Phase II trial of hu14.18-IL2 for patients with metastatic melanoma

Phase I testing of the hu14.18-IL2 immunocytokine in melanoma patients showed immune activation, reversible toxicities, and a maximal tolerated dose of 7.5?mg/m²/day. In this phase II study, 14 patients with measurable metastatic melanoma were scheduled to receive hu14.18-IL2 at 6?mg/m²/day as 4-h intravenous infusions on Days 1, 2, and 3 of each 28?day cycle. Patients with stable disease (SD) or regression following cycle 2 could receive two additional treatment cycles. The primary objective was to evaluate antitumor activity and response duration. Secondary objectives evaluated adverse events and immunologic activation. All patients received two cycles of treatment. One patient had a partial response (PR) [1 PR of 14 patients?=?response rate of 7.1?%; confidence interval, 0.2?C33.9?%], and 4 patients had SD and received cycles 3 and 4. The PR and SD responses lasted 3?C4?months. All toxicities were reversible and those resulting in dose reduction included grade 3 hypotension (2 patients) and grade 2 renal insufficiency with oliguria (1 patient). Patients had a peripheral blood lymphocytosis on Day 8 and increased C-reactive protein. While one PR in 14 patients met protocol criteria to proceed to stage 2 and enter 16 additional patients, we suspended stage 2 due to limited availability of hu14.18-IL2 at that time and the brief duration of PR and SD. We conclude that subsequent testing of hu14.18-IL2 should involve melanoma patients with minimal residual disease based on compelling preclinical data and the confirmed immune activation with some antitumor activity in this study.     

Increased reticulocyte count from cord blood samples using hypotonic lysis

Human reticulocytes are one of the fundamental components needed to study the in vitro invasion processes of the human malaria parasite Plasmodium vivax. Additionally examinations of reticulocytes and their binding proteins are difficult in areas of the world that do not have access to advanced equipment or stem cell lines. These issues are particularly relevant to malaria vaccine candidate studies that are directed against surface proteins that the parasites use to gain entry into erythrocytes. Described here is a simple and inexpensive method to increase the reticulocyte count of cord blood samples. Exposure of cord blood to hypotonic saline (0.2%) for 5 min selectively lyses the non-reticulocytes resulting in an average 3.6-fold increase in reticulocyte count. Our studies show that this enrichment process does not damage the hemoglobin of the remaining erythrocytes which are still capable of supporting Plasmodium falciparum invasion and growth. This economical and rapid method of enrichment could facilitate studies of in vitro laboratory culturing of other malaria parasite species which preferentially invade reticulocytes such as P. vivax.     

Intratumoral hu14.18-IL-2 (IC) Induces Local and Systemic Antitumor Effects That Involve Both Activated T and NK Cells As Well As Enhanced IC Retention

hu14.18-IL-2 (IC) is an immunocytokine consisting of human IL-2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of i.v. hu14.18-IL-2 (i.v.-IC) in neuroblastoma and melanoma are underway and have already demonstrated activity in neuroblastoma. We showed previously that intratumoral hu14.18-IL-2 (IT-IC) results in enhanced antitumor activity in mouse models compared with i.v.-IC. The studies presented in this article were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of intratumoral administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing s.c. NXS2 neuroblastomas treated with IT-IC compared with those treated with i.v.-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E(+) NKp46(+) NK cells and NKG2A/C/E(+) CD8(+) T cells compared with control mice or mice treated with i.v.-IC. NKG2D levels were augmented more in tumor-infiltrating lymphocytes compared with splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared with i.v.-IC. Overall, IT-IC resulted in increased numbers of activated T and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival compared with i.v.-IC.     

Species specificity of protein kinase r antagonism by cytomegalovirus TRS1 genes

The host antiviral protein kinase R (PKR) has rapidly evolved during primate evolution, likely in response to challenges posed by many different viral antagonists, such as the TRS1 gene of cytomegaloviruses (CMVs). In turn, viral antagonists have adapted to changes in PKR. As a result of this "arms race," modern TRS1 alleles in CMVs may function differently in cells derived from alternative species. We have previously shown that human CMV TRS1 (HuTRS1) blocks the PKR pathway and rescues replication of a vaccinia virus mutant lacking its major PKR antagonist in human cells. We now demonstrate that HuTRS1 does not have these activities in Old World monkey cells. Conversely, the rhesus cytomegalovirus homologue of HuTRS1 (RhTRS1) fulfills these functions in African green monkey cells, but not rhesus or human cells. Both TRS1 proteins bind to double-stranded RNA and, in the cell types in which they can rescue VVΔE3L replication, they also bind to PKR and prevent phosphorylation of the α-subunit of eukaryotic initiation factor 2. However, while HuTRS1 binds to inactive human PKR and prevents its autophosphorylation, RhTRS1 binds to phosphorylated African green monkey PKR. These studies reveal that evolutionary adaptations in this critical host defense protein have altered its binding interface in a way that has resulted in a qualitatively altered mechanism of PKR antagonism by viral TRS1 alleles from different CMVs. These results suggest that PKR antagonism is likely one of the factors that contributes to species specificity of cytomegalovirus replication.     

