Stromal vascular fraction in the treatment of myositis.

Muscle regeneration is a physiological process that converts satellite cells into mature myotubes under the influence of an inflammatory environment progressively replaced by an anti-inflammatory environment, with precise crosstalk between immune and muscular cells. If the succession of these phases is disturbed, the immune system can sometimes become auto-reactive, leading to chronic muscular inflammatory diseases, such as myositis. The triggers of these autoimmune myopathies remain mostly unknown, but the main mechanisms of pathogenesis are partially understood. They involve chronic inflammation, which could be associated with an auto-reactive immune response, and gradually with a decrease in the regenerative capacities of the muscle, leading to its degeneration, fibrosis and vascular architecture deterioration. Immunosuppressive treatments can block the first part of the process, but sometimes muscle remains weakened, or even still deteriorates, due to the exhaustion of its capacities. For patients refractory to immunosuppressive therapies, mesenchymal stem cells have shown interesting effects but their use is limited by their availability. Stromal vascular fraction, which can easily be extracted from adipose tissue, has shown good tolerance and possible therapeutic benefits in several degenerative and autoimmune diseases. However, despite the increasing use of stromal vascular fraction, the therapeutically active components within this heterogeneous cellular product are ill-defined and the mechanisms by which this therapy might be active remain insufficiently understood. We review herein the current knowledge on the mechanisms of action of stromal vascular fraction and hypothesise on how it could potentially respond to some of the unmet treatment needs of refractory myositis.

Death and non-lethal weapons: A case of homicide by penetrating injury without projectile.

Herein, we report the death of a man, approximately thirty years old, victim of a short-range shot to the thorax from a PIEXON JPX4, a hand weapon classed category D in France, said to be non-lethal. External examination of the lesion revealed characteristics similar to those of ballistic injuries. The autopsy found an intercostal wound that reached the pulmonary parenchyma and the pulmonary artery, with severe hemothorax. By the end of the autopsy, no projectile had been found in the body, nor any exit orifice. Death was caused by the effects of hemorrhagic shock. The PIEXON JPX4 has four cartridges, projecting a capsaicin gel that is designed to irritate the ENT area (ear nose throat) to incapacitate an assailant. The manufacturer recommends not to use it at distances of less than 1.5 m. Experimental shots were performed on gelatine blocks at point-blank range and at distances of 5, 10, 20, 30, 50, 100 and 150 cm to evaluate the distance necessary for the jet of gel to have a penetrating effect. Shots at 5-30 cm penetrated the structure. None of the other shots were penetrating. The autopsy and experiment data therefore show the penetrating potential of the jet of gel. Herein, we report the first death due to use of the PIEXON JPX4. From a forensic investigation viewpoint, we add a new exception to the "bullet rule". (The odd and even bullet rule states that if the number of gunshot wounds of entrance and exit found in the body is even, the presumption is that no bullet is lodged in the body. If the number of gunshot wounds of entrance and exit is odd, the presumption is that one or more bullets have been lodged in the body.).

Anti-desmoglein 1 antibodies in Tunisian healthy subjects: arguments for the role of environmental factors in the occurrence of Tunisian pemphigus foliaceus.

Pemphigus foliaceus is an autoimmune blistering skin disease mediated by autoantibodies directed against desmoglein 1 and occurs as a sporadic form throughout the world, or as an endemic form called fogo selvagem in Brazil. Healthy subjects living in Brazilian endemic areas produce antidesmoglein 1 antibodies, suggesting the role of environmental factors in the initiation of the autoimmune response. Tunisia was described recently as an endemic area where the disease is characterized by its high rate among young people, especially women. An enzyme-linked immunosorbent assay using recombinant desmoglein 1 as antigen was used to detect antibodies against desmoglein 1 and calibrated with sera from 67 French healthy blood donors, 20 French pemphigus foliaceus patients and patients with other bullous skin diseases. When sera from 179 healthy Tunisian blood donors were tested, 31 (17%) were found positive. The desmoglein 1 binding activity of these 31 sera was confirmed in 10 cases by indirect immunofluorescence analysis and/or immunoblotting using human epidermal extract. Subclass analysis of antidesmoglein 1 antibodies showed that they were almost exclusively of the IgG2 subclass in positive normal sera and of IgG4 subclass in patients with PF. Thus, antibodies against desmoglein 1 are prevalent in normal subjects living in Tunisia which, along with their IgG2 isotype, suggests the role of the environment in the pathogenesis of this endemic type of pemphigus foliaceus and the need for additional factors to switch from a subclinical to a clinical form of the disease.

