Practical Details for the Detection and Interpretation of Cryoglobulins.

BACKGROUND: Cryoglobulins are immunoglobulins that precipitate at low temperature. Strict preanalytical and analytical conditions are critical for the detection of cryoglobulins. CONTENT: This review will focus on practical recommendations for detection and characterization of cryoglobulins and the technical problems that may be encountered. A laboratory report format is proposed for presentation of these results that includes the parameters necessary for an optimal interpretation by clinicians. The first step of detection of cryoglobulins can be performed in any laboratory that has a 37 °C incubator and temperature-controlled centrifuge. The second step is the characterization of cryoglobulins, and this often must be performed in more specialized laboratories. Characterization includes immunoglobulin typing, for the classification of cryoglobulins and potential underlying disease(s); quantification of immunoglobulins and rheumatoid factor in the cryoprecipitate to define the pathogenicity; and quantification of serum complement, which is useful for diagnosis. SUMMARY: These practical recommendations will be useful for the accurate detection of cryoglobulins, an essential step for the diagnosis of cryoglobulinemic vasculitis, a rare but severe clinical manifestation of cryoglobulins.

Insights into the immunological description of cryoglobulins with regard to detection and characterization in Slovenian rheumatological patients.

The detection of cryoglobulins (CG) used to diagnose cryoglobulinemic vasculitis requires strict adherence to protocol, with emphasis on the preanalytical part. Our main objectives were to introduce a more sensitive and specific protocol for the detection of CG and to characterize CG in Slovenian patients diagnosed with cryoglobulinemic vasculitis, other vasculitides, connective tissue diseases or non-rheumatic diseases examined at the Department of Rheumatology (University Medical Centre Ljubljana). Samples were routinely analyzed for the presence of CG with the protocol using the Folin-Ciocalteu reagent. In the newly introduced protocol, the type of CG was determined by immunofixation on visually observed positive samples and the concentration of CG in the cryoprecipitate and rheumatoid factor (RF) activity were measured by nephelometry. RF, C3c and C4 were measured in patients` serum and a decision tree analysis was performed using all results. The agreement between negative and positive results between the two protocols was 86%. Of the 258 patient samples tested, we found 56 patients (21.7%) with positive CG (37.5% - type II, 62.5% - type III). The RF activity was observed in 21.4% of CG positive subjects. The median concentration of type II CG was significantly higher than that of type III CG (67.4 mg/L vs. 45.0 mg/L, p = 0.037). Patients with type II had lower C4 concentrations and higher RF compared to patients with type III CG. In the decision tree, C4 was the strongest predictor of cryoglobulinemia in patients. With the newly implemented protocol, we were able to improve the detection and quantification of CG in the samples of our rheumatology patients and report the results to adequately support clinicians.

Cryoglobulinemia in systemic lupus erythematosus: a retrospective study of 213 patients.

OBJECTIVES: The clinical value of cryoglobulinemia (CG) in systemic lupus erythematosus (SLE) is largely unknown. The aim of this retrospective study was to describe the characteristics of CG in SLE, its impact on SLE phenotype, and the features associated with cryoglobulinemic vasculitis (CryoVas) in SLE patients. METHODS: This retrospective study conducted in a French university hospital reviewed the data from 213 SLE patients having been screened for CG between January 2013 and December 2017. SLE patients positive for CG were compared to SLE patients without CG. Patients were classified as CryoVas using the criteria of De Vita et al. RESULTS: Of the 213 SLE patients included (mean age 29.2 years, female sex 85%), 142 (66%) had at least one positive CG in their history, 67% of them having a persistent CG at follow-up. CG was type III in 114 (80%) cases and type II in 27 (19%) cases. The mean concentration of the cryoprecipitate was 40mg/L (range 0-228). Patients with CG had significantly more C4 consumption. Among patients with CG, 21 (15%) developed a CryoVas. The clinical manifestations of patients with CryoVas were mainly cutaneous (purpura, ulcers, digital ischemia) and articular, without any death at follow-up. Severe manifestations of CG included glomerulonephritis in 1/21 (5%) patients and central nervous system involvement in 4/21 (19%) patients. A response to first-line treatments was observed in 12/13 (92%) patients, but relapses were observed for 3 of them. CONCLUSION: CG is frequent in SLE, but mostly asymptomatic. CryoVas features involve mostly joints, skin, and general symptoms. CryoVas in SLE appears to be a specific condition, with a low prevalence of neuropathy, membranoproliferative glomerulonephritis, and severe manifestations.

