Bruton’s tyrosine kinase (BTK) is involved in the regulation of B-cell growth, migration, and adhesion. The importance of BTK in cell trafficking is emphasized by the clonal contraction proceeded by lymphocytosis typical for the enzyme inhibitor, ibrutinib,in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Here, we investigated BTK regulation of leukemic B-cell trafficking in a mouse model of aggressive TCL1 CLL-like disease. Inhibiting BTK by ibrutinib reduced surface membrane (sm) levels of CXCR4 but not CXCR5, CD49d, and other adhesion/homing receptors. Decreased smCXCR4 levels resulted from blocking receptor signal transduction, which in turn aborted cycling from and to the membrane. This resulted in rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation. CLL cells with impaired smCXCR4 from BTK-inhibition failed to home to spleens. These functional changes mainly resulted from inhibition of CXCR4 phosphorylation at Ser339, mediated directly by blocking BTK enzymatic activity and indirectly by affecting the function of downstream targets PLCγ2 and PKCμ, and eventually synthesis of PIM-1 and BTK itself. Our data identify CXCR4 as a key regulator in BTK-mediated CLL cell retention and have elucidated a complex set of not previously described mechanisms responsible for these effects.

Respiratory syncytial virus is a common cause of acute respiratory infection in children. Previous reports have associated respiratory syncytial virus infection and acute neurologic symptoms, including apnea and seizures. This study examined the prevalence of acute neurologic symptoms associated with respiratory syncytial virus and non-respiratory syncytial virus respiratory infections in children requiring admission to a pediatric intensive care unit. We screened all admissions over a 4-year period at a single center, identifying children between birth and 2 years of age with bronchiolitis or acute upper respiratory infection. Children with a history of prematurity, preexisting medical conditions, or prior neurologic problems were excluded. Retrospective chart review of 236 admissions was conducted, identifying the etiology of respiratory infection and the presence or absence of acute neurologic signs or symptoms. This study demonstrated a prevalence of acute neurologic signs or symptoms in 36.4% of respiratory syncytial virus-negative patients (n = 115) and 39.1% in the respiratory syncytial virus-positive group (n = 121). Apnea, the most common symptom, was observed in 19.8% of respiratory syncytial virus-positive cases and 16.5% of negative patients. Seizures were reported in 6.6% of the positive cases and in 12.2% of the negative cohort. These differences showed no statistical significance. Although neurologic signs and symptoms are common in this population of children with acute respiratory infection, there was no significant difference in the prevalence of neurologic problems between respiratory syncytial virus-positive and -negative patients.

No abstract

Pulmonary edema complicating generalized tonic-clonic seizures has rarely been reported in children, although it has been well documented in adults. We report two patients, aged 8 and 9 years, who developed clinical and radiographic evidence of the condition. Fever, leukocytosis, and arterial hypoxemia are seen in the absence of cardiac dysfunction or infection. Rapid and complete recovery is to be expected if supportive therapy is instituted. Since these patients had no underlying cardiac pathology, control of the seizures will prevent further episodes.

No abstract

No abstract

A case of Gastrografin aspiration in a 3 3/4-year-old child admitted for multiple trauma is described.

975 We recently described a xenograft model of chronic lymphocytic leukemia (CLL) using NOD/SCID/γcnull (NSG) mice. Adoptive transfer of primary patient PBMCs into these mice results in engraftment and proliferation of CLL cells if autologous activated T cells are present. To date, such CLL-derived T cell proliferation has been achieved by co-transfer of third party antigen presenting cells (APCs). Unfortunately, in this setting, mice succumb to graft-versus-host disease, due to complex interactions between CLL cells, alloAPCs, T cells, and the xenogeneic host. We hypothesized that alternative strategies of autologous T cell activation might refine the model, ultimately providing longer CLL cell engraftment and animal survival. We describe 3 approaches to achieving engraftment and activation of CLL-derived T cells that support B cell growth in vivo. First, we activated CLL CD3+ cells isolated from PBMCs of 4 patients with anti-CD3/28 beads for 72 hours in vitro. Cells were then mixed with CFSE-labeled PBMCs from the same patient at varying ratios (1:50 to 1:1000 CD3+ cells:CLL PBMCs) and injected intraorbitally (io) into a total of 17 mice. CD4, CD8 and CD19 cell engraftment, identified by a human CD45 lymphocyte gate (hCD45), and proliferation, assessed by CFSE dilution of labeled cells, were monitored weekly. All mice demonstrated detectable CD3+ and CD5+CD19+ cells from week (wk) 1. The percent (%) CD3+ cells, as a proportion of hCD45, increased weekly in all mice receiving anti-CD3/28-activated cells. While overall % CD5+CD19+ cells decreased weekly, the % proliferating increased and strongly correlated with increasing % of T cells (r2=0.7799, p<0.0001, 45 evaluable data pairs). At the time of reporting (up to 5 wks follow up), 4 animals have died. Death correlated with high % circulating CD8+ (mean 57.7% vs. 14.2% prior to death, p<0.0001), but not CD4+ cells (mean 31.0% vs 36.3%, p=n/s). Spleen immunohistochemistry analyses revealed follicles containing CD20+ CLL cells, based on L chain restriction and RT-PCR for the leukemic IGHV-D-J. Follicles were infiltrated with both CD4+ and CD8+ cells. These findings were the same as found with our published model. Our second approach involved engraftment of solely CLL-derived peripheral blood T cells, without associated autologous PBMCs or alloAPCs. Positively selected CD3+ populations from 2 patients were activated with anti-CD3/28 beads, expanded in vitro, and 5–10×106 such cells injected into mice io. Cells from both patients yielded engraftment of CD4+ and CD8+ cells by wk 2. We then utilized two mice engrafted with cells from one patient, 1 with 70% and 1 with 30% CD4+ cells to compare the effect of CD4+ T cells in CLL cell growth. Injection of CFSE-labeled PBMCs into the mouse with high % of CD4 cells showed a clear circulating CFSE+CD5+CD19+ population at wk 1. By wk 2, 95% of the leukemic B cells had divided with 60% showing ≥6 divisions. In the mouse with low % CD4, CD5+CD19+ cell engraftment was suboptimal, never being more than 100 detectable events in analysis from any single bleed. No clear CFSE dilution pattern was apparent. Finally, we have assessed the development of CLL-derived T cells from autologous bone marrow CD34+ cells of 4 CLL cases. 48hr old neonatal NSG mice received CD34+ cells (≤1×106) intrahepatically. In 1 of the 4 animals, CD19+ cells emerged at 2 months but by month 4 the predominant hCD45+ population was CD3+ (60% of total hCD45). Subsequent io injection of CFSE-labeled PBMCs from the same subject demonstrated engraftment and immediate proliferation (detected by wk1 following injection) of both the io transferred CD3+ and CD5+CD19+ populations. In conclusion, we demonstrate that engraftment and expansion of CLL-derived T cells is feasible in NSG mice both as in vitro activated cells or as descendants of CD34+ cells and that the presence of CLL cells and third party APCs is not required. Such T cells are able to support engraftment and proliferation of CLL B cells from the same CLL subject. Strategies to reduce excessive numbers of T-cell subsets which may prevent early death of animals are underway. Elimination of third party APCs will permit easier dissection of the pathophysiology within this model, allowing for a better understanding of CLL cell kinetics and mechanism of action of novel therapeutics to treat this disease. Disclosures: No relevant conflicts of interest to declare.