Worakij Chalermskulrat

Non‐tuberculous mycobacteria in end stage cystic fibrosis: implications for lung transplantation

Background: WC and NS contributed equally. Non-tuberculous mycobacteria (NTM) frequently colonise patients with end stage cystic fibrosis (CF), but its impact on the course of the disease following lung transplantation is unknown. Methods: Lung transplant recipients with CF who underwent lung transplantation at our institution between January 1990 and May 2003 (n = 146) and CF patients awaiting lung transplantation in May 2003 (n = 31) were studied retrospectively. Results: The prevalence rate of NTM isolated from respiratory cultures in patients with end stage CF referred for lung transplantation was 19.7%, compared with a prevalence rate of 13.7% for NTM isolates in CF lung transplant recipients. The overall prevalence of invasive NTM disease after lung transplantation was low (3.4%) and was predicted most strongly by pre-transplant NTM isolation (p = 0.001, Fisher’s exact test, odds ratio (OR) 6.13, 95% CI 3.2 to 11.4). This association was restricted to Mycobacterium abscessus (p = 0.005, Fisher’s exact test, OR 7.45, 95% CI 2.9 to 16.9). While NTM disease caused significant morbidity in a small number of patients after transplantation, it was successfully treated and did not influence the post-transplant course of the disease. Conclusion: The isolation of NTM before transplantation in CF patients should not be an exclusion criterion for lung transplantation, but it may alert the clinician to patients at risk of recurrence following transplantation.

Abnormal bone turnover in cystic fibrosis adults.

Abstract: Cystic fibrosis (CF) patients often have low bone mineral density (BMD) and may suffer from fractures and kyphosis. The pathogenesis of low BMD in CF is multifactorial. To study bone metabolism, we collected fasting serum and urine from 50 clinically stable CF adults (mean age 28 years) and 53 matched controls to measure markers of bone formation and bone resorption. The CF subjects had moderate lung disease (FEV1: 46.1 ± 18.6% predicted) and malnutrition (BMI: 20.0 ± 3.3 kg/m2). Only 3 subjects had normal BMD. CF subjects had higher urinary N-telopeptides of type I collagen (81.0 ± 60.0 vs 49.0 ± 24.2 nm BCE/mmol creatinine, p= 0.0006) and free deoxypyridinoline (7.3 ± 5.0 vs 5.3 ± 1.9 nM/mM, p= 0.004) levels than controls. Serum osteocalcin levels were similar in the two groups, a result confirmed by two immunoassays that recognize different epitopes on osteocalcin. Serum bone-specific alkaline phosphatase levels were elevated in CF patients (32.0 ± 11.3 vs 21.8 ± 7.0 U/l, p<0.0001), but were much more closely associated with serum total alkaline phosphatase levels (r = 0.51, p = 0.001) than with age or gender. Parathyroid hormone levels were elevated (p= 0.007) and 25-hydroxyvitamin D levels were depressed (p= 0.0002) in the CF patients in comparison with controls. These results indicate that adults with CF have increased bone resorption with little change in bone formation. Medications that decrease bone resorption or improve calcium homeostasis may be effective therapies for CF bone disease.

Epithelial Kinetics in Mouse Heterotopic Tracheal Allografts

Obliterative bronchiolitis (OB) is the most important cause of graft dysfunction post‐lung transplantation. It is likely that the small airway epithelium is a target of the alloimmune response, and that epithelial integrity is a crucial determinant of airway patency. Our goals are to elucidate epithelial cell kinetics in the heterotopic mouse trachea model and to determine potential mechanisms of cell death in allografts. Allografts and isografts were obtained by transplanting BALB/c tracheas into C57BL/6 and BALB/c immunosuppressed and nonimmunosuppressed hosts, respectively and harvested from day 3–20. Morphometry, BrdU and TUNEL labeling, and EM studies were performed. Columnar epithelium in isografts and allografts sloughs during day 0–3, but regenerates in both sets of grafts by day 10. Subsequently, allografts become inflamed and denuded, while isografts retain an intact epithelium. Prior to airway denudation, allografts exhibited significantly increased epithelial cell density, BrdU labeling index (LI), and TUNEL positive cells. Epithelial apoptosis was confirmed by electron microscopy. Allograft percent ciliated columnar epithelium and lumenal circumference were significantly decreased. Cyclosporin delayed airway fibrosis but did not alter the progression of the allograft through the phases of early ischemic injury, airway epithelial cell regeneration, and eventual cell death. These studies quantitatively demonstrate that the allograft epithelium actively regenerates in the alloimmune environment, but succumbs to increased apoptotic cell death, underscoring the importance of the airway epithelium as a self‐renewing source of alloantigen.

