Palmitate-derivatized human IL-2: a potential anticancer immunotherapeutic of low systemic toxicity

Purpose and experimental design

Recombinant human IL-2 (rhIL-2) is a potent cytokine and FDA-approved anticancer drug. However, its clinical use has been limited by severe toxicity, associated primarily with systemic administration with excess protein distributing freely throughout the body. We hypothesized that rhIL-2 in alternate forms permitting more restricted localization may exert stronger antitumor efficacy and less toxicity. Here, we have tested the utility of palmitate-derivatized rhIL-2. rhIL-2 was reacted with N-hydroxysuccinimide palmitate ester. The resultant lipidated rhIL-2 (pIL-2), when mixed with cells, could spontaneously transfer from solution to cell surfaces. Next, anticancer efficacy of pIL-2 was assessed in two modalities. For adoptive T cell therapy, antitumor cytotoxic T cells (CTLs) were protein transferred (“painted”) with pIL-2 and injected into mice bearing lymphoma. For in situ therapy, pIL-2 was injected intratumorally into mice bearing melanoma. Tumor growth and IL-2-associated toxicity were determined.

Results

In the lymphoma model, painting of the antitumor CTLs with pIL-2 markedly increased their viability and titer. In the melanoma model, intratumoral injection of pIL-2, but not rhIL-2, increased the number of activated CD8⁺ T cells (IFN-γ⁺) in the spleen, reduced lung metastasis and prolonged the survival of treated mice. Moreover, while repeated intratumoral injection of rhIL-2 at an excessively high dose (10 injections of 10,000 IU/mouse) caused marked vascular leakage syndrome, the same regimen using pIL-2 caused no detectable toxicity.

Conclusions

Transferring spontaneously from solution to cell surfaces, pIL-2 may bypass the current limitations of rhIL-2 and, thus, serve as a more effective and tolerable anticancer drug.     

iRSpot-PseDNC: identify recombination spots with pseudo dinucleotide composition

Meiotic recombination is an important biological process. As a main driving force of evolution, recombination provides natural new combinations of genetic variations. Rather than randomly occurring across a genome, meiotic recombination takes place in some genomic regions (the so-called ‘hotspots’) with higher frequencies, and in the other regions (the so-called ‘coldspots’) with lower frequencies. Therefore, the information of the hotspots and coldspots would provide useful insights for in-depth studying of the mechanism of recombination and the genome evolution process as well. So far, the recombination regions have been mainly determined by experiments, which are both expensive and time-consuming. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapidly and effectively identifying the recombination regions. In this study, a predictor, called ‘iRSpot-PseDNC’, was developed for identifying the recombination hotspots and coldspots. In the new predictor, the samples of DNA sequences are formulated by a novel feature vector, the so-called ‘pseudo dinucleotide composition’ (PseDNC), into which six local DNA structural properties, i.e. three angular parameters (twist, tilt and roll) and three translational parameters (shift, slide and rise), are incorporated. It was observed by the rigorous jackknife test that the overall success rate achieved by iRSpot-PseDNC was >82% in identifying recombination spots in Saccharomyces cerevisiae, indicating the new predictor is promising or at least may become a complementary tool to the existing methods in this area. Although the benchmark data set used to train and test the current method was from S. cerevisiae, the basic approaches can also be extended to deal with all the other genomes. Particularly, it has not escaped our notice that the PseDNC approach can be also used to study many other DNA-related problems. As a user-friendly web-server, iRSpot-PseDNC is freely accessible at http://lin.uestc.edu.cn/server/iRSpot-PseDNC.     

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.     

Autocrine CCL3 and CCL4 Induced by the Oncoprotein LMP1 Promote Epstein-Barr Virus-Triggered B Cell Proliferation

Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.     

Revision of the genus Thermistis Pascoe 1867, with descriptions of three new species (Coleoptera: Cerambycidae: Lamiinae: Saperdini)

The genus Thermistis Pascoe 1867 is revised. T. croceocincta conjunctesignata Rondon & Breuning 1971 is upgraded to a species and newly recorded from China and Myanmar. T. xanthomelas Holzschuh 2007 is newly recorded from Vietnam, Laos and Myanmar. T. sulphureonotata Pu 1984 is newly recorded from Vietnam and Laos. Three new species are described from China: T. hainanensis Lin & Yang n. sp. from Hainan Island, T. kaiyuni Chou & Kurihara n. sp. from Taiwan Island and T. cheni Lin & Chou n. sp. from Yunnan and Sichuan provinces. Photographs of habitus and terminalia and a key to the eleven valid species of Thermistis are presented.     

