Liver perfusion changes occurring during pancreatic islet engraftment: a dynamic contrast-enhanced magnetic resonance study.

The aim of this study was to investigate liver microvascular adaptation following the intraportal infusion of pancreatic islets (pancreatic islet transplantation [islet-tx]) in diabetic patients using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). DCE-MRI was performed before and 7 days after islet-tx in six diabetic patients. Initial area under curve (AUC60) and volume transfer coefficient (Ktrans) were assessed as markers of liver perfusion. Clinical and metabolic monthly follow-up was performed in all patients, considering fasting C-peptide and ß-score as main indices of graft function. High variability in the response of liver microvasculature to islet infusion was observed: two patients showed a significant reduction in liver perfusion after transplantation (pt.2: AUC60 = -23.4%, Ktrans = -31.7%; pt.4: AUC60 = -23.7%, Ktrans = -27.9%); three patients did not show any significant variation of liver perfusion and one patient showed a significant increase (pt.3: AUC60 = +31%, Ktrans = +42.8%). Interestingly, a correlation between DCE-MRI parameters and indices of graft function was observed and, in particular, both patients with DCE-MRI evidence of posttransplantation liver perfusion reduction experienced premature graft failure. Our preliminary study demonstrated that DCE-MRI may identify different adaptive responses of liver microvasculature in patients submitted to islet-tx. These different responses could have an impact on islet engraftment, although reported findings need confirmation from larger studies.

Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients.

BACKGROUND: Antibodies against aquaporin-4 (AQP4), a water channel particularly expressed on perivascular astrocytic podocytes, are proposed as a marker for the diagnosis of neuromyelitis optica (NMO). However, a consensus on seroprevalence and optimal detection method has not yet been reached. OBJECTIVES: To investigate the performance of different assays to detect anti-AQP4 antibodies. METHODS: We set up five different assays. Two of them were capable to detect perivascular IgG reactivity on brain tissue by immunofluorescence (NMO-IgG). Other three assays have been set to detect anti-AQP4 antibodies: immunofluorescence and flow cytometry on AQP4-expressing cells, and a radioimmunoprecipitation assay. We assessed sensitivity and specificity of these assays by interrogating sera of 33 NMO patients, 13 patients at high risk to develop NMO (hrNMO), 6 patients affected by acute partial transverse myelitis (APTM), 20 patients with multiple sclerosis (MS), and 67 age- and sex-matched healthy controls. RESULTS: We found that the presence of serum NMO-IgG and anti-AQP4 reactivity is almost exclusively restricted to patients with NMO and hrNMO. Seroprevalence and sensitivity ranged from 30 to 47%, depending on the assay. Specificity ranged from 95 to 100%. Comparing results obtained in the five assays, we noticed lack of concordance in some samples. CONCLUSIONS: Detection of NMO-IgG or anti-AQP4 antibodies may represent a valuable tool to assist neurologists in the differential diagnosis between patients with NMO, hrNMO, APTM, or MS. The current lack of a gold standard to detect anti-AQP4 antibodies implies the necessity to standardize the detection of these antibodies.

Improving the procedure for detection of intrahepatic transplanted islets by magnetic resonance imaging.

Islet transplantation is an effective therapy for restoring normoglycemia in type-1 diabetes, but long-term islet graft function is achieved only in a minority of cases. Noninvasive magnetic resonance imaging of pancreatic islets is an attractive option for "real-time" monitoring of graft evolution. So far, previous studies have been performed in the absence of a standardized labeling procedure and, besides a feasibility study in patients, the effectiveness and safety of various labeling approaches were tested only with high field magnets (4.7 T). In this study, we addressed: (a) standardization of a labeling procedure for human islets with clinically-approved contrast agent Endorem, (b) safety aspects of labeling related to inflammation and (c) quality of imaging both at 7 T and 1.5 T. We have highlighted that the ratio of Endorem/islet is crucial for reproducible labeling, with a ratio of 2.24 ug/IEQ, allowing successful in vivo imaging both with 1.5 T and 7.0 T magnets up to 143 days after intrahepatic transplant. With this standardized labeling procedure, labeled islets are neither inflamed nor more susceptible to inflammatory insults than unlabeled ones. This report represents an important contribution towards the development of a standardized and safe clinical protocol for the noninvasive imaging of transplanted islets in humans.

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation.

