Effect of Monoclonal Antibody Blockade of Long Fragment Neurotensin on Weight Loss, Behavior, and Metabolic Traits After High-Fat Diet Induced Obesity.

Background: Obesity is a major public health problem of our time as a risk factor for cardiometabolic disease and the available pharmacological tools needed to tackle the obesity pandemic are insufficient. Neurotensin (NTS) is a 13 amino acid peptide, which is derived from a larger precursor hormone called proneurotensin or Long Form NTS (LF NTS). NTS modulates neuro-transmitter release in the central system nervous, and facilitates intestinal fat absorption in the gastrointestinal tract. Mice lacking LF NTS are protected from high fat diet (HFD) induced obesity, hepatic steatosis and glucose intolerance. In humans, increased levels of LF NTS strongly and independently predict the development of obesity, diabetes mellitus, cardiovascular disease and mortality. With the perspective to develop therapeutic tools to neutralize LF NTS, we developed a monoclonal antibody, specifically inhibiting the function of the LF NTS (LF NTS mAb). This antibody was tested for the effects on body weight, metabolic parameters and behavior in mice made obese by high-fat diet. Methods: C57bl/6j mice were subjected to high-fat diet (HFD) until they reached an obesity state, then food was switched to chow. Mice were treated with either PBS (control therapy) or LF NTS mAb at the dose of 5 mg/kg once a week (i.v.). Mice weight, plasma biochemical analysis, fat and muscle size and distribution and behavioral tests were performed during the losing weight period and the stabilization period. Results: Obese mice treated with the LF NTS mAb lost weight significantly faster than the control treated group. LF NTS mAb treatment also resulted in smaller fat depots, increased fecal cholesterol excretion, reduced liver fat and larger muscle fiber size. Moreover, mice on active therapy were also less stressed, more curious and more active, providing a possible explanation to their weight loss. Conclusion: Our results demonstrate that in mice subjected to HFD-induced obesity, a blockade of LF NTS with a monoclonal antibody results in reduced body weight, adipocyte volume and increased muscle fiber size, possibly explained by beneficial effects on behavior. The underlying mechanisms as well as any future role of LF NTS mAb as an anti-obesity agent warrants further studies.

IL-38 inhibits microglial inflammatory mediators and is decreased in amygdala of children with autism spectrum disorder.

Autism spectrum disorder (ASD) is characterized by impaired social interactions and communication. The pathogenesis of ASD is not known, but it involves activation of microglia. We had shown that the peptide neurotensin (NT) is increased in the serum of children with ASD and stimulates cultured adult human microglia to secrete the proinflammatory molecules IL-1ß and CXCL8. This process is inhibited by the cytokine IL-37. Another cytokine, IL-38, has been reported to have antiinflammatory actions. In this report, we show that pretreatment of cultured adult human microglia with recombinant IL-38 (aa3-152, 1-100 ng/mL) inhibits (P < 0.0001) NT-stimulated (10 nM) secretion of IL-1ß (at 1 ng/mL) and CXCL8 (at 100 ng/mL). In fact, IL-38 (aa3-152, 1 ng/mL) is more potent than IL-37 (100 ng/mL). Here, we report that pretreatment with IL-38 (100 ng/mL) of embryonic microglia (HMC3), in which secretion of IL-1ß was undetectable, inhibits secretion of CXCL8 (P = 0.004). Gene expression of IL-38 and its receptor IL-36R are decreased (P = 0.001 and P = 0.04, respectively) in amygdala from patients with ASD (n = 8) compared to non-ASD controls (n = 8), obtained from the University of Maryland NeuroBioBank. IL-38 is increased (P = 0.03) in the serum of children with ASD. These findings indicate an important role for IL-38 in the inhibition of activation of human microglia, thus supporting its development as a treatment approach for ASD.

Modulation of lung cancer cell plasticity and heterogeneity with the restoration of cisplatin sensitivity by neurotensin antibody.

