Absolute quantification (ml blood/sec ∗ mm2 tissue) of normal vs. diabetic foot skin microvascular blood perfusion: Feasibility of FM-PPG measurements under clinical conditions.

Fluorescence-mediated photoplethysmography (FM-PPG) is the first routine clinical methodology by which to quantifiably measure tissue blood perfusion in absolute terms (mL blood/sec ∗ mm2 tissue). The FM-PPG methodology has been described in detail previously in this journal (MVR 114, 2017, 92-100), along with initial proof-of-concept measurements of blood perfusion in both ocular and forearm skin tissues. The motivation for the current study was to investigate whether FM-PPG can be used readily and routinely under realistic clinical conditions. The vehicle for doing this was to measure medial foot capillary blood flow, i.e., tissue perfusion, in 7 normal subjects, mean = 6.76 ±â€¯2.29 E-005 mL/(sec ∙ mm2), and lesion-free areas of 8 type-2 diabetic patients with skin ulceration, mean = 4.67 + 3.15 E-005 mL/(sec ∙ mm2). Thus, perfusion in the diabetics was found to be moderately lower than that in the normal control subjects. Earlier skin perfusion measurements in medial forearms of 4 normal subjects, mean = 2.64 + 0.22 E-005 mL/(sec ∙ mm2), were lower than both the normal and diabetic foot perfusion measurements. Variability in the heartbeat-to-heartbeat blood perfusion pulses in the skin capillaries, defined as the ratio of the standard deviation among beat-to-beat pulses divided by the mean perfusion of those pulses, was determined for each subject. Average variability in foot skin was 21% in the diabetic population, versus 16% for normal subjects; and it was 18% in forearm skin. We conclude that absolute quantitative FM-PPG measurement of skin blood perfusion at the level of nutritive capillaries is feasible routinely under clinical conditions, allowing for quantitative measurement of skin tissue blood perfusion in absolute terms.

Emerging infectious disease outbreaks: estimating disease risk in Australian blood donors travelling overseas.

BACKGROUND AND OBJECTIVES: International travel assists spread of infectious pathogens. Australians regularly travel to South-eastern Asia and the isles of the South Pacific, where they may become infected with infectious agents, such as dengue (DENV), chikungunya (CHIKV) and Zika (ZIKV) viruses that pose a potential risk to transfusion safety. In Australia, donors are temporarily restricted from donating for fresh component manufacture following travel to many countries, including those in this study. We aimed to estimate the unmitigated transfusion-transmission (TT) risk from donors travelling internationally to areas affected by emerging infectious diseases. MATERIALS AND METHODS: We used the European Up-Front Risk Assessment Tool, with travel and notification data, to estimate the TT risk from donors travelling to areas affected by disease outbreaks: Fiji (DENV), Bali (DENV), Phuket (DENV), Indonesia (CHIKV) and French Polynesia (ZIKV). RESULTS: We predict minimal risk from travel, with the annual unmitigated risk of an infected component being released varying from 1 in 1·43 million to <1 in one billion and the risk of severe consequences ranging from 1 in 130 million to <1 in one billion. CONCLUSION: The predicted unmitigated likelihood of infection in blood components manufactured from donors travelling to the above-mentioned areas was very low, with the possibility of severe consequences in a transfusion recipient even smaller. Given the increasing demand for plasma products in Australia, the current strategy of restricting donors returning from select infectious disease outbreak areas to source plasma collection provides a simple and effective risk management approach.

Seroprevalence of antibodies to primate erythroparvovirus 1 (B19V) in Australia.

