Jayaraman Lakshmanan

Protective effect of epidermal growth factor in an experimental model of colitis in rats

BACKGROUND/AIMSnThe role of epidermal growth factor (EGF) in the maintenance of mucosal integrity in the lower gastrointestinal tract is unknown. The aim of this study was to determine the effect of EGF in experimental colitis.nnnMETHODSnColitis was induced with 2,4,6-trinitrobenzenesulfonic acid/ethanol enemas. Rats were pretreated with intraperitoneal administration of recombinant human EGF (600 micrograms/kg) or vehicle 1 hour before induction of colitis and daily thereafter until killed at 8 hours, 48 hours, and 1 week. A separate group received an identical dosage and administration of EGF or vehicle for 1 week with treatment initiated 24 hours after the induction of colitis. Colonic tissue was evaluated macroscopically, histologically, and for myeloperoxidase activity.nnnRESULTSnPretreatment with EGF reduced microscopic erosions at 8 and 48 hours by 74% and 54%, respectively (P < 0.05). At 1 week, microscopic ulcerations and myeloperoxidase activity were reduced by 65% in the EGF-pretreated group (P < 0.05). No significant difference in macroscopic injury, histological damage, or myeloperoxidase activity was noted when EGF treatment was initiated after the induction of colitis.nnnCONCLUSIONSnSystemic EGF administration reduces mucosal damage and inflammation in a trinitrobenzenesulfonic acid/ethanol model of colitis in rats through a mechanism involving mucosal protection.

Mice lacking transforming growth factor alpha have an increased susceptibility to dextran sulfate-induced colitis.

BACKGROUND & AIMSnThere is indirect evidence that transforming growth factor alpha (TGF-alpha) is an important mediator of mucosal defense and repair. TGF-alpha knockout mice and TGF-alpha-deficient mice (wa-1) provide novel approaches to evaluate the role of TGF-alpha in preserving the integrity of the colon.nnnMETHODSnColitis was induced by oral administration of dextran sodium sulfate (DSS, 5 g/dL) to knockout mice, their genetic controls (GC), wa-1 mice, and BALB/c mice. TGF-alpha was also administered intraperitoneally to wa-1 mice to evaluate the effect of exogenous TGF-alpha in DSS colitis.nnnRESULTSnIn response to DSS, nearly 60% of the entire colonic mucosa was destroyed in knockout and wa-1 mice, compared with 22% in GC mice and 16% in BALB/ c mice. Body weight loss was doubled in knockout (28%) and wa-1 mice (23%) compared with GC (11%) and Balb/c mice (12%). TGF-alpha application to wa-1 mice reduced the severity of mucosal injury by almost 70% compared with controls.nnnCONCLUSIONSnThe marked susceptibility of TGF-alpha knockout and wa-1 mice to DSS and the obvious amelioration of the colonic injury by exogenous TGF-alpha application in wa-1 mice suggest that TGF-alpha is a mediator of protection and/or healing mechanisms in the colon.

Comparison between epidermal growth factor, transforming growth factor-α and EGF receptor levels in regions of adult rat brain

Examination of adult rat brain regions by specific radioimmunoassays revealed a widespread distribution of transforming growth factor-alpha (TGF-alpha), but not epidermal growth factor (EGF), the peptide that had previously been reported to be present in rodent brain. Polyadenylated RNA samples from the different regions of rat brain were analyzed by Northern blot to identify mRNA species encoding precursor proteins for EGF (preproEGF), TGF-alpha (preproTGF-alpha), and the EGF/TGF-alpha receptor. The results indicate that TGF-alpha is the most abundant ligand for the EGF/TGF-alpha receptor in most parts of the brain analyzed. Message for preproEGF was only detectable after prolonged autoradiographic exposure; levels of preproEGF mRNA were between two and three orders of magnitude lower in brain than those expressed in control tissue (kidney), and one to two orders of magnitude lower than preproTGF-alpha mRNA levels in all brain regions. These results were confirmed by analysis of mRNA by RT/PCR, and support the hypothesis that expression of preproEGF mRNA in the brain is limited to smaller discrete areas, whereas preproTGF-alpha gene expression is almost ubiquitous.

