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Tineke Casneuf,1 Amy E Axel,2 Peter King,2 John D Alvarez,2 Jillian L Werbeck,3 Tinne Verhulst,1 Karin Verstraeten,1 Brett M Hall,2 A Kate Sasser2 1Janssen Research and Development, Beerse, Belgium; 2Janssen Research and Development, Spring House, PA, 3LabConnect LLC, Seattle, WA, USAIntroduction: Interleukin-6 (IL-6) is an important growth factor for estrogen receptor-α (ERα)-positive breast cancer, and elevated serum IL-6 is associated with poor prognosis.Methods: The role of the phosphorylated signal transducer and activator of transcription 3 pathway was investigated in ERα-positive breast cancer. A panel of cell lines was treated with exogenous IL-6. An IL-6 specific gene signature was generated by profiling ten ERα-positive breast cancer cell lines alone or following treatment with 10 ng/mL recombinant IL-6 or human marrow stromal cell-conditioned media, with or without siltuximab (a neutralizing anti-IL-6 antibody) and grown in three-dimensional tumor microenvironment-aligned cultures for 4 days, 5 days, or 6 days. The established IL-6 signature was validated against 36 human ERα-positive breast tumor samples with matched serum. A comparative MCF-7 xenograft murine model was utilized to determine the role of IL-6 in estrogen-supplemented ERa-positive breast cancer to assess the efficacy of anti-IL-6 therapy in vivo.Results: In eight of nine ERα-positive breast cancer cell lines, recombinant IL-6 increased phosphorylation of tyrosine 705 of STAT3. Differential gene expression analysis identified 17 genes that could be used to determine IL-6 pathway activation by combining their expression intensity into a pathway activation score. The gene signature included a variety of genes involved in immune cell function and migration, cell growth and apoptosis, and the tumor microenvironment. Validation of the IL-6 gene signature in 36 matched human serum and ERα-positive breast tumor samples showed that patients with a high IL-6 pathway activation score were also enriched for elevated serum IL-6 (≥10 pg/mL). When human IL-6 was provided in vivo, MCF-7 cells engrafted without the need for estrogen supplementation, and addition of estrogen to IL-6 did not further enhance engraftment. Subsequently, we prophylactically treated mice at MCF-7 engraftment with siltuximab, fulvestrant, or combination therapy. Siltuximab alone was able to blunt MCF-7 engraftment. Similarly, siltuximab alone induced regressions in 90% (9/10) of tumors, which were established in the presence which were established in the presence of hMSC expressing human IL-6 and estrogen.Conclusion: Given the established role for IL-6 in ERα-positive breast cancer, these data demonstrate the potential for anti-IL-6 therapeutics in breast cancer.Keywords: breast cancer, estrogen receptor, gene signature, paracrine IL-6, siltuximab

Wound healing in response to acute injury is mediated by the coordinated and transient activation of parenchymal, stromal, and immune cells that resolves to homeostasis. Environmental, genetic, and epigenetic factors associated with inflammation and aging can lead to persistent activation of the microenvironment and fibrosis. Here, we identify opposing roles of interleukin-4 (IL-4) cytokine signaling in interstitial macrophages and type II alveolar epithelial cells (ATIIs). We show that IL4Ra signaling in macrophages promotes regeneration of the alveolar epithelium after bleomycin-induced lung injury. Using organoids and mouse models, we show that IL-4 directly acts on a subset of ATIIs to induce the expression of the transcription factor SOX9 and reprograms them toward a progenitor-like state with both airway and alveolar lineage potential. In the contexts of aging and bleomycin-induced lung injury, this leads to aberrant epithelial cell differentiation and bronchiolization, consistent with cellular and histological changes observed in interstitial lung disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

On December 5, 2022, an indirect drive fusion implosion on the National Ignition Facility (NIF) achieved a target gain G_{target} of 1.5. This is the first laboratory demonstration of exceeding "scientific breakeven" (or G_{target}>1) where 2.05 MJ of 351 nm laser light produced 3.1 MJ of total fusion yield, a result which significantly exceeds the Lawson criterion for fusion ignition as reported in a previous NIF implosion [H. Abu-Shawareb et al. (Indirect Drive ICF Collaboration), Phys. Rev. Lett. 129, 075001 (2022)PRLTAO0031-900710.1103/PhysRevLett.129.075001]. This achievement is the culmination of more than five decades of research and gives proof that laboratory fusion, based on fundamental physics principles, is possible. This Letter reports on the target, laser, design, and experimental advancements that led to this result.

