sensitivity limit of 1 male cell per 1 million female cells. Allo-Ab production was measured by the PRA-STAT enzyme-linked immunosorbent assay (Sangstat) method. RESULTS: Chimerism was observed in 60% of groups 1 and 2, 83% of group 3, and 82% of group 4. Among all groups, graft existence, irrespective of its function, positively predicted chimerism in 92% with a sensitivity of 88% and a specificity of 78%. In group 3, all three patients with acute vascular rejection had chimerism and donor-specific allo-Abs. In group 4, eight of the nine patients with no rejection had chimerism. CONCLUSION: Chimerism relates to persistence of allogeneic stimulus irrespective of its function. Chimerism did not confer protection against allo-Ab production or vascular rejection, and its existence was not crucial for sustenance of allo-Ab production.

In vitro IgG1 alloreactive antibody production by circulating memory cells in humans.

This study was designed to investigate the presence of IgG1 alloreactive memory cells in the peripheral blood in humans and their in vitro activation requirements. Alloreactive antibody production was measured after cell activation with cytokines: Interleukin (IL) 2, IL-4 and IL-10, interferon gamma, alloantigens and/or OKT3, lipopolysaccharide or Pokeweed mitogen and Epstein-Barr virus transformation. The examined cells were taken from ten sensitized and five nonsensitized uremic patients with previous graft loss and five normal controls. The titers and percentage panel reactive IgG1 antibody reactivity present in the respective sera was further compared with the in vitro profile. Alloreactive antibody reactivity was measured by PRA-STAT ELISA method. The results show that: 1) Short term control T cell lines or nonsensitized cells were unable to provide the necessary help to autologous B cells to produce alloreactive antibodies of the IgG subclass. 2) Activated cells from sensitized patients produced low levels of alloreactive IgGl antibodies. 3) Stimulation with any of the cytokines and/or mitogens or alloantigens or allopeptides was not sufficient to produce consistent levels of alloreactive IgG 1 antibodies, in spite of its presence in the respective sera.

Alloantigen-blocking antibodies in sera from highly sensitized uremic patients: antibody class and relationship with lymphocytotoxins.

BACKGROUND: Previously, we identified an antimitogenic IgG antibody separated from sera of patients with known kidney transplant chronic rejection. This antibody inhibits individual patients' own unprimed T helper cell responses to alloantigens as well as a third-party mixed lymphocyte response, but does not inhibit autologous unprimed T helper cell proliferation to adherent anti-CD3 antibody. We suggest that the mechanism of inhibitory action is allogeneic-dependent. METHODS: We used a series of similar experimental designs to test the presence of this antibody in uremic, sensitized patients and have studied its relationship to sensitization as defined by the presence of lymphocytotoxins in four uremic groups: highly sensitized with or without previous graft loss, moderately sensitized with or without graft loss, nonsensitized without previous graft loss, and nonsensitized with graft loss. RESULTS: (1) Sensitization is associated with the presence of a potent antibody that blocks primary mixed lymphocyte response. Primed cells are less susceptible to its antimitogenic action. (2) The blocking antibody activity is present only in sensitized patients who have IgG lymphocytotoxic activity against the same HLA class I antigens. (3) The blocking activity is unequal in the following order: IgG 3 > IgG 1 > IgG 2. (4) Although IgG 1 and 2 fractions contain lymphocytotoxic activity against HLA class I antigens, the IgG 3 fraction does not. CONCLUSIONS: The differential effect of IgG antibodies on naive and memory T cells may explain why humeral responses to alloantigens can be maintained in the presence of blocking antibodies.

Immunoglobulin G subclasses and susceptibility to allosensitization in humans.

