Genentech

Apoptosis is a cell suicide mechanism that enables metazoans to control cell number in tissues and to eliminate individual cells that threaten the animal's survival. Certain cells have unique sensors, termed death receptors, on their surface. Death receptors detect the presence of extracellular death signals and, in response, they rapidly ignite the cell's intrinsic apoptosis machinery.

MOTIVATION: We introduce GMAP, a standalone program for mapping and aligning cDNA sequences to a genome. The program maps and aligns a single sequence with minimal startup time and memory requirements, and provides fast batch processing of large sequence sets. The program generates accurate gene structures, even in the presence of substantial polymorphisms and sequence errors, without using probabilistic splice site models. Methodology underlying the program includes a minimal sampling strategy for genomic mapping, oligomer chaining for approximate alignment, sandwich DP for splice site detection, and microexon identification with statistical significance testing. RESULTS: On a set of human messenger RNAs with random mutations at a 1 and 3% rate, GMAP identified all splice sites accurately in over 99.3% of the sequences, which was one-tenth the error rate of existing programs. On a large set of human expressed sequence tags, GMAP provided higher-quality alignments more often than blat did. On a set of Arabidopsis cDNAs, GMAP performed comparably with GeneSeqer. In these experiments, GMAP demonstrated a several-fold increase in speed over existing programs. AVAILABILITY: Source code for gmap and associated programs is available at http://www.gene.com/share/gmap SUPPLEMENTARY INFORMATION: http://www.gene.com/share/gmap.

Interleukin (IL)-17 is a pro-inflammatory cytokine that is produced by activated T cells. Despite increasing evidence that high levels of IL-17 are associated with several chronic inflammatory diseases including rheumatoid arthritis, psoriasis, and multiple sclerosis, the regulation of its expression is not well characterized. We observe that IL-17 production is increased in response to the recently described cytokine IL-23. We present evidence that murine IL-23, which is produced by activated dendritic cells, acts on memory T cells, resulting in elevated IL-17 secretion. IL-23 also induced expression of the related cytokine IL-17F. IL-23 is a heterodimeric cytokine and shares a subunit, p40, with IL-12. In contrast to IL-23, IL-12 had only marginal effects on IL-17 production. These data suggest that during a secondary immune response, IL-23 can promote an activation state with features distinct from the well characterized Th1 and Th2 profiles.

Lec13 cells, a variant Chinese hamster ovary cell line, were used to produce human IgG1 that were deficient in fucose attached to the Asn²⁹⁷-linked carbohydrate but were otherwise similar to that found in IgG1 produced in normal Chinese hamster ovary cell lines and from human serum. Lack of fucose on the IgG1 had no effect on binding to human FcγRI, C1q, or the neonatal Fc receptor. Although no change in affinity was found for the His¹³¹ polymorphic form of human FcγRIIA, a slight improvement in binding was evident for FcγRIIB and the Arg¹³¹ FcγRIIA polymorphic form. In contrast, binding of the fucose-deficient IgG1 to human FcγRIIIA was improved up to 50-fold. Antibody-dependent cellular cytotoxicity assays using purified peripheral blood monocytes or natural killer cells from several donors showed enhanced cytotoxicity, especially evident at lower antibody concentrations. When combined with an IgG1 Fc protein variant that exhibited enhanced antibody-dependent cellular cytotoxicity, the lack of fucose was synergistic.

HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease. A variation on antibody-targeted therapy is utilization of antibodies to deliver cytotoxic agents specifically to antigen-expressing tumors. We determined in vitro and in vivo efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid (microtubule-depolymerizing agents) conjugates using disulfide and thioether linkers. Antiproliferative effects of trastuzumab-maytansinoid conjugates were evaluated on cultured normal and tumor cells. In vivo activity was determined in mouse breast cancer models, and toxicity was assessed in rats as measured by body weight loss. Surprisingly, trastuzumab linked to DM1 through a nonreducible thioether linkage (SMCC), displayed superior activity compared with unconjugated trastuzumab or trastuzumab linked to other maytansinoids through disulfide linkers. Serum concentrations of trastuzumab-MCC-DM1 remained elevated compared with other conjugates, and toxicity in rats was negligible compared with free DM1 or trastuzumab linked to DM1 through a reducible linker. Potent activity was observed on all HER2-overexpressing tumor cells, whereas nontransformed cells and tumor cell lines with normal HER2 expression were unaffected. In addition, trastuzumab-DM1 was active on HER2-overexpressing, trastuzumab-refractory tumors. In summary, trastuzumab-DM1 shows greater activity compared with nonconjugated trastuzumab while maintaining selectivity for HER2-overexpressing tumor cells. Because trastuzumab linked to DM1 through a nonreducible linker offers improved efficacy and pharmacokinetics and reduced toxicity over the reducible disulfide linkers evaluated, trastuzumab-MCC-DM1 was selected for clinical development.

