A Tangled Autoimmune Trio: Multiple Sclerosis, Systemic Lupus Erythematosus and Antineutrophil Cytoplasmic Antineutrophil Cytoplasmic Antibody Vasculitis.

The coexistence of multiple autoimmune diseases in the same individual is unusual and has received little attention in the literature. We present a young female patient with multiple sclerosis, systemic lupus erythematosus, and biopsy-proven renal proteinase 3 antineutrophil cytoplasmic antibodyassociated vasculitis who responded well to intravenous rituximab clinically and serologically.

Consensus statements for pharmacological management, monitoring of therapies, and comorbidity management of psoriatic arthritis in the United Arab Emirates.

OBJECTIVE: Psoriatic arthritis (PsA), a chronic inflammatory disease characterized by heterogeneous clinical manifestations, substantially impacts the quality of life of affected individuals. This article aims at developing consensus recommendations for the management of PsA and associated comorbidities and screening and monitoring requirements of PsA therapies in the United Arab Emirates (UAE) population. METHODS: An extensive review of present international and regional guidelines and publications on the pharmacological management, monitoring of therapies in the context of PsA was performed. Key findings from guidelines and literature were reviewed by a panel of experts from the UAE at several meetings to align with current clinical practices. Consensus statements were formulated based on collective agreement of the experts and members of Emirates Society for Rheumatology. RESULTS: The consensus recommendations were developed to aid practitioners in clinical decision-making with respect to dosage recommendations for pharmacological therapies for PsA, including conventional drugs, non-biologic, and biologic therapies. Consensus recommendations for therapeutic options for the treatment of PsA domains, including peripheral arthritis, axial disease, enthesitis, dactylitis, psoriasis, and nail disease, were developed. The panel emphasized the importance of monitoring PsA therapies and arrived at a consensus on monitoring requirements for PsA therapies. The expert panel proposed recommendations for the management of common comorbidities associated with PsA. CONCLUSION: These consensus recommendations can guide physicians and healthcare professionals in the UAE in making proper treatment decisions, as well as efficiently managing comorbidities and monitoring therapies in patients with PsA.

Consensus statements for evaluation and nonpharmacological Management of Psoriatic Arthritis in UAE.

OBJECTIVE: Psoriatic arthritis (PsA), a chronic inflammatory arthropathy, is often underdiagnosed in Middle Eastern countries, substantially impacting the treatment of affected individuals. This article aims to highlight current unmet clinical needs and provide consensus recommendations for region-specific evaluation methods and nonpharmacological therapies in the United Arab Emirates (UAE). METHOD: An extensive literature review was conducted, focusing especially on global and regional guidelines for the evaluation and treatment of PsA. These form the basis of the consensus statements formulated. Additionally, an expert panel of key opinion leaders from the UAE reviewed these guidelines and available literature at an advisory board meeting to identify unmet needs, bridge clinical gaps in the UAE, and develop consensus statements for the evaluation and treatment of PsA. RESULT: The consensus statements were developed based on overarching principles for the management of PsA, evaluation of patients with PsA, and nonpharmacological approaches for the management of PsA. The overarching principles included adopting a targeted, multidisciplinary approach, along with collaboration between rheumatologists and dermatologists in cases of clinically significant skin involvement. The panel also highlighted the value of composite disease severity measures for characterizing clinical manifestations of PsA. In terms of nonpharmacological management approaches, lifestyle modification (comprising dietary change, exercise, and cessation of smoking) and psychotherapy were recommended. CONCLUSION: The consensus statements will aid healthcare professionals in clinical decision-making in the context of PsA.

Predictors of not Achieving Remission or Low Disease Activity in Axial Spondyloarthritis Patients from Middle Eastern Countries: A Prospective, Multicenter, Real-world Study.