Maternal exposure to air pollution before and during pregnancy related to changes in newborn's cord blood lymphocyte subpopulations. The EDEN study cohort.

BACKGROUND: Toxicants can cross the placenta and expose the developing fetus to chemical contamination leading to possible adverse health effects, by potentially inducing alterations in immune competence. Our aim was to investigate the impacts of maternal exposure to air pollution before and during pregnancy on newborn's immune system. METHODS: Exposure to background particulate matter less than 10 µm in diameter (PM10) and nitrogen dioxide (NO2) was assessed in 370 women three months before and during pregnancy using monitoring stations. Personal exposure to four volatile organic compounds (VOCs) was measured in a subsample of 56 non-smoking women with a diffusive air sampler during the second trimester of pregnancy. Cord blood was analyzed at birth by multi-parameter flow cytometry to determine lymphocyte subsets. RESULTS: Among other immunophenotypic changes in cord blood, decreases in the CD4+CD25+ T-cell percentage of 0.82% (p = 0.01), 0.71% (p = 0.04), 0.88% (p = 0.02), and 0.59% (p = 0.04) for a 10 µg/m3 increase in PM10 levels three months before and during the first, second and third trimester of pregnancy, respectively, were observed after adjusting for confounders. A similar decrease in CD4+CD25+ T-cell percentage was observed in association with personal exposure to benzene. A similar trend was observed between NO2 exposure and CD4+CD25+ T-cell percentage; however the association was stronger between NO2 exposure and an increased percentage of CD8+ T-cells. CONCLUSIONS: These data suggest that maternal exposure to air pollution before and during pregnancy may alter the immune competence in offspring thus increasing the child's risk of developing health conditions later in life, including asthma and allergies.

[Accreditation of cryoglobulins: Experience of CHU of Lyon]. / Accréditation des cryoglobulines : retour d'expérience du CHU de Lyon.

Research and identification of a serum cryoglobulin is a current laboratory test. This analysis is complex to accredit due to, firstly, very strict pre-analytical requirements, secondly, lack of standardization between laboratories in carrying out the different phases of sampling until results validation. The method validation of this analysis, carried out to meet requirements of the NF EN ISO 15189 standard, is a complex process divided into three sub-processes: cryoglobulin research including detection and isolation of cryoprecipitate, typing by immunofixation and quantification of cryoglobulin by immunoglobulin assays. This work made it possible, thanks to the development of a risk matrix taking into account all pre-analytical, analytical and post-analytical phases, to stress the importance of technical proficiency and management of critical equipment . With this approach, the laboratory also checked homogeneity of results validation practices.

A soluble form of the triggering receptor expressed on myeloid cells-1 modulates the inflammatory response in murine sepsis.

The triggering receptor expressed on myeloid cells (TREM)-1 is a recently discovered receptor expressed on the surface of neutrophils and a subset of monocytes. Engagement of TREM-1 has been reported to trigger the synthesis of proinflammatory cytokines in the presence of microbial products. Previously, we have identified a soluble form of TREM-1 (sTREM-1) and observed significant levels in serum samples from septic shock patients but not controls. Here, we investigated its putative role in the modulation of inflammation during sepsis. We observed that sTREM-1 was secreted by monocytes activated in vitro by LPS and in the serum of animals involved in an experimental model of septic shock. Both in vitro and in vivo, a synthetic peptide mimicking a short highly conserved domain of sTREM-1 appeared to attenuate cytokine production by human monocytes and protect septic animals from hyper-responsiveness and death. This peptide seemed to be efficient not only in preventing but also in down-modulating the deleterious effects of proinflammatory cytokines. These data suggest that in vivo modulation of TREM-1 by sTREM peptide might be a suitable therapeutic tool for the treatment of sepsis.

Contribution of rare and predicted pathogenic gene variants to childhood-onset lupus: a large, genetic panel analysis of British and French cohorts.

