IntroductionViperin (virus inhibitory protein, endoplasmic reticulumassociated, interferon-inducible) was first cloned from interferontreated human macrophages, 1 and identified as an antiviral protein that is protective against human cytomegalovirus and influenza A virus. 1,2 In the following studies using oligonucleotide microarray chip assays, viperin has been recognized as a highly inducible candidate gene in response to a wide range of viruses and microbial products such as LPS and double-stranded RNA, 3,4 implying that viperin is an important component of innate immunity to diverse pathogens. Viperin induction upon stimulation has been reported in human fetal astrocytes, 5 fibroblasts, Huh-7 and HepG2 cells 6,7 in vitro, and in disease conditions such as vascular cells of atherosclerosis 8 and liver tissue of patients with chronic hepatitis C. 3 Nevertheless, the cell types that are capable of producing viperin are still enigmatic, and the functions of viperin other than its antiviral activity have remained unexplored.In order to delineate the physiologic role of viperin in the immune system, a mouse model defective in viperin was generated by gene targeting. We assessed here whether viperin deficiency affects T-cell development and function. T-cell responses are intimately dependent upon survival-promoting signals induced by T-cell receptors (TCRs), the costimulatory molecule CD28, and cytokine receptors. Several transcription factors have been shown to contribute to these processes, including those belonging to NFAT, AP-1, NF-B, and STAT families. 9 We found that viperin Ϫ/Ϫ CD4 ϩ T cells produced a normal amount of IL-2 and were able to respond to polarizing cytokines for T helper 2 (Th2) differentiation. However, there was reduced Th2 cytokine production (IL-4, IL-5, and IL-13) in association with impaired GATA-3 induction in viperin-deficient CD4 ϩ T cells after stimulation with anti-CD3 in the presence or absence of anti-CD28 antibodies. In addition, viperin Ϫ/Ϫ T cells showed decreased DNA binding activities of NF-B1/p50 and JunB in response to TCR ligation in gel-shift assays. These data suggest that viperin induction is required for TCR-mediated NF-B and AP-1 activities, GATA-3 activation, and subsequent Th2 cell development for optimal Th2 response. Methods Generation of viperin-deficient miceMice deficient in viperin was generated using standard procedures. The viperin targeting vector consisted of a 1.8-kb short fragment in putative promoter region, a neomycin resistance gene cassette that replaced exons 1 and 2 as well as an upstream 2.2-kb putative promoter, and a 5.4-kb-long genomic fragment containing exons 3 and 4 (Figure 1 A). One embryonic stem cell clone derived from 129/Sv mice, with a targeted viperin allele detected by Southern blot, was injected into C57BL/6 blastocysts to produce chimeric mice, and germline transmission of the mutation was verified by polymerase chain reaction (PCR) analysis of tail DNA. Reverse transcription (RT)-PCR with primers spanning exons 2 and 3 in enriched...

Abstract. Bcl-2 defines a new class of proto-oncogenes that block cell death without promoting cell proliferation. To elucidate the role of Bcl-2 in the development of glomerular lesions in human IgA nephropathy (IgAN), we applied immunohistochemistry coupled with in situ hybridization to detect the expression of Bcl-2 products and their association with Bax, p27 kip1 , and p57 kip2 in modulating the apoptotic, proliferative, and sclerotic events in progressive glomerular injury. Glomerular cell apoptosis was examined by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining. A total of 51 IgAN cases were categorized into four subgroups (A to D) according to the severity of their histopathological lesions. Creatinine levels, creatinine clearance, and magnitude of proteinuria based on 24-h urine collections at the time of diagnostic renal biopsy were available for the majority of subjects.

TTP is an anti-inflammatory protein that acts by binding to AREs in its target mRNAs, such as Tnf mRNA, and promoting their deadenylation and decay. TNF released from inflammatory cells can then stimulate gene expression in tissue cells, such as fibroblasts. To determine whether TTP could affect the decay of TNF-induced transcripts in fibroblasts, we exposed primary embryonic fibroblasts and stable fibroblast cell lines, derived from WT and TTP KO mice, to TNF. The decay rates of transcripts encoded by several early-response genes, including Cxcl1, Cxcl2, Ier3, Ptgs2, and Lif, were significantly slowed in TTP-deficient fibroblasts after TNF stimulation. These changes were associated with TTP-dependent increases in CXCL1, CXCL2, and IER3 protein levels. The TTP-susceptible transcripts contained multiple, conserved, closely spaced, potential TTP binding sites in their 3'-UTRs. WT TTP, but not a nonbinding TTP zinc finger mutant, bound to RNA probes that were based on the mRNA sequences of Cxcl1, Cxcl2, Ptgs2, and Lif. TTP-promoted decay of transcripts encoding chemokines and other proinflammatory mediators is thus a critical post-transcriptional regulatory mechanism in the response of secondary cells, such as fibroblasts, to TNF released from primary immune cells.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFb signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-na€ ve tumors. Tumors with high AXL, TGFb, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelialto-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-na€ ve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFb, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition.Significance: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that can bind to AU-rich element-containing mRNAs and promote their decay. TTP knockout mice develop a severe inflammatory syndrome, largely due to excess tumor necrosis factor (TNF), whose mRNA is a direct target of TTP binding and destabilization. TTP's RNA binding activity and its ability to promote mRNA decay are lost when one of the zinc-coordinating residues of either zinc finger is mutated. To address several long-standing questions about TTP activity in intact animals, we developed a knock-in mouse with a cysteine-to-arginine mutation within the first zinc finger. Homozygous knock-in mice developed a severe inflammatory syndrome that was essentially identical to that of complete TTP deficiency, suggesting that TTP's critical anti-inflammatory role in mammalian physiology is secondary to its ability to bind RNA. In addition, there was no evidence for a "dominant-negative" effect of the mutant allele in heterozygotes, as suggested by previous experiments. Finally, mRNA decay experiments in mutant macrophages demonstrated that TTP can regulate the stability of its own mRNA, albeit to a minor extent. These studies suggest that RNA binding is an essential first step in the physiological activities of members of this protein family.