BACKGROUD: Primate erythroparvovirus 1 (B19V) is a globally ubiquitous DNA virus. Infection results in a variety of clinical presentations including erythema infectiosum in children and arthralgia in adults. There is limited understanding of the seroprevalence of B19V antibodies in the Australian population and therefore of population-wide immunity. This study aimed to investigate the seroprevalence of B19V antibodies in an Australian blood donor cohort, along with a cohort from a paediatric population. METHODS: Age/sex/geographical location stratified plasma samples (n = 2221) were collected from Australian blood donors. Samples were also sourced from paediatric patients (n = 223) in Queensland. All samples were screened for B19V IgG using an indirect- enzyme-linked immunosorbent assay. RESULTS: Overall, 57.90% (95% CI: 55.94%-59.85%) of samples tested positive for B19V IgG, with the national age-standardized seroprevalence of B19V exposure in Australians aged 0 to 79 years estimated to be 54.41%. Increasing age (p < 0.001) and state of residence (p < 0.001) were independently associated with B19V exposure in blood donors, with the highest rates in donors from Tasmania (71.88%, 95% CI: 66.95%-76.80%) and donors aged 65-80 years (78.41%, 95% CI: 74.11%-82.71%). A seroprevalence of 52.04% (95% CI: 47.92%-56.15%) was reported in women of child-bearing age (16 to 44 years). Sex was not associated with exposure in blood donors (p = 0.547) or in children (p = 0.261) screened in this study. CONCLUSIONS: This study highlights a clear association between B19V exposure and increasing age, with over half of the Australian population likely to be immune to this virus. Differences in seroprevalence were also observed in donors residing in different states, with a higher prevalence reported in those from the southern states. The finding is consistent with previous studies, with higher rates observed in countries with a higher latitude. This study provides much needed insight into the prevalence of B19V exposure in the Australian population, which has implications for public health as well as transfusion and transplantation safety in Australia.

Hepatitis E virus RNA in Australian blood donors: prevalence and risk assessment.

BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is a known transfusion-transmissible agent. HEV infection has increased in prevalence in many developed nations with RNA detection in donors as high as 1 in 600. A high proportion of HEV infections are asymptomatic and therefore not interdicted by donor exclusion criteria. To manage the HEV transfusion-transmission (TT) risk some developed nations have implemented HEV RNA screening. In Australia, HEV is rarely notified; although locally acquired infections have been reported, and the burden of disease is unknown. The purpose of this study was to determine the frequency of HEV infection in Australian donors and associated TT risk. MATERIALS AND METHODS: Plasma samples (n = 74 131) were collected from whole blood donors during 2016 and screened for HEV RNA by transcription-mediated amplification (TMA) in pools of six. Individual TMA reactive samples were confirmed by RT-PCR and, if positive, viral load determined. Prevalence data from the study were used to model the HEV-TT risk. RESULTS: One sample in 74 131 (95% CI: 1 in 1 481 781 to 1 in 15 031) was confirmed positive for HEV RNA, with an estimated viral load of 180 IU/ml, which is below that typically associated with TT. Using a transmission-risk model, we estimated the risk of an adverse outcome associated with TT-HEV of approximately 1 in 3·5 million components transfused. CONCLUSION: Hepatitis E virus viremia is rare in Australia and lower than the published RNA prevalence estimates of other developed countries. The risk of TT-HEV adverse outcomes is negligible, and HEV RNA donor screening is not currently indicated.

Diverse and novel RHD variants in Australian blood donors with a weak D phenotype: implication for transfusion management.

BACKGROUND AND OBJECTIVES: Variant RHD genes associated with the weak D phenotype can result in complete or partial D-epitope expression on the red cell. This study examines the genetic classification in Australian blood donors with a weak D phenotype and correlates RHD variants associated with the weak D phenotype against D-epitope profile. MATERIALS AND METHODS: Following automated and manual serology, blood samples from donors reported as 'weak D' (n = 100) were RHD genotyped by a commercial SNP-typing platform and Sanger sequencing. Two commercial anti-D antibody kits were used for extended serological testing for D-epitope profiles. RESULTS: Three samples had wild-type RHD exonic sequences, and 97 samples had RHD variants. RHD*weak D type 1, RHD*weak D type 2 or RHD*weak D type 3 was detected in 75 donors. The remaining 22 samples exhibited 17 different RHD variants. One donor exhibited a novel RHD*c.939+3A>C lacking one D-epitope. Weak D types 1·1, 5, 15, 17 and 90 showed a partial D-epitope profile. CONCLUSION: The array of RHD variants detected in this study indicated diversity in the Australian donor population that needs to be accommodated for in future genotyping strategies.