Long interspersed nuclear element-1 (LINE-1 or L1), the most abundant family of autonomous retrotransposons occupying over 17% of human DNA, is epigenetically silenced in normal tissues by the mechanisms involving p53 but is frequently derepressed in cancer, suggesting that L1-encoded proteins may act as tumor-associated antigens recognized by the immune system. In this study, we established an immunoassay to detect circulating autoantibodies against L1 proteins in human blood. Using this assay in >2,800 individuals with or without cancer, we observed significantly higher IgG titers against L1-encoded ORF1p and ORF2p in patients with lung, pancreatic, ovarian, esophageal, and liver cancers than in healthy individuals. Remarkably, elevated levels of anti-ORF1p-reactive IgG were observed in patients with cancer with disease stages 1 and 2, indicating that the immune response to L1 antigens can occur in the early phases of carcinogenesis. We concluded that the antibody response against L1 antigens could contribute to the diagnosis and determination of immunoreactivity of tumors among cancer types that frequently escape early detection., Significance: The discovery of autoantibodies against antigens encoded by L1 retrotransposons in patients with five poorly curable cancer types has potential implications for the detection of an ongoing carcinogenic process and tumor immunoreactivity., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Activation of endogenous retrotransposons frequently occurs in cancer cells and contributes to tumor genomic instability. To test whether inhibition of retrotranspositions has an anticancer effect, we used treatment with the nucleoside reverse transcriptase inhibitor (NRTI) stavudine (STV) in mouse cancer models, MMTV-HER2/Neu and Th-MYCN, that spontaneously develop breast cancer and neuroblastoma, respectively. In both cases, STV in drinking water did not affect tumor incidence nor demonstrate direct antitumor effects. However, STV dramatically extended progression-free survival in both models following an initial complete response to chemotherapy. To approach the mechanism underlying this phenomenon, we analyzed the effect of NRTI on the selection of treatment-resistant variants in tumor cells in culture. Cultivation of mouse breast carcinoma 4T1 in the presence of STV dramatically reduced the frequency of cells capable of surviving treatment with anticancer drugs. Global transcriptome analysis demonstrated that the acquisition of drug resistance by 4T1 cells was accompanied by an increase in the constitutive activity of interferon type I and NF-κB pathways and an elevated expression of LINE-1 elements, which are known to induce inflammatory responses via their products of reverse transcription. Treatment with NRTI reduced NF-κB activity and reverted drug resistance. Furthermore, the inducible expression of LINE-1 stimulated inflammatory response and increased the frequency of drug-resistant variants in a tumor cell population. These results indicate a mechanism by which retrotransposon desilencing can stimulate tumor cell survival during treatment and suggest reverse transcriptase inhibition as a potential therapeutic approach for targeting the development of drug-resistant cancers.

The immune response to an allograft activates lymphocytes with the capacity to cause rejection. Activation of CD4 + CD25 + Foxp3 + T regulatory cells (Treg) can down-regulate allograft rejection and can induce immune tolerance to the allograft. Treg represent <10% of peripheral CD4 + T cells and do not markedly increase in tolerant hosts. CD4 + CD25 + Foxp3 + T cells include both resting and activated Treg that can be distinguished by several markers, many of which are also expressed by effector T cells. More detailed characterization of Treg to identify increased activated antigen-specific Treg may allow reduction of non-specific immunosuppression. Natural thymus derived resting Treg (tTreg) are CD4 + CD25 + Foxp3 + T cells and only partially inhibit alloantigen presenting cell activation of effector cells. Cytokines produced by activated effector cells activate these tTreg to more potent alloantigen-activated Treg that may promote a state of operational tolerance. Activated Treg can be distinguished by several molecules they are induced to express, or whose expression they have suppressed. These include CD45RA/RO, cytokine receptors, chemokine receptors that alter pathways of migration and transcription factors, cytokines and suppression mediating molecules. As the total Treg population does not increase in operational tolerance, it is the activated Treg which may be the most informative to monitor. Here we review the methods used to monitor peripheral Treg, the effect of immunosuppressive regimens on Treg, and correlations with clinical outcomes such as graft survival and rejection. Experimental therapies involving ex vivo Treg expansion and administration in renal transplantation are not reviewed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cheung, Zahorowska, Suranyi, Wong, Diep, Spicer, Verma, Hodgkinson and Hall.)