Overall survival of patients with metastatic non-small cell lung cancer (NSCLC) has significantly improved with platinum-based salt treatments and recently with targeted therapies and immunotherapies. However, treatment failure occurs due to acquired or emerging tumor resistance. We developed a monoclonal antibody against the proform of neurotensin (LF-NTS mAb) that alters the homeostasis of tumors overexpressing NTSR1. Neurotensin is frequently overexpressed along with its high affinity receptor (NTSR1) in tumors from epithelial origins. This ligand/receptor complex contributes to the progression of many tumor types by activation of the cellular effects involved in tumor progression (proliferation, survival, migration, and invasion). We demonstrate that LF-NTS mAb operates on the plasticity of tumor cells overexpressing NTSR1 and lowers their aggressiveness. The mAb enables the restoration of platinum-based therapies responsiveness, while also decreasing metastatic processes. Efficacy dosage with long-term treatment showed no obvious adverse events, while demonstrating improvement in the performance status. Our data suggests that LF-NTS mAb is an ideal candidate to be safely added to the conventional standard of care in order to improve its efficacy.

Potent antitumor effect of neurotensin receptor-targeted oncolytic adenovirus co-expressing decorin and Wnt antagonist in an orthotopic pancreatic tumor model.

Pancreatic cancer is highly aggressive, malignant, and notoriously difficult to cure using conventional cancer therapies. These conventional therapies have significant limitations due to excessive extracellular matrix (ECM) of pancreatic cancer and poor cancer specificity. The excess ECM prevents infiltration of drugs into the inner layer of the solid tumor. Therefore, novel treatment modalities that can specifically target the tumor and degrade the ECM are required for effective therapy. In the present study, we used ECM-degrading and Wnt signal-disrupting oncolytic adenovirus (oAd/DCN/LRP) to achieve a desirable therapeutic outcome against pancreatic cancer. In addition, to overcome the limitations in systemic delivery of oncolytic Ad (oAd) and to specifically target pancreatic cancer, neurotensin peptide (NT)-conjugated polyethylene glycol (PEG) was chemically crosslinked to the surface of Ad, generating a systemically injectable hybrid system, oAd/DCN/LRP-PEG-NT. We tested the targeting and therapeutic efficacy of oAd/DCN/LRP-PEG-NT toward neurotensin receptor 1 (NTR)-overexpressing pancreatic cancer cells, both in vitro and in vivo. The oAd/DCN/LRP-PEG-NT elicited increased NTR-selective cancer cell killing and transduction efficiency when compared with a cognate control lacking NT (oAd/DCN/LRP-PEG). Furthermore, systemic administration of oAd/DCN/LRP-PEG-NT significantly decreased induction of innate and adaptive immune responses against Ad, and blood retention time was markedly prolonged by PEGylation. Moreover, NTR-targeting oAd elicited greater in vivo tumor growth suppression when compared with naked oAd and 9.5 × 10(6)-fold increased tumor-to-liver ratio. This significantly enhanced antitumor effect of oAd/DCN/LRP-PEG-NT was mediated by active viral replication and viral spreading, which was facilitated by ECM degradation and inhibition of Wnt signaling-related factors (Wnt, ß-catenin, and/or vimentin) in the tumor tissues. Taken together, these results demonstrate that oAd/DCN/LRP-PEG-NT has strong therapeutic potential for systemic treatment of NTR-overexpressing pancreatic cancer due to its NTR-targeting ability, enhanced therapeutic efficacy, and safety.

Investigation of proNT/NMN secretion from small cell lung carcinoma cells using a mouse xenograft model.

