Gene therapy delivered micro-dystrophins co-localize with transgenic utrophin in dystrophic skeletal muscle fibers.

Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by the absence of functional dystrophin. There are multiple ongoing clinical trials for DMD that are testing gene therapy treatments consisting of adeno-associated viral (AAV) vectors carrying miniaturized versions of dystrophin optimized for function, termed micro-dystrophins (µDys). Utrophin, the fetal homolog of dystrophin, has repeatedly been reported to be upregulated in human DMD muscle as a compensatory mechanism, but whether µDys displaces full-length utrophin is unknown. In this study, dystrophin/utrophin-deficient mice with transgenic overexpression of full-length utrophin in skeletal muscles were systemically administered low doses of either AAV6-CK8e-Hinge3-µDys (µDysH3) or AAV6-CK8e-µDys5 (µDys5). We used immunofluorescence to qualitatively assess the localization of µDys with transgenic utrophin and neuronal nitric oxide synthase (nNOS) in quadriceps muscles. µDys protein resulting from both gene therapies co-localized at myofiber membranes with transgenic utrophin. We also confirmed the sarcolemmal co-localization of nNOS with µDys5, but not with transgenic utrophin expression or µDysH3. Transgenic utrophin expression and µDys proteins produced from both therapies stabilize the dystrophin-glycoprotein complex as observed by sarcolemmal localization of ß-dystroglycan. This study suggests that µDys gene therapy will likely not inhibit any endogenous compensation by utrophin in DMD muscle.

Micro-dystrophin gene therapy demonstrates long-term cardiac efficacy in a severe Duchenne muscular dystrophy model.

Micro-dystrophin gene replacement therapies for Duchenne muscular dystrophy (DMD) are currently in clinical trials, but have not been thoroughly investigated for their efficacy on cardiomyopathy progression to heart failure. We previously validated Fiona/dystrophin-utrophin-deficient (dko) mice as a DMD cardiomyopathy model that progresses to reduced ejection fraction indicative of heart failure. Adeno-associated viral (AAV) vector delivery of an early generation micro-dystrophin prevented cardiac pathology and functional decline through 1 year of age in this new model. We now show that gene therapy using a micro-dystrophin optimized for skeletal muscle efficacy (AAV-µDys5), and which is currently in a clinical trial, is able to fully prevent cardiac pathology and cardiac strain abnormalities and maintain normal (>45%) ejection fraction through 18 months of age in Fiona/dko mice. Early treatment with AAV-µDys5 prevents inflammation and fibrosis in Fiona/dko hearts. Collagen in cardiac fibrotic scars becomes more tightly packed from 12 to 18 months in Fiona/dko mice, but the area of fibrosis containing tenascin C does not change. Increased tight collagen correlates with unexpected improvements in Fiona/dko whole-heart function that maintain impaired cardiac strain and strain rate. This study supports micro-dystrophin gene therapy as a promising intervention for preventing DMD cardiomyopathy progression.

Mineralocorticoid receptor antagonists and glucocorticoids differentially affect skeletal muscle inflammation and pathology in muscular dystrophy.

Mineralocorticoid receptor antagonists (MRAs) slow cardiomyopathy in patients with Duchenne muscular dystrophy (DMD) and improve skeletal muscle pathology and function in dystrophic mice. However, glucocorticoids, known antiinflammatory drugs, remain a standard of care for DMD, despite substantial side effects. Exact mechanisms underlying mineralocorticoid receptor (MR) signaling contribution to dystrophy are unknown. Whether MRAs affect inflammation in dystrophic muscles and how they compare with glucocorticoids is unclear. The MRA spironolactone and glucocorticoid prednisolone were each administered for 1 week to dystrophic mdx mice during peak skeletal muscle necrosis to compare effects on inflammation. Both drugs reduced cytokine levels in mdx quadriceps, but prednisolone elevated diaphragm cytokines. Spironolactone did not alter myeloid populations in mdx quadriceps or diaphragms, but prednisolone increased F4/80hi macrophages. Both spironolactone and prednisolone reduced inflammatory gene expression in myeloid cells sorted from mdx quadriceps, while prednisolone additionally perturbed cell cycle genes. Spironolactone also repressed myeloid expression of the gene encoding fibronectin, while prednisolone increased its expression. Overall, spironolactone exhibits antiinflammatory properties without altering leukocyte distribution within skeletal muscles, while prednisolone suppresses quadriceps cytokines but increases diaphragm cytokines and pathology. Antiinflammatory properties of MRAs and different limb and respiratory muscle responses to glucocorticoids should be considered when optimizing treatments for patients with DMD.

