An immunologically relevant rodent model demonstrates safety of therapy using a tumour-specific IgE.

BACKGROUND: Designing biologically informative models for assessing the safety of novel agents, especially for cancer immunotherapy, carries substantial challenges. The choice of an in vivo system for studies on IgE antibodies represents a major impediment to their clinical translation, especially with respect to class-specific immunological functions and safety. Fcε receptor expression and structure are different in humans and mice, so that the murine system is not informative when studying human IgE biology. By contrast, FcεRI expression and cellular distribution in rats mirror that of humans. METHODS: We are developing MOv18 IgE, a human chimeric antibody recognizing the tumour-associated antigen folate receptor alpha. We created an immunologically congruent surrogate rat model likely to recapitulate human IgE-FcεR interactions and engineered a surrogate rat IgE equivalent to MOv18. Employing this model, we examined in vivo safety and efficacy of antitumour IgE antibodies. RESULTS: In immunocompetent rats, rodent IgE restricted growth of syngeneic tumours in the absence of clinical, histopathological or metabolic signs associated with obvious toxicity. No physiological or immunological evidence of a "cytokine storm" or allergic response was seen, even at 50 mg/kg weekly doses. IgE treatment was associated with elevated serum concentrations of TNFα, a mediator previously linked with IgE-mediated antitumour and antiparasitic functions, alongside evidence of substantially elevated tumoural immune cell infiltration and immunological pathway activation in tumour-bearing lungs. CONCLUSION: Our findings indicate safety of MOv18 IgE, in conjunction with efficacy and immune activation, supporting the translation of this therapeutic approach to the clinical arena.

Harnessing engineered antibodies of the IgE class to combat malignancy: initial assessment of FcɛRI-mediated basophil activation by a tumour-specific IgE antibody to evaluate the risk of type I hypersensitivity.

BACKGROUND: IgE antibodies, sequestered into tissues and retained locally by the high-affinity IgE receptor, FcɛRI, on powerful effector cells such as mast cells, macrophages and eosinophils, may offer improvements in the therapy of solid tumours. The chimeric antibody, MOv18 IgE, against the human ovarian carcinoma antigen, folate receptor α (FRα), is more effective than its IgG1 counterpart in xenograft models of ovarian cancer. Although MOv18 IgE binds to a single epitope on FRα and cannot cross-link IgE receptors on basophils, there remains a risk that components in the circulation of ovarian cancer patients might cross-link FRα-MOv18-IgE-receptor-FcɛRI complexes on basophils to cause type I hypersensitivity. OBJECTIVE: To assess the propensity for MOv18 used in a therapeutic setting to cause FcɛRI-mediated type I hypersensitivity. METHODS: As validated readouts of the potential for MOv18 to cause FcɛRI-mediated type I hypersensitivity we measured release of a granule-stored mediator from a rat basophilic leukaemia cell line RBL SX-38 stably transfected with human tetrameric (αßγ2) FcɛRI, and induction of CD63 on blood basophils from patients with ovarian carcinoma and healthy controls ex vivo. RESULTS: Serum FRα levels were increased in ovarian cancer patients compared with healthy controls. MOv18 IgE alone, or in the presence of its antigen recombinant human FRα, or of healthy volunteer (n=14) or ovarian carcinoma patient (n=32) sera, did not induce RBL SX-38 cell degranulation. Exposure to FRα-expressing ovarian tumour cells at target-to-effector ratios expected within tumours induced degranulation. MOv18 IgE did not induce expression of CD63 in blood basophils from either healthy volunteers (n=6), or cancer patients, despite detectable levels of circulating FRα (n=5). CONCLUSION AND CLINICAL RELEVANCE: These encouraging data are compatible with the hypothesis that, when ovarian carcinoma patients are treated with MOv18, FcɛRI-mediated activation of effector cells occurs within the tumour mass but not in the circulation mandating, with due caution, further pre-clinical studies.

The control of microvascular permeability and blood pressure by neutral endopeptidase.

