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.

Directional gamma sensing from covariance processing of inter-detector Compton crosstalk energy asymmetries.

Energy asymmetry of inter-detector crosstalk from Compton scattering can be exploited to infer the direction to a gamma source. A covariance approach extracts the correlated crosstalk from data streams to estimate matched signals from Compton gammas split over two detectors. On a covariance map the signal appears as an asymmetric cross diagonal band with axes intercepts at the full photo-peak energy of the original gamma. The asymmetry of the crosstalk band can be processed to determine the direction to the radiation source. The technique does not require detector shadowing, masking, or coded apertures, thus sensitivity is not sacrificed to obtain the directional information. An angular precision of better than 1° of arc is possible, and processing of data streams can be done in real time with very modest computing hardware.

The serum proteome of Atlantic salmon, Salmo salar, during pancreas disease (PD) following infection with salmonid alphavirus subtype 3 (SAV3).

Salmonid alphavirus is the aetological agent of pancreas disease (PD) in marine Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, with most outbreaks in Norway caused by SAV subtype 3 (SAV3). This atypical alphavirus is transmitted horizontally causing a significant economic impact on the aquaculture industry. This histopathological and proteomic study, using an established cohabitational experimental model, investigated the correlation between tissue damage during PD and a number of serum proteins associated with these pathologies in Atlantic salmon. The proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting. A number of humoral components of immunity which may act as biomarkers of the disease were also identified. For example, creatine kinase, enolase and malate dehydrogenase serum concentrations were shown to correlate with pathology during PD. In contrast, hemopexin, transferrin, and apolipoprotein, amongst others, altered during later stages of the disease and did not correlate with tissue pathologies. This approach has given new insight into not only PD but also fish disease as a whole, by characterisation of the protein response to infection, through pathological processes to tissue recovery. BIOLOGICAL SIGNIFICANCE: Salmonid alphavirus causes pancreas disease (PD) in Atlantic salmon, Salmo salar, and has a major economic impact on the aquaculture industry. A proteomic investigation of the change to the serum proteome during PD has been made with an established experimental model of the disease. Serum proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting with 72 protein spots being shown to alter significantly over the 12week period of the infection. The concentrations of certain proteins in serum such as creatine kinase, enolase and malate dehydrogenase were shown to correlate with tissue pathology while other proteins such as hemopexin, transferrin, and apolipoprotein, altered in concentration during later stages of the disease and did not correlate with tissue pathologies. The protein response to infection may be used to monitor disease progression and enhance understanding of the pathology of PD.

Covariance analysis of gamma ray spectra.

The covariance method exploits fluctuations in signals to recover information encoded in correlations which are usually lost when signal averaging occurs. In nuclear spectroscopy it can be regarded as a generalization of the coincidence technique. The method can be used to extract signal from uncorrelated noise, to separate overlapping spectral peaks, to identify escape peaks, to reconstruct spectra from Compton continua, and to generate secondary spectral fingerprints. We discuss a few statistical considerations of the covariance method and present experimental examples of its use in gamma spectroscopy.

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.

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.

Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system.

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.

Non-toxic ubiquitous over-expression of utrophin in the mdx mouse.

Duchenne muscular dystrophy (DMD) is an inherited, severe muscle wasting disease caused by the loss of the cytoskeletal protein, dystrophin. Patients usually die in their late teens or early twenties of cardiac or respiratory failure. We have previously demonstrated that the dystrophin related protein, utrophin is able to compensate for the loss of dystrophin in the mdx mouse, the mouse model of the disease. Expression of a utrophin transgene under the control of an HSA promoter results in localization of utrophin to the sarcolemma and prevents the muscle pathology. Here we show that the over-expression of full-length utrophin in a broad range of tissues is not detrimental in the mdx mouse. These findings have important implications for the feasibility of the up-regulation of utrophin in therapy for DMD since they suggest that tissue specific up-regulation may not be necessary.

Protein transfection of intact microvessels specifically modulates vasoreactivity and permeability.

Precise regulation of microvascular tone and barrier function is essential for proper coronary perfusion and performance. Agonist-induced alterations in either or both of these functions ultimately lead to microcirculatory dysfunction and cardiac insufficiency. Two important pathways involved in regulating vasomotor response and barrier function are the activation of nitric oxide synthase (NOS) and upregulation of protein kinase C (PKC). To date, studies of these two signaling proteins have relied mainly on pharmacological approaches. Unfortunately, the specificity of various inhibitors can be cause for concern. In order to address this problem, a protein transfection technique we developed for cultured endothelial cells has been modified and applied to isolated, intact coronary microvessels. Our results from green fluorescent protein transfection in arterioles and venules showed that this procedure could be used to introduce proteins into the microvascular wall. By transfecting inhibitor peptides against NOS and PKC into coronary arterioles and venules, we have been able to determine the specific roles of these two enzymes in vasodilation and hyperpermeability responses.

iNOS expression in dystrophinopathies can be reduced by somatic gene transfer of dystrophin or utrophin.

BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy.

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization.

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.

Myosin light chain kinase transference induces myosin light chain activation and endothelial hyperpermeability.

The actomyosin complex is the major cytoskeletal component that controls cell contraction. In this study, we investigated the effects of actomyosin interaction on endothelial barrier function and gap formation. Activated myosin light chain kinase (MLCK) protein was transferred into coronary venular endothelial cell (CVEC) monolayers. Uptake of the activated protein resulted in a significant shift in myosin light chain (MLC) from an unphosphorylated to a diphosphorylated form. In addition, MLCK induced a hyperpermeability response of the monolayer as measured by albumin transendothelial flux. Microscopic examination of MLCK-treated CVECs revealed widespread gap formation in the monolayer, loss of peripheral beta-catenin, and increases in actin stress fibers. Inhibition of all of the above responses by a specific MLCK inhibitor suggests they are the direct result of exogenously added MLCK. These data suggest that activation of MLCK in CVECs causes phosphorylation of MLC and contraction of CVECs, resulting in gap formation and concomitant increases in permeability. This study uses a novel technique to measure the effects of an activated kinase on both its substrate and cellular morphology and function through direct transference into endothelial cells.

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.