Mineralocorticoid Receptor Signaling in the Inflammatory Skeletal Muscle Microenvironments of Muscular Dystrophy and Acute Injury.

Duchenne muscular dystrophy (DMD) is a striated muscle degenerative disease due to loss of functional dystrophin protein. Loss of dystrophin results in susceptibility of muscle membranes to damage, leading to muscle degeneration and continuous inflammation and fibrosis that further exacerbate pathology. Long-term glucocorticoid receptor (GR) agonist treatment, the current standard-of-care for DMD, modestly improves prognosis but has serious side effects. The mineralocorticoid receptor (MR), a ligand-activated transcription factor present in many cell types, has been implicated as a therapeutic target for DMD. MR antagonists (MRAs) have fewer side effects than GR agonists and are used clinically for heart failure. MRA efficacy has recently been demonstrated for DMD cardiomyopathy and in preclinical studies, MRAs also alleviate dystrophic skeletal muscle pathology. MRAs lead to improvements in muscle force and membrane stability and reductions in degeneration, inflammation, and fibrosis in dystrophic muscles. Myofiber-specific MR knockout leads to most of these improvements, supporting an MR-dependent mechanism of action, but MRAs additionally stabilize myofiber membranes in an MR-independent manner. Immune cell MR signaling in dystrophic and acutely injured normal muscle contributes to wound healing, and myeloid-specific MR knockout is detrimental. More research is needed to fully elucidate MR signaling in striated muscle microenvironments. Direct comparisons of genomic and non-genomic effects of glucocorticoids and MRAs on skeletal muscles and heart will contribute to optimal temporal use of these drugs, since they compete for binding conserved receptors. Despite the advent of genetic medicines, therapies targeting inflammation and fibrosis will be necessary to achieve optimal patient outcomes.

Influence of crystallite size on acetone hydrogenation over copper catalysts.

Acetone hydrogenation was studied over a family of Cu/SiO2 catalysts as well as UHP Cu powder and a Cu chromite catalyst. Oxygen chemisorption via dissociative N2O adsorption was used to count surface Cu atoms and calculate crystallite sizes, and a microwave absorption technique was used to measure the electrical conductivity of these Cu particles. Under differential reaction conditions at 423 K and 1 atm, all catalysts exhibited deactivation on stream and activities were typically 10-20% of their initial values after 3-4 h on stream. However, initial turnover frequencies (TOFs) varied from 0.056 s(-1) on the most highly dispersed Cu catalyst to 0.50 s(-1) on Cu powder, with the highest TOF of 2.4 s(-1) occurring on 110 nm crystallites. A similar trend with a broader (80-fold) variation was observed in the "steady-state" TOF values. Apparent activation energies varied between 11 and 14 kcal/mol. These initial TOF values are in good agreement with previous results, and a correlation exists between TOF and Cu crystallite size in this reaction, which appears to be structure sensitive. In addition, the electrical conductivity of these dispersed Cu nanoparticles, which was always less than that of bulk Cu, also increased with increasing Cu crystallite size; consequently, the change in this parameter may offer a possible explanation for the increase in TOF.

V delta repertoire during thymic ontogeny suggests three novel waves of gamma delta TCR expression.

