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Background: Hypoparathyroidism-retardation-dysmorphism (HRD) syndrome is a disease composed of hypoparathyroidism, growth retardation, severe developmental delay, and typical dysmorphic features caused by the tubulin-specific chaperone E gene variant. Many patients succumb in infancy to HRD due to overwhelming infections mainly caused by Pneumococcus spp. Knowledge related to the immune system in these patients is scarce., Purpose: To define the immune phenotype of a cohort of HRD patients including their cellular, humoral, and neutrophil functions., Methods: The study included HRD patients followed at Soroka University Medical Center. Clinical and immunological data were obtained, including immunoglobulin concentrations, specific antibody titers, lymphocyte subpopulations, lymphocyte proliferation, and neutrophil functions., Results: Nine patients (5 females and 4 males) were enrolled, aged 6 months to 15 years. All received amoxicillin prophylaxis as part of a routine established previously. Three patients had bacteremia with Klebsiella, Shigella spp., and Candida. Three patients had confirmed coronavirus disease 19 (COVID-19), and two of them died from this infection. All patients had normal blood counts. Patients showed high total IgA and IgE levels, low anti-pneumococcal antibodies in spite of a routine vaccination schedule, and reduced frequency of naive B cells with increased frequency of CD21lowCD27- B cells. All patients had abnormal T-cell population distributions, including reduced terminally differentiated effector memory CD8, inverted CD4/CD8 ratios, and impaired phytohemagglutinin (PHA)-induced lymphocyte proliferation. Neutrophil superoxide production and chemotaxis were normal in all patients tested., Conclusion: HRD is a combined immunodeficiency disease with syndromic features, manifesting in severe invasive bacterial and viral infections., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Previous studies have demonstrated that cytosolic phospholipase A 2 α (cPLA 2 α) is required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA 2 α translocates to the plasma membranes by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the cytosolic oxidase factor, p47 phox in human phagocytes. Intravenous administration of antisense against cPLA 2 α that significantly inhibited its expression in mouse peritoneal neutrophils and macrophages also inhibited superoxide production, in contrast to cPLA 2 α knockout mice that showed normal superoxide production. The present study aimed to determine whether there is a binding between cPLA 2 α-C2 domain and p47 phox -PX in mouse macrophages, to further support the role of cPLA 2 α in oxidase regulation also in mouse phagocytes., Methods and Results: A significant binding of mouse GST-p47 phox -PX domain fusion protein and cPLA 2 α in stimulated mouse phagocyte membranes was demonstrated by pull-down experiments, although lower than that detected by the human p47 phox -PX domain. Substituting the amino acids Phe98, Asn99, and Gly100 to Cys98, Ser99, and Thr100 in the mouse p47 phox -PX domain (present in the human p47 phox -PX domain) caused strong binding that was similar to that detected by the human p47 phox -PX domain CONCLUSIONS: The binding between cPLA 2 α-C2 and p47 phox -PX domains exists in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice, probably due to the absence of amino acids Cys98, Ser 99, and Thr100in the p47 phox -PX domain that facilitate the binding to cPLA 2 α., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Amyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by the selective death of motor neurons. Cytosolic phospholipase A 2 alpha (cPLA 2 α) upregulation and activation in the spinal cord of ALS patients has been reported. We have previously shown that cPLA 2 α upregulation in the spinal cord of mutant SOD1 transgenic mice (SOD1 G93A ) was detected long before the development of the disease, and inhibition of cPLA 2 α upregulation delayed the disease's onset. The aim of the present study was to determine the mechanism for cPLA 2 α upregulation., Methods: Immunofluorescence analysis and western blot analysis of misfolded SOD1, cPLA 2 α and inflammatory markers were performed in the spinal cord sections of SOD1 G93A transgenic mice and in primary motor neurons. Over expression of mutant SOD1 was performed by induction or transfection in primary motor neurons and in differentiated NSC34 motor neuron like cells., Results: Misfolded SOD1 was detected in the spinal cord of 3 weeks old mutant SOD1 G93A mice before cPLA 2 α upregulation. Elevated expression of both misfolded SOD1 and cPLA 2 α was specifically detected in the motor neurons at 6 weeks with a high correlation between them. Elevated TNFα levels were detected in the spinal cord lysates of 6 weeks old mutant SOD1 G93A mice. Elevated TNFα was specifically detected in the motor neurons and its expression was highly correlated with cPLA 2 α expression at 6 weeks. Induction of mutant SOD1 in primary motor neurons induced cPLA 2 α and TNFα upregulation. Over expression of mutant SOD1 in NSC34 cells caused cPLA 2 α upregulation which was prevented by antibodies against TNFα. The addition of TNFα to NSC34 cells caused cPLA 2 α upregulation in a dose dependent manner., Conclusions: Motor neurons expressing elevated cPLA 2 α and TNFα are in an inflammatory state as early as at 6 weeks old mutant SOD1 G93A mice long before the development of the disease. Accumulated misfolded SOD1 in the motor neurons induced cPLA 2 α upregulation via induction of TNFα., (© 2021. The Author(s).)

