Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation

Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 mu g/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by similar to 40%), whereas PDIred diminished (by similar to 60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by similar to 70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47(phox) in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47(phox). PDI-p47(phox) association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47(phox) in cytosol. Incubation of purified PDI (> 80% reduced) and p47(phox) in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re) cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer. J. Leukoc. Biol. 90: 799-810; 2011.

The p.A2215D Thyroglobulin Gene Mutation Leads to Deficient Synthesis and Secretion of the Mutated Protein and Congenital Hypothyroidism with Wide Phenotype Variation

Context: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. Objectives: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p. A2215D mutation. Design: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. Results: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p. A2215D, p. R277X, and g. IVS30 + 1G > T), and two novel mutations (p. Q2142X and g. IVS46-1G > A). Two known (g. IVS30 + 1G/p. A2215D and p. A2215D/p. R277X) and one novel (p. R277X/g. IVS46-1G > A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. Conclusion: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p. A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone. (J Clin Endocrinol Metab 94: 2938-2944, 2009)

Heterozygosity for the S180L Variant of MAL/TIRAP, a Gene Expressing an Adaptor Protein in the Toll-Like Receptor Pathway, Is Associated with Lower Risk of Developing Chronic Chagas Cardiomyopathy

Background. Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Among T. cruzi-infected individuals, only a subgroup develops severe chronic Chagas cardiomyopathy (CCC); the majority remain asymptomatic. T. cruzi displays numerous ligands for the Toll-like receptors (TLRs), which are an important component of innate immunity that lead to the transcription of proinflammatory cytokines by nuclear factor-kappa B. Because proinflammatory cytokines play an important role in CCC, we hypothesized that single-nucleotide polymorphisms (SNPs) in the genes that encode proteins in the TLR pathway could explain differential susceptibility to CCC among T. cruzi-infected individuals. Methods. For 169 patients with CCC and 76 T. cruzi-infected, asymptomatic individuals, we analyzed SNPs by use of polymerase chain reaction-restriction fragment length polymorphism analysis for the genes TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP, which encodes an adaptor protein. Results. Heterozygous carriers of the MAL/TIRAP variant S180L were more prevalent in the asymptomatic group (24 [32%] of 76 subjects) than in the CCC group (21 [12%] of 169) (chi(2) = 12.6; P = .0004 [adjusted P (P(c)) = .0084]; odds ratio [OR], 0.31 [95% confidence interval {CI}, 0.16-0.60]). Subgroup analysis showed a stronger association when asymptomatic patients were compared with patients who had severe CCC (i.e., patients with left-ventricular ejection fraction <= 40%) (chi(2) = 11.3; P = .0008 [P(c) = .017]; OR, 0.22 [95% CI, 0.09-0.56]) than when asymptomatic patients were compared with patients who had mild CCC (i.e., patients with left-ventricular ejection fraction >40%) (chi(2) = 7.7; P = .005 [P(c) = .11]; OR, 0.33 [95% CI, 0.15-0.73]). Conclusion. T. cruzi-infected individuals who are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may have a lower risk of developing CCC.

p63 Protein expression in high risk diffuse large B-cell lymphoma

Background: p63 gene is a p53 homologue that encodes proteins with transactivation, DNA-binding and tetramerisation domains. The isoforms TAp63 and TAp73 transactivate p53 target genes and induce apoptosis, whereas the isoforms Delta Np63 and Delta Np73 lack transactivation and might have dominant-negative effects in p53 family members. p63 is expressed in germinal centre lymphocytes and can be related to the development of the lymphoma, but the prognostic significance of its expression in the survival of patients with diffuse large B-cell lymphoma (DLBCL) remains unclear. Aims: To determine whether quantitative immunohistochemical (IHC) analysis of p63 protein expression correlates with CD10 antigen, Bcl-6 antigen and IRF4 antigen expression and to determine whether p63 is a surrogate predictor of overall survival in high-intermediate and high risk DLBCL populations. Methods: CD10, Bcl-6 and IRF4 expression were retrospectively evaluated by IHC in 73 samples of high intermediate and high risk DLBCL and were used to divide the lymphomas into subgroups of germinal centre B-celllike (GCB) and activate B-cell-like (ABC) DLBCL. Similarly, p63 expression was evaluated by IHC and the results were compared with subgroups of DLBCL origin and with the survival rates for these patients. Results: p63 was expressed in more than 50% of malignant cells in 11 patients and did not show correlation with subgroups of GCB-like DLBCL or ABC-like DLBCL, but p63(+) patients had better disease-free survival (DFS) than those who were negative (p = 0.01). Conclusions: p63(+) high-intermediate and high risk DLBCL patients have a better DFS than negative cases.