Understanding the cellular basis of allosensitization is important because persistence of cytotoxic alloreactive antibodies significantly decreases the chances for receiving a second kidney graft and has a detrimental effect on graft survival rates. In this study, serum levels of IgG subclasses were compared within three groups of uremic patients with different levels of allosensitization (panel reactive antibody level > or = 70%, 10-65% and < or = 10%). All the non-sensitized patients had already lost at least one graft indicating resistance to allosensitization by the previous graft. In addition, the in vitro T-cell proliferation and immunoglobulin M and G subclass production were studied after activation by pokeweed mitogen and alloantigens. The patients' demographics were comparable. The results show that all serum IgG subclass levels in the three groups were comparable and within the range of normal control. Similarly, T-cell proliferation and the in vitro production of IgM was not significantly different. The lymphocytotoxic activity present in each IgG subclass was not associated with an increase in the respective serum subclass level or the in vitro production of the same subclass in the sensitized patients. The data indicate that humoral immunity, as reflected by subclass immunoglobulin levels, is, in fact, normal, in the three groups and that sustenance of cytotoxic antibody production reflects a specific immune response controlled by factors other than intrinsic B-cell abnormality.

Inhibition of human lymphocyte function by organic solvents.

UNLABELLED: We studied the direct effect of reactive hydroxyl precursors and inhibitors on CD4+ T-cell function. We used hydrogen peroxide plus ferrous chloride as the hydroxyl radical-generating system and di-methyl sulphourea, di-methyl sulfoxide, pyrrolidine dithiocarbonate, methanol, and ethanol, at a noncytotoxic concentration, as inhibitors. The immune parameter studies were proliferation and interleukin-2 production by peripheral blood lymphocytes stimulated with anti-CD3 antibody, phytohemagglutinin and alloantigens; proliferation, interleukin-2 production and mRNA expression of interleukin-4 and interferon gamma by allogeneic CD4+ T-cell clones stimulated with alloantigens. The results show that lymphocytes produce significant amounts of reactive oxygen species as measured by malondialdehyde produced in cultures. The hydroxyl radical-generating system did not change any of the cellular responses studied although it doubled Malondialdehyde production. Hydroxyl radical scavengers significantly inhibited all responses at doses that didn't significantly decrease malondialdehyde production. DNA analysis failed to show evidence for apoptosis. CONCLUSION: Hydroxyl radical scavengers inhibit lymphocyte mitogenesis by a process that is independent of scavenging hydroxyl radicals.

Analysis of the in vitro effect of exogenous nitric oxide on human lymphocytes.

We investigated the role of endogenous or exogenous nitric oxide (NO) on human lymphocyte function. We used sodium nitroprusside, nitroglycerine, S-nitroso-N-acetylpenicillamine, sodium nitrite and S-nitroso-L-glutathione as NO-generating compounds. All agents were used at doses that do not produce direct cytotoxicity as measured by trypan blue exclusion as well as chromium-51 release assay. The immune responses examined were peripheral blood lymphocytes (PBL) proliferation and IL-2 production after activation with OKT3 and PHA; allogeneic mediated proliferation and cell mediated cytotoxicity (CML) in MLR; IgG and IgM production after PBL activation with Con-A; proliferation and expression of IFN-gamma and IL-4 mRNA after activation of allogeneic CD4+T cell clones. Cytokine mRNA expression was measured by reverse transcriptase PCR. Our results show that proliferating lymphocytes do not produce a detectable amount of NO as measured by the Griess reaction. In separate experiments, the addition of NG-monomethyl-L-arginine (L-NMMA) did not affect lymphocyte proliferation. Sodium nitroprusside and nitroglycerine exerted a dose dependent antimitogenic effect, inhibited cytokine production and expression, CML generation and antibody production. DNA gel electrophoresis showed no evidence for enhanced programmed cell death. The antimitogenic effect could not be blocked by the NO scavengers, hemoglobin or methylene blue. In contrast, the other nitric oxide generating compounds did not inhibit lymphocyte mitogenesis. The results suggest that human lymphocytes do not produce appreciable amounts of NO to affect lymphocyte mitogenesis. Sodium nitroprusside and nitroglycerine have a potent but nonspecific immunoinhibitory effect on human lymphocyte function by a mechanism other than NO production. In addition, pharmacological levels of NO do not inhibit human lymphocyte mitogenesis.

Protein malnutrition and decreased protein turnover in chronic renal allograft failure.

