Sylvain Mukenge

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum

We investigated the effect of the cannabinoid agonist (+)WIN‐55212‐2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or co‐incubated, reduced twitch responses to a similar degree (85%). (+)WIN‐55212‐2 concentration‐dependently inhibited twitch responses (IC50 73 nm), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN‐55212‐2. Atropine but not (+)WIN‐55212‐2 or TTX prevented carbachol‐induced tonic contraction.  These results provide functional evidence of the existence of prejunctional cannabinoid CB1‐receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.

Functional assessment of neuronal cannabinoid receptors in the muscular layers of human ileum and colon

BACKGROUND & AIMS The notion that specific receptors account for the ability of natural and synthetic cannabinoids to alter physiological functions, prompted this study aimed at assessing their functional presence in the human gut. METHODS The effects have been studied of cannabinoids and selective antagonists of their receptors on chemically or electrically evoked contractions in preparations of human intestinal smooth muscle in vitro. RESULTS Atropine prevented the contractions of longitudinal and circular muscle strips of ileum and colon induced by carbachol or electrical field stimulation; tetrodotoxin abolished only the latter which suggests they do involve activation of cholinergic neurons. The synthetic cannabinoid (+)WIN 55,212-2 had no effect on carbachol contractions, but in a concentration-dependent fashion prevented those elicited by electrical field stimulation - which were insensitive to the putative endogenous cannabinoid anandamide - more potently in longitudinal than in circular strips. The selective CB1 receptor antagonist SR141716, which had no effect in the absence of (+)WIN 55,212-2, competitively antagonised its inhibition of electrical field stimulation contractions, unlike the selective CB2 antagonist SR144528. CONCLUSIONS Cannabinoid CB1 receptors are functionally present in the human ileum and colon; their pharmacological activation apparently results in inhibition of excitatory cholinergic pathways subserving smooth muscle contraction.

In vitro characterization of tachykinin NK2-receptors modulating motor responses of human colonic muscle strips

1 Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose‐dependently contracted by neurokinin A (EC50, 4.9 nm), the tachykinin NK2‐receptor selective agonist [β‐Ala8]neurokinin A (4–10) ([β‐Ala8]NKA (4–10)) (EC50, 5.0 nm), neurokinin B (EC50, 5.3 nm) and substance P (EC50, 160 nm), but not by the tachykinin NK1‐receptor selective agonist [Sar9Met(O2)11] substance P, or the NK3‐receptor selective agonists, senktide and [MePhe7] neurokinin B. No regional differences between transverse and distal colon were observed in response to [β‐Ala8]NKA (4–10). 2 Atropine (1 μm) and tetrodotoxin (1 μm) did not significantly inhibit responses to [β‐Ala8]NKA (4–10), neurokinin A, substance P or neurokinin B. 3 The newly developed non‐peptide antagonists for tachykinin NK2‐receptors SR 48968, SR 144190 and its N‐demethyl (SR 144743) and N,N‐demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [β‐Ala8]NKA (4–10), with similar potency (Schild plot pA2 values 9.4, 9.4 and 9.3, slope = 1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept∼9.3) and a low (X intercept 8.4, slope 1.6) affinity site. Co‐incubation of SR 48968 (10, 100 nm) and SR 144782 (10 nm) produced additive effects; in this experimental condition, SR 48968 apparent affinity (pKB) was 8.2. In addition, SR 144782 (0.1 μm) antagonized responses to neurokinin A, substance P and neurokinin B, with pKB consistent with its affinity for tachykinin NK2‐receptors. The potent and selective NK1 and NK3‐receptor antagonists, SR 140333 and SR 142801 (both 0.1 μm), failed to inhibit contractions induced by SP or NKB. 4 In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non‐neuronal tachykinin NK2‐receptors, but not tachykinin NK1‐ or NK3‐receptors. Our findings with SR 48968 suggest the existence of two tachykinin NK2‐receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric effects at the tachykinin NK2‐receptor might well account for the complexity of the observed interaction.

