Neutrophils Enable Local and Non-Invasive Liposome Delivery to Inflamed Skeletal Muscle and Ischemic Heart

Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease, and cancer. Improving localized delivery of immunosuppressive drugs to inflamed tissue in a non-invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Here, a neutrophil-mediated delivery system able to transport drug-loaded nanocarriers to inflamed tissue by exploiting the inherent ability of neutrophils to migrate to inflammatory tissue is reported. This hybrid system (neutrophils loaded with liposomes ex vivo) efficiently migrates in vitro following an inflammatory chemokine gradient. Furthermore, the triggered release of loaded liposomes and reuptake by target macrophages is studied. The migratory behavior of liposome-loaded neutrophils is confirmed in vivo by demonstrating the delivery of drug-loaded liposomes to an inflamed skeletal muscle in mice. A single low-dose injection of the hybrid system locally reduces inflammatory cytokine levels. Biodistribution of liposome-loaded neutrophils in a human-disease-relevant myocardial ischemia reperfusion injury mouse model after i.v. injection confirms the ability of injected neutrophils to carry loaded liposomes to inflammation sites. This strategy shows the potential of nanocarrier-loaded neutrophils as a universal platform to deliver anti-inflammatory drugs to promote tissue regeneration in inflammatory diseases.     

Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1beta and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1beta and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal-Wallis analysis of variance with Dunn's multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences ( P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1beta determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.     

Comparison of polarized and depolarized arrest in the isolated rat heart for long-term preservation

BACKGROUND: Hypothermic hyperkalemic cardioplegic solutions are currently used for donor heart preservation. Hyperkalemia-induced depolarization of the resting membrane potential (Em) may predispose the heart to Na+ and Ca2+ loading via voltage-dependent "window currents," thereby exacerbating injury and limiting the safe storage duration. Alternatively, maintaining the resting Em with a polarizing solution may reduce ionic movements and improve postischemic recovery; we investigated this concept with the reversible sodium channel blocker tetrodotoxin (TTX) to determine (1) whether polarized arrest was more efficacious than depolarized arrest during hypothermic long-term myocardial preservation and (2) whether TTX induces and maintains polarized arrest. METHODS AND RESULTS: The isolated crystalloid-perfused working rat heart preparation was used in this study. Preliminary studies determined an optimal TTX concentration of 22 micromol/L and an optimal storage temperature of 7.5 degrees C. To compare depolarized and polarized arrest, hearts were arrested with either Krebs-Henseleit (KH) buffer (control), KH buffer containing 16 mmol/L K+, or KH buffer containing 22 micromol/L TTX and then stored at 7.5 degrees C for 5 hours. Postischemic recovery of aortic flow was 13+/-4%, 38+/-2%, and 48+/-3%* (*P<.05 versus control and 16 mmol/L K+), respectively. When conventional 3 mol/L KCl-filled intracellular microelectrodes were used, Em gradually depolarized during control unprotected ischemia to approximately -55 mV before reperfusion, whereas arrest with 16 mmol/L K+ caused rapid depolarization to approximately -50 mV, where it remained throughout the 5-hour storage period. In contrast, in 22 micromol/L TTX-arrested hearts, Em remained more polarized, at approximately -70 mV, for the entire ischemic period. CONCLUSIONS: Blockade of cardiac sodium channels by TTX during ischemia maintained polarized arrest, which was more protective than depolarized arrest, possibly because of reduced ionic imbalance.     

Developmental differences in superoxide production in isolated guinea-pig hearts during reperfusion

The production of free radicals on reperfusion has been implicated as an important factor governing post-ischemic recovery of cardiac function. Although the response of the heart to ischemia and reperfusion is known to change during cardiac development, it is not known if different rates of free radical production play a role in these altered responses. The aim of this investigation was to determine if the production of the superoxide anion (O2-) on reperfusion differs in the immature and mature heart. Immature hearts, obtained from 3-day premature guinea pigs (delivered by cesarean section) were compared with those from adults (7 weeks old). Using the isolated Langendorff preparation. O2- production was measured during reperfusion following ischemic durations [0 (aerobic control), 15, 20, 30, and 60 min, n = 6/group] by the reduction of succinylated ferricytochrome c in the perfusate. Both immature and mature hearts exhibited bell-shaped relationship between ischemic duration and peak O2- production on reperfusion: (13.4 +/- 5.9; 22.2 +/- 5.4; 23.0 +/- 7.8; 59.3 +/- 16.2; 33.7 +/- 15.1; 32.6 +/- 8.5 nmol/min/g wet weight in the immature heart and 15.7 +/- 1.9; 55.0 +/- 30.2; 82.8 +/- 14.0; 78.8 +/- 33.8; 40.6 +/- 16.4; 45.4 +/- 13.1 nmol/min/g wet weight in the mature heart after 0; 15; 20; 30; 45 and 60 min of ischemia, respectively). A similar relationship was also demonstrated with O2- production over the 20-min reperfusion period: (134.0 +/- 57.1; 106.5 +/- 46.2; 199.3 +/- 50.6; 362.0 +/- 99.5; 375.0 +/- 60.9; 221.0 +/- 73.0 nmol/20 min/g wet weight in the immature heart and 97.8 +/- 54; 282.0 +/- 139.0; 933.3 +/- 210.3; 964.0 +/- 374.0; 443.0 +/- 106.0; 352.0 +/- 1551.0 nmol/20 min/g wet weight in the mature heart after 0, 15, 20, 30, 45 and 60 min of ischemia, respectively). Mature hearts consistently produced more O2- than immature hearts on reperfusion, while there was no significant difference in their capacity to produce O2- during aerobic perfusion. We conclude that the immature heart may be at less risk from the free radical component of reperfusion injury than the mature heart.     