Plasma extravasation from postcapillary venules is one of the earliest steps of inflammation. Substance P (SP) and bradykinin (BK) mediate extravasation and cause hypotension. The cell-surface enzyme neutral endopeptidase (NEP) inactivates both peptides. Thus, absence of NEP may predispose development of inflammation and hypotension. We examined these possibilities in mice in which the NEP gene was deleted by homologous recombination. There was widespread basal plasma extravasation in postcapillary venular endothelia in NEP-/- mice, which was reversed by recombinant NEP and antagonists of SP (NK1) and BK (B2) receptors. Mean arterial blood pressure was 20% lower in NEP-/- animals, but this was unaffected by reintroduction of recombinant NEP and the kinin receptor antagonists. The hypotension was also independent of nitric oxide (NO), because NEP-/- mice treated with a NO synthase inhibitor remained hypotensive relative to the wild type. Thus, NEP has important roles in regulating basal microvascular permeability by degrading SP and BK, and may regulate blood pressure set point through a mechanism that is independent of SP, BK and NO. The use of NEP antagonists as candidate drugs in cardiovascular disease is suggested by the blood pressure data reported herein.

Enhancing ovarian cancer conventional chemotherapy through the combination with cannabidiol loaded microparticles.

In this work, we evaluated, for the first time, the antitumor effect of cannabidiol (CBD) as monotherapy and in combination with conventional chemotherapeutics in ovarian cancer and developed PLGA-microparticles as CBD carriers to optimize its anticancer activity. Spherical microparticles, with a mean particle size around 25 µm and high entrapment efficiency were obtained. Microparticles elaborated with a CBD:polymer ratio of 10:100 were selected due to the most suitable release profile with a zero-order CBD release (14.13 ± 0.17 µg/day/10 mg Mps) for 40 days. The single administration of this formulation showed an in vitro extended antitumor activity for at least 10 days and an in ovo antitumor efficacy comparable to that of CBD in solution after daily topical administration (≈1.5-fold reduction in tumor growth vs control). The use of CBD in combination with paclitaxel (PTX) was really effective. The best treatment schedule was the pre + co-administration of CBD (10 µM) with PTX. Using this protocol, the single administration of microparticles was even more effective than the daily administration of CBD in solution, achieving a ≈10- and 8- fold reduction in PTX IC50 respectively. This protocol was also effective in ovo. While PTX conducted to a 1.5-fold tumor growth inhibition, its combination with both CBD in solution (daily administered) and 10-Mps (single administration) showed a 2-fold decrease. These results show the promising potential of CBD-Mps administered in combination with PTX for ovarian cancer treatment, since it would allow to reduce the administered dose of this antineoplastic drug maintaining the same efficacy and, as a consequence, reducing PTX adverse effects.

Anti-PSMA 124I-scFvD2B as a new immuno-PET tool for prostate cancer: preclinical proof of principle.

BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer-related death in the Western population. The use in oncology of positron emission tomography/computed tomography (PET/CT) with emerging radiopharmaceuticals promises accurate staging of primary disease, restaging of recurrent disease and detection of metastatic lesions. Prostate-specific membrane antigen (PSMA) expression, directly related to androgen-independence, metastasis and progression, renders this tumour associate antigen a good target for the development of new radiopharmaceuticals for PET. Aim of this study was to demonstrate in a preclinical in vivo model (PSMA-positive versus PSMA-negative tumours) the targeting specificity and sensitivity of the anti-PSMA single-chain variable fragment (scFv) labelled with 124I. METHODS: The 124I-labeling conditions of the antibody fragment scFvD2B were optimized and assessed for purity and immunoreactivity. The specificity of 124I-scFvD2B was tested in mice bearing PSMA-positive and PSMA-negative tumours to assess both ex-vivo biodistribution and immune-PET. RESULTS: The uptake fraction of 124I-scFvD2B was very high on PSMA positive cells (range 75-91%) and highly specific and immuno-PET at the optimal time point, defined between 15 h and 24 h, provides a specific localization of lesions bearing the target antigen of interest (PSMA positive vs PSMA negative tumors %ID/g: p = 0.0198 and p = 0.0176 respectively) yielding a median target/background ratio around 30-40. CONCLUSIONS: Preclinical in vivo results of our immuno-PET reagent are highly promising. The target to background ratio is improved notably using PET compared to SPECT previously performed. These data suggest that, upon clinical confirmation of sensitivity and specificity, our anti-PSMA 124I-scFvD2B may be superior to other diagnostic modalities for PCa. The possibility to combine in patients our 124I-scFvD2B in multi-modal systems, such as PET/CT, PET/MR and PET/SPECT/CT, will provide quantitative 3D tomographic images improving the knowledge of cancer biology and treatment.