Objectives: We sought to identify the predictors of not achieving remission or low disease activity (LDA) among axial spondyloarthritis (SpA) patients in four Middle Eastern countries. Methods: In this multicenter prospective real-world study, adult patients with axial SpA diagnosed clinically during January-June 2019, and who met the Assessment of SpondyloArthritis International Society classification criteria for axial SpA, were enrolled from the participating centers of four countries-Lebanon, Oman, Qatar, and the UAE. Patient demographics, disease history, comorbidities, treatment, and compliance data were obtained at baseline. The primary outcome was to determine the percentage of patients who did not achieve the clinical target of remission or LDA as indicated by Ankylosing Spondylitis Disease Activity Score-C-reactive protein (ASDAS-CRP) < 2.1 after a three-month follow-up period. Secondary outcomes were assessing the demographic and clinical characteristics of 'achievers' and 'non-achievers' and to study the predictors of ASDAS-CRP ≥ 2.1 in different clinical subsets. Results: The participants were 309 patients of both sexes, with a median age of 43 years. Women had a slight majority (53.7%). At the end of the study, 72.2% of patients achieved the clinical target of ASDAS-CRP < 2.1. Non-achievers were significantly more likely to have enthesitis, positive human leukocyte antigen B 27 status, psoriasis, peripheral involvement, fibromyalgia, and a lower score on Compliance Questionnaire for Rheumatology (CQR). Multiple regression analysis showed that low CQR score, enthesitis, psoriasis, and family history of SpA were independent predictors of ASDAS-CRP ≥ 2.1. Conclusions: This real-world study suggests that low compliance, positive human leukocyte antigen B 27 status, peripheral involvement, and presence of enthesitis, psoriasis, and fibromyalgia are predictors of not achieving remission or LDA in axial SpA patients.

Primary Sjögren's Syndrome Presenting as Recurrent Ischemic Strokes.

Primary Sjogren's Syndrome (PSS) is an autoimmune exocrinopathy, with protean manifestations affecting multiple organ systems. Neurological manifestations are documented in about 20% of PSS cohorts in literature, with peripheral manifestations being commoner. Central nervous system manifestations of PSS (CNS-SS) encompass ischemic strokes, demyelinating lesions, aseptic meningitis, encephalitis, cerebellar ataxia, cognitive impairment and movement disorders. Ischemic stroke as presenting manifestation of PSS is extremely rare. We hereby describe a 50-year-old male, who presented for evaluation of 2 episodes of discrete focal neurological deficits over a duration of 6 weeks, with neuro-imaging findings revealing evidence of acute-subacute bihemispheric infarcts. Further evaluation revealed evidence of strongly positive anti phospholipid antibodies (aPL), indirect immunofluorescence antinuclear antibody (IIF-ANA), anti Sjögren's syndrome-A (SS-A/Ro) and anti-Ribonuclear protein (RNP) antibodies, with histopathological evidence of periductal and periacinar lymphocytic infiltration as well as acinar atrophy and interstitial fibrosis of minor salivary glands on lip biopsy, consistent with a diagnosis of Sjögren's syndrome, constituting a diagnosis of Antiphospholipid syndrome (APS) associated with PSS.

Hand Impairment in Systemic Sclerosis: Various Manifestations and Currently Available Treatment.

Systemic sclerosis (SSc) is an autoimmune disease initially recognized by hand involvement due to characteristic Raynaud's phenomenon (RP), puffy hands, skin thickening, and contractures resembling claw deformities. SSc contributes to hand impairment through inflammatory arthritis, joint contractures, tendon friction rubs (TFRs), RP, digital ulcers (DU), puffy hands, skin sclerosis, acro-osteolysis, and calcinosis. These manifestations, which often co-exist, can contribute to difficulty with occupational activities and activities of daily living (ADL), which can result in impaired quality of life. However, despite this knowledge, most diagnostic and treatment principles in SSc are focused on visceral manifestations due to known associations with morbidity and mortality. Treatment of inflammatory arthritis is symptom based and involves corticosteroids ≤10mg daily, methotrexate, tumor necrosis factor inhibitors, tocilizumab, and abatacept. Small joint contractures are managed by principles of occupational hand therapy and rarely surgical procedures. TFRs may be treated similar to inflammatory arthritis with corticosteroids. All patients with RP and DU should keep digits covered and warm and avoid vasoconstrictive agents. Pharmacologic management of RP begins with use of calcium channel blockers, but additional agents that may be considered are fluoxetine and phosphodiesterase 5 (PDE5) inhibitors. DU management also involves vasodilators including calcium channel blockers and PDE5 inhibitors; bosentan has also been shown to prevent DU. In patients with severe RP and active DU, intravenous epoprostenol or iloprost can be used and surgical procedures, such as botulinum injections and digital sympathectomies, may be considered. For those with early diffuse cutaneous SSc needing immunosuppression for skin sclerosis, methotrexate or mycophenolate mofetil can be used, but the agent of choice depends on co-existing manifestations, such as inflammatory arthritis and/or lung involvement. Various pharmacologic agents for calcinosis have been considered but are generally ineffective; however, surgical options, including excision of areas of calcinosis, can be considered. Overall management of hand impairment for all patients with SSc should include occupational hand therapy techniques such as range of motion exercises, paraffin wax, and devices to assist in ADL. Thus, treatment options for the various manifestations contributing to hand impairment in SSc are limited and often modestly efficacious at best. Robust studies are needed to address the manifestations of SSc that contribute to hand impairment.

