A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates.

Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.

Polyautoimmunity in Patients With Anticyclic Citrullinated Peptide Antibody-Positive and -Negative Rheumatoid Arthritis: a Nationwide Cohort Study From Denmark.

OBJECTIVE: This study aimed to compare the prevalence and incidence of polyautoimmunity between anticyclic citrullinated peptide antibody (anti-CCP)-positive and anti-CCP-negative patients with rheumatoid arthritis (RA). METHODS: In a nationwide register-based cohort study, patients with RA (disease duration ≤ 2 yrs) in the DANBIO rheumatology register with an available anti-CCP test in the Register of Laboratory Results for Research were identified. The polyautoimmunity outcome included 21 nonrheumatic autoimmune diseases identified by linkage between the Danish Patient Registry and Prescription Registry. The age- and sex-adjusted prevalence ratio (PR) was calculated by modified Poisson regression to estimate the prevalence at diagnosis in anti-CCP-positive vs anti-CCP-negative patients. The hazard ratio (HR) of polyautoimmunity within 5 years of entry into DANBIO was estimated in cause-specific Cox regression models. RESULTS: The study included 5839 anti-CCP-positive and 3799 anti-CCP-negative patients with RA. At first visit, the prevalence of prespecified polyautoimmune diseases in the Danish registers was 11.1% and 11.9% in anti-CCP-positive and anti-CCP-negative patients, respectively (PR 0.93, 95% CI 0.84-1.05). The most frequent autoimmune diseases were autoimmune thyroid disease, inflammatory bowel disease, and type 1 diabetes mellitus. During a mean follow-up of 3.5 years, only a few (n = 210) patients developed polyautoimmunity (HR 0.6, 95% CI 0.46-0.79). CONCLUSION: Polyautoimmunity as captured through the Danish National Patient Registry occurred in approximately 1 in 10 patients with RA at time of diagnosis regardless of anti-CCP status. In the years subsequent to the RA diagnosis, only a few and mainly anti-CCP-negative patients developed autoimmune disease.

Markers of intestinal mucositis to predict blood stream infections at the onset of fever during treatment for childhood acute leukemia.

Despite chemotherapy-induced intestinal mucositis being a main risk factor for blood stream infections (BSIs), no studies have investigated mucositis severity to predict BSI at fever onset during acute leukemia treatment. This study prospectively evaluated intestinal mucositis severity in 85 children with acute leukemia, representing 242 febrile episodes (122 with concurrent neutropenia) by measuring plasma levels of citrulline (reflecting enterocyte loss), regenerating islet-derived-protein 3α (REG3α, an intestinal antimicrobial peptide) and CCL20 (a mucosal immune regulatory chemokine) along with the general neutrophil chemo-attractants CXCL1 and CXCL8 at fever onset. BSI was documented in 14% of all febrile episodes and in 20% of the neutropenic febrile episodes. In age-, sex-, diagnosis- and neutrophil count-adjusted analyses, decreasing citrulline levels and increasing REG3α and CCL20 levels were independently associated with increased odds of BSI (OR = 1.6, 1.5 and 1.7 per halving/doubling, all p < 0.05). Additionally, higher CXCL1 and CXCL8 levels increased the odds of BSI (OR = 1.8 and 1.7 per doubling, all p < 0.0001). All three chemokines showed improved diagnostic accuracy compared to C-reactive protein and procalcitonin. These findings underline the importance of disrupted intestinal integrity as a main risk factor for BSI and suggest that objective markers for monitoring mucositis severity may help predicting BSI at fever onset.

Citrullination of myelin basic protein induces a Th17-cell response in healthy individuals and enhances the presentation of MBP85-99 in patients with multiple sclerosis.