The quest to understand how allogeneic transplanted tissue is not rejected and how tolerance is induced led to fundamental concepts in immunology. First, we review the research that led to the Clonal Deletion theory in the late 1950s that has since dominated the field of immunology and transplantation. At that time many basic mechanisms of immune response were unknown, including the role of lymphocytes and T cells in rejection. These original observations are reassessed by considering T regulatory cells that are produced by thymus of neonates to prevent autoimmunity. Second, we review "operational tolerance" induced in adult rodents and larger animals such as pigs. This can occur spontaneously especially with liver allografts, but also can develop after short courses of a variety of rejection inhibiting therapies. Over time these animals develop alloantigen specific tolerance to the graft but retain the capacity to reject third-party grafts. These animals have a "split tolerance" as peripheral lymphocytes from these animals respond to donor alloantigen in graft versus host assays and in mixed lymphocyte cultures, indicating there is no clonal deletion. Investigation of this phenomenon excludes many mechanisms, including anti-donor antibody blocking rejection as well as anti-idiotypic responses mediated by antibody or T cells. This split tolerance is transferred to a second immune-depleted host by T cells that retain the capacity to effect rejection of third-party grafts by the same host. Third, we review research on alloantigen specific inhibitory T cells that led to the first identification of the CD4 + CD25 + T regulatory cell. The key role of T cell derived cytokines, other than IL-2, in promoting survival and expansion of antigen specific T regulatory cells that mediate transplant tolerance is reviewed. The precise methods for inducing and diagnosing operational tolerance remain to be defined, but antigen specific T regulatory cells are key mediators., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hall, Verma, Tran and Hodgkinson.)

CD4 + CD25 + Foxp3 + T cell population is heterogenous and contains three major sub-groups. First, thymus derived T regulatory cells (tTreg) that are naïve/resting. Second, activated/memory Treg that are produced by activation of tTreg by antigen and cytokines. Third, effector lineage CD4 + CD25 + T cells generated from CD4 + CD25 - T cells' activation by antigen to transiently express CD25 and Foxp3. We have shown that freshly isolated CD4 + CD25 + T cells are activated by specific alloantigen and IL-4, not IL-2, to Ts2 cells that express the IL-5 receptor alpha. Ts2 cells are more potent than naïve/resting tTreg in suppressing specific alloimmunity. Here, we showed rIL-5 promoted further activation of Ts2 cells to Th2-like Treg, that expressed foxp3, irf4, gata3 and il5. In vivo , we studied the effects of rIL-5 treatment on Lewis heart allograft survival in F344 rats. Host CD4 + CD25 + T cells were assessed by FACS, in mixed lymphocyte culture and by RT-PCR to examine mRNA of Ts2 or Th2-like Treg markers. rIL-5 treatment given 7 days after transplantation reduced the severity of rejection and all grafts survived ≥60d whereas sham treated rats fully rejected by day 31 (p<0.01). Treatment with anti-CD25 or anti-IL-4 monoclonal antibody abolished the benefits of treatment with rIL-5 and accelerated rejection. After 10d treatment with rIL-5, hosts' CD4 + CD25 + cells expressed more Il5ra and responded to specific donor Lewis but not self. Enriched CD4 + CD25 + cells from rIL-5 treated rats with allografts surviving >60 days proliferated to specific donor only when rIL-5 was present and did not proliferate to self or third party. These cells had more mRNA for molecules expressed by Th2-like Treg includin g Irf4, gata3 and Il5. These findings were consistent with IL-5 treatment preventing rejection by activation of Ts2 cells and Th2-like Treg., Competing Interests: BH and SH hold patents related to production of antigen specific Treg and tests of tolerance related to this work. The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2021 Hall, Hall, Tran, Robinson, Wilcox, Rakesh, Wang, Sharland, Verma and Hodgkinson.)

Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double-ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen Pseudomonas aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP1 7 P2 7 tetradecamer that is required for proper biofilm development. PaClpP1 14 and PaClpP1 7 P2 7 complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non-canonical features in its N- and C-terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late-stage P. aeruginosa development., (© 2020 John Wiley & Sons Ltd.)

Resting and activated subpopulations of CD4 + CD25 + CD127 lo T regulatory cells (Treg) and CD4 + CD25 + CD127 + effector T cells in MS patients and in healthy individuals were compared. Peripheral blood mononuclear cells isolated using Ficoll Hypaque were stained with monoclonal antibodies and analysed by flow cytometer. CD45RA and Foxp3 expression within CD4 + cells and in CD4 + CD25 + CD127 lo T cells identified Population I; CD45RA + Foxp3 + , Population II; CD45RA - Foxp3 hi and Population III; CD45RA - Foxp3 + cells. Effector CD4 + CD127 + T cells were subdivided into Population IV; memory /effector CD45RA - CD25 - Foxp3 - and Population V; effector naïve CD45RA + CD25 - Foxp3 - CCR7 + and terminally differentiated RA + (TEMRA) effector memory cells. Chemokine receptor staining identified CXCR3 + Th1-like Treg, CCR6 + Th17-like Treg and CCR7 + resting Treg. Resting Treg (Population I) were reduced in MS patients, both in untreated and treated MS compared to healthy donors. Activated/memory Treg (Population II) were significantly increased in MS patients compared to healthy donors. Activated effector CD4 + (Population IV) were increased and the naïve/ TEMRA CD4 + (Population V) were decreased in MS compared to HD. Expression of CCR7 was mainly in Population I, whereas expression of CCR6 and CXCR3 was greatest in Populations II and intermediate in Population III. In MS, CCR6 + Treg were lower in Population III. This study found MS is associated with significant shifts in CD4 + T cells subpopulations. MS patients had lower resting CD4 + CD25 + CD45RA + CCR7 + Treg than healthy donors while activated CD4 + CD25 hi CD45RA - Foxp3 hi Treg were increased in MS patients even before treatment. Some MS patients had reduced CCR6 + Th17-like Treg, which may contribute to the activity of MS.