Taking advantage of a PCR technique that allows amplification of all variable region genes with equal efficiency, we defined three novel waves of TCR delta-chain transcription during thymic ontogeny. The canonical DV101-D2-J2 rearrangement was confined to a narrow window from days 14 to 18 of gestation, indicating that the postulated two consecutive gamma delta precursor waves bearing this canonical DV101 rearrangement will coincide on day 16. Neonatal delta-chain transcripts used a second wave of diverse V alpha gene segments that are exclusively located in the delta locus-proximal gene cluster of intermingled single members of different V alpha subfamilies. In the adult, only expression of a clan of three homologous subfamilies, ADV7, DV104, and ADV17, persists. The members of the ADV7 subfamily are also scattered across the alpha locus, but their usage does not show the position-dependent bias of the other V alpha-to-delta rearrangements.

Ligand recognition by alpha beta T cell receptors.

While still incomplete, the first data concerning the biochemistry of T cell receptor-ligand interactions in cell-free systems seem to have considerable predictive value regarding whether a T cell response is strong or weak or suppressive. This data will help considerably in elucidating the mechanisms behind T cell responsiveness. Also of great interest are the first structures of T cell receptor molecules and, particularly, TCR-ligand complexes. These appear to confirm earlier suggestions of a fixed orientation for TCR engagement with peptide/MHC and should form the basis for understanding higher oligomers, evidence for which has also just emerged. We conclude with an analysis of the highly diverse CDR3 loops found in all antigen receptor molecules and suggest that such regions form the core of both TCR and antibody specificity.

Conserved motifs in T-cell receptor CDR1 and CDR2: implications for ligand and CD8 co-receptor binding.

Recent X-ray crystallographic structures of the T-cell receptor (TCR) alpha and beta chains, as well as their trimolecular complexes with peptide-MHC ligand, have established their structural similarity with the immunoglobulin molecules. The complementarity-determining region (CDR1) and CDR2 encoded within the TCR germline variable (V) sequence genes are well conserved across different TCR V alpha and V beta subfamilies. Multiple sequence alignments have been made based on structural information; they indicate that there will be only a limited number of canonical conformations for the first and second CDR loops. The limited diversity shown by CDRs 1 and 2 contrasts with the extreme junctional CDR3 diversity. Furthermore, CDR2 alignments have revealed conservation of a positive net charge in V alpha subfamilies. A model has been proposed for a direct interaction of the lateral part of CDR2 alpha with the negatively charged membrane-proximal 'stalk' region of the CD8 molecule.

Control of TCR V alpha-mediated positive repertoire selection and alloreactivity by differential J alpha usage and CDR3 alpha composition.

In rats expressing the f allele of the rat MHC (RT1f), CD8 T cells utilizing the V alpha 8.2 segment are 10-fold overselected during thymic development, resulting in V alpha 8.2 expression by 14% of mature CD8 T cells as compared to 1-2% in MHC congenic strains. In the alloreactive responses of CD8 T cells from RT1f-negative rats against RT1f, V alpha 8.2+ CD8 T cells are also preferentially expanded. Neither overselection nor alloreactivity of V alpha 8.2+ TCR require selective V beta pairing. However, RT1f alloreactive V alpha 8.2+ TCR preferentially use a related set of J alpha segments which contribute short homogeneous CDR3 alpha loops, with features suggesting peptide promiscuity, and little N additions. In contrast, only few overselected V alpha 8.2+ CD8 T cells showed an imprint of positive selection on J usage or CDR3 composition. The results demonstrate that a single V alpha segment can promote both MHC allele-specific positive selection and alloreactivity, and that the latter is more dependent on an additional contribution of CDR3 alpha, possibly by promoting reactivity with a diverse set of MHC-bound peptides or by providing additional MHC contacts.

TCR-delta gene rearrangement and selection during fetal thymocyte development.