Panning phage antibody libraries on cells: isolation of human Fab fragments against ovarian carcinoma using guided selection.

The display of repertoires of human antibody (Ab) fragments on filamentous phages and selection by binding of the phage to antigen (Ag) have provided a ready means of deriving human Ab against purified Ag. However, it has been more difficult to obtain phage Ab against an individual Ag of a complex mixture, such as cell surface Ag. Using the technique of "guided selection," we generated human Ab against the high-affinity folate-binding protein (FBP), a cell surface Ag that is overexpressed in many human ovarian carcinomas. The guiding Ab template was provided by the light chain of mouse monoclonal Ab Mov19 (K[aff], 10[8] M[-1]) directed against FBP; this was paired with repertoires of human heavy chains displayed on phages, and the hybrid Ab fragments were selected by binding to an ovarian carcinoma cell line (OVCAR3). The selected human heavy chains were then paired with repertoires of human light chains. Further panning led to the isolation of a human Fab fragment, C4, with a binding affinity of 0.2 x 10(8) M(-1). This was highly specific for FBP, as demonstrated by ELISA and flow cytometry data and by immunoprecipitation of the relevant molecule from the cell surface of ovarian carcinoma cells. Moreover, C4 targeted the same or a closely related epitope of the Ag, as did the template rodent monoclonal Ab Mov19. These results suggest the usefulness of guided selection as a simple means to deriving human Ab against cell surface Ag for which a rodent Ab is available.

Targeting of interleukin 2 to human ovarian carcinoma by fusion with a single-chain Fv of antifolate receptor antibody.

To provide a new tool for the immunotherapy of human ovarian carcinoma, we constructed a fusion protein between interleukin-2 (IL-2) and the single-chain Fv (scFv) of MOV19, a monoclonal antibody directed against alpha-folate receptor (alpha-FR), known to be overexpressed on human nonmucinous ovarian carcinoma. This was accomplished by fusing the coding sequences in a single open reading frame and expressing the IL-2/MOV19 scFv chimera under the control of the murine immunoglobulin K promoter in J558L plasmacytoma cells. The design allowed the construction of a small molecule combining the specificity of MOV19 with the immunostimulatory activity of IL-2. This might improve the tissue penetration and distribution of the fusion protein within the tumor, reduce its immunogenicity, and avoid the toxicity related to the systemic administration of IL-2. The IL-2/MOV19 fusion protein was stable on purification from the cell supernatant and was biologically active. Importantly, this construct was able to target IL-2 onto the surface of alpha-FR-overexpressing tumor cells and stimulated the proliferation of the IL-2-dependent CTLL-2 cell line as well as that of human resting peripheral blood lymphocytes. In a syngeneic mouse model, IL-2/MOV19 scFv specifically targeted a-FR gene-transduced metastatic tumor cells without accumulating in normal tissues, due to its fast clearance from the body. Prolonged release of IL-2/MOV19 scFv by in vivo transplanted J558-EF6.1 producer cells protected 60% of mice from the development of lung metastases caused by an i.v. injection of a-FR gene-transduced tumor cells. Moreover, treatment with IL-2/MOV19 scFv, but not with recombinant IL-2, significantly reduced the volume of s.c. tumors. The pharmacokinetics and biological characteristics of IL-2/NMOV19 scFv might allow us to combine the systemic administration of this molecule with the adoptive transfer of in vitro retargeted T lymphocytes for the treatment of ovarian cancer, thereby providing local delivery of IL-2 without toxicity.

Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to alpha-folate receptor overexpression.

Caveolin (cav-1) and the GPI-anchored alpha-folate receptor (alphaFR) are membrane proteins both found associated to caveolar structures. Several studies in tumor cells independently reported cav-1 downregulation and alphaFR overexpression. Here we analysed the expression of the two molecules in normal and tumor ovarian samples derived from fresh specimens and from cultured cell lines. Whereas normal ovary surface epithelial cells displayed only cav-1 expression, ovarian tumor surgical samples and cell lines (COR, IGROV1, OVCAR3 and OVCA432) displayed high alphaFR and low-level or no cav-1 expression, except those cell lines (SKOV3 and SW626) with the lower alphaFR expression. SKOV3, but not two alphaFR-negative non-ovarian cell lines, exhibited down-regulation of cav-1 expression following stable alphaFR cDNA transfection. Conversely, cav-1 transfection in IGROV1 cells led to downregulated alphaFR expression, together with formation of caveolar structures and reduction of growth capability. Moreover, cav-1 expression was induced in IGROV1 cells by transfection with intracellular anti-alphaFR antibodies to downmodulate alphaFR expression. In cav-1 transfected cells, transcriptional activity of the alphaFR-specific promoter P1 was reduced by 70% and an additional specific DNA-protein complex was identified by gel-shift assay, indicating that cav-1 expression influences alphaFR gene transcription. Together these results support the notion that alphaFR and cav-1 protein expression is reciprocally regulated in ovary cancer cells.

In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation.

Antibodies can be made from repertoires of associated heavy and light chains displayed on the surface of bacteriophage, and are readily diversified by random point mutation or by chain shuffling. To make extensive variations around the "core" antigen binding contacts of a crystallographically solved mouse antibody NQ10/12.5 (gamma l, kappa), the NQ10 light chain was assembled in vitro with a repertoire of about 10(7) human heavy chains displayed on the surface of phage, and selected by binding to hapten. An antibody with a much improved affinity was isolated from the repertoire (K(a) = 10(9) M-1 compared with 10(8) M-1 for NQ10). The sequence of the human heavy chain (VH-IL) was highly related to NQ10. It conserved the same folds for the H1, H2 and H3 loops, six of the seven contact residues for hapten, and also a phOx binding motif (Asp-X-Gly-X-X) in the H3 loop. It appears that the new heavy chain partners for the NQ10 light chain often retain many critical antigen binding features found in the NQ10 heavy chain.

Acute ACE inhibition causes plasma extravasation in mice that is mediated by bradykinin and substance P.

The use of angiotensin-converting enzyme (ACE) has been associated with the occurrence of adverse effects, including cough and angioneurotic edema. Accumulation of kinins has been suggested to play a major role in these adverse effects of ACE inhibitor, although conclusive evidence for such a role is lacking. We investigated whether ACE inhibition increases plasma extravasation in mice (Swiss, C57Bl/6J, and J129Sv/Ev strains) via inhibition of bradykinin metabolism and stimulation of neurogenic inflammatory mechanisms. Intravenous captopril and enalapril increased the extravasation of Evans blue dye in all tissues examined (trachea, stomach, duodenum, and pancreas). This effect was evident 15 minutes after drug administration. The particulate dye Monastral blue identified the sites of captopril-induced leakage in the microvasculature. Pretreatment with the bradykinin B2 receptor antagonist Hoe 140 or with the tachykinin NK1 receptor antagonist SR 140333 inhibited captopril-evoked increase in plasma extravasation. In mice in which the gene encoding the bradykinin B2 receptor was disrupted by gene targeting, neither bradykinin nor captopril increased plasma extravasation. Pretreatment with Hoe 140 did not reduce the hypotensive response induced by captopril. The present findings suggest that ACE inhibition increases kinin levels in tissues and/or plasma. These increased kinin levels increase microvascular leakage in mouse airways and digestive tract via the release of tachykinins from terminals of primary sensory neurons. Exaggerated kinin production and the subsequent stimulation of peptide release from sensory nerves may be involved in adverse effects of ACE inhibitors.