AIMS/HYPOTHESIS: We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. METHODS: The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. RESULTS: Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin +/- high glucose release of chromogranin B predominated. Weak, Ca(2+)-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca(2+) sensitivity of the specific granules. CONCLUSIONS/INTERPRETATION: The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology.

Neurosecretory cells without neurosecretion: evidence of an independently regulated trait of the cell phenotype.

Neurosecretion competence is a fundamental property that enables differentiated neurones and professional neurosecretory cells to store neurotransmitters and hormones in specialized organelles, the synaptic-like vesicles and dense granules, and to release them by regulated exocytosis. In our laboratory, the study of rat phaeochromocytoma (PC12) clones that fail to express the above organelles or any other components involved in neurosecretion, whilst maintaining most of the general markers of the parental population, has served to demonstrate that this trait is controlled independently from the rest of the phenotype. The present review focuses on recent advances in elucidating the molecular mechanisms governing neurosecretion competence. Moreover, the opportunities that such neurosecretion-defective PC12 clones offer for the investigation of new aspects of regulated exocytosis and the localization of its components are summarized.

Blockade of membrane transport and disassembly of the Golgi complex by expression of syntaxin 1A in neurosecretion-incompetent cells: prevention by rbSEC1.

The t-SNAREs syntaxin1A and SNAP-25, i.e. the members of the complex involved in regulated exocytosis at synapses and neurosecretory cells, are delivered to their physiological site, the plasma membrane, when transfected into neurosecretion-competent cells, such as PC12 and AtT20. In contrast, when transfection is made into cells incompetent for neurosecretion, such as those of a defective PC12 clone and the NRK fibroblasts, which have no endogenous expression of these t-SNAREs, syntaxin1A (but neither two other syntaxin family members nor SNAP-25) remains stuck in the Golgi-TGN area with profound consequences to the cell: blockade of both membrane (SNAP-25, GAT-1) and secretory (chromogranin B) protein transport to the cell surface; progressive disassembly of the Golgi complex and TGN; ultimate disappearance of the latter structures, with intermixing of their markers (mannosidase II; TGN-38) with those of the endoplasmic reticulum (calreticulin) and with syntaxin1A itself. When, however, syntaxin 1A is transfected together with rbSec1, a protein known to participate in neurosecretory exocytosis via its dynamic interaction with the t-SNARE, neither the blockade nor the alterations of the Golgi complex take place. Our results demonstrate that syntaxin1A, in addition to its role in exocytosis at the cell surface, possesses a specific potential to interfere with intracellular membrane transport and that its interaction with rbSec1 is instrumental to its physiological function not only at the plasma membrane but also within the cell. At the latter site, the rbSec1-induced conversion of syntaxin1A into a form that can be transported and protects the cell from the development of severe structural and membrane traffic alterations.

Differential expression of distinct members of Rho family GTP-binding proteins during neuronal development: identification of Rac1B, a new neural-specific member of the family.

Previous studies on small GTP-binding proteins of the Rho family have revealed their involvement in the organization of cell actin cytoskeleton. The function of these GTPases during vertebrate development is not known. With the aim of understanding the possible role of these proteins during neuronal development, we have cloned and sequenced five members expressed in developing chick neural retinal cells. We have identified four chicken genes, cRhoA, cRhoB, cRhoC, and cRac1A, homologous to known human genes, and a novel Rac gene, cRac1B. Analysis of the distribution of four of the identified transcripts in chicken embryos shows for the first time high levels of expression of Rho family genes in the vertebrate developing nervous system, with distinct patterns of distribution for the different transcripts. In particular, cRhoA and cRac1A gene expression appeared ubiquitous in the whole embryo, and the cRhoB transcript was more prominent in populations of neurons actively extending neurites, whereas the newly identified cRac1B gene was homogeneously expressed only in the developing nervous system. Temporal analysis of the expression of the five genes suggests a correlation with the morphogenetic events occurring within the developing retina and the retinotectal pathway. Expression of an epitope-tagged cRac1B in retinal neurons showed a diffuse distribution of the protein in the cell body and along neurites. Taken as a whole, our results suggest important roles for ubiquitous and neural-specific members of the Rho family in the acquisition of the mature neuronal phenotype.

Exposure to perinatal morphine promotes developmental changes in rat striatum.

