Angela Cadoni

Evidence That Human Oral Epithelium Reconstituted In Vitro And Transplanted Onto Patients With Defects In The Oral Mucosa Retains Properties Of The Original Donor Site1

Normal human skin—derived keratinocytes cultured in vitro reconstitute a stratified epidermis suitable for grafting onto burn patients and patients with skin defects such as giant nevi or chronic leg ulcers. In vitro experiments and long-term studies of patients receiving cultured epidermis autografts on muscular fascia suggest that skin keratinocytes possess an intrinsic site specific differentiation program that is fully expressed only when the reconstituted epidermis is transplanted in vivo to different body sites. In this study we cultivated for the first time palate-derived epithelial cells that were able to reconstitute a palatal epithelium. We also demonstrate that this epithelium can be successfully transplanted onto patients presenting lack of adherent keratinizing gingival mucosa and is able, in a relatively short time, to fully express the differentiation program typical of the original donor site. The possibility of obtaining large quantities of cultured epithelium, able to retain properties of the original donor site, starting from 1–3-mm2 biopsies, could prove extremely useful in the reconstructive surgery of the mouth and of other mucosal body areas.

HLA‐DR Schwann cell reactivity in peripheral neuropathies of different origins

HLA-DR antigens have been found on Schwann cells in peripheral neuropathies of different origins but not in normal control cases. Class II antigen reactivity was more intense in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and hereditary motor and sensory neuropathy type 1 (HMSN), but was also observed in toxic or metabolic neuropathies. The expression of HLA-DR antigen on Schwann cells does not appear to be related to the inflammatory or autoimmune origin of the disease.

Different cellular and molecular mechanisms for early and late-onset myelin protein zero mutations

Mutations in the gene MPZ, encoding myelin protein zero (MPZ), cause inherited neuropathies collectively called Charcot-Marie-Tooth type 1B (CMT1B). Based on the age of onset, clinical and pathological features, most MPZ mutations are separable into two groups: one causing a severe, early-onset, demyelinating neuropathy and a second, causing a late-onset neuropathy with prominent axonal loss. To investigate potential pathomechanisms underlying the two phenotypes, we transiently transfected HeLa cells with two late-onset (T95M, H10P) and two early-onset (H52R, S22_W28 deletion) mutations and analyzed their effects on intracellular protein trafficking, glycosylation, cell viability and intercellular adhesion. We found that the two late-onset mutations were both transported to the cell membrane and moderately reduced MPZ-mediated intercellular adhesion. The two early-onset mutations caused two distinct abnormalities. H52R was correctly glycosylated and trafficked to the plasma membrane, but strongly affected intercellular adhesion. When co-expressed with wild-type MPZ (wtMPZ), a functional dominant negative effect was observed. Alternatively, S22_W28 deletion was retained within the cytoplasm and reduced both adhesion caused by wtMPZ and cellular viability. Since the same trafficking patterns were observed in transfected murine Schwann cells, they are not an artifact of heterologous cell expression. Our results suggest that at least some late-onset mutations cause a partial loss of function in the transfected cells, whereas multiple abnormal gain of function pathways can result in early-onset neuropathy. Further characterization of these pathways will lead to a better understanding of the pathogenesis of CMT1B and a rational basis for treating these debilitating inherited neuropathies.

Reliability of side-to-side ultrasound cross-sectional area measurements of lower extremity nerves in healthy subjects

Introduction: In peripheral nerve ultrasound, the healthy contralateral side may be used as internal control. Therefore, inherent side‐to‐side differences must be minimal. The goal of this study was to assess intrastudy, intraobserver, and interobserver reproducibility of ultrasound in comparative side‐to‐side evaluation of lower limb nerves. Methods: Lower limb nerves of 60 normal subjects were evaluated by 3 radiologists. Bilateral sciatic, tibial, common fibular, sural, lateral femoral cutaneous, femoral, obturator, and saphenous nerves were evaluated. Results: Overall, side‐to‐side differences were not statistically significant at any level. In the lower limb nerves, in a between‐limb comparison, the minimum detectable difference of cross‐sectional area ranged from 16.4 mm2 (sciatic nerve at the level of piriformis muscle) to 0.4 mm2 (saphenous nerve). Conclusion: In general, the healthy contralateral side can be used as an internal control. Muscle Nerve 46: 717–722, 2012