The post-translational modification citrullination has been proposed to play a role in the pathogenesis of multiple sclerosis (MS). Myelin basic protein (MBP) is a candidate autoantigen which is citrullinated to a minor extent under physiological conditions and hypercitrullinated in MS. We examined immune cell responses elicited by hypercitrullinated MBP (citMBP) in cultures of mononuclear cells from 18 patients with MS and 42 healthy donors (HDs). The immunodominant peptide of MBP, MBP85-99, containing citrulline in position 99, outcompeted the binding of native MBP85-99 to HLA-DR15, which is strongly linked to MS. Moreover, using the monoclonal antibody MK16 as probe, we observed that B cells and monocytes from HLA-DR15+ patients with MS presented MBP85-99 more efficiently after challenge with citMBP than with native MBP. Both citMBP and native MBP induced proliferation of CD4+ T cells from patients with MS as well as TNF-α production by their B cells and CD4+ T cells, and citrullination of MBP tended to enhance TNF-α secretion by CD4+ T cells from HLA-DR15+ patients. Unlike native MBP, citMBP induced differentiation into Th17 cells in cultures from HDs, while neither form of MBP induced Th17-cell differentiation in cultures from patients with MS. These data suggest a role for citrullination in the breach of tolerance to MBP in healthy individuals and in maintenance of the autoimmune response to MBP in patients with MS.

Gastrointestinal barrier integrity and mucosal inflammation as risk factors of blood stream infections in children treated for acute lymphoblastic leukaemia.

Chemotherapy-induced mucositis increases the risk of blood stream infections (BSI) due to translocation of bacteria across the intestinal epithelium. Our study investigated if quantitative measures of intestinal mucositis severity, including plasma citrulline (a marker of functional enterocytes) and CCL20 (an intestinal immune homeostatic chemokine), could identify patients at risk of BSI. A total of 106 children with ALL undergoing induction treatment (NOPHO ALL 2008) were included and information regarding BSI episodes was collected from the patients' medical records. Twenty-seven patients (25%) developed BSI during induction. Patients with BSI had a larger decrease in citrulline after chemotherapy than patients without BSI, and nearly all BSI episodes (25/27) occurred in the group of patients exhibiting a drop in citrulline (OR = 6.4 [95% CI: 1.4-29.3], P = .008). Patients who developed BSI had higher plasma CCL20 levels on days 8, 15 and 22 than patients without BSI (all P < .05), and elevated CCL20 levels on day 8 increased the risk of subsequent BSI (OR = 1.57 [1.11-2.22] per doubling of CCL20 level, P = .01) in a multivariable logistic regression analysis. These findings suggest that children with ALL who develop BSI during chemotherapy are characterised by more severe intestinal mucositis, as measured by plasma citrulline and CCL20. These markers may be useful in early risk stratification to guide treatment decisions.

Interferon alpha-2 treatment reduces circulating neutrophil extracellular trap levels in myeloproliferative neoplasms.

Neutrophil extracellular traps (NETs) may play a pathogenic role in the thrombosis associated with myeloproliferative neoplasms (MPNs). We measured serum NET levels in 128 pretreatment samples from patients with MPNs and in 85 samples taken after 12 months of treatment with interferon alpha-2 (PEG-IFNα-2) formulations or hydroxyurea (HU). No differences in NET levels were observed across subdiagnoses or phenotypic driver mutations. In PV, a JAK2V617F+ allele burden ≥50% associated with increased NET levels (p = 0.006). Baseline NET levels correlated with neutrophil count (r = 0.29, p = 0.001), neutrophil-to-lymphocyte ratio (r = 0.26, p = 0.004) and JAK2V617F allele burden (r = 0.22, p = 0.03), particularly in patients with PV and with allele burden ≥50% (r = 0.50, p = 0.01, r = 0.56, p = 0.002 and r = 0.45, p = 0.03 respectively). In PV, after 12 months of treatment, NET levels decreased on average by 60% in patients with allele burden ≥50%, compared to only 36% in patients with an allele burden <50%. Overall, treatment with PEG-IFNα-2a or PEG-IFNα-2b reduced NETs levels in 77% and 73% of patients, respectively, versus only 53% of HU-treated patients (average decrease across treatments: 48%). Normalization of blood counts did not per se account for these reductions. In conclusion, baseline NET levels correlated with neutrophil count, NLR and JAK2V617F allele burden, and IFNα was more effective at reducing prothrombotic NET levels than HU.

Markers of neutrophil chemotaxis for identification of blood stream infections in children with acute lymphoblastic leukemia undergoing induction treatment.