BACKGROUND: Although combination treatment with bevacizumab (humanized monoclonal antibody against vascular endothelial growth factor) and chemotherapy improves survival of patients with various metastatic carcinomas, an increased risk of arterial thromboembolic events has been observed in some trials. We characterized this risk by performing post hoc analyses of randomized controlled trials that evaluated combination treatment with bevacizumab and chemotherapy versus chemotherapy alone. Low-dose aspirin was permitted in these trials, and its safety was also analyzed. METHODS: Data were pooled from five randomized controlled trials that included a total of 1745 patients with metastatic colorectal, breast, or non-small-cell lung carcinoma. The risk of an arterial or venous thromboembolic event was assessed by simple incidence rates, rates per 100 person-years, and/or hazard ratios (HRs). The association between patient characteristics and risk of an arterial thromboembolic event was investigated primarily by Cox proportional hazards regression. The relationship between low-dose aspirin and bleeding was explored by incidence rates and rates per 100 person-years. RESULTS: Combined treatment with bevacizumab and chemotherapy, compared with chemotherapy alone, was associated with increased risk for an arterial thromboembolic event (HR = 2.0, 95% confidence interval [CI] = 1.05 to 3.75; P = .031) but not for a venous thromboembolic event (HR = 0.89, 95% CI = 0.66 to 1.20; P = .44). The absolute rate of developing an arterial thromboembolism was 5.5 events per 100 person-years for those receiving combination therapy and 3.1 events per 100 person-years for those receiving chemotherapy alone (ratio = 1.8, 95% CI = 0.94 to 3.33; P = .076). Development of an arterial thromboembolic event was associated with a prior arterial thromboembolic event (P<.001) or age of 65 years or older (P = .01). Baseline or on-study aspirin use was associated with modest increases in grade 3 and 4 bleeding events in both treatment groups, from 3.6% to 4.7% for bevacizumab-treated patients and from 1.7% to 2.2% for control subjects. CONCLUSIONS: Combination treatment with bevacizumab and chemotherapy, compared with chemotherapy alone, was associated with an increased risk of arterial thromboembolism but not venous thromboembolism.

IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing.

The 9,213-nucleotide structure of the AIDS/lymphadenopathy virus has been determined from molecular clones representing the integrated provirus and viral RNA. The sequence reveals that the virus is highly polymorphic and lacks significant nucleotide homology with type C retroviruses characterized previously. Together with an analysis of the two major viral subgenomic RNAs, these studies establish the coding frames for the gag, pol and env genes and predict the expression of a novel gene at the 3' end of the genome unrelated to the X genes of HTLV-1 and -II.

Vascular endothelial growth factor (VEGF) was identified as a heparin-binding polypeptide mitogen with a target cell specificity restricted to vascular endothelial cells. Molecular cloning reveals the existence of four species of VEGF having 121, 165, 189, and 206 amino acids. These have strikingly different secretion patterns, which suggests multiple physiological roles for this family of polypeptides. The two shorter forms are efficiently secreted, while the longer ones are mostly cell-associated. Alternative splicing of mRNA, rather that transcription from different genes, is the mechanism for their generation. In situ hybridization reveals that the VEGF mRNA is widely distributed in most tissues and organs and expressed at particularly high levels in areas of active vascular proliferation, like the ovarian corpus luteum. Ligand autoradiography on rat tissue sections demonstrates that VEGF binding sites are associated with vascular endothelial cells of both fenestrated and non-fenestrated capillaries and with the endothelium of large vessels, while no displaceable binding is evident on non-endothelial cell types. These findings support the hypothesis that VEGF plays a highly specific role in the maintenance and in the induction of growth of vascular endothelial cells.

Almost all modern rotation of factor loadings is based on optimizing a criterion, for example, the quartimax criterion for quartimax rotation. Recent advancements in numerical methods have led to general orthogonal and oblique algorithms for optimizing essentially any rotation criterion. All that is required for a specific application is a definition of the criterion and its gradient. The authors present the implementations of gradient projection algorithms, both orthogonal and oblique, as well as a catalogue of rotation criteria and corresponding gradients. Software for these is downloadable and free; a specific version is given for each of the computing environments used most by statisticians. Examples of rotation methods are presented by applying them to a loading matrix from Wehmeyer and Palmer.

