Silvia Stringara

Alteration of Th17 and Treg cell subpopulations co-exist in patients affected with systemic sclerosis.

Aim of the study has been to understand the relationship between TH17 and Treg cell subsets in patients affected with systemic sclerosis (SSc). Phenotypes and functions of Th17 and Treg cell subsets were analyzed in a series of 36 SSc patients. Th17 cell concentration in the peripheral blood was found to be increased in SSc patients with respect to healthy controls independently from type or stage of disease. After PBMC stimulation with a polyclonal stimulus or Candida albicans antigens the frequency of Th17 T cell clones was significantly higher in SSc patients with respect to controls suggesting the skewing of immune response in SSc patients toward Th17 cell generation/expansion. Concerning the Treg compartment, both CD4+CD25+ and CD8+CD28- Treg subsets showed quantitative and qualitative alteration in the peripheral blood of SSc patients. Collectively, these data highlight the existence of an imbalanced ratio between Th17 and Treg cell subsets in SSc patients.

CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes.

CD39 is an ectoenzyme, present on different immune cell subsets, which mediates immunosuppressive functions catalyzing ATP degradation. It is not known whether CD39 is expressed and implicated in the activity of CD8+ regulatory T lymphocytes (Treg). In this study, CD39 expression and function was analyzed in both CD8+ and CD4+CD25hi Treg from the peripheral blood of healthy donors as well as from tumor specimens. CD39 was found expressed by both CD8+ (from the majority of healthy donors and tumor patients) and CD4+CD25hi Treg, and CD39 expression correlated with suppression activity mediated by CD8+ Treg. Importantly, CD39 counteraction remarkably inhibited the suppression activity of CD8+ Treg (both from peripheral blood and tumor microenvironment) suggesting that CD39-mediated inhibition constitutes a prevalent hallmark of their function. Collectively, these findings, unveiling a new mechanism of action for CD8+ Treg, provide new knowledge on intratumoral molecular pathways related to tumor immune escape, which could be exploited in the future for designing new biological tools for anticancer immune intervention.

Caveolin-3 T78M and T78K missense mutations lead to different phenotypes in vivo and in vitro.

Caveolins are the principal protein components of caveolae, invaginations of the plasma membrane involved in cell signaling and trafficking. Caveolin-3 (Cav-3) is the muscle-specific isoform of the caveolin family and mutations in the CAV3 gene lead to a large group of neuromuscular disorders. In unrelated patients, we identified two distinct CAV3 mutations involving the same codon 78. Patient 1, affected by dilated cardiomyopathy and limb girdle muscular dystrophy (LGMD)-1C, shows an autosomal recessive mutation converting threonine to methionine (T78M). Patient 2, affected by isolated familiar hyperCKemia, shows an autosomal dominant mutation converting threonine to lysine (T78K). Cav-3 wild type (WT) and Cav-3 mutations were transiently transfected into Cos-7 cells. Cav-3 WT and Cav-3 T78M mutant localized at the plasma membrane, whereas Cav-3 T78K was retained in a perinuclear compartment. Cav-3 T78K expression was decreased by 87% when compared with Cav-3 WT, whereas Cav-3 T78M protein levels were unchanged. To evaluate whether Cav-3 T78K and Cav-3 T78M mutants behaved with a dominant negative pattern, Cos-7 cells were cotransfected with green fluorescent protein (GFP)-Cav-3 WT in combination with either mutant or WT Cav-3. When cotransfected with Cav-3 WT or Cav-3 T78M, GFP-Cav-3 WT was localized at the plasma membrane, as expected. However, when cotransfected with Cav-3 T78K, GFP-Cav-3 WT was retained in a perinuclear compartment, and its protein levels were reduced by 60%, suggesting a dominant negative action. Accordingly, Cav-3 protein levels in muscles from a biopsy of patient 2 (T78K mutation) were reduced by 80%. In conclusion, CAV3 T78M and T78K mutations lead to distinct disorders showing different clinical features and inheritance, and displaying distinct phenotypes in vitro.

