Urinary biomolecular indicators of exercise-induced over exertion injury

Poor health and injury represent major obstacles to the future economic security of Australia. The national economic cost of work-related injury is estimated at $57.5 billion p/a. Since exposure to high physical demands is a major risk factor for musculoskeletal injury, monitoring and managing such physical activity levels in workers is a potentially important injury prevention strategy. Current injury monitoring practices are inadequate for the provision of clinically valuable information about the tissue specific responses to physical exertion. Injury of various soft tissue structures can manifest over time through accumulation of micro-trauma. Such micro-trauma has a propensity to increase the risk of acute injuries to soft-tissue structures such as muscle or tendon. As such, the capacity to monitor biomarkers that result from the disruption of these tissues offers a means of assisting the pre-emptive management of subclinical injury prior to acute failure or for evaluation of recovery processes. Here we have adopted an in-vivo exercise induced muscle damage model allowing the application of laboratory controlled conditions to assist in uncovering biochemical indicators associated with soft-tissue trauma and recovery. Importantly, urine was utilised as the diagnostic medium since it is non-invasive to collect, more acceptable to workers and less costly to employers. Moreover, it is our hypothesis that exercise induced tissue degradation products enter the circulation and are subsequently filtered by the kidney and pass through to the urine. To test this hypothesis a range of metabolomic and proteomic discovery-phase techniques were used, along with targeted approaches. Several small molecules relating to tissue damage were identified along with a series of skeletal muscle-specific protein fragments resulting from exercise induced soft-tissue damage. Each of the potential biomolecular markers appeared to be temporally present within urine. Moreover, the regulation of abundance seemed to be associated with functional recovery following the injury. This discovery may have important clinical applications for monitoring of a variety of inflammatory myopathies as well as novel applications in monitoring of the musculoskeletal health status of workers, professional athletes and/or military personnel to reduce the onset of potentially debilitating musculoskeletal injuries within these professions.

Bcl-X(L) translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

BACKGROUND: The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X(L), pro-apoptotic Bax and Bad). METHODS: Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (Western immunoblots, densitometry, immunoelectron microscopy). RESULTS: Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X(L) and Bax, but not Bcl-2 or Bad, was identified in control distal cells. Bcl-X(L) and Bax had nonsignificant increases (P> 0.05) in these cells. Bcl-2, Bax, and Bcl-X(L), but not Bad, were endogenously expressed in control proximal cells. Bcl-X(L) was significantly decreased in treated proximal cultures (P < 0.05), with Bax and Bcl-2 having nonsignificant increases (P> 0.05). Immunoelectron microscopy localization indicated that control and treated but surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X(L) from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-X(L) expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. CONCLUSION: The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X(L) in proximal cells, as well as translocation of Bcl-X(L) protein to mitochondria within the surviving distal cells.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

Acute injuries in track and field athletes: A 3-year observational study at the Penn Relays Carnival with epidemiology and medical coverage implications

Background Few studies have examined acute injuries in track and field in both elite and sub-elite athletes. Purpose To observe the absolute and relative rates of injury in track and field athletes across a wide range of competition levels and ages during three years of the Penn Relays Carnival to assist with future medical coverage planning and injury prevention strategies. Study design: Descriptive epidemiology study. Methods Over a 3-year period all injuries treated by the medical staff were recorded on a standardised injury report form. Absolute injury rates (absolute number of injuries) and relative injury rates (number of injuries per 1000 participants) were determined and odds ratios (OR) of injury rates were calculated between sexes, competition levels and events. Injuries were also broken down into major or minor medical or orthopedic injuries. Results Throughout the study period 48,473 competing athletes participated in the Penn Relays Carnival, and 436 injuries were sustained. For medical coverage purposes, the relative rate of injury subtypes was greatest for minor orthopedic injuries (5.71 injuries per 1000 participants), followed by minor medical injuries (3.42 injuries per 1000 participants), major medical injuries (0.69 injuries per 1000 participants) and major orthopedic injuries (0.18 injuries per 1000 participants). College/elite level athletes displayed the lowest relative injury rate (7.99 injuries per 1000 participants), which was significantly less than high school (9.87 injuries per 1000 participants) and masters level athletes (16.33 injuries per 1000 participants). Males displayed a greater likelihood of suffering a minor orthopedic injury compared to females (OR = 1.36, 95% CI = 1.06 to 1.75; χ2 = 5.73, p = 0.017) but were less likely to sustain a major medical injury (OR = 0.33, 95% CI = 0.15 to 0.75; χ2 = 7.75, p = 0.005). Of the three most heavily participated in events, the 4 x 400m relay displayed the greatest relative injury rate (13.6 injuries per 1000 participants) compared to the 4 x 100 and 4 x 200m relay. Conclusions Medical coverage teams for future large scale track and field events need to plan for at least two major orthopedic and seven major medical injuries per 1000 participants. Male track and field athletes, particularly masters level male athletes, are at greater risk of injury compared to other genders and competition levels.

