Randomized comparison of single dose of recombinant human IL-12 versus placebo for restoration of hematopoiesis and improved survival in rhesus monkeys exposed to lethal radiation

BACKGROUND: The hematopoietic syndrome of the acute radiation syndrome (HSARS) is a life-threatening condition in humans exposed to total body irradiation (TBI); no drugs are approved for treating this condition. Recombinant human interleukin-12 (rHuIL-12) is being developed for HSARS mitigation under the FDA Animal Rule, where efficacy is proven in an appropriate animal model and safety is demonstrated in humans. METHODS: In this blinded study, rhesus monkeys (9 animals/sex/dose group) were randomized to receive a single subcutaneous injection of placebo (group 1) or rHuIL-12 at doses of 50, 100, 250, or 500 ng/kg (groups 2–5, respectively), without antibiotics, fluids or blood transfusions, 24–25 hours after TBI (700 cGy). RESULTS: Survival rates at Day 60 were 11%, 33%, 39%, 39%, and 50% for groups 1–5, respectively (log rank p < 0.05 for each dose vs. control). rHuIL-12 also significantly reduced the incidences of severe neutropenia, severe thrombocytopenia, and sepsis (positive hemoculture). Additionally, bone marrow regeneration following TBI was significantly greater in monkeys treated with rHuIL-12 than in controls. CONCLUSIONS: Data from this study demonstrate that a single injection of rHuIL-12 delivered one day after TBI can significantly increase survival and reduce radiation-induced hematopoietic toxicity and infections. These data significantly advance development of rHuIL-12 toward approval under the Animal Rule as an effective stand-alone medical countermeasure against the lethal effects of radiation exposure

The Transmembrane Isoform of Plasmodium falciparum MAEBL Is Essential for the Invasion of Anopheles Salivary Glands

Malaria transmission depends on infective stages in the mosquito salivary glands. Plasmodium sporozoites that mature in midgut oocysts must traverse the hemocoel and invade the mosquito salivary glands in a process thought to be mediated by parasite ligands. MAEBL, a homologue of the transmembrane EBP ligands essential in merozoite invasion, is expressed abundantly in midgut sporozoites. Alternative splicing generates different MAEBL isoforms and so it is unclear what form is functionally essential. To identify the MAEBL isoform required for P. falciparum (NF54) sporozoite invasion of salivary glands, we created knockout and allelic replacements each carrying CDS of a single MAEBL isoform. Only the transmembrane form of MAEBL is essential and is the first P. falciparum ligand validated as essential for invasion of Anopheles salivary glands. MAEBL is the first P. falciparum ligand experimentally determined to be essential for this important step in the life cycle where the vector becomes infectious for transmitting sporozoites to people. With an increasing emphasis on advancing vector-based transgenic methods for suppression of malaria, it is important that this type of study, using modern molecular genetic tools, is done with the agent of the human disease. Understanding what P. falciparum sporozoite ligands are critical for mosquito transmission will help validate targets for vector-based transmission-blocking strategies

HemaMax™, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8–9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit–expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD50/30) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12–14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin

A Systematic Review of Immunological Studies of Erythema Nodosum Leprosum.

Erythema nodosum leprosum (ENL) is a painful inflammatory complication of leprosy occurring in 50% of lepromatous leprosy patients and 5-10% of borderline lepromatous patients. It is a significant cause of economic hardship, morbidity and mortality in leprosy patients. Our understanding of the causes of ENL is limited. We performed a systematic review of the published literature and critically evaluated the evidence for the role of neutrophils, immune complexes (ICs), T-cells, cytokines, and other immunological factors that could contribute to the development of ENL. Searches of the literature were performed in PubMed. Studies, independent of published date, using samples from patients with ENL were included. The search revealed more than 20,000 articles of which 146 eligible studies were included in this systematic review. The studies demonstrate that ENL may be associated with a neutrophilic infiltrate, but it is not clear whether it is an IC-mediated process or that the presence of ICs is an epiphenomenon. Increased levels of tumor necrosis factor-α and other pro-inflammatory cytokines support the role of this cytokine in the inflammatory phase of ENL but not necessarily the initiation. T-cell subsets appear to be important in ENL since multiple studies report an increased CD4+/CD8+ ratio in both skin and peripheral blood of patients with ENL. Microarray data have identified new molecules and whole pathophysiological pathways associated with ENL and provides new insights into the pathogenesis of ENL. Studies of ENL are often difficult to compare due to a lack of case definitions, treatment status, and timing of sampling as well as the use of different laboratory techniques. A standardized approach to some of these issues would be useful. ENL appears to be a complex interaction of various aspects of the immune system. Rigorous clinical descriptions of well-defined cohorts of patients and a systems biology approach using available technologies such as genomics, epigenomics, transcriptomics, and proteomics could yield greater understanding of the condition