SINGLE MEASUREMENTS OF C-REACTIVE PROTEIN AND DISEASE ACTIVITY SCORES ARE NOT PREDICTORS OF CAROTID ATHEROSCLEROSIS IN RHEUMATOID ARTHRITIS PATIENTS

Background: Although inflammation has a defined role in the pathogenesis of atherosclerosis, the link between rheumatoid arthritis (RA) parameters of disease activity and atherosclerotic findings are not defined. Objective: To investigate the association between subclinical carotid atherosclerosis and clinical/laboratorial parameters of RA systemic inflammatory activity. Methods: Seventy-one RA patients were consecutively selected and compared to 53 healthy controls. Smoking, diabetes and hypertension were excluded, as well as the use of statins or fibrates. B-mode carotid ultrasound was performed in all subjects. CRP, ESR and fibrinogen were determined in both groups. Clinical assessment of RA activity included DAS 28 and SDAI. Correlation between plaques and intima-media thickness (IMT) of common carotid arteries and inflammatory parameters was evaluated. Results: Carotid plaques were more prevalent in RA patients than in controls (14.1% vs. 1.9 %, p=0.02) and marginally increased IMT was observed (0.72 +/- 0.17 vs. 0.67 +/- 0.15mm, p=0.07). RA patients with plaques had older age (p=0.001) and increased IMT (p<0.001), but low SDAI (p=0.025) compared to those without plaques. RA patients with plaques had also longer disease duration, although this difference did not reach statistical significance (p=0.06). No significant correlations were found between IMT and ESR (p=0.80), CRP (p=0.75), fibrinogen (p=0.94), HAQ (p=0.89) and DAS 28 (p=0.13). Conclusions: Carotid atherosclerosis is more frequently detected in RA but its prevalence was not correlated with isolated inflammatory markers measurement or noncumulative activity scores. These findings reinforce the need to evaluate subclinical atherosclerosis in RA patients, and to find predictors of atherosclerotic lesions.

Aerobic Exercise Improves Reverse Cholesterol Transport in Cholesteryl Ester Transfer Protein Transgenic Mice

We analyzed the effect of a 6-week aerobic exercise training program on the in vivo macrophage reverse cholesterol transport (RCT) in human cholesteryl ester transfer protein (CETP) transgenic (CETP-tg) mice. Male CETP-tg mice were randomly assigned to a sedentary group or a carefully supervised exercise training group (treadmill 15 m/min, 30 min sessions, five sessions per week). The levels of plasma lipids were determined by enzymatic methods, and the lipoprotein profile was determined by fast protein liquid chromatography (FPLC). CETP activity was determined by measuring the transfer rate of (14)C-cholesterol from HDL to apo-B containing lipoproteins, using plasma from CETP-tg mice as a source of CETP. The reverse cholesterol transport was determined in vivo by measuring the [(3)H]-cholesterol recovery in plasma and feces (24 and 48 h) and in the liver (48 h) following a peritoneal injection of [(3)H]-cholesterol labeled J774-macrophages into both sedentary and exercise trained mice. The protein levels of liver receptors were determined by immunoblot, and the mRNA levels for liver enzymes were measured using RT-PCR. Exercise training did not significantly affect the levels of plasma lipids or CETP activity. The HDL fraction assessed by FPLC was higher in exercise-trained compared to sedentary mice. In comparison to the sedentary group, a greater recovery of [(3)H]-cholesterol from the injected macrophages was found in the plasma, liver and feces of exercise-trained animals. The latter occurred even with a reduction in the liver CYP7A1 mRNA level in exercised trained animals. Exercise training increased the liver LDL receptor and ABCA-1 protein levels, although the SR-BI protein content was unchanged. The RCT benefit in CETP-tg mice elicited by exercise training helps to elucidate the role of exercise in the prevention of atherosclerosis in humans.