Peripheral blood lymphocytes genetically modified to express the self/tumor antigen MAGE-A3 induce antitumor immune responses in cancer patients

Dendritic cell (DC) targeting in vivo has recently been shown to confer strong and protective cytotoxic T lymphocyte (CTL)-based immunity in tumor murine models. Our group has recently demonstrated in preclinical models that the infusion of genetically modified lymphocytes (GMLs) expressing the self/tumor antigen TRP-2 is able to elicit functional TRP-2-specific effectors with antitumor activity by targeting DCs in vivo. Here we have analyzed vaccine- and tumor-specific immune responses of 10 melanoma patients treated with autologous GMLs expressing the cancer germline gene MAGE-A3. Three of 10 patients treated with MAGE-A3-GML showed an increase of circulating anti-MAGE-A3 T cells, and developed skin delayed-type hypersensitivity to MAGE-A3. Interestingly, in 2 of these patients, with progressive and measurable tumors at study entry, anti-MAGE-A3 T cells were detected not only in the blood but also within tumors resected after vaccination. These results demonstrate that the infusion of MAGE-A3-GML elicits antitumor T cells, which are capable of trafficking to inflamed tissues and of infiltrating tumors. Clinical studies on a larger group of patients are needed to evaluate the clinical efficacy of such a strategy.

Functional assessment of β adrenoceptor subtypes in human colonic circular and longitudinal (taenia coli) smooth muscle

BACKGROUND AND AIMS The subtype and species related heterogeneity of β adrenoceptors prompted a functional reappraisal of these molecular targets of motility inhibition in the human colon. METHODS Relaxation of muscle strips was measured in vitro. RESULTS The following agonists had decreasing relaxing potency (effective concentration range 10−8–10−4 mol/l): (−)isoprenaline (non-selective), terbutaline (β2 selective), CGP 12177 (β3 selective, also β1, β2antagonist), and SR 58611A (β3 selective). Isoprenaline and terbutaline were more potent on circular than taenia strips; CGP 12177 and SR 58611A weakly and partially relaxed taenia but had little effect on circular strips. The potency of isoprenaline on circular strips was greatly reduced by the β1 selective antagonist CGP 20712 (10−7 mol/l), and less so by ICI 118551 (10−7 mol/l, β2 selective). CGP 20712 and ICI 118551 together (both 3×10-6 mol/l) had no effect on taenia relaxation by SR 58611A and rendered isoprenaline and terbutaline virtually inactive on circular strips, although not on taenia, which was relaxed at higher than control concentrations and maximally by isoprenaline. Propranolol, a β1, β2 non-selective antagonist, at high concentrations (10-5 mol/l) prevented taenia relaxation by CGP 12177 and SR 58611A; its quantitative antagonism of isoprenaline (in common with that of CGP 12177 used as an antagonist) was competitive in circular strips but not on taenia. CONCLUSIONS β1, β2, and β3 adrenoceptors are functionally detectable in the human colon; agonist stimulation of any one type relaxed taenia but only isoprenaline was fully effective at the β3 subtype.

Lymphocytes genetically modified to express tumor antigens target DCs in vivo and induce antitumor immunity

The exploitation of the physiologic processing and presenting machinery of DCs by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. Here we show that lymphocytes genetically modified to express self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of tyrosinase-related protein 2-transduced (TRP-2-transduced) lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice. Analysis of the mechanism responsible for the induction of such an immune response allowed us to demonstrate that cross-presentation of the antigen mediated by the CD11c(+)CD8alpha(+) DC subset had occurred. Furthermore, we demonstrated in vivo and in vitro that DCs had undergone activation upon phagocytosis of genetically modified lymphocytes, a process mediated by a cell-to-cell contact mechanism independent of CD40 triggering. Targeting and activation of secondary lymphoid organ-resident DCs endowed antigen-specific T cells with full effector functions, which ultimately increased tumor growth control and animal survival in a therapeutic tumor setting. We conclude that the use of transduced lymphocytes represents an efficient method for the in vivo loading of tumor-associated antigens on DCs.

