Margherita Massa

Epitope-specific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis

Modulation of epitope-specific immune responses would represent a major addition to available therapeutic options for many autoimmune diseases. The objective of this work was to induce immune deviation by mucosal peptide-specific immunotherapy in rheumatoid arthritis (RA) patients, and to dissect the related immunological mechanisms by using a technology for the detection of low-affinity class II-restricted peptide-specific T cells. A group of patients with early RA was treated for 6 months orally with dnaJP1, a peptide that induces proinflammatory T cell responses in naive RA patients. Immunological analysis at initial, intermediate and end treatment points showed an intriguing change from proinflammatory to regulatory T cell function. In fact, dnaJP1-induced T cell production of IL-4 and IL-10 increased significantly when initial and end treatment points were compared, whereas dnaJP1-induced T cell proliferation and production of IL-2, IFN-γ, and tumor necrosis factor-α decreased significantly. The total number of dnaJP1-specific cells did not change over time, whereas expression of foxP3 by CD4+CD25bright cells increased, suggesting that the treatment affected regulatory T cell function. Thus, rather than clonal deletion, the observed change in immune reactivity to dnaJP1 was the outcome of treatment-induced emergence of T cells with a different functional phenotype. This study contributes to our knowledge of mechanisms and tools needed for antigen-specific immune modulation in humans, thus laying the foundation for exploitation of this approach for therapeutic purposes.

Tolerogenic immune responses to novel T-cell epitopes from heat-shock protein 60 in juvenile idiopathic arthritis

BACKGROUND Juvenile idiopathic arthritis is a heterogeneous autoimmune disease characterised by chronic inflammation of one or more joints. In patients with this disease, T-cell reactivity to autologous heat-shock protein 60 (HSP60) is associated with a favourable prognosis. We sought to identify HSP60 T-cell epitopes to find potential targets for HSP60 immunotherapy and to assess whether immune responses to these epitopes contribute to the distinct clinical outcome of this disease. METHODS We identified eight potential epitopes using a computer algorithm from both self and microbial HSP60 binding to many HLA-DR molecules. We analysed the pattern of T-cell responses induced by these HSP60 peptides in peripheral-blood mononuclear cells (PBMC) of 57 patients with juvenile idiopathic arthritis, 27 healthy controls, and 20 disease controls. We undertook in-vitro MHC binding studies with the identified peptides, and HLA class II typing of a subset of patients with juvenile idiopathic arthritis. FINDINGS Five of the eight peptides identified yielded proliferative T-cell responses in 50-70% of PBMC from patients with juvenile idiopathic arthritis irrespective of MHC genotype, but not in PBMC from healthy or disease controls. Although PBMC from both patients with juvenile idiopathic arthritis and healthy controls produced interferon gamma in response to these peptides, only PBMC from patients with the disease produced interleukin 10. INTERPRETATION The recorded T-cell-induction in juvenile idiopathic arthritis is tolerogenic. In patients with oligoarticular disease, the immune responses to the HSP60 epitopes identified could contribute to disease remission. RELEVANCE TO PRACTICE The broad recognition of these HSP60 epitopes in a population of patients with polymorphic MHC genotypes opens the way for HSP60-peptide immunotherapy, representing a novel treatment option to specifically modulate the immune system in patients with juvenile idiopathic arthritis.

Remote ischemic post-conditioning of the lower limb during primary percutaneous coronary intervention safely reduces enzymatic infarct size in anterior myocardial infarction: A randomized controlled trial

