Fructose biphosphate aldolase: A new cassava allergen.

Background: Food allergy has considerably increased in recent years and this situation has been aggravated mainly by the consumption of more processed and complex foods, since minor or potentially allergenic foods are not required to be labeled. Manihot esculenta (cassava) is a widely consumed food in South America, Africa, and Asia and can be used in the production of flour and starch, as well as several other products. This root can cause allergic reactions with symptoms ranging from mild to severe. Methods: Thus, the aim of this study was the characterization of the immunogenic cassava proteins responsible for sensitizing patients allergic to it. Using a 2D-SDS-PAGE based proteomic approach, six proteins were identified, including Fructose Bisphosphate Aldolase (FBA). Recombinant FBA was produced in Expi293 cells and evaluated by immunoblotting with the serum of 10 individual study subjects. Results: Our results showed six cassava IgE-reactive proteins. From those, recombinant fructose bisphosphate aldolase (FBA) showed a positivity of 80% among tested sera, proving to be a highly sensitizing protein. Conclusion: The recombinant FBA molecule obtained in this study can be important for in vivo diagnostic assays, by producing more accurate results, and for desensitization protocols, in which the use of the isolated molecule produces more precise results by avoiding secondary sensitization. Trial registration: All patients signed a consent form approved by the internal ethics committee CAPPesq, Comissão de Ética para Análise de Projetos de Pesquisa do HC FMUSP (CAAE: 10420619.6.0000.0068).

The oldest unvaccinated Covid-19 survivors in South America.

BACKGROUND: Although older adults are at a high risk of severe or critical Covid-19, there are many cases of unvaccinated centenarians who had a silent infection or recovered from mild or moderate Covid-19. We studied three Brazilian supercentenarians, older than 110 years, who survived Covid-19 in 2020 before being vaccinated. RESULTS: Despite their advanced age, humoral immune response analysis showed that these individuals displayed robust levels of IgG and neutralizing antibodies (NAbs) against SARS-CoV-2. Enrichment of plasma proteins and metabolites related to innate immune response and host defense was also observed. None presented autoantibodies (auto-Abs) to type I interferon (IFN). Furthermore, these supercentenarians do not carry rare variants in genes underlying the known inborn errors of immunity, including particular inborn errors of type I IFN. CONCLUSION: These observations suggest that their Covid-19 resilience might be a combination of their genetic background and their innate and adaptive immunity.

Nitric oxide and interactions with reactive oxygen species in the development of melanoma, breast, and colon cancer: A redox signaling perspective.

Cancer development is closely related to chronic inflammation, which is associated with identifiable markers of tumor progression, such as uncontrolled cell proliferation, angiogenesis, genomic instability, chemotherapeutic resistance, and metastases. Redox processes mediated by reactive oxygen species (ROS) and nitric oxide (NO) within the inflammatory tumor microenvironment play an essential role in directly influencing intercellular and intracellular signaling. These reactive species originating in the cancer cell or its microenvironment, mediate the epithelial-mesenchymal transition (EMT) and the mesenchymal-epithelial transition (MET). However, intracellular interactions between NO and ROS must be controlled to prevent cell death. Melanoma, breast, and colon cancer cells have developed a mechanism to survive and adapt to oxidative and nitrosative stress. The mechanism involves a spatial-temporal fine adjustment of the intracellular concentrations of NO and ROS, thereby guaranteeing the successful development of cancer cells. Physiological concentrations of NO and supra physiological concentrations of ROS are prevalent in cancer cells at the primary site. The situation reverses in cancer cells undergoing the EMT prior to being released into the blood stream. Intracellular supra physiological concentrations of NO found in circulating cancer cells endow them with anoikis resistance. When the anoikis-resistant cancer cells arrive at a metastatic site they undergo the MET. Endogenous supra physiological concentrations of ROS and physiological NO concentrations are prevalent in these cells. Understanding tumor progression from the perspective of redox signaling permits the characterization of new markers and approaches to therapy. The synthesis and use of compounds with the capacity of modifying intracellular concentrations of NO and ROS may prove effective in disrupting a redox homeostasis operative in cancer cells.