The workshop entitled 'Interventions to Slow Aging in Humans: Are We Ready?' was held in Erice, Italy, on October 8-13, 2013, to bring together leading experts in the biology and genetics of aging and obtain a consensus related to the discovery and development of safe interventions to slow aging and increase healthy lifespan in humans. There was consensus that there is sufficient evidence that aging interventions will delay and prevent disease onset for many chronic conditions of adult and old age. Essential pathways have been identified, and behavioral, dietary, and pharmacologic approaches have emerged. Although many gene targets and drugs were discussed and there was not complete consensus about all interventions, the participants selected a subset of the most promising strategies that could be tested in humans for their effects on healthspan. These were: (i) dietary interventions mimicking chronic dietary restriction (periodic fasting mimicking diets, protein restriction, etc.); (ii) drugs that inhibit the growth hormone/IGF-I axis; (iii) drugs that inhibit the mTOR-S6K pathway; or (iv) drugs that activate AMPK or specific sirtuins. These choices were based in part on consistent evidence for the pro-longevity effects and ability of these interventions to prevent or delay multiple age-related diseases and improve healthspan in simple model organisms and rodents and their potential to be safe and effective in extending human healthspan. The authors of this manuscript were speakers and discussants invited to the workshop. The following summary highlights the major points addressed and the conclusions of the meeting.

To understand the role of the 75-kDa tumor necrosis factor (TNF) receptor in non-lymphoid cells, the cytotoxic signaling and ligand binding activities of the 55-kDa (TNF-R1) and 75-kDa (TNF-R2) TNF receptors were investigated using agonist and antagonist antibodies specific for the two receptor types. This study indicates that although TNF-R2 can significantly reduce the TNF concentration required for cell killing, the mechanism by which this is accomplished is not through the generation of an intracellular signal by TNF-R2. Instead, TNF-R2 regulates the rate of TNF association with TNF-R1, possibly by increasing the local concentration of TNF at the cell surface through rapid ligand association and dissociation. We propose that other cell-surface receptors, such as the low affinity p75 nerve growth factor receptor, may utilize an analogous "ligand passing" mechanism.