Transfer of chimeric receptor gene made of variable regions of tumor-specific antibody confers anticarbohydrate specificity on T cells.

The antitumor specificity of T cells can be induced by gene transfer using a recently developed therapeutic approach (T body). In this work, we genetically conferred anticarbohydrate specificity onto T cells using the variable regions of monoclonal antibody MLuC1, which binds the Lewis(Y) (LeY) tumor-associated antigen that is overexpressed on several human carcinomas. The variable regions of MLuC1, which are in a single-chain Fv (ScFv) configuration, were cloned and spliced in a eukaryotic expression vector with both the gene encoding the signal-transducing gamma-chain of the human Fcgamma receptor and a flexible hinge domain. The chimeric ScFv-gamma gene was expressed in a murine cytotoxic T-cell hybridoma. Transfectants receiving vector only served as a negative control (mock). Screening for functional transfectants was carried out using a tumor growth inhibition assay. The soluble form of MLuC1 ScFv was recovered from bacteria periplasm and tested for binding to LeY-expressing cells by the fluorescence-activated cell sorter analysis. Despite the low binding ability of the soluble MLuC1 ScFv, 7 of 13 genetically engineered cytotoxic T lymphocyte clones inhibited the growth of LeY-positive cells and did not affect growth of LeY-negative cells. None of the mock clones tested specifically inhibited tumor growth. These data indicate that, by chimeric MLuC1 ScFv-gamma gene transfer, it is possible to confer anticarbohydrate specificity onto T cells and extend the applicability of the T-body approach to tumor-associated antigens that are naturally not recognized by T cells.

Involvement of tachykinins in plasma extravasation induced by bradykinin and low pH medium in the guinea-pig conjunctiva.

1. The effect of bradykinin, capsaicin, substance P and low pH medium on plasma extravasation in the guinea-pig conjunctiva has been studied. Evans blue dye was measured in the conjunctiva after local instillation of the agents into the conjunctival sac. 2. Bradykinin (2-50 nmol), capsaicin (20-50 nmol) and substance P (0.5-5 nmol) caused a dose-dependent increase in plasma extravasation with the following order of potency: substance P > bradykinin = capsaicin. The effect of capsaicin (50 nmol) and substance P (5 nmol) was abolished by the tachykinin NK1 receptor antagonist, CP-99,994 (8 mumol kg-1, i.v.) (P < 0.01), whereas CP-100,263 (8 mumol kg-1, i.v.) the inactive enantiomer of CP-99,994 was without effect. CP-99,994 inhibited by 70% (P < 0.01) the effect of bradykinin. 3. The kinin B2 receptor antagonist, Hoe 140 (icatibant, 10 nmol kg-1, i.v.) abolished the response to bradykinin (50 nmol) (P < 0.01), but did not affect the responses to capsaicin (50 nmol) or substance P (5 nmol). Plasma extravasation induced by low pH medium (pH 1) was abolished by CP-99,994 (P < 0.01) and by Hoe 140 (P < 0.01). 4. The present findings suggest that: endogenous or exogenous tachykinins increase plasma extravasation in the guinea-pig conjunctiva by activation of NK1 receptors; bradykinin-induced plasma extravasation is mediated by tachykinin release from sensory nerve endings; low pH media cause plasma extravasation via release of kinins that by activation of B2 receptors release tachykinins from sensory nerve endings.

Inhibition of bradykinin-evoked trigeminal nerve stimulation by the non-peptide bradykinin B2 receptor antagonist WIN 64338 in vivo and in vitro.