This study shows that perinatal exposure to morphine promotes developmental changes (up to 8 months of life) in the striatum by up-regulating concentrations of substance P and met-enkephalin with changes of prometenkephalin A mRNA expression at the day of birth only. Dopamine metabolism (up to 60 days) is also increased as suggested by the reduced concentrations of dopamine and increased content of 3,4-dihydroxyphenylacetic acid. Tyrosine hydroxylase mRNA expression is selectively reduced only in the substantia nigra by perinatal morphine. Serotonin content is reduced only during the early postnatal days and is unaffected thereafter. Supplementation of naltrexone to morphine-exposed rats prevents monoaminergic and neuropeptidergic changes in the striatum, which directly implicates opioid receptors in the developmental changes caused by morphine. The data suggest that perinatal morphine may inhibit met-enkephalin release, causing accumulation of the peptide without corresponding changes in specific mRNA. Dopamine release may also be increased as indicated by a higher metabolism and consequent reduction of tyrosine hydroxylase mRNA expression in the substantia nigra.

Tyrosine phosphorylation induced by integrin-mediated adhesion of retinal neurons to laminin.

Integrin alpha 6 beta 1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to understand some of the intracellular mechanisms which allow integrin-mediated neurite extension on laminin in primary neuronal cultures. Both integrin-mediated adhesion to laminin and antibody-induced integrin clustering resulted in the increased tyrosine phosphorylation of a 120 kDa polypeptide which was identified as the focal adhesion kinase. The kinetics of phosphorylation and dephosphorylation of this kinase were dramatically different in neurons plated on laminin, than in neurons in which the receptors were clustered with anti-integrin antibodies. To look at possible interactions of the focal adhesion kinase with integrins, we made use of sucrose velocity gradients, which have allowed the identification of a large complex containing the alpha 6 beta 1 laminin receptor. Analysis of the gradients showed that the focal adhesion kinase was not associated with the integrin receptors under these experimental conditions, while about 26% of the c-Src kinase codistributed with the integrin receptor complex, and showed a molecular size and a distribution similar to that of a 59 kDa phosphoprotein comigrating with the alpha 6 beta 1 receptor. Our results suggest that integrin-induced tyrosine phosphorylation is an early intracellular event during neuronal adhesion, and that the integrin-mediated increase in tyrosine phosphorylation of the focal adhesion kinase is not sufficient per se for the induction of neurite outgrowth. Furthermore, our data indicate that Src kinase may be involved in integrin-mediated neuronal interactions with laminin.

Perinatal exposure to morphine: reactive changes in the brain after 6-hydroxydopamine.

The effects of neonatal 6-hydroxydopamine treatment on the brain of control rats and of rats perinatally exposed to morphine were examined. Noradrenaline levels were increased in the pons-medulla, mesencephalon and caudate of 8-week-old control rats lesioned with neonatal 6-hydroxydopamine; perinatal morphine treatment prevented such an increase. In the caudate, there was a loss of dopamine and an increase of serotonin following the neurotoxic lesion; exposure to perinatal morphine prevented the serotonin increase. Brain expression of synapsin I mRNA was particularly abundant in cerebral cortex, hippocampus, dentate gyrus and olfactory bulb. In perinatal morphine-treated rats, the expression of synapsin I mRNA was significantly reduced; interestingly, the neonatal treatment with 6-hydroxydopamine normalized its expression. Therefore, brain-reactive neurochemical changes triggered by 6-hydroxydopamine were suppressed by perinatal morphine exposure whereas the association of morphine exposure and 6-hydroxydopamine lesion promoted the normal mRNA expression of the synaptic marker synapsin I.

In situ hybridization study of myelin protein mRNA in rats with an experimental diabetic neuropathy.

Distribution of protein zero (P0) and myelin basic protein (MBP) mRNAs in the sciatic nerve from rats with alloxan-induced diabetes was analyzed at two different time points using in situ hybridization. Some animals of each diabetic group were treated with insulin. Densitometric quantitation of silver clusters revealed that 5 weeks after diabetes induction P0 mRNA only is significantly increased, while at 14 weeks both P0 and MBP mRNA contents are markedly higher than controls. Insulin treatment normalizes glycemia levels and slightly counteracts increased P0 mRNA at both stages of diabetes. An increase in MBP mRNA is observed in chronic diabetic animals only, and is unaltered by the normoglycemic effect of insulin. The increased transcript levels of P0 and MBP suggest that Schwann cells can modulate gene expression of myelin-specific proteins in response to diabetic-induced metabolic derangement. Such a change may represent a higher turnover of myelin proteins as an attempt by the Schwann cells to repair the diabetes-induced nerve damage. The observed pattern of transcript amount is only slightly influenced by insulin treatment.