OBJECTIVES This study sought to evaluate whether remote ischemic post-conditioning (RIPC) could reduce enzymatic infarct size in patients with anterior ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention (pPCI). BACKGROUND Myocardial reperfusion injury may attenuate the benefit of pPCI. In animal models, RIPC mitigates myocardial reperfusion injury. METHODS One hundred patients with anterior ST-segment elevation myocardial infarction and occluded left anterior descending artery were randomized to pPCI + RIPC (n = 50) or conventional pPCI (n = 50). RIPC consisted of 3 cycles of 5 min/5 min ischemia/reperfusion by cuff inflation/deflation of the lower limb. The primary endpoint was infarct size assessed by the area under the curve of creatinine kinase-myocardial band release (CK-MB). Secondary endpoints included the following: infarct size assessed by cardiac magnetic resonance delayed enhancement volume; T2-weighted edema volume; ST-segment resolution >50%; TIMI (Thrombolysis In Myocardial Infarction) frame count; and myocardial blush grading. RESULTS Four patients (2 RIPC, 2 controls) were excluded due to missing samples of CK-MB. A total of 96 patients were analyzed; median area under the curve CK-MB was 8,814 (interquartile range [IQR]: 5,567 to 11,325) arbitrary units in the RIPC group and 10,065 (IQR: 7,465 to 14,004) arbitrary units in control subjects (relative reduction: 20%, 95% confidence interval: 0.2% to 28.7%; p = 0.043). Seventy-seven patients underwent a cardiac magnetic resonance scan 3 to 5 days after randomization, and 66 patients repeated a second scan after 4 months. T2-weighted edema volume was 37 ± 16 cc in RIPC patients and 47 ± 22 cc in control subjects (p = 0.049). ST-segment resolution >50% was 66% in RIPC and 37% in control subjects (p = 0.015). We observed no significant differences in TIMI frame count, myocardial blush grading, and delayed enhancement volume. CONCLUSIONS In patients with anterior ST-segment elevation myocardial infarction, RIPC at the time of pPCI reduced enzymatic infarct size and was also associated with an improvement of T2-weighted edema volume and ST-segment resolution >50%. (Remote Postconditioning in Patients With Acute Myocardial Infarction Treated by Primary Percutaneous Coronary Intervention [PCI] [RemPostCon]; NCT00865722).

Circulating Endothelial Progenitor Cells in Preterm Infants with Bronchopulmonary Dysplasia

RATIONALE The new form of bronchopulmonary dysplasia (BPD) is characterized by lung immaturity with disrupted alveolar and capillary development after extremely premature birth, but the mechanism of impaired lung vascular formation is still not completely understood. OBJECTIVES We tested the hypothesis that reduced numbers of circulating endothelial progenitor cells at birth are associated with the development of BPD. METHODS We studied ninety-eight preterm infants with gestational age of less than 32 weeks or a birth weight less than 1,500 g. Endothelial colony-forming cells (ECFCs) were assessed by clonogenic analysis in infants for whom cord blood was available. The proportion of circulating endothelial and hematopoietic cells was measured by flow cytometry at birth, at 48 hours, and at 7 days of life. MEASUREMENTS AND MAIN RESULTS ECFCs in cord blood were lower in infants who later developed BPD (median [range]: 0.00 [0.00-0.48] vs. 2.00 [0.00-21.87]; P = 0.002). ECFCs decreased with decreasing gestational age (r = 0.41; P = 0.02), but even at extremely low gestational ages, infants with higher numbers of ECFCs were protected from BPD. The endothelial and hematopoietic cell subsets studied by flow cytometry were comparable in infants with and without BPD and rapidly decreased after birth. CONCLUSIONS ECFCs are low at extremely low gestational ages and increase during gestation; extremely preterm infants who display lower numbers at birth have an increased risk of developing BPD. Our findings suggest that decreased ECFCs following extremely preterm birth may be associated with the risk for developing lung vascular immaturity characteristic of new BPD.

Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.

Background Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca2+ entry (SOCE), which is activated by a depletion of the intracellular Ca2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca2+-sensor, Stim1, and the plasmalemmal Ca2+ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients. Methodology/Principal Findings The present study employed Ca2+ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La3+ and Gd3+. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca2+ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca2+ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs. Conclusions SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.

Circulating CD34+, CD133+, and vascular endothelial growth factor receptor 2-positive endothelial progenitor cells in myelofibrosis with myeloid metaplasia.