TCR delta-chain gene rearrangements were analyzed at different stages of thymic ontogeny. The VDJ delta junctional sequences of the dominantly expressed TCR delta-chain V7 subfamily are highly conserved in fetal and neonatal thymocytes. They are equally conserved in C delta-targeted mice lacking cell surface expression of gamma delta receptors, indicating evolutionary selection acting at the level of DNA rearrangement. Yet, in C delta-mutant mice, the frequency of in-frame transcripts is reduced to 61%, from 88% in wild-type mice, suggesting cellular selection of this DV7-overexpressing gamma delta thymocyte subset. In contrast, in genomic DNA rearrangements, no difference was found in the frequency of productive rearrangements between mutant (26%) and wild-type mice (31%). This in-frame rate, characteristic of random rearrangements, indicates that positive selection is not required for thymic maturation of gamma delta T cells. The results are discussed with regard to models of alpha beta/gamma delta lineage commitment.

Identification of the complete expressed human TCR V gamma repertoire by flow cytometry.

Five of the six expressed human TCR V gamma regions (V gamma 2, 3, 4, 8 and 9) can be detected by available mAb. No mAb with specificity for the remaining V gamma 5 region has been described. In this study, we have characterized mAb 56.3, which was obtained after immunization with V gamma 2/3/4/9-negative thymic gamma delta cells. V gamma transcripts were analyzed by inverse PCR and RT-PCR in 56.3+ cell lines and clones derived from peripheral blood. mAb 56.3 recognized all cells that expressed in-frame V gamma 5 transcripts. In addition, mAb 56.3 recognized some but not all cells expressing V gamma 3, but did not react with a large panel of clones expressing V gamma 2, 4, 8 or 9. In combination with anti-V gamma 2/3/4 mAb 23D12, V gamma 5-expressing cells could be clearly identified as 56.3+23D12(-). In some donors, mAb 56.3 also recognized a small fraction of TCR alpha beta cells. At the clonal level, these cells expressed in-frame V gamma 5-J beta-C beta or V gamma 3-J beta-C beta trans-rearrangements. When mAb 56.3 was combined with V gamma 2/3/4-, V gamma 8- and V gamma 9-specific mAb, all peripheral blood gamma delta T cells were stained. Thus, mAb 56.3 supplements the panel of available TCR V gamma-specific mAb. It is now possible to analyze the complete expressed human V gamma repertoire by flow cytometry.

Clonal expansion of Vgamma3/Vdelta3-expressing gammadelta T cells in an HIV-1/2-negative patient with CD4 T-cell deficiency.

Immunological investigations were carried out in an HIV-1/2//HTLV-1-negative patient with CD4 T-cell deficiency (0.357-0.6 x 10(9)/l) and expansion of gammadelta T cells which accounted for 26-42% of peripheral blood lymphocytes during an observation period of 3 years. Flow cytometry analyses with a panel of available Vgamma/Vdelta-specific monoclonal antibodies indicated that the pathologically expanded gammadelta population expressed Vgamma2 or Vgamma3 paired with Vdelta3 on the surface but lacked the expression of activation antigens such as CD38 or CD71. Cloning and sequencing of RT-PCR products obtained after amplification of cDNA with Vgamma-Cgamma and Vdelta-Cdelta specific primers confirmed the presence of a clonally expanded Vgamma3/Vdelta3 population in the peripheral blood of this patient. Cytotoxicity assays performed with purified gammadelta T cells as effectors and resting or preactivated autologous CD4 T cells as targets failed to reveal evidence for autoreactive cytotoxicity of Vgamma3/Vdelta3 cells as a possible mechanism of CD4 T-cell deficiency in this patient.

Inverse PCR amplification of low-abundancy message of gamma delta T cell receptor genes.

The technique of inverse PCR permits the rapid amplification and identification of unknown DNA segments adjacent to well characterized core regions. In the field of immunology anchored PCR and inverse PCR are useful methods for examining junctional diversity and unknown variable gene segments of rearranged T cell receptor genes. We have applied an improved inverse PCR protocol to study the repertoire of gamma delta T cell receptor genes in the developing thymus of the mouse.

