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Objectives: To contribute to the understanding of the biochemical changes associated with the RBC storage lesion., Aim: To investigate changes in O(2) equilibrium and on/off kinetic rates during routine cold storage., Background: As RBCs are stored between 1 and 6°C numerous biochemical changes occur within the RBCs, including changes in the properties of the haemoglobin itself. This study serially analysed for the first time the O(2) equilibrium and on/off kinetic rates across the RBC membrane during routine storage., Methods/materials: The oxygen binding (k(on) ) and offloading (k(off) ) constants were measured in fresh RBCs and then in AS-5-preserved RBCs at weekly intervals, along with oxygen equilibrium curves (OECs), 2,3-Diphosphoglycerate (2,3-DPG), p50 and the Hill number (n)., Results: The k(on) increased slightly as the 2,3-DPG and p50 decreased during storage, whereas the k(off) remained largely unchanged. The OECs demonstrated the expected increase in O(2) affinity, whereas the Hill number was unchanged during storage., Conclusion: In spite of the biochemical, structural and functional changes associated with the storage of RBCs, their in vitro interactions with oxygen were largely preserved through 42 days of storage., (Transfusion Medicine © 2010 British Blood Transfusion Society. No claim to original US government works.)

Background: Endothelial membrane microparticles (EMP) in plasma are elevated in several vascular diseases., Objectives: To test the hypothesis that EMP would be increased in patients with acute ischemic stroke and would correlate with stroke severity, brain lesion volume and outcome., Patients and Methods: Forty-one patients were studied and divided into two groups based on the National Institutes of Health Stroke Scale (NIHSS) score: 20 patients with mild stroke (NIHSS score < 5) and 21 patients with moderate-severe stroke (NIHSS score > or = 5). Lesion volume was measured using diffusion-weighted magnetic resonance imaging and discharge outcome was based on the discharge Barthel and Rankin scores. Twenty-three age-matched control subjects were also studied. Using flow cytometry, endoglin-positive EMP: CD105+ CD41a-CD45- (E(+)EMP), specific endothelial EMP expressing VE-cadherin and endoglin: CD105+CD144+ (C(+)EMP), EMP expressing phosphatidylserine: CD105+PS+ CD41a- (PS(+)EMP) and EMP expressing ICAM-1: CD105+CD54+ CD45- (I(+)EMP) were analyzed., Results: Significantly higher PS(+)EMP counts were observed in the group of acute ischemic stroke patients [median 59 (25th-75th percentile: 28-86) MP microL(-1)] relative to the controls [28 (14-36) MP microL(-1)] (P = 0.002). All four EMP phenotypes studied were elevated in the subgroup of moderate-severe stroke patients relative to the controls (all P < 0.05). In the patients with acute ischemic stroke three EMP phenotypes (E(+)EMP, PS(+)EMP and I(+)EMP) correlated significantly with brain lesion volume, with I(+)EMP (P = 0.002) showing the strongest correlation. Admission counts of C(+)EMP (P = 0.0003) and E(+)EMP (P = 0.003) correlated significantly with discharge clinical outcome., Conclusions: Certain circulating EMP phenotypes may be associated with severity, lesion volume and outcome of acute ischemic stroke. EMP analysis shows promising contribution to understanding stroke pathophysiology.

Lens-associated uveitis (LAU), a severe inflammatory eye disease, is thought to be mediated by autoimmunity against lens crystallins. Previously described animal models for this disease are antibody-mediated, since no cellular response to self crystallins could be induced in experimental animals. Here, we describe a new model for LAU, in which lymphocytes from knockout mice deficient in alphaB-crystallin are sensitized against the deleted protein and induce severe ocular inflammation when adoptively transferred into wild type recipients. Similar to LAU, the experimental disease developed only following rupture of the lens capsule, produced in this study by capsulotomy; no disease was detected in recipient eyes with no capsulotomy, or in those treated with cautery, or in eyes affected by systemic treatment with sodium iodate, lipopolysaccharide or X-irradiation. The ocular changes in affected eyes included heavy cellular infiltration and proteinaceous exudate in both the anterior and posterior segments of the eye, that reached their peak on day 4 following cell transfer and subsided quite rapidly thereafter.

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzyme deficiency leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal alpha-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human alpha-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, alpha-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20-35% of that of the normal mice. The transduced animals continued to show higher alpha-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or alpha-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Mammalian interphotoreceptor retinoid-binding proteins (IRBPs) are highly uveitogenic in Lewis rats. Xenopus laevis IRBP resembles mammalian IRBP in its four-fold structure, and has approximately 70% amino acid sequence identity with the bovine protein. This study investigated the uveitogenicity of recombinant Xenopus IRBP and two of its derived peptides in Lewis rats. Rats immunized with Xenopus IRBP developed uveoretinitis as well as pineal inflammation. The Xenopus molecule was, however, less immunopathogenic than the bovine IRBP. Of the two Xenopus IRBP peptides tested, 1180-1191 was remarkably uveitogenic, whereas sequence 521-540 exhibited low activity. It is assumed, therefore, that as with bovine IRBP, peptide 1180-1191 is the major uveitogenic sequence in Xenopus IRBP. The role individual residues of these peptides play in the immunopathogenic process is discussed. Our data thus demonstrate that despite its being phylogenetically remote, Xenopus IRBP is uveitogenic in Lewis rats, (Copyright 2000 Academic Press.)

