Kenneth R. McCurry

Nitrate and nitrite in biology, nutrition and therapeutics

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Transgenic pigs expressing human CD59 and decay-accelerating factor produce an intrinsic barrier to complement-mediated damage.

We characterize a line of transgenic pigs that express the human complement-regulatory proteins human CD59 and human decay-accelerating factor. These genes, under the control of heterologous promoters, are expressed in a variety of organs, including the vasculature of the heart, kidney, and liver. We demonstrate that moderate levels of these gene products are sufficient to protect peripheral blood cells from human or baboon complement. Using pig to baboon heterotopic heart transplants, we show that expression of these proteins is sufficient to block the complement-mediated damage that is the hallmark of such xenografts, when nontransgenic organs are used. These results indicate that there is significant species specificity of intrinsic complement regulatory protein function. This specificity is evident in transgenic organs in which low levels of human CD59 and human decay-accelerating factor expression significantly effect the humoral immune response that causes xenograft rejection. This result suggests that transgenic organs with high levels of human complement-regulatory protein expression will be sufficient to alleviate the humoral immunological barriers that currently block the use of xenogeneic organs for human transplantation.

Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury.

Ischemia/reperfusion (I/R) injury during small intestinal transplantation (SITx) frequently causes complications including dysmotility, inflammation and organ failure. Recent evidence indicates hydrogen inhalation eliminates toxic hydroxyl radicals. Syngeneic, orthotopic SITx was performed in Lewis rats with 3 h of cold ischemic time. Both donor and recipient received perioperative air or 2% hydrogen inhalation. SITx caused a delay in gastrointestinal transit and decreased jejunal circular muscle contractile activity 24 h after surgery. Hydrogen treatment resulted in significantly improved gastrointestinal transit, as well as jejunal smooth muscle contractility in response to bethanechol. The transplant induced upregulation in the inflammatory mediators CCL2, IL‐1β, IL‐6 and TNF‐α were mitigated by hydrogen. Hydrogen significantly diminished lipid peroxidation compared to elevated tissue malondialdehyde levels in air‐treated grafts demonstrating an antioxidant effect. Histopathological mucosal erosion and increased gut permeability indicated a breakdown in posttransplant mucosal barrier function which was significantly attenuated by hydrogen treatment. In recipient lung, hydrogen treatment also resulted in a significant abatement in inflammatory mRNA induction and reduced neutrophil recruitment. Hydrogen inhalation significantly ameliorates intestinal transplant injury and prevents remote organ inflammation via its antioxidant effects. Administration of perioperative hydrogen gas may be a potent and clinically applicable therapeutic strategy for intestinal I/R injury.

Carbon Monoxide Induces Cytoprotection in Rat Orthotopic Lung Transplantation via Anti-Inflammatory and Anti-Apoptotic Effects

Successful lung transplantation has been limited by the high incidence of acute graft rejection. There is mounting evidence that the stress response gene heme oxygenase-1 (HO-1) and/or its catalytic by-product carbon monoxide (CO) confers cytoprotection against tissue and cellular injury. This led us to hypothesize that CO may protect against lung transplant rejection via its anti-inflammatory and antiapoptotic effects. Orthotopic left lung transplantation was performed in Lewis rat recipients from Brown-Norway rat donors. HO-1 mRNA and protein expression were markedly induced in transplanted rat lungs compared to sham-operated control lungs. Transplanted lungs developed severe intraalveolar hemorrhage, marked infiltration of inflammatory cells, and intravascular coagulation. However, in the presence of CO exposure (500 ppm), the gross anatomy and histology of transplanted lungs showed marked preservation. Furthermore, transplanted lungs displayed increased apoptotic cell death compared with the transplanted lungs of CO-exposed recipients, as assessed by TUNEL and caspase-3 immunostaining. CO exposure inhibited the induction of IL-6 mRNA and protein expression in lung and serum, respectively. Gene array analysis revealed that CO also down-regulated other proinflammatory genes, including MIP-1alpha and MIF, and growth factors such as platelet-derived growth factor, which were up-regulated by transplantation. These data suggest that the anti-inflammatory and antiapoptotic properties of CO confer potent cytoprotection in a rat model of lung transplantation.

