Laura Caponi

Anti-ribosomal P protein antibodies

Anti-ribosomal P protein antibodies (Abs) recognize three specific ribosomal proteins located in the large ribosomes subunit. The term anti-ribosomal P protein Abs, often shortened in anti-P Abs, is due to the fact that these three proteins, P0, P1 and P2, of 38, 19 and 17 Kd molecular weight, respectively, are phosphorylated. One of the major points of interest of these autoAbs derives from their high specificity for systemic lupus erythematosus (SLE), their description in other connective tissue diseases (CTDs) being only occasional. In SLE patients, their association with renal and hepatic involvement has been proposed, while the possible association with psychiatric and/or neurological involvement is still a matter of debate. From a serological point of view, a preferential association of anti-P with anti-Sm and/or anti-DNA Abs, possibly due, at least in part, to cross-reactivity, has been suggested. This observation is intriguing since all these autoAbs are considered specific serological indicators of SLE. This review will summarize clinical and serological data on anti-P Abs provided by the main studies published in the last few years and the more recent findings about proteins constituting their targets.

Anti‐ribosomal antibodies bind the Sm proteins D and B/B′

In order to analyse the specificity of human anti‐ribosomal P protein antibodies, anti‐ribosomal P protein antibodies were affinity‐purified from the sera of lupus patients. Their binding capacity towards recombinant SmD protein and recombinant SmB/B′ protein was evaluated by immunoblot and ELISA. Epitope mapping of SmD was performed by means of synthetic peptides. Anti‐ribosomal P protein antibodies bound recombinant SmD (5/10) and SmB/B′ (4/10) on immunoblot; 6/10 showed binding capacity to SmD on ELISA. Inhibition experiments using ELISA confirmed the specificity of this binding. Our data indicate the cross‐reactivity of spontaneously developed anti‐ribosomal P protein antibodies with the B/B′ and D constituents of the Sm complex. The coexistence of anti‐Sm and anti‐ribosomal antibodies in lupus sera may therefore be due, at least in part, to the reactivity of a single autoantibody population.

Antibodies directed against ribosomal P proteins cross-react with phospholipids

Anti‐ribosomal P protein (anti‐P) antibodies are marker antibodies in systemic lupus erythematosus (SLE). Their association with psychiatric or neurological manifestations has been proposed, but remains controversial. Anti‐phospholipid antibodies are the hallmark of a syndrome that may comprise a number of neurological manifestations. Thus, anti‐P and anti‐phospholipid antibodies have both been associated with central nervous system involvement and their co‐existence in the same sera was reported. We verified the ability of purified anti‐P antibodies to bind different phospholipids and phospholipid‐binding proteins in solid‐phase assays. Anti‐P antibodies from five of eight patients bound cardiolipin (CL) when saturated with fetal calf serum (FCS); in three cases anti‐CL antibodies were also detected in the flow‐through. No anti‐P eluate, nor any corresponding flow‐through, bound β2‐glycoprotein I alone or prothrombin. Moreover, no anti‐P eluate bound CL when the plates were blocked with bovine serum albumin in the absence of FCS. Anti‐P antibodies with anti‐CL activity bound both ssDNA and dsDNA and also nucleosomes in three patients. Our data indicate a great heterogeneity of anti‐P antibodies that appear to be overlapped partially with the other autoantibody populations detected frequently in SLE.

Role of matrix metalloproteinases and their tissue inhibitors as potential biomarkers of left ventricular remodelling in the athlete's heart

The aim of the present study was to verify whether plasma MMPs (matrix metalloproteinases) and TIMPs (tissue inhibitors of MMPs) could be used as potential markers of paraphysiological remodelling in the athletes heart, and to correlate these matrix parameters with echocardiographic signs of LV (left ventricular) remodelling. Plasma MMP-2 and MMP-9 were measured by zymography, and TIMP-1 and TIMP-2 were measured by ELISA in 42 veteran marathoners with AH (athletes heart), and in 25 sedentary healthy subjects (CTL). All subjects were submitted to a clinical examination and two-dimensional colour Doppler echocardiography together with the measurement of circulating NT-proBNP (N-terminal pro-B-type natriuretic peptide); GGT (gamma-glutamyl transpeptidase) was evaluated as a marker of cardiovascular disease. Veteran athletes had a significant elevation in LV dimensions and calculated LV mass index. Diastolic and systolic functions were normal for both groups. MMP-9 levels were significantly lower in AH than in CTL subjects (56.9+/-4.3 compared with 119.4+/-21.5 m-units/l, P<0.01). There were significant differences in MMP-2 between the two groups, with a down-regulation in the AH subjects (182.5+/-16.8 units/ml in CTL compared with 117.1+/-9.1 units/ml in AH, P<0.01). MMP-2 and MMP-2/TIMP-2 were inversely correlated with myocardial indices of hypertrophy in AH and CTL subjects. AH and CTL subjects showed similar TIMP values. The results of the present study indicate that MMPs and TIMPs could represent potential biomarkers of adaptive heart remodelling in the athletes. In addition, the inverse correlation of the MMP-2/TIMP-2 system with echocardiographic signs of myocardial hypertrophy could represent a new diagnostic and prognostic indicator useful in the evaluation of cardiovascular risk in athletes.

