Obesity-associated T-cell and macrophage activation improve partly after a lifestyle intervention.

BACKGROUND: The relation between low-grade inflammation and metabolic dysfunction in obesity is not fully explored. OBJECTIVE: To evaluate immune parameters in the obese state and after a lifestyle intervention program. METHODS: Patients with obesity (n = 87) from an academic obesity clinic were compared with controls with regard to macrophage and T-cell activation (reflected by serum levels of soluble CD163 (sCD163) and soluble IL-2 receptor (sIL-2R), respectively), and an array of cytokines, chemokines, and growth factors. In addition, these parameters and regulatory T-cells (Treg), were studied in 27 patients who followed a 75-week lifestyle intervention (dietary advice, exercise, and psychoeducation). RESULTS: Mean sIL-2R and sCD163 levels were higher in patients than controls (sIL-2R:2884 ± 936 pg/ml vs. 2207 ± 813 pg/ml, p = 0.001; sCD163:1279 ± 580 pg/ml vs. 661 ± 271 pg/ml, p < 0.0001 respectively). Patients with metabolic syndrome (MetS) had higher sCD163 than those without (1467 ± 656 pg/ml vs. 1103 ± 438 pg/ml). Patients had higher IL-1ß, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-15, IL-17A, MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, G-CSF, GM-CSF, FGF, IFN-γ, and TNF-α than controls, whereas VEGF-A, PDGF-BB, and eotaxin were lower. Upon intervention, sIL-2R decreased while peripheral Treg frequencies increased within the reference range (p = 0.042 and p = 0.005 respectively). The sIL-2R decrease correlated to a decrease in waist circumference (rho = 0.388, p = 0.045) and in trend to a decrease in MetS components (rho = 0.345, p = 0.078). The Treg increase was unrelated to weight loss or metabolic improvement. Mean sCD163 did not change significantly upon intervention, nor did the cytokines, chemokines, and growth factors (except IP-10/CXCL10). CONCLUSION: In obesity, T-cell homeostasis improves after a lifestyle intervention. Immunologic alterations can occur independently of metabolic improvement.

A multicentre cohort study of serum and peritoneal biomarkers to predict anastomotic leakage after rectal cancer resection.

AIM: Anastomotic leakage (AL) is one of the most feared complications after rectal resection. This study aimed to assess a combination of biomarkers for early detection of AL after rectal cancer resection. METHOD: This study was an international multicentre prospective cohort study. All patients received a pelvic drain after rectal cancer resection. On the first three postoperative days drain fluid was collected daily and C-reactive protein (CRP) was measured. Matrix metalloproteinase-2 (MMP2), MMP9, glucose, lactate, interleukin 1-beta (IL1ß), IL6, IL10, tumour necrosis factor alpha (TNFα), Escherichia coli, Enterococcus faecalis, lipopolysaccharide-binding protein and amylase were measured in the drain fluid. Prediction models for AL were built for each postoperative day using multivariate penalized logistic regression. Model performance was estimated by the c-index for discrimination. The model with the best performance was visualized with a nomogram and calibration was plotted. RESULTS: A total of 292 patients were analysed; 38 (13.0%) patients suffered from AL, with a median interval to diagnosis of 6.0 (interquartile ratio 4.0-14.8) days. AL occurred less often after partial than after total mesorectal excision (4.9% vs 15.2%, P = 0.035). Of all patients with AL, 26 (68.4%) required reoperation. AL was more often treated by reoperation in patients without a diverting ileostomy (18/20 vs 8/18, P = 0.03). The prediction model for postoperative day 1 included MMP9, TNFα, diverting ileostomy and surgical technique (c-index = 0.71). The prediction model for postoperative day 2 only included CRP (c-index = 0.69). The prediction model for postoperative day 3 included CRP and MMP9 and obtained the best model performance (c-index = 0.78). CONCLUSION: The combination of serum CRP and peritoneal MMP9 may be useful for earlier prediction of AL after rectal cancer resection. In clinical practice, this combination of biomarkers should be interpreted in the clinical context as with any other diagnostic tool.