Cell-surface HSP70 associates with thrombomodulin in endothelial cells.

Heat shock protein-70 (HSP70) is crucial for proteostasis and displays cell-protective effects. Meanwhile, enhanced levels of cell-surface (cs) and secreted HSP70 paradoxically associate with pathologic cardiovascular conditions. However, mechanisms regulating csHSP70 pool are unknown. We hypothesized that total and csHSP70 expressions are modulated by hemodynamic forces, major contributors to endothelial pathophysiology. We also investigated whether thrombomodulin, a crucial thromboresistance cell-surface protein, is a csHSP70 target. We used proteomic/western analysis, confocal microscopy, and cs-biotinylation to analyze the pattern and specific characteristics of intracellular and csHSP70. HSP70 interaction with thrombomodulin was investigated by confocal colocalization, en face immunofluorescence, proximity assay, and immunoprecipitation. Thrombomodulin activity was assessed by measured protein C activation two-step assay. Our results show that csHSP70 pool in endothelial cells (EC) exhibits a peculiar cluster-like pattern and undergoes enhanced expression by physiological arterial-level laminar shear stress. Conversely, total and csHSP70 expressions were diminished under low shear stress, a known proatherogenic hemodynamic pattern. Furthermore, total HSP70 levels were decreased in aortic arch (associated with proatherogenic turbulent flow) compared with thoracic aorta (associated with atheroprotective laminar flow). Importantly, csHSP70 co-localized with thrombomodulin in cultured EC and aorta endothelium; proximity ligation assays and immunoprecipitation confirmed their physical interaction in EC. Remarkably, immunoneutralization of csHSP70 enhanced thrombomodulin activity in EC and aorta ex vivo. Overall, proatherogenic hemodynamic forces promote reduced total HSP70 expression, which might implicate in disturbed proteostasis; meanwhile, the associated decrease in cs-HSP70 pool associates with thromboresistance signaling. Cell-surface HSP70 (csHSP70) expression regulation and csHSP70 targets in vascular cells are unknown. We showed that HSP70 levels are shear stress-modulated and decreased under proatherogenic conditions. Remarkably, csHSP70 binds thrombomodulin and inhibits its activity in endothelial cells. This mechanism can potentially explain some deleterious effects previously associated with high extracellular HSP70 levels, as csHSP70 potentially could restrict thromboresistance and support thrombosis/inflammation in stress situations.

Conserved Gene Microsynteny Unveils Functional Interaction Between Protein Disulfide Isomerase and Rho Guanine-Dissociation Inhibitor Families.

Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIα were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.

Conserved gene microsynteny unveils functional interaction between protein disulfide isomerase and rho guanine-dissociation inhibitor families

Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIa were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.

Preventive and therapeutic moderate aerobic exercise programs convert atherosclerotic plaques into a more stable phenotype.

UNLABELLED: The mechanisms by which exercise affects atherosclerotic plaque stability remain incompletely understood. We evaluated the effects of two training protocols on both atherosclerotic plaque structure and the signaling pathways involved in plaque rupture. METHODS: Male low-density lipoprotein (LDL) receptor knockout mice were fed a high-fat, high-cholesterol diet (HFD). One group was subjected to moderate exercise using a treadmill for 14weeks (preventive protocol). The other group started an exercise regimen after 16weeks of the HFD (therapeutic group). Atherosclerotic plaques within the aorta were evaluated for lipid and collagen contents, as well as for inflammatory markers. Plasma cholesterol and cytokine levels were also determined. RESULTS: The mice receiving a HFD developed hypercholesterolemia and atherosclerotic plaques within the aorta. The aortas from the animals in the preventive protocol exhibited smaller lipid cores and higher collagen content. These animals also exhibited lower CD40 expression within the plaques. The aortas of the mice in the therapeutic group exhibited higher collagen content, but no differences in either lipid core size or plaque size were noted. No differences in blood pressure, plasma cholesterol, cytokine levels, plaque size or metalloproteinase 9 expression were observed in the trained animals compared with the sedentary animals. CONCLUSION: Moderate aerobic exercise modified atherosclerotic plaque characteristics and converted the plaques into a more stable phenotype, increasing the collagen content in response to both exercise programs. Furthermore, moderate aerobic exercise reduced the animals' fat content and decreased the activity of the CD40-CD40L signaling pathway in the preventive group.