Perinatal morphine. I: Effects on synapsin and neurotransmitter systems in the brain.

We have previously shown that rat perinatal exposure to morphine causes dopaminergic and met-enkephalin (ME) and substance P (SP) changes in the striatum during the early postnatal period (Tenconi et al.: Int J Dev Neurosci 10: 517 - 526, 1992); in addition it increases the susceptibility to neurotoxic lesions and impairs regenerative capacity of the serotoninergic system (Gorio et al.: J Neurosci Res 34: 462 - 471, 1993). Our study shows that ME and SP levels increase postnatally in several areas of the rat brain, reaching the highest values between 30 and 60 days, after which the peptide content subsides to lower levels. Perinatal exposure to morphine increases such ME and SP levels during the early stages of postnatal life. No effect of morphine on 5-HT and NE is observed, while the dopaminergic system is mainly affected in the mesencephalon. The pre- and postnatal brain expression of synapsin I mRNA is gradually and progressively localized in discrete areas of the brain. In the brain of rats perinatally exposed to morphine, the abundance of synapsin I mRNA expression is markedly reduced. Therefore, perinatal exposure to morphine affects early postnatal synaptic development in the brain as shown by the altered peptidergic and monoaminergic content and by the reduced synapsin I mRNA expression.

Myelin protein transcripts increase in experimental diabetic neuropathy.

A Northern blot analysis of P0 and MBP myelin protein transcripts in the sciatic nerve from rats with alloxan-induced diabetes at two different time points is described. After 5 weeks of diabetes induction, only P0 mRNA is significantly increased by 39%, while at 14 weeks both P0 and MBP mRNA contents are markedly higher than controls. Insulin treatment normalizes glycemia levels, partially counteracts P0 mRNA increase at both stages of diabetes and delays MBP mRNA increase present only in chronic animals. We suggest that increased transcript levels of P0 and MBP in Schwann cells may represent a higher turnover of myelin sheath specific proteins in diabetic syndrome, as attempt to repair the hyperglycemia-induced nerve damage, which is partially prevented by insulin treatment.

Distribution of gephyrin transcripts in the adult and developing rat brain.

The peripheral membrane protein gephyrin copurifies with the inhibitory glycine receptor of mammalian spinal cord. It binds with high affinity to polymerized tubulin and has been implicated in the anchoring of the glycine receptor to cytoskeletal elements. Recently, cDNA cloning has identified variants of the gephyrin mRNA, which originate from alternative splicing of four exonic regions (cassettes 1-4). In this study, the expression patterns of gephyrin splice variants were determined in the adult and developing rat brain by in situ hybridization with synthetic oligonucleotide probes. Gephyrin transcripts were detected throughout the brain and spinal cord, with mRNAs containing cassette 2 (C2 transcripts) being predominant in adult animals. C3 and C4 transcripts were seen in cerebellar granule cells and in the dentate gyrus, whereas a C1 probe did not produce detectable hybridization signals. During development, C2 and C3 mRNAs were found in most brain regions. Generally, the spatial and temporal distribution of gephyrin transcripts is similar to that of the glycine receptor beta subunit mRNA reported previously.

Diabetes-induced alterations of central nervous system G proteins. ADP-ribosylation, immunoreactivity, and gene-expression studies in rat striatum.

Previous studies from our laboratory have suggested that diabetes-associated central nervous system abnormalities are characterized by progressive alterations of neurotransmitters and of transductional Gi/Go proteins. In this study, we have further characterized these abnormalities in the striatum of alloxan-diabetic rats by means of adenosine 5'-diphosphate (ADP)-ribosylation, and Western and Northern blotting techniques. Fourteen weeks after diabetes induction, pertussis-toxin (PTX) catalyzed ADP-ribosylation of Gi/Go proteins was markedly reduced in diabetic animals, as shown by a clear decrease of 32P-ADPribose incorporation into G protein alpha subunits. In agreement with our previous pharmacological studies that showed a reduction of Gi-mediated modulation of adenylate cyclase activity only at this stage of diabetes, no changes in PTX-mediated ADP-ribosylation were observed earlier (5-wk diabetes). Immunoblotting studies performed by using antibodies selectively raised against Gi-2, Go, and Gs proteins did not reveal any differences between control and diabetic animals at any stage of diabetes. Similarly, the mRNAs corresponding to the alpha subunits of Gi-2, Go, and Gs proteins did not show any marked changes in chronic diabetic rats with respect to control animals. It is therefore concluded that diabetes is associated with development of a time-related alteration of cerebral Gi/Go proteins and that this defect is not owing to gross changes in either content of G proteins or mRNA level, but probably reflects modifications of G protein's structure or physiological status affecting the coupling with membrane effector systems and the sensitivity to PTX.