Proneurotensin/neuromedin N (proNT/NMN), the precursor of neurotensin (NT) and neuromedin N (NMN), is produced by cancer tissues derived from the pancreas and colon. NT stimulates tumor growth and proliferation through its receptors; however, little is known about the precursor molecule in cancer tissues. We previously demonstrated that proNT/NMN is secreted from small cell lung carcinoma (SCLC) cell lines in serum-free conditioned medium, but not from non-small cell lung carcinoma (NSCLC) cell lines. It was suggested that this precursor may serve as a tumor marker for SCLC. In this study, we established in vivo xenograft models to evaluate the possibility of proNT/NMN as a specific tumor marker. SBC3 cells, derived from human SCLC, were inoculated into mice, and the proNT/NMN levels in plasma and tumor tissues were detected using specific antibodies. In contrast to control mouse plasma, the proNT/NMN levels in tumor-bearing mice increased as the tumors grew, and the elevated plasma proNT/NMN levels were decreased by tumor resection. Moreover, proNT/NMN was expressed in SBC3 tumors, suggesting that proNT/NMN was secreted into blood from the tumor, and this secretion may be specific to SCLC.

HMC-1 human mast cells synthesize neurotensin (NT) precursor, secrete bioactive NT-like peptide(s) and express NT receptor NTS1.

OBJECTIVE AND DESIGN: To determine if mast cells synthesize the inflammatory peptide, neurotensin (NT), secrete immunoreactive and bioactive NT, and express the NT receptor NTS1. MATERIALS: HMC-1 cells, pleural mast cells from Sprague-Dawley rats, LAD2 mast cells, and human cord blood mast cells were used. TREATMENT: HMC-1 cells were stimulated with NT, C48/80, mastoparan, or PGE(2). For changes in cutaneous vascular permeability, anesthetized rats were injected intravenously with Evans Blue dye and intradermally with saline, NT, histamine, diphenhydramine, and C48/80. METHODS: RT-PCR was used to identify RNA transcripts. Histamine was measured by fluorometric assay. In vivo cutaneous vascular permeability assays, radio-immunoassays for NT, Western blotting for the NT precursor protein and NTS1 protein from HMC-1 cells and tissues from rats were used. Immunohistochemistry was used to identify NT precursor-like proteins in HMC-1 mast cells. RESULTS: HMC-1 cells express mRNAs for NT precursor, PC5A processing enzyme and NTS1 receptor. Human cord blood mast cells and LAD2 mast cells express mRNA transcripts for NT precursor and NTS1. Western blotting showed NT precursor and NTS1 receptor in HMC1. Rat tissues with high numbers of mast cells contained NT precursor proteins. NT-like peptides from HMC-1 displayed NT-like bioactivity. CONCLUSIONS: HMC-1 mast cells synthesize and secrete immunoreactive and bioactive NT-like peptide(s) and express the NT receptor, suggesting that NT from mast cells might serve autocrine and paracrine roles.

Contribution of neurotensin in the immune and neuroendocrine modulation of normal and abnormal enteric function.

Among various hormones, which are synthesized by intestinal cells and influence enteric function, neurotensin (NT) has gained scientific attention the last three decades. This neuropeptide, mainly located in neuronal synaptic vesicles of hypothalamus and in neuroendocrine cells of the small bowel, participates in enteric digestive processes, gut motility and intestinal inflammatory mechanisms by cooperating with other regulators such as histamine, substance P and somatostatin. NT plays an important role mainly in intestinal lipid metabolism by cooperating with cholecystokinin and establishes a hormonal brain-gut-adipose tissue connection, which could adjust appetite, weight status and generally eating behavior with the amount and the content (particularly fat) of food intake. Moreover, NT achieves a multi-level control of intestinal motility by cooperating with the enteric- and central nervous system, and other enteric hormones (such as somatostatin). NT regulates motility patterns related to the efficiency of the digestive process, stool emptying, transition from the fasted to the postprandial state and reestablishment of the fasted status. In addition, NT possesses a long-term enteroprotective role towards the intestinal tract, despite the fact that under certain circumstances NT may participate in short-term subcellular pathways promoting an acute inflammatory response. The aim of this review is two-fold. First, is to provide an up-to-date synopsis of the available knowledge regarding the involvement of neurotensin in enteric functional status, and highlight its significance in physiological and pathological conditions. Second, is to propose new research directions concerning the role of neurotensin and other intestinal regulatory peptides in the establishment of the brain-gut axis and in the development of functional disorders of the abdominal tract. Conclusively, to clarify the areas, in which an experimental therapeutic intervention, based on NT analogs, may lead to encouraging results.