Meeting the nutrition challenge of stage 3 renal failure : considerations for nursing practice

Self-care management is needed for effective management of chronic kidney disease. The main aim for treatment or management of chronic kidney disease is to delay the worsening of kidney function, and to prevent or to manage the co-morbidities. Selfcare management is not easy, and patients will face many challenges, especially when they cannot get use to the new treatment plan. One of the challenges they face is dietary restriction, which is a very important aspect in any self-care management programme. Chronic kidney disease patients require a low-protein, low-sodium, low-potassium, and low-phosphorus diet. There are several strategies patients can undertake to ensure adherence, such as self-monitoring their dietary habits and type of food consumed using a food diary; involving social support, such as family members and spouse to help them to adhere to their diet restrictions; setting goals and providing positive reinforcement when they achieved the targeted goals; joining self-management programmes to equip themselves with the necessary skills so that they can better adhere to the treatment regimes, including diet restriction; and lastly, having the knowledge about their regime, and using this knowledge to help them understand and improve their adherence.

Patients Undergoing Subacute Physical Rehabilitation following an Acute Hospital Admission Demonstrated Improvement in Cognitive Functional Task Independence

Objective. This study investigated cognitive functioning among older adults with physical debility not attributable to an acute injury or neurological condition who were receiving subacute inpatient physical rehabilitation. Design. A cohort investigation with assessments at admission and discharge. Setting. Three geriatric rehabilitation hospital wards. Participants. Consecutive rehabilitation admissions () following acute hospitalization (study criteria excluded orthopaedic, neurological, or amputation admissions). Intervention. Usual rehabilitation care. Measurements. The Functional Independence Measure (FIM) Cognitive and Motor items. Results. A total of 704 (86.5%) participants (mean age = 76.5 years) completed both assessments. Significant improvement in FIM Cognitive items (-score range 3.93–8.74, all ) and FIM Cognitive total score (-score = 9.12, ) occurred, in addition to improvement in FIM Motor performance. A moderate positive correlation existed between change in Motor and Cognitive scores (Spearman’s rho = 0.41). Generalized linear modelling indicated that better cognition at admission (coefficient = 0.398, ) and younger age (coefficient = −0.280, ) were predictive of improvement in Motor performance. Younger age (coefficient = −0.049, ) was predictive of improvement in FIM Cognitive score. Conclusions. Improvement in cognitive functioning was observed in addition to motor function improvement among this population. Causal links cannot be drawn without further research.

Common values in assessing health outcomes from disease and injury: Disability weights measurement study for the Global Burden of Disease Study 2010