Commercial Recombinant Human β‐Nerve Growth Factor and Adult Rat Dorsal Root Ganglia Contain an Identical Molecular Species of Nerve Growth Factor Prohormone

Abstract: Examination of commercial recombinant human β‐nerve growth factor (rh‐β‐NGF) preparations with polyclonal antibodies specific to 13‐kDa NGF and pro‐NGF‐specific domains revealed the presence of high‐molecular‐mass immunoreactive proteins, including a 60‐kDa NGF prohormone. On incubation with a mixture of N‐ and O‐specific glycosidases, the 60‐kDa NGF prohormone generated a 32‐kDa protein corresponding to the molecular size of NGF precursor predicted by the cloned human NGF cDNA. Highly sensitive chemiluminescence immunoblot analysis of adult rat dorsal root ganglia, spinal cord, and colon tissues with NGF‐ and pro‐NGF domain‐specific antibodies also revealed the presence of high‐molecular‐mass proteins, including the 60‐kDa NGF prohormone. Based on the presence of the 60‐kDa NGF prohormone in dorsal root ganglia and its efferent tissues, we suggest that proteolytically unprocessed, glycosylated NGF prohormone may mediate interactions between neurons and the tissues they innervate.

Epidermal Growth Factor in Synaptosomal Fractions of Mouse Cerebral Cortex

Abstract: Using a specific and sensitive epidermal growth factor radioimmunoassay (EGF‐RIA) we measured EGF concentrations in whole brain, cerebral cortex, and cerebral cortical synaptosomal (pinched‐off presynaptic nerve terminals) fractions of 26‐day‐old mouse brain. The relative EGF concentration in synaptosomal fractions was significantly greater than the growth factor concentrations in whole brain or cerebral cortex. Intracerebral injection, in an amount of EGF, several‐fold greater than whole brain EGF content, did not appreciably increase synaptosomal EGF concentration, suggesting that no artifact was involved. The high synaptosomal EGF content suggests a neurotransmitter or a neuromodulator role for EGF in the CNS.

Expression of nerve growth factor mRNA and its translation products in the anagen hair follicle

Abstract: The cellular localization of NGF mRNA and its translation products have been identified in ovine hair follicles. NGF mRNA was detected in the proliferating cells of the follicle bulb and differentiating cells of the suprabulbar region, but was absent from the outer root sheath. Western analysis revealed the presence of a 73 kDaNGF prohormone in extracts of ovine flank skin, but the mature 13 kDaNGF was absent. Immunohistochemical analysis with antibodies specific to mouse NGF and a pro‐ NGF specific domain localized the NGF prohormone to outer root sheath cells in the upper bulb region of the follicle, adjacent to the zone of keratinization. Antibody binding was also associated with the luminal epithelium of the apocrine sweat gland and the pilary canal of the follicle at its junction with the epidermis. These observations, together with the reported presence of high‐ and low‐affinity NGF receptors in the follicle, implicate the NGF prohormone‐responsive neuronal system in the regulation of hair growth.

Development of ovine fetal ileal motility: role of muscarinic receptor subtypes

OBJECTIVEnIn the preterm human fetus, immaturity of gastrointestinal (GI) motility contributes to impairment of oral feeding and an increased risk of necrotizing enterocolitis. In view of the limited knowledge of fetal GI motility development, and the primary role of the muscarinic system in adult GI motility, we examined the development of GI muscarinic receptor subtypes associated with ileal motility.nnnSTUDY DESIGNnOvine term fetal, newborn, and pregnant adult ileal longitudinal muscle contractile responses to muscarinic agonists (bethanechol) and muscarinic nonspecific (atropine) and subtype specific-antagonists (M1-M4) were examined in organ baths. Immunohistochemical analysis of ileal muscle muscarinic receptor subtypes was correlated with contractile responses.nnnRESULTSnBethanechol induced a concentration-dependent ileal contraction at all 3 age groups. Adult ileal maximal tension was 2-fold higher than that of the fetus and newborn, while 50% effective concentration (EC(50)) was similar at all ages. Atropine (10(-6)mol/L) inhibited contractility in fetal (67%+/-7%), newborn (82%+/-5%), and adult (97%+/-2%) in an age-dependent manner. The M3 antagonist exhibited robust inhibition at all age groups while the M2 antagonist demonstrated enhanced inhibition in the fetus. Immunohistochemical analysis indicated coexpression of subtype receptors in fetal, newborn, and adult ileal smooth muscle with increasing expression with advancing age.nnnCONCLUSIONnThese results demonstrate a specific developmental pattern of muscarinic receptor subtype expression. Knowledge and/or alterations of GI motility regulation may aid in the treatment of the preterm fetus or newborn.

