Identification of a C-reactive protein like homologue from black rockfish (Sebastes schlegelii) evidencing its potent anti-microbial properties at molecular level.

Pentraxins are a family of evolutionary conserved proteins that contains two main members, namely c-reactive proteins (CRPs) and serum amyloid P (SAP), which are involved in acute phase responses in animals. In this study, a cDNA sequence of a CRP-like molecule was identified from a previously constructed black rockfish cDNA database (RfCRP) and subsequently characterized at its molecular level. The complete coding region of RfCRP is 672 bp in length, and encodes a protein containing 224 amino acids with a predicted molecular mass of 25.19 kDa. Analysis of its derived amino acid sequence enabled typical features of pentraxin family members to be identified, including the pentraxin family signature in RfCRP. Results from multiple sequence alignment suggest the conservation of functionally important residues in RfCRP. According to the phylogenetic reconstruction that was generated using different pentraxin counterparts from different taxa, RfCRP shares a common vertebrate ancestral origin and most closely clusters with marine teleostan CRP. Furthermore, recombinant RfCRP demonstrated Ca(2+)-dependent agglutination activity against Escherichia coli, which could be completely inhibited in the presence of carbohydrate based ligands. Moreover, recombinant RfCRP also exhibited anti-bacterial activity against both E. coli and Streptococcus iniae. In addition, qPCR analysis indicated that RfCRP is ubiquitously expressed in physiologically important tissues, with pronounced expression in the spleen. After healthy fish were treated with polysaccharides or live S. iniae, basal expression of RfCRP was significantly upregulated in spleen and head kidney tissues. Collectively, our results suggest that RfCRP may be important in host anti-bacterial defense, and it might potentially participate in the acute phase of infection.

Importância da proteína C-reativa no diagnóstico e no prognóstico intra-hospitalar em pacientes com dor torácica na sala de emergência / C-reactive protein diagnostic and prognostic value in patients presenting at the emergency room with chest pain / C-reactive protein diagnostic and prognostic value in patients presenting at the emergency room with chest pain

OBJETIVO: Testar os valores diagnóstico e prognóstico imediatos da proteína C-reativa (PCR) nos pacientes admitidos na sala de emergência (SE) com dor torácica (DT) e sem elevação do segmento ST no eletrocardiograma (ECG). MÉTODOS: De janeiro de 2002 a dezembro de 2003, 980 pacientes consecutivos foram atendidos com DT suspeita de síndrome coronariana aguda na SE (idade = 64,9 ± 14,3 anos, homens = 55 por cento, diabéticos = 18 por cento, ECG normal = 84 por cento). Dosou-se a PCR na admissão, a creatinofosfoquinase MB fração massa (CKMB) e a troponina I seriadas, além de se registrar ECG seriados. As medidas da PCR foram padronizadas (PCR-p) pelo valor do limite superior da normalidade (LSN) do teste utilizado (3,0 mg/L para a PCR de alta sensibilidade-PCR-AS e 0,1 mg/dl para PCR titulada-PCR-t). RESULTADOS: Foi diagnosticado infarto agudo do miocárdio (IAM) em 125 pacientes, e seus valores para a PCR-p foram 1,31 ± 2,90 (mediana = 0,47) versus 0,79 ± 1,39 (0,30) nos sem IAM (p = 0,031). A PCR-p > 1,0 apresentou sensibilidade de 30 por cento, especificidade de 80,4 por cento, valores preditivos positivo e negativo de 6,1 por cento e de 96,7 por cento, para o diagnóstico de IAM. Houve quarenta eventos cardíacos intra-hospitalares (óbitos = dezesseis, revascularizações de urgência = 22, IAM = dois). No 1° quartil da PCR-p (< 0,10) registraram-se três eventos, enquanto no 4° quartil (> 0,93) ocorreram quinze eventos (p = 0,003). Na regressão logística foram preditores independentes para eventos cardíacos a insuficiência ventricular esquerda, o sexo masculino e a PCR-p > 0,32, com razão de chances de 7,6, 2,8 e 2,2, respectivamente. CONCLUSÃO: Nos pacientes atendidos com DT na SE, a PCR-p: 1) Não foi um bom marcador de IAM, apesar de um valor normal praticamente afastar esse diagnóstico; 2) Um valor superior a um terço do seu limite superior da normalidade (LSN) (>1 mg/L da PCR-AS ou >0,33 mg/dl da PCR-t) foi preditor de eventos cardíacos adversos intra-hospitalares.

