Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection

Dengue virus infection is an increasingly important tropical disease, causing 100 million cases each year. Symptoms range from mild febrile illness to severe hemorrhagic fever. The pathogenesis is incompletely understood, but immunopathology is thought to play a part, with antibody-dependent enhancement and massive immune activation of T cells and monocytes/macrophages leading to a disproportionate production of proinflammatory cytokines. We sought to investigate whether a defective population of regulatory T cells (T reg cells) could be contributing to immunopathology in severe dengue disease. CD4(+)CD25(high)FoxP3(+) T reg cells of patients with acute dengue infection of different severities showed a conventional phenotype. Unexpectedly, their capacity to suppress T cell proliferation and to secrete interleukin-10 was not altered. Moreover, T reg cells suppressed the production of vasoactive cytokines after dengue-specific stimulation. Furthermore, T reg cell frequencies and also T reg cell/effector T cell ratios were increased in patients with acute infection. A strong indication that a relative rise of T reg cell/effector T cell ratios is beneficial for disease outcome comes from patients with mild disease in which this ratio is significantly increased (P < 0.0001) in contrast to severe cases (P = 0.2145). We conclude that although T reg cells expand and function normally in acute dengue infection, their relative frequencies are insufficient to control the immunopathology of severe disease.     

Intraoperative hypoxia secondary to pneumothorax: The role of lung ultrasound

Intraoperative pneumothorax during general anesthesia is a dangerous event. It is a possible cause of sudden intraoperative hypoxia, which can be critical especially in high‐risk patients such as those with end‐stage heart failure. Early diagnosis and effective treatment are essential. We describe the case of a pneumothorax during cardiac transplantation, diagnosed by ultrasound and immediately treated. A good skill in lung ultrasound is advantageous in the management of intraoperative hypoxia, particularly for prompt diagnosis of pneumothorax.     

Telomerase inhibition abolishes the tumorigenicity of pediatric ependymoma tumor-initiating cells

Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.     

The prothrombotic paradox of severe obesity after cardiac surgery under cardiopulmonary bypass

Background

Obesity is suggested to reduce postoperative bleeding in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) but perioperative hemostasis variations have not been studied. Therefore, we investigated the effects of severe obesity (body mass index [BMI] ≥ 35 kg/m²) on chest tube output (CTO) and hemostasis in patients undergoing cardiac surgery with CPB.

Materials and Methods

We prospectively investigated 2799 consecutive patients who underwent coronary and/or valve surgery using CPB between 2008 and 2012. 204 patients (7.3%) presented a severe obesity.

Results

In the severe obesity group, the 6-h and 24-h CTO were significantly reduced by -21.8% and -14.8% respectively (P < 0.0001) compared with the control group. A significant reduction of the mean number of red blood cell units transfused at 24 h was observed in the severe obesity groups (P = 0.01). On admission to the intensive care unit, a significant increase of platelet count (+ 9.2%; P < 0.0001), fibrinogen level (+ 12.2%; P < 0.0001) and prothrombin time (+ 4.1%; P < 0.01) and a significant decrease of the activated partial thromboplastin time (-4.2%; P < 0.01) were observed in the severe obesity group compared with the control group.In multivariate analysis, severe obesity was significantly associated to a decreased risk of excessive bleeding (24-h CTO > 90th percentile; Odds ratio: 0.37, 95% CI: 0.17 to 0.82). No significant differences were observed regarding postoperative thromboembolic events between the two groups.

Conclusions

Severe obesity is associated with a prothrombotic postoperative state that leads to a reduction of postoperative blood loss in patients undergoing cardiac surgery with CPB.     

Mycophenolic acid quantification in human peripheral blood mononuclear cells using liquid chromatography–tandem mass spectrometry

Background and objectives

Mycophenolic acid (MPA) is an immunosuppressant widely used to prevent organ rejection after organ transplantation. MPA therapeutic drug monitoring (TDM) is currently recommended by plasma C₀ – or even better – by AUC determinations, but little is known about the potential interest of measuring the intra-lymphocyte MPA concentrations, representing the target site of action. The aim of this study is to develop and validate a selective and sensitive analytical method for quantification of MPA levels in human peripheral blood mononuclear cells (PBMCs).

Methods

PBMCs were extracted from heparin blood samples by Leucosep®. Methanol and MPA-d3 were used as extraction solvent and internal standard, respectively. Chromatographic separation was obtained on a XBridge BEH C18 column (2.1 mm × 75 mm, 2.5 μm) maintained at 50 °C on a Waters Alliance 2795 coupled to a QuattroMicro tandem mass-spectrometer. The multiple reaction monitoring transitions used for quantification were m/z 320.97 → 207.0 for MPA, and 324.2 → 210.1 for MPA-d3, in positive ESI mode.