Collagen is the major structural protein in the extracellular matrix of skin produced by fibroblasts. UV exposure results in infiltration of neutrophils within the epidermis and dermis, inducing collagen damage and contributing to the process of photo-aging. Collagen-3 is an integral structural component with collagen-1, and is an important regulator of collagen-1 fibrillogenesis. Addition of neutrophils activated with TNFα to normal human dermal fibroblast cultures, but not their supernatant, caused significant collagen-3 damage. To study whether Lumenato can protect from collagen-3 damage, it was added to co-cultures of Normal human dermal fibroblasts and neutrophils activated with TNFα. Lumenato prevented collagen-3 damage induced by activated neutrophils in a dose-dependent manner in the co-cultures. Lumenato also induced a low rate of collagen-3 synthesis in a dose-dependent manner detected by pro-collagen-3 secretion, but did not affect fibroblast cell number. Although Lumenato inhibited MMP-8, MMP-9, and elastase secreted from neutrophils, its main effect was in inhibiting both NADPH oxidase-producing superoxides and MPO activity-producing halides in a dose-dependent manner that correlated with protection from collagen-3 damage. In conclusion, the results suggest that Lumenato induces low levels of collagen-3 that may contribute for skin health and is very effective in defending the co-cultures from collagen-3 damage by inhibiting free radicals secreted from neutrophils, thus, indicating Lumenato's possible potential for skin protection., Competing Interests: The authors have read the journal's policy, and the authors of the study have the following competing interests to declare: Lycored Ltd. partially supported the costs of materials of the research in the form of a grant (number 2016-2020) awarded to RL. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors would like to declare the following patents/patent applications associated with this research: A patent Application No. PCT/IL2020/051086 was submitted based on the research results.

Biallelic mutations in the zeta-associated protein 70 (ZAP70) gene cause combined immunodeficiency (CID). Neonatal screening for severe CID in Israel is implemented since 2015. We report on clinical, flow cytometry, and genetic data of an unusual ZAP70 deficiency patient. A 10-week-old Bedouin female presented with severe autoimmune hemolytic anemia. Cytomegalovirus (CMV) negative packed cell therapy was given without improvement; indexes of hemolysis worsened. At this time, thrombocytopenia was noted. The patient was treated with single dose of 1 g/kg intravenous immunoglobulin with rapid resolution of hemolysis. Serum immunoglobulin concentrations were normal; flow cytometry revealed severe CD8 lymphocytopenia. Lymphocyte proliferation test demonstrated reduced response to concanavalin A and phytohemagglutinin. Gated T cells were negative for intracellular ZAP70. A genetic analysis revealed a missense homozygous c.1388C > T (p.A463V) mutation, confirming the diagnosis of ZAP70 deficiency. She later on developed urinary tract infection due to ESBL producing E. coli treated with amikacin and severe CMV infection that partially responded to ganciclovir therapy and at 7 months of age, she successfully underwent allogeneic hematopoietic stem cell transplantation. Neonatal screening by T cell receptor excision circles (TRECs) for SCID was normal, yet very low TRECs were recorded at the time of CID diagnosis. Normal neonatal screening for SCID does not rule out the diagnosis of CID due to ZAP70 deficiency. This type of CID can present with autoimmunity as the sole initial manifestation of the disease.

