Nlrp1 inflammasome is downregulated in trauma patients.

UNLABELLED: After a major trauma, IL-1ß-producing capacity of monocytes is reduced. Generation of IL-1ß is important for appropriate immune response after trauma and requires not only synthesis and transcription of inflammasome components but also their activation. Altered IL-1ß-processing due to deregulated NLRP inflammasomes assembly is associated with several inflammatory diseases. However, the precise role of NLRP1 inflammasome in monocytes after trauma is unknown. Here, we investigated if NLRP1 inflammasome components are responsible for depressed monocyte function after trauma. We found in ex vivo in vitro assays that LPS-stimulation of CD14(+)-isolated monocytes from healthy volunteers (HV) results in remarkably higher capacity of the IL-1ß-release compared to trauma patients (TP). During the 10-day time course, this monocyte depression was highest immediately after admission. Inflammasome activation correlating with this inflammatory response was demonstrated by enhanced protein production of cleaved IL-1ß and caspase-1. Furthermore, we found that the gene expression of IL-1ß, caspase-1, and ASC was comparable in TP and HV after LPS-stimulation during the 10-day course, while NLRP1 was markedly reduced in TP. We demonstrated that transfected monocytes from TP, which expressed the lacking components, were recovered in their LPS-induced IL-1ß-release and that lacking of NLRP1 is responsible for the suppressed monocyte activity after trauma. The restoration of NLRP1 inflammasome suggests new mechanistic target for the recovery of dysbalanced immune reaction after trauma. KEY MESSAGE: Suppression in monocyte function occurs early after a major trauma or surgery. Reduced gene expression abrogates NLRP1 inflammasome assembly after trauma. Limited availability of inflammasome components may cause reduced host defense. Restoring NLRP1 in immune-suppressed monocytes recovers NLPR1 activity after trauma. Recovered inflammasome activity may improve the immune response to PAMPs/DAMPs.

Decreased inflammatory responses of human lung epithelial cells after ethanol exposure are mimicked by ethyl pyruvate.

BACKGROUND AND PURPOSE: Leukocyte migration into alveolar space plays a critical role in pulmonary inflammation resulting in lung injury. Acute ethanol (EtOH) exposure exerts anti-inflammatory effects. The clinical use of EtOH is critical due to its side effects. Here, we compared effects of EtOH and ethyl pyruvate (EtP) on neutrophil adhesion and activation of cultured alveolar epithelial cells (A549). EXPERIMENTAL APPROACH: Time course and dose-dependent release of interleukin- (IL-) 6 and IL-8 from A549 were measured after pretreatment of A549 with EtP (2.5-10 mM), sodium pyruvate (NaP, 10 mM), or EtOH (85-170 mM), and subsequent lipopolysaccharide or IL-1beta stimulation. Neutrophil adhesion to pretreated and stimulated A549 monolayers and CD54 surface expression were determined. KEY RESULTS: Treating A549 with EtOH or EtP reduced substantially the cytokine-induced release of IL-8 and IL-6. EtOH and EtP (but not NaP) reduced the adhesion of neutrophils to monolayers in a dose- and time-dependent fashion. CD54 expression on A549 decreased after EtOH or EtP treatment before IL-1beta stimulation. CONCLUSIONS AND IMPLICATIONS: EtP reduces secretory and adhesive potential of lung epithelial cells under inflammatory conditions. These findings suggest EtP as a potential treatment alternative that mimics the anti-inflammatory effects of EtOH in early inflammatory response in lungs.

Vascular endothelial growth factor--marker for proliferation in thyroid diseases?

UNLABELLED: Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis and is involved in tumor development. To date, the role of VEGF in benign diseases of the thyroid is not well known. The purpose of the present study is to determine the expression of VEGF and its receptors in primary cultures of human thyrocytes. METHODS: 50 patients with uninodular (n=11), multinodular (n=15), recurrent goiter (n=14) and Graves' disease (n=10) were enrolled. Nodular and corresponding paranodular tissue was obtained after surgery and investigated. RNA and protein were extracted from primary thyrocyte cultures. PCR, western blot and ELISA were performed to evaluate VEGF isoforms and VEGF receptor 1 and 2. RESULTS: Significantly increased transcription and protein expression of VEGF and its receptors were detected in nodular tissue of uninodular and recurrent goiter compared to the corresponding normal tissue. Active secretion of VEGF by thyrocytes was confirmed by ELISA. In multinodu-lar goiter, no difference could be found between nodular and corresponding paranodular tissue in terms of expression of VEGF or its receptors. Furthermore, we found the highest levels of VEGF and its receptors in tissue obtained from patients with Graves' disease. CONCLUSION: Increased expression of VEGF and its receptors might be crucial in the proliferation of thyrocytes and therefore may contribute to the development of goiter and goiter recurrence.

[Laser-induced Interstitial thermotherapy (LITT) in hepatic metastases of various sizes in an animal model]. / Laserinduzierte interstitielle Thermotherapie (LITT) von Lebermetastasen unterschiedlicher Grösse im Kleintiermodell.