The distribution of MNS hybrid glycophorins with Mur antigen expression in Chinese donors including identification of a novel GYP.Bun allele.

BACKGROUND AND OBJECTIVES: MNS hybrid glycophorins are identified by characteristic antigen profiles. One of these is the Mur antigen, which is expressed on red cell hybrid glycophorins of several phenotypes of the 'Miltenberger' series found predominantly in East Asian population. The aim of this study was to investigate the distribution of Mur-positive hybrid glycophorins and clarify the genetic basis in the donors from southern China. MATERIALS AND METHODS: Blood samples from 528 donors were collected for Mur antigen serological typing. Sequencing of GYPB pseudoexon 3 and MNS phenotyping were conducted in Mur-positive samples. The multiplex ligation-dependent probe amplification (MLPA) was used to confirm the zygosity of the GYP.Mur allele and determine the MNSs genotype. The expression of Mur antigen was evaluated by flow cytometry. RESULTS: Fifty-one Mur-positive samples were identified by serological testing. Sequencing analysis showed 50 donors (50/528, 9.5%) with the GYP.Mur allele (48 heterozygotes and two homozygotes), which were confirmed by the MLPA genotyping analysis, and one donor (1/528, 0.19%) with a novel GYP.Bun allele. Flow cytometry analysis revealed higher Mur antigen expression on GP.Mur (Mi.III) homozygotes than heterozygotes. For the GYP.Mur homozygotes, an incorrect 'N' positive typing with anti-N lectin was obtained. CONCLUSION: GP.Mur (Mi.III) is the main Mur-positive hybrid glycophorin in Guangzhou donors. The dosage effect of Mur antigen observed provides a basis for selecting the homozygous GP.Mur RBCs as the reagent cells to avoid neglecting weak antibodies. A separate GYP.Bun lineage found in the southern China provides evidence for further complexity in the MNS system.

A novel FY*A allele with the 265T and 298A SNPs formerly associated exclusively with the FY*B allele and weak Fy(b) antigen expression: implication for genotyping interpretative algorithms.

BACKGROUND AND OBJECTIVES: An Australian Caucasian blood donor consistently presented a serology profile for the Duffy blood group as Fy(a+b+) with Fy(a) antigen expression weaker than other examples of Fy(a+b+) red cells. Molecular typing studies were performed to investigate the reason for the observed serology profile. MATERIAL AND METHODS: Blood group genotyping was performed using a commercial SNP microarray platform. Sanger sequencing was performed using primer sets to amplify across exons 1 and 2 of the FY gene and using allele-specific primers. RESULTS: The propositus was genotyped as FY*A/B, FY*X heterozygote that predicted the Fy(a+b+(w) ) phenotype. Sequencing identified the 265T and 298A variants on the FY*A allele. This link between FY*A allele and 265T was confirmed by allele-specific PCR. CONCLUSION: The reduced Fy(a) antigen reactivity is attributed to a FY*A allele-carrying 265T and 298A variants previously defined in combination only with the FY*B allele and associated with weak Fy(b) antigen expression. This novel allele should be considered in genotyping interpretative algorithms for generating a predicted phenotype.

Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms.

BACKGROUND AND OBJECTIVES: Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fy(a) /Fy(b) polymorphism, for weak Fy(b) antigen, and for the red cell null Fy(a-b-) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. MATERIALS AND METHODS: Samples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a-b-) n = 47 (one with anti-Fy3 antibody). RESULTS: Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fy(b) expression discrepant with Fy(b-) (Group 1 and 3). For three, SNP genotyping predicted Fy(a) , discrepant with Fy(a-b-) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null. CONCLUSION: Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status.

On the Eicosanoid Trail with John Vane and Jack McGiff: 1974-1976. A personal reminiscence.

In this brief personal reminiscence I comment upon the friendship and mutual understanding that arose between two great scientists and co-travellers, John Vane and Jack McGiff. I relate the events that led up to their meeting and focus on the brief period of time when they worked together on eicosanoid pharmacology in the UK.

Malaria antibody persistence correlates with duration of exposure.