BACKGROUND Measurement of the global burden of disease with disability-adjusted life-years (DALYs) requires disability weights that quantify health losses for all non-fatal consequences of disease and injury. There has been extensive debate about a range of conceptual and methodological issues concerning the definition and measurement of these weights. Our primary objective was a comprehensive re-estimation of disability weights for the Global Burden of Disease Study 2010 through a large-scale empirical investigation in which judgments about health losses associated with many causes of disease and injury were elicited from the general public in diverse communities through a new, standardised approach. METHODS We surveyed respondents in two ways: household surveys of adults aged 18 years or older (face-to-face interviews in Bangladesh, Indonesia, Peru, and Tanzania; telephone interviews in the USA) between Oct 28, 2009, and June 23, 2010; and an open-access web-based survey between July 26, 2010, and May 16, 2011. The surveys used paired comparison questions, in which respondents considered two hypothetical individuals with different, randomly selected health states and indicated which person they regarded as healthier. The web survey added questions about population health equivalence, which compared the overall health benefits of different life-saving or disease-prevention programmes. We analysed paired comparison responses with probit regression analysis on all 220 unique states in the study. We used results from the population health equivalence responses to anchor the results from the paired comparisons on the disability weight scale from 0 (implying no loss of health) to 1 (implying a health loss equivalent to death). Additionally, we compared new disability weights with those used in WHO's most recent update of the Global Burden of Disease Study for 2004. FINDINGS 13,902 individuals participated in household surveys and 16,328 in the web survey. Analysis of paired comparison responses indicated a high degree of consistency across surveys: correlations between individual survey results and results from analysis of the pooled dataset were 0·9 or higher in all surveys except in Bangladesh (r=0·75). Most of the 220 disability weights were located on the mild end of the severity scale, with 58 (26%) having weights below 0·05. Five (11%) states had weights below 0·01, such as mild anaemia, mild hearing or vision loss, and secondary infertility. The health states with the highest disability weights were acute schizophrenia (0·76) and severe multiple sclerosis (0·71). We identified a broad pattern of agreement between the old and new weights (r=0·70), particularly in the moderate-to-severe range. However, in the mild range below 0·2, many states had significantly lower weights in our study than previously. INTERPRETATION This study represents the most extensive empirical effort as yet to measure disability weights. By contrast with the popular hypothesis that disability assessments vary widely across samples with different cultural environments, we have reported strong evidence of highly consistent results.

Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013

Background Up-to-date evidence about levels and trends in disease and injury incidence, prevalence, and years lived with disability (YLDs) is an essential input into global, regional, and national health policies. In the Global Burden of Disease Study 2013 (GBD 2013), we estimated these quantities for acute and chronic diseases and injuries for 188 countries between 1990 and 2013. Methods Estimates were calculated for disease and injury incidence, prevalence, and YLDs using GBD 2010 methods with some important refi nements. Results for incidence of acute disorders and prevalence of chronic disorders are new additions to the analysis. Key improvements include expansion to the cause and sequelae list, updated systematic reviews, use of detailed injury codes, improvements to the Bayesian meta-regression method (DisMod-MR), and use of severity splits for various causes. An index of data representativeness, showing data availability, was calculated for each cause and impairment during three periods globally and at the country level for 2013. In total, 35 620 distinct sources of data were used and documented to calculated estimates for 301 diseases and injuries and 2337 sequelae. The comorbidity simulation provides estimates for the number of sequelae, concurrently, by individuals by country, year, age, and sex. Disability weights were updated with the addition of new population-based survey data from four countries. Findings Disease and injury were highly prevalent; only a small fraction of individuals had no sequelae. Comorbidity rose substantially with age and in absolute terms from 1990 to 2013. Incidence of acute sequelae were predominantly infectious diseases and short-term injuries, with over 2 billion cases of upper respiratory infections and diarrhoeal disease episodes in 2013, with the notable exception of tooth pain due to permanent caries with more than 200 million incident cases in 2013. Conversely, leading chronic sequelae were largely attributable to non-communicable diseases, with prevalence estimates for asymptomatic permanent caries and tension-type headache of 2∙4 billion and 1∙6 billion, respectively. The distribution of the number of sequelae in populations varied widely across regions, with an expected relation between age and disease prevalence. YLDs for both sexes increased from 537∙6 million in 1990 to 764∙8 million in 2013 due to population growth and ageing, whereas the age-standardised rate decreased little from 114∙87 per 1000 people to 110∙31 per 1000 people between 1990 and 2013. Leading causes of YLDs included low back pain and major depressive disorder among the top ten causes of YLDs in every country. YLD rates per person, by major cause groups, indicated the main drivers of increases were due to musculoskeletal, mental, and substance use disorders, neurological disorders, and chronic respiratory diseases; however HIV/AIDS was a notable driver of increasing YLDs in sub-Saharan Africa. Also, the proportion of disability-adjusted life years due to YLDs increased globally from 21·1% in 1990 to 31·2% in 2013. Interpretation Ageing of the world’s population is leading to a substantial increase in the numbers of individuals with sequelae of diseases and injuries. Rates of YLDs are declining much more slowly than mortality rates. The non-fatal dimensions of disease and injury will require more and more attention from health systems. The transition to nonfatal outcomes as the dominant source of burden of disease is occurring rapidly outside of sub-Saharan Africa. Our results can guide future health initiatives through examination of epidemiological trends and a better understanding of variation across countries.