Aquaporin-4 expression is severely reduced in human sarcoglycanopathies and dysferlinopathies

Aquaporin-4 (AQP4) is the major water channel expressed in fast-twitch skeletal muscle fibers. AQP4 is reduced in Duchenne and Becker Muscular Dystrophies, but not in caveolinopathies, thus suggesting an interaction with dystrophin or with members of the dystrophin-glycoprotein complex (DGC) rather than a nonspecific effect due to muscle membrane damage. To establish the role of sarcoglycans in AQP4 decrease occurring in muscular dystrophy, AQP4 expression was analyzed in muscle biopsies from patients affected by Limb Girdle Muscular Dystrophies (LGMDs) 2C-F genetically confirmed. In all the LGMD 2C-F (2α-, 1β-, 2γ-, 1δ-deficiency), AQP4 was severely decreased. This effect was associated to a marked reduction in α1-syntrophin levels. In control muscle AQP4 did not show a direct interaction with any of the four sarcoglycans but, it co-immunoprecipitated with α1-syntrophin, indicating that this modular protein may link AQP4 levels with the DGC complex. To determine whether AQP4 expression could be affected in other LGMDs due to the defect of a membrane protein not associated to the dystrophin complex, we examined AQP4 expression in 6 patients affected by dysferlin deficiency genetically confirmed. All the patients displayed a reduction of the water channel, and AQP4 expression appeared to correlate with the severity of the muscle histopathological lesions. However, differently from what observed in the sarcoglycans, α1-syntrophin expression was normal or just slightly reduced. These results seem to indicate an additional mechanism of regulation of AQP4 levels in muscle cells.In accordance with a specific effect of membrane muscle disorders, AQP4 protein levels were not changed in 3 mitochondrial and 3 metabolic myopathies. In conclusion, AQP4 expression and membrane localization are markedly reduced in LGMD 2B-2F. The role of AQP4 in the degenerative mechanism occurring in these diseases will be the object of our future research.

Mitochondrial DNA Deletion in a Child With Mitochondrial Encephalomyopathy, Growth Hormone Deficiency, and Hypoparathyroidism

We report an 11-year-old boy with short stature, bilateral ptosis, sensorineural hearing loss, muscle weakness, and endocrine abnormalities. Brain magnetic resonance imaging (MRI) showed a bilateral abnormal signal in the globus pallidus and in the midbrain tegment. Muscle biopsy specimens showed ragged red and cytochrome c oxidase negative fibers, and biochemical analysis of muscle homogenate showed a partial defect of complex I and IV activities of the respiratory chain enzymes. Analysis of mitochondrial DNA by a polymerase chain reaction screening procedure and Southern blot revealed a novel heteroplasmic single mitochondrial DNA deletion of 7.8 kb in different tissues. This deletion was absent in the blood DNA of his mother and brother. This case further expands and confirms the wide clinical spectrum of mitochondrial disorders associated with single large-scale mitochondrial DNA deletions. (J Child Neurol 2006;21:983—985; DOI 10.2310/7010.2006.00218).

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

GDAP1 mutation in autosomal recessive Charcot-Marie-Tooth with pyramidal features