Background: The American College of Surgeons Resources for Optimal Care of the Injured Patient recommends using hypotension, defined as systolic blood pressure ≤90 mm Hg, as an indicator of a full team trauma activation. We hypothesized that an elevated shock index (SI) predicts significant traumatic injuries better than hypotension alone., Methods: This is a retrospective cohort study analyzing full team trauma activations between February 2018 and January 2020, excluding transfers and those who had missing values for prehospital blood pressure or heart rate. We reviewed patients' demographics, prehospital and emergency department vitals, injury pattern, need for operation, and clinical outcomes. The primary outcome was rate of significant injury defined as identified injured liver, spleen, or kidney, pelvis fracture, long bone fracture, significant extremity soft tissue damage, hemothorax, or pneumothorax., Results: Among 544 patients, 82 (15.1%) had prehospital hypotension and 492 had normal blood pressure. Of the patients with prehospital hypotension, 34 (41.5%) had a significant injury. There was no difference in age, gender, medical history, or injury pattern between the two groups. There was no difference between the two groups in rate of serious injury (41.5% vs. 46.1%, NS), need for emergent operation (31.7% vs. 28.1%, NS) or death (20.7% vs. 18.8%, NS). On the other hand, SI ≥1 was associated with increased rate of serious injury (54.6% vs. 43.4%, p=0.04). On a logistic regression analysis, prehospital hypotension was not associated with significant injury or need for emergent operation (OR 0.83, 95% CI 0.51 to 1.33 and OR 1.32, 95% CI 0.79 to 2.25, respectively). SI ≥1 was associated with both increased odds of significant injury and need for emergent operation (OR 1.57, 95% CI 1.01 to 2.44 and OR 1.64, 95% CI 1.01 to 2.66)., Discussion: SI was a better indicator and could replace hypotension to better categorize and triage patients in need of higher level of care., Level of Evidence: Prognostic and epidemiologic, level III., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Adipose tissue is recognized as a major source of systemic inflammation with age, driving age-related tissue dysfunction and pathogenesis. Macrophages (Mφ) are central to these changes yet adipose tissue Mφ (ATMs) from aged mice remain poorly characterized. To identify biomarkers underlying changes in aged adipose tissue, we performed an unbiased RNA-seq analysis of ATMs from young (8-week-old) and healthy aged (80-week-old) mice. One of the genes identified, V-set immunoglobulin-domain-containing 4 (VSIG4/CRIg), encodes a Mφ-associated complement receptor and B7 family-related immune checkpoint protein. Here, we demonstrate that Vsig4 expression is highly upregulated with age in perigonadal white adipose tissue (gWAT) in two mouse strains (inbred C57BL/6J and outbred NIH Swiss) independent of gender. The accumulation of VSIG4 was mainly attributed to a fourfold increase in the proportion of VSIG4 + ATMs (13%-52%). In a longitudinal study, VSIG4 expression in gWAT showed a strong correlation with age within a cohort of male and female mice and correlated strongly with physiological frailty index (PFI, a multi-parameter assessment of health) in male mice. Our results indicate that VSIG4 is a novel biomarker of aged murine ATMs. VSIG4 expression was also found to be elevated in other aging tissues (e.g., thymus) and was strongly induced in tumor-adjacent stroma in cases of spontaneous and xenograft lung cancer models. VSIG4 expression was recently associated with cancer and several inflammatory diseases with diagnostic and prognostic potential in both mice and humans. Further investigation is required to determine whether VSIG4-positive Mφ contribute to immunosenescence and/or systemic age-related deficits., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4 + CD25 + T cells both from sensitized hosts or from naïve hosts after ex-vivo activation by PNM and rIL-2. Transfer of naïve CD4 + CD25 + T cells has no effect on EAN, nor did naïve CD4 + CD25 + T cells activated with rIL-2 and renal tubular antigen. Culture of naive CD4 + CD25 + Treg with rIL-2 and PNM induced mRNA for the IFN-gamma receptor. We showed naïve CD4 + CD25 + T cells activated by specific auto-antigen and rIL-2 produced more potent antigen-specific Treg that may have therapeutic potential., Competing Interests: Declaration of Competing Interest SJH and BMH hold patents related to activation of T regulatory cells. All other authors have no conflicts of interest., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Background: Lymphocytes have become the target of cancer interventions through engineering or immune checkpoint antibodies. We previously found decreased lymphocyte counts to be a predictor of mortality and complications in trauma and cardiac surgery patients. We hypothesized lack of lymphocyte count recovery postoperatively would predict outcomes in esophagectomy patients., Methods: A retrospective review of all patients undergoing esophagectomy for adenocarcinoma performed over 13 y at our center by a single surgeon after institutional review board approval was performed. Patients were grouped by postoperative lymphocytes counts: never low, low with recovery, and low without recovery. Resolution of lymphopenia was assessed by day 4. Primary end points were overall and recurrence-free survival., Results: In total, 198 patients were included with a minimum 6-mo follow-up. Collectively the 5-y recurrence and overall survival rates were 36% and 50%, respectively. Recurrence was significantly higher at 5 y in patients with persistent lymphopenia (43%) compared with those who recovered (14% P = 0.0017) and those who never dropped (0% P = 0.0009). The persistent lymphopenia group had significantly lower survival (45%) compared with the two other groups (67% P = 0.0232)., Conclusions: There is a significant decrease in the overall and recurrence-free survival in those patients whose lymphocyte count drops without recovery after their esophagectomy. These data imply differences in immune responses to the stress of surgery that can be measured with routine postoperative laboratory values and are indicative of overall outcomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Therapy with alloantigen-specific CD4 + CD25 + T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rβ2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4 + CD25 + Foxp3 + Treg. After culture of naïve DA CD4 + CD8 - CD25 + T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10-20% and CD8β on <5% of these cells. These cells expressed ifngr and Il12rb2 . CD8α + cells had increased Ifngr that characterizes Ts1 cells as well was Irf4 , a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4 + CD8α + CD25 + Treg consistent with the CD8α + cells expressing IL-12R. In MLC, the CD8α + fraction suppressed responses against allogeneic stimulators more than the mixed Ts1 population, whereas the CD4 + CD8 - CD25 + T cells were less potent. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8α + cells were much less effective. This study demonstrated that expression of CD8α by rIL-2 and alloantigen activation of CD4 + CD8 - CD25 + Foxp3 + T cells was a marker of activated and potent Treg that included alloantigen-specific Treg., (Copyright © 2019 Verma, Robinson, Carter, Wilcox, Tran, Wang, Sharland, Nomura, Plain, Bishop, Hodgkinson and Hall.)

