Impact of COVID-19 & Response Measures on HIV-HCV Prevention Services and Social Determinants in People Who Inject Drugs in 13 Sites with Recent HIV Outbreaks in Europe, North America and Israel.

HIV/HCV prevention among people who inject drugs (PWID) is of key public health importance. We aimed to assess the impact of COVID-19 and associated response measures on HIV/HCV prevention services and socio-economic status of PWID in high-HIV-risk sites. Sites with recent (2011-2019) HIV outbreaks among PWID in Europe North America and Israel, that had been previously identified, were contacted early May 2020. Out of 17 sites invited to participate, 13 accepted. Semi-structured qualitative site reports were prepared covering data from March to May 2020, analyzed/coded and confirmed with a structured questionnaire, in which all sites explicitly responded to all 103 issues reported in the qualitative reports. Opioid maintenance treatment, needle/syringe programs and antiretroviral treatment /hepatitis C treatment continued, but with important reductions and operational changes. Increases in overdoses, widespread difficulties with food and hygiene needs, disruptions in drug supply, and increased homelessness were reported. Service programs rapidly reformed long established, and politically entrenched, restrictive service delivery policies. Future epidemic control measures should include mitigation of negative side-effects on service provision and socio-economic determinants in PWID.

RESUMEN: La prevención del VIH/VHC entre las personas que se inyectan drogas (PWID) es de vital importancia para la salud pública. Nuestro objetivo fue evaluar el impacto de COVID-19 y las medidas de respuesta asociadas en los servicios de prevención del VIH/VHC y el estado socioeconómico de las PWID en sitios de alto riesgo de VIH. Se contactó con sitios con brotes recientes (2011­2019) de VIH entre PWID en Europa, América del Norte e Israel, que habían sido previamente identificados, a principios de mayo de 2020. De los 17 sitios invitados a participar, 13 aceptaron. Se prepararon informes cualitativos semiestructurados del sitio que cubrían los datos de marzo a mayo de 2020, analizados/codificados y confirmados con un cuestionario estructurado, en el que todos los sitios respondieron explícitamente a los 103 asuntos reportados en los informes cualitativos. El tratamiento de mantenimiento con opiáceos, los programas de agujas/jeringas y el tratamiento antirretroviral/tratamiento de la hepatitis C continuaron, pero con importantes reducciones y cambios operativos. Se reportaron aumentos en las sobredosis, dificultades generalizadas con las necesidades alimentarias y de higiene, interrupciones en el suministro de medicamentos y aumento de personas sin hogar. Los programas de servicios reformaron rápidamente las políticas restrictivas de prestación de servicios, establecidas desde hace mucho tiempo y políticamente arraigadas. Las futuras medidas de control de epidemias deben incluir la mitigación de los efectos secundarios negativos en la prestación de servicios y los determinantes socioeconómicos en las PWID.

Arginine, ornithine and citrulline supplementation in rainbow trout: Free amino acid dynamics and gene expression responses to bacterial infection.

Supplementing the diet with functional ingredients is a key strategy to improve fish performance and health in aquaculture. The amino acids of the urea and nitric oxide (NO) cycles - arginine, ornithine and citrulline - perform crucial roles in the immune response through the generation of NO and the synthesis of polyamine used for tissue repair. We previously found that citrulline supplementation improves and maintains circulating free arginine levels in rainbow trout more effectively than arginine supplementation. Here, to test whether supplementation of urea cycle amino acids modulates the immune response in rainbow trout (Oncorhynchus mykiss), we supplemented a commercial diet with high levels (2% of total diet) of either arginine, ornithine or citrulline during a 7-week feeding trial, before challenging fish with the bacterium Aeromonas salmonicida. We carried out two separate experiments to investigate fish survival and 24 h post-infection to investigate the immediate response of free amino acid levels, and transcriptional changes in genes encoding urea cycle, NO cycle and polyamine synthesis enzymes. There were no differences in percentage fish mortality between diets, however there were numerous highly significant changes in free amino acid levels and gene expression to both dietary supplementation and infection. Out of 26 amino acids detected in blood plasma, 8 were significantly changed by infection and 9 by dietary supplementation of either arginine, ornithine or citrulline. Taurine, glycine and aspartic acid displayed the largest decreases in circulating levels in infected fish, while ornithine and isoleucine were the only amino acids that increased in concentration. We investigated transcriptional responses of the enzymes involved in arginine metabolism in liver and head kidney; transcripts for polyamine synthesis enzymes showed highly significant increases in both tissues across all diets following infection. The paralogous arginase-encoding genes, Arg1a, Arg1b, Arg2a and Arg2b, displayed complex responses across tissues and also due to diet and infection. Overall, these findings improve our understanding of amino acid metabolism following infection and suggests new potential amino acid targets for improving the immune response in salmonids.