Antibody Usage in the Lab

Introduction ELISA assays Immunoblotting Immunoprecipitation Immunofluorescence Immunohistochemistry Antibody specificity analysed by peptides synthesised on cellulose membranes Appendices.

PADI4 gene in multiple sclerosis: A family-based association study

In multiple sclerosis (MS) MBP is heavily citrullinated by peptidylarginine deiminase (PAD). This post-translational modification may be crucial for its pathogenesis. PADI4 is the isoform expressed in inflammatory infiltrates. The aim of this study was to analyse the role of PADI4 gene in conferring susceptibility to MS, by means of a family-based association study, testing three SNPs by RFLP. No association was found either with single SNPs or haplotypes. Similarly no significant association was detected partitioning the patients according to DRB1*15 positivity or disease severity. These results do not support a major role of the PADI4 gene, but further studies may contribute to clarify the genetic factors that regulate deimination.

C-type natriuretic peptide is closely associated to obesity in Caucasian adolescents.

CNP is a natural regulator of adipogenesis playing a role in the development of obesity in childhood. Aim of the study was to evaluate CNP plasma levels in normal-weight (N), overweight (OW) and obese adolescents (O). Eighty two subjects (age:12.8±2.4, years) without cardiac dysfunction were enrolled and CNP plasma levels were measured by RIA. NT-proBNP, MR-proANP, AGEs, reactive hyperemia index (RHI) and standard clinical chemistry parameters were also measured. O and OW adolescents had higher values of BMI and fat mass than N. CNP levels were significantly lower in OW:4.79[3.29-21.15] and O:3.81[1.55-13.4] than in N:13.21[7.6-37.8]; p<0.0001N vs O, p=0.0003N vs OW). LogCNP values correlated significantly and inversely with BMI z-score, FM%, TF% and circulating levels of CRP, insulin, total cholesterol, LDL, and triglycerides, in addition to an inverse relationship with skin AGEs and a direct correlation with RHI. LogCNP was also inversely associated with LogNT-proBNP and LogMR-proANP values. Using ROC analysis the risk of obesity resulted significantly (p≪0.0001) associated with CNP values (AUC=0.9724). These results suggest that CNP may play a more important role than BNP and ANP related peptides, as risk marker of obesity, in addition to its involvement in adipogenesis and endothelial dysfunction.

β2-Microglobulin and TIMP1 Are Linked Together in Cardiorenal Remodeling and Failure

Background/Aims: The cardiorenal syndrome is a complication in patients hospitalized with chronic heart failure (CHF). The β2-microglobulin (b2M) level is an index of decreased glomerular filtration rate (GFR), tissue turnover and inflammation. It is an emerging new predictive marker of cardiovascular events and mortality, but its role as a biomarker of cardiorenal remodeling and failure is still unknown. TIMP1, an endogenous tissue inhibitor of activated matrix metalloproteinases, is a biomarker of heart remodeling and failure. We aimed to evaluate the circulating profile of b2M and TIMP1 in CHF patients, in sedentary controls with no tissue remodeling and in veteran athletes with physiological cardiorenal remodeling and athletes heart (AH). Methods: We investigated the plasma levels of b2M and TIMP1 in 24 subjects with CHF without primitive renal disease, in 25 sedentary controls and in 30 veteran marathoners with AH over 50 years. Results: The b2M and TIMP1 levels were higher in CHF patients, and there was a correlation between them (r = 0.5287, p < 0.0095). The b2M level correlated with the severity of cardiorenal impairment: with proBNP (r = 0.66, p > 0.0007), percent ejection fraction (r = -0.56, p = 0.0162) and GFR (r = 0.83, p < 0.0001). b2M was also correlated with TIMP1 in AH subjects (r = 0.7548, p < 0.0001) but not in controls. This correlation was independent from GFR in both CHF patients and sedentary controls. Conclusions: In CHF patients, the plasma levels of b2M and TIMP1 were linked together and correlated with the severity of cardiorenal failure. Moreover, a strong correlation between b2M and TIMP1 characterized cardiovascular remodeling not only in CHF patients but also in AH subjects. These findings suggest that clinicians should use b2M and TIMP1 as associated biomarkers of cardiorenal remodeling and failure.