PURPOSE: To assess the effect of laser-induced thermotherapy (LITT) on liver metastases of various size from colon carcinoma in an animal model. MATERIALS AND METHODS: Liver metastases were implanted in 20 Wistar Albino Glaxo (WAG) rats by subcapsular injection of cells from a colorectal strain (CC531) (day 0). The animals were divided into two groups with regard to the measured tumor size of 0.05 - 0.06 cm (3) (group A) and 0.10 - 0.12 cm (3) (group B). On day 14 after laparotomy, the tumors were exposed to 1064 nm Nd:YAG laser light at 2 watts for 5 minutes after intratumoral placement of the laser applicator set. The tumor volumes before (V1, at day 13) and after treatment (V2, at day 28) were determined by MRI and the mean tumor growth ratio (V2/V1) was calculated. RESULTS: The mean tumor volumes V1 and V2 were 0.05 +/- 0.003 cm (3) and 0.23 +/- 0.016 cm (3) in group A, and 0.11 +/- 0.006 cm (3) and 0.68 +/- 0.037 cm (3) in group B. The mean tumor growth ratio (V2/V1) was 4.31 +/- 0.19 in group A and 6.11 +/- 0.14 in group B. The mean volume of the induced necrosis (0.15 +/- 0.01 cm (3)) was the same for both groups ( p > 0.05). Compared to group B, liver metastases of group A showed a significant slower tumor growth velocity (paired t-test, p < 0.0001). CONCLUSION: The interventional treatment of large hepatic tumors with LITT leads to faster tumor growth compared to smaller lesions.

Transarterial chemoembolization alone and in combination with other therapies: a comparative study in an animal HCC model.

The purpose of this study is to compare transarterial chemoembolization (TACE) alone and in combination with other therapies in an animal model. Subcapsular implantation of a solid Morris hepatoma 3924A in the liver was carried out in 50 male ACI rats (day 0). Tumor volume (V1) was measured by MRI (day 13). After laparotomy and retrograde placement of a catheter into the gastroduodenal artery (day 14), the following protocols of the interventional procedure were applied: TACE (mitomycin C + lipiodol) + immunotherapy (group A: TNFalpha + IL-2, group B: OK-432 + IL-2); TACE + antiangiogenesis therapy (group C: TNP-470, group D: endostatin); TACE alone in group E (control group). Tumor volume (V2) was assessed by MRI and the mean ratio of x (V2/V1) was calculated. Data were analyzed using Dunnett's t test (comparing therapeutic groups with the control group) and the Student-Newman-Keuls test (comparing significant therapeutic groups). Multivariate analysis showed a significant reduction in the tumor growth rate (P<0.05) in groups B (x=6.53) and C (x=4.01) compared to the mean ratio of the control group E (x=9.14). Significant results were observed in group C (P<0.05) in comparison with the other therapeutic groups. TACE combined with immunotherapy (OK-432) and antiangiogenesis therapy (TNP-470) retards tumor growth compared with TACE alone in an HCC animal model.

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells.

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.

Prostaglandin E2 induces expression of P-selectin (CD62P) on cultured human umbilical vein endothelial cells and enhances endothelial binding of CD4-T-cells.

Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.

Mycophenolate mofetil decreases endothelial prostaglandin E2 in response to allogeneic T cells or cytokines.

BACKGROUND: Prostaglandin E2 (PGE2) is a powerful endogenous immune suppressant and interferes with various T-cell functions. However, it is not known in detail whether immunosuppressive drugs influence the PGE2-driven immune response in transplant patients. Therefore, we investigated the effect of several immunosuppressive compounds, in particular the novel drug mycophenolate mofetil (MMF), on endothelial PGE2 release. METHODS: Endothelial cells (HUVEC) were activated by either allogeneic CD4+ or CD8+ T cells, or by the cytokines interleukin-1 or gamma-interferon. Using an enzyme-linked immunosorbent assay, we analyzed PGE2 release of the activated HWEC in the presence of MMF, cyclosporine, or tacrolimus. As verapamil and mibefradil also possess immunosuppressive properties, they were included in the study as well. RESULTS: Activation of HUVEC with interleukin-1 or T cells resulted in a drastic accumulation of PGE2 in the supernatant. Cyclosporine or tacrolimus had no effect on PGE2 release. However, Ca2+ channel blockers, when applied at higher dosages, caused a significant increase in PGE2. Interestingly, MMF strongly diminished the PGE2 level in the cell culture supernatant in a concentration-dependent manner. CONCLUSION: The results demonstrate an inhibitory effect of MMF on PGE2 production, which may lower the benefits of the PGE2-triggered immune response after organ transplantation.

In vitro analysis of verapamil-induced immunosuppression: potent inhibition of T cell motility and lymphocytic transmigration through allogeneic endothelial cells.