Transmission of a homogenous hepatitis B virus population of A1896-containing strains leading to mild resolving acute hepatitis and seroconversion to hepatitis B e antigen antibodies in an adult

The pre-core variant, A1896, which switches off hepatitis B e antigen (HBeAg) production, is common in hepatitis B e antigen antibody (anti-HBe)-positive chronic hepatitis patients. It has been observed in occasional case reports of acute hepatitis. However, transmission in the absence of HBeAg-producing strains, leading to acute nonfulminant hepatitis and clearance in adults, has not been reported. Here, we show that this event can occur, further confirming that A1896 strains are "wild-type" and can lead to all the same outcomes as G1896 strains. This is in keeping with phylogenetic evidence that A1896 is transmitted independently on a large scale in the population and explains anti-HBe- positive persons who have not had an HBeAg-positive phase documented.     

UKPDS 25: autoantibodies to islet-cell cytoplasm and glutamic acid decarboxylase for prediction of insulin requirement in type 2 diabetes. UK Prospective Diabetes Study Group

BACKGROUND: Autoantibodies to islet-cell cytoplasm (ICA) and glutamic acid decarboxylase (GADA) can occur in apparently typical, non-insulin dependent diabetes mellitus (type 2). We investigated whether the presence of either or both antibodies characterises a subtype of diabetes and provides better prediction of requirement for insulin therapy by 6 years' follow-up than clinical variables. METHODS: We measured ICA and GADA at diagnosis of diabetes in a representative population of 3672 white patients with type 2 diabetes, aged between 25 and 65 years. The phenotype was assessed by age of onset, body-mass index, percentage haemoglobin A1c (HbA1c), and islet beta-cell function. We investigated the need for insulin therapy among 1538 patients not assigned insulin and followed up for 6 years from diagnosis. FINDINGS: The proportion of patients with ICA and GADA decreased with increasing age at diagnosis (from 33 [21%] of 157 patients aged 25-34 [corrected] to 66 [4%] of 1769 aged 55-65 for ICA; from 53 [34%] to 122 [7%] for GADA). Among patients younger than 35 at diagnosis, those with ICA or GADA had lower body-mass index than those without (mean 24.9 [SD 6.0] vs 31.7 [7.3] kg/m2; p < 0.0001 and had higher percentage of HbA1c (9.7 vs 8.7%, p < 0.05). 94% of patients with ICA and 84% of those with GADA required insulin therapy by 6 years, compared with 14% of those without the antibodies (p < 0.0001). Among patients older than 55 at diagnosis, the difference between those with and without antibodies in body-mass index was smaller (27.2 [5.4] vs 28.6 [4.8] kg/m2, p < 0.001); 44% of those with ICA, 34% of those with GADA, and 5% with neither antibody required insulin therapy by 6 years (p < 0.0001). Among patients older than 45 years, body-mass index and HbA1c provided little predictive information for insulin requirement, whereas the positive predictive values of GADA (> or = 60 U/L) alone, or both GADA (> or = 20 U/L) and ICA (> 5 U/L), for insulin therapy were 52% and 68%. INTERPRETATION: Among young adults with type 2 diabetes, the phenotype of those with ICA or GADA antibodies was similar to that of classic juvenile-onset insulin-dependent diabetes, and either phenotype or antibodies predicted insulin requirement. In older adults, the phenotype was closer to that of patients without antibodies and only the presence of antibodies predicted an increased likelihood of insulin requirement.     

A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.     

Capture immunoassay for the diagnosis of bovine mastitis using a monoclonal antibody to polymorphonuclear granulocytes.

A direct capture enzyme-linked immunosorbent assay (ELISA) was developed to measure elevated polymorphonuclear granulocyte (PMN) antigens using horseradish peroxidase (EC 1.11.1.7) conjugated rabbit polyclonal anti-PMN antisera and a monoclonal antibody specific for PMN cells. Optical densities obtained in the ELISA were used to predict the cell counts of milk samples. Predicted counts were not significantly different from actual somatic cell counts (SCC). In a total of 156 bovine milk samples the correlation coefficient between somatic cell counting, taking greater than 500,000 cells/ml as being indicative of mastitis, and the assay was 0.94, yielding an assay sensitivity of 95.2% and a specificity of 97.3%. In further trials the ELISA could detect elevated PMN antigens in milk with SCC as low as 100,000 cells/ml. The results indicate that the monoclonal antibody based direct ELISA has excellent potential in the detection and determination of bovine mastitis.