Serum Soluble Interleukin-2 Receptor Does Not Differentiate Complex Regional Pain Syndrome from Other Pain Conditions in a Tertiary Referral Setting.

Previously, we showed that serum soluble interleukin-2 receptor (sIL-2R) levels, a marker for T-cell activation, were higher in complex regional pain syndrome (CRPS) patients than in healthy controls, suggesting pathogenic T-cell activation in CRPS. Additionally, sIL-2R levels discriminated well between CRPS and healthy controls with a high sensitivity (90%) and specificity (89.5%), suggesting a possible role for sIL-2R in the diagnosis of CRPS. In order to further validate this marker in the diagnostic workup of CRPS, we conducted this prospective cohort study in which we determined sIL-2R levels in patients that were referred to our tertiary referral center with a suspicion of CRPS in a limb, and subsequently compared sIL-2R levels between the patients that were diagnosed with CRPS (CRPS group) and those who were not (no CRPS group). A group of anonymous blood bank donors were used as a healthy control group. Furthermore, we explored the relationship between sIL-2R and CRPS disease severity using the CRPS severity score. Median sIL-2R levels of both the CRPS group (2809.0 pg/ml; Q3-Q1: 3913.0-1589.0) and no CRPS group (3654.0 pg/ml; Q3-Q1: 4429.0-2095.5) were significantly higher than that of the control group (1515.0 pg/ml; Q3-Q1: 1880.0-1150.0): CRPS vs. controls, p < .001; no CRPS vs. controls, p < 0.001. Serum sIL-2R levels did not differ significantly between the CRPS and no CRPS group. A statistically significant negative correlation was observed between sIL-2R levels and the CRPS severity score (r s = -0.468, p = 0.024). Our results confirm our previous findings of higher sIL-2R levels in CRPS patients than in healthy controls. We further showed that serum sIL-2R cannot differentiate between CRPS and other pain conditions of a limb in a tertiary referral setting. Interestingly, a negative correlation was found between sIL-2R and CRPS disease severity; this finding warrants further research into the relationship between sIL-2R and CRPS disease severity.

Soluble Interleukin-2 Receptor: A Potential Marker for Monitoring Disease Activity in IgG4-Related Disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a fibroinflammatory condition. T-cells play a crucial role in the pathogenesis, and therefore, serum soluble interleukin-2 receptor (sIL-2R) may be a potential biomarker. METHOD: We studied the levels of sIL-2R in 26 histologically proven IgG4-RD patients with available serum sIL-2R and compared them to those in newly diagnosed and untreated sarcoidosis patients (n = 78) and controls (n = 101) and the serum sIL-2R levels in patients after treatment of IgG4-RD (n = 15). The disease activity was measured using the IgG4-Related Disease Responder Index (IgG4-RD RI). RESULTS: Median serum sIL-2R in IgG4-RD patients was 4667 pg/ml compared to 1515 pg/ml in controls (P < 0.001) and 6050 pg/ml in sarcoidosis patients (P = 0.004 compared to IgG4-RD). All IgG4-RD patients had elevated serum sIL-2R levels compared to the reference value of <2500 pg/ml in controls and 85% elevated serum IgG4; however, these did not correlate with each other. Both serum sIL-2R and IgG4 levels declined significantly after treatment (P = 0.001 and P = 0.01, resp.). Before treatment, serum sIL-2R level and IgG4-RD RI did not correlate with each other. However, the decrease in serum sIL-2R upon treatment did correlate significantly (P = 0.04) with the decrease in disease activity assessed by IgG-RD RI. CONCLUSION: Serum sIL-2R is elevated in IgG4-RD reflecting the inflammatory process with enhanced T-cell activation. Furthermore, serum sIL-2R might serve as a potential marker of response to treatment in IgG4-RD.

Mycophenolate mofetil hampers antibody responses to a broad range of vaccinations in kidney transplant recipients: Results from a randomized controlled study.

