hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33

The completion of the Human Genome Project resulted in discovery of many unknown novel genes. This feat paved the way for the future development of novel therapeutics for the treatment of human disease based on novel biological functions and pathways. Towards this aim, we undertook a bioinformatics analysis of in-house microarray data derived from purified hematopoietic stem cell populations. This effort led to the discovery of HSS1 (Hematopoietic Signal peptide-containing Secreted 1) and its splice variant HSM1 (Hematopoietic Signal peptide-containing Membrane domain-containing 1). HSS1 gene is evolutionarily conserved across species, phyla and even kingdoms, including mammals, invertebrates and plants. Structural analysis showed no homology between HSS1 and known proteins or known protein domains, indicating that it was a truly novel protein. Interestingly, the human HSS1 (hHSS1) gene is located at chromosome 19q13.33, a genomic region implicated in various cancers, including malignant glioma. Stable expression of hHSS1 in glioma-derived A172 and U87 cell lines greatly reduced their proliferation rates compared to mock-transfected cells. hHSS1 expression significantly affected the malignant phenotype of U87 cells both in vitro and in vivo. Further, preliminary immunohistochemical analysis revealed an increase in hHSS1/HSM1 immunoreactivity in two out of four high-grade astrocytomas (glioblastoma multiforme, WHO IV) as compared to low expression in all four low-grade diffuse astrocytomas (WHO grade II). High-expression of hHSS1 in high-grade gliomas was further supported by microarray data, which indicated that mesenchymal subclass gliomas exclusively up-regulated hHSS1. Our data reveal that HSS1 is a truly novel protein defining a new class of secreted factors, and that it may have an important role in cancer, particularly glioma

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

Human papillomavirus type 16 variants in cervical cancer from an admixtured population in Brazil

Several studies indicate that molecular variants of HPV-16 have different geographic distribution and risk associated with persistent infection and development of high-grade cervical lesions. In the present study, the frequency of HPV-16 variants was determined in 81 biopsies from women with cervical intraepithelial neoplasia grade III or invasive cervical cancer from the city of Belem, Northern Brazil. Host DNAs were also genotyped in order to analyze the ethnicity-related distribution of these variants. Ninie different HPV-16 LCR variants belonging to four phylogenetic branches were identified. Among these, two new isolates were characterized. The most prevalent HPV-16 variant detected was the Asian-American B-2,followed by the European B-12 and the European prototype. Infections by multiple variants were observed in both invasive cervical cancer and cervical intraepithelial neoplasia grade III cases. The analysis of a specific polymorphism within the E6 viral gene was performed in a subset of 76 isolates. The E6-350G polymorphism was significantly more frequent in Asian-American variants. The HPV-16 variability detected followed the same pattern of the genetic ancestry observed in Northern Brazil, with European, Amerindian and African roots. Although African ancestry was higher among women infected by the prototype, no correlation between ethnical origin and HPV-16 variants was found. These results corroborate previous data showing a high frequency of Asian-American variants in cervical neoplasia among women with multiethnic origin

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

Plasma Pharmacokinetic Characteristics of HemaMax in Non-Irradiated Rhesus Monkeys.

<p>Animals received HemaMax subcutaneously at a dose of either 250 ng/Kg or 1000 ng/Kg in the absence of irradiation. The plasma concentrations of HemaMax were determined by ELISA.</p><p>AUC = area under the curve; C_max = maximum plasma concentrations; T_max = time to achieve the maximum plasma concentration; t_1/2 = half life.</p

NHP and human bone marrow and small intestine express IL-12Rβ2.

<p>Tissues from NHP and human femoral bone marrow (a) and jejunum/ileum (b) were immunohistochemically stained for IL-12Rβ2. (a) Progenitor cells and megakaryocytes expressing IL-12Rβ2 are shown. Adipocytes did not express IL-12Rβ2. (b) Intestinal crypts expressing IL-12Rβ2 are shown. Lymphoid cells in the lamina propria and submucosal regions also expressed IL-12Rβ2. C = crypt; LP = lamina propria. Magnification was 40× in (a) and 100× in (b).</p

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

Plasma PK Characteristics of m-HemaMax in Irradiated and Non-Irradiated Mice.

<p>Animals received m-HemaMax subcutaneously at a dose of 10 ng/mouse, 20 ng/mouse, 40 ng/mouse, or 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 were determined by ELISA.</p><p>AUC = area under the curve; C_max = maximum plasma concentrations; NR = no irradiation; R = irradiation; TBI = total body irradiation; T_max = time to achieve the maximum plasma concentration; t_1/2 = half life.</p