Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells

Myeloid leukemias can express interleukin-3 receptors (IL-3R). Therefore, as an antileukemia drug, a fusion immunotoxin was synthesized consisting of the murine IL-3 (mIL-3) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mIL-3 hybrid gene was cloned into a vector under the control of an inducible promote. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was potent because it inhibited FDC-P1, an IL-3R- expressing murine myelomonocytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL). Kinetics were rapid and cell-free studies showed that DT390-mIL- 3 was as toxic as native DT. DT390-mIL-3 was selective because anti-mIL- 3 monoclonal antibody, but not irrelevant antibody, inhibited its ability to kill. Cell lines not expressing IL-3R were not inhibited by the fusion protein. Because the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, bone marrow (BM) progenitor assays were performed. DT390-mIL-3 selectively inhibited committed BM progenitor cells as measured by in vitro colony-forming unit-granulocyte- macrophage and in vivo colony-forming unit-spleen colony assays. To determine if this fusion protein was reactive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed. Eight million DT390-mIL-3-treated C57BL/6 Ly5.2 BM cells, but not 4 million, were able to rescue lethally irradiated congenic C57BL/6 Ly5.1 recipients, suggesting that progenitor cells might be heterogenous in their expression of IL-3R. This idea was supported in competitive repopulation experiments in which DT390-mIL-3 treated C57BL/6 Ly5.2 BM cells were mixed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57BL/6 Ly5.1 mice. A significant reduction, but not elimination, of Ly5.2- expressing cells 95 days post-BM transplantation and secondary transfer experiments indicated that IL-3R is not uniformly expressed on all primitive progenitor cells. The fact that some early progenitor cells survived DT390-mIL-3 treatment indicates that this fusion toxin may be useful in the treatment of myeloid leukemias that express the IL-3R.     

Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid

The recent finding that retinoic acid induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line, HL-60, prompted an investigation of the sensitivity to this inducer of human myelocytic leukemia cells in primary suspension culture. Of the 21 leukemic specimens, only cells from the two patients with acute promyelocytic leukemia differentiated in response to retinoic acid. After an incubation period of 5--7 days in 1 microM retinoic acid, the cells from these two patients showed extensive morphological and functional maturation. Thus, because it appears that retinoic acid specifically induces granulocytic differentiation of leukemic promyelocytes, this compound may have therapeutic utility in the treatment of acute promyelocytic leukemia.     

Isolated thrombocytopenia after allogeneic bone marrow transplantation: existence of transient and chronic thrombocytopenic syndromes

Isolated thrombocytopenia after bone marrow transplantation was investigated in 65 fully engrafted patients surviving at least 60 days posttransplant. Twenty-four patients (37%) developed this complication, which occurred most frequently in patients receiving pretransplant preparation with total body irradiation or busulfan. Two distinct thrombocytopenic syndromes were identified: (1) transient thrombocytopenia (nine patients), in which a normal platelet count (greater than 100,000/microL) was initially established by day +40 but then diminished to less than 10,000 to 45,000/microL on day +40 to +70, with subsequent resolution of the thrombocytopenia by day +90; (2) chronic thrombocytopenia (15 patients), in which a platelet count greater than 100,000/microL was not achieved at any time during the first four months posttransplant, despite the simultaneous presence of normal granulocyte and reticulocyte counts. Although the transient syndrome did not adversely affect prognosis, the chronic syndrome carried a high mortality (21% actuarial survival at 1,000 days posttransplant compared with 67% survival for all patients, P less than .01) and had a high association with both severe (grades 3 to 4) acute graft-versus-host disease (GVHD) and chronic GVHD. In three of nine patients with transient thrombocytopenia, a temporal association with trimethoprim-sulfamethoxazole administration was observed, whereas in all other patients, no drug association could be found. Bone marrow biopsies in those patients with drug-associated thrombocytopenia showed decreased numbers of megakaryocytes, whereas biopsies in the remainder of the transiently thrombocytopenic patients demonstrated adequate numbers of platelet precursors, suggesting peripheral platelet destruction or ineffective thrombopoiesis. Biopsies in the chronic thrombocytopenic patients included those with and without adequate numbers of platelet precursors, although the association with chronic GVHD was strongest in patients demonstrating normal numbers of megakaryocytes. We conclude that isolated thrombocytopenia represents a significant complication of bone marrow transplantation, particularly in patients receiving hematopoietic ablative preparatory regimens, and that it is the chronic, not the transient, thrombocytopenic syndrome that is associated with an adverse patient prognosis.     

