FcαRI Dynamics Are Regulated by GSK-3 and PKCζ During Cytokine Mediated Inside-Out Signaling.

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics.

In Vitro Effects of Pantoprazole on Platelet Aggregation in Blood Samples From Clopidogrel and Aspirin-treated Patients.

Platelet inhibition during treatment with the antiplatelet drug clopidogrel is prone to great interindividual variability and is believed to be affected by several factors such as genetics and drug-drug interactions. Proton pump inhibitors have been shown to interfere with the liver metabolism of clopidogrel. However, there are limited data on any direct effects proton pump inhibitors may have on clopidogrel. The aim of the study was to evaluate whether the in vitro addition of pantoprazole affects platelet aggregation in blood samples from clopidogrel and aspirin-treated patients. Blood samples were drawn from 66 patients on clopidogrel and aspirin who underwent coronary angiography. Platelet aggregation was analyzed using the bed-side Plateletworks assay before and after the addition of 2 different amounts of pantoprazole. The addition of 2.5 µL (4 mg/mL) pantoprazole, final concentration 0.01 mg/mL, was followed by a significant reduction (26%, P ≤ 0.001) of platelet aggregation, which was further reduced (39%, P ≤ 0.001) when a higher dose, 10 µL (4 mg/mL), final concentration 0.04 mg/mL, was added. In conclusion, platelet aggregation was significantly decreased by in vitro addition of pantoprazole. To explore the clinical relevance of this, future studies are needed.

Physiological concentrations of leptin do not affect human neutrophils.

Leptin is an adipokine that is thought to be important in many inflammatory diseases, and is known to influence the function of several leukocyte types. However, no clear consensus is present regarding the responsiveness of neutrophils for this adipokine. In this study a 2D DIGE proteomics approach was used as an unbiased approach to identify leptin-induced effects on neutrophils. Additionally chemotaxis and survival experiments were performed to reproduce results from literature showing putative effects of leptin on these neutrophil responses. Leptin did not induce any significant changes in the proteome provided leptin was added at physiologically relevant concentrations (250 ng). Our leptin batches were biologically active as they induced proliferation in LeptinR expressing Ba/F3 cells. At high concentrations (25000 ng) leptin induced a change in neutrophil proteome. Seventeen differently regulated spots were identified of which twelve could be characterized by mass spectrometry. Two of these identified proteins, SerpinB1 and p40 phox, were chosen for further analysis but leptin-induced expression analyzed by western blot were highly variable. Additionally leptin also induced neutrophil survival at these high concentrations. No leptin-induced chemotaxis of human neutrophils was detected at any concentration. In conclusion, physiological concentrations of leptin do not affect neutrophils. High leptin concentrations induced survival and changes in the neutrophils proteome, but this was most likely mediated by an indirect effect. However, it cannot be ruled out that the effects were mediated by a yet not-identified leptin receptor on human neutrophils.

Intraoperative local insufflation of warmed humidified CO2 increases open wound and core temperatures: a randomized clinical trial.

BACKGROUND: The open surgical wound is exposed to cold dry ambient air, resulting in substantial heat loss through radiation, evaporation, and convection. At the same time, anesthesia decreases the patient's core temperature. Despite preventive measures, mild intraoperative hypothermia has been associated with postoperative morbidity. We hypothesized that local insufflation of warmed humidified carbon dioxide (CO(2)) would maintain wound and core temperature. METHODS: Eighty patients undergoing open colon surgery were randomized to standard warming measures, or to additional local wound insufflation of warmed (30 °C) humidified (93 % rH) CO(2) via a gas diffuser. Surface temperature of the open abdominal wound was measured with a heat-sensitive infrared camera, and core temperature was measured with an ear thermometer. RESULTS: Mean operative time was 219 ± 104 and 205 ± 85 min in the CO(2) group and the control group, respectively (p = 0.550). Clinical variables did not differ significantly between the groups. The median wound area and wound edge temperatures were 1.2 °C (p < 0.001) and 1.0 °C (p = 0.002) higher in the CO(2) group, respectively, than in the control group. The mean core temperature after intubation was the same (35.9 °C) in both groups, but at end of surgery core temperature in the two groups differed, with a mean of 36.2 ± 0.5 °C in the CO(2) group and a mean of 35.8 ± 0.5 °C in the control group (p = 0.003). CONCLUSIONS: Insufflation of warmed, humidified CO(2) in an open surgical wound cavity prevents intraoperative decrease in surgical wound temperature as well as core temperature.