Neutrophil chemotactic defects have been reported previously in patients with hyper-IgE syndrome. Bi-allelic mutations in dedicator of cytokinesis 8 (DOCK8) gene usually cause an autosomal recessive hyper-IgE syndrome phenotype. Data are lacking about expression of DOCK8 protein in neutrophils or the possible role of DOCK8 in neutrophil function. We sought to determine if DOCK8 protein is expressed in neutrophils and if DOCK8 plays a role in neutrophil function. The expression of DOCK8 protein was assessed in neutrophils from healthy volunteers with and without activators. Neutrophil chemotaxis, phagocytosis and superoxide generation were studied in neutrophils from DOCK8-deficient patients compared to neutrophils from healthy controls before and after stimulation with activators: phorbol 12-myristate 13-acetate (PMA) or N-Formylmethionyl-leucyl-phenylalanine (fMLP). DOCK8 protein is expressed in resting neutrophils from healthy controls, with a significant increase in DOCK8 expression after stimulation. Neutrophil functions were assessed in 6 DOCK8-deficient patients. All patients had the same non-sense mutation (c.C5134A, p.S1711X). Normal chemotaxis was recorded in 4/6 patients while a mild to moderate chemotaxis defect was recorded in 2/6. Superoxide generation was mainly normal in neutrophils from all six patients and phagocytosis was normal in five patients tested. We conclude that DOCK8 protein is expressed in resting human neutrophils and DOCK8 expression is increased after stimulation with either PMA or fMLP. Most patients with a disease-causing mutation in DOCK8 have normal neutrophil functions, while a minority showed a mild to moderate chemotactic defect.

Microglia activation toward the M1 phenotype has been reported to contribute to the neurodegenerative processes and cognition alterations due to the release of pro-inflammatory mediators and cytokines. The aim of the present research was to assess the effectiveness of free fatty acids omega-3 preparations: eicosapentaenoic acid (EPA) or/and docosahexaenoic acid (DHA), carotenoids and phenolics combinations, in inhibiting the release of inflammatory mediators from activated microglia. Preincubation of BV-2 microglia cells with each of the FFAs omega-3 preparations in a range of 0.03-2 μM together with Lyc-O-mato ® (0.1 μM), Carnosic acid (0.2 μM) with or without Lutein (0.2 μM), 1 h before addition of lipopolysaccharide (LPS) for 16 h caused a synergistic inhibition of nitric oxide (NO) production with a rank order of EPA > Ropufa (EPA/DHA 2/1) > Krill (EPA/DHA 1.23/1). The optimal inhibitory combinations of EPA (0.125 μM) with the phytonutrients caused a synergistic inhibition of prostaglandin E 2 (PGE 2 ) release, IL-6 secretion, superoxide and NO production and prevention of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) upregulation and elevated CD40 expression in microglia exposed to LPS or interferon-γ (IFN-γ), representing infection or inflammation, respectively. The presence of the combination caused a synergistic increase in the release of the anti-inflammatory cytokine IL-10. The inhibitory effects by the combinations of EPA with the phytonutrients were mediated by the inhibition of the redox-sensitive NF-κB activation and detected by its phosphorylated p-65 on serine 536 in microglia stimulated by either LPS or IFN-γ. In addition, phosphorylated CREB on serine 133 which was shown to be involved in the induction of iNOS was inhibited by the combinations in stimulated cells. In conclusion, the results suggest that low concentrations of EPA with the phytonutrients are very efficient in inhibiting the transformation of microglia to M1 phenotype and may prevent cognition deficit.

Mutations in the dedicator of cytokinesis 8 (DOCK8) gene cause a combined immunodeficiency usually diagnosed as autosomal recessive hyper IgE syndrome. We sought to reveal the varying manifestations in patients with a unique mutation in DOCK8 gene by a retrospective medical record review. Ten patients from five consanguineous families and three tribes were included. Seven patients were homozygous for the c.C5134A, p.S1711X mutation, and the remaining three patients were their siblings manifesting hyper IgE syndrome features without a genetic diagnosis. Prior to the genetic diagnosis, the clinical diagnosis was "hyper IgE syndrome" in six patients and "anti-pneumococcal antibody deficiency," "recurrent pneumonia with bronchiectasis," and "asthma with hypereosinophilic syndrome" each diagnosed once. One additional patient was diagnosed due to family history. The age of presentation varied from 1 to 16 months. Eczema was diagnosed in all patients, food allergies in three, and severe herpes keratitis or malignancy or autoimmunity in two patients. Elevated IgE was recorded in nine patients; however, in six patients, the initial serum IgE concentration was equal to or less than three times the normal concentration for age, and in these patients, the median age at IgE evaluation was 7.5 months compared with 21.5 months in patients with an initial IgE concentration above three times the normal concentration for age (P = 0.067). The spectrum of disease manifestations in patients with a unique mutation in DOCK8 is variable. The genotype-phenotype correlations may be modified by genetic and/or epigenetic modifiers beyond the monogenic effect. Younger patients tend to have lower IgE concentrations at the initial measurement of IgE.