Neurotensin receptor-2 and -3 are crucial for the anti-apoptotic effect of neurotensin on pancreatic beta-TC3 cells.

The neuropeptide neurotensin (NT) has been recently shown to protect pancreatic beta cells from toxic agents-induced apoptosis through interaction with the NT receptor-2 (NTSR2) and activation of the phosphatidylinositol-3 kinase pathway. However, expression of the NT receptor-3/sortilin (NTSR3) in the mouse pancreatic beta cell line -TC3 led us to investigate its possible functional role in these cells. By using siRNA, immunoprecipitation, co-localization and caspase-3 assays,we provide evidence for a functional endogenous interaction between NTSR2 and NTSR3. Expression of both receptors is necessary for the protective action of NT on staurosporine-induced caspase-3 activity in -TC3 cells. Moreover, NTSR2 and NTSR3 co-immunoprecipitate and are co-localized at the plasma membrane. Thus, the NT response in beta cells is controlled by the formation of a functional complex between NTSR2 and NTSR3.

Biology of neurotensin: revisited study.

The tridecapeptide neurotensin (NT) acts in the mammalian brain as a primary neurotransmitter or neuromodulator of classical neurotransmitters. Morphological and functional in vitro and in vivo studies have demonstrated the existence of close interactions between NT and dopamine both in limbic and in striatal brain regions. Additionally, biochemical and neurochemical evidence indicates that in these brain regions NT also plays a crucial role in the regulation of the aminoacidergic signalling. Immune cells, such as lymphocytes, macrophages and mast cells are reported to be activated by neuropeptides, such as neurotensin; this activation leads to cytokine and immunoglobulin production. In addition, neurotensin increases calcium level and the production of nitric oxide. Therefore neurotensin is deeply involved in immunity and inflammation but its real function still remains to be elucidated.

Localization of neurotensin immunoreactivity in neurons and organ of Corti of rat cochlea.

The distribution of neurotensin-containing cell bodies and fibers has been observed in the central and peripheral nervous system, including sensory ganglia, but no description has been found in the peripheral auditory system. Here, we investigated the presence of neurotensin immunoreactivity in the cochlea of the adult Wistar rat. Strong neurotensin immunoreactivity was detected in the cytoplasm of the inner hair cells (IHC) and Deiters' cells of the organ of Corti. Outer hair cells (OHC) show weak immunoreaction. Neurotensin immunoreactivity was also found in the neurons and fibers of the spiral ganglia. Quantitative microdensitometric image analysis of the neurotensin immunoreactivity showed a strong immunoreaction in the hair cells of organ of Corti and a moderate to strong labeling in the spiral ganglion neurons. A series of double immunolabeling experiments demonstrated a strong neurotensin immunoreactivity in the parvalbumin immunoreactive IHC and also in the calbindin immunoreactive Deiters' cells. Weak neurotensin immunoreactivity was seen in the calbindin positive OHC. Neurofilament and parvalbumin immunoreactive neurons and fibers in the spiral ganglia showed neurotensin immunoreactivity. Calbindin immunoreactivity was not detected in the spiral ganglion neurons, which are labeled by neurotensin immunoreactivity. The presence of neurotensin in the cochlea may be related to its modulation of neurotransmission in the peripheral auditory pathway.

Paraventricular nucleus neurons producing neurotensin after lipopolysaccharide treatment project to the median eminence.