OBJECTIVES: To study the effect of mycophenolate mofetil (MMF) on various vaccination responses in kidney transplant recipients. METHODS: In a randomized controlled trial (EudraCT nr.: 2014-001372-66), low immunologically risk kidney transplant recipients were randomized to TAC/MMF or TAC-monotherapy (TACmono), six months post-transplantation. One year after transplantation, in a pre-specified sub-study, recipients were vaccinated against pneumococcus, tetanus and influenza. Blood was sampled before and 21 days after vaccination. Adequate vaccination responses were defined by international criteria. A post-hoc analysis was conducted on SARS-CoV-2 vaccination responses within the same cohort. RESULTS: Seventy-one recipients received pneumococcal and tetanus vaccines (TAC/MMF: n = 37, TACmono: n = 34), with 29 also vaccinated against influenza. When vaccinated, recipients were 60 (54-66) years old, with median eGFR of 54 (44-67) ml/min, tacrolimus trough levels 6.1 (5.4-7.0) ug/L in both groups and TAC/MMF daily MMF dose of 1000 (500-2000) mg. Adequate vaccination responses were: pneumococcal (TAC/MMF 43%, TACmono 74%, p = 0.016), tetanus (TAC/MMF 35%, TACmono 82%, p < 0.0001) and influenza (TAC/MMF 20%, TACmono 71%, p = 0.0092). Only 7% of TAC/MMF responded adequately to all three compared to 36% of TACmono (p = 0.080). Additionally, 40% of TAC/MMF responded inadequately to all three, whereas all TACmono patients responded adequately to at least one vaccination (p = 0.041). Lower SARS-CoV-2 vaccination antibody responses correlated with lower pneumococcal antibody vaccination responses (correlation coefficient: 0.41, p = 0.040). CONCLUSIONS: MMF on top of tacrolimus severely hampers antibody responses to a broad range of vaccinations.

The peripheral blood compartment in patients with Graves' disease: activated T lymphocytes and increased transitional and pre-naive mature B lymphocytes.

Graves' disease (GD) is an autoimmune disease that involves aberrant B and T lymphocyte responses. Detailed knowledge about lymphocyte subpopulation composition will therefore enhance our understanding of the pathogenesis of GD and might support the development of new immunomodulatory treatment approaches. The aim of this study was to gain detailed insight into the composition of the peripheral blood lymphocyte compartment in GD before and during anti-thyroid drug therapy. Major B and T lymphocyte subpopulations were investigated by flow cytometry in peripheral blood from newly diagnosed GD patients (n = 5), GD patients treated with anti-thyroid drugs (n = 4), patients with recurrent GD (n = 7) and healthy controls (HC; n = 10). In GD patients, numbers of activated T lymphocytes [human leucocyte antigen D-related (HLA-DR)⁺ and CD25⁺] were increased. The B lymphocyte compartment in GD was characterized by significantly higher numbers of transitional (CD38(high) CD27⁻, P < 0.03) and pre-naive mature (CD38(low) CD27⁻ IgD⁺ CD5⁺, P < 0.04) B lymphocytes, while memory populations were slightly decreased. The increased numbers of CD5⁺, transitional and pre-naive mature B lymphocytes correlated positively with fT4 plasma levels. GD is associated with increased numbers of activated T lymphocytes and transitional and pre-naive mature CD5⁺ B lymphocytes within the peripheral blood. The increase in CD5⁺ B lymphocytes was due mainly to an increase in transitional and pre-naive mature B lymphocytes. Increased fT4 plasma levels might be associated with this increase in transitional and pre-naive mature CD5⁺ B lymphocytes.

Baseline TGF-ß correlates with protection after immunization with Plasmodium falciparum sporozoites in the controlled human malaria infection model.

BACKGROUND: Here we assessed a possible relationship between baseline TGF-ß concentrations and acquisition of sterile immunity after Plasmodium falciparum sporozoite immunization. METHODS: TGF-ß concentrations were determined in samples of 65 malaria-naive volunteers in 4 studies either prior to and after challenge infection, or prior to and after first immunizing infection under chemoprophylaxis with P. falciparum sporozoites. RESULTS: High baseline TGF-ß concentrations were associated with rapid acquisition of sterile protection (p = 0.028). CONCLUSION: Baseline TGF-ß concentrations predict the efficiency of acquisition of sterile immunity following sporozoite immunization and may represent a steady-state regulatory mechanism to keep in check immune systems with a low threshold for activation.

