Multiple immunofluorescence technology.
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Animals↗
Biomedical subjects
Gian-Luca Ferri
Publications and source records attributed to Gian-Luca Ferri.
Median eminence dopaminergic nerve terminals: a novel target in autoimmune polyendocrine syndrome?
CONTEXT: Autoantibodies to adenohypophyseal endocrine cells or to vasopressin neurohypophyseal neurons have long been known. Conversely, autoimmune targeting of further hypothalamic-hypophyseal structures, such as the blood-brain barrier-deprived median eminence, has been little studied. OBJECTIVE AND METHODS: We studied a case of autoimmune polyendocrine syndrome type I with GH secretory deficiency, a distinctly rare event in autoimmune polyendocrine syndrome type I. We used rat and bovine tissue substrates to study autoantibodies against hypothalamic-hypophyseal nerve structures and endocrine cells. RESULTS: In the study case, circulating autoantibodies selectively decorated median eminence dopaminergic nerve terminals, as well as pituitary gonadotropes, but not GHRH nerve terminals or pituitary somatotropes. Such autoantibodies appeared de novo in parallel with the onset of GH secretory deficiency, whereas no median eminence labeling was found in patients suffering of idiopathic GH deficiency (n = 7) or in healthy controls (n = 23). CONCLUSIONS: The pathophysiological significance of our patient's autoantibodies remains to be confirmed. Nonetheless, the heterogeneous neuroendocrine structures of the median eminence are pointed out as potential immune targets, relevant to autoimmune polyendocrinopathy, as well as to a wide range of other conditions.
Adolescent↗
Differential expression and seasonal modulation of VGF peptides in sheep pituitary.
The inducible gene vgf and its peptide products are relevant to the neuroendocrine regulation of homeostasis and reproduction in rodents. We show here that in the anterior pituitary of female sheep the somatotrope, gonadotrope, and lactotrope/thyrotrope cell populations each expressed vgf mRNA, but displayed a distinct profile of VGF immunoreactive peptides. ProVGF C-terminus and VGF(443-588) immunoreactivities were found in lactotropes and thyrotropes, often in a subcellular location restricted to the Golgi area and suggestive of rapid peptide (or proVGF) release upon biosynthesis, while high molecular weight bands consistent with proVGF were shown in pituitary extracts. Distinct seasonal changes were revealed, proVGF C-terminus immunoreactive cells being largely identified as lactotropes during the summer (83.7 +/- 2.1% (mean +/-s.e.m.) versus 27.0 +/- 1.9% during the winter), as opposed to thyrotropes during the winter (73.0 +/- 1.9% versus 16.3 +/- 2.1% during the summer). Conversely, antisera to peptides adjacent to the 'Arg-Pro-Arg' cleavage site, and to the VGF(553-555) N-terminus of the proVGF-derived peptide V, selectively labeled gonadotropes, indicating processing to small peptides not retaining the proVGF C-terminus in such cells. Finally, a peptide related to the VGF(4-240) region was immunostained in somatotropes, shown in a Western blot as a band of relative molecular mass of approximately 16,000. In conclusion, a complex, endocrine cell-type-specific processing of proVGF was revealed. Further to the known inducibility of vgf mRNA upon a range of stimuli, discreet, selective modulations of VGF-peptide profile/s are suggested, possibly involved in specific neuro/endocrine or modulatory mechanisms.
Animals↗
Processing, distribution, and function of VGF, a neuronal and endocrine peptide precursor.
1. The vgf gene encodes a neuropeptide precursor with a restricted pattern of expression that is limited to a subset of neurons in the central and peripheral nervous systems and to specific populations of endocrine cells in the adenohypophysis, adrenal medulla, gastrointestinal tract, and pancreas. In responsive neurons, vgf transcription is upregulated by neurotrophins. the basis for the original identification of VGF as nerve growth factor- (NGF) inducible in PC12 cells (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985). 2. In this review, we shall summarize data concerning the transcriptional regulation of vgf in vitro, the structural organization of the vgf promoter as well as the transcription factors which regulate its activity. 3. On the basis of in situ hybridization and immunohistochemical studies, the in vivo tissue-specific expression of VGF during differentiation and in the adult will be summarized. 4. Parallel biochemical data will be reviewed, addressing the proteolytical processing of the pro-VGF precursor within the secretory compartment of neuroendocrine cells. 5. Finally, analysis of the phenotype of VGF knockout mice will be discussed, implying a nonredundant role of VGF products in the regulation of energy storage and expenditure.