Sirs: Two Italian siblings, a 18year-old man and a 12-year-old girl, were admitted to our hospital with peripheral neuropathy. The symptoms had started at the age of 5 and 6 years with abnormal gait and weakness in distal leg muscles. The two sibs showed a clinical picture characterized by wasting and severe weakness of the small hand muscles (MRC strength scale of III/V in the abductor brevis and opponens pollicis, abductor digiti minimi, interossei and lumbricali), gross atrophy and weakness of distal leg muscles, pes cavus, steppage gait with bilateral foot drop. Tendon reflexes in the upper limbs and knee reflexes were brisk (4+) and tendon tap to quadriceps showed thigh adductor muscle activation; ankle reflexes were absent. The plantar reflex was difficult to assess owing to the marked weakness of dorsiflexor muscles of the toe. Vibration sense was slightly reduced at ankles. Cranial nerves were intact. Neither patient showed hoarseness or vocal cord paralysis. Nerve conduction studies and needle EMG indicated the presence of an axonal sensory-motor neuropathy in both patients. In the man, compound muscle action potential (CMAP) was unrecordable in the abductor hallucis muscle and showed a marked amplitude reduction in the abductor pollicis brevis and abductor digiti minimi. In the girl, CMAP amplitude was markedly reduced in the median and ulnar nerves and within the normal range in the tibial nerve. Sensory action potential amplitude was markedly reduced in all the explored nerves (Table 1). EMG showed clear signs of chronic denervation in the explored muscles and some fibrillation potentials in tibialis anterior muscle of the older patient. Sural nerve biopsy, performed in the older sibling, revealed segmental demyelination and remyelination with onion bulb formation, a pattern typically seen in inherited demyelinating neuropathies. Transcranial magnetic stimulation (TMS) indicated corticospinal damage in the man: motor evoked potentials (MEPs) were normal in upper limbs and unrecordable from abductor hallucis muscle. MEPs recorded from rectus femoris muscle showed a prolonged central motor conduction time (CMCT, 18ms: nv <16ms, figure 1). In the girl, MEPs were normal both for upper and lower limbs. Brain and cervical spine MRI were normal. Six exons of ganglioside-induced differentiation-associated protein 1 (GDAP1) were amplified by PCR using primers, sequenced and electrophoresed as previously described [3]. Molecular analysis revealed a homozygous missense mutation (c.347 T>G) in exon 3 of GDAP1 gene, predicting a methionine to arginine change at codon 116 (p.M116R). The mutation was not found in 196 controls by minisequencing analysis of exon 3 amplificons (extended oligonucleotide 5’-atgttgtacccgtgggtaatac-3’; SNaPshot kit, Applied Biosystems). The genetic basis of CharcotMarie-Tooth disease (CMT) with pyramidal features remains largely undefined so far [1,7]. To date, a single MFN2 (mitofusin 2) mutation has been found in a family with CMT2A and pyramidal signs [8]. Thus, the identification of additional mutations causing both upper and lower neuron damage is a step toward the genetic dissection of CMT. Our patients presented with CMT and pyramidal features due to a M116R mutation of GDAP1 gene. GDAP1 belongs to the subfamily of glutathione S-transferases. It is localized in mitochondria with a possible role in the maintenance of the R. Biancheri (&) Æ F. Zara Æ P. Striano M. Pedemonte Æ D. Cassandrini S. Stringara Æ C. Minetti Dept. of Neuroscience and Rehabilitation Giannina Gaslini Institute Muscular and Neurodegenerative Disease Unit, University of Genova Largo G. Gaslini 5 16147 Genova, Italy Tel.: +39-010-563/6603 Fax: +39-010-353/8265 E-Mail: roberta@biancheri.com

Middle interhemispheric variant of holoprosencephaly: A very mild clinical case

Middle interhemispheric variant (MIH) of holoprosencephaly or syntelencephaly was described in 19931 as a fourth subtype of holoprosencephaly (HPE), in addition to the three classic types of alobar, semilobar, and lobar HPE.2 MIH consists of an abnormal midline continuity of the posterior frontal and parietal regions of the cerebral hemispheres, with separation of the basal forebrain, anterior frontal lobes, and occipital regions. Although MIH and classic HPE share a number of similarities, they are related to different embryologic mechanisms. Classic HPE is caused by a defect in the formation of the embryonic floor plate, whereas MIH is secondary to a disturbance of formation of the roof plate.3 The ZIC2 gene plays a critical role in differentiation of the roof plate of the developing embryo in the dorsal midline of the neural tube.3 In mice, decreased levels of ZIC2 result in the failure to form midline CNS structures. In humans, mutations in the ZIC2 gene have been found in ∼3 to 4% of HPE cases, including individuals with MIH, confirming that MIH is a variant of HPE.4 …