BACKGROUND: Although neutropenic fever is frequently observed during chemotherapy, only a minor proportion is caused by blood stream infections (BSI). This study investigated measurements of neutrophil chemotaxis as risk markers for BSI in children with acute lymphoblastic leukemia (ALL). METHODS: The chemokines CXCL1 and CXCL8 were measured weekly in 106 children with ALL during induction treatment. Information regarding BSI episodes was collected from the patients' medical records. RESULTS: During induction treatment, 102 (96%) patients developed profound neutropenia and 27 (25%) were diagnosed with BSI, debuting on median day 12 (range: 4-29). Patients developing BSI had increased levels of CXCL1 on days 8 and 15 as well as increased CXCL8 on days 8, 15, 22, and 29 compared to patients without BSI (all p < 0.05). Patients with BSI < day 12 exhibited increased CXCL1 and CXCL8 levels as early as day 8 (81 vs. 4 pg/mL, p = 0.031 and 35 vs. 10 pg/mL, p < 0.0001, respectively), while CXCL1 and CXCL8 were increased on day 15 (215 vs. 57 pg/mL, p = 0.022 and 68 vs. 17 pg/mL, p = 0.0002) and after (all p < 0.01) in patients with BSI ≥ day 12. CONCLUSION: The markers of neutrophil chemotaxis, CXCL1, and CXCL8 may help to identify patients at increased risk of BSI during chemotherapy-induced neutropenia.

Plasma Levels of MRP-8/14 Associate With Neutrophil Recovery, Bacterial Bloodstream Infections, and Engraftment Syndrome Following Pediatric Allogeneic Hematopoietic Stem Cell Transplantation.

Neutrophil engraftment is essential for the successful outcome after allogeneic hematopoietic stem cell transplantation (HSCT), but neutrophil activation may also induce transplant-related complications. Myeloid-related protein (MRP)-8/14 is expressed in granulocytes during inflammatory conditions and secreted in response to tissue damage along with the release of pro-inflammatory cytokines together with leukocyte recruitment and activation. In this study, we investigated associations between levels of the neutrophil activition marker MRP-8/14, neutrophil recovery and toxicities after pediatric HSCT. We included 73 children undergoing allogeneic HSCT using bone marrow or peripheral blood stem cell grafts from matched sibling or unrelated donors. Plasma levels of MRP-8/14 were measured by enzyme-linked immunosorbent assay from preconditioning until 6 months after transplantation. Overall, MRP-8/14 levels decreased from pre-conditioning to a nadir at day 7 and then rose again until day 28, preceding the reappearance of neutrophils. MRP-8/14 levels were significantly reduced at day 14 in patients with delayed neutrophil engraftment compared with patients who engrafted by day 21 (0.20 versus 0.48 µg/mL, P = .0012) and in patients who developed bacterial bloodstream infections compared to patients without this complication (0.2 versus 0.36 µg/mL, P = .048). Patients developing engraftment syndrome had significantly elevated MRP-8/14 levels at day 7 and 21 compared to patients without engraftment syndrome (0.32 versus 0.2 µg/mL, P = .042 and 1.9 versus 0.80 µg/mL, P = .039, respectively), as well as increased neutrophil counts from day 9 to 25 (P ≤ .016). Similarly, neutrophil counts were increased at day 13 to 17 in patients with acute graft-versus-host disease grade III-IV compared with grade 0-II. This study is the first to monitor neutrophil activation by MRP-8/14 in HSCT patients in relation to infectious, as well as noninfectious post-transplantation complications. Our results provide increased insights into the pathophysiology of these complications, and further studies should explore the potential use of MRP-8/14 as a clinically useful biomarker.

B-cell cytokine responses to Porphyromonas gingivalis in patients with periodontitis and healthy controls.