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 LIPS because death rates 5 days after intraperitoneal inoculation of LIPS were higher in wild-type than in huCETP(+/-) mice, whereas all huCETP(+/+) mice remained alive. After LIPS 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 LIPS binding to plasma high-density lipoprotein/low-density lipoprotein. The liver uptake of intravenous infused C-14-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.

Cutaneous vasculitis in systemic lupus erythematosus: association with anti-ribosomal P protein antibody and Raynaud phenomenon

Ninety-one consecutive systemic lupus erythematosus (SLE) patients (American College of Rheumatology criteria) with a history of cutaneous vasculitis were compared to 163 SLE controls without this clinical manifestation from July to December 2007 in order to determine the possible clinical and serological association of this manifestation. Data were obtained in an ongoing electronic database protocol and autoantibodies to anti-double-stranded DNA, anti-Sm, anti-RNP, anti-Ro/SS-A, anti-La/SS-B, and anticardiolipin and ribosomal P protein antibody (anti-P) were detected by standard techniques. Exclusion criteria were the presence of anti-phospholipid syndrome or antibodies, Sjogren syndrome, and a history of thrombosis. The mean age (38.5 +/- 11.5 vs. 37.8 +/- 11.6 years, p = 0.635), disease duration (12.5 +/- 7.8 vs. 11.8 +/- 7.9 years, p = 0.501), and frequency of white race (71.4% vs. 70.5%, p = 0.872) and female sex (96.8% vs. 93.7%, p = 0.272) were comparable in both groups. The vasculitis group had a higher frequency of malar rash (97.9% vs. 87.4%, p = 0.004), photosensitivity (91.4% vs. 81.6%, p = 0.030), and Raynaud phenomenon (RP; 27.7% vs. 7.5%, p < 0.001), whereas all other clinical manifestation including renal and central nervous system involvements were similar to the control group. Laboratorial data revealed that only anti-P (35.1% vs. 12.1%, p < 0.001) was more frequent in patients with vasculitis. In a multivariate logistic regression model, cutaneous vasculitis was associated to the presence of RP (OR = 3.70; 95% confidence interval [CI] = 1.73-8.00) and anti-P (OR = 3.42; 95% CI = 1.76-6.66). In summary, SLE cutaneous vasculitis characterizes a subgroup of patients with more RP and anti-P antibodies but not accompanied by a higher frequency of renal and central nervous system involvements.

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.

Most of the patients presenting myocardial infarction would not be eligible for intensive lipid-lowering based on clinical algorithms or plasma C-reactive protein

Objective: The study we assessed how often patients who are manifesting a myocardial infarction (MI) would not be considered candidates for intensive lipid-lowering therapy based on the current guidelines. Methods: In 355 consecutive patients manifesting ST elevation MI (STEMI), admission plasma C-reactive protein (CRP) was measured and Framingham risk score (FRS), PROCAM risk score, Reynolds risk score, ASSIGN risk score, QRISK, and SCORE algorithms were applied. Cardiac computed tomography and carotid ultrasound were performed to assess the coronary artery calcium score (CAC), carotid intima-media thickness (cIMT) and the presence of carotid plaques. Results: Less than 50% of STEMI patients would be identified as having high risk before the event by any of these algorithms. With the exception of FRS (9%), all other algorithms would assign low risk to about half of the enrolled patients. Plasma CRP was <1.0 mg/L in 70% and >2 mg/L in 14% of the patients. The average cIMT was 0.8 +/- 0.2 mm and only in 24% of patients was >= 1.0 mm. Carotid plaques were found in 74% of patients. CAC > 100 was found in 66% of patients. Adding CAC >100 plus the presence of carotid plaque, a high-risk condition would be identified in 100% of the patients using any of the above mentioned algorithms. Conclusion: More than half of patients manifesting STEMI would not be considered as candidates for intensive preventive therapy by the current clinical algorithms. The addition of anatomical parameters such as CAC and the presence of carotid plaques can substantially reduce the CVD risk underestimation. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