1. This study investigated the effect of the recently described non-peptide bradykinin B2 receptor antagonist, WIN 64338 ([[4-[[2- [[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl)-1-oxopropyl] amino]phenyl]methyl]tributylphosphoniumchloride monohydrochloride), in experimental models of bradykinin-evoked sensory nerve stimulation. 2. In the rabbit isolated iris sphincter in vitro, bradykinin-evoked contractile responses are mediated via tachykinins released from peripheral endings of the trigeminal sensory nerve. WIN 64338 (1-10 microM) competitively antagonised contractile responses to bradykinin with a pKB estimate of 6.6 +/- 0.1 (n = 11). The antagonism was selective since WIN 64338 (10 microM) did not significantly inhibit submaximal contractile responses to the direct-acting spasmogens substance P (10 nM), neurokinin A (3 nM), substance P methyl ester (10 nM) or senktide (100 nM); nor by sensory non-adrenergic non-cholinergic nerve stimulation evoked by capsaicin (10 microM), or electrical field-stimulation (3, 10, 30 Hz) (P > 0.05; n = 3-11). 3. Topical application of bradykinin to the conjunctiva and to the nasal mucosa of the guinea-pig in vivo causes plasma extravasation predominantly via the release of tachykinins from peripheral endings of the trigeminal nerve. The increases in plasma extravasation (measured by extravasation of Evans blue dye) induced by bradykinin in the guinea-pig conjunctiva (20 nmol) and nasal mucosa (50 nmol) were markedly reduced (by 81 +/- 3% and 69 +/- 5%, respectively) following pretreatment with WIN 64338 (30 nmol kg-1, i.v.) (n = 5-6; P < 0.05), with almost complete inhibition at a higher dose of WIN 64338 (300 nmol kg-1, i.v.; n = 5-6). This inhibition was selective since at 300 nmol kg-1, WIN 64338 did not inhibit plasma extravasation evoked by substance P in the conjunctiva (5 nmol; P > 0.05; n = 6) or in the nasal mucosa (50 nmol; P > 0.05; n = 5). 4. This study demonstrates that WIN 64338 is a selective and competitive bradykinin B2 receptor antagonist and can be useful for analysing bradykinin-evoked trigeminal nerve stimulation both in vitro and in vivo.

Evidence that tachykinins relax the guinea-pig trachea via nitric oxide release and by stimulation of a septide-insensitive NK1 receptor.

1. This study investigated the possibility that tachykinins relax the guinea-pig isolated trachea by releasing nitric oxide (NO) from the epithelium. The types of tachykinin receptor mediating both relaxation and contraction of the trachea were also studied. Isometric tension was recorded in isolated tracheal tube preparations precontracted with acetylcholine (10 microM) in which compounds were administered intraluminally in the presence of phosphoramidon and indomethacin (both 1 microM) and the tachykinin NK2 receptor antagonist, SR 48,968 ((S)-N-methyl-N[4-(4-acetyl amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide), 0.1 microM). 2. In the presence of the inactive enantiomer of an NO-synthase inhibitor, NG-monomethyl-D-arginine (D-NMMA, 100 microM), substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and the selective NK1 receptor agonist, [Sar9, Met(O2)11]-SP, (0.1-10 nM) relaxed tracheal tube preparations. This relaxation was changed into a contraction by pretreatment with the NO-synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA, 100 microM). The effect of L-NMMA on SP- and [Sar9, Met(O2)11]-SP-induced responses was reversed by L-arginine (L-Arg, 1 mM), but not by D-Arg (1 mM). After removal of the epithelium SP, NKA and NKB and [Sar9, Met(O2)11]-SP (0.1-10 nM) evoked contractile responses in the presence of either L-NMMA (100 microM) or D-NMMA (100 microM). The effects of SP and [Sar9, Met(O2)11]-SP obtained in the presence of another NO-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) or its inactive enantiomer, NG-nitro-D-arginine methyl ester (D-NAME, 100 microM) were similar to those observed with L-NMMA or D-NMMA, respectively. 3. The selective NK1 receptor agonist, [pGlu6, Pro9]-SP(6-11) (septide, 0.1-10 nM) evoked contractile responses of tracheal tube preparations in the presence of either D-NMMA (100 microM) or L-NMMA (100 microM). The log concentration-response curve to septide obtained in the presence of L-NMMA was similar to that obtained in the presence of D-NMMA. [Sar9, Met(O2)11]-SP (0.1-10 nM) relaxed tracheal tube preparations precontracted with septide (1 microM), whereas septide (0.1 nM-1 microM) further contracted tracheal tube preparations precontracted with [Sar9, Met(O2)11]-SP (1 microM). 4. Relaxant and contractile responses evoked by SP, NKA, NKB and by [Sar9, Met(O2)11]-SP (0.1-10 nM) were not affected by a combination of the histamine H1 (pyrilamine, 1 microM) and H2 (cimetidine, 1 microM) receptor antagonists, but were abolished by the tachykinin NK1 receptor antagonist, CP-99,994 ((2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine, 1 microM), though not by its inactive enantiomer CP-100,263 (1 microM). Contractile responses evoked by septide (10 nM and 1 microM) were also abolished by CP-99,994 (1 microM) but not by CP-100,263 (1 microM). 5. These results demonstrate that tachykinins relax guinea-pig tracheal tube preparations by releasing NO via the stimulation of epithelial NK1 receptors by a mechanism independent of histamine release. The NK1 receptor type involved is sensitive to SP, NKA, NKB and [Sar9, Met(O2)11]-SP but not to septide, and is pharmacologically distinct from the NK1 receptor that mediates contraction, which is stimulated by all the agonists, including septide.