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.     

Concurrent habitat and life history influences on effective/census population size ratios in stream-dwelling trout

Lower effective sizes (N(e)) than census sizes (N) are routinely documented in natural populations, but knowledge of how multiple factors interact to lower N(e)/N ratios is often limited. We show how combined habitat and life-history influences drive a 2.4- to 6.1-fold difference in N(e)/N ratios between two pristine brook trout (Salvelinus fontinalis) populations occupying streams separated by only 750 m. Local habitat features, particularly drainage area and stream depth, govern trout biomass produced in each stream. They also generate higher trout densities in the shallower stream by favoring smaller body size and earlier age-at-maturity. The combination of higher densities and reduced breeding site availability in the shallower stream likely leads to more competition among breeding trout, which results in greater variance in individual reproductive success and a greater reduction in N(e) relative to N. A similar disparity between juvenile or adult densities and breeding habitat availability is reported for other species and hence may also result in divergent N(e)/N ratios elsewhere. These divergent N(e)/N ratios between adjacent populations are also an instructive reminder for species conservation programs that genetic and demographic parameters may differ dramatically within species.     

The effects of a low vitamin E diet on dichloroacetate- and trichloroacetate-induced oxidative stress in the livers of mice

Groups of mice were fed either a standard (Std) diet or a diet not supplemented with vitamin E (Low-E) and were divided into three subgroups that were treated subchronically by gavage, with water (control), dichloroacetate (DCA), or trichloroacetate (TCA). The livers of the animals were assayed for various biomarkers of oxidative stress (OS), antioxidant enzyme activities, and total glutathione (GSH). In general, livers from the low-E diet group expressed lower levels of biomarkers of OS associated with greater increases in various antioxidant enzymes activities and GSH when compared with the corresponding treatments in the Std diet group. These results suggest that vitamin E supplementation to the diet, while essential to maintain certain body functions, can compromise the effectiveness of the hepatic antioxidant enzymes and GSH resulting in an increase in DCA- and TCA-induced OS and a possible increase in the compounds-induced hepatotoxic/hepatocarcinogenic effects in mice.     

Alpha4beta7/MAdCAM-1 interactions play an essential role in transitioning cryptopatches into isolated lymphoid follicles and a nonessential role in cryptopatch formation

The alpha(4) integrins alpha(4)beta(7) and alpha(4)beta(1), and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of alpha(4) integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express alpha(4)beta(7) and alpha(4)beta(1), including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal beta(7)(-/-) mice and adult and neonatal mice given blocking Abs to alpha(4)beta(7), MAdCAM-1, or VCAM-1 did not identify a role for alpha(4) integrins in cryptopatch (CP) development; however, these studies demonstrated that alpha(4)beta(7) and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that beta(7)(-/-) LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that beta(7) expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for alpha(4)beta(7)/MAdCAM-1 in ILF development corresponding to the influx of beta(7)-expressing lymphocytes and a nonessential role for beta(7)-localizing LTi-like cells to the small intestine.     

Human tumor-derived exosomes down-modulate NKG2D expression

NKG2D is an activating receptor for NK, NKT, CD8(+), and gammadelta(+) T cells, whose aberrant loss in cancer is a key mechanism of immune evasion. Soluble NKG2D ligands and growth factors, such as TGFbeta1 emanating from tumors, are mechanisms for down-regulating NKG2D expression. Cancers thereby impair the capacity of lymphocytes to recognize and destroy them. In this study, we show that exosomes derived from cancer cells express ligands for NKG2D and express TGFbeta1, and we investigate the impact of such exosomes on CD8(+) T and NK cell NKG2D expression and on NKG2D-dependent functions. Exosomes produced by various cancer cell lines in vitro, or isolated from pleural effusions of mesothelioma patients triggered down-regulation of surface NKG2D expression by NK cells and CD8(+) T cells. This decrease was rapid, sustained, and resulted from direct interactions between exosomes and NK cells or CD8(+) T cells. Other markers (CD4, CD8, CD56, CD16, CD94, or CD69) remained unchanged, indicating the selectivity and nonactivatory nature of the response. Exosomal NKG2D ligands were partially responsible for this effect, as down-modulation of NKG2D was slightly attenuated in the presence of MICA-specific Ab. In contrast, TGFbeta1-neutralizing Ab strongly abrogated NKG2D down-modulation, suggesting exosomally expressed TGFbeta as the principal mechanism. Lymphocyte effector function was impaired by pretreatment with tumor exosomes, as these cells exhibited poor NKG2D-dependent production of IFN-gamma and poor NKG2D-dependent killing function. This hyporesponsiveness was evident even in the presence of IL-15, a strong inducer of NKG2D. Our data show that NKG2D is a likely physiological target for exosome-mediated immune evasion in cancer.