Analysis of peripheral blood T-cell subsets in active thyroid-associated ophthalmopathy: absence of effect of octreotide-LAR on T-cell subsets in patients with thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is thought to be a T-cell-mediated autoimmune disorder. We sought to characterize abnormalities in the peripheral blood T-cell subsets in patients with TAO, and examine whether the long-acting somatostatin analogue, octreotide-LAR, treatment affects these cells. We analyzed peripheral blood T-cell subsets by flow cytometry in 26 euthyroid patients with moderately severe active TAO and 24 controls. Twenty-five of the patients with TAO were enrolled in a randomized trial to receive either 30 mg of octreotide-LAR (n = 11) or placebo (n = 14) every 4 weeks for 16 weeks; all 25 patients subsequently received octreotide-LAR 30 mg every 4 weeks from week 16 to 32. T-cell subsets were analysed at baseline, week 16, and week 32. At baseline, the relative percentage of CD4+ helper T-cells (p = 0.0003) and the CD4+/CD8+ ratio (p = 0.008) were significantly higher in patients with TAO compared to controls. Patients with TAO had higher naïve active T cells (CD45RA+, CD45RA+ CD4+) and lower memory T cells (CD45RO+, CD45RO+ CD4+) than controls. At weeks 16 and 32, there were no significant differences in any T-cell subsets between the octreotide-LAR-treated and placebo groups. These results support a role of T cell in the pathogenesis of TAO, and show that octreotide-LAR has no effect on T-cell subsets during 32-weeks of treatment.

How to improve the quality of kidneys from non-heart-beating donors: a randomised controlled trial of thrombolysis in non-heart-beating donors.

BACKGROUND: The growth in the prevalence of end-stage renal failure has been accompanied with a rise in the waiting list for renal transplantation, which has not been matched by an increase in the kidney donor pool. Non-heart-beating donors (NHBD) offer a potential source of kidneys that are not currently being significantly used. Cardiac arrest for a protracted period of time leads to in situ thrombosis, and, as a consequence, the discard rates for harvested kidneys is higher than brain-stem-dead donors. METHODS: A double-blinded, randomised, controlled trial of streptokinase preflush or placebo for NHBD was performed. An initial 30 donors were entered into the study. After routine nephrectomy, NHBD kidneys were machine perfused as part of viability screening before transplantation. Kidneys were then transplanted within 24 hours of cardiac arrest. The primary objectives were the improvements of viability parameters (perfusion, enzyme levels, and histopathology) of the kidneys. The secondary objective was to increase the number of kidneys passing the viability tests and thus transplanted. RESULTS: The two groups of NHBD donors and their kidneys were similar in their descriptive epidemiologic characteristics. The NHBD kidneys from the streptokinase-treated donors had a better appearance at procurement (P<0.001) and performed better during machine preservation (P<0.001). Enzyme biomarkers present in the kidney perfusate were all significantly reduced by the use of streptokinase. These included glutathione S-transferase (P<0.001), fatty acid binding protein (P<0.001), and alanine aminopeptidase (P<0.001). However, although there was a higher proportion of kidneys transplanted through the use of streptokinase (63.6% with streptokinase vs. 42.6% with placebo), this did not achieve significance. There was no difference with respect to postoperative bleeding and transfusion requirements in the recipient whether streptokinase preflush or placebo was used. CONCLUSION: This study using streptokinase preflush in the NHBD was found to improve the condition of the kidneys retrieved. The improvement in the quality of the donor kidneys was not associated with an increased morbidity in the recipient.

Improving the quality of kidneys from non-heart-beating donors, using streptokinase: an animal model.