We report the identification of a novel human cytokine, distantly related to interleukin (IL)-10, which we term IL-22. IL-22 is produced by activated T cells. IL-22 is a ligand for CRF2–4, a member of the class II cytokine receptor family. No high affinity ligand has yet been reported for this receptor, although it has been reported to serve as a second component in IL-10 signaling. A new member of the interferon receptor family, which we term IL-22R, functions as a second component together with CRF2–4 to enable IL-22 signaling. IL-22 does not bind the IL-10R. Cell lines were identified that respond to IL-22 by activation of STATs 1, 3, and 5, but were unresponsive to IL-10. In contrast to IL-10, IL-22 does not inhibit the production of proinflammatory cytokines by monocytes in response to LPS nor does it impact IL-10 function on monocytes, but it has modest inhibitory effects on IL-4 production from Th2 T cells. We report the identification of a novel human cytokine, distantly related to interleukin (IL)-10, which we term IL-22. IL-22 is produced by activated T cells. IL-22 is a ligand for CRF2–4, a member of the class II cytokine receptor family. No high affinity ligand has yet been reported for this receptor, although it has been reported to serve as a second component in IL-10 signaling. A new member of the interferon receptor family, which we term IL-22R, functions as a second component together with CRF2–4 to enable IL-22 signaling. IL-22 does not bind the IL-10R. Cell lines were identified that respond to IL-22 by activation of STATs 1, 3, and 5, but were unresponsive to IL-10. In contrast to IL-10, IL-22 does not inhibit the production of proinflammatory cytokines by monocytes in response to LPS nor does it impact IL-10 function on monocytes, but it has modest inhibitory effects on IL-4 production from Th2 T cells. interleukin interferon signal transducers and activators of transcription reverse transcriptase-polymerase chain reaction The class II cytokine receptor family, also known as the interferon receptor family, includes the IL-10R,1 tissue factor, the two subunits of the IFNγ receptor, the two subunits of the IFNα/β receptor, and CRF2–4. Additional members of this family also exist in human genomic sequence (1Adams, R. L., Farrah, T. M., Jelmberg, A. C., Lok, S., and Whitemore, T. E. (1999) Patent WO9907848-A1 and U. S. Patent 5,965,704.Google Scholar). 2M-H. Xie, S. Aggarwal, W-H. Ho, J. Foster, Z. Zhang, J. Stinson, W. I. Wook, A. D. Goddard and A. L. Gurney, unpublished observations. The known biological actions mediated by these molecules are diverse and include the antiviral actions of IFNα, IFNβ, and IFNγ, the immunomodulatory effects of IFNγ, a TH1 cytokine that potentiates inflammatory responses and promotes cell-mediated immune responses, the multiple and generally immunosuppressive actions of IL-10, and the role of tissue factor as a high affinity receptor for plasma factor VII/VIIa involved in cellular initiation of the coagulation cascade. The class II cytokine receptors are evolutionarily related and are characterized by the presence of a single transmembrane domain and an extracellular domain that contains several fibronectin-type three repeats. Given the great importance of members of the interferon receptor family, it is of interest to identify proteins that interact with this system as they are likely to perform significant immune functions. In this report we identify a new human cytokine, IL-22, that signals through a receptor complex that contains CRF2–4 and a new member of the class II cytokine receptor family. CRF2–4 has previously been demonstrated to be a functional component of the IL-10 signaling complex. This is the first example within the class II cytokine receptor family of a receptor being utilized as a component of multiple distinct cytokine signaling complexes. Antibodies used for STAT supershift experiments were purchased from Santa Cruz Biotechnology. Antibodies for STAT tyrosine phosphorylation were purchased from Upstate Biotechnology. Antibodies for isolation of T cells were purchased from Pharmingen. Recombinant human IL-4, IFNγ, IL-10, and IL-12 was purchased from R&D Systems. Human TNF, IL-1, IL-4, IL-6, IL-13, and IFNγ enzyme-linked immunosorbent assay were purchased from R&D Systems. A cDNA clone encoding IL-22 was identified in the Lifeseq EST data base (Incyte Pharmaceuticals) and sequenced in its entirety. Coding sequence for IL-22, IL-10, and the interferon family receptors were obtained by PCR amplification. Fc fusion proteins (immunoadhesins) were prepared by fusion of the entire open reading frames of IL-22 and IL-10 in-frame with the Fc region of human IgG1 in the eukaryotic expression vector pRK5tkNEO and the baculovirus vector pHIF, a derivative of pVL1393 purchased from Pharmingen. Fusion proteins were transiently expressed in human 293 cells or Sf9 insect cells and purified over a protein A column. IL-22 was also expressed as a C-terminal 8 × His tag fusion in baculovirus and purified by nickel affinity column. The identity of the purified protein was verified by N-terminal sequence analysis. IL-22 was also expressed with an N-terminal gD epitope tag as described (2Xie M.H. Holcomb I. Deuel B. Dowd P. Huang A. Vagts A. Foster J. Liang J. Brush J. Gu Q. Hillan K. Goddard A. Gurney A.L. Cytokine. 1999; 11: 729-735Crossref PubMed Scopus (232) Google Scholar). The sequences of the DNA constructs were confirmed by DNA sequencing. STAT activation and supershift assays using the SIE sequence and Western blot analysis of STAT tyrosine phosphorylation were performed as described previously (3Gurney A.L. Wong S.C. Henzel W.J. de Sauvage F.