OBJECTIVE: To test immediate diagnostic and prognostic values of C-reactive protein (CRP) in patients admitted to the emergency room (ER) with chest pain (CP) without ST-segment elevation on the electrocardiogram (ECG). METHODS: From January 2002 to December 2003, 980 patients were consecutively seen in the ER with CP suggestive of acute coronary syndrome (ACS) (age = 64.9 ± 14.3, men = 55 percent, diabetic = 18 percent, normal ECG = 84 percent). Serial CRP, creatine kinase MB mass (CKMB-mass) and troponin I determinations were performed on admission, in addition to serial ECG. CRP measurements were standardized (s-CRP) by the upper limit of normal (ULN) of the test used (3.0 mg/L for high-sensitivity C-reactive protein [hs-CRP] and 0.1 mg/dL for titrated CRP [t-CRP]). RESULTS: One hundred and twenty-five patients were diagnosed with acute myocardial infarction (AMI), and their s-CRP values were 1.31 ± 2.90 (median = 0.47) compared to 0.79 ± 1.39 (0.30) in no-AMI patients (p = 0.031). The s-CRP > 1.0 showed 30 percent sensitivity and 80 percent specificity, plus negative and positive predictive values of 6.1 percent and 96.7 percent, respectively, for AMI diagnosis. There were forty in-hospital cardiac events (16 deaths, 22 urgent revascularizations, and 2 acute myocardial infarction). In the first quartile of the s-CRP (< 0.10), three events were recorded, while in the fourth quartile (> 0.93) 15 events (p = 0.003) occurred. In the logistic regression model, masculine gender and s-CRP > 0.32 (odds ratio 7.6, 2.8 and 2.2, respectively) were independent predictors of cardiac events and left ventricular failure. CONCLUSION: In patients with chest pain presenting at the emergency room, s-CRP was not a good marker of AMI, although this diagnosis is virtually excluded by a normal value; in addition, values one-third above the upper limit of normal (>1 mg/L for hs-CRP or >0.33 mg/dL for t-CRP) were predictive of in-hospital adverse cardiac events.

Análise de marcadores de estabilização da placa aterosclerótica após evento coronariano agudo / Inflammatory markers of atherosclerotic plaque stabilization after acute coronary event - temporal trends

OBJETIVO: Avaliar o tempo para a estabilização da placa aterosclerótica nas síndromes coronarianas agudas (SCA) utilizando-se marcadores inflamatórios. MÉTODOS: Estudo prospectivo, quarenta pacientes com SCA sem supradesnivelamento de ST versus quarenta indivíduos sem doença coronariana. Proteína C-reativa (PCR), fibrinogênio, fator VIIIc, interleucina-6 e TNF (fator de necrose tumoral)-alfa foram coletados na internação, na alta hospitalar e após três e seis meses. RESULTADOS: Comparada ao controle, a PCR foi significativamente maior na internação e na alta, mas não após três e seis meses. Os níveis de fibrinogênio não apresentaram variações, exceto aos seis meses, quando foi significativamente menor que o controle. O fator VIIIc não diferiu do controle na internação, mas foi significativamente maior na alta, e sem diferenças aos três e seis meses. A IL-6 foi significativamente maior que o controle em todos os períodos. Entretanto, houve queda significativa dos seus níveis entre a alta e três meses. O TNF-alfa não foi significativamente diferente do controle em nenhum momento. Somente a IL-6 se correlacionou significativa e independentemente com eventos cardiovasculares futuros. CONCLUSÃO: Quanto a PCR e fator VIIIc, sugere-se estabilização da placa em até três meses; a análise da IL-6 sugere estabilização a partir do terceiro mês, apesar de permanecer elevada em relação ao controle em até seis meses. Apenas a IL-6 mostrou valor prognóstico de eventos futuros em um ano.

Leptin is not associated independently with hypertension in Japanese-Brazilian women

We evaluated the relationship of leptin with hypertension adjusted for body mass index (BMI) and/or waist circumference in a population of Japanese-Brazilian women aged > or = 30 years with centrally distributed adiposity. After excluding diabetic subjects, the study subjects - who participated in a population-based study on the prevalence of metabolic syndrome - showed prevalence rates of obesity (BMI > or = 25 kg/m²) and central adiposity (waist > or = 80 cm) of 32.0 and 37.8 percent, respectively. The hypertensive group (N = 162) was older, had higher BMI (24.9 ± 4.2 vs 23.3 ± 3.4 kg/m², P < 0.001), waist circumference (81.1 ± 10.1 vs 76.3 ± 8.2 cm, P < 0.001) and insulin levels (8.0 ± 6.2 vs 7.1 ± 4.9 æU/mL, P < 0.05) than the normotensive group (N = 322) and showed an unfavorable metabolic profile (higher 2-h plasma glucose, C-reactive protein and non-HDL cholesterol levels). Leptin did not differ between groups (8.2 ± 6.8 vs 7.2 ± 6.6 ng/mL, P = 0.09, for hypertensive vs normotensive, respectively) and its levels correlated significantly with anthropometric variables but not with blood pressure. Logistic regression analysis indicated that age and waist were independently associated with hypertension but not with homeostasis model assessment of insulin resistance or leptin levels. The lack of an independent association of hypertension with metabolic parameters (2-h glucose, C-reactive protein and non-HDL cholesterol) after adjustment for central adiposity suggested that visceral fat deposition may be the common mediator of the disturbances of the metabolic syndrome. Our data indicate that age and waist are major determinants of hypertension in this population of centrally obese (waist > or = 80 cm) Japanese-Brazilian women, but do not support a role for leptin in the elevation of blood pressure.