Linomide is a potent immunomodulator that either enhances or suppresses certain immunological processes. Of particular interest is this compound's capacity to inhibit a variety of organ-specific autoimmune diseases. Here, we report on the effects of linomide on several immunological reactions elicited by endotoxin (LPS), both in vivo and in vitro. In rats and mice linomide inhibited the elicitation of endotoxin-induced uveitis (EIU), an acute inflammatory eye disease that develops within 24 h following footpad injection of LPS. Linomide also inhibited the production of TNF-alpha and IL-6 by LPS-stimulated rat and mouse macrophage monolayers. On the other hand, treatment with linomide significantly increased the levels of IL-1beta (mice and less in rats), IL-6 (rats), and TNF-alpha (mice) in serum samples collected 2 h following injection with LPS. The increased production of proinflammatory cytokines in linomide-treated mice was also indicated by the enhanced lethal effect of LPS in these mice. The finding of elevated levels of these cytokines in animals with suppressed EIU is also in line with previous observations of an inverse relationship between EIU severity and levels of TNF-alpha. Data recorded here underscore the unique capacity of linomide to both enhance and suppress the immune system., (Copyright 1999 Academic Press.)

Rheumatoid arthritis is a systemic disease of unknown etiology. The purpose of this study was to elucidate an unrecognized interaction between neutrophilic myeloperoxidase (MPO) and macrophages (Mphi) which could perpetuate the inflammatory response associated with arthritis. A monoarticular arthritis was induced by intra-articular injection of group A streptococcus cell wall fragments (PG-APS) into the ankle joint of female Lewis rats. After swelling/erythema subsided, joints were reinjected with either recombinant MPO or enzymatically inactive MPO (iMPO). Joint measurements were made daily and arthritis was confirmed by histology. Neither iMPO nor MPO could initiate "clinical" arthritis; however, either form of the enzyme injected after PG-APS induced a dose-dependent increase in erythema and swelling. Mannans, which block the binding of MPO to Mo, ablated clinical symptoms. Also, the presence of tumor necrosis factor alpha was observed only in diseased joints using immunocytochemistry., (Copyright 1999 Academic Press.)

Macrophages (Mphi) are involved in host defenses against opportunistic pathogens. Previous studies by the present investigators indicate that Mphi exposed to enzymatically active myeloperoxidase (MPO), exhibited both increased phagocytosis and killing of Candida albicans. The purpose of this study was to determine if enzymatically inactive Mphi-mannose receptor (MMR) ligands could function similarly. Resident murine peritoneal Mphi were exposed to the MMR ligands, mannosylated bovine serum albumin (mBSA), and enzymatically inactive myeloperoxidase (iMPO), followed by exposure to opsonized C. albicans. Both mBSA and iMPO induced a slight increase in the number of phagocytizing cells; however, candidacidal activity was significantly higher in treated cultures compared to controls (P < or = 0.001). The production of reactive oxygen intermediates (ROI) was detected using chemiluminescence. After employment of ROI scavengers, a decrease in candidacidal activity was observed. The data suggest that MMR-ligand interaction alone is sufficient to significantly enhance the candidacidal activity of Mphi via ROI, and that iMPO which is released at a site of inflammation induces Mphi-mediated killing of microorganisms. These findings indicate a previously unrecognized role of iMPO.

Rheumatoid arthritis (RA) is characterized by an abnormal cellular and cytokine infiltration of inflamed joints. This study addresses a previously unrecognized interaction between neutrophilic-myeloperoxidase (MPO) and macrophages (Mphi) which could explain the perpetuation of inflammation associated with RA. A monoarticular arthritis was induced in female Lewis rats by injection of streptococcal cell wall extracts (PG-APS). After swelling and erythema subsided, joints were re-injected with one of the following: porcine MPO or partially inactivated MPO (iMPO). Injection with either MPO or iMPO induced a 'flare' of experimental RA. Blocking the Mphi-mannose receptor by mannans, ablated exacerbation of disease. These results indicate that MPO or iMPO can play a pivotal role in the perpetuation but not initiation of this RA model.

Previous studies by these investigators have shown that mannosylated bovine serum albumin (m-BSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans by resident murine peritoneal macrophages (MO). Upregulation of the above MO functions was associated with binding of m-BSA to the MO-mannose receptor. The present study was done to determine if the immunostimulant, acemannan prepared from aloe vera, could stimulate MO in a similar manner. Resident peritoneal MO collected from C57BL/6 mice were exposed to acemannan for 10 min. The RB was measured using chemiluminescence and demonstrated approximately a two-fold increase above the media controls. In studies involving phagocytosis, MO were exposed to acemannan, washed and exposed to Candida at a ratio of 1:5. The percent phagocytosis and Candida killing were determined using fluorescence microscopy. There was a marked increase in phagocytosis in the treated cultures (45%) compared to controls (25%). Macrophages exposed to acemannan for 10 min resulted in ca 38% killing of Candida albicans compared with 0-5% killing in controls. If MO were incubated with acemannan for 60 min, 98% of the yeast were killed compared to 0-5% in the controls. The results of the present study indicate that short term exposure of MO to acemannan upregulates the RB, phagocytosis and candidicidal activity. Further studies are needed to clarify the potential use of this immunostimulant as an anti-fungal agent.

Candida albicans is an opportunistic pathogen whose resurgence coincides with the rising number of AIDS patients. Neutrophils are known to be involved in the clearance of Candida infections; however, the role of macrophages in host defenses against this organism is not well understood. The present study was undertaken to examine an unrecognized interaction between neutrophils and macrophages resulting in enhanced killing of candidae in vitro. Murine peritoneal macrophages exposed to recombinant myeloperoxidase exhibited enhancement of the respiratory burst, increased phagocytosis, and a dose-dependent increase in intracellular killing of Candida species. Radical scavengers reduced the killing, indicating a role of reactive oxygen intermediates in the candidacidal activity observed. These data suggest that at the site of infection, myeloperoxidase released from neutrophils activates macrophages and induces microbicidal activity.