Results

The total HPLC run time was 6 min. The retention times for MPA-d3 and MPA were 2.55 for both compounds. The method was linear from 0.1 to 50 ng/mL MPA. The coefficient r² ranged from 0.996 to 0.998. Intra-assay and inter-assay imprecisions were < 10% in the whole range of concentrations, and < 20% at the LLOQ, and accuracy level was > 90%. The matrix and ion suppression effects were < 6%. The MPA limit of quantification was 0.1 ng/mL. No interference was identified in the assay. From the preliminary results, intra-cellular MPA concentrations in kidney transplant patients ranged from 0.69 to 3.39 ng/10⁷ cells.

Conclusion

We described a robust, rapid and simple method suitable for the determination of MPA concentrations in PBMCs. This method is currently used in pharmacokinetics–pharmacodynamics (PK–PD) clinical trials, in comparison to standard plasma TDM.     

CD47 fusion protein targets CD172a+ cells in Crohn’s disease and dampens the production of IL-1β and TNF

In mice, the transfer of CD172a⁺ (SIRP-α) dendritic cells (DCs) elicits T cell–driven colitis, whereas treatment with CD47-Fc protein, a CD172a-binding agent, confers protection. The aim of this study was to elucidate the nature and functional properties of human CD172a⁺ DCs in chronic intestinal inflammation. Here, we show that CD172a⁺CD11c⁺ cells accumulate in the mesenteric lymph nodes (mLNs) and inflamed intestinal mucosa in patients with Crohn’s disease (CD). These cells are distinct from resident DCs and may coexpress markers typically associated with monocyte-derived inflammatory DCs such as CD14 and/or DC-SIGN, E-Cadherin, and/or CX₃CR1. Spontaneous IL-1β and TNF production by HLA-DR⁺ cells in CD tissues is restricted to those expressing CD172a. An avidity-improved CD47 fusion protein (CD47-Var1) suppresses the release of a wide array of inflammatory cytokines by CD172a⁺ cells, which may include HLA-DR⁻CD172a⁺ neutrophils, in inflamed colonic explant cultures and impairs the ability of HLA-DR⁺CD172a⁺ cells to activate memory Th17 but not Th1 responses in mLNs. In conclusion, targeting CD172a⁺ cells may represent novel therapeutic perspectives for patients with CD.The gastrointestinal tract is lined with a single layer of epithelial cells that separates the gut lumen from the connective tissue and the immune system (Kaser et al., 2010; MacDonald et al., 2011). Because it is constantly exposed to dietary and environmental antigens and to an estimated community of 10¹⁴ commensal bacteria, the immune system is confronted with the difficult task of enforcing tolerance to innocuous environmental antigens while also protecting against invading pathogens. An aberrant immune response to the intestinal microbiota contributes to the pathogenesis of Crohn’s disease (CD), a chronic inflammatory bowel disease (IBD) that affects genetically predisposed individuals (Chassaing and Darfeuille-Michaud, 2011; Maloy and Powrie, 2011).Mononuclear phagocytes, which include a large population of macrophages (MΦ) and rare subsets of DCs, are critical for the establishment and maintenance of gut homeostasis (Coombes and Powrie, 2008; Varol et al., 2010). However, myeloid cell heterogeneity in phenotype, origin, and function has led to confusion over the classification between MΦ and DCs, especially in mucosal tissues (Gautier et al., 2012; Miller et al., 2012). In murine tissues, CD11c is not an adequate marker to identify DCs because it is also expressed in varying levels on F4/80⁺ MΦ (Medina-Contreras et al., 2011; Rivollier et al., 2012). This is in contrast to resident MΦ in human lamina propria (LP), which do not express CD11c (Smith et al., 2011). In mice, macrophage–dendritic cell progenitors (MDPs) give rise to dedicated common DC precursors (precDCs) and monocytes via developmental pathways that are governed by Flt3L and M-CSF, respectively (Liu et al., 2009). Both the CD103⁺CD11b⁺ and CD103⁺CD11b⁻ DC subsets originate from precDCs. Tissue-resident CD103⁻CX₃CR1⁺ mononuclear phagocytes, which are the dominant population in the murine gut LP, derive from Ly6C^high circulating monocytes. Murine intestinal homeostasis has been demonstrated to critically depend on a delicate equilibrium between tolerogenic migratory CD103⁺CX₃CR1⁻ DCs and pathogenic CD103⁻CX₃CR1⁺ mononuclear phagocytes (Jaensson et al., 2008; Bogunovic et al., 2009; Varol et al., 2009). In fact, mice genetically depleted of CD103⁺ DCs and CX₃CR1⁺ MΦ do not develop spontaneous inflammation (Birnberg et al., 2008). Animals that have a predominance of CX₃CR1⁺ cells in the LP develop exacerbated colitis (Varol et al., 2009). However, both CX₃CR1⁺F4/80⁺CD103⁻ LP MΦ and CD103⁺ DCs can induce gut tolerance through the generation and/or maintenance of the suppressive activity of Foxp3⁺ regulatory T cells, and CX₃CR1 deficiency leads to exacerbated DSS-induced colitis (Denning et al., 2007; Sun et al., 2007; Medina-Contreras et al., 2011).Recent studies have independently demonstrated that CD103⁻E-Cadherin⁺ and CD103⁻SIRP-α⁺ (CD172a) cells induce experimental colitis in mice (Fortin et al., 2009; Siddiqui et al., 2010). These pathogenic cells accumulate in the inflamed colons and/or LNs. The CD103⁻E-Cadherin⁺ cells originate from Ly6C^high circulating monocytes that migrate in a CCR7-independent manner to the mesenteric LNs (mLNs), whereas the CD103⁻CD172a⁺ DCs accumulate in the inflamed colons and mLNs via a CD47-dependent process. These cell populations promote T cell driven anti-CD40–mediated colitis and drive Th17-associated TNBS colitis in mice; the latter can be ameliorated by the administration of a CD47-Fc fusion protein that putatively targets the CD172a⁺ cells. Whether human equivalents of the colitogenic CD103⁻CD172a⁺ cells exist and whether they can be targeted by CD47-Fc in the mLNs (inductive site) and/or intestinal tissues (effector site) of CD patients remains unknown.Previous studies have reported the presence of CD14⁺ MΦ in situ in the colons of CD patients (Grimm et al., 1995a). Imaging analyses of intestinal mucosal tissues of CD patients have also revealed the existence of several distinct DC populations including DC-SIGN (CD209)⁺CD11c⁺ DCs, CD83⁺ DCs, CD103⁺ DCs, plasmacytoid DCs (pDCs) and Slan⁺ monocytes/DCs (de Baey et al., 2003; Jaensson et al., 2008; te Velde et al., 2003; Verstege et al., 2008). In addition, a CD33⁺CD14⁺ intermediate MΦ/DC subset has been detected at similar frequencies throughout the nonlesional and lesional gut mucosa in CD patients (Kamada et al., 2008).In this study, we provide compelling evidence for the accumulation of proinflammatory cytokine-producing HLA-DR⁺CD172a⁺ cells that coexpress or not E-Cadherin and CX₃CR1 in the mLNs and inflamed mucosa of CD patients. These cells are a major source of IL-1β, IL-6, and TNF and can be targeted by an avidity-improved CD47 fusion protein (CD47-Var1) in inflamed CD tissues.     