Supplementation of arginine, ornithine and citrulline in rainbow trout (Oncorhynchus mykiss): Effects on growth, amino acid levels in plasma and gene expression responses in liver tissue.

Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, growth and development. Arginine is a FAA with crucial roles in protein deposition and the immune response. In mammals, supplementation of arginine's precursor amino acid, citrulline, is known to increase circulating arginine to levels beyond direct arginine supplementation, however, citrulline supplementation is poorly studied in fish. To address this knowledge gap, we supplemented the diet of rainbow trout with arginine and its precursor amino acids, ornithine and citrulline, at 3 levels (0.5%, 1% and 2% of the total diet) during a 14-week experiment. We sampled fish at 3 h and 24 h post-feeding to investigate immediate and steady-state effects, respectively. There were no differences in fish growth for any of the diets across a range of indicators. In blood plasma, out of 26 amino acids detected, 11 and 6 displayed significant changes 24 h and 3 h post-prandial, respectively. Arginine, ornithine and citrulline levels were all significantly increased by the citrulline supplemented diets. In muscle, 8 amino acids were significantly altered by supplemented diets, while there were no significant changes in liver. Arginine was increased by 2% citrulline supplementation in muscle tissue. We also investigated the transcriptional responses of urea cycle, nitric oxide cycle and rate-limiting polyamine synthesis enzymes, related to arginine's metabolism, in liver. At both time points, only 2 enzymes were significantly altered by the supplemented diets, however several significant changes were observed comparing 3 h and 24 h post-prandial expression levels. Of these, the paralogous polyamine synthesis enzyme encoding genes ODC1 and ODC2 displayed the largest increases in 3 h post-prandial fish. These findings demonstrate that endogenous synthesis of arginine is possible from a citrulline supplemented diet and improve our understanding of arginine metabolism in fish.

Rainbow trout (Oncorhynchus mykiss) urea cycle and polyamine synthesis gene families show dynamic expression responses to inflammation.

The urea cycle is an endogenous source of arginine that also supports removal of nitrogenous waste following protein metabolism. This cycle is considered inefficient in salmonids, where only 10-15% of nitrogenous waste is excreted as urea. In rainbow trout, arginine is an essential amino acid that has attracted attention due to its many functional roles. These roles include the regulation of protein deposition, immune responses and polyamine synthesis; the latter is directly linked to the urea cycle and involved in tissue repair. The key enzymes used in the urea cycle, namely arginase, ornithine transcarbamylase, argininosuccinate synthase and argininosuccinate lyase, in addition to two rate limiting enzymes required for polyamine synthesis (ornithine decarboxylase and s-adenosylmethionine decarboxylase) are poorly studied in fishes, and their responses to inflammation remain unknown. To address this knowledge gap, we characterised these gene families using phylogenetics and comparative genomics, investigated their mRNA distribution among a panel of tissues and established their transcriptional responses to an acute inflammatory response caused by bacterial infection in liver and muscle. Gene duplicates (paralogues) were identified for arginase (ARG1a, 1b, 2a and 2b), ornithine decarboxylase (ODC1 and 2) and s-adenosylmethionine decarboxylase (SAMdc1 and 2), including paralogues retained from an ancestral salmonid-specific whole genome duplication. ARG2a and 2b were highly upregulated following bacterial infection in liver, whereas ARG1b was downregulated, while both paralogues of SAMdc and ODC were upregulated in liver and unchanged in muscle. Overall, these findings improve our understanding of the molecules supporting the urea cycle and polyamine synthesis in fish, highlighting major changes in the regulation of these systems during inflammation.