The role of catechol-O-methyltransferase (COMT) and the effects of COMT inhibition in brain and in cardiovascular and renal pathophysiology

Catechol-O-methyltransferase (COMT) metabolizes catecholamines such as dopamine (DA), noradrenaline (NA) and adrenaline, which are vital neurotransmitters and hormones that play important roles in the regulation of physiological processes. COMT enzyme has a functional Val158Met polymorphism in humans, which affects the subjects COMT activity. Increasing evidence suggests that this functional polymorphism may play a role in the etiology of various diseases from schizophrenia to cancers. The aim of this project was to provide novel biochemical information on the physiological and especially pathophysiological roles of COMT enzyme as well as the effects of COMT inhibition in the brain and in the cardiovascular and renal system. To assess the roles of COMT and COMT inhibition in pathophysiology, we used four different study designs. The possible beneficial effects of COMT inhibition were studied in double-transgenic rats (dTGRs) harbouring human angiotensinogen and renin genes. Due to angiotensin II (Ang II) overexpression, these animals exhibit severe hypetension, cardiovascular and renal end-organ damage and mortality of approximately 25-40% at the age of 7-weeks. The dTGRs and their Sprague-Dawley controls tissue samples were assessed with light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography (HPLC) to evaluate the tissue damages and the possible protective effects pharmacological intervention with COMT inhibitors. In a second study, the consequence of genetic and pharmacological COMT blockade in blood pressure regulation during normal and high-sodium was elucidated using COMT-deficient mice. The blood pressure and the heart rate were measured using direct radiotelemetric blood pressure surveillance. In a third study, the effects of acute and subchronic COMT inhibition during combined levodopa (L-DOPA) + dopa decarboxylase inhibitor treatment in homocysteine formation was evaluated. Finally, we assessed the COMT enzyme expression, activity and cellular localization in the CNS during inflammation-induced neurodegeneration using Western blotting, HPLC and various enzymatic assays. The effects of pharmacological COMT inhibition on neurodegeneration were also studied. The COMT inhibitor entacapone protected against the Ang II-induced perivascular inflammation, renal damage and cardiovascular mortality in dTGRs. COMT inhibitors reduced the albuminuria by 85% and prevented the cardiovascular mortality completely. Entacapone treatment was shown to ameliorate oxidative stress and inflammation. Furthermore, we established that the genetic and pharmacological COMT enzyme blockade protects against the blood pressure-elevating effects of high sodium intake in mice. These effects were mediated via enhanced renal dopaminergic tone and suggest an important role of COMT enzyme, especially in salt-sensitive hypertension. Entacapone also ameliorated the L-DOPA-induced hyperhomocysteinemia in rats. This is important, since decreased homocysteine levels may decrease the risk of cardiovascular diseases in Parkinson´s disease (PD) patients using L-DOPA. The Lipopolysaccharide (LPS)-induced inflammation and subsequent delayed dopaminergic neurodegeneration were accompanied by up-regulation of COMT expression and activity in microglial cells as well as in perivascular cells. Interestingly, similar perivascular up-regulation of COMT expression in inflamed renal tissue was previously noted in dTGRs. These results suggest that inflammation reactions may up-regulate COMT expression. Furthermore, this increased glial and perivascular COMT activity in the central nervous system (CNS) may decrease the bioavailability of L-DOPA and be related to the motor fluctuation noted during L-DOPA therapy in PD patients.