Animals↗
The endogenous cannabinoid system protects against colonic inflammation.
Excessive inflammatory responses can emerge as a potential danger for organisms' health. Physiological balance between pro- and anti-inflammatory processes constitutes an important feature of responses against harmful events. Here, we show that cannabinoid receptors type 1 (CB1) mediate intrinsic protective signals that counteract proinflammatory responses. Both intrarectal infusion of 2,4-dinitrobenzene sulfonic acid (DNBS) and oral administration of dextrane sulfate sodium induced stronger inflammation in CB1-deficient mice (CB1(-/-)) than in wild-type littermates (CB1(+/+)). Treatment of wild-type mice with the specific CB1 antagonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716A) mimicked the phenotype of CB1(-/-) mice, showing an acute requirement of CB1 receptors for protection from inflammation. Consistently, treatment with the cannabinoid receptor agonist R(-)-7-hydroxy-Delta(6)-tetra-hydrocannabinol-dimethylheptyl (HU210) or genetic ablation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) resulted in protection against DNBS-induced colitis. Electrophysiological recordings from circular smooth muscle cells, performed 8 hours after DNBS treatment, revealed spontaneous oscillatory action potentials in CB1(-/-) but not in CB1(+/+) colons, indicating an early CB1-mediated control of inflammation-induced irritation of smooth muscle cells. DNBS treatment increased the percentage of myenteric neurons expressing CB1 receptors, suggesting an enhancement of cannabinoid signaling during colitis. Our results indicate that the endogenous cannabinoid system represents a promising therapeutic target for the treatment of intestinal disease conditions characterized by excessive inflammatory responses.
Amidohydrolases↗
Embedding media for cryomicrotomy: an applicative reappraisal.
We reassess here the formulation of cryoembedding media in connection with recent developments in commercial cryomicrotomes. Water-based solutions of polyvinyl alcohols were our starting media, and each of 2 different polymers (56-98, MW approximately 195000; and 6-98, MW approximately 47000) showed a critical concentration for optimum sectioning. At higher or lower polymer concentrations, wrinkles and folds became apparent in tissue areas of sections, or in the sectioned embedding medium areas between tissues, respectively. Addition of polyethylene glycol (MW 380-420) further facilitated and improved sectioning, resulting in frozen tissue blocks that cut well in the 2 to 100 microm range and further, using disposable blades throughout. Applying a wide temperature differential between tissue specimen (-11 degrees C to -13 degrees C) and cutting knife (-33 degrees C to -35 degrees C), serial adjacent sections were reproducibly obtained at a 2-microm setting, singly or in short ribbons. Embedding media of low and high viscosity were obtained, depending on the polyvinyl alcohol polymer used, and could be applied sequentially for tissue infiltration followed by embedding with precise sample orientation. When required, media were made semisolid by addition of carboxymethylcellulose.
Animals↗
Equipment testing and tuning: the cold-knife cryomicrotome microm HM-560.
A major innovative feature of the Microm HM-560 cryomicrotome is the independent control of specimen and knife temperatures. We used such equipment with a variety of tissues, and fixation and freezing procedures. High-quality sectioning was reproducibly obtained using 1) a low temperature setting for the sectioning blade ("cold knife," about -33 degrees C); 2) a comparatively high temperature for the specimen; and 3) a suitable mounting medium, which would remain solid up to about 0 degrees C. Specimen temperature was set between -8 degrees and -15 degrees C for 4-microm sectioning, higher temperatures (-1 degrees to -8 degrees C) being appropriate when cutting at 10 to 20 microm. Under such conditions, disposable blades were effective throughout, while a modified antiroll plate profile further enhanced usability. After intensive use for almost 3 years, by more than 15 different users, the cryomicrotome is in excellent working order.
Animals↗