Fibrillin-1 mgΔ(lpn) Marfan syndrome mutation associates with preserved proteostasis and bypass of a protein disulfide isomerase-dependent quality checkpoint.

Fibrillin-1 mutations promote Marfan syndrome (MFS) via complex yet unclear pathways. The roles of endoplasmic reticulum (ER) and the major ER redox chaperone protein disulfide isomerase-A1 in the processing of normal and mutated fibrillin-1 and ensuing protein secretion and/or intracellular retention are unclear. Our results in mouse embryonic fibroblasts bearing the exon-skipping mgΔ(lox-P-neo) (mgΔ(lpn)) mutation, which associates in vivo with MFS and in vitro with disrupted microfibrils, indicate a preserved ER-dependent proteostasis or redox homeostasis. Rather, mutated fibrillin-1 is secreted normally through Golgi-dependent pathways and is not intracellularly retained. Similar results occurred for the C1039G point mutation. In parallel, we provide evidence that PDIA1 physically interacts with fibrillin-1 in the ER. Moreover, siRNA against PDIA1 augmented fibrillin-1 secretion rates in wild-type cells. However, fibrillin-1 with the mgΔ(lpn) mutation bypassed PDI checkpoint delay, while the C1039G mutation did not. This heretofore undisclosed PDIA1-mediated mechanism may be important to control the extracellular availability of function-competent fibrillin-1, an important determinant of disease phenotype. Moreover, our results may reveal a novel, holdase-like, PDI function associated with ER protein quality control.

Effect of Previous High Glutamine Infusion on Inflammatory Mediators and Mortality in an Acute Pancreatitis Model.

Parenteral glutamine supplementation in acute inflammatory conditions is controversial. We evaluated the inflammatory and survival responses after parenteral glutamine infusion in sodium taurocholate-induced acute pancreatitis (AP) model. Lewis rats received 1 g/kg parenteral glutamine (n = 42), saline (n = 44), or no treatment (n = 45) for 48 h before AP induction. Blood, lung, and liver samples were collected 2, 12, and 24 h after AP to measure serum cytokines levels and tissue heat shock protein (HSP) expression. From each group, 20 animals were not sacrificed after AP for a 7-day mortality study. Serum cytokine levels did not differ among groups at any time point, but the intragroup analysis over time showed higher interferon-γ only in the nontreatment and saline groups at 2 h (versus 12 and 24 h; both p ≤ 0.05). The glutamine group exhibited greater lung and liver HSP90 expression than did the nontreatment group at 2 and 12 h, respectively; greater liver HSP90 and HSP70 expression than did the saline group at 12 h; and smaller lung HSP70 and liver HSP90 expression than did the nontreatment group at 24 h (all p ≤ 0.019). The 7-day mortality rate did not differ among groups. In experimental AP, pretreatment with parenteral glutamine was safe and improved early inflammatory mediator profiles without affecting mortality.

Inducible nitric oxide synthase inhibition increases MMP-2 activity leading to imbalance between extracellular matrix deposition and degradation after polypropylene mesh implant.

