Beneficial effects of levobupivacaine regional anaesthesia on postoperative opioid induced hyperalgesia in diabetic mice.

BACKGROUND: Diabetic neuropathy is one of the most common complications of diabetes and causes various problems in daily life. The aim of this study was to assess the effect of regional anaesthesia on post surgery opioid induced hyperalgesia in diabetic and non-diabetic mice. METHODS: Diabetic and non-diabetic mice underwent plantar surgery. Levobupivacaine and sufentanil were used before surgery, for sciatic nerve block (regional anaesthesia) and analgesia, respectively. Diabetic and non-diabetic groups were each randomly assigned to three subgroups: control, no sufentanil and no levobupivacaine; sufentanil and no levobupivacaine; sufentanil and levobupivacaine. Three tests were used to assess pain behaviour: mechanical nociception; thermal nociception and guarding behaviours using a pain scale. RESULTS: Sufentanil, alone or in combination with levobupivacaine, produced antinociceptive effects shortly after administration. Subsequently, sufentanil induced hyperalgesia in diabetic and non-diabetic mice. Opioid-induced hyperalgesia was enhanced in diabetic mice. Levobupivacaine associated to sufentanil completely prevented hyperalgesia in both groups of mice. CONCLUSION: The results suggest that regional anaesthesia can decrease opioid-induced hyperalgesia in diabetic as well as in non-diabetic mice. These observations may be clinically relevant for the management of diabetic patients.

Genetic manipulation and genetic variation of the kallikrein-kinin system: impact on cardiovascular and renal diseases.

Genetic manipulation of the kallikrein-kinin system (KKS) in mice, with either gain or loss of function, and study of human genetic variability in KKS components which has been well documented at the phenotypic and genomic level, have allowed recognizing the physiological role of KKS in health and in disease. This role has been especially documented in the cardiovascular system and the kidney. Kinins are produced at slow rate in most organs in resting condition and/or inactivated quickly. Yet the KKS is involved in arterial function and in renal tubular function. In several pathological situations, kinin production increases, kinin receptor synthesis is upregulated, and kinins play an important role, whether beneficial or detrimental, in disease outcome. In the setting of ischemic, diabetic or hemodynamic aggression, kinin release by tissue kallikrein protects against organ damage, through B2 and/or B1 bradykinin receptor activation, depending on organ and disease. This has been well documented for the ischemic or diabetic heart, kidney and skeletal muscle, where KKS activity reduces oxidative stress, limits necrosis or fibrosis and promotes angiogenesis. On the other hand, in some pathological situations where plasma prekallikrein is inappropriately activated, excess kinin release in local or systemic circulation is detrimental, through oedema or hypotension. Putative therapeutic application of these clinical and experimental findings through current pharmacological development is discussed in the chapter.

Bradykinin inhibits high glucose- and growth factor-induced collagen synthesis in mesangial cells through the B2-kinin receptor.

Mesangial matrix expansion is an early lesion leading to glomeruloclerosis and chronic renal diseases. A beneficial effect is achieved with angiotensin I-converting enzyme inhibitors (ACEI), which also favor bradykinin (BK) B2 receptor (B2R) activation. To define the underlying mechanism, we hypothesized that B2R activation could be a negative regulator of collagen synthesis in mesangial cells (MC). We investigated the effect of BK on collagen synthesis and signaling in MC. Inflammation was evaluated by intercellular adhesion molecule-1 (ICAM-1) expression. BK inhibited collagen I and IV synthesis stimulated by high glucose, epithelial growth factor (EGF), and transforming growth factor-ß (TGF-ß) but did not alter ICAM-1. Inhibition of collagen synthesis was B2R but not B1R mediated. PKC or phosphatidylinositol 3-kinase (PI3K) inhibitors mimicked the BK effect. B2R activation inhibited TGF-ß- and EGF-induced Erk1/2, Smad2/3, Akt S473, and EGFR phosphorylation. A phosphatase inhibitor prevented BK effects. The in vivo impact of B2R on mesangial matrix expansion was assessed in streptozotocin-diabetic rodents. Deletion of B2R increased mesangial matrix expansion and albuminuria in diabetic mice. In diabetic rats, matrix expansion and albuminuria were prevented by ACEI but not by ACEI and B2R antagonist cotreatment. Consistently, the lowered BK content of diabetic glomeruli was restored by ACEI. In conclusion, deficient B2R activation aggravated mesangial matrix expansion in diabetic rodents whereas B2R activation reduced MC collagen synthesis by a mechanism targeting Erk1/2 and Akt, common pathways activated by EGF and TGF-ß. Taken together, the data support the hypothesis of an antifibrosing effect of B2R activation.

Antidiabetic Effects of Aqueous and Dichloromethane/Methanol Stem Bark Extracts of Pterocarpus soyauxii Taub (Papilionaceae) on Streptozotocin-induced Diabetic Rats.