Adalimumab (antitumour necrosis factor-α) treatment of hidradenitis suppurativa ameliorates skin inflammation: an in situ and ex vivo study.

BACKGROUND: Hidradenitis suppurativa (HS) is a difficult-to-manage disease. Randomized controlled trials with antitumour necrosis factor (TNF)-α biologics have been conducted and in most studies disease activity was reduced. However, the mechanism of action in HS skin is so far unknown. OBJECTIVES: To assess whether anti-TNF-α treatment affects in situ cytokine production and frequency of inflammatory cell populations in HS lesional skin. METHODS: Nine patients with HS, participating in a larger placebo-controlled, double-blind phase IIb clinical trial on the efficacy and safety of adalimumab in patients with moderate to severe HS (M10-467), were randomized and treated for 16weeks. In a mechanism-of-action substudy, biopsies were obtained at fixed time points pre- and post-treatment. One part of the biopsy was cultured for 24h for cytokine release in the culture medium, while another part was used for in situ analysis. RESULTS: Secretion of cytokines, including interleukin (IL)-1ß, CXCL9 [monokine induced by interferon-γ (MIG)], IL-10, IL-11, B-lymphocyte chemoattractant (BLC) and IL-17A, was significantly elevated in HS. Adalimumab treatment was associated with decreased production of cytokines in HS skin, especially IL-1ß, CXCL9 (MIG) and BLC. Treatment significantly reduced the number of CD11c+,CD14+ and CD68+ cells in HS lesional skin. The numbers of CD3+ and CD4+ T cells, and CD20+ and CD138+ B cells were also reduced by adalimumab treatment. CONCLUSIONS: Adalimumab treatment inhibits important cytokines and inflammatory cell numbers in lesional HS skin, especially levels of IL-1ß and numbers of inflammatory CD11c+ dendritic cells.

Elevated levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-10 in hidradenitis suppurativa skin: a rationale for targeting TNF-α and IL-1ß.

BACKGROUND: The pathogenesis of hidradenitis suppurativa (HS) is largely unknown and the disease is difficult to treat. Patients are in high need of an effective treatment. Although it is not known whether the levels of tumour necrosis factor (TNF)-α are aberrant in HS skin, anti-TNF-α biologics are used, with variable clinical efficacy. OBJECTIVES: To determine the cytokine profile in lesional and perilesional HS skin. METHODS: We cultured 20 lesional and 10 normal-appearing perilesional HS skin samples, seven psoriasis and six healthy control skin samples in a transwell culture system. Two distinct cytokine bead arrays were used to measure the spectrum of inflammatory cytokines in the culture supernatant. Results from HS skin samples were compared with those of healthy and psoriasis skin. RESULTS: The proinflammatory cytokines interleukin (IL)-1ß and TNF-α as well as the anti-inflammatory cytokine IL-10 were significantly elevated in HS skin. Elevated levels of these cytokines were also found in perilesional HS skin. Fold increases relative to control skin of IL-1ß, TNF-α and IL-10 in HS were 31, 5 and 34, compared with psoriasis: 4, 1 and 2, respectively. Levels of all three cytokines showed a trend towards a positive correlation with disease severity. IL-2, IL-4, IL-5 and interferon-γ were hardly detectable in HS or healthy control skin. CONCLUSIONS: This study shows for the first time that IL-1ß, TNF-α and IL-10 levels are elevated in HS skin. These data provide a rationale for therapies with biologics targeting cytokines such as TNF-α and IL-1.

Breakfast partly restores the anti-inflammatory function of high-density lipoproteins from patients with type 2 diabetes mellitus.

