Osteoarthritis-related biomarkers profile in chronic anterior cruciate ligament injured knee.

OBJECTIVE: Increasing evidence has shown the involvement of the innate immune system and inflammatory response in osteoarthritis (OA) pathogenesis; however, anterior cruciate ligament (ACL) tears are recognized risk factors for development of post-traumatic OA. We investigated (1) whether inflammatory mediators involved in OA pathogenesis are also present at significant concentrations in the knee joint sometime after ACL complete tear and may be considered as prognostic biomarkers of progression to secondary OA; and (2) whether quantification in serum may surrogate synovial fluid (SF) measurements in both cases. METHODS: Thirty-seven end-stage OA patients and 33 patients with ACL complete tear that were included on the waiting list for knee surgery were consecutively recruited. Serum and SF samples were taken before surgery, and tumor necrosis factor-alpha, (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), matrix metalloproteinase-1 (MMP1), matrix metalloproteinase-3 (MMP3), tissue inhibitor of metalloproteinase-1 (TIMP1), bone morphogenetic protein-7 (BMP7), regulated upon activation normal t-cell expressed and secreted (RANTES), cytokine interferon-γ-induced protein 10 (IP-10) and heat shock protein family A (Hsp70) member 1A (HSPA1A) were quantified by enzyme-linked immunosorbent assay (ELISA.) Normally distributed data were compared using a one-way analysis of variance (ANOVA) test. Data not normally distributed were analyzed using a nonparametric Mann-Whitney rank sum test. Differences in means were compared using a Student's t-test. Correlations were analyzed using Pearson's coefficient of variation. RESULTS: Eighty-seven percent of patients with OA and 86% of those with ACL tear had quantifiable levels of biomarkers in SF. SF levels of IL-6, IL-8, MMP1, MMP3, RANTES, IP-10, BMP7 and HSPA1A were significantly lower in ACL injury knees compared with those with OA, but much higher than those reported in control subjects. Serum levels of IL-6, IP-10, and MMP1 were also lower in patients with ACL tears, who had, however, significantly higher TNF-α, HSPA1A, and TIMP1 levels when compared with OA patients. Levels of biomarkers tested in serum and SF samples were significantly different. CONCLUSIONS: Our data propose that cytokines IL-6 and IL-8 and the chemokines RANTES, IP-10, MMP1, MMP3, and HSPA1A may be involved in the inflammatory process leading to synovitis, the central lesion in OA onset and development; persistent high levels of these substances sometime after ACL injury suggest that they could play an etiopathogenic role in the maintenance of the inflammatory environment leading to post-traumatic OA. Serum biomarker levels do not appear to faithfully reflect what occurs inside the joint. Thus, most biomarkers cannot yet be considered as useful inflammatory biomarkers of knee joint diseases.

Gene expression, protein profiling, and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint.

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.

An overview of cytokines and heat shock response in polytraumatized patients.

Early after injury, local tissue damage induces a local and systemic inflammatory response that activates the immune system and leads to the development of systemic inflammatory response syndrome (SIRS). This post-traumatic response often results in uncontrolled release of inflammatory mediators and over-activation of the immune system, which occasionally results in multiple organ dysfunction syndrome (MODS). In parallel, a state of immunosuppression develops. This counter-regulating suppression of different cellular and humoral immune functions has been termed "compensatory anti-inflammatory response syndrome (CARS)." Both SIRS and CARS occur simultaneously even in the initial phase after injury. Pro- and anti-inflammatory cytokines have been suggested to play a major role in development of SIRS, although the degree of involvement of the different cytokines is quite disparate. While TNF-α and IL-1ß are quite irrelevant for predicting organ dysfunction, IL-6 is the parameter that best predicts mortality. The hyperinflammatory state seems to be the cause of post-traumatic immunosuppression and heat shock proteins (HSPs), which have been proposed as one of the endogenous stimuli for the deterioration of the immune system acting as danger-associated molecular patterns (DAMPs). Extracellular HSPA1A released from injured tissues increase up to ten times immediately after trauma and even more in patients with MODS. It has powerful immune properties that could contribute to post-traumatic immunosuppression through several mechanisms that have been previously described, so HSPs could represent trauma-associated immunomodulatory mediators. For this reason, HSPA1A has been suggested to be a helpful early prognostic biomarker of trauma after severe injury: serial quantification of serum HSPA1A and anti-Hsp70 concentrations in the first hours after trauma is proposed to be used as a predictive biomarker of MODS and immunosuppression development in polytraumatized patients.

