Search

Video 1 shows the real time phagocytosis of cancer cells in presence of NI-1701

CD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal “don't eat me” signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701–induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20–targeted therapy. Mol Cancer Ther; 17(8); 1739–51. ©2018 AACR.

Supplementary Table S3 shows the ratio of AUC0-inf low/high dose for the PK single dose study.

Supplementary material and methods

Supplementary Table S5 shows the hematological results of cynomolgus monkeys treated with vehicle (Ctr.) or NI-1701 (Treat.) at 30 and 100 mg/kg over 4 weeks.

Supplementary Figure S1 shows shows the percentage of in vitro phagocytosis of Raji cells by macrophages with NI-1701 or CD19/CD47hi biAb.

Supplementary Table S2 shows the cell surface density (expressed as number of receptors per cell) for CD19, CD47, CD20 on different human tumor cell lines.

Video 2 shows the real time phagocytosis of cancer cells in presence of hIgG1 control

Supplementary Table S4 shows the mean Cmax values from the DRF and the single dose study.

Supplementary Table S1 shows the cell surface density (expressed as number of receptors per cell) for CD19, CD47, CD20 on different subpopulations in human whole blood

Supplementary Figure S2 shows in vitro hemagglutination in human or cynomolgus monkeys whole blood, or platelet aggregation in human platelet rich-plasma (PRP)in presence of NI-1701

PD-1/PD-L1 blockade has improved survival across many types of cancer, but only in a minority of patients. Co-targeting PD-1/PD-L1 and the CD47/SIRPα myeloid checkpoint with monoclonal antibody (mAb) combinations showed increased antitumor responses in preclinical studies. However, CD47 mAbs are hindered by ubiquitous CD47 expression leading to rapid target-mediated clearance and safety concerns, including anemia and thrombocytopenia. Consequently, dual-targeting CD47xPD-L1 bispecific antibodies (bsAbs) enabling selective inhibition of CD47 on PD-L1-positive tumors offer an alternative approach. A fully human bsAb pairing a high affinity PD-L1 arm to a low affinity CD47 arm was generated using the κλ-body platform. The latter is also used in our CD47xCD19 bispecific antibody NI-1701/TG-1801, currently in phase I clinical trials (NCT03804996, NCT04806035). The resulting CD47xPD-L1 bsAb of human IgG1 isotype (NI-2601) was evaluated in various binding and receptor-blocking assays, and then tested for its capacity to enhance T-cell activation in vitro and induce Fc-mediated killing of tumor cells through phagocytosis (ADCP) and antibody-dependent cell cytotoxicity (ADCC). A surrogate bsAb was also evaluated in vivo in a syngeneic mouse model. NI-2601 demonstrated effective blockade of PD-1/PD-L1 interaction, and T-cell activation in vitro, similar to the anti-PD-L1 clinical benchmarks atezolizumab and avelumab. Consistent with its low-affinity CD47 arm, the bsAb did not bind to red blood cells (RBC) and CD47 blockade was driven by PD-L1 co-engagement. Using a panel of tumor cell lines, expressing various PD-L1 levels, NI-2601 showed superior activity in ADCP and ADCC as compared to the anti-PD-L1 IgG1 mAb, avelumab. The anti-tumor activity of this approach using surrogate CD47xPD-L1 bsAb was confirmed in a syngeneic MC38 colon carcinoma model. Thus, NI-2601 is able to harness Fc-effector function to eliminate PD-L1-positive tumor cells while sparing PD-L1-negative cells, such as RBC or platelets. Pharmacokinetic and tolerability studies in non-human primate are planned for 2022. Citation Format: Xavier Chauchet, Nicolas Bosson, Margaux Legrand, Laura Cons, Sébastien Calloud, Alizée Viandier, Françoise Richard, Pauline Malinge, Tereza Bautzova, Jérémie Bourguignon, Guillemette Pontini, Elise Penarrieta, Mengzhu Sun, Ulla Ravn, Valéry Moine, Giovanni Magistrelli, Yves Poitevin, Stéphanie Hugues, Limin Shang, Walter Ferlin, Krzysztof Masternak. NI-2601, an Fc-active CD47xPD-L1 bispecific antibody that selectively targets CD47 on PD-L1-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3429.

CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.

BackgroundPD-1/PD-L1 blockade can significantly improve survival across many types of cancer, but only in a minority of patients. To broaden its therapeutic efficacy, several combination partners are now being evaluated together with PD-1/PD-L1 blockade. Agents blocking CD47/SIRPα innate immune checkpoint are one such example, and co-targeting PD-1/PD-L1 and CD47 with monoclonal antibody (mAb) combinations showed increased antitumor responses in preclinical studies. However, CD47 mAbs are hindered by ubiquitous CD47 expression leading to rapid target-mediated clearance and safety concerns. Consequently, dual-targeting CD47xPD-L1 bispecific antibodies (bsAbs) enabling preferential inhibition of CD47 on PD-L1-positive cells are being tested as an alternative approach. We compare here two distinct bsAbs, based on a common PD-L1 antibody arm, with differing FcgR-enabling effector functions and CD47-binding arm affinities.MethodsAn array of fully human bsAbs associating a high affinity PD-L1 arm to CD47 arms with varying affinities were generated using the κλ-body platform.1 CD47xPD-L1 bsAbs of human IgG1 isotype (CD47 low affinities) or IgG4 isotype (CD47 high affinities) were screened in various binding assays (including to red blood cells (RBC)) and in receptor-blocking assays, and then tested for their Fc-mediated killing and T-cell activation activity (SEA-stimulated PBMC assay). Selected molecules were evaluated in vivo.ResultsBoth bsAb approaches demonstrated strong blockade of PD-1/PD-L1 interaction and significantly enhanced T-cell activation in vitro. CD47lowxPD-L1 IgG1 bsAbs did not bind to RBC and showed PD-L1-guided inhibition of CD47. ADCP and ADCC experiments with a panel of tumor cell lines expressing various target levels showed superior killing activity with CD47lowxPD-L1 IgG1 bsAbs as compared to the anti-PD-L1 IgG1 mAb, avelumab. On the other hand, CD47highxPD-L1 IgG4 bsAbs showed residual RBC binding and PD-L1-independent blocking of CD47/SIRPα. These CD47high IgG4 bsAbs were able to enhance the anti-tumor activity of anti-tumor-associated antigen (TAA) mAbs in vitro (phagocytosis), and in vivo (Raji lymphoma xenograft model). In addition, anti-tumor activity of mouse CD47xPD-L1 bsAbs in a syngeneic MC38 colon carcinoma model was demonstrated.ConclusionsWith the objective of finding the optimal CD47xPD-L1 bsAb design, two approaches targeting CD47 and PD-L1 inhibition were tested. Both the CD47lowxPD-L1 IgG1 bsAbs and CD47highxPD-L1 IgG4 bsAbs were able to mediate enhanced antitumor responses, the former as a standalone treatment, the latter in conjunction with an anti-TAA mAb. To further characterize the CD47lowxPD-L1 and CD47highxPD-L1 bsAbs, lead candidates will be tested in PK and tolerability studies in non-human primates.ReferencesFischer N, Elson G, Magistrelli G, Dheilly E, Fouque N, Laurendon A, et al. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat Commun 2015 May;6(1):6113.

Richard Françoise. Des pionnières au Moyen-Âge La communauté des béguines. In: Diplômées, n°270-271, 2020. Les Pionnières. pp. 7-17.

Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in several solid malignancies, including gastric, lung, mesothelioma, pancreatic and ovarian cancers. While several MSLN-targeting therapeutic approaches are in development, only limited efficacy has been achieved in patients. A potential shortcoming of several described antibody-based approaches is that they target the membrane distal region of MSLN and, additionally, are known to be handicapped by the high levels of circulating soluble MSLN in patients. We show here, using monoclonal antibodies (mAbs) targeting different MSLN-spanning epitopes, that the membrane-proximal region resulted in more efficient killing of MSLN-positive tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Surprisingly, no augmented killing was observed in antibody-dependent cellular phagocytosis (ADCP) by mAbs targeting this membrane-proximal region. To further increase the ADCP potential, we, therefore, generated bispecific antibodies (bsAbs) coupling a high-affinity MSLN binding arm to a blocking CD47 arm. Here, targeting the membrane-proximal domain of MSLN demonstrated enhanced ADCP activity compared to membrane-distal domains when the bsAbs were used in in vitro phagocytosis killing assays. Importantly, the superior anti-tumor activity was also translated in xenograft tumor models. Furthermore, we show that the bsAb approach targeting the membrane-proximal epitope of MSLN optimized ADCC activity by augmenting FcγR-IIIA activation and enhanced ADCP via a more efficient blockade of the CD47/SIRPα axis.

In this paper, we give new characterizations for the eigenvalues of the prolate wave equation as limits of the zeros of some families of polynomials: the coefficients of the formal power series appearing in the solutions near 0, 1 or ∞ (in the variables x, x − 1 or 1/x respectively). The result, which seems to be true for all values of the parameter τ , according to our numerical experiments, is here proved for small values of the parameter τ .

CD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal “don't eat me” signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701–induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20–targeted therapy. Mol Cancer Ther; 17(8); 1739–51. ©2018 AACR.

Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.

In this paper we will present the family of Newton algorithms. From the computer algebra point of view, the most basic of them is well known for the local analysis of plane algebraic curves f(x,y)=0 and consists in expanding y as Puiseux series in the variable x. A similar algorithm has been developped for multi-variate algebraic equations and for linear differential equations, using the same basic tools: a “regular” case, associated with a “simple” class of solutions, and a “simple” method of calculus of these solutions; a Newton polygon; changes of variable of type ramification; changes of unknown function of two types y=ctμ+ϕ or y=exp (c/tμ)ϕ.

Proneurotensin/neuromedin N (pro-NT/NN) is the common precursor of two biologically active related peptides, neuromedin N (NN) and neurotensin (NT). It undergoes a tissue-specific processing leading to the formation in some tissues and cancer cell lines of large peptides ending with the NT (large NT) or NN (large NN) sequence. In this study, we prepared and purified high amounts of recombinant large NT and large NN using the Drosophila S2 cell expression system. The binding and pharmacological properties of recombinant large peptides were characterized and compared to those of NT and NN using either COS cells transfected with the human subtype-1 NT receptor (hNTS1) or the human colon adenocarcinoma HT29 cell line that endogenously expresses hNTS1. Furthermore, the metabolic stability of the large peptides, when exposed to HT29 cells, was compared to that of NT and NN. Both large NT and large NN were able to bind to and activate hNTS1 with potencies that were approximately 10 times lower than that of their small counterpart. In addition, the large forms proved to be far less sensitive to degradation than the small peptides. Taken together, these data suggest that the large forms might represent endogenous, long-lasting activators of hNTS1 in a number of physiopathological situations.