Background and aims: High-density lipoproteins (HDL) of patients with type 2 diabetes mellitus (T2DM) have impaired anti-inflammatory activities. The anti-inflammatory activity of HDL has been determined ex vivo after isolation by different methods from blood mostly obtained after overnight fasting. We first determined the effect of the HDL isolation method, and subsequently the effect of food intake on the anti-inflammatory function of HDL from T2DM patients. Methods: Blood was collected from healthy controls and T2DM patients after an overnight fast, and from T2DM patients 3 h after breakfast (n = 17 each). HDL was isolated by a two-step density gradient ultracentrifugation in iodixanol (HDLDGUC2), by sequential salt density flotation (HDLSEQ) or by PEG precipitation (HDLPEG). The anti-inflammatory function of HDL was determined by the reduction of the TNFα-induced expression of VCAM-1 in human coronary artery endothelial cells (HCAEC) and retinal endothelial cells (REC). Results: HDL isolated by the three different methods from healthy controls inhibited TNFα-induced VCAM-1 expression in HCAEC. With apoA-I at 0.7 µM, HDLDGUC2 and HDLSEQ were similarly effective (16% versus 14% reduction; n = 3; p > 0.05) but less effective than HDLPEG (28%, p < 0.05). Since ultracentrifugation removes most of the unbound plasma proteins, we used HDLDGUC2 for further experiments. With apoA-I at 3.2 µM, HDL from fasting healthy controls and T2DM patients reduced TNFα-induced VCAM-1 expression in HCAEC by 58 ± 13% and 51 ± 20%, respectively (p = 0.35), and in REC by 42 ± 13% and 25 ± 18%, respectively (p < 0.05). Compared to preprandial HDL, postprandial HDL from T2DM patients reduced VCAM-1 expression by 56 ± 16% (paired test: p < 0.001) in HCAEC and by 34 ± 13% (paired test: p < 0.05) in REC. Conclusions: The ex vivo anti-inflammatory activity of HDL is affected by the HDL isolation method. Two-step ultracentrifugation in an iodixanol gradient is a suitable method for HDL isolation when testing HDL anti-inflammatory function. The anti-inflammatory activity of HDL from overnight fasted T2DM patients is significantly impaired in REC but not in HCAEC. The anti-inflammatory function of HDL is partly restored by food intake.

Mitigation of septic shock in mice and rhesus monkeys by human chorionic gonadotrophin-related oligopeptides.

The marked improvement of several immune-mediated inflammatory diseases during pregnancy has drawn attention to pregnancy hormones as potential therapeutics for such disorders. Low molecular weight fractions derived from the pregnancy hormone human chorionic gonadotrophin (hCG) have remarkable potent immunosuppressive effects in mouse models of diabetes and septic shock. Based on these data we have designed a set of oligopeptides related to the primary structure of hCG and tested these in models of septic shock in mice and rhesus monkeys. We demonstrate that mice exposed to lipopolysaccharide (LPS) and treated subsequently with selected tri-, tetra-, penta- and hepta-meric oligopeptides (i.e. MTR, VVC, MTRV, LQGV, AQGV, VLPALP, VLPALPQ) are protected against fatal LPS-induced septic shock. Moreover, administration of a cocktail of three selected oligopeptides (LQGV, AQGV and VLPALP) improved the pathological features markedly and nearly improved haemodynamic parameters associated with intravenous Escherichia coli-induced septic shock in rhesus monkeys. These data indicate that the designed hCG-related oligopeptides may present a potential treatment for the initial hyperdynamic phase of septic shock in humans.

Clinical significance of soluble interleukin-2 receptor measurement in immune-mediated diseases.

A soluble form of the interleukin-2 receptor (sIL-2R) is secreted upon T-cell activation. Increased blood levels of sIL-2R occur in a variety of immunological diseases. Although the biological function of sIL-2R is incompletely understood, both in health and disease, sIL-2R serum measurements are commonly conducted in clinical practice as it may help to facilitate diagnosis of specific immune-mediated diseases, such as haemophagocytic lymphohistiocytosis and sarcoidosis. In these, and in other immune-diseases, sIL-2R levels may be used as a biomarker to monitor/predict disease activity and treatment response. In this review, we will give a brief overview of the biology of the IL-2/IL-2R system and will subsequently discuss the clinical utility of sIL-2R measurement, especially in the context of haemophagocytic lymphohistiocytosis, sarcoidosis, rheumatoid arthritis, systemic lupus erythematosus, juvenile idiopathic arthritis, adult-onset Still's disease, ANCA-associated vasculitis, and IgG4-related disease.

