Nocturnal hypoxaemia is common in adults with sickle cell anaemia.

The symptoms and sequelae of sickle cell anaemia (SCA) are caused by the polymerization of deoxygenated sickle haemoglobin, and people with SCA may be uniquely susceptible to adverse outcomes from hypoxia and haemoglobin desaturation. We examined by oximetry adults (aged 18-45 years) with SCA presenting symptoms indicative of polysomnography, at a single institution, irrespective of treatment, for nocturnal hypoxaemia. Clinical labs and blood for in vitro assessments were taken upon enrolment and after 8-12 weeks of oxygen therapy or observation. Of 21 screened participants, nine (43%) had sufficient nocturnal hypoxaemia to warrant oxygen therapy (≥5 min at SpO2 ≤ 88%). Time spent at SpO2 ≤ 88% associated with age (p = 0.0092), annual hospitalizations (p = 0.0018) and anaemia (p = 0.0139), as well as plasma levels of TNFα (p = 0.0019) and IL-4 (p = 0.0147). Longitudinal analysis showed that WBC significantly decreased during the follow-up period in hypoxic individuals but not in non-hypoxic individuals (p = 0.0361 and p = 0.6969 respectively). Plasma levels of CCL2 and IL-1ra tended to increase, while levels of red blood cell reactive oxygen species tended to decrease with oxygen therapy. Overall, nocturnal hypoxaemia was common in this pilot study population and associated with plausible clinical comorbidities; oxygen therapy may decrease inflammation and oxidative damage in hypoxic individuals.

Persistent albuminuria and chronic kidney disease in adults with sickle cell anaemia: Results from a multicenter natural history study.

Clinical and laboratory correlates of chronic kidney disease (CKD) in sickle cell anaemia remain incompletely defined. In a multicenter cohort study, we evaluated the prevalence of persistent albuminuria (PA) and characteristics associated with PA, albumin-creatinine ratio (ACR) and decreased estimated glomerular filtration rate (eGFR) using logistic, linear and multinomial regression models, respectively. Of 269 participants (median age: 30 years; 57.2% females), the prevalence of PA was 35.7%. Using baseline ACR values of <100 and ≥100 mg/g, the probabilities of PA were 30.0% and 94.6%, respectively. In multivariable logistic regression analyses, male sex (ß = 0.80 [SE = 0.36], p = 0.024) and ACE inhibitors/ARBs use (ß = 1.54 [SE = 0.43], p < 0.001) were associated with higher likelihoods of PA, while higher haemoglobin (ß = -0.33 [SE = 0.13], p = 0.009) and HbF (ß = -0.04 [SE = 0.02], p = 0.041) were associated with lower likelihoods of PA. In multivariable multinomial regression analyses, older age (ß = 0.06 [SE = 0.02], p = 0.004) and higher alkaline phosphatase (ß = 0.01 [SE = 0.00], p = 0.004) were associated with higher odds of having eGFR 60-90 versus eGFR>90 mL/min/1.73 m2 using the cystatin C-based CKD-EPI-2012 equation. Additionally, higher systolic blood pressure (ß = 0.11 [SE = 0.03], p = 0.001) and blood urea nitrogen (ß = 0.45 [SE = 0.12], p < 0.001) were associated with higher odds, while higher haemoglobin (ß = -1.22 [SE = 0.43], p = 0.004) was associated with lower odds of having eGFR<60 versus eGFR>90 mL/min/1.73 m2. PA and decreased eGFR are associated with measures of disease severity and comorbid conditions (Clinicaltrials.gov Identifier: NCT03277547).

A pilot study of the effect of rivaroxaban in sickle cell anemia.

