TXU (Anti-CD7)-Pokeweed Antiviral Protein as a Potent Inhibitor of Human Immunodeficiency Virus

We have evaluated the clinical potential of TXU (anti-CD7)-pokeweed antiviral protein (PAP) immunoconjugate (TXU-PAP) as a new biotherapeutic anti-human immunodeficiency virus (anti-HIV) agent by evaluating its anti-HIV type 1 (anti-HIV-1) activity in vitro, as well as in a surrogate human peripheral blood lymphocyte-severe combined immunodeficient (Hu-PBL-SCID) mouse model of human AIDS. The present report documents in a side-by-side comparison the superior in vitro anti-HIV-1 activity of TXU-PAP compared to the activities of zidovudine, 2′,3′-didehydro-2′,3′-dideoxythymidine, unconjugated PAP, and B53-PAP, an anti-CD4-PAP immunoconjugate. Notably, TXU-PAP elicited potent anti-HIV activity in the Hu-PBL-SCID mouse model of human AIDS without any side effects and at doses that were very well tolerated by cynomolgus monkeys. Furthermore, plasma samples from TXU-PAP-treated cynomolgus monkeys showed potent anti-HIV-1 activity in vitro.     

Limitations of the human-PBL-SCID mouse model for vaginal transmission of HIV-1

PROBLEM: SCID mice reconstituted with human peripheral blood lymphocytes (PBL) are amenable to vaginal transmission of HIV-1. We investigated the effectiveness of this model to establish systemic HIV-1 infection. METHOD OF STUDY: Eighty progesterone-primed C.B-17 SCID mice were reconstituted with human-PBLs and intravaginally inoculated with CCR5 HIV-1 (BaL or 92BR09) infected human-PBLs in the presence of human semen. After two weeks, viral RNA load in spleen, peritoneal lavage (PL), and serum was quantitated by the nucleic acid sequence-based amplification method. RESULTS: In five independent experiments, spleen from 8/60 (13.3%), PL from 7/60 (11.6%), and serum from 16/56 (28.5%) mice were positive for BaL HIV-1 infection. Similarly, spleen from 4/20 (20%), PL from 1/20 (5%) and serum from 5/20 (25%) mice vaginally inoculated with 92BR09-infected human-PBLs were positive for HIV-1. A one-sided power analysis using normal approximation revealed that at 5% significance level, the overall response rate need to increase form 0.29 to 0.9 and 80% of the control groups needs to achieve a response rate between 6/10 and 9/10 to make the assay feasible. CONCLUSION: The incidence of vaginal transmission of CCR5 HIV-1 in the human-PBL-SCID mouse was low and variable, which constitutes a major disadvantage for preclinical evaluation of vaginal microbicides.     

Inducing apoptosis in chemotherapy-resistant B-lineage acute lymphoblastic leukaemia cells by targeting HSPA5, a master regulator of the anti-apoptotic unfolded protein response signalling network

We present previously unknown evidence that the immunoglobulin heavy chain binding protein BIP/HSPA5, also known as glucose regulated protein (GRP)78, serving as a pivotal component of the pro-survival axis of the unfolded protein response (UPR) signalling network, is abundantly expressed in relapsed B-lineage acute lymphoblastic leukaemia (ALL) and contributes to chemotherapy resistance of leukaemic B-cell precursors. The resistance of B-lineage ALL cells to the standard anti-leukaemic drug vincristine was overcome by the HSPA5 inhibitor epigallocatechin gallate, which inhibits the anti-apoptotic function of HSPA5 by targeting its ATP-binding domain. Notably, chemotherapy-resistant B-lineage ALL cells underwent apoptosis within 48 h of exposure to a doxorubicin-conjugated cell-penetrating cyclic anti-HSPA5 peptide targeting surface-expressed HSPA5 molecules on leukaemia cells. The identification of the HSPA5 as a chemoresistance biomarker and molecular target for B-lineage ALL may lead to new anti-leukaemic treatment strategies that are much needed.     