Nontuberculous mycobacteria in women, young and old

Lady Windermere syndrome is a unique entity within the spectrum of pulmonary NTM diseases. There are differences in several clinical aspects between Lady Windermere syndrome and the classic pulmonary NTM disease, including manifestations, pathogenesis, and natural history. Recently, emerging pieces of information provide a more scientific explanation of why women are more susceptible to this form of infection and how they develop clinical disease. As the result, these patients probably require quite different diagnostic and therapeutic approaches compared with those with the classic presentation. Studies exclusive to LWS are lacking and are absolutely necessary as they will enhance our understanding of, and hence successful management strategies for, this increasingly recognized disease.

Calcineurin inhibitor effects on growth and phenotype of human airway epithelial cells in vitro

Calcineurin inhibitors (CIs) cyclosporin and tacrolimus form the basis for immunosuppression in lung transplantation, yet also exert biological effects on nonlymphoid tissue. With the advent of inhaled cyclosporin, we hypothesize that the airway epithelium is also subject to CI effects at high doses. The aim of this study was to identify human tracheobronchial epithelial cell (hTBEC) calcineurin gene expression and quantify effects of CIs on hTBEC growth, interleukin‐1‐β stimulated IL‐8 production and hTBEC phenotype. Cyclophillin B and FK‐associated binding protein, calcineurin A (α and β), and NFATC3 and NFAT5 were detected in hTBEC cultures by RT‐PCR. Acute and chronic cyclosporine treatment 1000 ng/mL significantly inhibited hTBEC proliferation, while tacrolimus did not (range of 10 ng/mL to 1000 ng/mL for acute treatment, 50 ng/mL for chronic treatment). Cyclosporin at 10 000 ng/mL significantly increased LDH release by well‐differentiated hTBEC cultures (n = 6) and trended towards significance at 1000 ng/mL. IL1‐β stimulated IL‐8 production was significantly increased in rapidly growing hTBEC cultures (n = 8) treated with cyclosporin (p = 0.049). Prolonged treatment of well‐differentiated hTBECs at air‐liquid‐interface (ALI) with cyclosporin 1000 ng/mL significantly reduced intact multilayered mucociliary epithelium (p = 0.009). Inhibition of hTBEC growth, stimulation of IL‐8 production and long‐term effects on mucociliary phenotype and intact multi‐layered epithelium suggest that cyclosporin may have a direct toxic effect on airway epithelium after transplantation.

Combined donor specific transfusion and anti‐CD154 therapy achieves airway allograft tolerance

Background: The state of tolerance allows long term graft survival without immunosuppressants. Lung transplantation tolerance has not been consistently achieved in either small or large animal models. Methods: The mechanisms and effectiveness of a tolerance induction protocol consisting of donor specific transfusion (DST; day 0) and a short course of co-stimulatory blockade (anti-CD154 antibody; days −7, −4, 0 and +4) were studied in the mouse heterotopic tracheal transplant model of chronic lung rejection. C57BL/6 mice received BALB/c tracheal grafts (day 0) and were treated with DST alone, anti-CD154 alone, the combination (DST/anti-CD154), or no treatment. No non-specific immunosuppressants were used. Results: DST/anti-CD154 in combination, but neither treatment alone, markedly prolonged the lumen patency and survival (>100 days) of fully histo-incompatible allografts (p<0.05 versus control allografts at every time point studied up to 16 weeks) without immunosuppression. This protocol was donor antigen specific as third party grafts (C3H) were promptly rejected. In addition, DST/anti-CD154 did not result in mixed chimerism but induced transplantation tolerance via a peripheral mechanism(s), which included significantly reduced cytotoxic T cell activity (p<0.001) and a significantly increased percentage of CD4+CD25+ cells (p = 0.03). Conclusions: The DST/anti-CD154 protocol successfully induced and maintained long term, donor specific tolerance in the mouse heterotopic airway graft model of chronic lung rejection. This finding may lead us closer to successful tolerance induction in lung transplantation.