Prosthetic mesh implants are commonly used to correct abdominal wall defects. However, success of the procedure is conditioned by an adequate inflammatory response to the device. We hypothesized that nitric oxide produced by nitric oxide synthase 2 (NOS2) and MMP-2 and -9 participate in response induced by mesh implants in the abdominal wall and, consequently, affect the outcome of the surgical procedure. In the first step, temporal inflammatory markers profile was evaluated. Polypropylene meshes were implanted in the peritoneal side of the abdominal wall of C57Black mice. After 2, 4, 7, 15, and 30 days, tissues around the mesh implant were collected and inflammatory markers were analyzed. In the second step, NOS2 activity was inhibited with nitro-L-arginine methyl ester (L-NAME). Samples were collected after 15 days (when inflammation was reduced), and the inflammatory and tissue remodeling markers were investigated. Polypropylene mesh implant induced a pro-inflammatory environment mediated by intense MMP-2 and -9 activities, NO release, and interleukin-1ß production peaking in 7 days and gradually decreasing after 15 days. NOS2 inhibition increased MMP-2 activity and resulted in a higher visceral adhesion incidence at the mesh implantation site when compared with non-treated animals that underwent the same procedure. We conclude that NOS2-derived NO is crucial for adequate response to polypropylene mesh implant integration in the peritoneum. NO deficiency results in an imbalance between extracellular matrix deposition/degradation contributing to visceral adhesions incidence.

Low-level laser therapy (808 nm) reduces inflammatory response and oxidative stress in rat tibialis anterior muscle after cryolesion.

BACKGROUND AND OBJECTIVE: Muscle regeneration is a complex phenomenon, involving coordinated activation of several cellular responses. During this process, oxidative stress and consequent tissue damage occur with a severity that may depend on the intensity and duration of the inflammatory response. Among the therapeutic approaches to attenuate inflammation and increase tissue repair, low-level laser therapy (LLLT) may be a safe and effective clinical procedure. The aim of this study was to evaluate the effects of LLLT on oxidative/nitrative stress and inflammatory mediators produced during a cryolesion of the tibialis anterior (TA) muscle in rats. MATERIAL AND METHODS: Sixty Wistar rats were randomly divided into three groups (n = 20): control (BC), injured TA muscle without LLLT (IC), injured TA muscle submitted to LLLT (IRI). The injured region was irradiated daily for 4 consecutive days, starting immediately after the lesion using a AlGaAs laser (continuous wave, 808 nm, tip area of 0.00785 cm(2) , power 30 mW, application time 47 seconds, fluence 180 J/cm(2) ; 3.8 mW/cm(2) ; and total energy 1.4 J). The animals were sacrificed on the fourth day after injury. RESULTS: LLLT reduced oxidative and nitrative stress in injured muscle, decreased lipid peroxidation, nitrotyrosine formation and NO production, probably due to reduction in iNOS protein expression. Moreover, LLLT increased SOD gene expression, and decreased the inflammatory response as measured by gene expression of NF-kß and COX-2 and by TNF-α and IL-1ß concentration. CONCLUSION: These results suggest that LLLT could be an effective therapeutic approach to modulate oxidative and nitrative stress and to reduce inflammation in injured muscle.

Nitric oxide modulates metalloproteinase-2, collagen deposition and adhesion rate after polypropylene mesh implantation in the intra-abdominal wall.

Prosthetic meshes are commonly used to correct abdominal wall defects. However, the inflammatory reaction induced by these devices in the peritoneum is not completely understood. We hypothesized that nitric oxide (NO), produced by nitric oxide synthase 2 (NOS2) may modulate the response induced by mesh implants in the abdominal wall and, consequently, affect the outcome of the surgical procedure. Polypropylene meshes were implanted in the peritoneal side of the abdominal wall in wild-type and NOS2-deficient (NOS2(-/-)) mice. After 15 days tissues around the mesh implant were collected, and inflammatory markers (the cytokine interleukin 1ß (IL-1ß) and NO) and tissue remodeling (collagen and metalloproteinases (MMP) 2 and 9) were analyzed. The lack of NOS2-derived NO induced a higher incidence of visceral adhesions at the mesh implantation site compared with wild-type mice that underwent the same procedure (P<0.05). Additionally, higher levels of IL-1ß were present in the mesh-implanted NOS2(-/-) animals compared with control and wild-type mice. Mesh implantation induced collagen I and III deposition, but in smaller amounts in NOS2(-/-) mice. MMP-9 activity after the surgical procedure was similarly increased in both groups. Conversely, MMP-2 activity was unchanged in mesh-implanted wild-type mice, but was significantly increased in NOS2(-/-) mice (P<0.01), due to decreased S-nitrosylation of the enzyme in these animals. We conclude that NOS2-derived NO is crucial for an adequate response to and integration of polypropylene mesh implants in the peritoneum. NO deficiency results in a prolonged inflammatory reaction to the mesh implant, and reduced collagen deposition may contribute to an increased incidence of visceral adhesions.