AIM OF THE STUDY: The aim is to evaluate the hypoglycemic and antidiabetic effects of aqueous and CH2Cl2/CH3OH stem bark extracts of Pterocarpus soyauxii Taub in normal and diabetic rats. MATERIALS AND METHODS: Streptozotocin (STZ)-induced diabetic and normal adult Wistar rats were orally administered with aqueous and CH2Cl2/CH3OH plant extracts of P. soyauxii at various doses (38-300 mg/kg) in a single administration. In addition, STZ-induced diabetic rats received prolonged daily administration for 14 days. Glibenclamide (GB) (10 mg/kg) was used as reference treatment. In acute test, fasting blood glucose was followed for 5 h. In subacute test, body weight, food and water intakes, and blood glucose were followed weekly and serum biochemical parameters evaluated after 14 days treatment. RESULTS: Acute administration of aqueous and CH2Cl2/CH3OH stem bark extracts moderately decreased fasting blood glucose compared to GB, significantly in normal rats (P < 0.05 to P < 0.01) but, as GB, not significantly in diabetic rats. Prolonged treatments in diabetic rats with aqueous and CH2Cl2/CH3OH extracts reduced blood glucose to an extent, respectively, superior or similar to GB. Moreover, P. soyauxii also significantly (P < 0.01) reduced weight loss, and diabetes increased serum triglycerides, total cholesterol, and transaminases (alanine aminotransferase/aspartate aminotransferase) elevations. CONCLUSION: P. soyauxii Taub stem bark extracts have possible value for antidiabetic oral medication. SUMMARY: Aqueous and Dichloromethane/Methanol stem bark extracts of Pterocarpus soyauxii Taub have potent (compared to Glibenclamide) antidiabetic effects in STZ-diabetic rats, with specific kinetics and dose-responses.Moderate hypoglycemia effects upon acute P. soyauxii administration.Potent anti-hyperglycemic effects of sub-acute P. soyauxii administration in STZ-diabetic rats.Potent anti-hyperlipidemic effects of sub-acute P. soyauxii administration in STZ-diabetic rats.Improved hepatic and renal serum parameters after sub-acute P. soyauxii administration in STZ-diabetic rats.P. soyauxii extracts may be useful for oral treatment of diabetes and related metabolic disorders. Abbreviations Used: CH2Cl2/CH3OH: Dichloromethane/Methanol; STZ: Streptozotocin; GB: Glibenclamide; AE: Aqueous extract; OE: Organic extract; FeCl3: Iron (III) chloride; NaCl: Sodium chloride; K3Fe(CN)6: Potassium ferricyanide; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; H: Hour; BW: Body weight, W1 and W2: Weeks 1 and 2; CHOD: Cholesterol oxidase; GPO: Glycerol-3 Phosphate oxidase; PAP: Phenol + Aminophenazone.

Expression of membrane-bound and soluble FasL in Fas- and FADD-dependent T lymphocyte apoptosis induced by mildly oxidized LDL.

Apoptosis plays an essential role in atherosclerosis. Oxidized low-density lipoproteins (oxLDL) and activated T lymphocytes are present in atherosclerotic lesions, and we have previously reported that oxLDL induce apoptosis of activated T lymphocytes. We now show that this is preceded by an increase of Fas and FasL expression. Fas and FasL overexpression was dependent on reactive oxygen species (ROS) production as well as ERK and JNK activation. In addition, oxLDL triggered an early production of soluble FasL by T lymphocytes. Blocking anti-Fas antibody or Fas-Fc protein, but also antioxidant molecules and inhibitors of ERK and JNK, decreased oxLDL-mediated apoptosis. Moreover, PHA-activated murine lymphocytes lacking a functional Fas receptor were partially resistant to oxLDL. Finally, Jurkat T cells deficient for FADD, an adaptor protein required for Fas signaling, resisted oxLDL-induced apoptosis. OxLDL triggered caspase 8 and 3 activation as well as ceramide production in PHA-activated lymphocytes and in Jurkat cells. Caspase activation was completely impaired in FADD-deficient cells, but ceramide production was not affected. Altogether, our results highlight the putative role of both membrane-bound and soluble FasL in oxLDL-induced Fas and FADD-dependent apoptosis of T lymphocytes and suggest an involvement of ROS, ERK, and JNK in this process.

Engraftment of human T, B and NK cells in CB.17 SCID/beige mice by transfer of human spleen cells.