The role of mast cells in ischemic and hemorrhagic brain injury

Stroke, ischemic or hemorrhagic, belongs among the foremost causes of death and disability worldwide. Massive brain swelling is the leading cause of death in large hemispheric strokes and is only modestly alleviated by available treatment. Thrombolysis with tissue plasminogen activator (TPA) is the only approved therapy in acute ischemic stroke, but fear of TPA-mediated hemorrhage is often a reason for withholding this otherwise beneficial treatment. In addition, recanalization of the occluded artery (spontaneously or with thrombolysis) may cause reperfusion injury by promoting brain edema, hemorrhage, and inflammatory cell infiltration. A dominant event underlying these phenomena seems to be disruption of the blood-brain barrier (BBB). In contrast to ischemic stroke, no widely approved clinical therapy exists for intracerebral hemorrhage (ICH), which is associated with poor outcome mainly due to the mass effect of enlarging hematoma and associated brain swelling. Mast cells (MCs) are perivascularly located resident inflammatory cells which contain potent vasoactive, proteolytic, and fibrinolytic substances in their cytoplasmic granules. Experiments from our laboratory showed MC density and their state of granulation to be altered early following focal transient cerebral ischemia, and degranulating MCs were associated with perivascular edema and hemorrhage. (I) Pharmacological MC stabilization led to significantly reduced ischemic brain swelling (40%) and BBB leakage (50%), whereas pharmacological MC degranulation raised these by 90% and 50%, respectively. Pharmacological MC stabilization also revealed a 40% reduction in neutrophil infiltration. Moreover, genetic MC deficiency was associated with an almost 60% reduction in brain swelling, 50% reduction in BBB leakage, and 50% less neutrophil infiltration, compared with controls. (II) TPA induced MC degranulation in vitro. In vivo experiments with post-ischemic TPA administration demonstrated 70- to 100-fold increases in hemorrhage formation (HF) compared with controls HF. HF was significantly reduced by pharmacological MC stabilization at 3 (95%), 6 (75%), and 24 hours (95%) of follow-up. Genetic MC deficiency again supported the role of MCs, leading to 90% reduction in HF at 6 and 24 hours. Pharmacological MC stabilization and genetic MC deficiency were also associated with significant reduction in brain swelling and in neutrophil infiltration. Importantly, these effects translated into a significantly better neurological outcome and lower mortality after 24 hours. (III) Finally, in ICH experiments, pharmacological MC stabilization resulted in significantly less brain swelling, diminished growth in hematoma volume, better neurological scores, and decreased mortality. Pharmacological MC degranulation produced the opposite effects. Genetic MC deficiency revealed a beneficial effect similar to that found with pharmacological MC stabilization. In sum, the role of MCs in these clinically relevant scenarios is supported by a series of experiments performed both in vitro and in vivo. That not only genetic MC deficiency but also drugs targeting MCs could modulate these parameters (translated into better outcome and decreased mortality), suggests a potential therapeutic approach in a number of highly prevalent cerebral insults in which extensive tissue injury is followed by dangerous brain swelling and inflammatory cell infiltration. Furthermore, these experiments could hint at a novel therapy to improve the safety of thrombolytics, and a potential cellular target for those seeking novel forms of treatment for ICH.