PURPOSE Endothelial progenitor cells (EPCs) are present in circulation and contribute to vasculogenesis in adults. We measured the number of circulating EPCs in patients with myelofibrosis with myeloid metaplasia (MMM), and we examined the relationship between the number of EPCs and severity of the MMM disease process. PATIENTS AND METHODS The number of EPCs was measured by assaying the CD34+CD133+ vascular endothelial growth factor receptor 2 (VEGFR2) -positive cell phenotype in 110 MMM patients, 16 patients with other Philadelphia-negative chronic myeloproliferative disorders (Ph-negative CMPDs), and 14 healthy participants. In four MMM patients, the capacity of selected CD34+ cells to form endothelial colonies (CFU-End) in vitro was tested. RESULTS CD34+, CD133+, and VEGFR2-positive EPCs were detectable in unselected peripheral-blood cells of 50.9% MMM patients, 37.5% control patients, and 21% healthy participants. Patients with MMM had a median of 0.26% EPCs, significantly higher than that in healthy controls (median, 0%) and in patients with other Ph-negative CMPDs (median, 0.1%). In 14.5% of MMM patients, the numbers of EPCs were greater than the highest value found in patients with other Ph-negative CMPDs. CD34+ selected cells produced colony-forming unit-endothelial (CFU-End), which were vascular endothelial (VE) -cadherin positive, CD31+, von Willebrand factor positive, and CD45-. In MMM patients, the larger the number of EPCs, the smaller the number of circulating immature myeloid cells and circulating CD45+CD34+ hematopoietic progenitor cells. Increased numbers of EPCs were associated with younger age and a diagnosis of prefibrotic MMM. CONCLUSION Circulating EPCs are elevated in MMM patients in the early stage of the disease. Heightened mobilization of EPCs may represent an important mechanism for development of neoangiogenesis in MMM.

High-dose erythropoietin in patients with acute myocardial infarction: A pilot, randomised, placebo-controlled study

BACKGROUND Mortality and morbidity after acute myocardial infarction (AMI) remain high even when myocardial reperfusion is successful. Erythropoietin (EPO) protects against experimental MI. METHODS The aim of this single-centre study was to investigate the effects of short-term high-dose erythropoietin on peripheral blood cells (PBCs) and infarct size in 30 patients with a first uncomplicated AMI undergoing percutaneous coronary intervention (PCI) who were randomly assigned to treatment with EPO (33 × 10(3)IU before PCI, and 24 and 48 h after admission), or placebo. We considered short-term CD34+ cell mobilisation, quantitative PBC gene expression in the apoptotic, angiogenic and inflammatory pathways, and enzymatically estimated infarct size. Echocardiographic and cardiac magnetic resonance studies were performed in the acute phase and six months later. RESULTS CD34+ cell mobilisation 72 h after admission was greater in the EPO-treated patient group (93 cells/μl [36-217] vs 22 cells/μl [6-51]; p = 0.002), who also showed higher expression of the anti-apoptotic AKT and NFkB, the pro-angiogenic VEGFR-2, and the EPO-R genes, and lower expression of the pro-apoptotic CASP3 and TP53 and pro-inflammatory IL12a genes. Moreover, they showed smaller infarct size (30% reduction in CK-MB release; p = 0.025), and a favourable pattern of left ventricular remodelling. CONCLUSIONS Short-term high-dose EPO administration in patients with AMI treated by PCI and standard anti-platelet therapy increases the levels of circulating CD34+ cells, shifts PBC gene expression towards anti-apoptotic, pro-angiogenic and anti-inflammatory pathways, and decreases infarct size. The clinical relevance of these results needs to be confirmed in specifically tailored trials.

Immune responses to the Escherichia coli dnaJ heat shock protein in juvenile rheumatoid arthritis and their correlation with disease activity

Patients with juvenile rheumatoid arthritis frequently have abnormal immune responses to the hsp65 class of bacterial heat shock proteins. However, lymphocytes from children with other inflammatory diseases may also recognize hsp65, and the role of these antigens in juvenile rheumatoid arthritis remains controversial. We have studied humoral and cellular immune responses to a distinct, recently described bacterial heat shock protein, designated dnaJ. The Escherichia coli dnaJ gene was cloned and expressed, and the purified recombinant protein was used as an antigen. Neither normal children nor children with various chronic inflammatory diseases had lymphocyte proliferative responses to recombinant dnaJ. However, lymphocytes from patients with polyarticular, pauciarticular, and systemic manifestations of juvenile rheumatoid arthritis responded strongly to the antigen. Cellular immune responses to dnaJ were higher in synovial fluid than in blood and higher in children with active disease than in children in remission. These data show that increased immune reactivity to dnaJ is characteristic of juvenile rheumatoid arthritis and that the magnitude of the immune response is linked to disease activity. The results suggest that an abnormal immune response to antigens on commensal gut bacteria may contribute to the generation of chronic inflammation in juvenile rheumatoid arthritis.

Evidence that prefibrotic myelofibrosis is aligned along a clinical and biological continuum featuring primary myelofibrosis.

Purpose In the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking. Patients and Methods We investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF. Results As compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis. Conclusion Pre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.

Endothelial colony-forming cells from patients with chronic myeloproliferative disorders lack the disease-specific molecular clonality marker

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit-endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.