Models of severe combined immuno-deficient (SCID) mice reconstituted with a competent human immune system represent a valuable tool for the study of human immune responses in vivo. Reconstitution with human cells can be achieved using large numbers of peripheral blood lymphocytes, but levels of engraftment are poor and graft versus host disease (GVHD) frequently occurs. SCID/beige mice are at the same time deficient for adaptive and innate immunity and the objective of this study was to develop a safe and efficient way to achieve human lymphocyte engraftment in these mice using human spleen cells. After institutional authorisations and informed consent of relatives, a piece of spleen was obtained from cadaveric organ donors and the splenocytes were isolated and cryopreserved for later use. Single intraperitoneal injections of 5-100 x10(6) splenocytes were performed into SCID/beige mice. Reconstitution of a human immune system was monitored weekly by the presence of human cells and IgG in peripheral blood. The mice were sacrificed 4 weeks after the injection and the engraftment in lymphoid organs was studied. A reproducible reconstitution was obtained with intraperitoneal injection of 30-40 x10(6) spleen cells. Human T, B and NK cells as well as human IgG were present in peripheral blood. In lymphoid tissues, the same lymphocytic subpopulations were detected and in addition some antigen presenting cells. The reconstitution was functional because graft rejection was observed after transplantation of human allogeneic tissues. When less than 30 x10(6) cells were injected, the reconstitution was variable. When more than 40 x10(6) cells were injected, GVHD occurred with increasing frequency. In conclusion, we show that intraperitoneal injection of 30-40 x10(6) human splenocytes into SCID/beige mice induces a quick and functional engraftment of human T, B and NK cells with no risk of GVHD. This model may be used to study human transplantation immunobiology in vivo.

Kinin receptors in vascular biology and pathology.

Endogenous kinins are important vasoactive peptides whose effects are mediated by two G-Protein-coupled receptors (R), named B2R (constitutive) and B1R (inducible). They are involved in vascular homeostasis, ischemic pre- and post- conditioning, but also in cardiovascular diseases. They contribute to the therapeutic effects of angiotensin-1 converting enzyme inhibitors (ACEI) and angiotensin AT1 receptor blockers. Benefits derive primarily from vasodilatory, antiproliferative, antihypertrophic, antifibrotic, antithrombic and antioxidant properties, which are associated with the release of endothelial factors such as nitric oxide, prostacyclin and tissue plasminogen activator. Uncontrolled production of kinins or the inhibition of their metabolism may lead to unwanted pro-inflammatory side effects. Thus, B2R antagonism is salutary in angioedema, septic shock, stroke, and Chagas vasculopathy. B1R is virtually absent in healthy tissues, yet this receptor is induced by the cytokine pathway and the oxidative stress via the transcriptional nuclear factor NF-κB. The B1R may play a compensatory role for the lack of B2R, and its up-regulation during tissue damage may be a useful mechanism of host defense. Activation of both receptors may be beneficial, notably in neovascularisation, angiogenesis, heart ischemia and diabetic nephropathy. At the same time, B1R is a potent activator of inducible nitric oxide and NADPH oxidase, which are associated with vascular inflammation, increased permeability, insulin resistance, endothelial dysfunction and diabetic complications. The dual beneficial and deleterious effects of kinin receptors and, particularly B1R, raise an unsettled issue on the therapeutic value of B1R/B2R agonists versus antagonists in cardiovascular diseases. Hence, the Janus-face of kinin receptors needs to be seriously addressed in the upcoming clinical trials for each pathological setting.

Targeting the 'Janus face' of the B2-bradykinin receptor.

INTRODUCTION: Kinins are main active mediators of the kallikrein-kinin system (KKS) via bradykinin type 1 inducible (B1R) and type 2 constitutive (B2R) receptors. B2R mediates most physiological bradykinin (BK) responses, including vasodilation, natriuresis, NO, prostaglandins release. AREAS COVERED: The article summarizes knowledge on kinins, B2R signaling and biological functions; highlights crosstalks between B2R and renin-angiotensin system (RAS). The double role (Janus face) in physiopathology, namely the beneficial protection of the endothelium, which forms the basis for the therapeutical utilization of B2 receptor agonists, on the one side, and the involvement of B2R in inflammation or infection diseases and in pain mechanisms, which justifies the use of B2R antagonists, on the other side, is extensively analyzed. EXPERT OPINION: For decades, the B2R has been unconsciously activated during angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) treatments. Whether direct B2R targeting with stable agonists could bring additional therapeutic benefit to RAS inhibition should be investigated. Efficacy, established in experimental models, should be confirmed by translational studies in cardiovascular pathologies, glaucoma, Duchenne cardiopathy and during brain cancer therapy. The other face of B2R is targeted by antagonists already approved to treat hereditary angioedema. The use of antagonists could be extended to other angioedema and efficacy tested against acute pain and inflammatory diseases.

Distribution and expression of B2-kinin receptor on human leukocyte subsets in young adults and elderly using flow cytometry.