Opportunistic Infections in 547 Organ Transplant Recipients Receiving Alemtuzumab, a Humanized Monoclonal CD-52 Antibody

BACKGROUND Alemtuzumab is being increasingly used for the prevention and/or treatment of acute allograft rejection in organ transplant recipients. We assessed the risks of infection in, to our knowledge, the largest cohort and broadest range of organ transplant recipients yet reported to have received alemtuzumab. METHODS All patients who received alemtuzumab from September 2002 through March 2004, either as induction therapy at the time of transplantation or for the treatment of rejection, were evaluated for the development of an opportunistic infection (OI) until death or for 12 months after receipt of the last dose of alemtuzumab. RESULTS A total of 547 recipients were included, 65% of whom received alemtuzumab for induction therapy only. Overall, 56 recipients (10%) developed 62 OIs, including cytomegalovirus disease (n = 16), BK virus infection (n=12), posttransplantation lymphoproliferative disease (n=5), human herpesvirus 6 infection (n=1), parvovirus infection (n=1), esophageal candidiasis (n=12), cryptococcosis (n=2), invasive mold infection (n=4), Nocardia infection (n=4), mycobacterial infection (n=3), Balamuthia mandrillaris infection (n=1), and toxoplasmosis (n=1). Patients who received alemtuzumab for induction therapy were significantly less likely to develop an OI, compared with patients who received alemtuzumab for rejection therapy (4.5% vs. 21%; P<.001). Independent predictors of the development of an OI were administration of alemtuzumab for rejection therapy (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.8-6.8; P<.001), allograft failure (OR, 2.1; 95% CI, 1.1-4.4; P=.04), and receipt of a lung transplant (OR, 3.7; 95% CI, 1.7-8.0; P=.001) or an intestinal transplant (OR, 8.3; 95% CI, 3.5-19.5; P<.001). CONCLUSIONS Patients who received alemtuzumab for the treatment of allograft rejection were significantly more likely to develop an OI, compared with patients who received alemtuzumab for induction therapy only. Such data have implications for new antimicrobial prophylactic strategies.

Influenza virus infection in adult solid organ transplant recipients.

Background: Solid organ transplant (SOT) recipients have been reported to be more susceptible to influenza virus. However, little is known about the clinical epidemiology and the implications of influenza viral infection among SOT recipients.

Voriconazole Prophylaxis in Lung Transplant Recipients

Lung transplant recipients have one of the highest rates of invasive aspergillosis (IA) in solid organ transplantation. We used a single center, nonrandomized, retrospective, sequential study design to evaluate fungal infection rates in lung transplant recipients who were managed with either universal prophylaxis with voriconazole (n = 65) or targeted prophylaxis (n = 30) with itraconazole ± inhaled amphotericin in patients at high risk (pre‐ or posttransplant Aspergillus colonization [except Aspergillus niger]). The rate of IA at 1 year was better in lung transplant recipients receiving voriconazole prophylaxis as compared to the cohort managed with targeted prophylaxis (1.5% vs. 23%; p = 0.001). Twenty‐nine percent of cases in the targeted prophylaxis group were in patients colonized with A. niger who did not receive itraconazole. A threefold or higher increase in liver enzymes was noted in 37–60% of patients receiving voriconazole prophylaxis as compared to 15–41% of patients in the targeted prophylaxis cohort. Fourteen percent in the voriconazole group as compared to 8% in the targeted prophylaxis group had to discontinue antifungal medications due to side effects. Voriconazole prophylaxis can be used in preventing IA in lung transplant recipients. Regular monitoring of liver enzymes and serum concentrations of calcineurin inhibitors are required to avoid hepatotoxicity and nephrotoxicity.