Proteínas de choque térmico en la cardioprotección del consumo moderado regular de alcohol / Role of heat shock proteins in the cardioprotection of regular moderate alcohol consumption

Fundamento y objetivos: Estudiar si el efecto cardioprotector del consumo regular de alcohol puede explicarse a través de las heat shock proteins (HSP, «proteínas de choque térmico»), dado su papel etiopatogénico en la ateroesclerosis. Material y métodos: Estudio epidemiológico trasversal en 452 sujetos de 40-60 años de ambos sexos. Se realizó la historia clínica incluyendo frecuencia del consumo medio de alcohol y análisis bioquímicos, y se estatificó el grado de riesgo coronario según la Task Force. Se cuantificaron HSPA1A intracelular, HSPA1A y HSPD1 séricas y anti-Hsp70 y anti-Hsp60 por ELISA. Resultados: Doscientos treinta y ocho (52,7%) sujetos eran abstemios o bebedores de < 20 g/d de alcohol; 123 (27,2%) bebían 20-40 g/d, 66 (14,6%) 40-60 g/d y 25 > 60 g/d (5,5%). Doscientos treinta y nueve carecían de factores de riesgo vascular (RV) o tenían un RV < 5%; 161 tenían RV moderado (10-20%) y 52 presentaban enfermedad ateroesclerótica instaurada. Los bebedores de 40-60 g/d presentaron máximas concentraciones de HSPA1A intracelular, no significativas en RV moderado. HSPA1A sérica no presentó diferencias y HSPD1 fue indetectable. Los bebedores de 40-60 g/d y RV moderado o enfermedad ateroesclerótica presentaron las menores concentraciones de anti-Hsp70. Los anti-Hsp60 fueron máximos en varones bebedores de > 60 g/d y en mujeres bebedoras de 40-60 g/d, especialmente en RV moderado. Conclusiones: El efecto cardioprotector del consumo de 40-60 g/d de alcohol podría deberse, al menos en parte, al incremento de HSPA1A intracelular, potente proteína antiinflamatoria. El consumo excesivo regular de alcohol se asocia a un aumento de anticuerpos anti-Hsp60, estimulantes de citocinas proinflamatorias; ello podría explicar la mortalidad por enfermedad cardiovascular en estos pacientes. Se ha propuesto la aplicación clínica del seguimiento de anticuerpos anti-Hsp en pacientes en riesgo para detectar enfermedad ateroesclerótica (AU)

Background and objectives: To study whether the cardioprotective effect of regular alcohol consumption can be explained by the heat shock proteins (HSP), given their pathogenic role in atherosclerosis. Material and methods: Cross-sectional epidemiological study on 452 men and women aged 40-60. Clinical history, epidemiological survey (frequency of average alcohol consumption) and biochemical analysis was performed; Task Force Chart was applied for classification according to the risk of vascular disease. Intracellular HSPA1A, circulating HSPA1A and HSPD1, and anti-Hsp70/anti-Hsp60 antibodies were quantified by ELISA. Results: Two hundred and thirty-eight (52.7%) were abstemious or drank < 20 g/d of alcohol; 123 (27.2%) drank 20-40 g/d, 66 (14.6%) 40-60 g/d and 25 > 60 g/d (5.5%). Two hundred and thirty-nine had no vascular risk (VR) factor or a risk < 5%, 161 had moderate VR (10-20%) and 52 had established atherosclerotic disease. Drinkers of 40-60 g/d showed the highest concentrations of intracellular HSPA1A, which were not significant in subjects with moderate VR. Extracellular HSPA1A didn’t differ and HSPD1 was undetectable. Drinkers of 40-60 g/d and moderate VR or atherosclerotic disease presented the lowest concentrations of anti-Hsp70. The highest levels of serum anti-Hsp60 were shown in heavy male drinkers of > 60 g/d especially in subjects with moderate VR, and female drinkers of 40-60 g/d. Conclusions: The cardioprotective effect of 40-60 g/d of alcohol consumption could be due in part, to increased intracellular HSPA1A, a potent anti-inflammatory protein. Excessive intake of alcohol increases antibodies anti-Hsp60, stimulating proinflammatory cytokines. This fact may explain the mortality from cardiovascular disease in heavy drinkers. The clinical application of antibody anti-Hsps quantification has been proposed in patients at risk in order to detect atherosclerotic disease (AU)

[Role of heat shock proteins in the cardioprotection of regular moderate alcohol consumption]. / Proteínas de choque térmico en la cardioprotección del consumo moderado regular de alcohol.