PX3.102, a novel chinese herb extract, diminishes chronic airway allograft rejection.

More effective immunosuppressants are needed to improve lung-transplantation survival. PX3.102 is a novel immunosuppressant isolated from a mixture of traditional Chinese herbs. We tested its protective role on chronic lung rejection in the heterotopic tracheal transplant model. C57BL/6 mice received BALB/c tracheal grafts and were treated with PX3.102, cyclosporine A, or vehicle. PX3.102 improved tracheal allograft lumen patency (*P<0.01 vs. vehicle and P=0.14 vs. cyclosporine A) but not epithelialization (P>0.2 vs. vehicle). Subsequent in vitro studies demonstrated that PX3.10 was toxic to fully differentiated human tracheal epithelial cells in a dose-dependent manner. PX3.102 markedly suppressed antigen-specific lymphocyte proliferation in vitro at a concentration 10 times lower than cyclosporine A. In conclusion, PX3.102, a promising and potent immunosuppressant, although exhibiting toxicity to airway epithelial cells at high doses, is effective in inhibiting chronic airway allograft rejection.

Immunity of Allograft Rejection: An Overview

Transplantation between genetically disparate individuals of the same species evokes numerous changes in the host and the graft. In general, the outcome of a transplanted organ is determined by two closely interrelated components. The first component is the biology of the foreign graft (i.e., type of organ and its macro-and microenvironments, the anatomic location, the type of vascular connection, the circumstance surrounding organ harvest and transplant, and the genetic differences between the donor and the recipient). The second component is the nature and intensity of immune responses that develop in the recipient after encountering that specific foreign tissue. The immunity of allograft rejection involves a number of powerful, complex, interrelated, and well-orchestrated immune mechanisms and is only partially understood. This destructive immune response, if left unchecked, can and almost universally does, lead to complete destruction of the transplanted organ over time. This chapter offers an overview of the features and mechanisms of the immune system that work in concert to interact with and reject the organ allograft. Subsequent chapters will describe the role of distinct components that affect the immunology characteristic of the rejection response.

Alloantigen recognition in the mouse heterotopic model of OB

(CyA) has been linked to the development of accelerated cardiac allograft vasculopathy (CAV). CyA may promote CAV by (i) impairing nitric oxide (NO) mediated vasodilatation, (ii) stimulating oxidant production and (iii) downregulating Fas-Ligand (FasL) expression thereby leading to neutrophil migration. In the present study, we hypothesized that the NO synthase cofactor, tetrahydrobiopterin (BH4), would attenuate the deleterious effects of CyA via the aforementioned pathways. Methods: The effects of BH4 (0.1mM) on endothelium dependent and independent relaxation were assessed in rat aortae with and without exposure to CyA (25mg/kg/day for 7 days). Lucigenin chemiluminiscence was employed to examine the effects of CyA BH4 on coronary artery superoxide production. FasL expression was measured in cultured human coronary endothelial cells exposed to CyA BH4. Results: CyA treated aortae displayed impaired endothelium dependent vasodilation (%Emax 67 7 vs 91 9, p 0.05), with no change in endothelium independent vasodilation. BH4 attenuated the CyA-induced endothelial dysfunction (%Emax 76 5, p 0.05). BH4 also attenuated the CyA-induced increase in superoxide production (BH4 1250 56 cpm/mg vs. CYA: 1845 81 vs control 848 86, p 0.05). Coronary endothelial FasL expression was not affected by either CyA or BH4. Conclusions: BH4 attenuates CyA induced endothelial dysfunction and superoxide production. BH4 may be an important cellular target for the prevention of cardiac allograft vasculopathy related to cyclosporin induced endothelial injury.