BACKGROUND: Systemic lupus erythematosus (SLE) is a rare immunological disorder and genetic factors are considered important in its causation. Monogenic lupus has been associated with around 30 genotypes in humans and 60 in mice, while genome-wide association studies have identified more than 90 risk loci. We aimed to analyse the contribution of rare and predicted pathogenic gene variants in a population of unselected cases of childhood-onset SLE. METHODS: For this genetic panel analysis we designed a next-generation sequencing panel comprising 147 genes, including all known lupus-causing genes in humans, and potentially lupus-causing genes identified through GWAS and animal models. We screened 117 probands fulfilling American College of Rheumatology (ACR) criteria for SLE, ascertained through British and French cohorts of childhood-onset SLE, and compared these data with those of 791 ethnically matched controls from the 1000 Genomes Project and 574 controls from the FREX Consortium. FINDINGS: After filtering, mendelian genotypes were confirmed in eight probands, involving variants in C1QA, C1QC, C2, DNASE1L3, and IKZF1. Seven additional patients carried heterozygous variants in complement or type I interferon-associated autosomal recessive genes, with decreased concentrations of the encoded proteins C3 and C9 recorded in two patients. Rare variants that were predicted to be damaging were significantly enriched in the childhood-onset SLE cohort compared with controls; 25% of SLE probands versus 5% of controls were identified to harbour at least one rare, predicted damaging variant (p=2·98 × 10-11). Inborn errors of immunity were estimated to account for 7% of cases of childhood-onset SLE, with defects in innate immunity representing the main monogenic contribution. INTERPRETATION: An accumulation of rare variants that are predicted to be damaging in SLE-associated genes might contribute to disease expression and clinical heterogeneity. FUNDING: European Research Council.

Predictive factors of renal involvement in cryoglobulinaemia: a retrospective study of 153 patients.

BACKGROUND: The course of cryoglobulinaemia varies widely, from asymptomatic patients to severe vasculitis syndrome. Renal involvement (RI) is the major prognostic factor, and frequently occurs several years after diagnosis. However, predictive factors for RI are not well known. The aim of our study was to identify RI predictive factors during cryoglobulinaemia. METHODS: We retrospectively reviewed the clinical charts of a consecutive series of 153 patients positive for cryoglobulinaemia in the University Hospital of Lyon (France). RI was defined either histologically or biologically if cryoglobulinaemia was the only possible cause of nephropathy. RESULTS: Among the 153 positive patients (mean age 55 years, 37% male), cryoglobulinaemia was associated with RI in 45 (29%) patients. Sixty-five percent of patients had Type II cryoglobulinaemia, 28% had Type III and 7% had Type I. Autoimmune diseases were the most common aetiology (48%), followed by infectious diseases (18%) and lymphoproliferative disorders (13%). Membranoproliferative glomerulonephritis was the main histological pattern (93% of the 14 histological analyses). A multivariable logistic regression showed that Type II cryoglobulinaemia, a high serum cryoglobulin concentration, the presence of an IgG kappa monoclonal component and diabetes were independently associated with the risk for developing RI. CONCLUSION: We identified several factors predictive of RI in patients with cryoglobulinaemia, which were different from the diagnostic criteria for cryoglobulinaemic vasculitis. This could suggest a specific pathophysiology for RI. We suggest performing an extensive renal monitoring and ensure nephroprotection when a diagnosis of cryoglobulinaemia is made in patients with these predictive factors.

Better Control of Holder Pasteurization Results in Higher Retention of Human Milk Lactoferrin, IgA, and Lysozyme.

Background: Holder pasteurization is commonly used in milk banks. We previously reported that the pattern of temperature and time may be different according to the pasteurizer used. Aim: The aim of our study was to assess the variances in pasteurization using two different devices: a standard pasteurizer (Past STD) and an optimized pasteurizer (Past OPTI). Methods: Immunoglobulin A (IgA), lactoferrin (LF), and lysozyme (LZ) content were assessed before and after pasteurization of 24 donor human milk samples. The impact of the pasteurization device was evaluated by testing 50- to 200-mL samples. Results: Mean temperature and duration of the plateau were 1.5°C lower and 11 min shorter, respectively, with Past OPTI vs. Past STD. The loss of IgA, LF, and LZ was 17.6, 5.6, and 9.8% lower, respectively, with Past OPTI than with Past STD. Conclusions: Accurate control of temperature enabled better preservation of IgA, LF, and LZ in donor milk. Holder pasteurization should be optimized, and new techniques proposed to treat donor milk should be compared with Holder pasteurization performed with a well-controlled device under realistic conditions.

Cryoglobulins: An update on detection, mechanisms and clinical contribution.

Cryoglobulins are immunoglobulins precipitating in cold condition. They are classified in 3 types according to the Brouet classification and may lead to vasculitis of small and medium size vessels. Vasculitis is related to vessel obstruction by monoclonal cryoglobulin aggregates in type I cryoglobulins and immune complex deposition in type II and III mixed cryoglobulins. This phenomenon is favored by low temperature, especially in skin, joints, and peripheral nerves, or increased cryoglobulin concentration in kidneys. For their detection, collection and clotting at 37°C are critical pre-analytical conditions. Cryoglobulin characterization and quantification are important to identify the underlying disease. Since detection and identification of cryoglobulins lack standardization, a protocol for such detection, characterization and quantification is proposed.