Individualized immunosuppression after renal transplantation in childhood

Pediatric renal transplantation (TX) has evolved greatly during the past few decades, and today TX is considered the standard care for children with end-stage renal disease. In Finland, 191 children had received renal transplants by October 2007, and 42% of them have already reached adulthood. Improvements in treatment of end-stage renal disease, surgical techniques, intensive care medicine, and in immunosuppressive therapy have paved the way to the current highly successful outcomes of pediatric transplantation. In children, the transplanted graft should last for decades, and normal growth and development should be guaranteed. These objectives set considerable requirements in optimizing and fine-tuning the post-operative therapy. Careful optimization of immunosuppressive therapy is crucial in protecting the graft against rejection, but also in protecting the patient against adverse effects of the medication. In the present study, the results of a retrospective investigation into individualized dosing of immunosuppresive medication, based on pharmacokinetic profiles, therapeutic drug monitoring, graft function and histology studies, and glucocorticoid biological activity determinations, are reported. Subgroups of a total of 178 patients, who received renal transplants in 1988 2006 were included in the study. The mean age at TX was 6.5 years, and approximately 26% of the patients were <2 years of age. The most common diagnosis leading to renal TX was congenital nephrosis of the Finnish type (NPHS1). Pediatric patients in Finland receive standard triple immunosuppression consisting of cyclosporine A (CsA), methylprednisolone (MP) and azathioprine (AZA) after renal TX. Optimal dosing of these agents is important to prevent rejections and preserve graft function in one hand, and to avoid the potentially serious adverse effects on the other hand. CsA has a narrow therapeutic window and individually variable pharmacokinetics. Therapeutic monitoring of CsA is, therefore, mandatory. Traditionally, CsA monitoring has been based on pre-dose trough levels (C0), but recent pharmacokinetic and clinical studies have revealed that the immunosuppressive effect may be related to diurnal CsA exposure and blood CsA concentration 0-4 hours after dosing. The two-hour post-dose concentration (C2) has proved a reliable surrogate marker of CsA exposure. Individual starting doses of CsA were analyzed in 65 patients. A recommended dose based on a pre-TX pharmacokinetic study was calculated for each patient by the pre-TX protocol. The predicted dose was clearly higher in the youngest children than in the older ones (22.9±10.4 and 10.5±5.1 mg/kg/d in patients <2 and >8 years of age, respectively). The actually administered oral doses of CsA were collected for three weeks after TX and compared to the pharmacokinetically predicted dose. After the TX, dosing of CsA was adjusted according to clinical parameters and blood CsA trough concentration. The pharmacokinetically predicted dose and patient age were the two significant parameters explaining post-TX doses of CsA. Accordingly, young children received significantly higher oral doses of CsA than the older ones. The correlation to the actually administered doses after TX was best in those patients, who had a predicted dose clearly higher or lower (> ±25%) than the average in their age-group. Due to the great individual variation in pharmacokinetics standardized dosing of CsA (based on body mass or surface area) may not be adequate. Pre-Tx profiles are helpful in determining suitable initial CsA doses. CsA monitoring based on trough and C2 concentrations was analyzed in 47 patients, who received renal transplants in 2001 2006. C0, C2 and experienced acute rejections were collected during the post-TX hospitalization, and also three months after TX when the first protocol core biopsy was obtained. The patients who remained rejection free had slightly higher C2 concentrations, especially very early after TX. However, after the first two weeks also the trough level was higher in the rejection-free patients than in those with acute rejections. Three months after TX the trough level was higher in patients with normal histology than in those with rejection changes in the routine biopsy. Monitoring of both the trough level and C2 may thus be warranted to guarantee sufficient peak concentration and baseline immunosuppression on one hand and to avoid over-exposure on the other hand. Controlling of rejection in the early months after transplantation is crucial as it may contribute to the development of long-term allograft nephropathy. Recently, it has become evident that immunoactivation fulfilling the histological criteria of acute rejection is possible in a well functioning graft with no clinical sings or laboratory perturbations. The influence of treatment of subclinical rejection, diagnosed in 3-month protocol biopsy, to graft function and histology 18 months after TX was analyzed in 22 patients and compared to 35 historical control patients. The incidence of subclinical rejection at three months was 43%, and the patients received a standard rejection treatment (a course of increased MP) and/or increased baseline immunosuppression, depending on the severity of rejection and graft function. Glomerular filtration rate (GFR) at 18 months was significantly better in the patients who were screened and treated for subclinical rejection in comparison to the historical patients (86.7±22.5 vs. 67.9±31.9 ml/min/1.73m2, respectively). The improvement was most remarkable in the youngest (<2 years) age group (94.1±11.0 vs. 67.9±26.8 ml/min/1.73m2). Histological findings of chronic allograft nephropathy were also more common in the historical patients in the 18-month protocol biopsy. All pediatric renal TX patients receive MP as a part of the baseline immunosuppression. Although the maintenance dose of MP is very low in the majority of the patients, the well-known steroid-related adverse affects are not uncommon. It has been shown in a previous study in Finnish pediatric TX patients that steroid exposure, measured as area under concentration-time curve (AUC), rather than the dose correlates with the adverse effects. In the present study, MP AUC was measured in sixteen stable maintenance patients, and a correlation with excess weight gain during 12 months after TX as well as with height deficit was found. A novel bioassay measuring the activation of glucocorticoid receptor dependent transcription cascade was also employed to assess the biological effect of MP. Glucocorticoid bioactivity was found to be related to the adverse effects, although the relationship was not as apparent as that with serum MP concentration. The findings in this study support individualized monitoring and adjustment of immunosuppression based on pharmacokinetics, graft function and histology. Pharmacokinetic profiles are helpful in estimating drug exposure and thus identifying the patients who might be at risk for excessive or insufficient immunosuppression. Individualized doses and monitoring of blood concentrations should definitely be employed with CsA, but possibly also with steroids. As an alternative to complete steroid withdrawal, individualized dosing based on drug exposure monitoring might help in avoiding the adverse effects. Early screening and treatment of subclinical immunoactivation is beneficial as it improves the prospects of good long-term graft function.