GFAP expression of human Schwann cells in tissue culture

We have studied the expression of the intermediate filament (IF) proteins, vimentin and glial fibrillary acidic protein (GFAP), in cultured human Schwann cells (SC) from patients with different neuropathies and normal control cases. SC cultures from sural nerve biopsies of 8 subjects with axonal neuropathies, 8 with demyelinating neuropathies and 3 normal controls were included in this study and processed with double immunofluorescence technique, using anti-vimentin and anti-GFAP antibodies, during the 2nd, 4th and 6th week of culture. Five cultures incubated with anti-GFAP antibodies were also processed for immunoelectron microscopy. Specificity tests of the used antibodies were performed. We have found that: (1) cultured human SC constantly express vimentin; (2) SC from normal controls are GFAP-negative in the first period of culture; (3) SC from pathologic nerves can contain GFAP-immunoreactive IF and the percentage of GFAP-positive SC is higher in axonal than in demyelinating neuropathies; (4) during the permanence in culture human SC from both normal and pathologic cases acquire the ability to synthesize GFAP. The obtained data suggest that the removal from axonal contact and the resulting loss of myelinating function induce a cytoskeletal cellular response in human SC characterized by the cytoplasmic accumulation of GFAP-immunoreactive IF.

Schwann cell GFAP expression increases in axonal neuropathies

We studied the Schwann cell (SC) GFAP immunoreactivity in normal human peripheral nerves and in neuropathies of different origin. Immunofluorescence and immunocytochemistry were carried out on serial frozen sections of 58 peripheral nerve biopsies using monoclonal antibodies (mabs) antivimentin and anti GFAP, and antiserum anti S-100 and anti GFAP. To test the specificity of the mabs and antiserum used, proper competition controls on tissue sections of 2 selected cases, tissue cultures studies of human fibroblasts and immunoblotting of homogenates of human fibroblasts, 3 normal and 5 pathologic nerves were carried out. In order to evaluate a possible correlation between SC GFAP positivity and neuropathologic findings a quantitative study was performed, evaluating the SC GFAP reactivity in all the 58 cases, and relating the SC GFAP positivity to the index of nerve pathology (IP) in 9 selected cases, and to the percentage of teased fibers showing axonal degeneration or demyelination and remyelination in 25 representative cases. We demonstrate that in normal human sural nerves and in demyelinating neuropathies only a few scattered SC are recognized by the mabs or antiserum anti GFAP. On the contrary in axonal neuropathies the majority of SC gain the property to express intermediate filaments which show common antigenic properties with GFAP.

Forebrain white matter in spontaneously hypertensive rats: a quantitative image analysis study.

The volume and the morphology of brain white matter as well as the number and the size of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were investigated in 6-month-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats. The volume of frontal and occipital cortex and of hippocampus was decreased in SHR in comparison with normotensive rats, whereas the volume of neostriatum was unchanged. A remarkable decrease of the volume of internal capsule and striosomes, a moderate reduction of that of corpus callosum and no changes of the volume of external capsule and of white matter of hippocampus were also observed in SHR. In SHR the number of astrocytes was higher in the frontal and occipital cortex and in the white matter of the CA1 and CA3 subfields of the hippocampus, but not in the corpus callosum or in the grey matter of the CA1 and CA3 subfields. Staining for myelin did not reveal alterations in single fibre sheath morphology. These findings indicate the occurrence of changes of forebrain white matter in SHR, consisting in the reduction of it without qualitative modifications of myelinated fibres. The development of gliosis apparently not related with changes of volume of white matter was also found.

Nerves of the hand beyond the carpal tunnel.