BACKGROUND: Cytokine-producing B cells play a well-established role in modifying immune responses in chronic inflammatory diseases. We characterized B-cell cytokine responses against periodontitis-associated bacteria in patients with periodontitis. METHODS: Blood and saliva samples were collected from patients with periodontitis grade B (N = 31) or grade C (N = 25), and 25 healthy controls (HCs). Mononuclear cells were stimulated with Porphyromonas gingivalis, Fusobacterium nucleatum, Staphylococcus epidermidis, or Cutibacterium acnes, and B-cell production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-γ, IL-10 and transforming growth factor (TGF)-ß by B cells was assessed by flow cytometry. RESULTS: HCs had higher baseline frequencies of B cells producing IFN-γ or TNF-α than grade B patients, but only B cells from grade B patients showed significant differentiation into IFN-γ-, TNF-α-, TGF-ß-, or IL-10-producing cells after challenge with P. gingivalis and into IFN-γ-, TGF-ß-, or IL-10-producing cells after challenge F. nucleatum. Notably, the baseline frequency of IL-10-producing B cells from grade C patients correlated inversely with clinical attachment loss (AL). The major proportion of the IFN-γ- and TGF-ß-producing B cells were CD27+ memory cells, while the IL-10-producing B cells were mainly CD27- CD5- . CONCLUSIONS: B cells from grade B patients, particularly those harboring P. gingivalis, showed proinflammatory B-cell responses to P. gingivalis. Moreover, the baseline frequency of IL-10-producing B cells in the grade C group correlated inversely with AL, suggesting a diminished immunoregulatory capacity of IL-10-producing B cells in these patients.

Insulin-like growth factor-1 and insulin-like growth factor binding protein-3: impact on early haematopoietic reconstitution following allogeneic haematopoietic stem cell transplantation.

OBJECTIVES: The aim of the study was to investigate whether high endogenous levels of insulin-like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3) were related to a faster reconstitution of different blood cell populations in the early phase after allogeneic myeloablative haematopoietic stem cell transplantation (HSCT). METHODS: We measured IGF-1 and IGFBP-3 by chemiluminescence during the first three weeks after transplantation in 35 adult patients undergoing myeloablative HSCT and calculated area under the curve divided by time (AUC/t) for each patient. RESULTS: Circulating levels of IGF-1 and IGFBP-3 correlated with counts of reticulocytes (rs  = 0.44, p = .011 and r = 0.41, p = .017, respectively) and thrombocytes (rs  = 0.38, p = .030 and rs  = 0.56, p = .0008) three weeks post-transplant. Furthermore, high IGFBP-3 levels correlated with absolute lymphocyte counts 3 weeks post-HSCT (rs  = 0.54, p = .012) and were associated with shorter time to neutrophil engraftment (rs  = -0.35, p = .043). Both IGF-1 and IGFBP-3 levels were associated with the number of circulating natural killer cells one month after HSCT (rs  = 0.42, p = .032 and rs  = 0.57, p = .0026). CONCLUSION: These data indicate that high levels of IGF-1 and IGFBP-3 relate to a faster haematopoietic reconstitution after HSCT and suggest a biological influence of these mediators in haematopoietic homeostasis in these patients.

Circulating antibodies against leukotoxin A as marker of periodontitis Grades B and C and oral infection with Aggregatibacter actinomycetemcomitans.

BACKGROUND: The facultative bacterium Aggregatibacter actinomycetemcomitans (Aa) is strongly associated with periodontitis and is occasionally found in periodontally healthy subjects. We aimed to determine the prevalence of salivary Aa among patients with either periodontitis Grade B (periodontitis-B) or Grade C (periodontitis-C), periodontally healthy controls (HCs), and to determine if systemic antibodies against Aa or its virulence factor leukotoxin A (LtxA) may serve as biomarkers that reveal the oral presence of the bacterium and discriminate subjects with periodontitis-C, periodontitis-B, or no periodontitis from each other. METHODS: Serum and unstimulated saliva samples were collected from patients with periodontitis-C (n = 27), patients with periodontitis-B (n = 34), and HCs (n = 28). Serum level of immunoglobulin G antibodies to fragmented whole Aa and to LtxA were quantified using a bead-based assay. Aa was identified in saliva using quantitative polymerase chain reaction (qPCR). All analyses were adjusted for age, sex, and current smoking status. RESULTS: Aa was present in saliva from 11% of HCs, in 32% of patients with periodontitis-B (P = 0.04 versus HCs), and in 37% of patients with periodontitis-C (P = 0.02 versus HCs). Serum antibodies to fragments of Aa associated significantly with periodontitis-C (P = 0.03), while serum anti-LtxA antibodies associated with both periodontitis-B and periodontitis-C (P = 0.002 and P = 9×10-4 , respectively). Moreover, a significant association between serum anti-LtxA antibodies and Aa count in saliva was observed (P = 0.001). On the basis of serum anti-LtxA antibody levels, patients with periodontitis could be discriminated from HCs (AUC = 0.74 in ROC curve-analysis, P = 0.0003), and carriers of Aa could be discriminated from non-carriers (AUC = 0.78, P <0.0001). CONCLUSIONS: Aa is highly prevalent in saliva of patients with periodontitis-B or periodontitis-C. Systemic immunoglobulin G antibodies against LtxA distinguish patients with periodontitis, regardless of grade, from HCs, while their quantity reflects the concurrent bacterial burden in the oral cavity.