Temporal Patterns of Circulating Inflammation Biomarker Networks Differentiate Susceptibility to Nosocomial Infection Following Blunt Trauma in Humans.

BACKGROUND: Severe traumatic injury can lead to immune dysfunction that renders trauma patients susceptible to nosocomial infections (NI) and prolonged intensive care unit (ICU) stays. We hypothesized that early circulating biomarker patterns following trauma would correlate with sustained immune dysregulation associated with NI and remote organ failure. METHODS: In a cohort of 472 blunt trauma survivors studied over an 8-year period, 127 patients (27%) were diagnosed with NI versus 345 trauma patients without NI. To perform a pairwise, case-control study with 1:1 matching, 44 of the NI patients were compared with 44 no-NI trauma patients selected by matching patient demographics and injury characteristics. Plasma obtained upon admission and over time were assayed for 26 inflammatory mediators and analyzed for the presence of dynamic networks. RESULTS: Significant differences in ICU length of stay (LOS), hospital LOS, and days on mechanical ventilation were observed in the NI patients versus no-NI patients. Although NI was not detected until day 7, multiple mediators were significantly elevated within the first 24 hours in patients who developed NI. Circulating inflammation biomarkers exhibited 4 distinct dynamic patterns, of which 2 clearly distinguish patients destined to develop NI from those who did not. Mediator network connectivity analysis revealed a higher, coordinated degree of activation of both innate and lymphoid pathways in the NI patients over the initial 24 hours. CONCLUSIONS: These studies implicate unique dynamic immune responses, reflected in circulating biomarkers that differentiate patients prone to persistent critical illness and infections following injury, independent of mechanism of injury, injury severity, age, or sex.

A challenging twist in pulmonary renal syndrome.

Case. We report a rare case of hydralazine-induced anti-neutrophil cytoplasmic antibody-associated vasculitis. A 75-year-old African American woman with history of high blood pressure on hydralazine for 3 years presented with acute onset of shortness of breath and hemoptysis. Lab workup revealed a severe normocytic anemia and a serum creatinine of 5.09 mg/dL (baseline 0.9). Bronchoscopy demonstrated active pulmonary hemorrhage. A urine sample revealed red cell casts and a renal biopsy demonstrated pauci-immune, focally necrotizing glomerulonephritis with small crescents consistent with possible anti-neutrophil cytoplasmic antibody-positive renal vasculitis. Serologies showed high-titer MPO-ANCA and high-titer anti-histone antibodies. She was treated with intravenous steroids and subsequently with immunosuppression after cessation of hydralazine. The patient was subsequently discharged from hospital after a rapid clinical improvement. Conclusion. Hydralazine-induced anti-neutrophil cytoplasmic antibody-positive renal vasculitis is a rare adverse effect and can present as a severe vasculitic syndrome with multiple organ involvement. Features of this association include the presence of high titer of anti-myeloperoxidase anti-neutrophil cytoplasmic antibody with multiantigenicity, positive anti-histone antibodies, and the lack of immunoglobulin and complement deposition. Prompt cessation of hydralazine may be sufficient to reverse disease activity but immunosuppression may be needed.