Imaging studies including ultrasound (US) and magnetic resonance imaging may be required to evaluate the median nerve in patients with suspected carpal tunnel syndrome. However, the radial and ulnar nerves contribute to sensory and motor innervations to the hand as well. Compressive, traumatic, and iatrogenic events may damage the small terminal branches of these nerves. In the hand, US is able to identify injuries of the median, ulnar, radial nerve, and terminal branches. This article presents the role of imaging to evaluate the nerves of the hand with an emphasis on US. Due to its high-resolution capabilities, US is useful to determine the location, extent, and type of nerve lesion. Moreover, US is useful for a postsurgical assessment. The anterior interosseous nerve, Guyons tunnel syndrome, and Wartenbergs syndrome are also described.

Anatomical study of the iliohypogastric, ilioinguinal, and genitofemoral nerves using high-resolution ultrasound

Introduction: In this study we aimed to determine whether high‐resolution ultrasound (US) can identify the iliohypogastric (IH), ilioinguinal (II), and genitofemoral (GF) nerves and their relations. Methods: This investigation, initially undertaken in cadavers, was followed by a high‐resolution US study in 30 healthy adult volunteers (180 nerves) by 2 musculoskeletal radiologists on separate occasions, using 2 different approaches (proximal to distal and distal to proximal). A 0–3 scale was used to assess nerve visibility. Location and course of the IH, II, and GF nerves and their relations to adjacent anatomical structures were analyzed. Results: Nerves and their terminal branches were better visualized with the distal‐to‐proximal approach (P < 0.05). Visualization of the terminal branches was possible in up to 60% of volunteers. Conclusions: High‐resolution ultrasound (US) can identify the IH, II, and GF nerves at the level of the abdominal wall and the terminal branches in the majority of volunteers. Muscle Nerve 51: 42–48, 2015

Effects of Cholinergic Enhancing Drugs on Cholinergic Transporters in the Brain and Peripheral Blood Lymphocytes of Spontaneously Hypertensive Rats

Cholinergic hypofunction is a trait of Alzheimers disease and vascular dementia and countering it is one of the main therapeutic strategies available for these disorders. Cholinergic transporters control cellular mechanisms of acetylcholine (ACh) synthesis and release at presynaptic terminals. This study has assessed the influence of 4 week treatment with two different cholinergic enhancing drugs, the cholinergic precursor choline alphoscerate (alpha-glyceryl-phosphorylcholine) or the acetylcholinesterase (AChE) inhibitor galantamine on high affinity choline uptake transporter (CHT) and vesicular ACh transporter (VAChT) expression in the brain of spontaneously hypertensive rats (SHR). SHR represent an animal model of cerebrovascular injury characterized by cholinergic hypofunction. Analysis was performed by immunochemistry, ELISA and immunohistochemistry on frontal cortex, striatum and hippocampus. Immunochemical and ELISA analysis was extended to peripheral blood lymphocytes (PBL), used as a peripheral reference of changes of brain cholinergic markers. An increased expression of VAChT and CHT was observed in brain areas investigated and in PBL of SHR. The similar trend for cholinergic transporters observed in brain and PBL suggests these cells may represent a marker of brain cholinergic transporters. Treatment with choline alphoscerate increased CHT and to a greater extent VAChT expression. Treatment with galantamine countered the increase of CHT and VAChT. The different activity of the cholinergic precursor and of the AChE inhibitor on parameters investigated is likely related to their mechanism of action. Choline alphoscerate increases ACh synthesis and release. This requires an augmentation of systems regulating neurotransmitter uptake and storage. The effect of choline alphoscerate on CHT and VAChT observed in this study suggests an improved synaptic efficiency elicited by the compound. The AChE inhibitor slows-down ACh degradation in the synaptic cleft. A greater availability of neurotransmitter elicited by galantamine counters the enhanced activity of cholinergic transporters compensating cholinergic deficits. Differences in the activity of the cholinergic precursor and AChE inhibitor investigated on CHT and VAChT suggests that association between choline alphoscerate and AChE/cholinesterase inhibitors may represent a strategy for potentiating deficient cholinergic neurotransmission worthwhile of being investigated in clinical trials.