BACKGROUND AND OBJECTIVES: In Australia, the risk of transfusion-transmitted malaria is managed through the identification of 'at-risk' donors, antibody screening enzyme-linked immunoassay (EIA) and, if reactive, exclusion from fresh blood component manufacture. Donor management depends on the duration of exposure in malarious regions (>6 months: 'Resident', <6 months: 'Visitor') or a history of malaria diagnosis. We analysed antibody testing and demographic data to investigate antibody persistence dynamics. To assess the yield from retesting 3 years after an initial EIA reactive result, we estimated the proportion of donors who would become non-reactive over this period. MATERIALS AND METHODS: Test results and demographic data from donors who were malaria EIA reactive were analysed. Time since possible exposure was estimated and antibody survival modelled. RESULTS: Among seroreverters, the time since last possible exposure was significantly shorter in 'Visitors' than in 'Residents'. The antibody survival modelling predicted 20% of previously EIA reactive 'Visitors', but only 2% of 'Residents' would become non-reactive within 3 years of their first reactive EIA. CONCLUSION: Antibody persistence in donors correlates with exposure category, with semi-immune 'Residents' maintaining detectable antibodies significantly longer than non-immune 'Visitors'.

Annexin A1 modulates macula densa function by inhibiting cyclooxygenase 2.

Annexin A1 (ANXA1) exerts anti-inflammatory effects through multiple mechanisms including inhibition of prostaglandin synthesis. Once secreted, ANXA1 can bind to G protein-coupled formyl peptide receptors (Fpr) and activate diverse cellular signaling pathways. ANXA1 is known to be expressed in cells of the juxtaglomerular apparatus, but its relation to the expression of cyclooxygenase 2 (COX-2) in thick ascending limb and macula densa cells has not been elucidated. We hypothesized that ANXA1 regulates the biosynthesis of COX-2. ANXA1 abundance in rat kidney macula densa was extensively colocalized with COX-2 (95%). Furosemide, an established stimulus for COX-2 induction, caused enhanced expression of both ANXA1 and COX-2 with maintained colocalization (99%). In ANXA1-deficient mice, COX-2-positive cells were more numerous than in control mice (+107%; normalized to glomerular number; P < 0.05) and renin expression was increased (+566%; normalized to glomerular number; P < 0.05). Cultured macula densa cells transfected with full-length rat ANXA1 revealed downregulation of COX-2 mRNA (-59%; P < 0.05). Similarly, treatment with dexamethasone suppressed COX-2 mRNA in the cells (-49%; P < 0.05), while inducing ANXA1 mRNA (+56%; P < 0.05) and ANXA1 protein secretion. Inhibition of the ANXA-1 receptor Fpr1 with cyclosporin H blunted the effect of dexamethasone on COX-2 expression. These data show that ANXA1 exerts an inhibitory effect on COX-2 expression in the macula densa. ANXA1 may be a novel intrinsic modulator of renal juxtaglomerular regulation by inhibition of PGE(2) synthesis.

Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration.

Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.

Inhibition of poly (ADP-ribose) synthetase attenuates neutrophil recruitment and exerts antiinflammatory effects.

A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10-20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS-/- mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity.

Deformation behaviour of stomatocyte, discocyte and echinocyte red blood cell morphologies during optical tweezers stretching.

The red blood cell (RBC) deformability is a critical aspect, and assessing the cell deformation characteristics is essential for better diagnostics of healthy and deteriorating RBCs. There is a need to explore the connection between the cell deformation characteristics, cell morphology, disease states, storage lesion and cell shape-transformation conditions for better diagnostics and treatments. A numerical approach inspired from the previous research for RBC morphology predictions and for analysis of RBC deformations is proposed for the first time, to investigate the deformation characteristics of different RBC morphologies. The present study investigates the deformability characteristics of stomatocyte, discocyte and echinocyte morphologies during optical tweezers stretching and provides the opportunity to study the combined contribution of cytoskeletal spectrin network and the lipid-bilayer during RBC deformation. The proposed numerical approach predicts agreeable deformation characteristics of the healthy discocyte with the analogous experimental observations and is extended to further investigate the deformation characteristics of stomatocyte and echinocyte morphologies. In particular, the computer simulations are performed to investigate the influence of direct stretching forces on different equilibrium cell morphologies on cell spectrin link extensions and cell elongation index, along with a parametric analysis on membrane shear modulus, spectrin link extensibility, bending modulus and RBC membrane-bead contact diameter. The results agree with the experimentally observed stiffer nature of stomatocyte and echinocyte with respect to a healthy discocyte at experimentally determined membrane characteristics and suggest the preservation of relevant morphological characteristics, changes in spectrin link densities and the primary contribution of cytoskeletal spectrin network on deformation behaviour of stomatocyte, discocyte and echinocyte morphologies during optical tweezers stretching deformation. The numerical approach presented here forms the foundation for investigations into deformation characteristics and recoverability of RBCs undergoing storage lesion.