INTRODUCTION: The contribution of coagulation activation to the pathogenesis of sickle cell disease (SCD) remains incompletely defined. We evaluated the efficacy and safety of rivaroxaban, an oral direct factor Xa inhibitor, in subjects with sickle cell anemia. MATERIALS AND METHODS: In this pilot, single-center, randomized, double-blind, placebo-controlled, crossover study, eligible subjects with sickle cell anemia received rivaroxaban or placebo. The effect of rivaroxaban on coagulation activation, endothelial activation, inflammation, and microvascular blood flow was evaluated. RESULTS: Fourteen patients (HbSS - 14; females - 9) with mean age of 38 ± 10.6 years were randomized to receive rivaroxaban 20 mg daily or placebo for 4 weeks and, following a 2-week washout phase, were "crossed-over" to the treatment arm opposite to which they were initially assigned. Mean adherence to treatment with rivaroxaban, assessed by pill counts, was 85.6% in the first treatment period and 93.6% in the second period. Treatment with rivaroxaban resulted in a decrease from baseline of thrombin-antithrombin complex versus placebo (-34.4 ug/L [95% CI: -69.4, 0.53] vs. 0.35 ug/L [95% CI: -3.8, 4.5], p = .08), but the difference was not statistically significant. No significant differences were observed in changes from baseline of D-dimer, inflammatory, and endothelial activation markers or measures of microvascular blood flow. Rivaroxaban was well tolerated. CONCLUSIONS: Rivaroxaban was safe but did not significantly decrease coagulation activation, endothelial activation, or inflammation. Rivaroxaban did not improve microvascular blood flow. Adequately powered studies are required to further evaluate the efficacy of rivaroxaban in SCD. Clinicaltrials.gov Identifier: NCT02072668.

1α,25-dihydroxyvitamin D3 (vitamin D3) catalyzes suppressive activity on human natural regulatory T cells, uniquely modulates cell cycle progression, and augments FOXP3.

Human natural regulatory T cells (nTregs) show great promise for therapeutically modulating immune-mediated disease, but remain poorly understood. One explanation under intense scrutiny is how to induce suppressive function in non-nTregs and increase the size of the regulatory population. A second possibility would be to make existing nTregs more effective, like a catalyst raises the specific activity of an enzyme. The latter has been difficult to investigate due to the lack of a robust short-term suppression assay. Using a microassay described herein we demonstrate that nTregs in distinct phases of cell cycle progression exhibit graded degrees of potency. Moreover, we show that physiological concentrations of 1α,25-dihydroxyvitamin D3 (vitamin D3) boosts nTregs function. The enhanced suppressive capacity is likely due to vitamin D3's ability to uniquely modulate cell cycle progression and elevate FOXP3 expression. These data suggest a role for vitamin D3 as a mechanism for catalyzing potency of nTregs.

Therapeutic in vivo selection of thymic-derived natural T regulatory cells following non-myeloablative hematopoietic stem cell transplant for IPEX.

FOXP3 is critical for the development and function of CD4(+)CD25(bright) natural regulatory T cells (nTreg). Individuals harboring mutations in FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). We describe a child diagnosed with IPEX who underwent a reduced intensity, T and B cell depleted, matched unrelated donor bone marrow transplant followed by clinical resolution. Using lineage-specific donor chimerism studies, we demonstrate that non-myeloablative HSCT resolves disease in the context of low level donor hematopoietic stem cell (HSC) engraftment. Despite low-levels of donor HSC, thymically-derived nTreg and to a lesser extent CD4(+) and CD8(+) T cells, exhibit a selective in vivo growth advantage for populations containing a functional FOXP3 gene. Moreover, nTreg from this patient show regulatory function directly ex vivo. These results have implications for improving clinical therapy for patients with IPEX and provide mechanistic insight into the in vivo development of human nTreg and unexpectedly, non-regulatory T cells.

Efficient and reproducible large-scale isolation of human CD4+ CD25+ regulatory T cells with potent suppressor activity.

CD4+ CD25+ regulatory T cells have been the subject of intense investigation and have been shown to modulate immune responses in the settings of autoimmunity, cancer and transplantation. The assessment and optimization of purification schemes for specific cellular subtypes such as CD4+ CD25+ regulatory T cells is a critical consideration in developing cell-based therapies in the clinical setting. In the following studies, different strategies for magnetic isolation are compared and the parameters which affect the overall potency of purified human CD4+ CD25+ regulatory T cells are discussed. The data demonstrate that large-scale magnetic isolation can be used to efficiently and reproducibly purify human CD4+ CD25+ regulatory T cells capable of modulating alloreactive T cell responses. The ability to rapidly purify the desired cells from peripheral blood suggests that magnetic isolation may be a suitable alternative to cell sorting for clinical settings, where large numbers of CD4+ CD25+ regulatory T cells may be necessary.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100-500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).