Local insufflation of warm humidified CO2increases open wound and core temperature during open colon surgery: a randomized clinical trial.

BACKGROUND: The open surgical wound is exposed to cold and dry ambient air resulting in heat loss through radiation, evaporation, and convection. Also, general and neuraxial anesthesia decrease the patient's core temperature. Despite routine preventive measures mild intraoperative hypothermia is still common and contributes to postoperative morbidity and mortality. We hypothesized that local insufflation of warm fully humidified CO(2) would increase both the open surgical wound and core temperature. METHODS: Eighty-three patients undergoing open colon surgery were equally and parallelly randomized to either standard warming measures including forced-air warming, warm fluids, and insulation of limbs and head, or to additional local wound insufflation of warm (37°C) humidified (100% relative humidity) CO(2) at a laminar flow (10 L/min) via a gas diffuser. Wound surface and core temperatures were followed with a heat-sensitive infrared camera and a tympanic thermometer. RESULTS: The mean wound area temperature during surgery was 31.3°C in the warm humidified CO(2) group compared with 29.6°C in the control group (P < 0.001, 95% confidence interval [CI], 1.2°C to 2.3°C). Also, the mean wound edge temperature during surgery was 30.1°C compared with 28.5°C in the control group (P < 0.001, 95% CI, 0.2°C to 0.7°C). Mean core temperature before start of surgery was similar with 36.7°C ± 0.5°C in the warm humidified CO(2) group versus 36.6°C ± 0.5°C in the control group (95% CI, 0.4 to -0.1°C). At end of surgery, the 2 groups differed significantly with 36.9 ± 0.5°C in the warm humidified CO(2) group versus 36.3 ± 0.5°C in the control group (P < 0.001, 95% CI, 0.38°C to 0.82°C). Moreover, only 8 patients of 40 in the warm humidified CO(2) group had a core temperature <36.5°C (20%, 95% CI, 7 to 33%), whereas in the control group this was the case in 24 of 39 (62%, 95% CI, 46% to 78%, P = 0.001) patients (difference of the percentages between the groups 42%, 95% CI, 22% to 61%, P < 0.001). With a cutoff at <36.0°C none of the patients in the warm humidified CO(2) group compared with 7 patients (18%, 95% CI, 5% to 31%, P = 0.005) in the control group was hypothermic at end of surgery (difference of the percentages between the groups 18%, 95% CI, 6% to 30%, P = 0.005). The median (25th/75th percentile) operating time was 181.5 (147.5/288) minutes in the warm humidified CO(2) group versus 217 (149/288) minutes in the control group (P = 0.312). Clinical variables did not show any significant differences between the groups. CONCLUSIONS: Insufflation of warm fully humidified CO(2) in an open surgical wound cavity increases surgical wound and core temperatures and helps to maintain normothermia.

The immune cell composition in Barrett's metaplastic tissue resembles that in normal duodenal tissue.

BACKGROUND AND OBJECTIVE: Barrett's esophagus (BE) is characterized by the transition of squamous epithelium into columnar epithelium with intestinal metaplasia. The increased number and types of immune cells in BE have been indicated to be due to a Th2-type inflammatory process. We tested the alternative hypothesis that the abundance of T-cells in BE is caused by a homing mechanism that is found in the duodenum. PATIENTS AND METHODS: Biopsies from BE and duodenal tissue from 30 BE patients and duodenal tissue from 18 controls were characterized by immmunohistochemistry for the presence of T-cells and eosinophils(eos). Ex vivo expanded T-cells were further phenotyped by multicolor analysis using flowcytometry. RESULTS: The high percentage of CD4(+)-T cells (69±3% (mean±SEM/n = 17, by flowcytometry)), measured by flowcytometry and immunohistochemistry, and the presence of non-activated eosinophils found in BE by immunohistochemical staining, were not different from that found in duodenal tissue. Expanded lymphocytes from these tissues had a similar phenotype, characterized by a comparable but low percentage of αE(CD103) positive CD4(+)cells (44±5% in BE, 43±4% in duodenum of BE and 34±7% in duodenum of controls) and a similar percentage of granzyme-B(+)CD8(+) cells(44±5% in BE, 33±6% in duodenum of BE and 36±7% in duodenum of controls). In addition, a similar percentage of α4ß7(+) T-lymphocytes (63±5% in BE, 58±5% in duodenum of BE and 62±8% in duodenum of controls) was found. Finally, mRNA expression of the ligand for α4ß7, MAdCAM-1, was also similar in BE and duodenal tissue. No evidence for a Th2-response was found as almost no IL-4(+)-T-cells were seen. CONCLUSION: The immune cell composition (lymphocytes and eosinophils) and expression of intestinal adhesion molecule MAdCAM-1 is similar in BE and duodenum. This supports the hypothesis that homing of lymphocytes to BE tissue is mainly caused by intestinal homing signals rather than to an active inflammatory response.