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5292-5296Crossref PubMed Scopus (177) Google Scholar) and as recommended by antibody suppliers. Monocytes were enriched from fresh human blood by isolation of leukocytes using Lymphocyte Separation Medium purchased from ICN Biomedicals followed by enrichment of monocytes by adherence to tissue culture flask. Monocytes were then cultured for 24 h in 10% fetal bovine serum RPMI medium plus indicated cytokine treatment. For IL-22 mRNA expression experiments, resting T cells were isolated from leukocytes by negative selection with antibodies to CD14, CD19, CD56, CD11A, and HLA-DR purchased from Pharmingen. Isolated T cells were cultured in 10% fetal bovine serum RPMI medium with anti-CD3 coated plates for 48 h with concanavalin A (2.5 μg/ml). To drive T cell differentiation to Th1 and Th2 T cells, CD4 positive cells were first isolated with anti-CD4 magnetic beads (Pharmingen). Cells were then cultured RPMI with an equal number (∼10 million) of irradiated monocytes in the presence of concanavalin A (2.5 μg/ml) and IL-2 (4 ng/ml) and either IL-12 (8 ng/ml) + anti IL-4 (0.5 μg/ml) or IL-4 (4 ng/ml) + anti-IFNγ (0.6 μg/ml) for Th1 or Th2, respectively. After 3 days, T cells were collected and washed twice and cultured for 24 h in the presence or absence of IL-22-His (8 nm). As part of a larger effort to identify novel secreted proteins, we identified a novel human sequence that bears significant similarity to IL-10 (Fig. 1 A). This protein also strongly resembles a recently identified murine protein termed IL-TIFα, and is likely its human ortholog. Based on its similarity to the cytokine IL-10 and the data presented herein on the identification of its receptor as a member of an established cytokine receptor family and its production within leukocytes and action upon leukocytes, this molecule may appropriately be considered an interleukin. Following convention, we propose terming this molecule human IL-22. The isolated cDNA encodes a protein of 179 amino acids that is 23% similar to IL-10 and 78% identical to IL-TIFα (4Dumoutier L. Louahed J. Renauld J.C. J. Immunol. 2000; 164: 1814-1819Crossref PubMed Scopus (443) Google Scholar). The first 33 amino acids are predicted to function as a signal sequence. N-terminal amino acid analysis of IL-22 expressed and purified using baculovirus confirm the mature sequence begins at amino acid residue 34. Northern expression analysis showed only trace level expression in several peripheral tissues (not shown). RT-PCR analysis showed that IL-22 mRNA is up-regulated in T cells stimulated with anti-CD3 and further induced by exposure to concanavalin A (Fig. 1 B). To establish whether IL-22 was ligand for any of the members of the class II cytokine receptors, each receptor was tested for its ability to bind IL-22. Cells transiently expressing CRF2–4 showed strong binding of IL-22-fc fusion protein (immunoadhesion) (Fig.2 A). In contrast, IL-10-fc bound only to IL-10R. IL-22 also displayed low but detectable binding to a new member of the family of unknown function which we identified by searching sequences present in public sequence databases (1Adams, R. L., Farrah, T. M., Jelmberg, A. C., Lok, S., and Whitemore, T. E. (1999) Patent WO9907848-A1 and U. S. Patent 5,965,704.Google Scholar), which we term IL-22R (Fig. 2 B). IL-22R is a 574-amino acid protein most related to IL-10R and CRF2–4 (Fig. 2 C). To confirm the direct interaction of IL-22 with CRF2–4 and IL-22R, binding studies were conducted with epitope-tagged ligand and soluble receptor immunoadhesins. IL-22 bound to CRF2–4 but not to any of the other members of the interferon receptor family (Fig. 2 D). Direct binding was not observed by Western blot analysis with IL-22R, suggesting the interaction with this component may be of low affinity. Reasoning that IL-22 likely activated the JAK-STAT signaling pathway in a manner analogous to that observed with the interferons and members of the hematopoietic cytokine family, we surveyed a series of cell lines for activation of STAT transcription factors in response to IL-22. Two cell lines were observed to show rapid and robust STAT activation; TK-10, a renal cell carcinoma (Fig.3 A) and SW480, a colon adenocarcinoma (not shown). TK-10 did not induce STAT activity in response to IL-10. Interestingly, in this survey we observed another cell line, MOLT-4, a human lymphoblast cell line that did respond to IL-10 but did not respond to IL-22. The specific STAT proteins activated in response to IL-22 were examined using antibodies to the known STATs. In gel-shift assays, antibodies to STAT1, STAT3, and STAT5 were able to supershift IL-22 induced binding complexes to an SIE sequence (Fig. 3 B) and other related STAT-binding elements (not shown). Furthermore, using antibodies specific for tyrosine-phosphorylated STAT, clear tyrosine phosphorylation of these STATs was observed in response to IL-22 (Fig. 3 C). As IL-22 and IL-10 signal STAT activation in distinct cell lines, we explored the expression pattern of IL-10R, CRF2–4, and IL-22R in these lines. RT-PCR analysis indicated CRF2–4 is expressed in both TK-10 and MOLT-4 (Fig. 4 A). In contrast, expression of IL-10R was detected in MOLT-4 and not in TK-10 and IL-22R expression was detected in TK-10 but not MOLT-4. These results suggested that functional signaling complexes for IL-10 included IL-10R and CRF2–4 as previously reported, and that IL-22 signaled through a complex that included CRF2–4 and IL-22R. To test this hypothesis, these receptors were transfected into COS cells and the ability of IL-10 and IL-22 to mediate STAT activation was determined (Fig.4 B). In agreement with previous reports IL-10 was able to mediate STAT activation in cells transfected with both CRF2–4 and IL-10R. Somewhat weaker activation was also seen in cells transfected with IL-10R alone. In contrast, IL-22 was able to mediate STAT activation in cells transfected with both CRF2–4 and IL-22R but not with either receptor transfected alone. Given the distant homology between IL-22 and IL-10, we examined whether IL-22 had similar biological activities to IL-10. Monocytes from freshly isolated human blood were examined for the ability of IL-22 to effect production of cytokines known to be regulated by IL-10. LPS induces a large increase in TNF production, an effect which may be substantially repressed by the presence of IL-10 (Fig.5). We observed that IL-22 had little if any effect on TNF production in the presence or absence of LPS. Furthermore, at 10-fold molar excess IL-10, it did not appear to inhibit the action of IL-10. Similar results were observed measuring production of IL-1 and IL-6 (not shown). The effect of IL-22 on production of cytokines by T cells was studied. Human T cells were polarized in vitro to either Th1 or Th2 differentiation by activation with concanavalin A in the presence of IL-12 and anti-IL-4 antibodies or IL-4 and anti-IFNγ antibodies, respectively. Cells were then washed and cultured in the presence or absence of IL-22 for 24 h. IL-22 treatment had little effect on IFNγ production from Th1 cells, but modestly inhibited production of IL-4 from Th2 cells (TableI).Table IIFNγ and IL-4 production by human T cellsIFNγIL-4Th1Th2Th1Th2−+−+−+−+pg/mlpg/mlExperiment 122507 ± 743422114 ± 114731225 ± 390833 ± 1046.5 ± 0.65.4 ± 0.785.2 ± 1.913.6 ± 2.7Experiment 211356 ± 18038844 ± 5471014 ± 91660 ± 22276 ± 1068 ± 22476 ± 39356 ± 48Experiment 37009 ± 5577714 ± 792247 ± 21138 ± 1027 ± 1.26.6 ± 0.729.6 ± 1.519.6 ± 2.5Experiment 410022 ± 21639241 ± 1232397 ± 39465 ± 19.45.5 ± 0.25.0 ± 0.351 ± 7.510.4 ± 0.7T cells were isolated from