Epistasis between DSG1 and HLA class II genes in pemphigus foliaceus.

Pemphigus foliaceus (PF) is a rare and severe cutaneous autoimmune disease caused by autoantibodies directed against desmoglein 1 (DSG1), a desmosomal adhesion glycoprotein. We previously showed that the DSG1 gene is polymorphic and that a coding synonymous T/C single nucleotide polymorphism at position 809 is associated with PF. To determine whether the disease occurred as a consequence of complex genetic interactions, we simultaneously examined the contribution of major histocompatibility complex (MHC) class II and DSG1 polymorphisms to PF susceptibility. Our analysis performed in 31 PF patients and 84 healthy controls first confirmed the previously reported common DRB1*04 and DRB1*14 genetic background in PF and individualized DRB1*0102, DRB1*0402 and DRB1*0406, and DRB1*1404 as susceptibility MHC class II alleles in French Caucasian PF patients. It also showed that the C/C(809) genotype was associated with PF. Combined analysis of HLA class II and DSG1 polymorphisms with several distinct statistical methods including logistic regression, showed that the DRB1*04 allele and the C/C(809) genotype interact to confer a higher susceptibility to PF. These data demonstrate the role of epistasis between individual genes in PF susceptibility and illustrate the genetic complexity of organ-specific autoimmune diseases.

A polymorphic variant of the gene coding desmoglein 1, the target autoantigen of pemphigus foliaceus, is associated with the disease.

Two polymorphic markers were identified on the desmoglein 1 gene which encodes the autoantigen targeted by pathogenic antibodies in pemphigus foliaceus (PF), a cutaneous autoimmune blistering disease. The first marker, made of a variant haplotype of five mis-sense mutations located on the part of the gene encoding the fourth and fifth extracellular domains of the protein, is not associated with the disease. The second marker consists of a single silent T to C transition at position 809 and was found to be significantly more frequent (P = 0.015) in Caucasian PF patients (n = 36) than in controls (n = 98). Thus, pemphigus foliaceus constitutes another example of autoimmune disease in which the autoantigen polymorphism contributes to disease susceptibility.

Expansion of circulating V gamma 9/V delta 1 T cells in a patient with a syndrome of recurrent fever: evidence for an unusual antigen-driven process leading to selection of recurrent motifs within TCR junctional loops of diverse lengths.

Polyclonal expansions of human Vdelta1 T cells have been described in diverse physiopathological situations without strong TCR structural data for an antigen-driven selection. Here, we have analyzed the phenotype and TCR repertoire of gamma delta T cells obtained from the peripheral blood of a 19-year-old patient with a syndrome of recurrent fever, which accounted for up to 40% of CD3(+) T cells and expressed predominantly Vgamma9 and Vdelta1 TCR regions and a memory phenotype. Sequence analysis of Vdelta1-Jdelta1 transcripts derived from peripheral blood lymphocytes (PBL) indicated that, while Vdelta1-Jdelta1 junctional sequences were diverse in length, all but one contained several recurrent motifs at conserved positions from both the 5'- and 3'-ends of the complementarity-determining region (CDR)3 loop. Analysis of gamma delta T cell clones derived from patient PBL demonstrated that Vgamma9(+) but not Vgamma9(-) T cell clones frequently expressed Vdelta1 chains with these characteristics and unveiled a hierarchy between the constraints imposed on the 5'- vs. the 3' motifs of the Vdelta1 CDR3 loops. These results constitute the first strong evidence for a nominal antigen-driven selection of Vdelta1 T cells in vivo and also suggest that the hierarchy of the constraints imposed by antigens respectively on the length and amino acid composition of TCR CDR3 loops differs between alpha beta and gamma delta T cells.

The zinc fingers of HIV nucleocapsid protein NCp7 direct interactions with the viral regulatory protein Vpr.