BACKGROUND AND OBJECTIVES: To study whether the cardioprotective effect of regular alcohol consumption can be explained by the heat shock proteins (HSP), given their pathogenic role in atherosclerosis. MATERIAL AND METHODS: Cross-sectional epidemiological study on 452 men and women aged 40-60. Clinical history, epidemiological survey (frequency of average alcohol consumption) and biochemical analysis was performed; Task Force Chart was applied for classification according to the risk of vascular disease. Intracellular HSPA1A, circulating HSPA1A and HSPD1, and anti-Hsp70/anti-Hsp60 antibodies were quantified by ELISA. RESULTS: Two hundred and thirty-eight (52.7%) were abstemious or drank<20 g/d of alcohol; 123 (27.2%) drank 20-40 g/d, 66 (14.6%) 40-60 g/d and 25>60 g/d (5.5%). Two hundred and thirty-nine had no vascular risk (VR) factor or a risk<5%, 161 had moderate VR (10-20%) and 52 had established atherosclerotic disease. Drinkers of 40-60 g/d showed the highest concentrations of intracellular HSPA1A, which were not significant in subjects with moderate VR. Extracellular HSPA1A didn't differ and HSPD1 was undetectable. Drinkers of 40-60 g/d and moderate VR or atherosclerotic disease presented the lowest concentrations of anti-Hsp70. The highest levels of serum anti-Hsp60 were shown in heavy male drinkers of>60 g/d especially in subjects with moderate VR, and female drinkers of 40-60 g/d. CONCLUSIONS: The cardioprotective effect of 40-60 g/d of alcohol consumption could be due in part, to increased intracellular HSPA1A, a potent anti-inflammatory protein. Excessive intake of alcohol increases antibodies anti-Hsp60, stimulating proinflammatory cytokines. This fact may explain the mortality from cardiovascular disease in heavy drinkers. The clinical application of antibody anti-Hsps quantification has been proposed in patients at risk in order to detect atherosclerotic disease.

Amyloid precursor protein metabolism and inflammation markers in preclinical Alzheimer disease.

OBJECTIVE: To investigate CSF markers involved in amyloid precursor protein processing, neuronal damage, and neuroinflammation in the preclinical stages of Alzheimer disease (AD) and participants with suspected non-Alzheimer pathology (SNAP). METHODS: We collected CSF from 266 cognitively normal volunteers participating in a cross-sectional multicenter study (the SIGNAL study) to investigate markers involved in amyloid precursor protein processing (Aß42, sAPPß, ß-secretase activity), neuronal damage (total-tau [t-tau], phospho-tau [p-tau]), and neuroinflammation (YKL-40). We analyzed the relationship among biomarkers, clinical variables, and the APOE genotype, and compared biomarker levels across the preclinical stages of the National Institute on Aging-Alzheimer's Association classification: stage 0, 1, 2, 3, and SNAP. RESULTS: The median age in the whole cohort was 58.8 years (range 39.8-81.6). Participants in stages 2-3 and SNAP had higher levels of YKL-40 than those in stages 0 and 1. Participants with SNAP had higher levels of sAPPß than participants in stage 0 and 1. No differences were found between stages 0, 1, and 2-3 in sAPPß and ß-secretase activity in CSF. Age correlated with t-tau, p-tau, and YKL-40. It also correlated with Aß42, but only in APOE ε4 carriers. Aß42 correlated positively with t-tau, sAPPß, and YKL-40 in participants with normal Aß42. CONCLUSIONS: Our findings suggest that inflammation in the CNS increases in normal aging and is intimately related to markers of neurodegeneration in the preclinical stages of AD and SNAP. sAPPß and ß-secretase activity are not useful diagnostic or staging markers in preclinical AD.

A blood-based, 7-metabolite signature for the early diagnosis of Alzheimer's disease.