OBJECT: To define the longitudinal relationship of declining renal function to protein consumption and turnover in the failing renal allograft model of chronic renal failure. METHOD: The study group is our first eight consecutive cadaveric renal graft recipients who after attaining a normal creatinine clearance, then developed chronic renal failure. We analysed their urea and creatinine clearances (Cur, Ccr), serum urea (SU), urinary urea and creatinine (Ur, Ucr), serum albumin (SA), urinary protein (Upr), body weight (BW), and steroid dose. Steady state Uur is also dietary protein intake (DPI) and protein catabolic rate (PCR). Ucr measures body protein mass. Ucr/Uur measures the ratio of body protein mass to urea excretion. Mean follow-up 4.7 years, range 1.5-8.7 years. RESULTS: Mean changes: (1) Body weight (BW) rose from 56 to 65 and then fell to 61 kgms. (2) Cur fell 65 to 5 and Ccr 92 to 12 ml/min/70 kg. (3) Uur fell from 369 to 107 and Ucr from 16.8 to 9.5 mmols/day/70 kg. (4) Uur/Ucr indexed at 1:1 fell to 0.49. (5) SU rose from 8.8 to 34.9 mmol/1; SA fell from 36.1 to 31.0 gms/1; Upr rose from 1.4 to 2.3 gms/day. (6) Prednisone rose from 26 to 66 and then fell to 33 mgms/day. Correlations: (1) Cur and Uur(r = 0.99, p < 0.001). (2) Ccr and Uur (r = 0.99, p < 0.001). (3) Cur and Uur/Ucr (r = 0.88, p < 0.01) with a decelerating breakpoint at Cur 18 and Ccr 32 ml/min/70 kg (p < 0.01). (4) SU and Uur negatively (r = 0.90, p < 0.01. (5) Cur and SA albumin (r = 0.82, p < 0.05). (6) Cur and prednisone, Upr and SA do not correlate. CONCLUSIONS: In this model of chronic renal failure: (1) Renal function controls protein intake. (2) Body protein mass is relatively well preserved despite the decreased protein intake implying a decrease in the protein turnover rate and a consequent increase in body protein average age. (3) Protein malnutrition, protein ageing, and decreased protein turnover are likely pathophysiological reactions to chronic renal failure and may be part of the pathogenesis of chronic uremia.

Immunopharmacologic therapy in renal transplantation.

The long-term kidney allograft survival rate is still far from optimum. Conventional immunosuppressive drugs used to prevent allograft rejection are associated with significant side effects. Moreover, withdrawal of these agents is often associated with graft loss due to rejection. No treatment is available for chronic rejection. Graft tolerance is difficult to achieve in humans, and therefore a continued goal in organ transplantation is to develop immunosuppressive regimens that are associated with fewer side effects and decreased rates of rejection, and that promote graft tolerance. The advent of newer pharmacologic agents and bioreagents is expected to improve patient and graft survival rates.

HLA-identity--long-term renal graft survival, acute vascular, chronic vascular, and acute interstitial rejection.

The object is analysis of the impact of acute and chronic rejection on long-term function in HLA-identical renal transplants performed from 1967 to 1995 by the Saskatchewan Renal Transplant Unit. Forty-eight grafts in 46 patients were studied, of which 39 were first and nine second grafts. Forty-two were for primary and six for secondary renal disease. Thirty-five received azathioprine/prednisone prophylaxis, and 13 received cyclosporine/prednisone with/without azathioprine. Ten-year all graft actuarial survival was 84%, 10-year actuarial graft survival in patients with primary renal disease 90%, and with subsequent graft after first HLA graft failed 97.5%, for age-matched population 98.5% (P=NS). Overall death rate was 8.7% (4/46); in secondary renal disease patients 50% (3/6); in primary renal disease patients 2.5% (1/40, P=0.004). All (9/9) HLA-identical second grafts functioned. Acute rejection with azathioprine/prednisone prophylaxis occurred in 55% (9/17) of grafts treated with <6 pre-graft blood transfusions, with the same prophylaxis but >5 units in 12% (2/16, P=0.015), and with cyclosporine prophylaxis in 13% (2/15, P=0.021). Pulse steroids alone reversed all acute rejection. Grafts failed in 6.2% (3/48), all in primary renal disease patients and one from technical one noncompliance, and one chronic rejection. Graft cost/patient/year amortized over 9 years is $3,855 and comparable dialysis cost would be $35,650; cost for all patients on dialysis for 9 years would be $11,293,320 while comparable graft cost was 1,221,418, a savings of 89.2%. Our conclusions are that HLA-identity associates with the following: (1) a 10-year actuarial survival in primary renal disease that equals that of the age-matched population, (2) uniform success in repeat grafts, (3) virtual absence of chronic rejection despite a high incidence of acute rejection in azathioprine/prednisone grafts that (4) always reversed on pulse steroids, and (5) a cost reduction for grafting of 93.2% compared with dialysis therapy.