Commercial mouse and human nerve growth factors contain nerve growth factor prohormone isoforms

Highly sensitive chemiluminescence immunoblot analysis was utilized to examine the purity of mouse 2.5S-, beta- and 7S nerve growth factors as well as that of recombinant human beta-nerve growth factor obtained from commercial vendors. Three polyclonal antisera and two monoclonal antibodies to 13 kDa nerve growth factor (2.5S NGF and beta-NGF) were employed for assessing the purity of each preparation. In addition, polyclonal antisera against two prepro-NGF specific domains were used for immunoblotting analysis to ascertain the identity of high molecular weight nerve growth factor immunoreactive proteins as prohormones. Both the mouse and human NGF preparations contained 53 and 60 kDa immunoreactive proteins. Of these, the mouse 60 kDa and the human 53 kDa proteins strongly immunoreacted with both prepro-nerve growth factor specific domain antibodies suggesting that they are two NGF prohormone isoforms. In addition, both the mouse and human nerve growth factor preparations contained proteins that were immunoreactive to polyclonal antisera and monoclonal antibodies to mouse 2.5S and/or beta-NGF. High molecular weight aggregates of prohormones were also observed in mouse and human nerve growth factor samples. In summary, none of the ten NGF samples examined were pure as stated. Our study cautions investigators in the field to be aware of the presence of nerve growth factor prohormones and other proteins in various mouse and human nerve growth factors sold commercially.

Epidermal Growth Factor Binding to Neonatal Mouse Skin Explants and Membrane Preparations—Effect of Triiodothyronine

ABSTRACT: Daily treatment of newborn Swiss-Webster mice with triiodothyronine (T3, 500 ng/day) increased epidermal growth factor (EGF) content in whole skin (epidermis + dermis). Separation of the epidermis using 0.01 M dithiothreitol followed by processing for radioimmunoassay measurement reveals levels of EGF 2-to 3-fold higher in epidermis than in whole skin. In vitro flotation of circular skin sections from control and T3 treated neonatal mice in medium containing [I125]EGF showed increased uptake of label following 5 days of in vivo T3 treatment. Mouse skin membrane preparations exhibit saturable, specific binding of [I125]EGF. T3 treatment for 5 days in vivo significantly increased EGF binding capacity in skin membrane preparations but did not alter EGF receptor affinity (Kd 4.5 nM). Protein, RNA, and DNA concentrations were significantly increased in whole neonatal mouse skin following T3 administration. These results suggest one mechanism by which thyroid hormones increase skin EGF concentration is augmentation of skin EGF receptor binding.

Neonatal Hyperthyroidism Alters Hepatic Epidermal Growth Factor Receptor Ontogeny in Mice

ABSTRACT: Epidermal growth factor (EGF) liver receptor ontogeny and somatic growth were studied in mice from day 7 to day 70 postnatally to assess long-term effects of short-term postnatal thyroxine treatment. The mice were given 0.4μg thyroxine/g body weight/day for the 1st wk of life. EGF receptor binding in liver tissue was studied on days 7, 15, 20, 30, and 70 postnatally. Treated animals had accelerated eyelid opening and tooth eruption, and permanent growth retardation was obvious from the second week of life. Hepatic EGF receptor-binding capacity increased markedly in control mice with increasing age in contrast to a very slow increase in treated mice, making the difference statistically significant (P < 0.01) from day 30. The affinity of EGF receptor binding initially was similar in the two groups of animals (1.09 × 109 M−1 and 1.02 × 109 M−1) and increased by day 30 in controls (2.57 ± 109 M−1 an increase that was not observed in treated animals either at day 30 or 70. These results suggest a sensitive period of imprinting during the first 7 days postnatally, a period when thyroxine can exert a permanent effect on later growth and later hepatic EGF receptor number.