Diatoms, silicic acid and biogenic silica dynamics along the salinity gradient of the Scheldt estuary (Belgium/The Netherlands)

The Scheldt estuary (Belgium/The Netherlands) was sampled along the entire salinity gradient from 2003 to 2005 for silicic acid (DSi), biogenic silica (BSi), suspended particulate matter (SPM) and pigments. Net DSi consumption and/or release within the estuary were investigated by comparing measured DSi concentrations with (fully-transient) model simulations of the concentrations that would have been obtained in case of conservative transport. The DSi consumption was at maximum in May due to diatoms of presumably marine origin blooming in the lower estuary. DSi consumption decreased rapidly in July, probably because of the grazing pressure of copepods also of marine origin, and DSi was released from late summer onwards. Multiple regression analyses showed that most of the BSi did not follow the dynamics of the living diatoms but rather that of the SPM. They also suggested that diatoms were more silicified in the upper estuary than in the lower estuary. Phytoliths were not expected to contribute significantly to the BSi pool. As BSi dynamics strongly differed from those of diatoms and DSi, this study highlighted the importance of taking BSi into account when investigating estuarine silica dynamics. This study also revealed the fundamental role of the coupling between the biogeochemical and ecological functioning of the lower estuary and that of the adjacent coastal zone. This contrasts with the classical consideration that estuaries act as one-way filters for dissolved and particulate material of riverine origin.     

Discovery of 3-phenyl-1H-5-pyrazolylamine derivatives containing a urea pharmacophore as potent and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3)

Preclinical investigations and early clinical trials suggest that FLT3 inhibitors are a viable therapy for acute myeloid leukemia. However, early clinical data have been underwhelming due to incomplete inhibition of FLT3. We have developed 3-phenyl-1H-5-pyrazolylamine as an efficient template for kinase inhibitors. Structure–activity relationships led to the discovery of sulfonamide, carbamate and urea series of FLT3 inhibitors. Previous studies showed that the sulfonamide 4 and carbamate 5 series were potent and selective FLT3 inhibitors with good in vivo efficacy. Herein, we describe the urea series, which we found to be potent inhibitors of FLT3 and VEGFR2. Some inhibited growth of FLT3-mutated MOLM-13 cells more strongly than the FLT3 inhibitors sorafenib (2) and ABT-869 (3). In preliminary in vivo toxicity studies of the four most active compounds, 10f was found to be the least toxic. A further in vivo efficacy study demonstrated that 10f achieved complete tumor regression in a higher proportion of MOLM-13 xenograft mice than 4 and 5 (70% vs 10% and 40%). These results show that compound 10f possesses improved pharmacologic and selectivity profiles and could be more effective than previously disclosed FLT3 inhibitors in the treatment of acute myeloid leukemia.     

Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.     

Ser/Thr Kinase-Like Protein of Nicotiana benthamiana Is Involved in the Cell-to-Cell Movement of Bamboo mosaic virus

To investigate the plant genes affected by Bamboo mosaic virus (BaMV) infection, we applied a cDNA-amplified fragment length polymorphism technique to screen genes with differential expression. A serine/threonine kinase-like (NbSTKL) gene of Nicotiana benthamiana is upregulated after BaMV infection. NbSTKL contains the homologous domain of Ser/Thr kinase. Knocking down the expression of NbSTKL by virus-induced gene silencing reduced the accumulation of BaMV in the inoculated leaves but not in the protoplasts. The spread of GFP-expressing BaMV in the inoculated leaves is also impeded by a reduced expression of NbSTKL. These data imply that NbSTKL facilitates the cell-to-cell movement of BaMV. The subcellular localization of NbSTKL is mainly on the cell membrane, which has been confirmed by mutagenesis and fractionation experiments. Combined with the results showing that active site mutation of NbSTKL does not change its subcellular localization but significantly affects BaMV accumulation, we conclude that NbSTKL may regulate BaMV movement on the cell membrane by its kinase-like activity. Moreover, the transient expression of NbSTKL does not significantly affect the accumulation of Cucumber mosaic virus (CMV) and Potato virus X (PVX); thus, NbSTKL might be a specific protein facilitating BaMV movement.