Comparison of human and mouse T-cell receptor variable gene segment subfamilies.

Like the immunoglobulin Igh-V and Igk-V gene families, the human or mouse TCRV gene families may be grouped into subfamilies displaying > 75% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most mouse Tcr-V subfamilies exhibit clear homology to human TCRV subfamilies (> 60% amino acid sequence similarity). Homologous pairs of TCRV genes in mice and humans show higher sequence similarity than TCRV genes from different subfamilies within either species, indicating transspecies evolution of TCRV genes. Mouse and human homologues show conservation of their relative map order, particularly in the 3' region and a similar sequential and developmentally programmed expression. When the V regions from both species were analyzed together, local length differences and conserved residues in the loop regions were revealed, characteristic of each of the four TCRV families.

Human T-cell receptor variable gene segment families.

Multiple DNA and protein sequence alignments have been constructed for the human T-cell receptor alpha/delta, beta, and gamma (TCRA/D, B, and G) variable (V) gene segments. The traditional classification into subfamilies was confirmed using a much larger pool of sequences. For each sequence, a name was derived which complies with the standard nomenclature. The traditional numbering of V gene segments in the order of their discovery was continued and changed when in conflict with names of other segments. By discriminating between alleles at the same locus versus genes from different loci, we were able to reduce the number of more than 150 different TCRBV sequences in the database to a repertoire of only 47 functional TCRBV gene segments. An extension of this analysis to the over 100 TCRAV sequences results in a predicted repertoire of 42 functional TCRAV gene segments. Our alignment revealed two residues that distinguish between the highly homologous V delta and V alpha, one at a site that in VH contacts the constant region, the other at the interface between immunoglobulin VH and VL. This site may be responsible for restricted pairing between certain V delta and V gamma chains. On the other hand, V beta and V gamma appear to be related by the fact that their CDR2 length is increased by four residues as compared with that of V alpha/delta peptides.

Mouse T-cell receptor variable gene segment families.

All mouse T-cell receptor alpha/delta, beta, and gamma variable (Tcra/d-, b-, and g-V) gene segments were aligned to compare the sequences with one another, to group them into subfamilies, and to derive a name which complies with the standard nomenclature. It was necessary to change the names of some V gene segments because they conflicted with those of other segments. The traditional classification into subfamilies was re-evaluated using a much larger pool of sequences. In the mouse, most V gene segments can be grouped into subfamilies of closely related genes with significantly less similarity between different subfamilies.

T cell receptor gamma delta repertoire in HIV-1-infected individuals.

While V gamma 9/V delta 2 cells dominate among peripheral blood gamma delta T cells in healthy adults, the majority of gamma delta T cells in most HIV-1-infected individuals express V delta 1. We asked whether these elevated levels of V delta 1 T cells were due to clonal expansion. Three-color flow cytometry with monoclonal antibodies against V gamma 2/V gamma 3/V gamma 4, V gamma 4 and V gamma 9 was used to investigate V gamma usage in 27 patients with elevated numbers of V delta 1 T cells. While the relative proportion of V gamma 9 cells among gamma delta T cells was significantly reduced in HIV-1+ individuals (10 +/- 11% vs. 80 +/- 17%, p < 0.001), the fraction of gamma delta T cells using V gamma 5 or V gamma 8 was significantly increased (54 +/- 15% vs. 7 +/- 11%, p < 0.001). In 1 patient, 76% of the V delta 1 cells expressed V gamma 2 or V gamma 3, suggesting clonality of the V delta 1 population. In line with this assumption, analysis of the V delta 1-J delta junctional regions by reverse transcription-polymerase chain reaction (RT-PCR) resulted in products of only one junctional length, as demonstrated by electrophoresis on denaturing gels, and 12 out of 16 (75%) in-frame junctional sequences were identical in this patient. In other HIV-1+ patients, RT-PCR resulted in products of several distinct sizes, also indicating a highly restricted repertoire. After sequencing the V delta 1-J delta junctional regions of 3 additional patients, we found repeated but patient-specific in-frame junctions accounting for 10-30% of the sequenced clones. However, limited V delta 1-J delta junctional diversity was also seen in healthy donors. RT-PCR products from 10 healthy individuals resulted in distinct bands on denaturing gels. In 1 of them exhibiting a single prominent band, 10 out of 17 (58%) sequenced junctions were identical. Two other healthy donors displayed 2/14 and 5/18 identical junctional sequences, respectively. Taken together, our results reveal significant alterations of V gamma usage in HIV-1+ patients, while the V delta 1 junctional repertoire is similarly restricted in HIV-1+ and HIV-1- individuals. Therefore, these data argue against an obligatory clonal expansion of V delta 1-expressing cells during HIV-1 infection.