Diagnostic and Prognostic Performances Over 9 Years of a Selective Screening Strategy for Gestational Diabetes Mellitus in a Cohort of 18,775 Subjects

OBJECTIVE

We aimed to evaluate a selective screening strategy for gestational diabetes mellitus (GDM) based on the presence of risk factors: BMI ≥25 kg/m², age ≥35 years, family history of diabetes, personal history of GDM, or birth of a child with macrosomia.

RESEARCH DESIGN AND METHODS

Of 20,630 deliveries between 2002 and 2010, we selected 18,775 deliveries in women with no known diabetes and for whom all risk factors were known. GDM was universally screened and defined as fasting plasma glucose level ≥5.3 mmol/L and/or 2-h postload (75 g) glucose level ≥7.8 mmol/L.

RESULTS

The prevalence of at least one risk factor has increased since 2002 (P < 0.001) from 51.7 to 61.5%, with no change in the GDM prevalence (mean 14.4%, intention to screen). At least one risk factor was present in 58.5% of women who represented 65.3% of all those with GDM. The presence of risk factors was significantly associated with GDM (odds ratio 1.4 [95% CI 1.3–1.5], P < 0.001) and with GDM-related events (preeclampsia/large for gestational age/dystocia) (P < 0.001) with the following incidences: no GDM/no risk factor 8.8%, no GDM/risk factor 11.1%, GDM/no risk factor 16.7%, and GDM/risk factor 18.2%.

CONCLUSIONS

The presence of risk factors increased during the last decade. This condition is predictive of GDM and GDM-related events. However, a selective screening would lead to missing one-third of the women with GDM who, even without risk factors, had more events than women without GDM. Therefore, these data stand against the present selective screening currently proposed in the French guidelines.New international diagnostic criteria for gestational diabetes mellitus (GDM) (1) have been progressively adopted worldwide, leading to a dramatic increase in GDM prevalence. A universal screening has been recommended, but a selective screening, based on the presence of risk factors, has been used as well (2). Using selective screening may lead to missing up to 45% of GDM cases (3). On the other hand, selective screening could help to concentrate medical resources on subjects with the highest risk of complications (2), especially those with a high BMI (4). This is crucial, as the cost-efficacy ratio of GDM screening still needs to be evaluated in women with no risk factors (5). For example, the expert panel for French guidelines stated, as a professional agreement, that there was no sufficient evidence for recommending universal screening (5). Based on the review of literature on GDM risk factors (6), they recommended GDM screening if at least one of the following criteria is present: maternal age ≥35 years, BMI ≥25 kg/m², history of diabetes in a first-degree relative, personal history of GDM, or birth of a child with macrosomia.We therefore aimed to retrospectively evaluate this recommendation in a large cohort of women who had delivered in the previous 9 years in the obstetrics department of our university hospital located in an eastern suburb of Paris, France. More specifically, the aims were to evaluate 1) the screening value of risk factors for GDM and 2) the prognostic value of risk factors for GDM-related events. We also took the opportunity of this large cohort to look for changes in the prevalence of GDM and risk factors during the years after 1999.