Electrocardiographic repolarization variables in detecting myocardial infarction and ischemic injury : From body surface potential mapping to a single lead

The aim of the studies was to improve the diagnostic capability of electrocardiography (ECG) in detecting myocardial ischemic injury with a future goal of an automatic screening and monitoring method for ischemic heart disease. The method of choice was body surface potential mapping (BSPM), containing numerous leads, with intention to find the optimal recording sites and optimal ECG variables for ischemia and myocardial infarction (MI) diagnostics. The studies included 144 patients with prior MI, 79 patients with evolving ischemia, 42 patients with left ventricular hypertrophy (LVH), and 84 healthy controls. Study I examined the depolarization wave in prior MI with respect to MI location. Studies II-V examined the depolarization and repolarization waves in prior MI detection with respect to the Minnesota code, Q-wave status, and study V also with respect to MI location. In study VI the depolarization and repolarization variables were examined in 79 patients in the face of evolving myocardial ischemia and ischemic injury. When analyzed from a single lead at any recording site the results revealed superiority of the repolarization variables over the depolarization variables and over the conventional 12-lead ECG methods, both in the detection of prior MI and evolving ischemic injury. The QT integral, covering both depolarization and repolarization, appeared indifferent to the Q-wave status, the time elapsed from MI, or the MI or ischemia location. In the face of evolving ischemic injury the performance of the QT integral was not hampered even by underlying LVH. The examined depolarization and repolarization variables were effective when recorded in a single site, in contrast to the conventional 12-lead ECG criteria. The inverse spatial correlation of the depolarization and depolarization waves in myocardial ischemia and injury could be reduced into the QT integral variable recorded in a single site on the left flank. In conclusion, the QT integral variable, detectable in a single lead, with optimal recording site on the left flank, was able to detect prior MI and evolving ischemic injury more effectively than the conventional ECG markers. The QT integral, in a single-lead or a small number of leads, offers potential for automated screening of ischemic heart disease, acute ischemia monitoring and therapeutic decision-guiding as well as risk stratification.

Ischemia-reperfusion injury in human liver transplantation : Mechanisms and effects on graft function

Liver transplantation is an established therapy for both acute and chronic liver failure. Despite excellent long-term outcome, graft dysfunction remains a problem affecting up to 15-30% of the recipients. The etiology of dysfunction is multifactorial, with ischemia-reperfusion injury regarded as one of the most important contributors. This thesis focuses on the inflammatory response during graft procurement and reperfusion in liver transplantation in adults. Activation of protein C was examined as a potential endogenous anti-inflammatory mechanism. The effects of inflammatory responses on graft function and outcome were investigated. Seventy adult patients undergoing liver transplantation in Helsinki University Central Hospital, and 50 multiorgan donors, were studied. Blood samples from the portal and the hepatic veins were drawn before graft procurement and at several time points during graft reperfusion to assess changes within the liver. Liver biopsies were taken before graft preservation and after reperfusion. Neutrophil and monocyte CD11b and L-selectin expression were analysed by flow cytometry. Plasma TNF-α, IL-6, IL-8, sICAM-1, and HMGB1 were determined by ELISA and Western-blotting. HMGB1 immunohistochemistry was performed on liver tissue specimens. Plasma protein C and activated protein C were determined by an enzyme-capture assay. Hepatic IL-8 release during graft procurement was associated with subsequent graft dysfunction, biliary in particular, in the recipient. Biliary marker levels increased only 5 7 days after transplantation. Thus, donor inflammatory response appears to influence recipient liver function with relatively long-lasting effects. Hepatic phagocyte activation and sequestration, with concomitant HMGB1 release, occurred during reperfusion. Neither phagocyte activation nor plasma cytokines correlated with postoperative graft function. Thus, activation of the inflammatory responses within the liver during reperfusion may be of minor clinical significance. However, HMGB1 was released from hepatocytes and were also correlated with postoperative transaminase levels. Accordingly, HMGB1 appears to be a marker of hepatocellular injury.