m-HemaMax administration increased plasma m-HemaMax and IFN-γ levels in irradiated and non-irradiated mice.

<p>Animals received m-HemaMax subcutaneously at a dose of (a) 10 ng/mouse, (b) 20 ng/mouse, (c) 40 ng/mouse, or (d) 200 ng/mouse in the absence of irradiation or at 24 hours after an LD_90/30 of TBI. The plasma concentrations of m-HemaMax and IFN-γ were determined by ELISA in blood samples withdrawn at the indicated times. The y-axis scale in (d) is 8 times greater than those in (a) and (b) and 5 times greater than that in (c). n = 3 per timepoint in each group.</p

Irradiated rhesus monkeys receiving HemaMax had less body weights loss than animals receiving vehicle.

<p>Body weights in Kg (a and b) and in percentage (c and d) are shown for the 100 ng/Kg and 250 ng/Kg dose groups. Monkeys were subjected to an LD_50/30 of TBI at day 0 and subsequently received either vehicle (P5.6TT) or HemaMax subcutaneously at the indicated dosing regimens. Supportive care was prohibited during the study. Body weights were recorded every other day for up to day 30.</p

m-HemaMax promotes hematopoietic recovery in irradiated mice.

<p>Representative sections of femoral bone marrow from non-irradiated, untreated mice that were stained for IL-12Rβ2 (orange color) are shown in (a). Animals were subjected to TBI (8.0 Gy) and subsequently received vehicle (P5.6TT) or m-HemaMax (20 ng/mouse) subcutaneously at the indicated times post irradiation (b–f). An additional group of mice received HemaMax at 24 hours after TBI (g). Femoral bone marrow was immunohistochemically stained for IL-12Rβ2 (orange color) 12 days after irradiation. While bone marrow from mice treated with vehicle lacked IL-12Rβ2–expressing cells and showed no signs of hematopoietic regeneration (b), mice treated with m-HemaMax showed hematopoietic reconstitution and the presence of IL-12Rβ2–expressing megakaryocytes, myeloid progenitors, and osteoblasts (c–f). Mice treated with HemaMax showed IL-12Rβ2–expressing osteoblasts but lacked megakaryocytes (g). Magnification = 100×.</p

Similar exposures to m-HemaMax and HemaMax at species-specific equivalent doses in mice and rhesus monkeys.

<p>The plot of plasma AUC_last of m-HemaMax versus the dose administered to mice in the absence of irradiation was linear at doses from 10 ng/mouse to 40 ng/mouse. The plasma AUC_last of HemaMax at monkey equivalent doses of 20 ng/Kg and 80 ng/Kg was in good agreement with the extend of dose-dependent increases in m-HemaMax exposure in mice.</p

Efficacy of m-HemaMax in increasing survival is not dependent on radiation dose in mice.

<p>Animals were subjected to TBI at ascending radiation doses of 8.6 Gy (LD_70/30), 8.8 Gy (LD_90/30), and 9.0 Gy (LD_100/30) and subsequently received m-HemaMax at a dose of 20 ng/mouse 24 hours after irradiation. Mice were monitored for survival up to day 30. Vehicle was P5.6TT.</p