Selective precipitation reaction: a novel diagnostic test for tissue pathology in Atlantic salmon, Salmo salar, infected with salmonid alphavirus (SAV3).

While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on-farm qualitative test and an in-laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (Δ340 ). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the Δ340 in infected fish rose from undetectable to a maximum at 6 weeks post-infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.

Serum enolase: a non-destructive biomarker of white skeletal myopathy during pancreas disease (PD) in Atlantic salmon Salmo salar L.

Diseases which cause skeletal muscle myopathy are some of the most economically damaging diseases in Atlantic salmon, Salmo salar L., aquaculture. Despite this, there are limited means of assessing fish health non-destructively. Previous investigation of the serum proteome of Atlantic salmon, Salmo salar L., during pancreas disease (PD) has identified proteins in serum that have potential as biomarkers of the disease. Amongst these proteins, the enzyme enolase was selected as the most viable for use as a biomarker of muscle myopathy associated with PD. Western blot and immunoassay (ELISA) validated enolase as a biomarker for PD, whilst immunohistochemistry identified white muscle as the source of enolase. Enolase was shown to be a specific marker for white muscle myopathy in salmon, rising in serum concentration significantly correlating with pathological damage to the tissue.

Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease usually resulting in death of patients by their early twenties. In contrast, mice lacking dystrophin (Dmd(mdx)), appear physically normal despite their underlying muscle pathology. Mice deficient for both dystrophin and the dystrophin-related protein, utrophin, (Dmd(mdx);Utrn-/- mice) die between 6 and 20 weeks of age suffering from severe muscle weakness with joint contractures, pronounced growth retardation and kyphosis, suggesting that dystrophin and utrophin play complementary roles. The exact cause of death in these mice was not determined. Here we show that expression of a truncated utrophin transgene solely within the skeletal muscle of these mutants prevents premature death and the development of any clinical phenotype. In the absence of full-length dystrophin and utrophin, the presence of truncated utrophin also decreases muscle fibre regeneration, relocalizes the dystrophin protein complex to the sarcolemma and re-establishes a normal expression pattern of developmental muscle proteins. These data suggest that Dmd(mdx);Utrn-/- mice succumb to a skeletal muscle defect and that their reduced lifespan is not due to cardiac or neurogenic components. The phenotypic rescue observed demonstrates that the Dmd(mdx);Utrn-/- mice are an ideal model for testing gene delivery protocols for the expression of utrophin or dystrophin in skeletal muscle. To determine the cause of death of the Dmd(mdx):Utrn-/- mice.

Subcellular components of probiotics Kocuria SM1 and Rhodococcus SM2 induce protective immunity in rainbow trout (Oncorhynchus mykiss, Walbaum) against Vibrio anguillarum.

The efficacy of cellular components of probiotics Kocuria SM1 and Rhodococcus SM2 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against vibriosis was assessed. Groups of fish (average weight = 10-15 g) were immunized intraperitoneally (i.p.) with 0.1 ml of subcellular materials, i.e., 0.2 ± 0.05 mg protein per fish, comprising extracellular proteins (ECPs), cell wall proteins (CWPs) and whole cell proteins (WCPs) of SM1 and SM2, respectively, or with 0.1 ml of phosphate-buffered saline (PBS) to serve as the control. Seven days after administration, fish from each group were challenged i.p. with 0.1 ml of a suspension in PBS of 3 × 10(5) cells ml(-1) per fish of Vibrio anguillarum. Use of CWPs and WCPs demonstrated significantly (P < 0.05) better protection against V. anguillarum insofar as mortalities were reduced to 11-17% [relative percent survival (RPS) = 80-87%], although ECPs fared less well (mortalities = 33-38%; RPS = 56-62%; P > 0.05), compared to 86% mortalities of the controls. The mode of action reflected activation of innate immune factors by CWPs and WCPs, demonstrating significantly (P < 0.05) increased expression of respiratory burst (optical density; OD(550 nm)) from 0.039 to 0.043-0.045, peroxidase (OD(550 nm)) from 0.26 to 0.37-0.55, and bacterial killing activities (i.e., percentage of surviving bacteria reduced from 79% to 56-57% for SM2). Moreover, an elevation of leucocyte number (from 1.93% to 1.98-2.93%; P > 0.05) and immunoglubolin level (from 27 mg ml(-1) to 28.5-33 mg ml(-1); P > 0.05) were observed with the experimental groups. These results indicate that cell components of the probiotics stimulate an immune response.

