Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase

A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony- forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.     

Does a preemptive block of the great auricular nerve improve postoperative analgesia in children undergoing tympanomastoid surgery?

We performed a double-blinded randomized controlled trial to evaluate the efficacy of preemptive analgesia in children undergoing tympanomastoid surgery. Children were divided into two groups: group block-block (BB) received a preemptive great auricular nerve block (GAN-block) with 0.25% bupivacaine with 1:200,000 epinephrine before incision followed by a second GAN-block with 0.25% bupivacaine with 1:200,000 epinephrine 1 h before the end of the procedure. Group sham block-block (SB-B) received a preemptive GAN-block with normal saline before surgical incision followed by a GAN-block with 0.25% bupivacaine with 1:200000 epinephrine 1 h before the completion of the procedure. All patients were evaluated for pain with the objective pain score (OPS) by a blinded observer. There was no difference in pain rescue requirements in the postanesthesia care unit (BB versus SB-B, 1 of 20 versus 3 of 20, P= 0.60) or in the short-stay unit (BB versus SB-B, 5 of 20 versus 11 of 20, P = 0.107) or for the entire hospital stay (P = 0.20). There was no significant difference between groups in the time to first rescue pain medication (BB versus SB-B, 226 +/- 71 min versus 201 +/- 94 min). There was no significant difference between groups regarding vomiting in the postoperative period (P = 0.52). We conclude that a preoperative GAN-block does not offer significant advantages for postoperative pain relief in children undergoing tympanomastoid surgery. IMPLICATIONS: This double-blinded randomized controlled trial compared the efficacy of preemptive analgesia with a peripheral nerve block of the great auricular nerve for decreasing postoperative pain in children undergoing tympanomastoid surgery. Preemptive analgesia did not improve the quality or duration of postoperative analgesia in our cohort.     

Age-dependent neurological phenotypes in a mouse model of PRRT2-related diseases

Paroxysmal kinesigenic dyskinesia is an episodic movement disorder caused by dominant mutations in the proline-rich transmembrane protein PRRT2, with onset in childhood and typically with improvement or resolution by middle age. Mutations in the same gene may also cause benign infantile seizures, which begin in the first year of life and typically remit by the age of 2 years. Many details of PRRT2 function at the synapse, and the effects of mutations on neuronal excitability in the pathophysiology of epilepsy and dyskinesia, have emerged through the work of several groups over the last decade. However, the age dependence of the phenotypes has not been explored in detail in transgenic models. Here, we report our findings in heterozygous and homozygous Prrt2 knockout mice that recapitulate the age dependence of dyskinesia seen in the human disease. We show that Prrt2 deletion reduces the levels of synaptic proteins in a dose-dependent manner that is most pronounced at postnatal day 5 (P5), attenuates at P60, and disappears by P180. In a test for foot slippage while crossing a balance beam, transient loss of coordination was most pronounced at P60 and less prominent at age extremes. Slower traverse time was noted in homozygous knockout mice only, consistent with the ataxia seen in rare individuals with biallelic loss of function mutations in Prrt2. We thus identify three age-dependent phenotypic windows in the mouse model, which recapitulate the pattern seen in humans with PRRT2-related diseases.

     

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.     

Characteristics and outcomes of diffuse large B-cell lymphoma presenting in leukaemic phase

Diffuse large B‐cell lymphoma (DLBCL) occasionally presents with circulating malignant cells. The clinical characteristics and long‐term outcomes of these patients have not been described. Twenty‐nine newly diagnosed DLBCL presenting in leukaemic phase were identified between 1996 and 2010, at two institutions. Median age was 48 years, and patients presented with leucocytosis, high lactate dehydrogenase levels, B symptoms, and high International Prognostic Index score. Extra nodal site involvement was observed in all patients and affected the bone marrow (100%), spleen (62%), pleura/lung (41%), liver (21%), bone (17%), bowels (7%) and cerebrospinal fluid (14%). Blood lymphomatous cells co‐expressed CD19, CD20, CD22, CD38, CD45, HLA‐DR and FMC7 in >90%, and kappa or lambda light chain restriction in >50%. Ninety per cent received rituximab and anthracycline‐based chemotherapy. Overall, remission was complete in 54% and partial in 31%; 15% had resistant disease. Median follow‐up was 47 months; 13 (45%) patients remain alive in complete remission. Median progression‐free and overall survivals were 11·5 and 46·7 months, respectively. In summary, patients with DLBCL in leukaemic phase present with high tumour burden and frequent involvement of extra nodal sites. In this uncommon DLBCL subgroup, anthracycline‐based regimens with rituximab are associated with early morbidity and mortality, but yield approximately 50% 4‐year survival.