BACKGROUND: Non-heart-beating donors (NHBD) offer a promising potential to increase the cadaveric organ donor pool, especially for kidneys. However, almost half of NHBD kidneys are discarded after viability assessment. This wastage is costly in both human and monetary terms. Intravascular thrombosis at the time of donor death is an event leading to failure in the viability assessment. We have developed an animal model to investigate the effects of the addition of streptokinase to the in situ flush medium before transplant in an attempt to redress the situation. METHODS: Two groups of eight healthy young Landrace Yorkshire white pigs were entered into the study. Both groups were subjected to approximately 70 min warm ischemia. Both groups received an intravascular flush with 4 L of Marshall's solution with heparin (1000 IU/L); one group of pigs also had streptokinase (1.5 MIU/L) added. After donor nephrectomy, all kidneys were machine perfused for 4 hr. Data on perfusion characteristics were taken and samples of kidney effluent were assayed for tissue damage biomarkers, glutathione S-transferase (GST) and alanine aminopeptidase (Ala-AP). Wedge sections of porcine kidneys were taken at the end of perfusion, for histological analysis. RESULTS: Data on machine perfusion parameters (temperature, mean pressure index, resistance) indicate better cooling, lower resistance, and lower mean pressure index in the streptokinase-treated group of pigs. GST and Ala-AP levels were increased in the nonstreptokinase group perfusates. Histopathological analysis of porcine kidneys indicated more ischemic injury and tissue damage in the nonstreptokinase group. CONCLUSION: The use of streptokinase in this porcine NHBD model conferred benefits to donor kidneys when assessed by machine perfusion. Markers of biochemical injury indicated that less renal tissue damage occurred with the incorporation of streptokinase in the in situ flush medium.

Long-term renal function in kidneys from non-heart-beating donors: A single-center experience.

BACKGROUND: Cadaveric kidneys from brain-stem-dead donors continue to be limited because the number of donors has reached a plateau. Wide recruitment of non-heart-beating donors (NHBD) could significantly increase the donor pool. NHBD renal transplants are underused because of the concern of poor quality graft function from such donors. In response to this perception, we reviewed 46 NHBD renal transplants performed in our center since 1998. METHODS: All NHBD kidneys were machine-perfused using the Newcastle continuous-hypothermic pulsatile preservation system before transplantation. A control heart-beating-donor (HBD) group was taken as the next consecutive HBD renal transplant to the NHBD transplant. The outcome and quality of function of the groups of renal transplants were analyzed for short-term and long-term performance. RESULTS: The renal transplant patients were matched for donor and recipient factors. Survival rates for allografts and patients were similar for 1 to 3 years. There was an increased incidence of delayed graft function in the NHBD renal transplants in the perioperative period. The creatinine clearance was 22.8+/-2.3 mL/minute for NHBD patients and 44.4+/-2.9 mL/minute for HBD patients at the time of discharge from hospital. This difference equalized after 3 months and the creatinine clearance for NHBD was 44.2+/-2.4 mL/minute and for HBD 49.2+/-3.4 mL/minute. CONCLUSIONS: Our results for NHBD renal transplants confirm that such grafts suffer primary warm ischemic injury, shown by the increased incidence of acute tubular necrosis and consequent delayed graft function. This produced poor renal function at the time of hospital discharge. After 3 months, the renal function of NHBD cases improved to the level seen in HBD patients.

Neutrophil chemotaxis and receptor expression in clinical septic shock.

OBJECTIVE: To examine the hypothesis that neutrophil chemotaxis to interleukin-8 (IL-8) is reduced in septic shock. Surface expression of neutrophil CXC chemokine receptors and the adhesion molecule CD11b were also examined and associations between disease severity, gas exchange and receptor expression were studied. DESIGN: Prospective cohort clinical study. SETTING. Intensive care unit in a tertiary referral teaching hospital. PATIENTS: Patients with septic shock ( n=15) and healthy controls ( n=8) were studied. MEASUREMENTS AND RESULTS: Daily (for 5 consecutive days) flow cytometric measurements of chemokine and beta integrin surface expression. "In vitro" neutrophil chemotaxis to IL-8 was also compared between patients with sepsis and healthy controls. CXCR2 expression significantly fell, CD11b expression increased and CXCR1 expression was unchanged throughout the study in the septic group compared with healthy controls. CD11b positively correlated with increasing APACHE II scores ( p<0.0001) and worsening PaO(2)/FIO(2) ratios ( p<0.0001). CXCR2 expression negatively correlated with both APACHE II scores ( p=0.016) and PaO(2)/FIO(2) ratios ( p=0.01). There was no correlation between CXCR1 expression and either APACHE II score or PaO(2)/FIO(2) ratios. Chemotaxis to IL-8 was reduced in patients with sepsis compared with healthy volunteers. CONCLUSIONS: Surface expression of the chemokine receptor CXCR2 and the beta-integrin CD11b, but not CXCR1, were reduced on neutrophils isolated from patients with septic shock compared with healthy controls. Chemotaxis to IL-8 was also reduced in neutrophils from septic patients compared with healthy controls. The changes in receptor expression correlated with measures of disease severity.