The neurotensin receptor 1, NTS1, is a G protein-coupled receptor with seven transmembrane domains (TM) that mediates most of the known effects of the neuropeptide. Our previous studies have pointed to extracellular loop 3 and adjacent TM7 as being potentially involved in agonist-induced activation of the NTS1. Here we investigated residues in these domains that might be involved in transconformational activation of the rat NTS1. Single amino acid mutated receptors were expressed in COS cells and inositol phosphate (IP) and cyclic AMP productions were studied. The F358A mutation in TM7 resulted in a time- and receptor concentration-dependent increase in spontaneous IP production. At expression levels of 12 pmol mg−1, agonist-independent IP production was increased 10 fold over basal for the F358A mutant receptor whereas the wild type NTS1 exhibited virtually no spontaneous activity at expression levels of 7.5 pmol mg−1. Neurotensin remained agonist on the F358A mutant receptor with a maximal effect that amounted to greater than twice basal IP levels. SR 48692 was inverse agonist at the mutant receptor, reversing IP production almost back to the levels measured in wild type NTS1-transfected cells. Cyclic AMP production was not constitutively activated with the F358A mutant receptor but was stimulated by neurotensin with the same concentration dependence as that observed with the wild type NTS1. This is the first report, to our knowledge, of a constitutively active mutant of the NTS1. The data are consistent with TM7 being involved in the transconformational changes that lead to agonist-induced coupling of the NTS1 to Gq. British Journal of Pharmacology (2002) 135, 997–1002; doi:10.1038/sj.bjp.0704546

Experiments were performed with human plasma irradiated in vitro or in vivo in order to evaluate the extent to which clastogenic factors might disturb the adaptive response to DNA-damaging factors currently studied in our laboratory. The studies were carried out with plasma isolated from whole blood given 4 Gy of X-rays in vitro and with plasma from people receiving local radiotherapy at a total dose of about 60 Gy gamma rays. Addition of irradiated plasma to culture medium did not result in a statistically significant increase in structural aberrations in chromosomes of non-irradiated normal blood.

With the aim of limiting hospitalisation and a possible loss of autonomy, the paramedical team of the post-emergency and geriatric orientation unit at Nancy general hospital endeavours to anticipate the life programme as soon as the elderly patient arrives. A daily review and close collaboration with the social worker are key elements in the care.

The hydrogeochemistry of a small portion of a calcareous alluvial aquifer recharged by the Drac River (Grenoble, France) is characterized both in time and space. Significant changes in water chemistry are observed as the river water infiltrates into the aquifer. Processes occuring in the river bank (mostly biologically mediated) and in the aquifer (additional solid-liquid interactions) buffer some of the groundwater composition regardless of the variations in the river input. However, small zones (of enhanced biological activity due to low permeability and/ or high organic carbon content) located away from the river bank can induce significant localized changes in groundwater quality (such as locally high dissolved Mn due to the dissolutive reaction of MnO 2(s) and/or MnOOH (s) ).

Radiobiological data indicate that fast neutrons could bring a benefit in the treatment of some tumour types, and suggest mechanisms through which this benefit could be achieved. However, radiobiology also clearly indicates that there is a need for patient selection as well as for a high-physical selectivity. The main difficulty when interpreting the results of neutron therapy are the poor technical conditions in which the first treatments were applied. This explains why the value and the place of neutron therapy are not universally recognized, although more than 15,000 patients have been treated so far worldwide. There are, however, clinical indications of fast neutrons bringing a benefit for the following tumour sites: salivary glands, paranasal sinuses, soft tissue sarcomas, prostatic adenocarcinomas, palliative treatment of melanoma and rectum. These tumours represent about 10-15% of all patients currently referred to the radiation therapy departments.

International audience; We describe a general procedure for computing Stokes matrices for solutions of linear differential equations with polynomial coefficients. The algorithms developed make an automation of the calculations possible, for a wide class of equations. We apply our techniques to some classical holonomic functions and also for some new special functions that are interesting in their own right: Ecalle's accelerating functions.

The ontogenetic variations of tyrosine hydroxylase (TH) have been studied in locus coeruleus of developing rats. During the first 2 weeks after birth, a large increase in TH content (6.04–23.99 TH units) in the noradrenergic structure was observed, followed by a period of progressive increase of the protein concentration (42 TH units in adult rats). The expression of TH was studied in the same ontogenetic period after treatment by RU24722 (20 mg/kg, i.p.). The long-term increase in TH concentration produced by the drug was found to follow ontogenetic variations. It becomes significant around the middle of the second week after birth and gradually increases until the 24th day of postnatal development, indicating a maturation of the mechanisms involved in the inducing effect.