Effect of capsazepine on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) induced by low pH, capsaicin and potassium in rat soleus muscle.

1. We have determined the effect of the competitive antagonist capsazepine at the capsaicin receptor on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from rat isolated soleus muscle induced by capsaicin (1 microM), by superfusion with low pH medium (pH 5) or by KCl (80 mM). 2. Each one of the three stimuli tested produced a marked CGRP-LI release. Total evoked release (fmol g-1) was 482 +/- 69, 169 +/- 20 and 253 +/- 43 for capsiacin, low pH medium and KCL, respectively. 3. Prior application of capsiacin (10 microM for 30 min followed by 30 min of washout) to produce capasaicin desensitization in vitro abolished CGRP-LI release induced by the three stimuli. 4. Capsazepine (1-100 microM, 45 min preincubation) inhibited the evoked CGRP-LI release. Capsaicin-induced release was significantly inhibited by 77, 92 and 96% with 10, 30 and 100 microM capsazepine, respectively. Low pH-induced release was inhibited by 78, 84, 88 and 93% with 3, 10, 30 and 100 microM capsazepine, respectively. KCl-induced release was significantly inhibited by 55 and 93% with 30 and 100 microM (but not with 10 microM) capsazepine, respectively. 5. These findings demonstrate that capsazepine prevents low pH- and capsaicin-induced CGRP-LI release from rat soleus muscle at concentrations which do not affect the release evoked by KCl. These findings imply a relationship between the action of low pH and activation of the capsaicin receptor. At high concentrations, capsazepine produces a nonspecific inhibitory effect on CGRP-LI release from peripheral endings of the capsaicin-sensitive primary afferent neurone.

Differential activation of the epithelial and smooth muscle NK1 receptors by synthetic tachykinin agonists in guinea-pig trachea.