Do tissue damage biomarkers used to assess machine-perfused NHBD kidneys predict long-term renal function post-transplant?

BACKGROUND: Renal transplantation in many units is limited by the availability of donor organs. Kidneys obtained from non-heart-beating donors (NHBD) represent an important resource, with the potential to substantially increase the available donor organ pool. Such kidneys are associated with increased warm ischaemic tissue injury which may be assessed by hypothermic machine perfusion. Within our transplant centre, a key component of such damage assessment and viability screening involves the quantification of the tissue damage biomarkers glutathione S-transferase in kidney perfusates. METHODS: Since 1998, 126 NHBD kidneys were machine-perfused prior to implantation, resulting in 74 transplants. Kidney perfusate samples were assayed for glutathione S-transferase in "real time", and alanine aminopeptidase and fatty acid binding protein in "retrospect". RESULTS: The pre-transplant concentration of these tissue injury biomarkers determined pre-transplant did not correlate with subsequent longer-term renal function, as assessed by measurement of serum creatinine (tGST: Spearman correlation r=-0.02; Ala-AP: r=0.02; H-FABP: r=-0.05) and creatinine clearance (tGST: r=0.08; Ala-AP: r=-0.02; H-FABP: r=0.14) for those kidneys that had passed their viability tests. CONCLUSIONS: Thus whilst these biomarkers may represent reliable pre-transplant indicators of immediate kidney viability and short-term kidney function, they do not predict the efficacy of renal function in the longer term.

Comparison of perfusate activities of glutathione S-transferase, alanine aminopeptidase and fatty acid binding protein in the assessment of non-heart-beating donor kidneys.

BACKGROUND: With a universal shortage of donor organs, screening and selection of marginal kidneys from non-heart-beating donors (NHBDs) provides a valuable source for transplantation. Pre-transplant viability assessment is based on a combination of flow characteristics and assessment of ischaemic tissue injury during NHBD kidney machine perfusion by measurement of total glutathione S-transferase (GST) activity. Successful viability screening has facilitated 69 renal transplants from 60 NHBDs within our transplant centre since 1998, with a first-year graft survival of 90.5%. METHODS: We have investigated alanine aminopeptidase (Ala-AP) and fatty acid binding protein (FABP) as alternative biochemical markers to GST for pre-transplantation viability assessment. They were measured, together with GST, in tissue perfusate samples from machine-perfused kidneys from controlled and uncontrolled NHBDs. RESULTS: During machine perfusion, a parallel response was seen for each of the three markers in the perfusates of controlled and uncontrolled NHBD kidneys over the 4-h perfusion. A highly significant correlation was obtained between GST and Ala-AP activities (P<0.0001) and between GST activity and FABP concentration (P<0.0001) in corresponding samples. CONCLUSION: In this study, GST, Ala-AP and FABP represent equivalent biochemical markers in terms of their ability to quantitate renal tissue injury in human NHBD kidneys. However, there may be some advantage in using all three analytes to provide complementary information on kidney allograft viability.

Combination antiplatelet therapy in patients with peripheral vascular bypass grafts.