Elevated levels of oxidized nucleosides in individuals with the JAK2V617F mutation from a general population study.

It is unknown if the somatic mutations in chronic myeloproliferative neoplasms (MPNs), JAK2V617F and Calreticulin, are associated with oxidative stress, or impaired mitochondrial defense against reactive oxygen species. In the Danish General Suburban Population Study (GESUS), including 116 JAK2V617F-mutated, 8 CALR-mutated, and 3310 mutation-negative participants without overt MPN, and in a study of 39 patients with myelofibrosis, the most advances type of MPNs, and 179 matched controls, we compared the urinary concentration of oxidized nucleosides - 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) - as markers of oxidative stress. In GESUS, we performed Mendelian randomization analyses, using the Ala16Val single nucleotide polymorphism in the superoxide dismutase2 (SOD2) gene. In the multivariate analyses in GESUS, the 8-oxodG and 8-oxoGuo concentration were 13% (95%CI: 6-21%, p < 0.001) and 6% (95%CI: 0.4-11%, p = 0.035) higher in mutation-positive than in mutation-negative participants, respectively. Each SOD2 T allele was associated with an odds ratio of being mutation-positive of 1.69 (95%CI: 1.12-2.55, p = 0.013) through 8-oxodG. The 8-oxodG and 8-oxoGuo concentrations were 77% (95%CI: 49-110%, p < 0.001) and 105% (95%CI: 80-133%, p < 0.001) higher in myelofibrosis patients than in controls, respectively. In conclusion, an impaired mitochondrial antioxidative defense, that is causatively associated with markers of oxidative stress, may contribute to the development of mutations associated with MPNs.

Protein array-based companion diagnostics in precision medicine.

INTRODUCTION: The development of companion diagnostics (CDx) will increase efficacy and cost-benefit markedly, compared to the currently prevailing trial-and-error approach for treatment. Recent improvements in high-throughput protein technology have resulted in large amounts of predictive biomarkers that are potentially useful components of future CDx assays. Current high multiplex protein arrays are suitable for discovery-based approaches, while low-density and more simple arrays are suitable for use in point-of-care facilities. AREA COVERED: This review discusses the technical platforms available for protein array focused CDx, explains the technical details of the platforms and provide examples of clinical use, ranging from multiplex arrays to low-density clinically applicable arrays. We thereafter highlight recent predictive biomarkers within different disease areas, such as oncology and autoimmune diseases. Lastly, we discuss some of the challenges connected to the implementation of CDx assays as point-of-care tests. EXPERT OPINION: Recent advances in the field of protein arrays have enabled high-density arrays permitting large biomarker discovery studies, which are beneficial for future CDx assays. The density of protein arrays range from a single protein to proteome-wide arrays, allowing the discovery of protein signatures that may correlate with drug response. Protein arrays will undoubtedly play a key role in future CDx assays.

B-cell frequencies and immunoregulatory phenotypes in myeloproliferative neoplasms: Influence of ruxolitinib, interferon-α2, or combination treatment.

