Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell-related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group-namely, the tolerant recipients-were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS-independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross-validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

Quality of life of living kidney donors: a single-center experience.

Renal transplantation improves the quality of life (QoL) of patients with end-stage renal disease. The preservation of QoL of living kidney donors is paramount. The aim of this study was to assess the QoL pre- and postdonation using Medical Outcome Survey Short Form-36 (SF-36) and to compare with a control group of potential donors who did not proceed with donation. Over a period of 28 years (1978 to 2006), 82 living donor renal transplantations were performed. Of the 78 eligible donors, 66 (85%) participated in the survey. The median postdonation period was 4.6 years (range, 3 months to 27 years). Thirty eight individuals were assessed in the control group. The postdonation SF-36 scores of the donors were not statistically significantly different from those of the control group except in one out of eight dimensions, which was physical role. However, in 44/66 (66%) donors, the postdonation scores were significantly lower compared to their predonation scores because of development of comorbidities such as musculoskeletal pain, migraine, myocardial infarction, diabetes, and peptic ulcers as the time progressed since kidney donation. The age, sex, time since donation, and relationship to recipient did not affect QoL. Eighty three percentage of the donors would have donated again if possible, and 90.9% wished to encourage living kidney donation. We conclude that the QoL of living kidney donors was not different from the healthy controls, although with the passage of time, there was some deterioration of QoL due to development of comorbidities.

De novo membranous nephropathy associated with donor-specific alloantibody.

Recent evidence suggests that alloantibody may play an aetiological role in the pathogenesis of membranous glomerulopathy in native kidneys. There is an increased awareness of the significance of alloantibody on renal transplant outcome, particularly with the development of more sensitive assays. We describe a kidney transplant patient who developed de novo membranous glomerulopathy (DNMG) with heavy proteinuria in the context of a donor-specific alloantibody (DSA) directed against HLA DQ7. Proteinuria resolved and kidney function stabilized following treatment with mycophenolate mofetil and an angiotensin receptor blocker. The titre of the DSA fell in parallel with resolution of the proteinuria. This is the first reported case of DNMG after kidney transplantation clearly associated with a DSA. We hypothesize that de novo membranous glomerulopathy may be an atypical manifestation of acute antibody-mediated damage. Cases of DNMG should be screened for alloantibody and the presence of alloantibody may influence the choice of therapy.

Renal transplantation after endovascular repair of abdominal aortic aneurysm.

An increasing number of abdominal aortic aneurysms (AAA) occur in renal failure patients because of strong association between atherosclerosis and chronic kidney disease. Endovascular aneurysm repair (EVAR) has proven to be an effective modality to treat AAA, particularly in patients with renal disease, because of its several advantages over the standard open procedure, including lower morbidity, shorter operative time, and shorter hospital stay. A Medline search showed a single publication on renal transplantation (RT) following EVAR of AAA. In this context, we report our case of successful RT in a patient who had undergone EVAR 2 years prior for a 5.7-cm AAA. No stent-related complications, such as graft occlusion, dislodgement, dissection, or endoleak, were observed in the perioperative period. The transplanted kidney had primary function leading to a stable serum creatinine of 115 micromol/L at 6 months. Although the long-term outcome of RT after endovascular repair of AAA remains unknown, currently available evidence shows favorable outcomes of EVAR in the normal population, in patients with renal diseases, and in RT recipients; hence, RT should not be denied to renal failure patients who have undergone EVAR in the past.

Vacuum-assisted closure (VAC) therapy in the management of wound infection following renal transplantation.

OBJECTIVES: Wound infection in the setting of immunosuppressed state such as renal transplantation (RT) causes significant morbidity from sepsis, prolongs hospital stay and is expensive. Vacuum-assisted closure (VAC) therapy is a new technique of management of wound based on the principle of application of controlled negative pressure. The aim of this study was to assess the efficacy of VAC therapy in the management of wound infection following RT. MATERIALS AND METHODS: This is a prospective study of a cohort of 180 consecutive RTs performed over a period of 4 years, where the data were retrieved from a prospectively maintained computerised database and case-notes. RESULTS: 9 of 180 (5%) patients developed wound infection following RT which led to cavitations and dehiscence with copious discharge, and refused to heal with conventional treatment. All 9 cases were treated with VAC therapy. The VAC system was removed after a median of 9 (range 3-30) days when discharge from the wound ceased. Four patients were discharged home with portable VAC device and managed on an outpatient basis, where the system was removed after a median 5.5 (range 3-7) days. The median hospital stay after initiation of VAC therapy was significantly shorter (5, range 2-12 days) than on conventional treatment prior to VAC therapy (11, range, 5-20 days) (p=0.003). Complete healing was achieved in all cases. CONCLUSIONS: The use of VAC therapy is an effective and safe adjunct to conventional and established treatment modalities for the management of wound infection and dehiscence following RT. Key words: Renal transplantation, wound infection, vacuum-assisted closure therapy.