Peripheral bypass graft occlusion occurs in approximately 25% of cases in the 5 years following surgery; hence, therapies directed at reducing the tendency toward thrombotic occlusion are of clinical importance. This trial was conducted to determine if the addition of clopidogrel to aspirin enhances antiplatelet therapy in patients with infrainguinal bypass grafts. A randomized placebo-controlled study was performed on 20 patients over 3 months following infrainguinal bypass surgery. In addition to their regular aspirin therapy, patients were randomized to receive either clopidogrel (group 1) or placebo (group 2) for 1 week. Platelet activation was measured ex vivo by platelet aggregometry and flow cytometry. In group 1, there was a significant reduction in spontaneous (SP), adenosine diphosphate (ADP), and arachidonic acid (AA)-induced platelet aggregation compared with group 2; SP -17% (CI -33, -0.2 p = 0.048), ADP -39%, (CI -56, -22 p = 0.001), AA -21% (CI -39, -4 p = 0.023). Flow cytometry demonstrated a significant reduction in ADP-induced platelet P-selectin expression and GPIIb/IIIa activation following treatment with clopidogrel but not with placebo. This study demonstrated that the addition of clopidogrel to aspirin reduces platelet activation measured by platelet aggregation and flow cytometry, supporting a long-term trial with clinical endpoints.

Transplantation from non heart beating donors in Newcastle upon Tyne.

Donor shortage has led transplant surgeons to reevaluate the concept of non heart beating donation. Organs from such donors are exposed to anoxia prior to harvesting and a portion of them will consequently present with delayed graft function. There is a need for dependable viability testing of organs from less than ideal NHBD donors and machine perfusion of kidneys provides such a tool. Our experience with own design machine perfusion device, studied parameters, perfusion solutions and outcomes is presented. Emphasis is placed on the incomparability of results obtained from different perfusion systems and the need to establish local criteria of kidney viability.

KGF suppresses alpha2beta1 integrin function and promotes differentiation of the transient amplifying population in human prostatic epithelium.

Prostate epithelial stem cells are self-renewing cells capable of differentiation into prostate epithelium, and are thought to contribute towards both benign and malignant conditions in the human prostate. We have previously demonstrated that prostate epithelial basal cells express high levels of integrin alpha2beta1 and this population can be subdivided into stem (alpha2beta1(hi) CD133+) and transient-amplifying population (TAP) cells (alpha2beta1(hi) CD133-). However, the molecular mechanism(s) controlling the commitment and regulation of these cells towards differentiated epithelium remains unclear. Here, we demonstrate that beta1 integrin function is required for the maintenance of basal prostatic epithelial cells and suppression of its function by either methylcellulose or, more specifically, beta1-blocking antibody (80 microg/ml) induces differentiation, with associated expression of the differentiation-specific markers prostate acid phosphatase (PAP) and cytokeratin 18 (CK18). Keratinocyte growth factor (KGF), a stromal-derived growth factor, has previously been implicated in prostate organogenesis using in vitro tissue recombination experiments. We show that treatment with KGF (10 ng/ml) potently induces epithelial differentiation with concomitant suppression of alpha2beta1 integrin expression as well as the induction of androgen receptor expression. Specifically, p38-MAPK appears to be involved and the presence of SB202190, a p38 inhibitor, significantly blocks KGF-induced differentiation. Furthermore, the expression of the high-affinity receptor tyrosine kinase to KGF (FGFR2) is predominantly detectable in alpha2beta1(hi) CD133- TAP cells when compared with stem cells (alpha2beta1(hi) CD133+), which would therefore be relatively unresponsive to the differentiating effect of KGF. Taken together, using a human primary culture model, we have demonstrated key roles for interactions between KGF and integrin-mediated function in the regulation of prostate epithelial differentiation.

The effect of inadequate in situ perfusion in the non heart-beating donor.

In situ aortic perfusion in the nonheart-beating donors (NHBD) is an important procedure to reduce primary warm ischaemic injury prior to formal donor organ retrieval. It allows an interim period to obtain donor family consent and theatre preparation. This study describes our experience of inadequate aortic perfusions resulting from difficult aortic cannulations and associated adverse outcome despite reasonable viability tests. Since 1998, all NHBD in our institution are perfused in situ using a double balloon triple lumen (DBTL) catheter inserted through a femoral artery cut-down procedure. The DBTL catheter is positioned with distal occlusive balloon at the aortic bifurcation using the "pull-back" technique, the proximal occlusive balloon lies above the renal arteries. This provides selective aortic perfusion in particular the kidneys. Venous decompression using a femoral vein catheter enables a "two-way infusion system". Pre-transplant viability status of retrieved kidneys is determined by measuring pressure/resistance characteristics to the flow and biochemical markers for ischaemic injury. There were 90 NHBD renal transplants performed from 72 donors. Three renal transplants were carried out from three donors of ineffective in situ perfusion secondary to cannulation difficulties. Femoral cannulation was difficult as a result of extensive atherosclerosis of donor vessels. The comparison of allograft outcome from effective and ineffective in situ perfusion of donors showed high rate of primary nonfunction (PNF) from ineffective perfusion (chi-squared, P < 0.0001). The cases demonstrated poor outcome from ineffective perfusion related to the cannulation difficulties. Therefore a strict policy should be taken in cases where aortic cannulation and perfusion is inadequate, despite pretransplant assessment. In these circumstances, the primary warm ischaemia time should be extended to include this period of ineffective perfusion.