Are Patients at Risk for Recurrent Disease Activity After Switching From Remicade® to Remsima®? An Observational Study.

Background: Since the late '90s, infliximab (Remicade®) is being used successfully to treat patients with several non-infectious immune mediated inflammatory diseases (IMIDs). In recent years, infliximab biosimilars, including Remsima® were introduced in clinical practice. Aim: To investigate the interchangeability of Remicade® (originator infliximab) and its biosimilar Remsima® in patients with rare immune-mediated inflammatory diseases (IMIDs). Methods: This two-phased prospective open label observational study was designed to monitor the transition from Remicade® to Remsima® in patients with rare IMIDs. All included patients were followed during the first 2 years. The primary endpoint was the demonstration of non-difference in quality of life and therapeutic efficacy, as measured by parameters including a safety monitoring program, physicians perception of disease activity (PPDA) and patient self-reported outcomes (PSROs). Secondary outcomes included routine blood analysis, pre-infusion serum drug concentration values and anti-drug antibody formation. Results: Forty eight patients treated with Remicade® were switched to Remsima® in June-July 2016 and subsequently monitored during the first 2 years. The group consisted of patients with sarcoidosis (n = 17), Behçet's disease (n = 12), non-infectious uveitis (n = 11), and other diagnoses (n = 8). There were no significant differences in PPDA, PSROs, clinical and laboratory assessments and pre-infusion serum drug concentrations between the groups. De novo anti-drug antibodies were observed in two patients. Seven patients with sarcoidosis and five with another diagnosis developed a significant disease relapse (n = 7) or adverse events (n = 5) within 2 years; 10 of these patients discontinued Remsima® treatment, one withdrew from the study and one received additional corticosteroid therapy. Conclusions: We observed no significant differences in PSROs, PPDA and laboratory parameters after treatment was switched from Remicade® to Remsima®. However, disease relapse or serious events were observed in 12 out of 48 patients when treatment was switched from Remicade® to Remsima®. The choice to switch anti-TNF alpha biologics in patients with rare IMIDs, particularly in sarcoidosis, requires well-considered decision-making and accurate monitoring due to a possibly higher incidence of disease worsening.

An Intraocular Inflammatory Profile of Rubella Associated Uveitis.

Purpose: To analyze intraocular cytokine levels and cell profiles in patients with rubella virus-associated uveitis (RVU). Methods: We collected intraocular fluid samples from patients with RVU (n = 10), uveitis of other causes (n = 27), and cataract (n = 22). Levels of 15 cytokines (IL-1ß, IL-1ra, IL-2, IL-6, IL-6rα, IL-7, IL-8, IL-10, IL-17A, IL-23, TARC, MCP-1, TNF-α, PlGF, and VEGF) were measured using multiplex assay, and intraocular cell populations were determined by multiparameter flowcytometry. Clinical characteristics of RVU patients were collected and compared to laboratory outcomes. Results: RVU patients exhibited high intraocular levels of MCP-1, IL-6rα, and TARC, whilst patients with noninfectious uveitis were characterized by high levels of PlGF. Cataract patients showed high levels of IL-2 and IL-23. Intraocular cell population of RVU patients disclosed mainly T-cells and monocytes/macrophages and B-cells were scarcely detected. Conclusion: RVU patients exhibit a cytokine profile distinct from noninfectious uveitis and cataract.

Human T-cell lines with well-defined T-cell receptor gene rearrangements as controls for the BIOMED-2 multiplex polymerase chain reaction tubes.

The BIOMED-2 multiplex polymerase chain reaction (PCR) tubes for analysis of immunoglobulin and T-cell receptor (TCR) gene rearrangements have recently been introduced as a reliable and easy tool for clonality diagnostics in suspected lymphoproliferations. Quality and performance assessment of PCR-based clonality diagnostics is generally performed using human leukemia/lymphoma cell lines as controls. We evaluated the utility of 30 well-defined human T-cell lines for quality performance testing of the BIOMED-2 PCR primers and protocols. The PCR analyses of the TCR loci were backed up by Southern blot analysis. The clonal TCRB, TCRG and TCRD gene rearrangements were analyzed for gene segment usage and for the size and composition of their junctional regions. In 29 out of 30 cell lines, unique clonal TCR gene rearrangements could be easily detected. Besides their usefulness in molecular clonality diagnostics, these cell lines can now be authenticated based on their TCR gene rearrangement profile. This enables their correct use in molecular clonality diagnostics and in other cancer research studies.