Endothelial chimerism and vascular sequestration protect pancreatic islet grafts from antibody-mediated rejection.

Humoral rejection is the most common cause of solid organ transplant failure. Here, we evaluated a cohort of 49 patients who were successfully grafted with allogenic islets and determined that the appearance of donor-specific anti-HLA antibodies (DSAs) did not accelerate the rate of islet graft attrition, suggesting resistance to humoral rejection. Murine DSAs bound to allogeneic targets expressed by islet cells and induced their destruction in vitro; however, passive transfer of the same DSAs did not affect islet graft survival in murine models. Live imaging revealed that DSAs were sequestrated in the circulation of the recipients and failed to reach the endocrine cells of grafted islets. We used murine heart transplantation models to confirm that endothelial cells were the only accessible targets for DSAs, which induced the development of typical microvascular lesions in allogeneic transplants. In contrast, the vasculature of DSA-exposed allogeneic islet grafts was devoid of lesions because sprouting of recipient capillaries reestablished blood flow in grafted islets. Thus, we conclude that endothelial chimerism combined with vascular sequestration of DSAs protects islet grafts from humoral rejection. The reduced immunoglobulin concentrations in the interstitial tissue, confirmed in patients, may have important implications for biotherapies such as vaccines and monoclonal antibodies.

Surface triggering receptor expressed on myeloid cells 1 expression patterns in septic shock.

OBJECTIVE: To analyze the pattern of cell-surface expression of the triggering receptor expressed on myeloid cells (TREM) 1 during septic shock. DESIGN AND SETTING: Prospective clinical study in an adult 16-bed medical ICU. PATIENTS AND METHODS: 25 septic shock patients, 15 patients with shock of noninfectious origin and 7 healthy volunteers. Arterial blood was drawn within 12 h of admission and subjected to flow cytometry analysis after staining with anti-TREM-1 and anti-CD14 antibodies. Repeated sampling was performed on days 2, 3, 5, 7, and 14 in septic shock patients. RESULTS: Monocytic TREM-1 expression was significantly higher in septic shock patients (mean fluorescence intensity 2.3+/-0.2) than in nonseptic patients (1.0+/-0.1), and healthy volunteers (1.0+/-0.1). There was no difference in monocytic TREM-1 expression between nonseptic patients and healthy volunteers or between any of the three groups with respect to TREM-1 expression on neutrophils. The time course of TREM-1 expression on monocytes diverged significantly by day 3 between survivors and ns. CONCLUSIONS: The specificity of TREM-1 regulation by infection is highlighted. Moreover, surface TREM-1 expression on monocytes may prove useful in allowing the follow-up of septic patients during the course of the disease.

Plasma level of a triggering receptor expressed on myeloid cells-1: its diagnostic accuracy in patients with suspected sepsis.

BACKGROUND: Previous experimental studies have suggested that the triggering receptor expressed on myeloid cells-1 (TREM-1) is specifically upregulated in the presence of microbial products. OBJECTIVE: To evaluate the diagnostic value of plasma levels of the soluble form of TREM-1 in patients admitted with clinical suspicion of infection. DESIGN: Prospective, noninterventional study conducted between July and September 2003. SETTING: Medical adult intensive care unit at a university hospital in France. PARTICIPANTS: 76 consecutive newly admitted patients who presented with clinically suspected infection and fulfilled at least 2 criteria of the systemic inflammatory response syndrome. MEASUREMENTS: Sensitivity and specificity of plasma soluble TREM-1 levels at admission for the diagnosis of infection. Two independent intensivists blinded to the results of soluble TREM-1 assays retrospectively classified patients as having the systemic inflammatory response syndrome, sepsis, severe sepsis, or septic shock. RESULTS: The systemic inflammatory response syndrome was diagnosed in 29 patients (38%), and sepsis, severe sepsis, or septic shock was diagnosed in the remaining 47 (62%). A plasma soluble TREM-1 level higher than 60 ng/mL was more accurate than any other clinical or laboratory finding for indicating infection (sensitivity, 96% [95% CI, 92% to 100%]; specificity, 89% [CI, 82% to 95%]; positive likelihood ratio, 8.6 [CI, 3.8 to 21.5]; negative likelihood ratio, 0.04 [CI, 0.01 to 0.2]). LIMITATIONS: The study did not enroll patients with mild infections not requiring intensive care unit hospitalization, patients older than 80 years of age, or patients who were immunocompromised. CONCLUSION: In newly admitted critically ill patients, measurement of plasma levels of soluble TREM-1 could help to rapidly identify those with infection.