Effect of hepatitis C serology on C-reactive protein in a cohort of Brazilian hemodialysis patients

Hepatitis C (HCV) is not an uncommon feature in hemodialysis (HD) patients and may be a cause of systemic inflammation. Plasma cytokine interleukin-6 (IL-6) is mainly produced by circulating and peripheral cells and induces the hepatic synthesis of C-reactive protein (CRP), which is the main acute phase reactant. The aim of this study was to investigate the influence of HCV on two markers of systemic inflammation, serum CRP and IL-6, in HD patients. The study included 118 HD patients (47 percent males, age 47 ± 13 years, 9 percent diabetics) who had been treated by standard HD for at least 6 months. The patients were divided into two groups depending on the presence (HCV+) or absence (HCV-) of serum antibodies against HCV. Serum albumin (S-Alb), plasma high sensitivity CRP (hsCRP), IL-6, and alanine aminotransferase (ALT) were measured and the values were compared with those for 22 healthy controls. Median hsCRP and IL-6 values and hsCRP/IL-6 ratio were: 3.5 vs 2.1 mg/l, P < 0.05; 4.3 vs 0.9 pg/ml, P < 0.0001, and 0.8 vs 2.7, P < 0.0001, for patients and controls, respectively. Age, gender, S-Alb, IL-6 and hsCRP did not differ between the HCV+ and HCV- patients. However, HCV+ patients had higher ALT (29 ± 21 vs 21 ± 25 IU/l) and had been on HD for a longer time (6.1 ± 3.0 vs 4.0 ± 2.0 years, P < 0.0001). Moreover, HCV+ patients had a significantly lower median hsCRP/IL-6 ratio (0.7 vs 0.9, P < 0.05) compared to the HCV- group. The lower hsCRP/IL-6 ratio in HCV+ patients than in HCV- patients suggests that hsCRP may be a less useful marker of inflammation in HCV+ patients and that a different cut-off value for hsCRP for this population of patients on HD may be required to define inflammation.

Dialysis water treated by reverse osmosis decreases the levels of C-reactive protein in uremic patients

Atherosclerosis is a major complication of chronic renal failure. Microinflammation is involved in atherogenesis and is associated with uremia and dialysis. The role of dialysate water contamination in inducing inflammation has been debated. Our aim was to study inflammatory markers in patients on chronic dialysis, before and 3 to 6 months after switching the water purification system from deionization to reverse osmosis. Patients had demographic, clinical and nutritional information collected and blood drawn for determination of albumin, ferritin, C-reactive protein (CRP), interleukin-6, and tumor necrosis factor-alpha in both situations. Acceptable levels of water purity were less than 200 colony-forming units of bacteria and less than 1 ng/ml of endotoxin. Sixteen patients died. They had higher median CRP (26.6 vs 11.2 mg/dl, P = 0.007) and lower median albumin levels (3.1 vs 3.9 g/l, P < 0.05) compared to the 31 survivors. Eight patients were excluded because of obvious inflammatory conditions. From the 23 remaining patients (mean age ± SD: 51.3 ± 13.9 years), 18 had a decrease in CRP after the water treatment system was changed. Overall, median CRP was lower with reverse osmosis than with deionization (13.2 vs 4.5 mg/l, P = 0.022, N = 23). There was no difference in albumin, cytokines, subjective global evaluation, or clinical and biochemical parameters. In conclusion, uremic patients presented a clinically significant reduction in CRP levels when dialysate water purification system switched from deionization to reverse osmosis. It is possible that better water treatments induce less inflammation and eventually less atherosclerosis in hemodialysis patients.

The effect of ITF-1697 on reperfusion in patients undergoing primary angioplasty. Safety and efficacy of a novel tetrapeptide, ITF-1697.