Monitorização dos anticorpos anti-hla após transplante renal e sua correlação com episódios de rejeição aguda

Introdução: A associação entre a presença de anticorpo anti-HLA doador específico (DSA), em pacientes com prova cruzada negativa por citotoxicidade dependente de complemento (CDC), e a ocorrência de episódios de rejeição mediada por anticorpos (RMA) e menor sobrevida do enxerto já foi demonstrada por diversos autores. Entretanto,estimar a relevância clínica da presença desses anticorpos, em um determinado receptor, é um grande desafio e portanto novas estratégias de monitorização imunológicas são necessárias. Objetivo: O objetivo desse estudo foi monitorar a presença de DSA, bem como a variação dos seus títulos durante o primeiro ano após o transplante renal e correlacionar com episódios de rejeição aguda e função do enxerto ao final desse período. Metodologia: Foram analisados 389 soros de 71 pacientes incluídos no estudo. A pesquisa de DSA foi realizada utilizando os testes LABScreen single antigenbeads nas amostras correspondentes aos tempos: pré-transplante, 14, 30, 90, 180 e 365 dias após o transplante. Episódios de rejeição aguda comprovados por biópsia foram analisados de acordo com a classificação de Banff 2007. A taxa de filtração glomerular (TFG) ao final do primeiro ano foi estimada utilizando a fórmula Modificationof Diet in Renal Disease (MDRD). Os pacientes foram inicialmente separados em 3 grupos de diferentes riscos imunológicos (pré-transplante): A) DSA-, B) DSA+ com MFI >1000 e < 5000 e C) DSA+ com MFI > 5000. Num segundo momento, foram novamente agrupados de acordo com o perfil de mudança nos valores de MFI (intensidade de fluorescência média) ao longo do primeiro ano. Resultados: DSA estavam presentes pré-transplante em 15 pacientes. RMA foi mais frequente no grupo C (p = 0,02). De acordo com a variação dos títulos de DSA pós-transplante os pacientes foram novamente agrupados: grupo I) permaneceu DSA- durante todo acompanhamento = 50 pacientes, II) diminuiu ou manteve títulos de DSA em relação ao tempo zero = 13 pacientes e III) aumentou títulos em relação ao tempo zero = 8 pacientes (6 foram DSA de novo). Três pacientes dos grupos I e um paciente do grupo II apresentaram episódios de rejeição aguda celular. Não foi observada oscilação significativa nos títulos de anticorpos durante esses eventos. Nenhum paciente desse grupo apresentou episódio de RMA. Episódio de RMA ocorreu em dois pacientes do grupo III. Em ambos os pacientes foi detectado aumento significativo nos valores de MFI dos DSA em relação ao tempo zero. Não foi observada diferença significativa na TFG entre os grupos analisados nesse estudo. Entretanto, observou-se uma diferença estatisticamente significativa na TFG entre os pacientes que apresentaram episódio de rejeição aguda em relação aos que não tiveram, sendo menor nos primeiros (p = 0,04). Conclusão: A monitorização prospectiva dos anticorpos pode ajudar a identificar pacientes em maior risco para ocorrência de RMA e o aumento nos valores de MFI DSA deve ser interpretado como um sinal de alerta, sobretudo em pacientes previamente sensibilizados.