JAK3 pathway is constitutively active in B-lineage acute lymphoblastic leukemia

In this article, we report that primary leukemic B-cell precursors from B-lineage acute lymphoblastic leukemia (ALL) patients overexpress multiple JAK3-activating cytokines as well as their receptors. We also show that amplified expression of JAK3 pathway genes in B-lineage ALL is associated with steroid resistance and relapse. Our findings further demonstrate that several different diagnostic classes of B-lineage lymphoid malignancies exhibit upregulated expression of JAK3 pathway genes, which are associated with an overexpression of genes for JAK3-stimulatory cytokines with concomitant deficiency of JAK3-inhibitory signaling molecules. Thus, despite the rare occurrence of activating JAK3 mutations, JAK3 appears to be constitutively active and represents a viable molecular target in the treatment of a broad range of B-lineage lymphoid malignancies, including B-lineage ALL.     

Stimulation of Bruton's tyrosine kinase (BTK) and inositol 1,4,5-trisphosphate production in leukemia and lymphoma cells exposed to low energy electromagnetic fields.

We examined the effects of low energy electromagnetic field (EMF) exposure on the BTK kinase activity in B18-2 ([Btk-, rBTK(wt)] DT40) chicken lymphoma B cells and NALM-6 leukemic pre-B cells. Exposure of B 18-2 cells to EMF resulted in activation of BTK within 1 to 15 minutes in 8 of 8 independent experiments with stimulation indexes ranging from 1.2 to 13.3. While in some experiments the BTK stimulation was transient, in others the BTK activity continued to be significantly elevated for up to 4 hours. Similarly, exposure of NALM-6 cells to EMF resulted in activation of BTK within 30 minutes in 7 of 7 experiments with stimulation indexes ranging from 1.2 to 7.4. Stimulation of BTK activity in EMF exposed cells was associated with enhanced phosphoinositide turnover and increased inositol-1,4,5-trisphosphate (IP3) production in 7 of 13 experiments with DT40 cells and 7 of 13 experiments with NALM-6 cells. The likelihood and magnitude of an IP3 response after EMF exposure were similar to those after BCR ligation on DT40 cells and CD19 ligation on NALM-6 cells. These results confirm and extend our previous studies regarding EMF-induced biochemical signaling events in B-lineage lymphoid cells.     

Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation.     

Thermal sensitivity and thermal tolerance of human B-lineage acute lymphoblastic leukemia (ALL) cells

The thermal sensitivities of four B-cell precursor acute lymphoblastic (ALL) cell lines (REH and KM-3 = pre pre B-ALL; NALM-6 and HPB-NULL = pre B-ALL), and 1 B-cell ALL (NAMALWA) cell line were studied and compared to the thermal sensitivity of the T-lineage ALL cell line MOLT-3 using an in vitro clonogenic assay system by limiting dilution. B-lineage ALL cells were as sensitive to hyperthermia as were T-lineage ALL cells. D0 values at 42 degrees C ranged from 44.9 min (NALM-6) to 85.6 min (NAMALWA), D0 values at 43 degrees C ranged from 15.3 min (NALM-6) to 35.7 min (KM-3), and D0 values at 44 degrees C ranged from 11.1 min (NALM-6) to 23.8 min (HPB-NULL). By comparison, the D0 values of MOLT-3 cells were 95.1 min at 42 degrees C, 23.8 min at 43 degrees C, and 14.7 min at 44 degrees C. The maximum log kill values which were observed ranged from 0.8 log (KM-3 and HPB-NULL) to 1.3 logs (NALM-6) at 42 degrees C, from 1.4 logs (KM-3) to 4.2 logs (NALM-6) at 43 degrees C, and from 3.8 logs (HPB-NULL) to 4.8 logs (NALM-6) at 44 degrees C. A thermal tolerant plateau was observed in the hyperthermia survival curves of REH, NALM-6, and HPB-NULL cells, providing circumstantial evidence that thermal tolerance may develop in some B-cell precursor ALL cells after 90-120 min of continuous heating. In contrast, no thermal tolerant plateau was observed in the hyperthermia survival curves of pre-pre-B-ALL/KM-3 B-cell ALL/NAMALWA or T-lineage ALL/MOLT-3 cells. The kinetics of development and decay of thermotolerance was studied for NALM-6 cells. Thermotolerance after a priming heat exposure to 42 degrees C for 30 min was maximum at 8 hr with a maximum thermotolerance ratio of 2.0, and it decayed by 24 hr. These findings extend previous studies on the thermal sensitivity of human leukemia cells and provide new information on the thermal sensitivity and thermotolerance of B-lineage ALL cells.