The gene cro promotes lytic growth of phages through binding of Cro protein dimers to regulatory DNA sites. Most Cro proteins are one-to-one orthologs, yet their sequence, structure and binding site sequences are quite divergent across lambdoid phages. We report the cocrystal structure of bacteriophage N15 Cro with a symmetric consensus site. We contrast this complex with an orthologous structure from phage λ, which has a dissimilar binding site sequence and a Cro protein that is highly divergent in sequence, dimerization interface and protein fold. The N15 Cro complex has less DNA bending and smaller DNA-induced changes in protein structure. N15 Cro makes fewer direct contacts and hydrogen bonds to bases, relying mostly on water-mediated and Van der Waals contacts to recognize the sequence. The recognition helices of N15 Cro and λ Cro make mostly nonhomologous and nonanalogous contacts. Interface alignment scores show that half-site binding geometries of N15 Cro and λ Cro are less similar to each other than to distantly related CI repressors. Despite this divergence, the Cro family shows several code-like protein-DNA sequence covariations. In some cases, orthologous genes can achieve a similar biological function using very different specific molecular interactions., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In response to retinal damage, the Müller glial cells (MGs) of the zebrafish retina have the ability to undergo a cellular reprogramming event in which they enter the cell cycle and divide asymmetrically, thereby producing multipotent retinal progenitors capable of regenerating lost retinal neurons. However, mammalian MGs do not exhibit such a proliferative and regenerative ability. Here, we identify Hippo pathway-mediated repression of the transcription cofactor YAP as a core regulatory mechanism that normally blocks mammalian MG proliferation and cellular reprogramming. MG-specific deletion of Hippo pathway components Lats1 and Lats2, as well as transgenic expression of a Hippo non-responsive form of YAP (YAP5SA), resulted in dramatic Cyclin D1 upregulation, loss of adult MG identity, and attainment of a highly proliferative, progenitor-like cellular state. Our results reveal that mammalian MGs may have latent regenerative capacity that can be stimulated by repressing Hippo signaling., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by "knocking-in" a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16 INK4a locus. We used this allele ( p16 tdTom ) for the enumeration, isolation, and characterization of individual p16 INK4a -expressing cells (tdTom + ). The half-life of the knocked-in transcript was shorter than that of the endogenous p16 INK4a mRNA, and therefore reporter expression better correlated with p16 INK4a promoter activation than p16 INK4a transcript abundance. The frequency of tdTom + cells increased with serial passage in cultured murine embryo fibroblasts from p16 tdTom/+ mice. In adult mice, tdTom + cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16 INK4a and found that tdTom + macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated β-galactosidase (SA-β-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16 INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Background and Objectives: Australian national surveillance data was used to assess recognition, sequelae, and antiviral therapy for congenital cytomegalovirus (CMV) cases., Study Design: Data from congenital CMV cases reported through the Australian Paediatric Surveillance Unit born January 1999 to December 2016 were described and Chi-square tests used to characterise trends and associations in case reporting, maternal CMV serology testing, and antiviral therapy. Descriptive analyses for hearing loss and developmental delay were reported for cases born ≥2004, following introduction of universal neonatal hearing screening., Results: There were 302 congenital CMV cases (214 symptomatic, 88 asymptomatic). Congenital CMV was suspected in 70.6% by 30 days of age, with no differences across birth cohorts. Maternal CMV serology testing was associated with maternal illness during pregnancy but not birth cohort. There was increasing antiviral use for symptomatic cases, being used in 14% born 1999-2004, 19.6% born 2005-2010, and 44.4% born 2011-2016 (p < 0.001). For those born ≥2004, hearing loss was reported in 42.1% of symptomatic and 26.6% of asymptomatic cases; while developmental delay was reported in 16.9% of symptomatic and 1.3% of asymptomatic cases., Conclusion: There appears to be under-reporting and under-recognition of congenital CMV despite increasing use of antiviral therapy. Universal newborn CMV screening should be considered to facilitate follow-up of affected children and targeted linkage into hearing and developmental services, and to provide population-level infant CMV epidemiology to support research and evaluation of antiviral and adjunctive therapies., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