Integrins on neutrophils are dispensable for migration into three-dimensional fibrin gels.

The innate immune system and the blood haemostasis system function cooperatively in many pathological conditions such as acute respiratory distress syndrome, deep venous thrombosis, ischaemia/reperfusion injury and cardiovascular disease. Infiltration of neutrophils into thrombotic substrates such as fibrin clots supports fibrinolysis, tissue damage and inflammation. Despite the importance of integrins in neutrophil attachment to fibrin-coated surfaces under flow conditions, little is known about their role in migration processes in shear free two-dimensional (2D) and three-dimensional (3D) fibrin(ogen) environments. Therefore, the present study was designed to study the role of functional integrins in mediating neutrophil migration on and in fibrin matrices. Time lapse video sequences of neutrophil chemokinesis and chemotaxis were made under conditions of active- or non-active integrins. Interestingly, migration of neutrophils on 2D fibrinogen coated surfaces and 3D fibrin matrices is independent of integrins as the response is not sensitive to alphaM-(CD11b) and beta2-(CD18) blocking antibodies and/or chelation of Ca2+ and Mg2+ by EDTA in bivalent ion-free buffers. The blocking integrin antibodies were shown to be functionally active in regular adhesion assays. Our study shows that integrins are dispensable for migration on 2D and in 3D fibrin matrices, both when neutrophils enter into the fibrin matrix and when captured in the matrix.

CO2 insufflation influences the temperature of the open surgical wound.

In open surgery, heat is lost due to radiation and evaporation through the wound. Hypothermia causes tissue hypoxia and impairs various cellular immune functions that increases the risk for postoperative wound infections and delayed wound healing. The patient's body is usually well protected with heating arrangements, but the open wound is left unprotected and until now no practical method has been available to protect it thermically. We therefore investigated if insufflation of an open surgical wound with carbon dioxide would affect wound temperature. In 10 patients undergoing cardiac surgery, the sternotomy wound was insufflated with dry, room temperature carbon dioxide via a gas diffuser for 2 minutes. A heat-sensitive camera measured the wound temperature before, during, and after insufflation. Exposure to carbon dioxide increased the median temperature of the whole wound by 0.5 degrees C (p=0.01). The temperature of the area distant to the diffuser increased by 1.2 degrees C (p<0.01) whereas in the area close to the diffuser it decreased by 1.8 degrees C (p<0.01). In conclusion, short-term insufflation of dry room temperature carbon dioxide in an open wound increases the surface temperature significantly. Although a small increase, it may reduce the incidence of postoperative wound infections in the future.

Homing and clonogenic outgrowth of CD34(+) peripheral blood stem cells: a role for L-selectin?

OBJECTIVE: After transplantation of hematopoietic stem cells, adhesion molecules play a major role in the multistep process of engraftment in which L-selectin is suggested to be of relevance. A positive correlation previously was found between the number of reinfused L-selectin(+) stem cells and platelet recovery. In the present study, we determined the role of L-selectin in different engraftment steps, i.e., adhesion to endothelial cells, migration, and clonogenic outgrowth by in vitro assays that closely mimic the in vivo situation. MATERIALS AND METHODS: Flow adhesion and migration experiments were performed using the human bone marrow endothelial cell line 4LHBMEC and isolated peripheral CD34(+) cells with or without blocking of L-selectin-ligand interaction. Various clonogenic assays, including serum-free colony-forming unit-megakaryocytes (CFU-MK) and burst-forming unit-megakaryocytes (BFU-MK), were performed with sorted L-selectin(+)L-selectin(-) cells or in the presence of antibodies. RESULTS: Blocking of L-selectin on CD34(+) cells did not significantly affect rolling over and firm adhesion to 4LHBMEC. In addition, no role for L-selectin was found in transendothelial migration experiments. Finally, in clonogenic outgrowth of sorted or anti-L-selectin monoclonal antibody-incubated CD34(+) cells, no key role for L-selectin expression could be defined in BFU-MK and CFU-MK assays. CONCLUSION: Using in vitro assays for CD34(+) stem cell adhesion, migration, and clonogenic capacity, we were not able to define a major role for L-selectin.