Catecholamine metabolism was studied in vivo in the Cl adrenergic area of the rostral ventrolateral medulla oblongata in rats, using differential normal pulse voltammetry coupled with an activated carbon fiber microelectrode. Pharmacological evidence indicates that 3,4-dihydroxyphenylacetic acid, the major dopamine metabolite, is responsible for the electrochemical signal appearance in the C1 group, and that it reflects the Catecholamine synthesis rate, as previously reported in the locus coeruleus. Indeed, 3,4-dihydroxyphenylacetic acid was estimated to be formed from 77% of the intracellular dopamine, since its synthesis was increased by only 23%, after blockade of the dopamine-β-hydroxylase activity. Neuronal activation by retrograde electrical stimulation increased the electrochemical signal, as well as hemorrhage and hypotension, suggesting that the level of extracellular 3,4-dihydroxyphenylacetic acid is a good biochemical index of the C1 adrenergic cellular activity in baseline conditions and during cellular activation.

e14016 Background: Many cancers up-regulate the expression of CD47 in order to evade immune surveillance and killing. This ‘don’t eat me’ signal is a molecular means for healthy cells to limit thei...

Purpose: We analyzed the potential influence of adjuvant radiotherapy on urinary continence after radical prostatectomy.Materials and Methods: A total of 100 patients with N0M0 prostate cancer randomized in a prospective study on postoperative radiotherapy for locally advanced disease (positive surgical margin, capsular perforation and/or seminal vesicle infiltration) were studied. Objective pad weighing tests corroborated by direct personal interviews were used to evaluate urinary continence at regular postoperative intervals.Results: Of the patients 48 received 60 Gy. external radiotherapy with 18 MV photon beams between 12 and 16 weeks postoperatively, and 52 were followed expectantly. Risk factors were similar in both groups. With a mean followup of 24 months, no difference in complete urinary continence was observed. Of the irradiated group 77% and of the surveillance group 83% were totally dry. The fate of the bladder neck had no significant influence on final continence status, although the...

L'unite d'hebergement renforce (UHR), specifique des unites de soins de longue duree (USLD) est un lieu de vie et de soins qui fonctionne nuit et jour. Elle propose sur un meme lieu l'hebergement, les soins, les activites sociales et therapeutiques pour des personnes âgees repondant aux criteres d'admission en USLD et presentant des troubles du comportement perturbateurs severes dans le cadre de l'evolution d'une maladie d'Alzheimer ou de maladies apparentees.

PURPOSE: Between 1978 and 1998, 533 prostate adenocarcinoma patients were treated with mixed photon-neutron radiotherapy. We report on a retrospective series of patients for whom the side effects of the treatment and their impact on quality of life were assessed by a mailed questionnaire. METHODS AND MATERIALS: The European Organization for Research and Treatment of Cancer quality-of-life core questionnaire and a prostate-specific questionnaire were used. Between 1990 and 1996, 308 consecutive patients were treated. Two protocols were used: pelvic fields (50 Gy equivalent photons, 2 Gy/fraction) followed by a prostate boost (66 Gy) or prostate alone. The neutron/photon ratio varied. The questionnaire was mailed to 262 patients presumed to be alive. RESULTS: Of the 262 patients, 230 replied. Of the 230 patients, 73% had no trouble doing strenuous activities and 4% had trouble with taking a short walk. The overall physical condition and quality-of-life questions received a mean score of 5.2 and 5.3 on a 7-point scale, respectively. Twenty-two percent had bowel movements at least four times daily, and 6% did so six times or more. Retaining stool was a problem in 26%, and only 38% reported full continence; 17% urinated four times or more nightly. Urinary incontinence was scored as "quite a bit" or "very much" in 11% and 5%, respectively. Hematuria and dysuria (pain) were reported by 7% and 16%, respectively, mainly as moderate. Only 28% reported easy erections, but 75% judged the sexual change acceptable. A greater neutron/photon ratio was significantly associated with more bowel problems (p = 0.003). CONCLUSION: Mixed photon-neutron therapy for prostate cancer was associated with significant patient-reported side effects. Their significant effect on patients' quality of life is described.