An Unusual Case of Adult-Onset Still's Disease with Hemophagocytic Syndrome, Necrotic Leukoencephalopathy and Disseminated Intravascular Coagulation.

Case. A 34-year-old African-American female with a history of adult-onset Still's disease presented to an outside hospital with oligoarthritis. She experienced a generalized tonic-clonic seizure en route via ambulance, was intubated upon arrival, and transferred to the intensive care unit for treatment of suspected pneumonia and sepsis. She subsequently developed generalized cutaneous desquamation that progressed despite the cessation of antibiotics and other potential offending drugs which required transfer to our hospital's burn unit. She was suspected to have reactive hemophagocytic syndrome based on her clinical presentation of fever, rash, polyarthritis, elevated liver enzymes, coagulopathy, splenomegaly, normocytic anemia, thrombocytopenia, hypertriglyceridemia, hyperferritinemia, and hemophagocytosis visualized in bone marrow biopsy specimen. Magnetic resonance imaging demonstrated necrotic demyelination of the deep white matter and corona radiata. The patient developed multiorgan dysfunction and DIC without any other attributable etiology. Despite aggressive broad spectrum therapy and high dose of steroids she progressively deteriorated and eventually expired. Conclusion. Previous publications have highlighted the prevalence of necrotic leukoencephalopathy in children with familial hemophagocytic syndrome. Our patient demonstrated some uncommon features complicating her HLH including DIC and necrotic leukoencephalopathy, which are very rare entities in AOSD.

Identification of a novel pathway of transforming growth factor-ß1 regulation by extracellular NAD+ in mouse macrophages: in vitro and in silico studies.

Extracellular ß-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-ß1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-ß1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-ß1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-ß1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-ß1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-ß1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-ß1.

Hemoadsorption reprograms inflammation in experimental gram-negative septic peritonitis: insights from in vivo and in silico studies.

UNLABELLED: Improper compartmentalization of the inflammatory response leads to systemic inflammation in sepsis. Hemoadsorption (HA) is an emerging approach to modulate sepsis-induced inflammation. We sought to define the effects of HA on inflammatory compartmentalization in Escherichia coli-induced fibrin peritonitis in rats. HYPOTHESIS: HA both reprograms and recompartmentalizes inflammation in sepsis. Sprague Dawley male rats were subjected to E. coli peritonitis and, after 24 h, were randomized to HA or sham treatment (sepsis alone). Venous blood samples collected at 0, 1, 3 and 6 h (that is, 24-30 h of total experimental sepsis), and peritoneal samples collected at 0 and 6 h, were assayed for 14 cytokines along with NO(2)(-/)NO(3)(-). Bacterial counts were assessed in the peritoneal fluid at 0 and 6 h. Plasma tumor necrosis factor (TNF)-α, interleukin (IL)-6, CXCL-1, and CCL2 were significantly reduced in HA versus sham. Principal component analysis (PCA) suggested that inflammation in sham was driven by IL-6 and TNF-α, whereas HA-associated inflammation was driven primarily by TNF-α, CXCL-1, IL-10 and CCL2. Whereas -peritoneal bacterial counts, plasma aspartate transaminase levels and peritoneal IL-5, IL-6, IL-18, interferon (IFN)-γ and NO(2)(-)/NO(3)(-) were significantly lower, both CXCL-1 and CCL2 as well as the peritoneal-to-plasma ratios of TNF-α, CXCL-1 and CCL2 were significantly higher in HA versus sham, suggesting that HA-induced inflammatory recompartmentalization leads to the different inflammatory drivers discerned in part by PCA. In conclusion, this study demonstrates the utility of combined in vivo/in silico methods and suggests that HA exerts differential effects on mediator gradients between local and systemic compartments that ultimately benefit the host.

Hypoxia-induced overexpression of BNIP3 is not dependent on hypoxia-inducible factor 1α in mouse hepatocytes.