Long‐term high‐physiological‐dose growth hormone reduces intra‐abdominal fat in HIV‐infected patients with a neutral effect on glucose metabolism

Objectives

The aim of the study was to investigate the effect of long‐term high‐physiological‐dose recombinant human growth hormone (rhGH) therapy on fat distribution and glucose metabolism in HIV‐infected patients.

Methods

Forty‐six HIV‐infected Caucasian men on highly active antiretroviral therapy (HAART), with an age range of 21–60 years and no significant comorbidity, were included in this randomized, placebo‐controlled, double‐blind, single‐centre trial. Twenty‐eight subjects were randomized to 0.7 mg/day rhGH, and 18 subjects to placebo, administered as daily subcutaneous injections between 1 and 3 pm for 40 weeks. Endpoints included changes in visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), limb fat mass, percentage of limb fat, plasma lipids, insulin resistance and glucose tolerance.

Results

VAT and trunk fat mass decreased significantly in the GH group compared with the placebo group [−19 cm² (−11%) vs. 12 cm² (6%), P=0.03, and −548 g (−9%) vs. 353 g (6%), P<0.01, respectively]. The beneficial fat redistribution in the GH group occurred without concomitant changes in subcutaneous fat at the abdomen or extremities. rhGH therapy was well tolerated. Insulin resistance, glucose tolerance, and total plasma cholesterol and triglycerides did not significantly change during intervention.

Conclusions

Daily 0.7 mg rhGH treatment for 40 weeks reduced abdominal visceral fat and trunk fat mass in HIV‐infected patients. This treatment appeared to be safe with respect to glucose tolerance and insulin sensitivity.

     

Reconstruction of iliac crest with rib to prevent donor site complications: A prospective study of 26 cases

Background:

The tricortical bone graft from the iliac crest are used to reconstruct the post corpectomy spinal defects. The donor iliac area defect is large and may give rise to pain at donor site, instability of pelvis, fracture of ilium, donor site muscle herniation or abdominal content herniation. Rib removed during thoracotomy was used by us to reconstruct the iliac crest defect.

Materials and Methods:

Twenty-six patients who underwent thoracotomy for dorsal spine corpectomy or curettage for various spinal pathologies from June 2002 to May 2004 were included in the study. After adequate decompression the spine was reconstructed by tricortical bone graft from iliac crest and reconstruction of the iliac crest was done with the rib removed for exposure during thoracotomy.

Results:

The mean follow up was 15 months. All patients had good graft incorporation which was evaluated on the basis of local tenderness and radiographs. One patient had graft displacement.

Conclusion:

The reconstruction of iliac crest by rib is a simple and effective procedure to prevent donor site complications.     

Interdisciplinary interactions,social systems and technical infrastructure required for successful implementation of mobile stroke units: A qualitative process evaluation

Rationale

Mobile stroke units (MSUs) are increasingly being implemented to provide acute stroke care in the prehospital environment, but a comprehensive implementation evaluation has not been undertaken.

Aim

To identify successes and challenges in the pre- and initial operations of the first Australian MSU service from an interdisciplinary perspective.

Methods

Process evaluation of the Melbourne MSU with a mixed-methods design. Purposive sampling targeted key stakeholder groups. Online surveys (administered June–September 2019) and semistructured interviews (October–November 2019) explored experiences. Directed content analysis (raters' agreement 85%) and thematic analysis results are presented using the Interactive Sociotechnical Analysis framework.

Results

Participants representing executive/program operations, MSU clinicians and hospital-based clinicians completed 135 surveys and 38 interviews. Results converged, with major themes addressing successes and challenges: stakeholders, vehicle, knowledge, training/education, communication, work processes and working relationships.

Conclusions

Successes and challenges of establishing a new MSU service extend beyond technical, to include operational and social aspects across prehospital and hospital environments.