fresh human blood and polarized in vitro towards Th1 or Th2 differentiation for 3 days. Cells were then washed and incubated for 24 h in the presence of IL-22-His (8 nm) as indicated for 24 h. Production of IFNγ and IL-4 was measured by enzyme-linked immunosorbent assay. Shown are the results from four independent experiments. Open table in a new tab T cells were isolated from fresh human blood and polarized in vitro towards Th1 or Th2 differentiation for 3 days. Cells were then washed and incubated for 24 h in the presence of IL-22-His (8 nm) as indicated for 24 h. Production of IFNγ and IL-4 was measured by enzyme-linked immunosorbent assay. Shown are the results from four independent experiments. IL-22 signals through a receptor complex that includes CRF2–4 and a new member of the class II cytokine receptor family, IL-22R. Previous reports have demonstrated that CRF2–4 serves as a second component in IL-10 signaling (5Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar, 6Spencer S.D. Di Marco F. Hooley J. Pitts-Meek S. Bauer M. Ryan A.M. Sordat B. Gibbs V.C. Aguet M. J. Exp. Med. 1998; 187: 571-578Crossref PubMed Scopus (304) Google Scholar). CRF2–4 was initially discovered and identified as a member of the interferon receptor family on the basis of sequence similarity (7Lutfalla G. Gardiner K. Uze G. Genomics. 1993; 16: 366-373Crossref PubMed Scopus (76) Google Scholar, 8Gibbs V.C. Pennica D. Gene ( Amst. ). 1997; 186: 97-101Crossref PubMed Scopus (29) Google Scholar). The gene is located within a cluster that also includes IFN-αR1, IFN-αR2, and IFN-γR2 on human chromosome 21. A two-component receptor for IL-10 signaling parallels the IFNα/β and IFNγ systems which have each been shown to signal through two component receptors. The data presented here suggest that, in addition, CFR2–4 serves as a binding component of an IL-22 signaling complex. Thus CRF2–4 could serve in combination with IL-10R to transduce IL-10 signaling and also function within a signaling complex for IL-22 that likely includes the new member of this family, IL-22R. Overexpression of CRF2–4 alone or in combination with IL-10R does not appear to be sufficient to enable IL-22-dependent STAT activation, but combined with IL-22R does enable STAT activation. While shared receptor components have not been previously observed among the class II cytokine receptor family, within the large family of hematopoietic cytokine receptors such utilization of receptors in multiple distinct combinations to respond to different cytokines is common. Examples include the common β-chain utilized by IL-3, IL-5, and GM-CSF, the common γ-chain utilized by IL-2, -4, -7, –9, and -15, and gp130 utilized by multiple members of the IL-6 family of cytokines (reviewed in Refs. 9He Y.W. Malek T.R. Crit. Rev. Immunol. 1998; 18: 503-524Crossref PubMed Google Scholar, 10de Groot R.P. Coffer P.J. Koenderman L. Cell. Signal. 1998; 10: 619-628Crossref PubMed Scopus (187) Google Scholar, 11Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1749) Google Scholar). The LIF receptor serves both as a ligand binding subunit for LIF and CT-1 and also as a second component in signaling complexes for oncostatin M and ciliary neurotrophic factor (12Pennica D. Wood W.I. Chien K.R. Cytokine Growth Factor Rev. 1996; 7: 81-91Crossref PubMed Scopus (91) Google Scholar,13Grotzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins. 1997; 27: 96-109Crossref PubMed Scopus (96) Google Scholar). In each of these cases the ligands, as in the case of IL-10 and IL-22, are evolutionarily related. Activation of STATs 1, 3, and 5 has also been reported for both IL-10 and murine IL-TIFα (4Dumoutier L. Louahed J. Renauld J.C. J. Immunol. 2000; 164: 1814-1819Crossref PubMed Scopus (443) Google Scholar, 14Finbloom D.S. Winestock K.D. J. Immunol. 1995; 155: 1079-1090PubMed Google Scholar, 15Wehinger J. Gouilleux F. Groner B. Finke J. Mertelsmann R. Weber-Nordt R.M. FEBS Lett. 1996; 394: 365-370Crossref PubMed Scopus (141) Google Scholar). These same STATs have also been shown to be activated by a great number of hematopoietic cytokines, suggesting that other considerations such as the pattern of receptor expression influence the biological responses (reviewed in Ref.16Chatterjee-Kishore M. van den Akker F. Stark G.R. Trends Cell Biol. 2000; 10: 106-111Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Mice with targeted disruption of either CRF2–4 and IL-10 have been reported (6Spencer S.D. Di Marco F. Hooley J. Pitts-Meek S. Bauer M. Ryan A.M. Sordat B. Gibbs V.C. Aguet M. J. Exp. Med. 1998; 187: 571-578Crossref PubMed Scopus (304) Google Scholar, 17Kuhn R. Lohler J. Rennick D. Rajewsky K. Muller W. Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3668) Google Scholar, 18Rennick D.M. Fort M.M. Davidson N.J. J. Leukocyte Biol. 1997; 61: 389-396Crossref PubMed Scopus (260) Google Scholar). The phenotypes bear some similarity but also significant differences. Mice deficient in IL-10 developed chronic enterocolitis, were anemic, and were growth retarded when raised in a conventional environment. In comparison only 60% of the CRF2–4 mice raised under similar conditions had colitus but this was limited to the large intestine and did not involve the small intestine. The mice did not develop anemia and unlike the IL-10-deficient mice the CRF2–4 mice displayed a 4-fold increase in spleen to body weight with extramedullary hematopoiesis in the spleen of erythroid, myeloid, and megakaryocytic lineages. These differences may be due in part to contributions of environment or genetic background, however, in light of the existence of a specific ligand for CRF2–4, the function of IL-10 in the CRF2–4-deficient mice should be re-evaluated. The broad distribution of CRF2–4 expression is reminiscent of the expression patterns seen for the receptors for IFNα/β and IFNγ whereas the expression of the IL-10R is restricted mainly to hematopoietic cells. Preliminary data suggests IL-22R is also restricted in its expression, but is detectable by RT-PCR in CD3 positive T cells and at low levels in several peripheral tissues (not shown). The broad expression of CRF2–4 may reflect the existence of additional ligands yet to be discovered which also share CRF2–4 as a common subunit. Interestingly, IL-10 and IL-22 appear to have somewhat mirror actions acting to promote Th2 or Th1 type responses, respectively. The ability of IL-22 to suppress IL-4 production may have therapeutic potential, particularly in the treatment of asthma. We thank Diane Pennica, Sherman Fong, and Paul Godowski for helpful discussions and Amy Carlow, Jeffrey Hooly, Peter Ng, and Mark Vasser for technical assistance.