Colitis in a renal transplant patient with human herpesvirus-6 infection.

A male patient developed colitis and a thrombotic microangiopathy 3 weeks after renal transplantation. Immunosuppression at the time of presentation was with sirolimus, mycophenolate mofetil, and prednisolone, but without a calcineurin inhibitor. Cytomegalovirus infection was excluded. However, human herpesvirus-6 DNA was detected at high copy number in both blood and colonic epithelium. The patient recovered after reduction in immunosuppression, with nutritional support and ganciclovir therapy.

The advantages of intravenous renal anaemia treatment in an undernourished patient with chronic kidney disease.

The subcutaneous (SC) treatment of renal anaemia in undernourished patients has potential limitations. In this case report we demonstrate the value of intravenous (IV) darbepoetin alfa in such a patient who experienced difficulty tolerating SC recombinant human erythropoietin (rHuEPO) therapy due to severe malnutrition. Intravenous treatment of renal anaemia in a malnourished patient is preferred because the absence of SC fat makes SC administration difficult. In such patients, darbepoetin alfa is the treatment of choice as it is administered less frequently than other erythropoietic therapies and is more effective at maintaining target haemoglobin (Hb) concentrations. In contrast to rHuEPO, darbepoetin alfa also has the additional advantage of bioequivalent IV and SC dose requirements.

Wegener's granulomatosis presenting as acute suppurative interstitial nephritis.

A 78 year old man presented with acute renal failure following a prolonged respiratory illness. A renal biopsy demonstrated severe suppurative interstitial nephritis with normal glomeruli. After nine weeks of antibiotics he remained anuric and a second biopsy demonstrated pauci-immune, necrotising glomerulonephritis. His subsequent clinical course was consistent with a diagnosis of Wegener's granulomatosis and antineutrophil cytoplasmic antibodies (ANCA) were detected. This is the first reported case of Wegener's granulomatosis presenting with an isolated tubulointerstitial lesion.

IgG2 anti-Galalpha1-3Gal does not induce porcine aortic endothelial cell accommodation in vitro.

BACKGROUND: Xenografts that have been protected from hyperacute rejection (HAR) are termed accommodated if they are not then rejected despite the presence of xenoantibody. It has been proposed that IgG may confer resistance to complement dependent cytotoxicity (CDC), a conventional in vitro marker of accommodation. We hypothesized that noncytotoxic IgG2 anti-Galalpha1-3Gal was responsible for this effect. METHODS AND RESULTS: We purified IgG anti-Galalpha1-3Gal from pooled human normal immunoglobulin and three sera, by elution from protein G and Galalpha1-3Gal-R immunoadsorbents. The eluates were IgM free and > or =95% IgG2. They bound to Galalpha1-3Gal, porcine aortic endothelial cells (PAEC) and lymphocytes. It was not possible to block IgM binding to PAEC or lymphocytes using IgG anti-Galalpha1-3Gal (200 microg/ml). The eluates were noncytotoxic in micro-CDC assays. To investigate accommodation, PAEC were cultured with subsaturating doses of the four IgG eluates for up to 144 hr. Resistance of nontrypsinized PAEC to CDC by human serum was measured in a cell viability assay. PAEC were not rendered resistant to CDC in any of the experiments. To investigate the possibility that accommodation might be induced by non-Galalpha1-3Gal IgG, the experiments were repeated using HNIg, again with no protection demonstrated. CONCLUSIONS: Using primary PAEC monolayers, we were unable to induce resistance to CDC with human normal immunoglobulin and its IgG2 anti-Gabeta1-3Gal subset. This contradicts previous experiments using trypsinized, immortalized cells. Although resistance to CDC is not an ideal marker of accommodation, the detrimental effects of IgG make it unlikely that it will become a useful clinical means of inducing accommodation.

Chloramine-induced haemolysis presenting as erythropoietin resistance.