Clinical and immunologic responses in melanoma patients vaccinated with MAGE-A3-genetically modified lymphocytes.

Cancer vaccines have recently been shown to induce some clinical benefits. The relationship between clinical activity and anti‐vaccine T cell responses is somewhat controversial. Indeed, in many trials it has been documented that the induction of vaccine‐specific T cells exceeds the clinical responses observed. Here, we evaluate immunological and clinical responses in 23 MAGE‐A3+ melanoma patients treated with autologous lymphocytes genetically engineered to express the tumor antigen MAGE‐A3 and the viral gene product thymidine kinase of the herpes simplex virus (HSV‐TK). HSV‐TK was used as safety system in case of adverse events and as tracer antigen to monitor the immune competence of treated patients. The increase of anti‐TK and anti‐MAGE‐A3 T‐cells after vaccination was observed in 90 and 27% of patients, respectively. Among 19 patients with measurable disease, we observed a disease control rate of 26.3%, with one objective clinical response, and four durable, stable diseases. Three patients out of five with no evidence of disease (NED) at the time of vaccination remained NED after 73+, 70+ and 50+ months. Notably, we report that only patients experiencing MAGE‐A3‐specific immune responses showed a clinical benefit. Additionally, we report that responder and non‐responder patients activate and expand T cells against the tracer antigen TK in a similar way, suggesting that local rather than systemic immune suppression might be involved in limiting clinically relevant antitumor immune responses.

In vitro functional evidence of different neurotensin-receptors modulating the motor response of human colonic muscle strips

The newly developed non‐peptide neurotensin (NT)‐receptor antagonists SR 48692 and SR 142948 were used to challenge NT responses of human colonic circular smooth muscle strips in vitro. The presence of NT1 and NT2 receptor transcripts in this tissue was tested by reverse transcriptase polymerase chain reaction (RT–PCR) analysis. NT potently and dose‐dependently contracted muscle strips, with significant regional differences in potency and efficacy between the transverse and distal colon: EC50, 3.6 and 7.5 nM; the maximal effect was 70 and 55% of 0.1 mM carbachol. Colonic responses to NT in both segments were virtually the same in the presence of atropine (1 μM), levocabastine (10 μM) or tetrodotoxin (1 μM). SR 142948 (10 nM–1 μM) competitively antagonized NT responses in the transverse and distal colon with similar affinities: pA2 values 8.71 and 8.45, slopes 0.98 and 0.99. SR 48692 (10 nM–10 μM) antagonized the NT response competitively in the distal colon (pA2 6.55, slope 0.79) and non‐competitively in the transverse colon (pA2 8.0, slope 0.51). Neither compound had any agonist effect. The fact that the specific antagonists prevented NT‐evoked atropine‐ and tetrodotoxin‐insensitive mechanical responses of colonic muscle strips is highly consistent with the presence in these tissues of non‐neuronal NT receptors, whose heterogeneity in the transverse segment is supported by the non‐competitive antagonism of SR 48692. The finding of NT1 receptor transcript in both transverse and distal colon suggests its identity with the lower affinity site disclosed functionally by SR 48692 in these segments.

Update on vaccines for melanoma patients

Vaccination against melanoma has a long history but only recently has this therapeutic approach found a reliable scientific rationale. This review summarizes and updates the knowledge on the role of the main players in the vaccination strategy against metastatic melanoma, that is, the melanoma-associated antigens recognized by T and natural killer T lymphocytes, the conditions for the immune response to develop and the mechanisms that interfere with it. A commented list of the major vaccination trials whose results have been recently published or are ongoing is also presented. Reasons for failure and potential success are underlined to offer a balanced view of this scientifically exciting but still clinically unrewarding field of investigation.