Perinatal morphine exposure alters peptidergic development in the striatum.

It has been reported that perinatal exposure to opiates affects mRNA synthesis, body growth and brain development in mammals, including humans. We have observed that morphine administration in drinking water during the perinatal period alters peptide development in the striatum of the rat. There is a marked increase in substance P and met-enkephalin content, the latter is maintained even at 30 days postnatally. The transient increase or earlier maturation of substance P content is correlated by a more precocious axon terminal organization as revealed by immunocytochemical staining. The increased metenkephalin content is correlated by a higher abundance of preproenkephalin A mRNA and this correlation is particularly evident at 15 days postnatally. At earlier times both northern blotting and in situ hybridization techniques fail to show any significant difference between control and morphine exposed rats, likely because the peptide content is not very different in the two groups or at least the gap is not as wide as at later times.

Differential expression of synaptophysin and synaptoporin mRNAs in the postnatal rat central nervous system.

Synaptophysin and synaptoporin are two homologous integral membrane proteins of small synaptic vesicles. Here, the distribution of the corresponding transcripts in the CNS of the rat was investigated by in situ hybridization using sequence-specific oligonucleotide probes. Synaptophysin mRNA was abundantly distributed through all major brain regions, whereas synaptoporin transcripts displayed a more restricted localization in telencephalic structures. Resolution at the cellular level disclosed a differential labeling of distinct cell types in different areas, suggesting that synaptophysin and synaptoporin are expressed by specific subpopulations of central neurons. Consistent with this conclusion, relative synaptoporin mRNA contents were found to vary between different brain regions during postnatal development, whereas synaptophysin transcripts showed a more uniform increase during the same period.

Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain.

The inhibitory glycine receptor (GlyR) is a ligand-gated ion channel which mediates post-synaptic inhibition in spinal cord and other regions of the vertebrate central nervous system. Previous biochemical and molecular cloning studies have indicated heterogeneity of GlyRs during development. Here, the distribution of GlyR subunit transcripts in rat brain and spinal cord was investigated by in situ hybridization using sequence-specific oligonucleotide probes. In adult animals, GlyR alpha 1 subunit mRNA was abundant in spinal cord, but was also seen in a few brain areas, e.g. superior and inferior colliculi, whereas alpha 2 transcripts were found in several brain regions including layer VI of the cerebral cortex and hippocampus. GlyR alpha 3 subunit mRNA was expressed at low levels in cerebellum, olfactory bulb and hippocampus, while high amounts of beta subunit transcripts were widely distributed throughout spinal cord and brain. During development, alpha 2 mRNA accumulated already prenatally and decreased after birth, whereas alpha 1 and alpha 3 subunit transcripts appeared only in postnatal brain structures. Hybridization signals of beta subunit mRNA were seen already at early embryonic stages and continuously increased to high levels in adult rats. These data reveal unexpected differences in the regional and developmental expression of GlyR subunit mRNAs and point to novel functions of GlyR proteins in the mammalian central nervous system.

Alternative splicing generates two isoforms of the alpha 2 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein which displays developmental heterogeneity in the mammalian central nervous system. Here we describe 2 novel cDNA variants of the rat GlyR alpha 2 subunit and demonstrate that alternative splicing generates these 2 isoforms. The deduced protein sequences (alpha 2A and alpha 2B) exhibit 99% identity with the previously characterized human alpha 2 subunit. In situ hybridization revealed expression of both alpha 2A and alpha 2B mRNAs in the prenatal rat brain, suggesting that these variant proteins may have a role in synaptogenesis. Heterologous expression in Xenopus oocytes showed that the more abundantly expressed alpha 2A subunit forms strychnine-sensitive ion channels which resemble human alpha 2 subunit GlyRs in their electrophysiological properties.