Inflammation consists in secretion of cytokines that stimulate the hypothalamo-pituitary-adrenal (HPA) axis to release the anti-inflammatory corticosterone. Upstream in this axis are corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) whose multipeptidergic phenotype changes: both corticotropin-releasing hormone mRNAs and neurotensin mRNAs are up-regulated. Combining in situ hybridization with a retrograde neuronal marker, we demonstrated that neurotensin-containing neurons in the paraventricular nucleus project to the median eminence.

Progesterone and norethisterone have different effects on tachykinin-like immunoreactivity in rat cortex and striatum.

OBJECTIVE: The purpose of this study was to investigate the effects of progesterone and the most commonly prescribed synthetic progestogen, norethisterone, on regional immune-like reactivity of neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA) and neurotensin (NT) in brains of female ovariectomized estradiol-substituted rats. RESULTS: Norethisterone+estradiol-treated rats had 44% lower SP levels compared with estradiol-only-treated in frontal cortex and 20% lower NKA levels in comparison with progesterone+estradiol-treated in frontal cortex. Progesterone+estradiol-treated rats had 66% lower SP levels in striatum in comparison with both estradiol-only-treated and norethisterone+estradiol-treated. No significant results were found for NPY and NT. CONCLUSION: Progesterone and the synthetic progestogen, norethisterone, have different effects on SP- and NKA-like immunoreactivity in rat cortex and striatum. The effects of NET on SP- and NKA-like immunoreactivity in frontal cortex may contribute to the mood effects ascribed to this progestogen in clinical usage.

A highly sensitive and selective radioimmunoassay for the measurement of neurotensin.

A highly selective and sensitive radioimmunoassay (RIA) for the detection of endogenous neurotensin (NT) has been developed. We have raised a C-terminally-directed antibody (CAb) that specifically binds 'biologically active' NT (NT and NT(8-13)) and that does not significantly cross-react with inactive NT metabolites or other bioactive peptides in the CNS. By reducing the volume of the assay to a low volume-RIA (30 microl), such that in vivo measurements can be made, we have increased the sensitivity (<0.3 fmol per tube), with inter- and intra-assay variations of 11.2 and 5.8%, respectively. Comparisons with similar methods of detecting NT have demonstrated that this RIA has a higher sensitivity than previously used RIA's and ELISA's. The data presented suggests that this sensitive RIA is a reliable method ideal for the detection of small quantities of biologically active NT.

Co-localization of neuroendocrine hormones in the human fetal pancreas.

OBJECTIVE AND DESIGN: Co-localization of the four major pancreatic hormones, and also of islet amyloid polypeptide (IAPP), peptide tyrosine tyrosine (PYY), secretin and neurotensin, has been studied in the endocrine pancreas of human fetuses at 16, 18 and 22 weeks of gestation. METHODS: Double and triple immunofluorescence stainings have been used. RESULTS: All three fetal pancreata contained cells that showed insulin, glucagon, somatostatin, pancreatic polypeptide (PP), IAPP, secretin and PYY immunoreactivity. Neurotensin cells were found in the youngest fetus and gastric inhibitory polypeptide (GIP) in the two older fetuses. Co-localization of two hormones occurred in most of the endocrine cell types in the three fetuses examined, but three hormones occurred in only a few cells and especially in the youngest fetus. Somatostatin cells were the only cell type which was largely monohormonal. Our findings showed that there are two different co-localization patterns: insulin was co-localized mainly with IAPP and glucagon, while secretin and PYY occurred together with glucagon and PP. CONCLUSIONS: These data are the first to describe secretin and neurotensin in the fetal pancreas. Two different co-localization patterns could be distinguished: insulin, IAPP and glucagon, and glucagon, secretin, PP and PYY.

Acute immobilization stress triggers skin mast cell degranulation via corticotropin releasing hormone, neurotensin, and substance P: A link to neurogenic skin disorders.