AIM: ITF-1697 is a C-reactive protein-derived tetrapeptide that, based on pre-clinical studies, is thought to reduce reperfusion injury. We performed a dose-finding study to assess safety, preliminary efficacy and clinical outcome of prolonged i.v. infusion of ITF-1697 in patients with an acute myocardial infarction (AMI) who were eligible for percutaneous coronary intervention (PCI). METHODS AND RESULTS: This was a multicentre dose-finding study that was randomised, double blind, and placebo-controlled. Four hundred and two patients were enrolled. Intravenous infusion of four dosages of ITF-1697 (0.1, 0.5, 1.0 or 2.0 microg/kg/min) or placebo was started before PCI and continued for 24 h. After interim analysis of data from 242 patients the study continued with the 0.1 and 1.0 microg/kg/min ITF-1697 regimes. Analysis did not raise any safety concerns. Post-procedure perfusion, assessed by TIMI flow, corrected TIMI frame count, blushgrade and ST-segment resolution, was similar for the placebo, 0.1 and 1.0 microg/kg/min regimes. Furthermore, the results showed no differences between the treatment regimes in enzymatic infarct size or clinical outcome up to 30 days. CONCLUSION: ITF-1697 was well tolerated. However, neither a dose-relation nor improvement of perfusion, clinical outcome or reduction of myocardial damage could be demonstrated with ITF-1697 during and after primary PCI for AMI.

Effect of partially modified retro-inverso analogues derived from C-reactive protein on the induction of nitric oxide synthesis in peritoneal macrophages.

1. The ability of three modified tetrapeptides, representing fragments of the C-reactive protein (CRP) sequence and stabilized in the first peptide bond by retro-inverso modification, to affect the secretion of nitric oxide (NO) was studied in macrophages of BALB/c mice. 2. These tetrapeptides, resembling the aminoacid sequence of tuftsin (CRP 1, H-gThr-(R,S)mLys-Pro-Leu-OH, ITF 1192; CRP II, H-gGly-(R, S)mLys-Pro-Arg-OH, ITF 1127; CRP III, H-gThr-(R,S)mLys-Pro-Gln-OH. ITF 1193), were able to induce NO synthesis by peritoneal macrophages in a dose-dependent manner; the most stimulating dose was 1000 ng ml-1 for CRP II and 100 ng ml-1 for CRP I and CRP III. NO synthesis was not strictly dependent on lipopolysaccharide (LPS) activation. 3. The enhanced effect of retro-inverso CRP-related analogues on the expression of iNOS (inducible NO synthase) was confirmed by higher levels of iNOS activity in the cytosol and by the increase in iNOS protein, as evaluated by Western blot analysis, in macrophages stimulated by CPR compared with untreated ones. 4. The production of NO by retro-inverso CRP-peptide analogues was significantly inhibited by dexamethasone (20 microM), NG-monomethyl-L-arginine (L-NMMA) (500 microM) and pyrrolidine dithiocarbamate (PDTC) (100 microM). 5. Retro-inverso CRP-peptide analogues stimulated macrophages to produce high levels of interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) in the presence of LPS. 6. Retro-inverso CRP-peptide analogues stimulated NO synthesis by the enhancement of endogenously produced IL-1 and TNF-alpha, as the treatment of peritoneal macrophages with LPS in the presence of neutralizing anti-IL-1 and anti-TNF monoclonal antibodies (mAbs) reduced retro-inverso analogue-induced NO secretion. Data indicate a predominant role for IL-1 alpha in the induction of NO secretion by retro-inverso analogues. 7. These results suggest that retro-inverso CRP derived analogues act as costimulators of NO and cytokine synthesis in macrophages. The mechanisms by which they cause iNOS induction appear to be strongly dependent on the activation of nuclear factor-kappa B (NF-kappa B).

Use of type I and type IV hypersensitivity responses to define the immunopharmacological profile of drugs.

Administration of antigen suspended in incomplete Freund's adjuvant supplemented with either heat-killed Mycobacterium tuberculosis (complete Freund's adjuvant, CFA) or Bordetella pertussis toxin sensitizes animals so that subsequent antigen challenge leads to delayed-type (DTH) or immediate type hypersensitivity (ITH) responses, named type IV and type I, respectively. Appropriate timing of administration of drugs with respect to immunization or antigen challenge allowed to detect predominantly immunosuppressive, antiinflammatory or antianaphylactic activities. Among the reference drugs tested, only cyclosporin A (CsA) and dexamethasone (Dex) markedly inhibited DTH reaction, due to their immunosuppressive and antiinflammatory activities, respectively, whereas leflunomide and indomethacin resulted less potent. On the other hand, only dexchlorpheniramine, a histamine-receptor antagonist, afforded significant protection against anaphylactic shock, a form of ITH. Two new chemical entities were studied according to this protocol: ITF 1697, a chemically stabilized C-reactive protein-derived tetrapeptide, and ITF 2018, a leflunomide analogue. Data obtained with these new compounds showed that ITF 1697 has antianaphylactic activity, while ITF 2018 is endowed, mainly, with antiinflammatory activity. These results show that, through appropriate timing of administration, established in vivo models of immunologically mediated disease states allow an accurate profiling of the effects of pharmacologically active molecules and the detection of unsuspected activities for new drugs.