Renal transplants from non-heart beating paracetamol overdose donors.

INTRODUCTION: Non-heart beating donors (NHBD) are widely encouraged to avert the critical shortage in the kidney donor pool. Ischaemic injury at the time of cardiac arrest in the NHBD is more pronounced and therefore the kidneys resulting are considered marginal. This review describes our experience with four kidneys from two controlled NHBDs who were exposed to paracetamol intoxication and subsequently were treated with mannitol prior to organ donation. MATERIALS AND METHOD: Two patients with fulminant liver failure following paracetamol overdose were referred as 'withdrawal of treatment' NHBD. As the two patients had developed hepatic encephalopathy they were treated with mannitol to reduce intra-cerebral oedema. The two donors were oligoanuric for at least 24 h prior to cardiac arrest. Following cardiac arrest, in situ perfusion was carried out and the kidneys were removed. One pair of kidneys were machine perfused while the second pair of kidneys were cold stored prior to transplantation. RESULTS: Pre-transplant assessment of NHBD kidneys resulted in three of four kidneys being transplanted. The NHBD kidneys exhibited a period of delayed graft function (DGF). The early transplant biopsies showed evidence of diffuse cytoplasmic vacuolation. These histological features disappeared with time and the renal function improved until the time of discharge. DISCUSSION: Non-heart beating donor kidneys are considered marginal and the effect of mannitol and paracetamol drug intoxication will induce reversible sub-lethal injury. A period of dialysis is inevitable in clearing the reactive intermediates of mannitol and paracetamol. The kidneys behaved as traditional controlled NHBD at time of discharge.

Graft function after kidney transplantation from non-heartbeating donors according to maastricht category.

PURPOSE: Donor shortages have led to some groups using alternative sources such as non-heartbeating donors (NHBDs). Kidneys from NHBDs suffer from warm ischemia at cardiac arrest which is reflected by acute tubular necrosis of the allograft, resulting in a period of delayed graft function. NHBDs are categorized by the circumstances surrounding the agonal events of death which reflect differences in the likelihood of ischemic injury to the kidney. In this study we determined the impact of ischemic injury on the renal function of kidneys procured from different categories of NHBDs. MATERIALS AND METHODS: From 1998 to 2003, 144 kidneys were procured from 72 NHBDs resulting in 93 transplants characterized into Maastricht categories II, III and IV NHBD renal transplants. Renal function after transplant was evaluated from the last dialysis until discharge from hospital, and then at 3 monthly intervals thereafter. RESULTS: Primary warm ischemic time is more prolonged in the uncontrolled donor (category II) than controlled donor (category III greater than IV). Delayed graft function occurs more frequently (Maastricht category II 83.8%, III 67.4% and IV 0%, ANOVA p <0.05) and the return to normal function is more prolonged in uncontrolled donors. This is illustrated by the greater incidence of acute tubular necrosis (Maastricht category II 81.1%, III 65.2% and IV 50.0%, ANOVA p = nonsignificant) in the kidney allograft. There was no difference in year 1 allograft survival (Maastricht category II 83.9%, III 92.5% and IV 100%, ANOVA p = nonsignificant). CONCLUSIONS: Early graft function is poorest in kidneys derived from Maastricht category II donors and best in category IV with III in-between. However, after 3 months the function of kidneys from all donors is the same.