Purity for clarity: the need for purification of tumor cells in DNA microarray studies.

It is now well established that gene expression profiling using DNA microarrays can provide novel information about various types of hematological malignancies, which may lead to identification of novel diagnostic markers. However, to successfully use microarrays for this purpose, the quality and reproducibility of the procedure need to be guaranteed. The quality of microarray analyses may be severely reduced, if variable frequencies of nontarget cells are present in the starting material. To systematically investigate the influence of different types of impurity, we determined gene expression profiles of leukemic samples containing different percentages of nonleukemic leukocytes. Furthermore, we used computer simulations to study the effect of different kinds of impurity as an alternative to conducting hundreds of microarray experiments on samples with various levels of purity. As expected, the percentage of erroneously identified genes rose with the increase of contaminating nontarget cells in the samples. The simulations demonstrated that a tumor load of less than 75% can lead to up to 25% erroneously identified genes. A tumor load of at least 90% leads to identification of at most 5% false-positive genes. We therefore propose that in order to draw well-founded conclusions, the percentage of target cells in microarray experiment samples should be at least 90%.

CALM-AF10+ T-ALL expression profiles are characterized by overexpression of HOXA and BMI1 oncogenes.

The t(10;11)(p13;q14-21) is found in T-ALL and acute myeloid leukemia and fuses CALM (Clathrin-Assembly protein-like Lymphoid-Myeloid leukaemia gene) to AF10. In order to gain insight into the transcriptional consequences of this fusion, microarray-based comparison of CALM-AF10+ vs CALM-AF10- T-ALL was performed. This analysis showed upregulation of HOXA5, HOXA9, HOXA10 and BMI1 in the CALM-AF10+ cases. Microarray results were validated by quantitative RT-PCR on an independent group of T-ALL and compared to mixed lineage leukemia-translocated acute leukemias (MLL-t AL). The overexpression of HOXA genes was associated with overexpression of its cofactor MEIS1 in CALM-AF10+ T-ALL, reaching levels of expression similar to those observed in MLL-t AL. Consequently, CALM-AF10+ T-ALL and MLL-t AL share a specific HOXA overexpression, indicating they activate common oncogenic pathways. In addition, BMI1, located close to AF10 breakpoint, was overexpressed only in CALM-AF10+ T-ALL and not in MLL-t AL. BMI1 controls cellular proliferation through suppression of the tumor suppressors encoded by the CDKN2A locus. This locus, often deleted in T-ALL, was conserved in CALM-AF10+ T-ALL. This suggests that decreased CDKN2A activity, as a result of BMI1 overexpression, contributes to leukemogenesis in CALM-AF10+ T-ALL. We propose to define a HOXA+ leukemia group composed of at least MLL-t, CALM-AF10 and HOXA-t AL, which may benefit from adapted management.

Disruption of the BCL11B gene through inv(14)(q11.2q32.31) results in the expression of BCL11B-TRDC fusion transcripts and is associated with the absence of wild-type BCL11B transcripts in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is associated with chromosomal aberrations characterized by juxtaposition of proto-oncogenes to T-cell receptor gene loci (TCR), resulting in the deregulated transcription of these proto-oncogenes. Here, we describe the molecular characterization of a novel chromosomal aberration, inv(14)(q11.2q32.31), in a T-ALL sample, involving the recently described BCL11B gene and the TCRD locus. The inversion joined the 5' part of BCL11B, including exons 1-3, to the TRDD3 gene segment of the TCRD locus, whereas the reciprocal breakpoint fused the TRDV1 gene segment to the fourth exon of BCL11B. The TRDV1-BCL11B joining region was 1344 bp long and contained fragments derived from 20q11.22, 3p21.33 and from 11p12, indicating the complex character of this aberration. A strong expression of in-frame transcripts with truncated BCL11B and TCRD constant region (TRDC) were observed, but in contrast to normal T cells and other T-ALL samples, no wild-type BCL11B transcripts were detected in the T-ALL sample. Screening of 37 other T-ALLs revealed one additional case with expression of the BCL11B-TRDC fusion transcript. As BCL11B appears to play a key role in T-cell differentiation, BCL11B disruption and disturbed expression may contribute to the development of T-cell malignancies in man.