The role of p38 mitogen-activated protein kinase in serum-induced leukemia inhibitory factor secretion by bone marrow stromal cells from pediatric myelodysplastic syndromes

Stromal cells from pediatric myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) associated with MDS(MDS-AML) present high expression of leukemia inhibitor factor (LIF). We demonstrated using mitogen-activated protein kinase ( MAPK) inhibitors that in stromal cells from pediatric MDS and MDS-AML, p38MAPK was critical in serum-induced secretion of LIF. The serum induction of phosphorylated p38MAPK form was observed only in stromal cells from healthy children, whereas in MDS and MDS-AML basal levels were maintained suggesting constitutive p38MAPK activation. Our study suggested the possible importance in pediatric MDS of p38MAPK signaling pathway which may be a future therapeutic target. (C) 2009 Elsevier Ltd. All rights reserved.

Prion protein ablation increases cellular aggregation and embolization contributing to mechanisms of metastasis

Cellular Prion Protein (PrP(C)) is a cell surface protein highly expressed in the nervous system, and to a lesser extent in other tissues. PrP(C) binds to the extracellular matrix laminin and vitronectin, to mediate cell adhesion and differentiation. Herein, we investigate how PrP(C) expression modulates the aggressiveness of transformed cells. Mesenchymal embryonic cells (MEC) from wildtype (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were immortalized and transformed by co-expression of ras and myc. These cells presented similar growth rates and tumor formation in vivo. When injected in the tail vein, PrnP(0/0)raS/myc cells exhibited increased lung colonization compared with Prnp(+/+)ras/myc cells. Additionally, Prnp(0/0)ras/myc cells form more aggregates with blood components than Prnp(+/+)ras/myc cells, facilitating the arrest of Prnp(0/0)ras/myc cells in the lung vasculature. Integrin alpha(v)beta(3) is more expressed and activated in MEC and in transformed Prnp(0/0) cells than in the respective Prnp(+/+) cells. The blocking of integrin alpha(v)beta(3) by RGD peptide reduces lung colonization in transformed Prnp(0/0) cells to similar levels of those presented by transformed Prnp(+/+) cells. Our data indicate that PrP(C) negatively modulates the expression and activation of integrin alpha(v)beta(3) resulting in a more aggressive phenotype. These results indicate that PrP(C) may have main implications in modulating metastasis formation. (C) 2009 UICC

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family Hp1(Hs alpha), Hp1(Hs beta) and Hp1(Hs gamma) which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1(Hs alpha), HP1(Hs beta) and HP1(Hs gamma) was determined by real-time RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow. (Int J Biol Markers 2008; 23: 219-24)

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pl 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

Expression of p16 protein in acral lentiginous melanoma

Background Acral lentiginous melanoma (ALM) is a clinicopathologic subtype of cutaneous malignant melanoma. ALM is the most common type of melanoma amongst Asians, Africans, and patients with mixed ancestry. In Brazil, ALM comprises 10% of cutaneous melanoma. ALM develops on palmar, plantar, and subungual skin, and its biology is different from that of other cutaneous melanomas, where sunlight is the major known environmental determinant. Alterations and inactivation of the p16INK4 gene that encodes a specific inhibitor of cyclin-dependent kinase have been related to melanoma genesis and progression. Few studies, however, have addressed p16 expression in ALM. Methods In order to verify and compare p16 protein expression, 32 paraffin-embedded ALM specimens were subjected to a immunohistochemical technique using a monoclonal anti-p16 antibody. The tumors were classified according to thickness (up to 1.0 mm and thicker than 1.0 mm) and the presence of ulceration. Results Twenty-five (78%) ALMs displayed positive p16 protein expression: 21 of the 25 (84%) with a thickness of more than 1.0 mm, and four of the seven (57%) with a thickness of 1.0 mm or less. Thirteen of the 17 (76%) nonulcerated lesions and 12 of the 15 (80%) ulcerated lesions displayed positive p16 protein expression. Conclusion The data obtained suggest that p16 protein expression per se may not represent a marker of retinoblastoma protein (pRb) pathway disturbance in ALM tumorigenesis.