Background: The aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A 2 alpha (cPLA 2 α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA 2 α has a direct, participatory role in the molecular events leading to CD40 induction., Methods: Cultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA 2 α in the events leading to CD40 protein induction., Results: Addition of LPS or IFNγ caused a significant upregulation of cPLA 2 α and of CD40, while prevention of cPLA 2 α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA 2 α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA 2 α detected by its phosphorylated form, while addition of IFNγ induced cPLA 2 α activation at a later time scale (4 h). The activation of cPLA 2 α is mediated by ERK activity. Suppression of cPLA 2 α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-κB detected by the phosphorylation of p65 on serine 536 at 15 min by LPS and at 4 h by IFNγ. Inhibition of NOX2 prevented NF-κB activation and CD40 induction but did not affect cPLA 2 α activation, suggesting cPLA 2 α is located upstream to NOX2 and NF-κB. The activation of cPLA 2 by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA 2 α by IFNγ was mediated by the secreted TNF-α at 4 h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA 2 α inhibitors, indicating that STAT1α is not under cPLA 2 α regulation., Conclusions: Our results show for the first time that cPLA 2 upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-κB. Cumulatively, our results indicate that cPLA 2 α may serve as a pivotal amplifier of the inflammatory response in the CNS.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by selective death of motor neurons in the cortex, brainstem, and spinal cord. Cytosolic phospholipase A2 alpha (cPLA2α) upregulation and activation in the spinal cord of patients with sporadic ALS and in the spinal cord of human mutant SOD1G93A (hmSOD1) transgenic mice were recently reported., Methods: cPLA2α upregulation in the brainstem and spinal cord was reduced by brain infusion of a specific antisense oligonucleotide against cPLA2α (AS), and the effect was evaluated on disease progression and brain cell activation., Results: We found that the elevation of cPLA2α protein expression in the spinal cord was first detected at 6-week-old hmSOD1 mice and remained elevated during their whole life span. Reduction of the elevated expression of cPLA2α in the spinal cord of hmSOD1 mice by brain infusion of an AS at week 15 (shortly before the appearance of the disease symptoms), for a duration of 6 weeks, delayed the loss of motor neuron function in comparison with hmSOD1 mice and with sense brain-infused hmSOD1 mice. To characterize the effect of cPLA2α upregulation on different processes taking place at the appearance of the disease symptoms, mice were brain infused with AS or with sense at week 15 for 3-4 weeks. The AS treatment that reduced cPLA2α upregulation in the spinal cord of AS-treated hmSOD1 mice (as analyzed at week 18-19) prevented the reduction in the number of the neurons (detected by NeuN) and inhibited astrocyte activation (detected by GFAP) and microglia activation (detected by Iba-1 and by CD40). In addition, AS treatment blunted the upregulation of the proinflammatory enzyme-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) detected in hmSOD1 mice., Conclusions: Since specific reduction of cPLA2α in the brainstem and spinal cord significantly attenuated the development of the disease, cPLA2α may offer an efficient target for treatment of ALS.