We sought to investigate the expression of the cell death protein BNIP3 in hypoxic hepatocytes, as well as the role that hypoxia-inducible factor 1 (HIF-1α) plays in the upregulation of BNIP3 in hypoxic primary mouse hepatocytes and in the livers of mice subjected to ischemia-reperfusion. Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 1, 3, 6, 24, and 48 h, and the RNA and protein were isolated for reverse transcriptase-polymerase chain reaction and Western blot analysis. Similarly, livers from mice subjected to segmental (70%) hepatic warm ischemia for 30 min or 1 h, or to 1-h ischemia followed by 0.5- to 4-h reperfusion, were collected and subjected to Western blot analysis for HIF-1α protein. We showed that hypoxic stress increases the formation of the BNIP3 homodimer while decreasing the amount of the monomeric form of BNIP3 in primary mouse hepatocytes. In contrast to RAW264.7 macrophages, there is a basal expression of HIF-α protein in normoxic primary mouse hepatocytes that does not change significantly upon exposure to hypoxia. Using siRNA technology, we demonstrated that reduced HIF-1α protein levels did not block the hypoxia-induced overexpression of BNIP3. In contrast to the effect on BNIP3 expression reported previously, livers from ischemic animals demonstrated only a modest increase in HIF-1α protein as compared with resting livers from control animals; and this expression was not statistically different from sham controls. These results suggest that HIF-1α does not mediate the hypoxia-induced upregulation of BNIP3 in mouse hepatocytes in vitro and possibly in the liver in vivo.

A dynamic view of trauma/hemorrhage-induced inflammation in mice: principal drivers and networks.

BACKGROUND: Complex biological processes such as acute inflammation induced by trauma/hemorrhagic shock/ (T/HS) are dynamic and multi-dimensional. We utilized multiplexing cytokine analysis coupled with data-driven modeling to gain a systems perspective into T/HS. METHODOLOGY/PRINCIPAL FINDINGS: Mice were subjected to surgical cannulation trauma (ST) ± hemorrhagic shock (HS; 25 mmHg), and followed for 1, 2, 3, or 4 h in each case. Serum was assayed for 20 cytokines and NO(2) (-)/NO(3) (-). These data were analyzed using four data-driven methods (Hierarchical Clustering Analysis [HCA], multivariate analysis [MA], Principal Component Analysis [PCA], and Dynamic Network Analysis [DyNA]). Using HCA, animals subjected to ST vs. ST + HS could be partially segregated based on inflammatory mediator profiles, despite a large overlap. Based on MA, interleukin [IL]-12p40/p70 (IL-12.total), monokine induced by interferon-γ (CXCL-9) [MIG], and IP-10 were the best discriminators between ST and ST/HS. PCA suggested that the inflammatory mediators found in the three main principal components in animals subjected to ST were IL-6, IL-10, and IL-13, while the three principal components in ST + HS included a large number of cytokines including IL-6, IL-10, keratinocyte-derived cytokine (CXCL-1) [KC], and tumor necrosis factor-α [TNF-α]. DyNA suggested that the circulating mediators produced in response to ST were characterized by a high degree of interconnection/complexity at all time points; the response to ST + HS consisted of different central nodes, and exhibited zero network density over the first 2 h with lesser connectivity vs. ST at all time points. DyNA also helped link the conclusions from MA and PCA, in that central nodes consisting of IP-10 and IL-12 were seen in ST, while MIG and IL-6 were central nodes in ST + HS. CONCLUSIONS/SIGNIFICANCE: These studies help elucidate the dynamics of T/HS-induced inflammation, complementing other forms of dynamic mechanistic modeling. These methods should be applicable to the analysis of other complex biological processes.

An adequately robust early TNF-alpha response is a hallmark of survival following trauma/hemorrhage.