Increased susceptibility of annexin-A1 null mice to nociceptive pain is indicative of a spinal antinociceptive action of annexin-A1.

BACKGROUND AND PURPOSE: Annexin-A1 (ANXA1), a glucocorticoid-regulated protein, mediates several of the anti-inflammatory actions of the glucocorticoids. Previous studies demonstrated that ANXA1 is involved in pain modulation. The current study, using ANXA1 knockout mice (ANXA1-/-), is aimed at addressing the site and mechanism of the modulatory action of ANXA1 as well as possible involvement of ANXA1 in mediating the analgesic action of glucocorticoids. EXPERIMENTAL APPROACH: The acetic acid-induced writhing response was performed in ANXA1-/- and wild-type (ANXA1+/+) mice with spinal and brain levels of prostaglandin E2 (PGE2) examined in both genotypes. The effect of the ANXA1 peptomimetic Ac2-26 as well as methylprednisolone on the writhing response and on spinal cord PGE2 of ANXA1+/+ and ANXA1-/- was compared. The expression of proteins involved in PGE2 synthesis, cytosolic phospholipase A2 (cPLA2) and cyclooxygenases (COXs), in the spinal cord of ANXA1+/+ and ANXA1-/- was also compared. KEY RESULTS: ANXA1-/- mice exhibited a significantly greater writhing response and increased spinal cord levels of PGE2 compared with ANXA1+/+ mice. Ac2-26 produced analgesia and reduced spinal PGE2 levels in ANXA1+/+ and ANXA1-/- mice, whereas methylprednisolone reduced the writhing response and spinal PGE2 levels in ANXA1+/+, but not in ANXA1-/- mice. The expression of cPLA2, COX-1, COX-2 and COX-3 in spinal cord tissues was upregulated in ANXA1-/-compared with ANXA1+/+. CONCLUSIONS AND IMPLICATIONS: We conclude that ANXA1 protein modulates nociceptive processing at the spinal level, by reducing synthesis of PGE2 by modulating cPLA2 and/or COX activity. The analgesic activity of methylprednisolone is mediated by spinal ANXA1.

Annexin-A1: a pivotal regulator of the innate and adaptive immune systems.

The glucocorticoids are the most potent anti-inflammatory drugs that we possess and are effective in a wide variety of diseases. Although their action is known to involve receptor mediated changes in gene transcription, the exact mechanisms whereby these bring about their pleiotropic action in inflammation are yet to be totally understood. Whilst many different genes are regulated by the glucocorticoids, we have identified one particular protein-annexin A1 (Anx-A1)-whose synthesis and release is strongly regulated by the glucocorticoids in many cell types. The biology of this protein, as revealed by studies using transgenic animals, peptide mimetics and neutralizing antibodies, speaks to its role as a key modulator of both of the innate and adaptive immune systems. The mechanism whereby this protein exerts its effects is likely to be through the FPR receptor family-a hitherto rather enigmatic family of G protein coupled receptors, which are increasingly implicated in the regulation of many inflammatory processes. Here we review some of the key findings that have led up to the elucidation of this key pathway in inflammatory resolution.

Glucocorticoid treatment inhibits annexin-1 expression in rheumatoid arthritis CD4+ T cells.