Colitis, an inflammation of the colon, is a well-characterized massive tissue injury. Cytosolic phospholipase A2 α (cPLA2 α) upregulation plays an important role in the development of several inflammatory diseases. The aim of the present study was to define the role of cPLA2 α upregulation in the development of colitis. We used a mouse model of dextran sulfate sodium induced colitis. Immunoblotting analysis showed that cPLA2 α and NF-κB were upregulated and activated in the colon from day 2 of colitis induction. This molecular event preceded the development of the disease, as determined by Disease Activity Index score, body weight, colon length, and the expression of colonic inflammatory markers, including neutrophil infiltration detected by myeloperoxidase and by NIMP-R14, ICAM-1, COX-2, iNOS upregulation and LTB4 and TNF-α secretion. Prevention of cPLA2 α upregulation and activity in the colon by i.v. administration of specific antisense oligonucleotides against cPLA2 α 1 day prior and every day of exposure to dextran sulfate sodium significantly impeded the development of the disease and prevented NF-κB activation, neutrophils infiltration into the colonic mucosa, and expression of proinflammatory proteins in the colon. Our results demonstrate a critical role of cPLA2 α upregulation in inflammation and development of murine colitis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Amyloid-β peptides generated by proteolysis of the β-amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer's disease. The present study aimed to determine whether cytosolic phospholipase A2 α (cPLA2 α) plays a role in elevated APP protein expression induced by aggregated amyloid-β1-42 (Aβ) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA2 α and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Aβ for 24 h and in cortical neuron of human amyloid-β1-42 brain infused mice. Prevention of cPLA2 α up-regulation and its activity by oligonucleotide antisense against cPLA2 α (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA2 α in the signals leading to APP induction, increased cPLA2 α expression and activity induced by Aβ was prevented by means of AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase-2 and the production of prostaglandin E2 (PGE2 ) were prevented. Addition of PGE2 or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H-89) attenuated the elevation of APP induced by Aβ. Inhibition of elevated cPLA2 α by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Aβ which coincided with cPLA2 α dependent activation of CREB in the cortex of Aβ brain infused mice. Our results show that accumulation of Aβ induced elevation of APP protein expression mediated by cPLA2 α, PGE2 release, and CREB activation via protein kinase A pathway., (© 2014 International Society for Neurochemistry.)

Purpose: The role of the Bruton tyrosine kinase (Btk) protein in neutrophil function has been evaluated using neutrophils from healthy volunteers after incubation with a Btk inhibitor, leflunomide metabolite analog (LFM-A13), suggesting an important role for Btk in neutrophil function. We sought to determine the role of Btk protein on neutrophil superoxide generation and chemotaxis stimulated by N-formyl-methionine-leucine-phenylalanine (fMLP)., Methods: Chemotaxis was assayed on agarose gel and superoxide generation by cytochrome C reduction. The affects of LFM-A13 on chemotaxis and superoxide generation in unstimulated and fMLP stimulated neutrophils were studied in Btk deficient neutrophils from XLA patients compared with matched controls analyzed simultaneously., Results: Chemotaxis and stimulated superoxide production were similar in the normal and Btk deficient neutrophils and were similarly inhibited by LFM-A13. In one patient, LFMA13 had no effect on superoxide generation in Btk deficient neutrophils up to a concentration of 25 microM, while inhibited superoxide production by control neutrophils., Conclusions: Our results suggest that Btk does not have a specific role in neutrophil fMLP-stimulated superoxide generation and chemotaxis since these activities were similarly inhibited by LFM-A13 in Btk deficient and normal neutrophils. The lack of superoxide generation following Btk inhibition by LFM-A13 in Btk deficient neutrophils from one patient may suggest some heterogeneity in the role of Btk in fMLP induced neutrophil superoxide generation.