BACKGROUND: Trauma/hemorrhagic shock (T/HS) results in cytokine-mediated acute inflammation that is generally considered detrimental. METHODOLOGY/PRINCIPAL FINDINGS: Paradoxically, plasma levels of the early inflammatory cytokine TNF-alpha (but not IL-6, IL-10, or NO(2) (-)/NO(3) (-)) were significantly elevated within 6 h post-admission in 19 human trauma survivors vs. 4 non-survivors. Moreover, plasma TNF-alpha was inversely correlated with Marshall Score, an index of organ dysfunction, both in the 23 patients taken together and in the survivor cohort. Accordingly, we hypothesized that if an early, robust pro-inflammatory response were to be a marker of an appropriate response to injury, then individuals exhibiting such a response would be predisposed to survive. We tested this hypothesis in swine subjected to various experimental paradigms of T/HS. Twenty-three anesthetized pigs were subjected to T/HS (12 HS-only and 11 HS + Thoracotomy; mean arterial pressure of 30 mmHg for 45-90 min) along with surgery-only controls. Plasma obtained at pre-surgery, baseline post-surgery, beginning of HS, and every 15 min thereafter until 75 min (in the HS only group) or 90 min (in the HS + Thoracotomy group) was assayed for TNF-alpha, IL-6, IL-10, and NO(2) (-)/NO(3) (-). Mean post-surgery+/-HS TNF-alpha levels were significantly higher in the survivors vs. non-survivors, while non-survivors exhibited no measurable change in TNF-alpha levels over the same interval. CONCLUSIONS/SIGNIFICANCE: Contrary to the current dogma, survival in the setting of severe, acute T/HS appears to be associated with an immediate increase in serum TNF-alpha. It is currently unclear if this response was the cause of this protection, a marker of survival, or both. This abstract won a Young Investigator Travel Award at the SHOCK 2008 meeting in Cologne, Germany.

Activation of latent transforming growth factor-beta1 by nitric oxide in macrophages: role of soluble guanylate cyclase and MAP kinases.

The inducible nitric oxide (NO) synthase and the cytokine transforming growth factor-beta1 (TGF-beta1), both central modulators of wound healing, interact reciprocally: TGF-beta1 generally suppresses iNOS expression, while NO can induce and activate latent TGF-beta1. We have shown that chemical NO activates recombinant human latent TGF-beta1 by S-nitrosation of the latency-associated peptide (LAP), a cleaved portion of pro-TGF-beta1 that maintains TGF-beta1 in a biologically-inactive state. We hypothesized that cell-associated TGF-beta1 could be activated by NO via known NO-inducible signaling pathways (soluble guanylate cyclase [sGC] and mitogen-activated protein [MAP] kinases). Treatment of mouse RAW 264.7 macrophage-like cells with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) led to a dose- and time-dependent increase in cell-associated active and latent TGF-beta1, as assessed by quantitative immunocytochemistry for active TGF-beta1 vs. LAP and partially validated by western blot analysis. Treatment with the sGC inhibitor 1,H-[1,2,4]oxadiazole[4,3-a]quinoxalon-1-one (ODQ) reduced both active and latent TGF-beta1 dose-dependently. SNAP, in the presence or absence of ODQ or the MAP kinase inhibitors, did not affect steady-state TGF-beta1 mRNA levels. Treatment with inhibitors specific for JNK1/2, ERK1/2, and p38 MAP kinases suppressed SNAP-induced active and latent TGF-beta1. Treatment with the cell-permeable cGMP analog 8-Br-cGMP increased both active and latent TGF-beta1. However, TGF-beta1 activation induced by 8-Br-cGMP was not blocked by MAP kinase inhibitors. Our findings suggest that NO activates latent TGF-beta1 via activation of sGC and generation of cGMP and separately via MAP kinase activation, and may shed insight into the mechanisms by which both cGMP production and MAP kinase activation enhance wound healing.

Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress.

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1-4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

A simple, rapid, and convenient Luminex-compatible method of tissue isolation.

Various pathological conditions are associated with changes in multiple protein biomarkers, and these changes can be assessed using xMAP beads and the Luminex platform. Although this platform is most commonly utilized in the analysis of biomarkers in serum, plasma, or urine, it is often desirable to measure these analytes in tissues (e.g., biopsy specimens). We have developed a simple, rapid method of tissue isolation in which excised tissues are permeabilized in RNAlater at 4 degrees C overnight, followed by Luminex analysis for inflammatory biomarkers (cytokines). This method was compared with flash-freezing in both mouse liver and human debrided wound specimens, and may be of utility in other clinical settings.