Expression of full-length utrophin prevents muscular dystrophy in mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal, progressive muscle wasting disease caused by a loss of sarcolemmal bound dystrophin, which results in the death of the muscle fiber leading to the gradual depletion of skeletal muscle. The molecular structure of dystrophin is very similar to that of the related protein utrophin. Utrophin is found in all tissues and is confined to the neuromuscular and myotendinous junctions in mature muscle. Sarcolemmal localization of a truncated utrophin transgene in the dystrophin-deficient mdx mouse significantly improves the dystrophic muscle phenotype. Therefore, up-regulation of utrophin by drug therapy is a plausible therapeutic approach in the treatment of DMD. Here we demonstrate that expression of full-length utrophin in mdx mice prevents the development of muscular dystrophy. We assessed muscle morphology, fiber regeneration and mechanical properties (force development and resistance to stretch) of mdx and transgenic mdx skeletal and diaphragm muscle. The utrophin levels required in muscle are significantly less than the normal endogenous utrophin levels seen in lung and kidney, and we provide evidence that the pathology depends on the amount of utrophin expression. These results also have important implications for DMD therapies in which utrophin replacement is achieved by delivery using exogenous vectors.

Expression of truncated utrophin leads to major functional improvements in dystrophin-deficient muscles of mice.

Dystrophin-deficient mice (mdx) expressing a truncated (trc) utrophin transgene show amelioration of the dystrophic phenotype. Here we report a multifunctional study demonstrating that trcutrophin expression leads to major improvements of the mechanical performance of muscle (that is, force development, mechanical resistance to forced lengthenings and maximal spontaneous activity) and of the maintenance of the intracellular calcium homeostasis. These are two essential functions of muscle fibers, known to be impaired in mdx mouse muscles and Duchenne muscular dystrophy (DMD) patients. Our results bring strong support to the hypothesis that muscle wasting in dystrophin-deficient DMD patients could be prevented by upregulation of utrophin.

Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum).

AIMS: The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. METHODS AND RESULTS: The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole-cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron-regulated outer membrane proteins (IROMP) of c.90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. CONCLUSIONS: The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. SIGNIFICANCE AND IMPACT OF THE STUDY: There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.

Novel non-motile phenotypes of Yersinia ruckeri suggest expansion of the current clonal complex theory.

The biochemical and cell surface characteristics of 63 non-motile isolates of Yersinia ruckeri from various sources were compared using the API 20E rapid identification system and conventional phenotypic methods. Eight individual phenotypic groups from a variety of fish species were observed from the data set. Non-motile isolates were not exclusively observed from serogroup O1; membership of biotype 2 was recorded for representatives from serogroups O2-O7. Variations in phenotypes highlights that new clonal groups are arising and that the current typing scheme requires expansion. Previously, it was hypothesized that disease was caused by a few virulent clones; data in this paper suggests that this assumption is not the case. The lipopolysaccharide (O antigen) type in the non-motile biotype was different from other isolates of Y. ruckeri.

The emerging family of dystrophin-related proteins.

Duchenne and Becker muscular dystrophies are caused by mutations in the gene encoding dystrophin, a component of the subsarcolemmal cytoskeleton. Dystrophin-related proteins are identical or homologous to the cysteine-rich and C-terminal domains of dystrophin. This part of dystrophin binds to a membrane-spanning glycoprotein complex in muscle. At least five dystrophin-related proteins are encoded by the Duchenne muscular dystrophy locus. These proteins are found in many non-muscle tissues where dystrophin is not expressed and they are thought to be membrane-associated. Two other dystrophin-related proteins--utrophin and an 87 kDa postsynaptic protein--are encoded by separate loci and, like dystrophin, they are components of the neuromuscular junction.

Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi.

Cytoplasmic dynein is a multisubunit, microtubule-dependent mechanochemical enzyme that has been proposed to function in a variety of intracellular movements, including minus-end-directed transport of organelles. Dynein-mediated vesicle transport is stimulated in vitro by addition of the Glued/dynactin complex raising the possibility that these two complexes interact in vivo. We report here that a class of phenotypically identical mutants of the filamentous fungus Neurospora crassa are defective in genes encoding subunits of either cytoplasmic dynein or the Glued/dynactin complex. These mutants, defined as ropy, have curled hyphae with abnormal nuclear distribution. ro-1 encodes the heavy chain of cytoplasmic dynein, while ro-4 encodes an actin-related protein that is a probable homologue of the actin-related protein Arpl (formerly referred to as actin-RPV or centractin), the major component of the glued/dynactin complex. The phenotypes of ro-1 and ro-4 mutants suggest that cytoplasmic dynein, as well as the Glued/dynactin complex, are required to maintain uniform nuclear distribution in fungal hyphae. We propose that cytoplasmic dynein maintains nuclear distribution through sliding of antiparallel microtubules emanating from neighboring spindle pole bodies.

Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice.

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.

Dystrophin and related proteins.

During the past year significant progress has been made in understanding how dystrophin deficiency leads to muscle cell necrosis in Duchenne muscular dystrophy and Becker muscular dystrophy. Dystrophin interacts with a glycoprotein complex spanning the muscle sarcolemma, effectively linking the actin cytoskeleton to the extracellular matrix. The carboxyl terminus of dystrophin is required for glycoprotein binding. Interestingly, at least three mRNAs transcribed from the distal end of the DMD gene in tissues other than muscle have been shown to encode this domain. Deficiency of a second component of the dystrophin-associated glycoprotein complex has been shown to occur in another muscle-wasting disorder, severe childhood autosomal recessive muscular dystrophy. Sequence analysis of the entire cDNA for the autosomal dystrophin-related protein utrophin has shown that dystrophin and utrophin are closely related. Furthermore, both of these proteins have been shown to bind to the same or a similar glycoprotein complex in muscle.

p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae.

Protein kinase C activation contributes to microvascular barrier dysfunction in the heart at early stages of diabetes.

The functional disturbance of microvasculature is recognized as an initiating mechanism that underlies the development of various diabetic complications. Although a causal relationship between microvascular leakage and tissue damage has been well documented in diabetic kidneys and eyes, there is a lack of information regarding the barrier function of coronary exchange vessels in the disease state. The aim of the present study was to evaluate the permeability property of coronary microvessels during the early development of experimental diabetes with a focus on the protein kinase C (PKC)-dependent signaling mechanism. The apparent permeability coefficient of albumin (Pa) was measured in isolated and perfused porcine coronary venules. The administration of high concentrations of D-glucose induced a dose-dependent increase in the Pa value, which was prevented by blockage of PKC with its selective inhibitors bisindolylmaleimide and Goe 6976. More importantly, an elevated basal permeability to albumin was observed in coronary venules at the early onset of streptozotocin-induced diabetes. The hyperpermeability was corrected with bisindolylmaleimide and the selective PKCbeta inhibitor hispidin. Concomitantly, protein kinase assay showed a high PKC activity in isolated diabetic venules. Immunoblot analysis of the diabetic heart revealed a significant subcellular translocation of PKCbetaII and PKCepsilon from the cytosol to the membrane, indicating that the specific activity of these isoforms was preferentially elevated. The results suggest that endothelial barrier dysfunction attributed to the activation of PKC occurs at the coronary exchange vessels in early diabetes.

Expression of the human interferon-beta gene cloned in phage M13 mp7.

The single-stranded DNA phage, M13 mp7 was used in the construction of an expression vector containing the coding sequence for mature interferon-beta (IFN-beta). Two clones expressed a fused polypeptide showing the biological and physicochemical properties of IFN-beta, despite the fact that the N-terminal amino acid sequence had been changed; 10(6) I.U./l of culture were produced with a molecular weight of 20 000.

Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.