Hypertonic saline reduces metalloproteinase expression in liver during pancreatitis.

1. We recently demonstrated that hypertonic saline reduces inflammation and mortality in acute pancreatitis. The present study investigated the effects of hypertonic saline in metalloproteinase (MMP) regulation and pancreatitis-associated hepatic injury. 2. Wistar rats were divided into four groups: (i) control, not subjected to insult or treatment; (ii) no treatment (NT), induction of pancreatitis (retrograde infusion of 2.5% sodium taurocholate (1.0 mL/kg)), but no further treatment; (iii) normal saline (NS), induction of pancreatitis and treatment with normal saline (0.9% NaCl, 34 mL/kg, i.v. bolus, 1 h after the induction of pancreatitis); and (iv) hypertonic saline (HS), induction of pancreatitis and treatment with hypertonic saline (7.5% NaCl, 4 mL/kg administered over a period of 5 min, 1 h after the induction of pancreatitis). In all four groups, 4, 12 and 24 h after the induction of pancreatitis, liver tissue samples were assayed to determine levels of MMP-2, MMP-9, 47 kDa heat shock protein (HSP47) and collagen (Type I and III). 3. Compared with the control group, MMP-9 expression and activity was increased twofold in the NS and NT groups 4 and 12 h after the induction of pancreatitis, but remained at basal levels in the HS group. In contrast, MMP-2 expression was increased twofold 12 h after the induction of pancreatitis only in the NS group, whereas the expression of HSP47 was increased 4 h after the induction of pancreatitis in the NS and NT groups. Greater extracellular matrix remodelling occurred in the NS and NT groups compared with the HS group, probably as a result of the hepatic wound-healing response to repeated injury. However, the collagen content in hepatic tissue remained at basal levels in the HS group. 4. In conclusion, the results of the present study indicate that hypertonic saline is hepatoprotective and reduces hepatic remodelling, maintaining the integrity of the hepatic extracellular matrix during pancreatitis. Hypertonic saline-mediated regulation of MMP expression may have clinical relevance in pancreatitis-associated liver injury.

Human cholesteryl ester transfer protein expression enhances the mouse survival rate in an experimental systemic inflammation model: a novel role for CETP.

Mice expressing human cholesteryl ester transfer protein (huCETP) are more resistant to Escherichia coli bacterial wall LPS because death rates 5 days after intraperitoneal inoculation of LPS were higher in wild-type than in huCETP+/+ mice, whereas all huCETP+/+ mice remained alive. After LPS inoculation, plasma concentrations of TNF-alpha and IL-6 increased less in huCETP+/+ than in wild-type mice. LPS in vitro elicited lower TNF-alpha production by CETP expressing than by wild-type macrophages. In addition, TNF-alpha production by RAW 264.7 murine macrophages increased on incubation with LPS but decreased in a dose-dependent manner when human CETP was added to the medium. Human CETP in vitro enhanced the LPS binding to plasma high-density lipoprotein/low-density lipoprotein. The liver uptake of intravenous infused 14C-LPS from Salmonella typhimurium was greater in huCETP+/+ than in wild-type mice. Present data indicate for the first time that CETP is an endogenous component involved in the first line of defense against an exacerbated production of proinflammatory mediators.