The kallikrein-kinin system has been investigated in many experimental models. Dysregulations of the KKS are likely to be involved in pathologies such as inflammation, cancer and cardiovascular diseases. Previous works on the human KKS mostly rely on gene polymorphism and mRNA expression. In order to assess the KKS in human at the protein level, we have developed an approach based on flow cytometric analysis of leukocytes. Whole blood samples were collected and erythrocytes were lysed. Permeabilised leukocytes were incubated with anti-B2R (IgG2b), anti-IgG2b-PE, anti-CD3-PerCP (lymphocytes) and anti-CD14-APC (monocytes) antibodies. FACScalibur analyzed fluorescence intensities. Results were expressed as per cent of B2R-positive cells in each leukocyte subset and as B2R fluorescence intensity per positive cell. Detection of the B2R protein by this methodology was validated by (i) correlation with Western blotting using two different B2R antibodies, (ii) BK-induced Erk activation, (iii) B2R mRNA expression. The methodology was then applied to evaluate variations of B2R expression in a population including young healthy, elderly healthy, and elderly treated hypertensive men and women. In the young healthy subjects, B2R distribution was: monocytes>polymorphonuclear neutrophils (PMN)>lymphocytes and no difference with gender was observed. Moreover, no difference was observed on PMN B2R expression. B2R expression remained unchanged in the elderly healthy or hypertensive men. By contrast, monocytes and lymphocytes B2R expressions were decreased in the elderly healthy women. Finally, FACS analysis of B2R expression on leukocytes subsets provides single cell quantification of B2R expression allowing comparison of cellular sub-populations. This approach provides a new efficient tool to investigate B2R profiling of immune system in pathological states.

Interleukin-6 deficiency fails to prevent chronic rejection after aortic allografts in apolipoprotein E-deficient mice.

BACKGROUND: Chronic vascular rejection (CVR) is characterized by an intimal thickening in the arteries of allografts due to immunoinflammatory reactions and smooth muscle cell proliferation. Interleukin 6 (IL-6) levels are increased in patients with graft rejection, however the role of IL-6 in CVR is not known. We investigated if IL-6 deficiency in the recipient could prevent CVR after an aortic allograft. METHODS: Donor aortas from wild-type DBA/2 mice were transplanted into C57BL/6 recipients, either wild-type mice or mice deficient for IL-6 (IL-6(-/-)), apolipoprotein E (ApoE(-/-)), or both (IL-6(-/-)ApoE(-/-)). Alloantibody titers were determined at Day 30, 60, or 90 after grafting. The grafts were examined for CVR lesions by morphometry and immunohistology. RESULTS: All recipient allografts displayed lesions typical for CVR. The lesions were larger in IL-6-deficient strains, and significantly so in IL-6(-/-)ApoE(-/-) recipients. Early immunoglobulin (Ig) G1 alloantibody deposits were observed in the grafts of ApoE-deficient strains and late IgG2a deposits in the grafts of IL-6-deficient strains. A rapid and sustained type 1 helper T cell (Th1; IgG2a) alloresponse in IL-6(-/-) mice, and a strong type 2 helper T cell (Th2; IgG1) response in ApoE(-/-) mice were observed. IL-6(-/-)ApoE(-/-) mice displayed the highest alloantibody titer, with a Th1 dominance. CONCLUSIONS: Unexpectedly, IL-6 deficiency in the recipient mice did not prevent CVR lesions but even aggravated them in IL-6(-/-)ApoE(-/-) recipients. This was associated with increased local and systemic alloresponses.

Multiple human mesenteric arterial grafts from the same donor to study human chronic vascular rejection in humanized SCID/beige mice.

BACKGROUND: Chronic vascular rejection (CVR) is a major problem in clinical transplantation. Studies in experimental animals have been important to understand some of its mechanisms, but they are hampered by the difficulty of extrapolating the results into clinical practice. METHODS: We created a new experimental model for the study of human CVR by grafting multiple human mesenteric arteries from the same human donor into different SCID/beige mice in the infrarenal aortic position. Twenty-seven different mice were successfully grafted with a human artery from 6 donors. One week later, 23 of the mice received an intraperitoneal injection of 40 million human spleen cells, either from the same donor (autologous) or from another donor (allogeneic). RESULTS: In 81% of the mice an immune reconstitution was obtained, shown by the presence of human T, B and NK cells and IgG in circulating blood. At the time of sacrifice, 5 weeks after the arterial transplantation, a typical CVR with infiltration of human immune cells and deposit of human immunoglobulin was observed in the reconstituted mice that received allogeneic cells, whereas only minor lesions were noted in autologous combinations. No CVR was observed without injection of human splenocytes. We did not observe lymphoma or graft-vs-host reactions during the experiment. CONCLUSIONS: We show that it is feasible to graft multiple human arteries from the same donor into SCID/beige mice, and that a specific and typical CVR is observed after reconstitution with allogeneic spleen cells. Our method allows for pre-clinical testing of new therapeutics in controlled series.