Increased cytosolic phospholipase A2α (cPLA2α) immunoreactivity and transcript were observed in Alzheimer's disease (AD) brain associated with amyloid deposits. Thus, the present study examined whether cPLA2α upregulation participate in cortical neuron damage induced by aggregated Aβ1-42 and determined its role in the signaling events leading to damage, using an antisense technology. Exposure of primary cortical neurons to 1μM aggregated Aβ1-42 for 24h induced up-regulation and activation of cPLA2α and apoptotic cell death of about 30% as detected by: cell count, MTT reduction, caspases-3 and -8 activation, DAPI and TUNEL staining, that were prevented by inhibition of cPLA2α up-regulation and activity in the presence of antisense against cPLA2α (AS). cPLA2α was rapidly activated upon addition of aggregated Aβ1-42, as determined by its phosphorylated form on serine 505, and this activity was dependent on NADPH oxidase activity. NOX2- and NOX4-NADPH oxidase upregulation at 24h of aggregated Aβ1-42 exposure was not affected by the presence of AS, but superoxide production was reduced, probably due to NOX2 inhibition. cPLA2α upregulation led to activation of neutral sphingomyelinase (N-SMase) as its activity was inhibited in the presence of AS, and could be restored by addition of arachidonic acid. Addition of ceramide analog induced caspase-8 activation leading to caspase-3 activation and apoptotic neuronal death. In conclusion, our results suggest that cPLA2α activity plays a crucial role in the signaling cascade leading to apoptotic neuronal death by aggregated Aβ1-42 probably via activation of N-SMase, ceramide production and caspases-3 and -8., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In established obesity, inflammation and macrophage recruitment likely contribute to the development of insulin resistance. In the current study, we set out to explore whether adipose tissue infiltration by neutrophils that occurs early (3 days) after initiating a high-fat diet (HFD) could contribute to the early occurrence of hepatic insulin resistance and to determine the role of cytosolic phospholipase A2α (cPLA2α) in this process. The 3-day HFD caused a significant upregulation of cPLA2α in periepididymal fat and in the liver. A specific antisense oligonucleotide (AS) effectively prevented cPLA2α induction, neutrophil infiltration into adipose tissue (likely involving MIP-2), and protected against 3-day HFD-induced impairment in hepatic insulin signaling and glucose over-production from pyruvate. To sort out the role of adipose neutrophil infiltration independent of cPLA2α induction in the liver, mice were injected intraperitoneally with anti-intracellular adhesion molecule-1 (ICAM-1) antibodies. This effectively prevented neutrophil infiltration without affecting cPLA2α or MIP-2, but like AS, prevented impairment in hepatic insulin signaling, the enhanced pyruvate-to-glucose flux, and the impaired insulin-mediated suppression of hepatic glucose production (assessed by clamp), which were induced by the 3-day HFD. Adipose tissue secretion of tumor necrosis factor-α (TNF-α) was increased by the 3-day HFD, but not if mice were treated with AS or ICAM-1 antibodies. Moreover, systemic TNF-α neutralization prevented 3-day HFD-induced hepatic insulin resistance, suggesting its mediatory role. We propose that an acute, cPLA2α-dependent, neutrophil-dominated inflammatory response of adipose tissue contributes to hepatic insulin resistance and glucose overproduction in the early adaptation to high-fat feeding.

Inflammatory mediators and cytokines play important roles in the pathogenesis of a vast number of human diseases; therefore much attention is focused on blunting their proinflammatory modes of action. The aims of the present research were to assess the effectiveness of combinations of carotenoids and phenolics, at concentrations that can be achieved in blood, to inhibit the release of inflammatory mediators from macrophages exposed to lipopolysaccharide (LPS) and to determine what the anti-inflammatory effect of the phytonutrient combinations was in an in vivo mouse model of peritonitis. Preincubation of mouse peritoneal macrophages with lycopene (1 μM) or Lyc-O-Mato (1 μM) and carnosic acid (2 μM), lutein (1 μM), and/or β-carotene (2 μM) 1h before addition of LPS for 24 h caused a synergistic inhibition of NO, prostaglandin E(2), and superoxide production derived from downregulation of iNOS, COX-2, and NADPH oxidase protein and mRNA expression and synergistic inhibition of TNFα secretion. We surmise that the anti-inflammatory action of the phytonutrient combinations used probably resides in their antioxidant properties, because they caused an immediate, efficient, and synergistic inhibition of LPS-induced internal superoxide production leading to a marked decrease in ERK and NF-κB activation. The anti-inflammatory effects of the selected phytonutrient combinations were also demonstrated in a mouse model of peritonitis: their supplementation in drinking water resulted in attenuation of neutrophil recruitment to the peritoneal cavity and in inhibition of inflammatory mediator production by peritoneal neutrophils and macrophages., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cytosolic phospholipase A(2)α (cPLA(2)α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA(2)α which coincided with a significant increase in cell proliferation. The inhibition of cPLA(2)α activity by pyrrophenone or by antisense oligonucleotide against cPLA(2)α (AS) or inhibition of prostaglandin E(2) (PGE(2)) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE(2). The secreted PGE(2) activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE(2). But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE(2). AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA(2)α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA(2)α-dependent PGE(2) production. PGE(2)via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A(2)α (cPLA(2)α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA(2)α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA(2)α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA(2)α (AS) that reduced cPLA(2)α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA(2)α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 μM AS prevented cPLA(2)α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA(2)α activation, suggesting that NADPH oxidase acts upstream to cPLA(2)α. Attenuating cPLA(2)α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE(2) that were essential for ICAM-1 upregulation. Inhibition of cPLA(2)α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser(536) and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA(2)α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE(2)-mediated protein kinase A.