Two G protein-coupled neurotensin (NT) receptors, termed NTR1 and NTR2, have been identified so far. In contrast to the NTR1, which has been extensively studied, little is known about the pharmacological and biological properties of the NTR2. In the course of characterizing NT analogs that exhibited binding selectivity for the NTR2, we discovered that this receptor constitutively activated inositol phosphate (IP) production. Here, we report on the constitutive activity of the human NTR2 (hNTR2) transfected in COS cells and on compounds that exhibit agonism, inverse agonism, and neutral antagonism at this receptor. IP levels increased linearly with time, whereas they remained constant in mock-transfected cells. Furthermore, IP production was proportional to the amount of hNTR2 present at the cell membrane. SR 48692, a nonpeptide antagonist of the NTR1, stimulated IP production, whereas levocabastine, a nonpeptide histamine H1 antagonist that binds the NTR2 but not the NTR1, behaved as a weak partial inverse agonist. NT analogs modified at position 11 of the NT molecule, in particular by the introduction of bulky aromatic D amino acids, exhibited binding selectivity at the hNTR2 and also behaved as partial inverse agonists, reversing constitutive IP production up to 50%. Finally, NT barely affected constitutive IP production but antagonized the effects of both agonist and inverse agonist compounds, thus behaving as a neutral antagonist. The unique pharmacological profile of the hNTR2 is discussed in the light of its sequence similarity with the NTR1 and the known binding site topology of NT and SR 48692 in the NTR1.

PURPOSE: To retrospectively evaluate survival, progression-free survival (PFS) and biological response in a series of patients irradiated with mixed neutron/photon beams for locally advanced prostate cancer in our institution. PATIENTS AND METHODS: Three hundred and eight patients were treated between January 1990 and December 1996. Fifty-five of these were recruited for pT3 or pN1 tumors after radical prostatectomy. Neoadjuvant androgen deprivation was given in 106 patients. The treatment protocol consisted of a mixed photon/neutron irradiation in a two-to-three proportion, up to a total equivalent dose of 66 Gy (assuming a clinical RBE value of 2.8). Pre- and post-treatment PSA determinations were available in practically all cases. Study endpoints were overall survival (OAS) and progression-free survival (PFS). The Cox proportional hazard regression model was used to investigate the prognostic value of baseline characteristics on survival and progression-free survival were a progression was defined as local, regional, metastatic or biological progression. Mean age was 69 years (49-86); mean pretreatment PSA was 15 (0.5-330) in all patients and 14 (0.5-160) in those receiving neoadjuvant hormonotherapy; seven patients only had an initial PSA < or = 4 ng/mL; 15% were T1, 46% were T2, 28% were T3 or pT3 and 4% were T4 (7% unspecified); WHO grade of differentiation was I in 38%, II in 38% and III in 14% (5% unspecified). RESULTS: The median follow-up was 2.8 years (0-7.8). Five-year overall survival (OAS) was 79% (95% CI: 71-87%) and 5-year progression-free survival (PFS) was 64% (95% CI: 54-74%) for the entire series. PFS in patients with an initial PSA > or = 20 ng/mL was the same. PFS could be predicted by two optimal Cox regression models, one including histological grade (p = 0.003) and initial PSA (p = 0.0009) as cofactors, the other including histological grade (p = 0.003) and T stage (p = 0.02). The main prognostic factors for overall survival were PSA and age. Biological responses with PSA < 1.5 ng/mL, < 1 ng/mL and < 0.5 ng/mL at any time after treatment were documented in 70%, 61% and 47% of the patients, respectively. CONCLUSION: Five-year OAS was 79%, PFS was 64%, and biological response was 70% for prostate cancer patients treated with mixed photon/neutron beams as applied at Louvain-la-Neuve, which are good results as compared with the literature. The usual prognostic factors were confirmed.

