HLA class I depletion by citric acid, and irradiation of apheresis platelets for transfusion of refractory patients.

BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.

A young woman with a transfusion-related pregnancy complication. / En ung kvinne med transfusjonsutløst svangerskapskomplikasjon.

BACKGROUND: Individuals with the K0 phenotype are extremely rare. They may develop anti-Ku antibodies, which react with all antigens of the Kell blood group system, thereby leading to haemolytic transfusion reactions and haemolytic disease of the fetus and newborn. CASE PRESENTATION: A primigravida who was transfused with one unit of red blood cells due to iron deficiency anaemia developed anti-Ku antibodies. The pregnancy was closely monitored by ultrasound and antibody titres. Maternal autologous blood collection was performed twice during the last trimester as back-up in case of maternal peripartum bleeding, and a few frozen K0 red blood cell units were provided in case of severe fetal anaemia. At gestational week 36+6, labour was induced due to increasing antibody titres and high blood velocities in the fetal middle cerebral artery during systole. The woman was delivered vaginally without need for transfusion. The infant was diagnosed with haemolytic disease of the fetus and newborn and treated with phototherapy, repeated infusions of intravenous immunoglobulin and iron supplements until normalisation of haemoglobin at three months of age. INTERPRETATION: Iron deficiency anaemia should be treated primarily with iron supplementation before considering blood transfusions, which pose the risk of developing alloantibodies that can cause transfusion complications and haemolytic disease of the fetus and newborn.

Effect of strenuous exercise on mediators of inflammation in patients with coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) is considered a low-grade inflammatory disease. We aimed to identify effects of short-term strenuous exercise on mediators of systemic inflammation, endothelial and platelet activation in patients with angiographically verified CAD. We hypothesized that a more pronounced inflammatory response would be present in patients with CAD than in those without CAD. METHODS: In subjects with symptoms indicative of stable CAD, an exercise stress test on a bicycle ergometer was performed. Venous blood samples, taken at rest and within 5 min after end of exercise, were analyzed for the following markers by ELISAs: TNF-α, IL-6, MCP-1, ICAM-1, VCAM-1, E-selectin, P-selectin, CD40L and RANTES. All participants underwent conventional coronary angiography. CAD was defined as having any degree of atherosclerosis. RESULTS: A total of 110 patients were included, of whom 74 were found to have CAD. Mean exercise duration was 10:06 ±â€¯3:56 min with no significant difference between the two groups. All measured markers changed significantly during exercise (p ≤ 0.012). A significantly less pronounced increase in CD40L in the CAD group than in the no CAD group was observed (p = 0.050), however, after adjustment for hematocrit this difference was no longer significant. CONCLUSION: An instant inflammatory response was observed during short-term strenuous exercise in patients with symptoms of CAD. However, the exercise mediated response was not more pronounced in patients with CAD.

A Double-Blinded Randomized Study Investigating a Possible Anti-Inflammatory Effect of Saxagliptin versus Placebo as Add-On Therapy in Patients with Both Type 2 Diabetes And Stable Coronary Artery Disease.

BACKGROUND: Promising results regarding potential anti-inflammatory and antiatherosclerotic effects of gliptins have been reported. Our aim was to investigate whether saxagliptin treatment modifies expression of inflammatory markers, primarily in peripheral blood mononuclear cells (PBMCs) and in circulating leukocytes in patients with stable coronary artery disease (CAD) and T2DM. METHODS: Patients (n = 12) were randomized to saxagliptin 5 mg daily or placebo for 3 months. Samples were taken at baseline and end of study in fasting state prior to intake of medications. PBMCs were isolated and cryopreserved at -150°C until ex vivo exposed to 1 ng/mL of lipopolysaccharide (LPS) for 4 hours. Gene expression was performed with custom-designed TaqMan® Arrays and relative quantification by real-time PCR (RT-qPCR). RESULTS: HbA1c was reduced in the saxagliptin-treated group compared to that in the change with placebo (p = 0.042). In unstimulated PBMCs and in circulating leukocytes, we observed a significant increase in IL-10 expression in the saxagliptin group (p = 0.043, both), significantly different from that in the placebo (p = 0.009 and p = 0.032, resp.). No between group differences in changes were observed in any of the selected proinflammatory markers. CONCLUSION: In our small cohort of patients with combined T2DM and CAD, a possible anti-inflammatory effect of saxagliptin, observed in the present study by upregulation of IL-10 in leukocytes, needs to be confirmed in larger studies.

Effects on serum fractalkine by diet and omega-3 fatty acid intervention: relation to clinical outcome.