Diet-induced weight loss and markers of endothelial dysfunction and inflammation in treated patients with type 2 diabetes.

BACKGROUND & AIMS: Overweight and obesity increase cardiovascular mortality in patients with type 2 diabetes (T2D). In a recent trial, however, diet-induced weight loss did not reduce the cardiovascular risk of patients with T2D, possibly due to the parallel intensive medical treatment. We investigated the effect of diet-induced weight loss on cardiovascular risk factors in overweight and obese patients with T2D, and whether this effect was influenced by the use of statins, ACE inhibitors, metformin and duration of T2D. METHODS: Patients with T2D and BMI >27 were subjected to an energy-restricted diet during 4 months. Before and after intervention, plasma levels of sICAM-1, sVCAM-1, hsCRP, vWF and classical biomarkers were measured. The association of the change in biomarker levels with medication use and T2D history, corrected for age, sex and change in insulin dose, was tested by matched linear regression analyses. RESULTS: In 131 patients, the diet resulted in weight loss of 10.2 kg (95%CI 9.2, 11.3; p < 0.001), improved median levels of HbA1c (-7.0 mmol/mol (95%CI -8.5, -5.0); p < 0.001), LDL cholesterol (-0.2 mmol/L (95%CI -0.4, -0.1); p < 0.001), sICAM-1 (-22.4 ng/mL (95%CI -37.1, -8.7); p = 0.001), vWF (-3.9 IU/mL (95%CI -6.4, -1.4); p = 0.003) and hs-CRP (-0.6 mg/L (95%CI -1.2, -0.2); p = 0.007), but did not affect sVCAM-1 levels (1.6 ng/mL (95%CI -41.5, 48.6); p = 0.949). Duration of T2D and medical treatment were not associated with these effects, except for an association between statin use and change in sVCAM-1, where statin users improved more. CONCLUSION: Diet-induced weight loss reduced the levels of biomarkers of endothelial dysfunction and inflammation in overweight and obese patients with T2D independently of medication use and T2D duration. Even on intensive medical drug treatment as well as after a long history of T2D, patients may still profit from diet-induced weight reduction.

Lack of common TCRA and TCRB clonotypes in CD8(+)/TCRαß(+) T-cell large granular lymphocyte leukemia: a review on the role of antigenic selection in the immunopathogenesis of CD8(+) T-LGL.

Clonal CD8(+)/T-cell receptor (TCR)αß(+) T-cell large granular lymphocyte (T-LGL) proliferations constitute the most common subtype of T-LGL leukemia. Although the etiology of T-LGL leukemia is largely unknown, it has been hypothesized that chronic antigenic stimulation contributes to the pathogenesis of this disorder. In the present study, we explored the association between expanded TCR-Vß and TCR-Vα clonotypes in a cohort of 26 CD8(+)/TCRαß(+) T-LGL leukemia patients, in conjunction with the HLA-ABC genotype, to find indications for common antigenic stimuli. In addition, we applied purpose-built sophisticated computational tools for an in-depth evaluation of clustering of TCRß (TCRB) complementarity determining region 3 (CDR3) amino-acid LGL clonotypes. We observed a lack of clear TCRA and TCRB CDR3 homology in CD8(+)/TCRαß(+) T-LGL, with only low level similarity between small numbers of cases. This is in strong contrast to the homology that is seen in CD4(+)/TCRαß(+) T-LGL and TCRγδ(+) T-LGL and thus underlines the idea that the LGL types have different etiopathogenesis. The heterogeneity of clonal CD8(+)/TCRαß(+) T-LGL proliferations might in fact suggest that multiple pathogens or autoantigens are involved.