OBJECTIVE: Given a proposed role for PD-L1+ and IL-10-producing B-cell subsets in promoting certain cancers, we sought to characterize the frequency and phenotype of B cells in patients with chronic myeloproliferative neoplasms (MPNs) and the influence of ruxolitinib and interferon-α2 therapy. METHODS: We analyzed B-cell frequencies and phenotype in patients with MPNs (n = 107), before and during treatment with ruxolitinib (n = 29), interferon-α2 (n = 21), or the two drugs in combination (COMBI; n = 42) and healthy donors (HDs; n = 52) using flow cytometry. RESULTS: Myelofibrosis patients had lower lymphocyte counts and proportions of B cells than patients with essential thrombocythemia or polycythemia vera and HDs. The B-cell count correlated inversely with JAK2-V617F allele burden and spleen size and increased after ruxolitinib or COMBI treatment. The proportions of PD-L1+ B cells and PD-1+ B cells were significantly higher in patients with myelofibrosis or polycythemia vera than in HDs and decreased during ruxolitinib and COMBI treatment. The proportions of TNF-α+ and IL-6+ B cells were elevated in myelofibrosis patients. The proportion of IL-6+ B cells decreased, and the proportion of IL-10+ B cells increased during ruxolitinib treatment. CONCLUSION: B-cell frequency and phenotype were altered in MPN patients. Ruxolitinib therapy had marked effects on both frequency and phenotype.

Inflammatory functional iron deficiency common in myelofibrosis, contributes to anaemia and impairs quality of life. From the Nordic MPN study Group.

OBJECTIVES: The study investigates the hypothesis that inflammation in myelofibrosis (MF) like in myeloma and lymphoma, may disturb iron distribution and contribute to anaemia. METHODS: A cross-sectional study of 80 MF and 23 ET patients was performed. RESULTS: About 35% of anaemic MF patients had functional iron deficiency (FID) with transferrin saturation <20 and normal or elevated S-ferritin (<500 µg/L). In ET, FID was rare. In MF patients with FID, 70.6% were anaemic, vs 29.4% in patients without FID (P = 0.03). Hepcidin was significantly higher in MF patients with anaemia, including transfusion-dependent patients, 50.6 vs 24.4 µg/L (P = 0.01). There was a significant negative correlation between Hb and inflammatory markers in all MF patients: IL-2, IL-6 and TNF-α, (P < 0.01-0.03), LD (P = 0.004) and hepcidin (P = 0.03). These correlations were also seen in the subgroup of anaemic MF patients (Table ). Tsat correlated negatively with CRP (P < 0.001). Symptom burden was heavier in MF patients with FID, and MPN-SAF quality of life scores correlated with IL-6 and CRP. CONCLUSIONS: The inflammatory state of MF disturbs iron turnover, FID is common and contributes to anaemia development and impairment of QoL. Anaemic MF patients should be screened for FID.

Development of anti-drug antibodies is associated with shortened survival in patients with metastatic melanoma treated with ipilimumab.

Introduction: Checkpoint inhibitors, including the CTLA-4 blocking antibody ipilimumab, have become the new standard therapy for many metastatic cancers. Development of anti-drug antibodies (ADAs) after treatment with other biopharmaceuticals has been thoroughly described, but the induction of ADAs after treatment with checkpoint inhibitors has been inadequately investigated. In this retrospective study, we relate ipilimumab serum levels and anti-ipilimumab antibody levels to clinical outcomes in patients with metastatic melanoma (MM). Method: Serum samples from 31 patients with MM were analyzed for serum levels of ipilimumab and ADAs to ipilimumab at baseline, and before the 2nd and 4th infusion using an in-house bead-based assay. The results were correlated with progression-free survival (PFS) and overall survival (OS). Results: Low serum levels of ipilimumab before the 2nd infusion correlated significantly with a shorter OS (p = 0.01) and PFS (p = 0.02). Eight patients (26%) were ADA-positive at either timepoint. ADA positivity correlated significantly with a shorter OS (p = 0.03) with a hazard ratio (HR) of 3.0 (95% CI: 1.2-7.8). Four of 8 ADA-positive patients (50%) discontinued therapy before the 4th infusion due to disease progression, compared to three of 23 (13%) ADA-negative patients. Conclusion: We confirm that low serum levels of ipilimumab are associated with a shortened OS, and we show for the first time that ADAs to ipilimumab are associated with shorter OS in patients with MM.

Effects of rituximab and dexamethasone on regulatory and proinflammatory B-cell subsets in patients with primary immune thrombocytopenia.