Introduction. Fractalkine is a chemokine associated with atherosclerosis. Increased serum levels have been reported in unstable coronary artery disease (CAD) and to predict mortality in heart failure. Mediterranean-like diet and omega-3 fatty acids (n3-PUFA) have documented cardioprotective and anti-inflammatory effects. We have investigated the effect of Mediterranean-like dietary counseling and n-3 PUFA on serum fractalkine in an elderly population and its ability to predict cardiovascular disease (CVD). Materials and Methods. 563 men (age 64-75 yrs) at high risk of CAD were randomized into a 2 × 2 factorial designed trial for 3-year dietary counseling and/or n-3 PUFA supplementation (2.4 g/d). Circulating levels of fractalkine were measured at baseline and at end of study. Clinical events were recorded after 3 years. Results. Fractalkine levels were significantly reduced in all groups from baseline to 3 years (P < 0.001, all), but without between-group differences in changes. Fractalkine levels at baseline were not predictive for CVD events (n = 68) or total mortality. Lower fractalkine levels were observed in smokers (P = 0.019). Conclusions. Reduced levels of fractalkine from baseline to 3 years were observed, however, without any influence of Mediterranean-like diet or n-3 PUFA supplementation. Fractalkine levels at baseline were not predictive for later CVD events.

Fractalkine levels are elevated early after PCI-treated ST-elevation myocardial infarction; no influence of autologous bone marrow derived stem cell injection.

BACKGROUND: Fractalkine (CX3CL1) is a chemokine associated with atherosclerosis and inflammation. There is limited knowledge of fractalkine levels during acute myocardial infarction (AMI) and stem cell treatment. We aimed to investigate the time profile of circulating fractalkine and gene expression of its receptor CX3CR1 during AMI, and the influence of intracoronary autologous bone marrow stem cell (mBMC) transplantation (given 6 days after AMI) on fractalkine levels. METHODS: We examined fractalkine levels at different time points by enzyme-linked immunosorbent assay (ELISA) in 20 patients with AMI, and 10 patients with stable angina pectoris (AP) undergoing percutaneous coronary intervention (PCI), and in 100 patients included in the randomized Autologous Stem-Cell Transplantation in Acute Myocardial Infarction (ASTAMI) trial. RESULTS: Patients with AMI had significantly elevated levels 3- and 12 h after PCI compared to patients with stable AP. After 12 h levels were similar in the two groups. An inverse pattern was observed in gene expression levels. No correlation between fractalkine levels and myocardial injury or infarct size was seen. We could not demonstrate any influence of autologous mBMC transplantation on fractalkine levels. CONCLUSION: Fractalkine levels are elevated the first 12 h after PCI in patients with AMI, however, not correlated to infarct size. The inverse pattern in gene expression of fractalkine receptor (CX3CR1) might be a compensatory mechanism. No effect of autologous mBMC transplantation given 6 days after AMI on fractalkine levels was observed.

Effects of long-term exercise training on adipose tissue expression of fractalkine and MCP-1 in patients with type 2 diabetes and stable coronary artery disease: a substudy of a randomized controlled trial.

PURPOSE: Adipose tissue inflammation plays a role in atherosclerosis and type 2 diabetes (T2DM). We aimed to investigate whether 12 months of exercise training in patients with both T2DM and coronary artery disease (CAD) reduced the genetic expression of the proinflammatory markers fractalkine (CX3CL1) and its receptor (CX3CR1) and monocyte chemoattractant protein-1 (MCP-1) in the subcutaneous adipose tissue. Expression of the genes in the circulating leukocytes and circulating levels of the markers were also investigated. PATIENTS AND METHODS: A total of 137 patients with T2DM and CAD were included to study the effects of exercise on atherosclerosis progression and glucose control. Patients were randomized to exercise training (combined aerobic and strength training) or control. At inclusion and after 12 months, fasting blood samples and a subcutaneous adipose tissue sample were taken. RNA was extracted from the adipose tissue and circulating leukocytes, and the expression levels were examined by reverse transcription-polymerase chain reaction. Circulating fractalkine and MCP-1 were determined by enzyme-linked immunosorbent assay. RESULTS: The analyses were performed in 114 patients who completed the study and adhered to the intervention principle. At baseline, gene expression of fractalkine and CX3CR1 in the adipose tissue was similar in the two groups. There were no change within either group and no between-group differences in changes from baseline. Circulating fractalkine increased after 12 months in the exercise group (P=0.044), significantly more compared to controls (P=0.042), however only in the patients with advanced vascular disease. Neither the expression levels of MCP-1 nor the circulating levels changed significantly in either group. At baseline, CX3CR1 expression in the adipose tissue was associated with body mass index (P<0.001). CONCLUSION: No significant effects of long-term exercise training on adipose tissue expression of fractalkine, CX3CR1, or MCP-1 were found in our patients with combined CAD and T2DM. However, a slight increase in circulating fractalkine after the intervention was recorded. The association of CX3CR1 expression with body mass index might indicate increased immune activation in the adipose tissue.