BACKGROUND: Cyclosporine A (CsA) and tacrolimus prevent proliferation but not transendothelial migration of alloreactive lymphocytes into donor organs. As a result, serious adverse effects, such as nephrotoxicity and neurotoxicity, have been observed under CsA/tacrolimus therapy. The incorporation of new drugs with infiltration blocking properties might enhance the efficacy of the current immunosuppressive protocol, allowing lower CsA/tacrolimus dosage. Because Ca2+ plays a critical role in cell-cell interaction, the Ca2+-channel blocker verapamil might be a good cany. didate for supporting CsA/tacrolimus-based therapy. METHODS: A T-cell endothelial cell coculture model or immobilized immunoglobulin G globulin chimeras were employed to investigate how S- and R- verapamil interfere with the lymphocytic infiltration process. The expression and arrangement of membranous adhesion receptors and cytoskeletal F-actin filaments were analyzed by fluorometric method in the presence of. verapamil. RESULTS: Both verapamil enantiomers strongly inhibited lymphocyte infiltration. CD4+ and CD8+ T-cells were influenced to a similar extent with regard to horizontal locomotion (CD4+=CD8+), but to a different extent with regard to adhesion and penetration (CD4+ > CD8+). Moreover, penetration was blocked to a higher extent than was adhesion. ID50-values were 31 microM (CD4+-adhesion) and 11 microM (CD4+-penetration). Verapamil reduced P-selectin expression on endothelial cells and effectively down-regulated binding of T-cells to immobilized P-selectin immunoglobulin G globulins (ID50=4.4 microM; CD4+). A verapamil-induced reduction of intracellular F-actin in T-lymphocytes was proven to be mainly responsible for diminished cell locomotion. CONCLUSIONS: The prevention of CD4+ T-cell penetration by verapamil might argue for its use as an adjunct to CsA/tacrolimus-based immunosuppressive therapy.

Novel mode of action of the calcium antagonist mibefradil (Ro 40-5967): potent immunosuppression by inhibition of T-cell infiltration through allogeneic endothelium.

Cyclosporin A reduces the mitotic activity of allosensitized lymphocytes, but fails to limit emigration of these cells into the donor organ. However, the modulation of both lymphocyte proliferation and infiltration are desirable characteristics of immunosuppressive therapy. The calcium-channel blocker, verapamil, has recently been shown to effectively prevent the transmigration of CD4+ and CD8+ T cells through allogeneic endothelium. Mibefradil (Ro 40-5967) represents a new generation of calcium antagonists with high potency and long-term activity. To evaluate the immunosuppressive potential of this drug, the influence of mibefradil on lymphocyte adhesion to, horizontal locomotion along, and penetration through allogeneic endothelium (HUVEC) was performed. When lymphocytes were prestimulated for 24 hr with mibefradil, adhesion and penetration were dose-dependently reduced. The adhesion ID50 values were 3.4 microM (CD4+ T cells) versus 9.2 microM (CD8+ T cells) and 2.1 microM (CD4+ T cells) versus 3.9 microM (CD8+ T cells) with regard to penetration. Mibefradil also effectively blocked horizontal locomotion. Specific down-regulation of T-cell binding to the P-selection receptor (ID50: CD4+ T cells, 0.8 microM: CD8+ T cells, 1.2 microM) and to the intracellular adhesion molecule-1 (ICAM-1) receptor (ID50: CD4+ T cells, 1.9 microM; CD8+ T cells, 1.5 microM) by mibefradil seems to be responsible for the decreased adhesion and penetration rates. Reduction of intracellular F-actin in T lymphocytes could diminish cell locomotion. In conclusion, the potent suppressive properties of mibefradil support its use as a co-medication in cyclosporin A-based immunosuppressive therapy.

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil.

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

The mechanism of spinal cord injury after simple and double aortic cross-clamping.

Ischemic spinal cord injury after cross-clamping of the descending aorta can occur independently of aortic disease. In a previous study we had shown a precipitous uniform fall of spinal surface oxygen tension downstream to the clamping site irrespective of level. In the present paper, the hemodynamic changes in the spinal and aortic collateral circulation were investigated. Pressures were measured in the proximal, distal, and excluded aortic segments (descending thoracic and lumbar aorta) as well as in the intercostal and the lumbar arterial beds. Before high aortic occlusion, pressures in the intercostal and lumbar arterial beds were lower than aortic pressure. Along with the postclamping fall in distal arterial pressure, intercostal and lumbar arterial bed pressure decreased further but remained above aortic pressure. Exclusion of the thoracic aorta by double clamping restored intercostal bed pressure almost to control, whereas exclusion of the abdominal aorta hardly affected lumbar bed pressure. We conclude that spinal collateral circulation is more highly developed in the thoracic than in the lumbar region. After aortic cross-clamping, blood tends to drain away from the spinal cord rather than supplying it longitudinally. Under clinical conditions, therefore, retrograde bleeding into the opened aorta as well as into the aorta downstream to the distal clamp should be minimized and larger vessels originating from the aorta should promptly be anastomosed to the graft.

Variance analysis. Using standards to predict nurse staffing patterns.

The use of standards to predict required nurse staffing patterns has received attention in recent years because of pressures for cost containment, revenue limitations and the increased availability of data. The establishment of a standard hour system can lead to a comparison of actual hours to the standard hours predicted and adjusted for case mix and changes in volume. The authors previously developed nurse staffing prediction models based on information from a 220-bed short-term hospital. Further study in terms of variance analysis (standard hours to actual hours) is explored in this article.