Initial analysis of the Assessment of Proteasome Inhibition for Extending Remissions (APEX) trial of relapsed multiple myeloma patients showed significantly longer time to progression, higher response rate, and improved survival with single-agent bortezomib versus high-dose dexamethasone. In this updated analysis (median follow-up: 22 months), survival was assessed in both arms, and efficacy updated for the bortezomib arm. Median survival was 29.8 months for bortezomib versus 23.7 months for dexamethasone, a 6-month benefit, despite substantial crossover from dexamethasone to bortezomib. Overall and complete response rates with bortezomib were 43% and 9%, respectively; among responding patients, 56% improved response with longer therapy beyond initial response, leading to continued improvement in overall quality of response. Higher response quality (100% M-protein reduction) was associated with longer response duration; response duration was not associated with time to response. These data confirm the activity of bortezomib and support extended treatment in relapsed multiple myeloma patients tolerating therapy.

IL-19, IL-20, IL-22, IL-24, and IL-26 are members of the IL-10 family of cytokines that have been shown to be up-regulated in psoriatic skin. Contrary to IL-10, these cytokines signal using receptor complex R1 subunits that are preferentially expressed on cells of epithelial origin; thus, we henceforth refer to them as the IL-20 subfamily cytokines. In this study, we show that primary human keratinocytes (KCs) express receptors for these cytokines and that IL-19, IL-20, IL-22, and IL-24 induce acanthosis in reconstituted human epidermis (RHE) in a dose-dependent manner. These cytokines also induce expression of the psoriasis-associated protein S100A7 and keratin 16 in RHE and cause persistent activation of Stat3 with nuclear localization. IL-22 had the most pronounced effects on KC proliferation and on the differentiation of KCs in RHE, inducing a decrease in the granular cell layer (hypogranulosis). Furthermore, gene expression analysis performed on cultured RHE treated with these cytokines showed that IL-19, IL-20, IL-22, and IL-24 regulate many of these same genes to variable degrees, inducing a gene expression profile consistent with inflammatory responses, wound healing re-epithelialization, and altered differentiation. Many of these genes have also been found to be up-regulated in psoriatic skin, including several chemokines, beta-defensins, S100 family proteins, and kallikreins. These results confirm that IL-20 subfamily cytokines are important regulators of epidermal KC biology with potentially pivotal roles in the immunopathology of psoriasis.