A 1926-ins-T mutation in the TrkA gene encoding the tyrosine kinase receptor for nerve growth factor (NGF) was previously documented in patients with congenital insensitivity to pain with anhidrosis (CIPA). These patients suffer from skin lacerations which often evolve into deep tissue infections. Abnormality in neutrophil functions may explain this high rate of severe infections. In this study we show that chemotaxis was significantly (P<0.001) suppressed in patients' neutrophils, compared to healthy controls. Although NGF alone did not exert a chemotactic effect, its presence enhanced both migration toward fMLP and phosphorylation of MAP kinases (ERK and JNK) in neutrophils from healthy controls, but not in neutrophils from CIPA patients. The significantly impaired chemotactic activity of neutrophils from a CIPA patient, which has been attributed to the molecular defect in the TrkA receptor, may contribute to the high rate of infection.

We have previously demonstrated a physical interaction between cytosolic phospholipase A2alpha (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain.

Cytosolic phospholipase A2alpha (cPLA2) plays an important role in the development of several inflammatory diseases. The aim of the present study is to determine whether inhibition of cPLA2 expression, using specific antisense oligonucleotides against cPLA2 (antisense), is efficient in reducing inflammation after its development. Two mouse models of inflammation were included in the study: thioglicolate peritonitis and collagen-induced arthritis (CIA). The antisense was found to be specific and efficient in inhibiting cPLA2 expression and NADPH oxidase activity ex vivo in peritoneal phagocytes. Immunoblotting and immunohistochemistry analysis showed a significant elevation in cPLA2 expression in the inflamed joints of collagen-induced arthritis mice localized in cell infiltrate, chondrocytes and the surrounding skin and skeletal muscle. Similarly, the cPLA2 metabolite, leukotriene B4, accumulated in the peritoneal cavity of mice with peritonitis. Inhibition of elevated cPLA2 expression after development of inflammation by intravenous administration of antisense resulted in a dramatic reduction in inflammation and a significant reduction in neutrophils recruitment to the site of inflammation in both mouse models of inflammation. Our results demonstrate the critical role of cPLA2 for the duration of inflammation and suggest that inhibition of cPLA2 expression by antisense oligonucleotides may serve as an efficient treatment of inflammatory diseases.

Chronic inflammation of adipose tissue in obesity is by now an established phenomenon, but the initiating event(s) of the inflammatory cascade are still unknown. We hypothesized that neutrophil infiltration into adipose tissue may precede macrophage infiltration as in classical immune responses. Here we demonstrate that early (3 and 7 days) after initiating high-fat feeding of C57BL/6J mice, neutrophils transiently infiltrate the parenchyma of intra-abdominal adipose tissue. Mean periepdidymal fat myeloperoxidase expression (representing neutrophils) was significantly increased 3.5-fold (P < 0.01) and 2.9-fold (P < 0.03), at days 3 and 7 compared with day 0. Immunohistochemistry analysis demonstrated a physical binding between neutrophils and adipocytes, which was supported by in vitro adherence assay: mouse peritoneal neutrophils adhered to a monolayer of 3T3-L1 mouse adipocytes, in a manner dependent on their activation state, 41.9 +/- 3.7% or 29.5 +/- 2%, by PMA or the IL-8 analog CXCL1 (KC), respectively, compared with 24.8 +/- 1.5% in unstimulated neutrophils, respectively. The degree of surface exposure of CD11b (Mac-1) corresponded to the percentage of adhered neutrophils. The adherence was prevented by preincubating neutrophils or adipocytes with anti-CD11b or anti-ICAM-1 antibodies. Furthermore, immunoprecipitation of CD11b from lysates of a mixed neutrophil-adipocyte cell population resulted in coimmunoprecipitation of ICAM-1, indicating that the interaction is mediated by neutrophil CD11b and adipocyte ICAM-1.

Impairment of neutrophil functions and high levels of apoptotic neutrophils have been reported in human immunodeficiency virus (HIV) patients. The aim of the present study was to investigate the direct in vitro effects of the different HIV protease inhibitors (PIs) on neutrophil functions and apoptosis and to explore their mechanisms of action. The effects of nelfinavir (NFV), saquinavir (SQV), lopinavir (LPV), ritonavir (RTV), and amprenavir (APV) in the range of 5 to 100 microg/ml on neutrophil function, apoptosis, and mu-calpain activity were studied. The neutrophil functions studied included superoxide production stimulated by 5 ng/ml phorbol myristate acetate, 5 x 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, and 1 mg/ml opsonized zymosan; specific chemotaxis; random migration; and phagocytosis. Apoptosis was determined by DNA fragmentation, fluorescein isothiocyanate-annexin V binding, and nuclear morphology. All three neutrophil functions, as well as apoptosis, were similarly affected by the PIs. SQV and NFV caused marked inhibition and LPV and RTV caused moderate inhibition, while APV had a minor effect. mu-Calpain activity was not affected by the PIs in neutrophil lysate but was inhibited after its translocation to the membranes after cell stimulation. SQV, which was the most potent inhibitor of neutrophil functions and apoptosis, caused significant inhibition of calpain activity, while APV had no effect. The similar patterns of inhibition of neutrophil functions and apoptosis by the PIs, which coincided with inhibition of calpain activity, suggest the involvement of calpain activity in the regulation of these processes.

Hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome is the first reported disease caused by a defect in the tubulin folding and assembly pathway. We aimed to summarize our experience with a cohort of patients with HRD, analyze their growth, and evaluate patients' polymorphonuclear cell (PMN) functions. The records of 22 HRD patients in a single medical center were reviewed. Growth during infancy and early childhood were analyzed by the Infancy-Childhood-Puberty (ICP) growth model. PMN functions were compared with healthy controls. Twelve patients died and many hospitalizations due to infections and convulsions were recorded. Growth measurements, expressed as weight and height SD scores in boys at a mean age of 4 y were -13.1+/-3.8 and -8.7+/-1 and -16.6+/-4.4 and -9.5+/-2.4, respectively, in girls at a mean age of 6.4 y. Chemotactic migration, random migration, and phagocytosis of PMN from HRD patients were significantly lower than that of PMN from healthy controls. No significant differences were found in superoxide production of PMN from patients compared with controls. Functional hyposplenism has been demonstrated in most of the studied patients. The defect in the tubulin folding and assembly pathway, previously described in HRD, has grave consequences on growth and PMN functions.

Vitamin B(12) deficiency is a well-known cause of recurrent aphthous stomatitis (RAS). However, the mechanism by which this deficiency causes the stomatitis is not well understood. Imerslund-Grasbeck syndrome (IGS) causes vitamin B(12) deficiency and proteinuria due to a defect in the vitamin B(12) receptor. We sought to determine whether the RAS observed in IGS patients is associated with neutrophil dysfunction. We report 3 infants with vitamin B(12) deficiency due to IGS, who presented with borderline or normal hemoglobin concentrations, RAS, and a neutrophil function defect. All 3 patients were homozygous for a splice site mutation affecting exon 4 of the AMN gene. A direct correlation was observed between low serum vitamin B12 levels and defective neutrophil function (low chemotaxis and elevated superoxide production) in the patients. Vitamin B(12) therapy led to an immediate resolution of aphthous stomatitis and full correction of neutrophil function. We demonstrated that serum vitamin B(12) deficiency is associated with a neutrophil chemotactic defect and RAS in IGS patients. We suggest that the RAS observed in these patients is due to this defect.

Fc gammaRIIA expressed on neutrophils and monocytes has a fundamental role in combating bacterial infections. In the present study, the requirement of cytosolic phospholipase A2 (cPLA2) for induction of Fc gammaRIIA expression was studied in a model of cPLA2-deficient PLB-985 cells (PLB-D cells). Fc gammaRIIA was acquired only during differentiation of PLB but not of PLB-D cells induced by either 1,25-dihydroxyvitamin D3, retinoic acid, or interferon gamma. Addition of prostaglandin E2 (PGE2) to PLB-D cells undergoing differentiation restored the expression of Fc gammaRIIA protein, whereas addition of indomethacin to PLB cells during differentiation inhibited both the production of PGE2 and the expression of Fc gammaRIIA. Inhibition of PKA during PLB differentiation prevented Fc gammaRIIA expression, whereas dibutyryl cAMP (dbcAMP) induced its expression in both PLB and PLB-D cells. CREB phosphorylation and CREB-CRE interaction were detected only in differentiated PLB cells and not PLB-D cells and were inhibited by indomethacin. A reporter gene containing a Fc gammaRIIA gene promoter fragment with the CRE element was sufficient for CREB activation. Our results are the first to show that CREB activation is involved in up-regulation of Fc gammaRIIA expression in myeloid lineages. PGE2 formed via cPLA2 activates CREB through PKA and this process is dependent on development of PGE2 receptor 4.

In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [alpha-(32)P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5'-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [alpha-(32)P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of approximately 17- and approximately 14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotide-binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.