Prospective Assessment of Platelia™ Aspergillus Galactomannan Antigen for the Diagnosis of Invasive Aspergillosis in Lung Transplant Recipients

The clinical utility of Platelia™Aspergillus galactomannan antigen for the early diagnosis of invasive aspergillosis was prospectively assessed in 70 consecutive lung transplant recipients. Sera were collected twice weekly and tested for galactomannan. Invasive aspergillosis was documented in 17.1% (12/70) of the patients. Using the generalized estimating equation model, at the cutoff value of ≥ 0.5, the sensitivity of the test was 30%, specificity 93% with positive and negative likelihood ratios of 4.2 and 0.75, respectively. Increasing the cutoff value to ≥ 0.66 yielded a sensitivity of 30%, specificity of 95%, and positive and negative likelihood ratios of 5.5 and 0.74. A total of 14 patients had false‐positive tests, including nine who had cystic fibrosis or chronic obstructive pulmonary disease. False‐positive tests occurred within 3 days of transplantation in 43% (6/14) of the patients, and within 7 days in 64% (9/14). Thus, the test demonstrated excellent specificity, but a low sensitivity for the diagnosis of aspergillosis in this patient population. Patients with cystic fibrosis or chronic obstructive pulmonary disease may transiently have a positive test in the early post‐transplant period.

Aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients

Background. The clinical utility of Platelia Aspergillus enzyme immunoassay (EIA) for galactomannan (GM) antigen detection in bronchoalveolar lavage (BAL) for the diagnosis of invasive aspergillosis (IA) in lung transplant recipients is not known. Methods. BAL fluid samples from consecutive lung transplant recipients who underwent bronchoscopy were prospectively analyzed for GM. Results. A total of 333 BAL samples from 116 patients were tested. Invasive aspergillosis was documented in 5.2% (6/116) of the patients. Samples analyzed included 9 BALs from two patients with proven IA, 19 BALs from four patients with probable IA, and 305 BALs from 110 patients without IA. At the index cutoff value of ≥0.5, the sensitivity was 60%; specificity was 95%, with positive and negative likelihood ratios of 14 and 0.41, respectively. Increasing the index cutoff value to ≥1.0 yielded a sensitivity of 60%, a specificity of 98%, and the positive and negative likelihood ratios of 28 and 0.40, respectively. Two of six patients with IA receiving antifungal prophylaxis had false-negative results. Conclusions. A Platelia EIA index cut-off ≥1.0 in the BAL fluid in a lung transplant recipient with a compatible clinical illness may be considered as suggestive of IA.

Tacrolimus Dosing in Adult Lung Transplant Patients Is Related to Cytochrome P4503A5 Gene Polymorphism

Tacrolimus is a potent immunosuppressive agent used in lung transplantation and is a substrate for both P‐glycoprotein (P‐gp, encoded by the gene MDR1) and cytochrome (CYP) P4503A. A previous study by the authors identified a correlation between the tacrolimus blood level per dose with CYP3A5 and MDR1 gene polymorphisms in pediatric heart transplant patients. The objective of this study was to confirm the influence of these polymorphisms on tacrolimus dosing in adult lung transplant patients. Adult lung transplant patients who had been followed for at least 1 year after lung transplantation were studied. Tacrolimus blood level (ng/mL) per dose (mg/day) at 1, 3, 6, 9, and 12 months after transplantation was calculated as [L/D]. DNA was extracted from blood. MDR1 3435 CC, CT, and TT; MDR1 2677 GG, GT, and TT; and CYP3A5*1 (expressor) and *3 (nonexpressor) genotypes were determined by PCR amplification, direct sequencing, and sequence evaluation. Eightythree patients were studied. At 1, 3, 6, 9, and 12 months after the transplant, a significant difference in [L/D] was found between the CYP3A5 expressor versus nonexpressor genotypes (mean ± SD of 1.49 ± 0.88 vs. 3.11 ± 4.27, p = 0.01; 1.23 ± 0.82 vs. 3.44 ± 8.97, p = 0.05; 1.32 ± 0.96 vs. 3.81 ± 6.66, p = 0.005; 0.95 ±1.19 vs. 3.74 ±5.98, p = 0.0015; and 0.45 ± 0.2 vs. 3.76 ± 6.75, p = 0.0001, respectively). MDR1 G2677T and C3435T genotypes had only minimal effects on [L/D] at 1 and 3 months after transplantation. This study confirms the relationship of CYP3A5 polymorphisms to tacrolimus dosing in organ transplant patients. CYP3A5 expressor genotypes required a larger tacrolimus dose to achieve the same blood levels than the CYP3A5 nonexpressors at all time points during the first posttransplant year. This was not uniformly true for MDR1. The authors therefore conclude that tacrolimus dosing in adult lung transplant patients is associated with CYP3A5 gene polymorphisms.