OBJECTIVE: To investigate the cytokine production and surface marker composition of B cells in adult patients with newly diagnosed primary immune thrombocytopenia (ITP) before and 12 months after treatment with rituximab + dexamethasone (RTX+DXM) or dexamethasone (DXM). METHODS: Peripheral blood mononuclear cells were isolated from nine patients treated with RTX+DXM, seven patients treated with DXM, and seven healthy donors. Expression of the cell-surface markers CD5, CD27, CD25, and CD19, and intracellular content of IL-6 and IL-10 were measured by flow cytometry. RESULTS: PBMCs from ITP patients at baseline contained a lower proportion of IL-10+ B cells (P < .01) and IL-6+ B cells (P < .01) than healthy controls. All patients responded to therapy and levels were normalized at 12 months. The proportion of CD5+ B cells increased (P < .01) and CD27+ memory B cells decreased (P < .05) 12 months after treatment with RTX+DXM compared to baseline, with an inverse correlation between platelet numbers and the proportion of CD27+ B cells (R = -0.71; P < .05). CONCLUSION: Both treatment regimens normalized the frequencies of cytokine-producing B cells. The additional increase in CD5+ B cells after RTX+DXM is compatible with induction of Bregs.

Reconstitution of Th17, Tc17 and Treg cells after paediatric haematopoietic stem cell transplantation: Impact of interleukin-7.

Successful reconstitution of T lymphocytes after allogeneic haematopoietic stem cell transplantation (HSCT) is needed to establish the graft-versus-leukaemia effect and an effective anti-microbial defense, but the ratio between functionally different T-cell subsets needs to be balanced to avoid graft-versus-host disease (GVHD). IL-7 is essential for T-cell generation in the thymus and peripheral T-cell homeostasis. High IL-7 levels have been associated with impaired T-cell reconstitution, increased risk of acute GVHD and treatment-related mortality, but the underlying cellular mechanisms behind these associations have not been investigated previously. We hypothesized that increased levels of IL-7 post-transplant alters the balance between immune-regulatory T cell subsets during the post-transplant lymphocyte recovery towards a more pro-inflammatory profile. We quantified Th17 cells, Tc17 cells and Tregs in 29 children following HSCT. Th17 cell and Treg counts rose significantly from day +90 to +180 post-HSCT, and prior acute GVHD was associated with significant changes in the concentration of Tregs (9.4×106/L vs. 1.3×106/L, P=0.0052) and the Th17/Treg ratio (1.5 vs. 4.2, P=0.025). The plasma level of IL-7 at day +90 correlated inversely with Th17 cell counts (rs=-0.65, P=0.0002) and the proportion of Tc17 cells (rs=0.64, P=0.0005) at day +90, but not with Tregs. Furthermore, high IL-7 levels at day +7 were predictive of a less naïve T-cell phenotype at day +90. These findings add further evidence that IL-7 is a key regulatory factor that may tune the balance between functionally different T-cell subsets following HSCT.

Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation.

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.

Cholesterol crystals enhance TLR2- and TLR4-mediated pro-inflammatory cytokine responses of monocytes to the proatherogenic oral bacterium Porphyromonas gingivalis.

Cholesterol deposits and pro-inflammatory cytokines play an essential role in the pathogenesis of atherosclerosis, a predominant cause of cardiovascular disease (CVD). Epidemiological evidence has linked periodontal disease (PD) with atherosclerotic CVD. Accordingly, viable periodontal pathogens, including Porphyromonas gingivalis, have been found in atherosclerotic plaques in humans and mice. We aimed to determine whether cholesterol crystals (CHCs) and oral bacteria synergize in the stimulation of human monocytes. Incubation of human monocytes with CHCs induced secretion of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, and IL-8. Moreover, CHCs markedly enhanced secretion of IL-1ß by monocytes stimulated with the toll-like receptor (TLR) 4 agonist Escherichia coli lipopolysaccharide (LPS), and the TLR2 agonist Staphylococcus aureus lipoteichoic acid. Notably, CHCs also enhanced IL-1ß secretion induced by P. gingivalis LPS and IL-1ß secretion induced by whole P. gingivalis bacteria. This enhancement was abrogated by the NLRP3 inflammasome inhibitors Z-YVAD-FMK and glibenclamide. CHCs had no effect on cytokine production induced by P. gingivalis gingipains. Taken together, our findings support that CHCs, via stimulation of NLRP3 inflammasomes, act in synergy with the periodontal pathogen P. gingivalis to promote monocyte secretion of pro-atherogenic cytokines.