Vascular endothelial growth factor (VEGF) is an important mediator of the intense angiogenesis which is characteristic of glioblastoma. While genetic manipulation of VEGF/VEGF receptor expression has previously been shown to inhibit glioblastoma growth, to date, no study has examined the efficacy of pharmacologic blockade of VEGF activity as a means to inhibit intracranial growth of human glioblastoma. Using intraperitoneal administration of a neutralizing anti-VEGF antibody, we demonstrate that inhibition of VEGF significantly prolongs survival in athymic rats inoculated in the basal ganglia with G55 human glioblastoma cells. Systemic anti-VEGF inhibition causes decreased tumor vascularity as well as a marked increase in tumor cell apoptosis in intracranial tumors. Although intracranial glioblastoma tumors grow more slowly as a consequence of anti-VEGF treatment, the histologic pattern of growth suggests that these tumors adapt to inhibition of angiogenesis by increased infiltration and cooption of the host vasculature.

Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.

Immunoglobulins (IgG) are soluble serum glycoproteins in which the oligosaccharides play significant roles in the bioactivity and pharmacokinetics. Recombinant immuno-globulins (rIgG) produced in different host cells by recombinant DNA technology are becoming major therapeutic agents to treat life threatening diseases such as cancer. Since glycosylation is cell type specific, rIgGs produced in different host cells contain different patterns of oligosaccharides which could affect the biological functions. In order to determine the extent of this variation N-linked oligosaccharide structures present in the IgGs of different animal species were characterized. IgGs of human, rhesus, dog, cow, guinea pig, sheep, goat, horse, rat, mouse, rabbit, cat, and chicken were treated with peptide-N-glycosidase-F (PNGase F) and the oligosaccharides analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for neutral and acidic oligosaccharides, in positive and negative ion modes, respectively. The data show that for neutral oligosaccharides, the proportions of terminal Gal, core Fuc and/or bisecting GlcNAc containing oligosaccharides vary from species to species; for sialylated oligosaccharides in the negative mode MALDI-TOF-MS show that human and chicken IgG contain oligosaccharides with N-acetylneuraminic acid (NANA), whereas rhesus, cow, sheep, goat, horse, and mouse IgGs contain oligosaccharides with N-glycolylneuraminic acid (NGNA). In contrast, IgGs from dog, guinea pig, rat, and rabbit contain both NANA and NGNA. Further, the PNGase F released oligosaccharides were derivatized with 9-aminopyrene 1,4,6-trisulfonic acid (APTS) and analyzed by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-LIF results indicate that the proportion of the two isomers of monogalactosylated, biantennary, complex oligosaccharides vary significantly, suggesting that the branch specificity of beta1, 4-galactosyltransferase might be different in different species. These results show that the glycosylation of IgGs is species-specific, and reveal the necessity for appropriate cell line selection to express rIgGs for human therapy. The results of this study are useful for people working in the transgenic area.

p21(Cip1/WAF1) (p21), a p53-inducible protein, is a critical regulator of cell cycle and cell survival. p21 binds to and inhibits both the DNA synthesis regulator proliferating cell nuclear antigen and cyclin A/E-CDK2 complexes. Recently, p21 has also been shown to be a positive regulator of cell cycle progression as p21 is necessary for the assembly and activation of cyclin D1-CDK4/6 complexes. Furthermore, elevated p21 protein levels have been observed in various aggressive tumors as well as linked to chemoresistance. Here we demonstrate that p21 is directly phosphorylated by AKT/PKB, a survival kinase that is hyperactivated in many late stage tumors. Two sites (Thr(145) and Ser(146)) in the carboxyl terminus of p21 are phosphorylated by AKT/PKB in vitro and in vivo. Phosphorylation of Thr(145) inhibits PCNA binding, whereas phosphorylation of Ser(146) significantly increases p21 protein stability. Glioblastoma cell lines with activated AKT/PKB show enhanced p21 stability, and they are more resistant to taxol-mediated toxicity. Finally, AKT/PKB controls the assembly of cyclin D1-CDK4 complexes through modulation of p21 and cyclin D1 levels. These data imply that enhanced levels of p21 in tumors are due, in part, to phosphorylation by activated AKT/PKB. Furthermore, they suggest that one mechanism of AKT/PKB regulation of tumor cell survival and/or proliferation is to stabilize p21 protein.

Interleukin-22 (IL-22) has important functions in host defense at mucosal surfaces as well as in tissue repair. It is unique as a cytokine that is produced by immune cells, including T-helper (Th) cell subsets and innate lymphocytes, but acts only on non-hematopoietic stromal cells, in particular epithelial cells, keratinocytes, and hepatocytes. Although IL-22 is beneficial to the host in many infectious and inflammatory disorders, depending on the target tissue it can be pathogenic due to its inherent pro-inflammatory properties, which are further enhanced when IL-22 is released together with other pro-inflammatory cytokines, in particular IL-17. To avoid pathology, IL-22 and IL-17 production have to be controlled tightly and independently. While common factors such as signal transducer and activator of transcription 3 (STAT3) and retinoid orphan receptor γt (RORγt) drive the expression of both cytokines, other factors, such as c-Maf act specifically on IL-22 and enable the separate expression of either cytokine. Here, we discuss the production of IL-22 from various T-cell populations as well as protective versus pathogenic roles of IL-22. Finally, we focus on recent advances in our understanding of the molecular regulation of IL-22 in T cells.