OBJECTIVE: Annexin-1 (Anx-A1) has been recently shown to play a key role in T-cell activation and to be highly expressed in T cells from RA patients. Here, we investigated the effects of glucocorticoids (GCs) on Anx-A1 expression in T cells in vitro and in vivo. METHODS: To evaluate the effects of dexamethasone (Dex) on Anx-A1 expression, human peripheral blood T cells were incubated with Dex and then analysed by real-time PCR and western blotting. Similar experiments were carried out in vivo by measuring Anx-A1 levels in T cells from patients with RA before and after administration of steroids. RESULTS: Incubation of T cells with Dex decreased Anx-A1 levels in a time-dependent fashion and almost abolished its expression after 12 h. Stimulation of T cells pre-incubated with Dex for 12 h with anti-CD3/CD28 led to significant reduction of IL-2 production. Addition of human recombinant Anx-A1 to Dex-treated cells reversed the inhibitory effects of the steroids on anti-CD3/CD28-induced IL-2 production. Treatment of RA patients with steroid decreased Anx-A1 expression in T cells. CONCLUSIONS: GCs suppress Anx-A1 expression in T cells in vitro and in vivo. These results provide evidence for a novel pathway by which steroids regulate the adaptive immune response and suggest that Anx-A1 may represent a target for the treatment of autoimmune diseases.

Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides.

The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They thus provide the first evidence for a role of the FPR family in the regulation of neuroendocrine function.

Glutamate-stimulated peroxynitrite production in a brain-derived endothelial cell line is dependent on N-methyl-D-aspartate (NMDA) receptor activation.

There is accumulating and convincing evidence indicating a role for glutamate in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, demonstrate that pharmacological inhibition of specific glutamate receptors suppresses neurological symptoms and prevents blood-brain barrier (BBB) breakdown. The mechanisms through which glutamate influences BBB function during EAE remain unclear. Glutamate triggers the production of nitric oxide and superoxide, which can lead to the formation of peroxynitrite (ONOO(-)). Recent studies have implicated ONOO(-) in the loss of neurovascular integrity during EAE. We propose that glutamate contributes to BBB breakdown via the actions of ONOO(-). The present investigation examined glutamate-induced ONOO(-) formation in the b.End3 brain-derived endothelial cell line. b.End3 cells were incubated with a concentration range of glutamate and ONOO(-) production was assessed over time. Results showed a concentration- and time-dependent increase in ONOO(-) levels in glutamate-treated cells that were suppressed by selective and non-selective inhibitors of ONOO(-)-mediated reactions. Specific activation of b.End3-associated NMDA receptors also resulted in a concentration-dependent increase in ONOO(-) production. The ability of b.End3 cells to respond to the presence of glutamate was confirmed through the detection of NMDA receptor immnuoreactivity in cell extracts. In addition, the use of the NMDA receptor antagonists MK-801 and memantine reduced glutamate-mediated ONOO(-) generation from b.End3 cells. The data reinforce the important relationship between glutamate and the NMDA receptor, positioned at neurovascular sites, which may be of particular relevance to the pathogenesis of demyelinating disease.

Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface.

Annexin A1 (ANXA1) has an important role in cell-cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane-bound receptors on adjacent sites after translocation of the protein from the cytoplasm to the cell surface of adjacent cells. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo-stellate) cells. LPS caused serine-phosphorylation of ANXA1 (ANXA1-S27-PO4) and translocation of the phosphorylated protein to the cell membrane. The fundamental requirement of phosphorylation for membrane translocation was confirmed by immunofluorescence microscopy on cells transfected with wild-type or mutated (S27/A) ANXA1 constructs tagged with enhanced green fluorescence protein. The trafficking of ANXA1-S27-PO4 to the cell surface was dependent on PI3-kinase and MAP-kinase. It also required HMG-coenzyme A and myristoylation. The effects of HMG-coenzyme A blockade were overcome by mevalonic acid (the product of HMG-coenzyme A) and farnesyl-pyrophosphate but not by geranyl-geranylpyrophosphate or cholesterol. Together, these results suggest that serine-27 phosphorylation is essential for the translocation of ANXA1 across the cell membrane and also identify a role for isoprenyl lipids. Such lipids could target consensus sequences in ANXA1. Alternatively, they may target other proteins in the signal transduction cascade (e.g., transporters).