Lack of early acute rejection does not predict safe tapering of immunosuppression.

In an effort to reduce long-term complications of immunosuppressive medications, some investigators have devised protocols of reduced dosage [Kupin et al. 1988, Kelly et al. 1989, Reinsmoen et al. 1993] which are intended to maintain the stability of the transplant and at the same time prevent or retard immunosuppressive medication side effects. Implicit in this plan is the assumption that there has been the development of some degree of graft acceptance [Koene 1989] and/or tolerance [Goulmy et al. 1985, Muluk et al. 1991], yet it is also true that drug non-compliance is a common cause of late acute rejection and graft loss in stable kidney recipients [Rao et al. 1988, Didlake et al. 1988, Rao et al. 1989, Hricik et al. 1991]. The latter fact is a warning that immunosuppression should be cautiously and gradually reduced. Patients who reject grafts early tend to require more immunosuppression. It is not known, however, whether patients who do not reject early require less immunosuppression. Herein, we present a case of late acute cellular and vascular rejection that occurred 9 years after stable renal graft function, in the absence of early acute rejection episodes. This case provides evidence to support the notion that lack of early acute rejection may not predict safe reduction of immunosuppression therapy.

The differential effect of alloantigen-blocking antibodies on unprimed and memory T helper cells.

Responsiveness to recall antigens by memory and naive T helper cells is different. To study whether such a difference is also applicable to affinity for allorecognition, we analyzed the effect of an IgG MLR blocking antibody separated from sera of patients with known kidney transplant chronic rejection on primed and unprimed CD4+ T cell alloreactivity. The results show that addition of the IgG fraction inhibits the patient's own unprimed T helper cell responses to a panel of four different alloantigens as well as a third-party mixed lymphocyte response. The same IgG fraction inhibited third-party naive T helper cell, but not autologous unprimed T helper cell, proliferation to adherent anti-CD3 antibody, which suggests that the mechanism of inhibitory action of the IgG is allogeneic-dependent. This IgG also did not induce inhibition of any of the T helper cell clone responsiveness, raised from the same or other patients, when stimulated with the same alloantigens used for unprimed cell alloactivation. Differential responses of naive and memory CD4+ T cells to alloantigens may explain some differences between the in vivo and in vitro systems and why allograft rejection can proceed in the presence of allogeneic blocking antibodies.

Application of the clearance concept to hyponatremic and hypernatremic disorders: a phenomenological analysis.

The kidney and its response to the antidiuretic hormone (ADH) are the principal protective mechanisms necessary to maintain a normal plasma tonicity (osmolality). Hence, determination of the response of the ADH-renal axis to an abnormal plasma tonicity is an important step to understanding water homeostasis. Determination of free water clearance is the most direct clinical method to measure the ability of the kidney to reabsorb or excrete water; it can be used as a sensitive method to study water metabolism, describing the abnormal water homeostasis in simple quantitative terms. A positive electrolyte-free water clearance denotes the excretion of excess free water. A negative electrolyte-free water clearance indicates reabsorption of excess free water. During hypertonicity, an increased concentration of ADH enhances renal reabsorption of free water. With diminished ADH secretion and normal renal function, a substantial volume of free water is cleared in response to hypotonic stimuli. A positive free water clearance > 0.4 L/day in hypertonic conditions or a negative free water clearance during hypotonicity confirms an abnormal ADH-renal axis response.