1. The presence of tachykinin NK1 receptors have been shown in the epithelium and smooth muscle of guinea-pig airways. Previous data showed that substance P (SP), and the NK1 receptor agonist, [Sar9, Met (O2)11]-SP, relax guinea-pig tracheal tube preparations by stimulation of epithelial NK1 receptors and via nitric oxide (NO) release. However, the selective tachykinin NK1 receptor agonist, septide, was unable to produce this effect. The aim of the present study was to investigate the ability of a series of SP analogues to stimulate NK1 receptors of guinea-pig airway epithelium. 2. Isometric tension was recorded in isolated tracheal tube preparations in which compounds were administered intraluminally in the presence of phosphoramidon, indomethacin (both 1 microM) and the tachykinin NK2 receptor antagonist, SR 48,968 ((S)-N-methyl N-(4-acetyl-amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl)benzam ide) (0.1 microM). Cumulative concentration-response curves were obtained in preparations under resting tone or in preparations precontracted with acetylcholine (ACh, 10 microM). 3. Contractile responses to low concentrations (0.1-10 nM) of substance P (SP) and the selective agonist of NK1 receptors, [Pro9]-SP. in non precontracted tracheae were higher in preparations pretreated with the NO-synthase inhibitor, NG-monomethyl L-arginine (L-NMMA, 100 microM) than in preparations pretreated with its inactive enantiomer D-NMMA (100 microM). Tracheal tube preparations precontracted with ACh and pretreated with D-NMMA were relaxed by low concentrations of SP and [Pro9]-SP (0.1-10 nM). In contrast, after pretreatment with L-NMMA, SP and [Pro9]-SP contracted tracheae at all the concentrations tested. 4. Concentration-response curves to the NK1 receptor agonists, SP methyl ester, [Apa9-10]-SP and [pGlu6] SP (6-11) obtained in non-precontracted tracheae were similar in the presence of either D-NMMA or L-NMMA. SP methyl ester, [Apa9-10]-SP and [pGlu6] SP (6-11) did not produce any relaxation, but instead, cause contractions in tracheal tube preparations precontracted with ACh and pretreated with D-NMMA. Concentration-response curves produced by all these agonists were similar in preparations precontracted with ACh and pretreated with L-NMMA or D-NMMA. 5. In guinea-pig tracheal tube preparations two groups of NK1 receptor agonists can be distinguished: one group, including [Pro9]-SP, stimulator epithelial NK1 receptors, the other group, including SP methyl ester, [Apa9-10]-SP and [pGlu6] SP (6-11), does not. One possible explanation for these findings and for the existence of compounds with a peculiar 'septide-like' pharmacological profile in the guinea-pig trachea could be the recently proposed phenomenon referred to as 'agonist-directed receptor trafficking'.

Spectral analysis of muscular sound during isometric contraction of biceps brachii.

The frequency content of muscular sound (MS), detected by placing a contact sensor transducer over the belly of the biceps brachii during 10 isometric contractions of 4 s each [10-100% of maximal voluntary contraction (MVC)] in seven sedentary men, was analyzed by the maximum entropy spectral estimation and the fast Fourier transform methods. With increasing %MVC, the power spectrum of the MS enlarges and tends to be multimodal beyond 30% MVC. Independent of the method, the mean frequency is approximately 11 Hz at the lower tasks, and then it increases up to 15 Hz at 80% MVC and to 22 Hz at 100% MVC. When the effort is increased the relative power in the 15- to 45-Hz bandwidth (range of firing rate of the motor units with fast-twitch fibers) from 20% reaches 55% of the power in the 6- to 45-Hz bandwidth (firing rate range of motor units with slow- and fast-twitch fibers). Our results obtained by the two different modeling approaches confirm the reliability of the sound signal. Moreover, it appears that from the MS the motor unit activation pattern can be retrieved.

Single-step purification of immunotoxins containing a high ionic charge ribosome inactivating protein clavin by carboxymethyl high-performance membrane chromatography.

High-performance membrane chromatography (HPMC) and HPLC hydroxyapatite chromatography were compared for their efficiency in purifying immunotoxins (ITs) containing the ribosome-inactivating protein clavin, which is characterized by a high anionic charge and a low molecular mass. Both methods efficiently removed unreacted clavin from the conjugate crude mixture, but only the cation-exchange HPMC allowed efficient single-step separation of the unreacted monoclonal antibody (mAb) from ITs obtained by different coupling procedures.

Monoclonal antibodies by genetic engineering: novel reagents for in vivo diagnosis and therapy.

In only a few years, the technology of antibody engineering has demonstrated its power and a variety of recombinant monoclonal antibodies are now being developed. Recent developments in gene manipulation have allowed the isolation of antibodies, including human antibodies, with or without immunization, by displaying functional antibody fragments on the surface of bacteriophage particles and directly selecting with antigen. In the present review some recent achievements in these areas are highlighted.