The multileaf collimator of the fast neutron therapy facility at Louvain-la-Neuve is described, as well as some of the physics experiments performed in order to evaluate the attenuation of neutron beams in different materials and thus optimize the composition of the collimator leaves. The multileaf collimator consists of two sets of 22 leaves each, which can be moved independently. They are made of iron and their thickness is 95 cm. Seven borated polyethylene disks are located in the distal part of the leaves in order to absorb more efficiently the low-energy component of the neutron spectrum. The width of the leaves is 1 cm at their distal part. The leaves can move 11 cm outwards and 6 cm inwards from their reference position, and field sizes up to 25.7 x 24.8 cm, as well as irregular field shapes, can be obtained. The inner part of the leaves and their two sides are always focused on the target. The complete multileaf collimator can rotate around the beam axis, from -90 degrees to +90 degrees from the reference position. The width of the penumbra (80-20% isodoses) is 0.64 cm and 1.17 cm at the depth of the maximum buildup and at 10 cm in depth respectively, for a 10 x 10 cm field size. The collimator is adequate for the energy of the p(65)+Be neutron beam of Louvain-la-Neuve and has been adapted to the fixed vertical beam. It has been designed following the original plans of Scanditronix, adjusted and fully assembled at the workshop of the Centre de Recherches du Cyclotron (CRC). Systematic measurements were performed in order to optimize the design and the composition of the leaves. In particular the attenuations of the actual beam and of monoenergetic neutron beams were measured in different materials such as iron and polyethylene. Above (upstream) the multileaf collimator, a fixed pre-collimator (iron thickness 50 cm; section 1 x 1 m) defines a conical aperture aligned on the largest opening of the leaves. It contains the two transmission chambers and a 2 cm thick polyethylene layer used for hardening the beam.

A PET camera is used to visualize the positron activity induced during protonbeam therapy in order to verify directly the proton-treatment plans. The positron emitters created are predominantly the 15O and 11C, whose total activity amounts to 12 MBq after an irradiation with 85 MeV protons, delivering 3 Gy in a volume of approximately 300 cm3. Although this method is a useful verification of patient set-up, care must be taken when deriving dose distributions from activity distributions. Correlation between both quantities is difficult, moreover at the last millimeters of their range, protons will no longer activate tissue. Due to the short half-lives the PET camera must be located close to the treatment facility.

We reviewed retrospectively a series of 58 patients with deeply invasive bladder cancer treated with fast neutron therapy (p(65) + Be) in order to evaluate its tolerance and side effects. Patients were divided into three groups according to treatment technique. Patients of group A received whole pelvis irradiation up to 50 Gy photon equivalent followed by a boost to the bladder up to 57-66 Gy photon equivalent (40-56 days). Group B patients were treated by a split course regimen of 30 Gy photon equivalent on the whole pelvis at 3-4 weeks interval (66-108 days). Group C patients, not suitable for radical treatment, received only 40-54 Gy photon equivalent (26-70 days). The overall 5-year actuarial survival rate was 30% (SE 8%). As expected, T stage was a statistically significant prognostic factor. The overall local control rate reached 21% at 4 years. Acute and late side effects were minimal to moderate. These results suggest that high-energy neutron beam treatment is at least as effective as photon beam treatment for bladder carcinoma, without a higher incidence of major side effects.

Fast-neutron therapy has been used routinely on the cyclotron at the Catholic University of Louvain in Louvain-la-Neuve since March 1978. This paper reports the results obtained in bladder carcinoma, including local tumor control, survival, and radiation-induced side effects. Our data are compared with those reported in the literature after neutron or conventional photon-beam therapy.