BACKGROUND: In December 1996 we identified an outbreak of erythropoietin (rHuEpo) resistance requiring a substantial increase in rHuEpo dosage in one of our four haemodialysis (HD) units. The dialysate chloramine levels in this unit had risen from <0.1 p.p.m. in 1996 to 0.25-0.3 p.p.m. in 1997. In the other three HD units levels remained <0.1 p.p.m. Other parameters of water quality were within accepted standards. METHODS: Monthly records of haemoglobin level and rHuEpo dose were available for 148 patients between January 1996 and May 1998. Seventy-two patients, with no recognized cause of rHuEpo resistance, were analysed in detail (August 1997 to April 1998). A subgroup of 15 patients was examined for evidence of haemolysis during HD (methaemoglobin and haptoglobin levels, reticulocyte counts and Heinz bodies). Larger carbon columns were installed in December 1997 to effect chloramine removal. RESULTS: There was an increase in mean methaemoglobinaemia of 23% (P<0.01) and a 21% fall in mean haptoglobin (P<0.01) across HD, although no patient had a reticulocytosis and only one patient with G6PD deficiency had Heinz bodies. Following installation of larger carbon columns there was an 18.6% rise (P<0.001) in mean haemoglobin level and a subsequent 25.0% reduction (P<0.001) in mean rHuEpo dose. Intradialytic changes in methaemoglobin and haptoglobin were abolished. The dialysate chloramine levels fell to < 0.1 p.p.m. Water company records subsequently revealed a sustained twofold increase in mains water chloramine from November 1996. CONCLUSIONS: This is the first report linking chloramine exposure and rHuEpo resistance, with only subtle signs of haemolysis. Unheralded changes in mains water constituents can directly affect dialysate water quality and clinical outcomes.

Demonstration of IgM antibodies of high affinity within the anti-Galalpha1-3Gal antibody repertoire.

BACKGROUND: Human anti-Galalpha1-3Gal IgG and IgM xenoantibodies can distinguish between very similar epitopes with a high degree of selectivity. METHODS: Anti-Galalpha1-3Gal antibodies were affinity isolated using two separate Galalpha1-3Gal-based immunoadsorbents, Galalpha1-3Gal itself and Galalpha1-3Galbeta1-4Glc. IgG and IgM were separated using a protein G column. Antibody purity was achieved by serial adsorption/elutions from the columns. By this means, different antibody fractions were prepared that contained either IgG or IgM, reactive with either Galalpha1-3Gal, Galalpha1-3Galbeta1-4Glc, or both. The dissociation equilibrium constants (Kd) of these antibodies were then measured using an IAsys biosensor. RESULTS AND CONCLUSIONS: Sera from two individuals were used and Kd values for one IgG (fraction 1A) and two IgM (fractions 1B and 2A) fractions were obtained. The Kd for the IgG was 4.85 x 10(-7) M (fraction 1A). For IgM, the Kd values were higher at 7.8x10(-10) M (fraction 1B) and 1.07x10(-10) M (fraction 2A). Natural anti-pig antibodies include high affinity IgM that continue to be produced without class switch. The B cell mechanism behind this is not known. It may be possible to exploit this mechanism in future xenotransplantation strategies.

Polymorphism in the human anti-pig natural antibody repertoire: implications for antigen-specific immunoadsorption.

BACKGROUND: Anti-Galalpha1-3Gal antibodies cause hyperacute rejection (HAR) in pig-to-primate xenotransplantation. Long-term graft survival has not been achieved despite abrogation of HAR using transgenic pigs. IgG and IgM anti-Galalpha1-3Gal also play a role in the events following abrogation of HAR. Characterizing these antibodies and developing a system for their removal is therefore crucial to future success in xenotransplantation. METHODS AND RESULTS: We have developed a neoglycoprotein enzyme-linked immunosorbent assay to probe the precise antigenic requirements for the binding of anti-Galalpha1-3Gal and have analyzed 77 normal sera. Sixty-six percent of individuals have IgG that recognizes the Galalpha1-3Gal di-, tri-, and pentasaccharides (D, T, and P, respectively), termed DTP phenotype. The frequency of other phenotypes was - -P, 13%; -TP, 12%; D-P, 8%; and DT-, 1%. The IgG subclasses found were IgG2 (95%), IgG3 (34%), IgG1 (31%), and IgG4 (17%). IgM in 91% of individuals recognized all three antigens. Further antibody heterogeneity was demonstrated when immunoadsorbents derived from Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc (PENTA) were tested. Galalpha1-3Galbeta1-4Glc (TRI 6) or PENTA agarose were effective for IgG removal in all individuals. For IgM removal, two deoxy derivatives were completely successful in 73% of individuals. Combining the Galalpha1-3Gal (DI) and TRI 6 agarose produced an adsorbent that completely removed anti-Galalpha1-3Gal IgG and IgM in all individuals tested. CONCLUSIONS: Although the polymorphism in the anti-Galalpha1-3Gal repertoire, which we have demonstrated, represents a major obstacle to the development of an effective immunoadsorbent, the combination of DI and TRI 6 agarose appears sufficient for pig-to-human xenotransplantation.