Accurate blood-based biomarkers of Alzheimer's disease (AD) could constitute simple, inexpensive, and non-invasive tools for the early diagnosis and treatment of this devastating neurodegenerative disease. We sought to develop a robust AD biomarker panel by identifying alterations in plasma metabolites that persist throughout the continuum of AD pathophysiology. Using a multicenter, cross-sectional study design, we based our analysis on metabolites whose levels were altered both in AD patients and in patients with amnestic mild cognitive impairment (aMCI), the earliest identifiable stage of AD. UPLC coupled to mass spectrometry was used to independently compare the levels of 495 plasma metabolites in aMCI (n = 58) and AD (n = 100) patients with those of normal cognition controls (NC, n = 93). Metabolite alterations common to both aMCI and AD patients were used to generate a logistic regression model that accurately distinguished AD from NC patients. The final panel consisted of seven metabolites: three amino acids (glutamic acid, alanine, and aspartic acid), one non-esterified fatty acid (22:6n-3, DHA), one bile acid (deoxycholic acid), one phosphatidylethanolamine [PE(36:4)], and one sphingomyelin [SM(39:1)]. Detailed analysis ruled out the influence of potential confounding variables, including comorbidities and treatments, on each of the seven biomarkers. The final model accurately distinguished AD from NC patients (AUC, 0.918). Importantly, the model also distinguished aMCI from NC patients (AUC, 0.826), indicating its potential diagnostic utility in early disease stages. These findings describe a sensitive biomarker panel that may facilitate the specific detection of early-stage AD through the analysis of plasma samples.

Heat shock proteins, end effectors of myocardium ischemic preconditioning?

The purpose of this study was to investigate (1) whether ischemia-reperfusion increased the content of heat shock protein 72 (Hsp72) transcripts and (2) whether myocardial content of Hsp72 is increased by ischemic preconditioning so that they can be considered as end effectors of preconditioning. Twelve male minipigs (8 protocol, 4 sham) were used, with the following ischemic preconditioning protocol: 3 ischemia and reperfusion 5-minute alternative cycles and last reperfusion cycle of 3 hours. Initial and final transmural biopsies (both in healthy and ischemic areas) were taken in all animals. Heat shock protein 72 messenger ribonucleic acid (mRNA) expression was measured by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method using complementary DNA normalized against the housekeeping gene cyclophilin. The identification of heat shock protein 72 was performed by immunoblot. In our "classic" preconditioning model, we found no changes in mRNA hsp72 levels or heat shock protein 72 content in the myocardium after 3 hours of reperfusion. Our experimental model is valid and the experimental techniques are appropriate, but the induction of heat shock proteins 72 as end effectors of cardioprotection in ischemic preconditioning does not occur in the first hours after ischemia, but probably at least 24 hours after it, in the so-called "second protection window."

Proteínas de choque térmico en el estrés quirúrgico: toracotomía frente a herniorrafia / Heat shock proteins in surgical stress: thoracotomy vs herniorrhaphy

Introducción. El estrés quirúrgico y anestésico libera citocinas y especies reactivas de oxígeno capaces de inducir la síntesis de las proteínas de choque térmico (HSP), proteínas con propiedades inmunomoduladoras y potentes autoantígenos. Se pretende estudiar la biología de las HSP70 intraleucocitarias y la posible respuesta autoinmunitaria desencadenada por 2 tipos diferentes de agresión quirúrgica. Pacientes y método. Grupo I: grupo control con 3 pacientes. Grupo II: grupo de toracotomía, cirugía radical y anestesia general, con 6 pacientes. Grupo III: grupo de cirugía poco radical, herniorrafia y raquianestesia, con 4 pacientes. Se analizaron HSP70 intraleucocitarias y anticuerpos anti-HSP70i, antes (T0) y 24 h después de la intervención (T1).Resultados. El 50 por ciento de los pacientes expuestos a toracotomía presentó un significativo descenso del contenido de HSP intracelulares en el postoperatorio, simultáneo al incremento de los valores de autoanticuerpos anti-HSP70i. El grupo de pacientes expuestos a herniorrafia con anestesia locorregional no desarrolló respuesta autoinmunitaria. Conclusiones. En el limitado número de pacientes estudiados, la enfermedad neoplásica y la mayor agresividad de la toracotomía parecen asociarse con una reducción de las HSP en comparación con lo que sucede en los pacientes más sanos a los que se les realizó una herniorrafia. La disminución de las HSP fue simultánea a la presencia de autoanticuerpos circulantes. No se observó relación entre el estado inmunitario previo y la respuesta autoinmunitaria en el postoperatorio inmediato (AU)