Many skin disorders, such as atopic dermatitis and psoriasis, worsen during stress and are associated with increased numbers and activation of mast cells which release vasoactive, nociceptive, and proinflammatory mediators. Nontraumatic acute psychological stress by immobilization has been shown to induce mast cell degranulation in the rat dura and colon. Moreover, intradermal injection of corticotropin-releasing hormone (CRH) or its analogue urocortin (10(-5)-10(-7) M) induced skin mast cell degranulation and increased vascular permeability. Here, we investigated the effect of acute immobilization stress on skin mast cell degranulation by light microscopy and electron microscopy. Immobilization for 30 min resulted (P < 0.05) in degranulation of 40.7 +/- 9.1% of skin mast cells compared to 22.2 +/- 7.3% in controls killed by CO(2) or 17.8 +/- 2.4% in controls killed by pentobarbital. Pretreatment intraperitoneally (ip) with antiserum to CRH for 60 min prior to stress reduced (P < 0.05) skin mast cell degranulation to 21.0 +/- 3. 3%. Pretreatment with the neurotensin (NT) receptor antagonist SR48692 reduced (P < 0.05) mast cell degranulation to 12.5 +/- 3.4%, which was significantly (P < 0.05) below control levels. In animals treated neonatally with capsaicin to deplete their sensory neurons of their neuropeptides, such as substance P (SP), mast cell degranulation due to immobilization stress was reduced to about 15%. This is the first time that stress has been shown to trigger skin mast cell degranulation, an action not only dependent on CRH, but apparently also involving NT and SP. These findings may have implications for the pathophysiology and possible therapy of neuroinflammatory skin disorders such as atopic dermatitis, neurogenic pruritus, or psoriasis, which are induced or exacerbated by stress.

Apposition of enkephalin- and neurotensin-immunoreactive neurons by serotonin-immunoreactive varicosities in the rat spinal cord.

The descending serotonergic system provides a powerful inhibitory input to the dorsal horn of the spinal cord. Little is known about the chemical identity of the spinal neurons that the serotonergic system innervates, although spinal enkephalinergic neurons are likely candidates. This study investigated the apposition of serotonin-immunoreactive varicosities onto enkephalin- and neurotensin-immunoreactive neurons in the rat lumbosacral spinal cord. Using a double immunofluorescence technique, serotonin-immunoreactive varicosities were observed to abut the soma or proximal dendrites of [Met]enkephalin- and neurotensin-immunoreactive neurons. Nearly 75% of all [Met]enkephalin- and neurotensin-immunoreactive neurons were apposed by serotonin-immunoreactive varicosities in the marginal zone and dorsal gray commissure. In substantia gelatinosa, approximately half of the [Met]enkephalin- and neurotensin-immunoreactive neurons were juxtaposed by serotonin-immunoreactive varicosities. [Met]enkephalin-immunoreactive neurons also were bordered by serotonin-immunoreactive varicosities in the nucleus proprius (65%) and sacral parasympathetic nucleus (75%). The results of this study suggest that the descending serotonergic system mediates nociception via probable contacts with intrinsic enkephalin and neurotensin spinal systems. The mode of action of spinal serotonin on enkephalin and neurotensin neurons may be through "volume" transmission vs synaptic or "wiring" transmission.

Differential effect of haloperidol on release of neurotensin in extrapyramidal and limbic systems.