C-reactive protein (CRP) increases VEGF-A expression in monocytic cells via a PI3-kinase and ERK 1/2 signaling dependent pathway.

C-reactive protein (CRP) is an independent predictor of atherosclerosis and its complications. Monocytes/macrophages are implicated in this complex disease which is, among other mechanisms, characterised by angiogenesis. The aim of this study was to analyse whether CRP plays a role in VEGF-A regulation by monocytic cells. Our findings show that CRP up-regulates VEGF-A mRNA expression and protein excretion in THP-1 cells in a concentration- and time-dependent manner. Furthermore, we studied the signaling pathway underlying this effect. CRP increases VEGF-A expression via a PI3-kinase and an extracellular-signal-regulated kinase (ERK) 1/2 dependent pathway. Our results suggest that CRP could play a role in the angiogenesis process via immune cells such as monocytes.

Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock.

The triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as tumor necrosis factor alpha and interleukin-1beta, and improved survival. The protective effect of these peptides on arterial pressure could partly be explained by a decreased production of nitric oxide. These data suggest that in vivo modulation of TREM-1 might be a suitable therapeutic tool for the treatment of sepsis.

Time-course of sTREM (soluble triggering receptor expressed on myeloid cells)-1, procalcitonin, and C-reactive protein plasma concentrations during sepsis.

OBJECTIVE: To describe the course of plasma sTREM (soluble triggering receptor expressed on myeloid cells)-1, procalcitonin (PCT), and C-reactive protein (CRP) concentrations during sepsis and their clinical informative value in predicting outcome. DESIGN: Prospective, noninterventional study. SETTING: Medical adult intensive care unit at a university hospital in France. PATIENTS: Sixty-three critically ill patients with sepsis, severe sepsis, or septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Soluble TREM-1 concentrations were significantly lower at admission in nonsurvivors (n = 21) than in survivors (n = 42) (94 [30-258] vs. 154 [52-435] pg/mL, p = .02), whereas PCT levels were higher among nonsurvivors (19.2 [0.3-179] vs. 2.4 (0-254) pg/mL, p = .001). CRP levels did not differ between the two groups of patients. Plasma PCT and CRP decreased during the 14-day period of study in both survivors and nonsurvivors. Conversely, sTREM-1 plasma concentrations remained stable or even increased in nonsurviving patients and decreased in survivors. An elevated baseline sTREM-1 level was found to be an independent protective factor with an odds of dying of 0.1 (95% confidence interval, 0.1-0.8). CONCLUSION: A progressive decline of plasma sTREM-1 concentration indicates a favorable clinical evolution during the recovery phase of sepsis. In addition, baseline sTREM-1 level may prove useful in predicting outcome of septic patients.

Anti-HBs cellular immune response in kidney recipients before and 4 months after transplantation.

Patients with renal failure represent a population at risk for hepatitis B, since only 50 to 60% of them develop protective humoral responses after vaccination. As this could be due to an altered regulation of cellular immune responses, the objectives of the present study were to evaluate the proliferative abilities of lymphocytes from patients with chronic renal failure after stimulation in vitro with a mitogen (pokeweed mitogen [PWM]) or HBsAg. In order to differentiate between the immunodeficiency associated with renal failure and that due to immunosuppression posttransplantation, the same subjects were tested before and 4 months after kidney transplantation. The lymphoproliferation assay used was performed by flow cytometry, which is based on sequential analysis of the cell cycle and which allows analysis of cytokine production. Serologically, the group of 36 patients tested comprised 22% nonresponders, 30% poor responders, and 48% responders. Lymphocyte growth was observed for all patients after stimulation with PWM, indicating that these cells had the capacity to proliferate in vitro. The level of lymphoproliferation in response to PWM was significantly reduced after transplantation, yet both before and after transplantation, all serologic nonresponders developed cellular responses to at least two vaccines. No correlation between humoral and cellular responses was shown. Proliferating cells were lymphocytes, which mostly secreted interleukin 4 (IL-4) and IL-10 for the three serologic groups. This study suggests that even when repeated vaccination fails to induce significant antibody levels in patients with renal failure, specific HBs cellular responses develop, and these may prove to be efficient in protecting these patients against hepatitis B.