New monoclonal antibody (23D12) recognizing three different V gamma elements of the human gamma delta T cell receptor. 23D12+ cells comprise a major subpopulation of gamma delta T cells in postnatal thymus.

The gamma delta TCR is expressed on 1 to 5% of CD3+ human peripheral blood T lymphocytes. The majority of peripheral blood gamma delta T cells expresses V gamma 9 paired with V delta 2; this subset strongly responds to certain microbial ligands. Other gamma delta T cell subsets with unknown Ag specificity expressing different V gamma elements are present in peripheral blood and lymphoid tissue. We describe a new anti-human V gamma mAb termed 23D12 with unusual specificity. As revealed by analysis of a large number of T cell clones and transfectants expressing molecularly well-defined gamma delta TCR, mAb 23D12 recognized several, but not all, members of the human V gamma 1 family, specifically V gamma 2, V gamma 3, and V gamma 4 but not V gamma 5 or V gamma 8. In combination with available mAb against V gamma 4, mAb 23D12 was used to identify V gamma 2- or V gamma 3-bearing cells. On average, 23D12+ cells accounted for 18% of peripheral blood gamma delta T cells and 56% of postnatal gamma delta thymocytes. In combination with anti-V gamma 9 mAb, mAb 23D12 23D12 identified gamma delta cells expressing V elements other than V gamma 2, V gamma 3, V gamma 4, or V gamma 9. Such cells are detectable in peripheral blood and postnatal thymus. Using mAb 23D12, we also confirmed the appearance of two distinct TCR gamma-chains on the surface of some gamma delta T cells.

Diversity of functional T-cell receptor delta-chain transcripts from bone marrow cells of athymic nude mice.

CD3+ cells are detectable in bone marrow of athymic mice homozygous for the nude mutation. As previously shown, cells expressing the gamma delta T-cell receptor (TcR) represent 30-40% of this T-cell population. Using V delta-specific, V alpha 4-specific, and C delta-specific primers, TcR delta-chain transcripts were reverse transcribed and polymerase chain reaction (PCR)-amplified from total RNA prepared from bone marrow cells (BMC) of 6-month-old NMRI nu/nu mice. Amplified TcR delta-chain cDNA was cloned, and 49 randomly selected clones derived from seven amplification reactions were sequenced. Sequence analyses showed: (1) more than 80% of the sequenced clones represented in-frame transcripts of the TcR delta-chain; (2) in-frame transcripts containing V delta 1-, V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6- and V alpha 4-gene segments were detectable in nude BMC; (3) V delta 2-, V delta 4- and V delta 5-containing transcripts were more abundant and more diverse than V delta 1- and V delta 3-containing transcripts; (4) extensive N-region diversity was present in the V delta-D delta 2 (N1), D delta 2-D delta 1 (N2) and D delta 1-J delta 1 (N3) junctional regions; (5) P nucleotide additions were present in many transcripts; and (6) unusual truncated, in-frame transcripts with deleted D- and J-region genes were detected. A large potential TcR delta-chain repertoire is thus present in nude BMC.