The effect of the antipsychotic drug haloperidol on extracellular neurotensin-like immunoreactivity was investigated by microdialysis and compared with the time-dependent response of tissue neurotensin-like immunoreactivity content in brain structures containing dopamine nerve cell bodies and terminals. A single administration of haloperidol (1 mg/kg) increased the extracellular neurotensin-like immunoreactivity levels in nucleus accumbens as measured by microdialysis, but decreased its extracellular concentration in the caudate regions surrounding the probe. The same treatment increased the tissue content of neurotensin-like immunoreactivity in both the nucleus accumbens core and all caudate regions examined within 24 h after the injection. Interestingly, although the neurotensin-like immunoreactivity concentration in the substantia nigra was not altered by the haloperidol treatment, neurotensin-like immunoreactivity levels decreased significantly in the ventral tegmental area. These findings suggest that varied neurotensin systems are associated with nigrostriatal and mesolimbic dopamine pathways and these systems have different responses to haloperidol. The changes in the release of neurotensin may contribute to altered caudate and accumbens neurotensin-like immunoreactivity tissue content induced by haloperidol treatment, but other factors, such as variation in synthesis also likely influence these effects. Differential actions of haloperidol on neurotensin release might be due to regional differences in dopamine or sigma receptor subtypes associated with the neurotensin-containing neurons.

Evaluation of cell surface antigens as potential targets for recombinant tumor toxins.

Tumor toxins are recombinant polypeptides derived from the fusion of a single-chain antibody domain or a ligand specific for a cell surface receptor with the enzymatic domain of a bacterial or plant toxin. If these receptors are preferentially expressed on the surface of tumor cells, the tumor toxins can serve as efficient therapeutics. Examples in which growth factors, cytokines, hormones or antibody domains have been fused to toxins have been described and the bacterially expressed molecules have shown anti-tumor effects in vitro and in vivo. Since many tumors are not sensitive to the existing tumor toxins, the search continues for new antigens with tumor specific or tumor enhanced expression. We explored three antigenic structures for their possible use as targets for tumor toxins, i.e. (a) a glycosphingolipid with an Le(a) antigenic structure which is overexpressed in gastrointestinal carcinomas, e.g. colon carcinoma, (b) the epithelial glycoprotein episialin which shows altered expression in various tumor tissues such as breast, ovary and pancreas and (c) the neurotensin receptor which is found on NSLC, pancreatic and colonic cancer cells. For targeting the tumor toxin to the carbohydrate structures, we constructed recombinant single-chain antibody domains (scFv) fused to a truncated form of the exotoxin A from Pseudomonas aeruginosa (ETA). In the case of the neurotensin receptor, the ligand neurotensin was exploited to construct a recombinant fusion protein with exotoxin A as the effector domain. Binding experiments showed that the recombinant proteins are able to specifically recognize their respective target structures. In vitro cytotoxicity assays with neurotensin-ETA showed specific killing activity for receptor positive tumor cell lines. For the scFv-ETA constructs we could not observe in vitro tumor toxicity. We conclude that the antigenic targets for tumor toxins have to be carefully chosen and that specific cell binding activity is not a sufficient criterion for tumor cell killing. The neurotensin-ETA, a new anti-tumor agent, shows that seven transmembrane spanning receptors are potential targets which allow toxin binding and promote cell killing through the appropriate internalization.

Pattern of distribution of neuropeptides in the camel lacrimal gland.

The pattern of distribution of neuropeptides, including neuropeptide-Y (NPY), vasoactive intestinal polypeptide (VIP), neurotensin (NT), serotonin (5-HT), galanin (GAL), leucine-enkephalin (LEU-ENK) and calcitoningene-related-peptide (CGRP), in the nerves of the camel lacrimal gland was investigated using immunohistochemical techniques. Fresh lacrimal gland segments, obtained from adult camels slaughtered in the local abattoir, were used for the immunohistochemical techniques. NPY and LEU-ENK immunoreactivity was observed in the nerve cell bodies and nerve fibers of the camel lacrimal gland. VIP, GAL and CGRP were demonstrated predominantly in fine varicose nerve fibers lying on the basolateral surfaces of the lacrimal acinar cells. NT and 5-HT were identified mainly in neurons situated in the periacinar regions, close to the basal surfaces of the acinar cells. It is concluded that the camel lacrimal nerves contain several neuropeptides including NPY, VIP, NT, 5-HT, GAL, LEU-ENK and CGRP which may modulate lacrimal fluid and protein secretion.