Audit of the management of spontaneous pneumothorax.

This audit suggests that clinical practice in the management of spontaneous pneumothorax differs from guidelines issued by the British Thoracic Society. In particular simple aspiration was attempted in only seven out of 65 patients and clamping of an intercostal chest drain occurred in 12 out of 50 cases. Junior medical staff require more training in intercostal drainage.

Role of estrogen deficiency in pathogenesis of secondary hyperparathyroidism and increased bone resorption in elderly women.

Whether the increased bone resorption and secondary hyperparathyroidism in elderly women is due to aging or to estrogen deficiency is unclear. To address this issue, we measured serum intact parathyroid hormone (PTH) and biochemical markers in serum and urine samples from 30 premenopausal women (32 +/- 0.5 years, mean age +/- SE), 30 estrogen-deficient postmenopausal women (74.2 +/- 0.6 years), and 30 elderly women (73.8 +/- 0.6 years) receiving long-term estrogen treatment. Because of the first and third groups were comparable in estrogen status but not in age, whereas the second and third groups were comparable in age but not in estrogen status, the independent effects of age and estrogen deficiency could be assessed quantitatively. Mean values were higher in the estrogen-deficient postmenopausal women than in the premenopausal women for serum PTH (by 33%, p < .01) and for bone resorption markers [by 50% p < .001) for urine cross-linked N-teleopeptide of type I collagen (NTx); 34% (p < .001) for urine pyridinoline (Pyd); and 36% (p < .001) for urine deoxypyridinoline (Dpd)]. However, mean values for serum PTH in the postmenopausal women receiving estrogen treatment did not differ from those in the premenopausal women, and mean values for bone resorption markers were not different (urine NTx and Pyd) or were lower [urine Dpd, by -12%, (p < .005)]. These findings suggest that late consequences of estrogen deficiency rather than age-related processes per se are the principal causes of the secondary hyperparathyroidism and increased bone resorption in elderly women.

Role of calcium intake in modulating age-related increases in parathyroid function and bone resorption.

Serum parathyroid hormone (PTH) and bone resorption increase in elderly women and contribute to age-related bone loss. Whether these abnormalities are caused by calcium deficiency resulting from age-related decreases in absorption and renal conservation is unclear. We studied 28 normal elderly women (mean +/- SD, age 69.3 +/- 2.7 yr) who were maintained for 3 yr on usual calcium intake levels (20.4 +/- 7.2 mmol/day [815 +/- 289 mg/day]; n = 15) (known as the usual calcium group) or high calcium intake levels (60.4 +/- 6.5 mmol/day [2414+/260 mg/day]; n = 13) (known as the high calcium group) and a reference group of 12 normal young adult women (age 30.1 +/- 4.4 yr), whose calcium intake was 23.0 +/- 4.8 mmol/day (918 +/- 193 mg/day) (known as the young group). Serum PTH was measured every 2 h, and urinary excretion of deoxypyridinoline (Dpd), a new marker for bone resorption, was measured in 4 h collections. Parathyroid gland secretory capacity was assessed during induced hypocalcemia. The mean 24 h serum PTH was 40% lower (P < 0.001), and the mean 24 h urinary Dpd was 35% lower (P < 0.005) in the high than in the usual calcium group. Mean parathyroid gland secretory capacity also was 47% lower (P < 0.005) in the high calcium group than in the usual calcium group. However, the usual calcium group had a mean 24 h serum PTH level that was 70% higher (P < 0.001) and a mean 24 h urinary Dpd level that was 30% higher (P < 0.005) than the young group, whereas the high calcium group was indistinguishable from the young group. Thus, failure of elderly women to increase their calcium intake to offset age-related increases in calcium requirement contributes substantially to their development of increased parathyroid activity and increased bone resorption, whereas a high calcium intake can reverse both abnormalities.