Adult skeletal muscles in vertebrates are composed of different types of myofibers endowed with distinct metabolic and contraction speed properties. Genesis of this fiber-type heterogeneity during development remains poorly known, at least in mammals. Six1 and Six4 homeoproteins of the Six / sine oculis family are expressed throughout muscle development in mice, and Six1 protein is enriched in the nuclei of adult fast-twitch myofibers. Furthermore, Six1/Six4 proteins are known to control the early activation of fast-type muscle genes in myocytes present in the mouse somitic myotome. Using double Six1 : Six4 mutants ( SixdKO ) to dissect in vivo the genesis of muscle fiber-type heterogeneity, we analyzed here the phenotype of the dorsal/epaxial muscles remaining in SixdKO . We show by electron microscopy analysis that the absence of these homeoproteins precludes normal sarcomeric organization of the myofiber leading to a dystrophic aspect, and by immunohistochemistry experiments a deficiency in synaptogenesis. Affymetrix transcriptome analysis of the muscles remaining in E18.5 SixdKO identifies a major role for these homeoproteins in the control of genes that are specifically activated in the adult fast/glycolytic myofibers, particularly those controlling Ca 2+ homeostasis. Absence of Six1 and Six4 leads to the development of dorsal myofibers lacking expression of fast-type muscle genes, and mainly expressing a slow-type muscle program. The absence of restriction of the slow-type program during the fetal period in SixdKO back muscles is associated with a decreased HDAC4 protein level, and subcellular relocalization of the transcription repressor Sox6. Six genes thus behave as essential global regulators of muscle gene expression, as well as a central switch to drive the skeletal muscle fast phenotype during fetal development.

With conventional photon therapy, radiographs are widely used for the verification of patient positioning. This method cannot be used with neutrons because of the poor sensitivity of standard films to neutrons compared with γ rays. In addition, there is little difference in energy absorption between bone and soft tissue for these neutron beams (Bewley et al, 1973; Catterall & Bewley, 1979). The problem can be solved by introducing into the collimator, at the target position, a diagnostic X-ray tube (Risler & Wootton, 1988). Another approach entails visualizing the neutron-induced activity within the patient using positron emission tomography (PET). At high neutron energies, shortlived positron emitters are produced (Kleck et al, 1988; Vynckier et al, 1988) with activities sufficiently high to be visualized; the dose-equivalent rate close to the patient as a result of this activation is ∼ 30 μSv h−1 immediately after irradiation (Tatcher et al, 1987). The same approach was reported recently for pion radiot...

The effects of RU24722 (14,15-dihydro-20,21-di-noreburnamine-14-ol) on tyrosine hydroxylase in central catecholaminergic neurons were studied in rats treated with different quantities of the molecule, and a time course was done for the minimal dose that gave the maximal effect. RU24722 induced increases in tyrosine hydroxylase activities and specific protein content in noradrenergic cells of the locus ceruleus and decreased all these parameters in dopaminergic neurons of the substantia nigra and ventral tegmental area. The results pointed out that the specific activity of newly synthesized tyrosine hydroxylase in the loci cerulei was potentially greater but was not expressed “in vivo” except 7 days after injection. The phenotypic specificity and the time course pattern of the action could be considered as a consequence of an induction mechanism. The comparison of long-term change in tyrosine hydroxylase values after pipe-roxane, RU24722, clonidine, and combined RU24722-clon-idine treatment demonstrated that an activation during a few hours did not induce tyrosine hydroxylase in central noradrenergic neurons. Clonidine antagonized the activating effect of RU24722 following its injection but did not affect its long-term induction properties.

An improved quantitative immunochemical determination of brain tyrosine hydroxylase (TH) concentrations was designed using direct transfer into nitrocellulose from 20-microns thick brain sections, followed by immunodetection and quantitative radioautography in three reference brain structures (locus ceruleus, substantia nigra, and ventral tegmental area). Results obtained by this methodology were similar to those obtained after extraction and Western blotting of the TH protein in control and reserpine-treated animals. Moreover, this methodology allows the combination of high sensitivity and high anatomical resolution in the study of the distribution of pharmacological effects. The locus ceruleus exhibited a significant posteroanterior distribution of TH protein concentration in control and reserpine-treated animals.