High Levels of Circulating Chemokine (C-X-C motif) Ligand 11 Are Associated with Euthyroid or Subclinically Hypothyroid Autoimmune Thyroiditis and with Chemokine (C-X-C Motif) Ligand 10

No data are available for chemokine (C-X-C motif) ligand 11 (CXCL11), together with CXCL10, circulating levels in autoimmune thyroiditis (AT). We measured serum CXCL11 and CXCL10 in 158 patients with newly diagnosed AT (26% with subclinical hypothyroidism), 56 euthyroid controls, and 20 patients with nontoxic multinodular goiter, all similar in gender distribution and age. CXCL11 was significantly higher in patients with AT (113±56 pg/mL) than in controls (67±16 pg/mL) or patients with multinodular goiter (75±18 pg/mL; P<0.0001). Among patients with AT, CXCL11 was significantly higher in those with a hypoechoic ultrasonographic pattern and hypothyroidism. In a multiple linear regression (MLR) model including age, thyroid volume, hypoechogenicity, hypervascularity, thyroid-stimulating hormone (TSH), and anti-thyroid peroxidase, age (P=0.009) and TSH (P<0.008) were significantly related to serum CXCL11. In an MLR model of CXCL11 (ln[pg/mL]) versus age, TSH, CXCL10 (ln[pg/mL]), TSH (P=0.028), and CXCL10 (P=0.003) were significantly and independently related to CXCL11. We first show that circulating CXCL11, together with CXCL10, is increased in patients with thyroiditis and hypothyroidism, and is related to CXCL10 levels. These results underline the importance of a Th1 immune attack in the initiation of AT.

Anti‐inflammatory signaling during ex vivo liver perfusion improves the preservation of pig liver grafts before transplantation

We read with interest the article by Goldaracena et al., and we think we have some direct and independent evidence supporting their work and the idea that controlling the production of inflammatory cytokines is a key step for optimizing liver graft metabolism during normothermic machine perfusion (NMP) and to drive this technology in clinical practice. So far, the majority of reported experiences have focused on the role of NMP in promoting the salvage of discarded livers, but when NMP preservation is compared with standard cold storage (CS), the anticipated advantages are still limited and inconsistent. Moreover, randomized controlled trials comparing NMP to CS in marginal grafts raise ethical implications and can hardly be implemented. We have recently transplanted 2 liver grafts after NMP preservation. Both grafts were procured from brain dead deceased donors: the first donor was a 73year-old Caucasian male with normal liver function and unremarkable liver histology, whereas the second was a 33-year-old Caucasian male resuscitated after a 30-minute cardiac arrest and with preprocurement aspartate aminotransferase (AST)5 129 IU/L. The recipients were a 53-year-old man with hepatitis C virus (HCV)–related cirrhosis (Model for End-Stage Liver Disease [MELD]5 16), and a 53-year-old man with alcoholic cirrhosis (MELD5 23), respectively. Procurement was with en bloc liver and pancreas retrieval and dual perfusion. NMP was performed with the Liver Assist device and a blood-based solution (total volume5 2265mL) including among others 2000 U fast-acting insulin (Actrapid) and 1.5 g of glucose. Cold ischemia time was 226 and 285 minutes, and duration of NMP was 325 and 240 minutes, respectively. Transplantations were with the standard technique and simultaneous arterial and portal revascularization. Both transplants were successful; the AST peak was 451 and 329 IU/L, respectively, and patients were discharged in good clinical condition on postoperative days 12 and 14. Interestingly, during NMP of the first liver graft, we found steady high glucose levels in the perfusate, unresponsive to additional administration of 500 U insulin (Fig. 1A), whereas the second graft showed progressively decreasing glucose (Fig. 1B). Although the interleukin (IL) 6 levels were similar, the level of the anti-inflammatory cytokine IL10 was lower and that of the proinflammatory cytokine tumor necrosis factor a (TNF-a) was far higher in the first patient (Fig. 1C). The periodic acid–Schiff staining of tissue sections showed a comparable and relevant decreased glycogen content in hepatocytes (not shown). In line with the findings of Goldaracena et al., these preliminary results provide direct evidence that a less favorable cytokines profile in the perfusate may preclude an optimal energy recovery, and that controlling cytokine production is needed to improve the performance of NMP. The reason for the different cytokine profiles observed in the 2 grafts deserve future investigations, but ischemic preconditioning and the quality of livers should be considered. Effective graft reconditioning Address reprint requests to Davide Ghinolfi, M.D., Ph.D., Division of Hepatobiliary Surgery and Liver Transplantation, University of Pisa Medical School Hospital, Via Paradisa 2, Building 6, Room 11, 56124 Pisa, Italy. Telephone.: 1 39 050 995048; FAX: 1 39 050 995420; E-mail: d.ghinolfi@ao-pisa.toscana.it