Insulin levels and HOMA index are associated with exercise capacity in patients with type 2 diabetes and coronary artery disease.

BACKGROUND: Previous studies on type 2 diabetes have shown an association between exercise capacity and insulin resistance. In patients with coronary artery disease (CAD) exercise capacity is often reduced due to exercise-induced ischemia. We have investigated the association between glucometabolic control, including the homeostatic model assessment (HOMA) of insulin resistance, and exercise capacity in patients with type 2 diabetes and CAD with and without exercise-induced ischemia. METHODS: In 137 patients (age 63.1 ± 7.9) cardiopulmonary exercise testing on treadmill was performed using a modified Balke protocol. The highest oxygen uptake (VO2peak) was reported as 30-s average. Fasting blood samples were drawn for determination of glucose, insulin and HbA1c. Insulin resistance (IR) was assessed by the HOMA2-IR computer model. Exercise-induced ischemia was defined as angina and/ or ST-depression in ECG ≥ 0.1 mV during the exercise test. RESULTS: HOMA2-IR was inversely correlated to VO2peak (r = -0.328, p < 0.001), still significant after adjusting for age, gender, smoking and BMI. Patients with HOMA2-IR above the median value (1.3) had an adjusted odds ratio of 3.26 (95 % CI 1.35 to 7.83, p = 0.008) for having VO2peak below median (23.8 mL/kg/min). Insulin levels were inversely correlated to VO2peak (r = -0.245, p = 0.010), also after adjusting for age and gender, but not after additional adjustment for BMI. The correlation between HOMA2-IR and VO2peak was also significant in the subgroups with (n = 51) and without exercise-induced ischemia (n = 86), being numerically stronger in the group with ischemia (r = -0.430, p = 0.003 and r = -0.276, p = 0.014, respectively). Fasting glucose and HbA1c were not correlated with VO2peak or AT. CONCLUSIONS: Insulin resistance, as estimated by fasting insulin and the HOMA index, was inversely associated with exercise capacity in patients with type 2 diabetes and CAD, the association being more pronounced in the subgroup with exercise-induced ischemia. These results indicate that insulin resistance is related to exercise capacity in type 2 diabetic patients with CAD, possibly even more so in patients with exercise-induced ischemia compared to those without.

Fractalkine and its receptor (CX3CR1) in patients with stable coronary artery disease and diabetes mellitus.

BACKGROUND: Fractalkine and its receptor CX3CR1 are associated with atherosclerosis. In vitro studies have shown increased expression of fractalkine in endothelial and vascular smooth muscle cells when stimulated with a high concentration of glucose. Increased serum levels of fractalkine have been shown in patients with type 2 diabetes mellitus (T2DM) and also in unstable coronary artery disease (CAD) patients. We investigated whether CAD patients with T2DM or metabolic syndrome have increased circulating and gene expression levels of fractalkine compared to CAD patients without these conditions. METHODS: Serum levels of fractalkine were analyzed by the enzyme-linked immunosorbent assay (ELISA) method in 1001 patients with angiographically verified CAD, of which 200 had T2DM and 244 had metabolic syndrome. All patients were taking aspirin as an antithrombotic treatment. Gene expression of fractalkine and CX3CR1 in circulating leukocytes was explored in a subset of patients (n=168). RESULTS: We found no significant difference in circulating levels of fractalkine in patients with T2DM [653 (556, 775) pg/mL] compared to patients without T2DM [646 (553, 761) pg/mL], p=0.50. There was also no difference between patients with and without metabolic syndrome (p=0.60). Fractalkine was not expressed in circulating leukocytes, and CX3CR1 was not expressed differently between any of the groups (p=0.13 and p=0.32, respectively). Smokers had lower fractalkine levels (p<0.001), and patients on angiotensin II receptor blockers had higher levels (p=0.047) compared to nonaffected patients. CONCLUSIONS: In the present CAD population, no differences in circulating levels of fractalkine or expression levels of CX3CR1 were observed between patients with and without T2DM, or with and without metabolic syndrome, which may be related to their underlying disease.