The 96-amino acid protein Vpr functions as a regulator of cellular processes involved in human immunodeficiency virus, type 1 (HIV-1) life cycle, in particular by interrupting cells division in the G2 phase. Incorporation of Vpr in the virion was reported to be mediated by the C-terminal domain of the Pr55(Gag) polyprotein precursor, which includes NCp7, a protein involved in the genomic RNA encapsidation and p6, a protein required for particle budding. To precisely define the Gag and Vpr sequences involved in this protein-protein interaction, NCp7, p6, and Vpr as well as a series of derived peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Binding assays were carried out by Far Western experiments and by competition studies using (52-96)Vpr immobilized onto agarose beads. The results show that interaction between NCp7 and Vpr occurs in vitro by a recognition mechanism requiring the zinc fingers of NCp7 and the last 16 amino acids of Vpr. Moreover, NCp10, the equivalent of NCp7 in Moloney murine leukemia virus but not polysine inhibits Vpr-NCp7 complexation. Interestingly enough, Vpr was found to interact with Gag, NCp15, and NCp7 but not with mature p6 in vitro. In vivo mutations in NCp7 zinc fingers in an HIV-1 molecular clone led to viruses with important defects in Vpr encapsidation. Together, these results suggest that NCp7 cooperates with p6 to induce Vpr encapsidation in HIV-1 mature particles. The NCp7-Vpr complex could also be important for interaction of Vpr with cellular proteins involved in cell division.

Spectrum of mutations in the COL4A5 collagen gene in X-linked Alport syndrome.

Alport syndrome is a mainly X-linked hereditary disease of basement membranes that is characterized by progressive renal failure, deafness, and ocular lesions. It is associated with mutations of the COL4A5 gene located at Xq22 and encoding the alpha5 chain of type IV collagen. We have screened 48 of the 51 exons of the COL4A5 gene by SSCP analysis and have identified 64 mutations and 10 sequence variants among 131 unrelated Alport syndrome patients. This represents a mutation-detection rate of 50%. There were no hot-spot mutations and no recurrent mutations in our population. The identified mutations were 6 nonsense mutations, 12 frameshift mutations, 17 splice-site mutations, and 29 missense mutations, 27 of the latter being glycine substitutions in the collagenous domain. Two of these occurred on the same allele in one patient and segregated with the disease in the family. We showed that some of the glycine substitutions could be associated with the lack of immunological expression of the alpha3(IV)-alpha5(IV) collagen chains in the glomerular basement membrane.

Splice-mediated insertion of an Alu sequence in the COL4A3 mRNA causing autosomal recessive Alport syndrome.

Alport syndrome is a mainly X-linked hereditary disease of basement membranes characterized by progressive renal failure, deafness, and ocular lesions. The alpha 3(IV) and alpha 4(IV) collagen genes have been recently shown to be involved in the less frequent autosomal recessive form. When screening lymphocyte COL4A3 mRNAs from Alport patients, we found a mutant whose transcripts were disrupted by a 74 bp insertion at the junction of exons IV or V and VI. The insertion derives from an antisense Alu element in COL4A3 intron V, which has been spliced into the alpha 3(IV) mRNA due to a G to T transversion activating a cryptic acceptor splice site in this Alu element. There is complete segregation of this mutation with the disease in the family. Our findings provide the first evidence for the pathogenic role of abnormal splicing of COL4A3. Moreover, we demonstrate the superiority of mutation screening at the mRNA level to detect a hitherto poorly recognized mutation mechanism in humans, splice-mediated insertion of an Alu fragment into a coding sequence.

Deletions in the COL4A5 collagen gene in X-linked Alport syndrome. Characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.

The type IV collagen alpha 5 chain (COL4A5) gene of 88 unrelated male patients with X-linked Alport syndrome was tested for major gene rearrangements by Southern blot analysis, using COL4A5 cDNA probes. 14 different deletions were detected, providing a 16% deletion rate in the COL4A5 gene in the patient population. The deletions are dispersed all over the gene with different sizes, ranging from 1 kb to the complete absence of the gene (> 250 kb) in one patient. In four patients with intragenic deletions, absence of the alpha 3 (IV) chain in the glomerular basement membrane was demonstrated by immunohistochemical studies. This finding supports the hypothesis that abnormalities in the alpha 5 (IV) chain may prevent normal incorporation of the alpha 3 (IV) chain into the glomerular basement membrane. Direct sequencing of cDNA amplified from lymphoblast mRNA of four patients with internal gene deletions, using appropriate combinations of primers amplifying across the predicted boundaries of the deletions, allowed us to determine the effect of the genomic rearrangements on the transcripts and, by inference, on the alpha 5 (IV) chain. Regardless of the extent of deletion and of the putative protein product, the 14 deletions occur in patients with juvenile-type Alport syndrome.