Prospective analysis of TEL gene rearrangements in childhood acute lymphoblastic leukemia: a Children's Oncology Group study.

PURPOSE: To prospectively determine the prognostic significance of the TEL-AML1 fusion in children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: TEL gene status was determined for 926 patients with B-precursor ALL enrolled on the Pediatric Oncology Group ALinC 16 trials and patients were observed for a median time of 8 years. RESULTS: Rearrangements of the TEL gene were detected in 244 patients (26%). The estimated 5-year event-free survival rate (+/- SE) for patients with TEL rearrangements was 86% +/- 2%, compared with 72% +/- 2% for those with germline TEL (P < .0001). TEL rearrangements were associated with a superior outcome among patients with standard-risk ALL, high-risk ALL, and rapid early responses to therapy. In a multivariate analysis that included risk group, sex, and day 15 marrow status, TEL status was an independent predictor of outcome (P = .0002). CONCLUSION: We conclude that TEL gene status should be incorporated into risk classification schemes and suggest that patients who have standard-risk features, the TEL-AML1 fusion, and rapid early responses to therapy, should be treated with antimetabolite-based therapy designed to maintain their high cure rates and avoid late effects.

Effector CD8+ T cells recovered from an influenza pneumonia differentiate to a state of focused gene expression.

The restriction of influenza A virus replication to mouse respiratory epithelium means that this host response is anatomically compartmentalized, on the one hand, to sites of T cell stimulation and proliferation in the secondary lymphoid tissue and, on the other hand, to the site of effector T cell function and pathology in the pneumonic lung. Thus, it is hardly surprising that virus-specific CD8(+) T cells recovered by bronchoalveolar lavage (BAL) from the infected respiratory tract seem more "activated" in terms of both cytolytic activity and cytokine production than those cells isolated from the spleen. The present analysis uses Affymetrix microarray technology to compare profiles of gene expression in these two lineage-related, yet anatomically separate, lymphocyte populations. Ninety differentially expressed genes were identified for influenza-specific CD8(+)D(b)NP(366)(+) T cells obtained directly ex vivo by BAL or spleen disruption, with nine genes being further analyzed by quantitative, real-time PCR at the population level. Integrin alphaE, for example, was shown by Affymetrix and real-time mRNA analyses and then by single-cell PCR and protein staining to be present at a much higher prevalence on the BAL CD8(+)D(b)NP(366)(+) set. The unpredicted finding, however, was that mRNA expression for 75% of the 90 genes was lower in T cells from the BAL than from the spleen. Apparently, the localization of virus-specific CD8(+) T cells to the site of virus-induced pathology is associated with a narrowing, or "focusing," of gene expression that favors enhanced effector function in the damaged, "high-antigen load" environment of the pneumonic lung.

Human CD4+CD25+ regulatory T cells share equally complex and comparable repertoires with CD4+CD25- counterparts.

CD4(+)CD25(+) T cells are critical mediators of peripheral immune tolerance. However, many developmental and functional characteristics of these cells are unknown, and knowledge of human regulatory T cells is particularly limited. To better understand how human CD4(+)CD25(+) T cells develop and function, we examined the diversity of CD4(+)CD25(+) and CD4(+)CD25(-) T cell repertoires in both thymus and peripheral blood. Levels of T receptor excision circles (TREC) were comparable in purified CD4(+)CD25(+) and CD4(+)CD25(-) thymic populations, but were significantly higher than those in samples derived from peripheral blood, consistent with murine studies demonstrating thymic development of CD4(+)CD25(+) regulatory T cells. Surprisingly, CD4(+)CD25(-) T cells isolated from peripheral blood had greater TREC quantities than their CD4(+)CD25(+) counterparts, supporting the possibility of extrathymic expansion as well. CD4(+)CD25(+) and CD4(+)CD25(-) T cells from a given individual showed overlapping profiles with respect to diversity by Vbeta staining and spectratyping. Interestingly, CD4(+)CD25(+) T cells have lower quantities of CD3 than CD4(+)CD25(-) T cells. Collectively, these data suggest that human CD4(+)CD25(+) T cells recognize a similar array of Ags as CD4(+)CD25(-) T cells. However, reduced levels of TCR on regulatory T cells suggest different requirements for activation and may contribute to how the immune system regulates whether a particular response is suppressed or augmented.