The fundamental study of phase transition kinetics has motivated experimental methods toward achieving the largest degree of undercooling possible, more recently culminating in the technique of rapid, quasi-isentropic compression. This approach has been demonstrated to freeze water into the high-pressure ice VII phase on nanosecond timescales, with some experiments undergoing heterogeneous nucleation while others, in apparent contradiction, suggest a homogeneous nucleation mode. In this study, we show through a combination of theory, simulation, and analysis of experiments that these seemingly contradictory results are in agreement when viewed from the perspective of classical nucleation theory. We find that, perhaps surprisingly, classical nucleation theory is capable of accurately predicting the solidification kinetics of ice VII formation under an extremely high driving force (|Δμ/k&#95;{B}T|≈1) but only if amended by two important considerations: (i) transient nucleation and (ii) separate liquid and solid temperatures. This is the first demonstration of a model that is able to reproduce the experimentally observed rapid freezing kinetics.

New protein folds may evolve from existing folds through metamorphic evolution involving a dramatic switch in structure. To mimic pathways by which amino acid sequence changes could induce a change in fold, we designed two folded hybrids of Xfaso 1 and Pfl 6, a pair of homologous Cro protein sequences with ~40% identity but different folds (all-α vs. α + β, respectively). Each hybrid, XPH1 or XPH2, is 85% identical in sequence to its parent, Xfaso 1 or Pfl 6, respectively; 55% identical to its noncognate parent; and ~70% identical to the other hybrid. XPH1 and XPH2 also feature a designed hybrid chameleon sequence corresponding to the C-terminal region, which switched from α-helical to β-sheet structure during Cro evolution. We report solution nuclear magnetic resonance (NMR) structures of XPH1 and XPH2 at 0.3 Å and 0.5 Å backbone root mean square deviation (RMSD), respectively. XPH1 retains a global fold generally similar to Xfaso 1, and XPH2 retains a fold similar to Pfl 6, as measured by TM-align scores (~0.7), DALI Z-scores (7-9), and backbone RMSD (2-3 Å RMSD for the most ordered regions). However, these scores also indicate significant deviations in structure. Most notably, XPH1 and XPH2 have different, and intermediate, secondary structure content relative to Xfaso 1 and Pfl 6. The multistep progression in sequence, from Xfaso 1 to XPH1 to XPH2 to Pfl 6, thus involves both abrupt and gradual changes in folding pattern. The plasticity of some protein folds may allow for "polymetamorphic" evolution through intermediate structures., (© 2018 The Protein Society.)

Objective: To examine if the protective effect of parasite infection on experimental autoimmune encephalomyelitis (EAE) was due to interleukin (IL)-5, a cytokine produced by a type-2 response that induces eosinophilia. We hypothesize that, in parasite infections, IL-5 also promotes expansion of antigen-specific T regulatory cells that control autoimmunity., Methods: Nippostrongylus brasiliensis larvae were used to infect Lewis rats prior to induction of EAE by myelin basic protein. Animals were sham treated, or given blocking monoclonal antibodies to interleukin 4 or 5 or to deplete CD25 + T cells. Reactivity of CD4 + CD25 + T regulatory cells from these animals was examined., Results: Parasite-infected hosts had reduced severity and length of EAE. The beneficial effect of parasitic infection was abolished with an anti-IL-5 or an anti-CD25 monoclonal antibody (mAb), but not anti-IL-4 mAb. Parasite-infected animals with EAE developed antigen-specific CD4 + CD25 + T regulatory cells earlier than EAE controls and these expressed more Il5ra than controls. Treatment with IL-5 also reduced the severity of EAE and induced Il5ra expressing CD4 + CD25 + T regulatory cells., Interpretation: The results of this study suggested that IL-5 produced by the type-2 inflammatory response to parasite infection promoted induction of autoantigen-specific CD25 + Il5ra + T regulatory cells that reduced the severity of autoimmunity. Such a mechanism may explain the protective effect of parasite infection in patients with multiple sclerosis where eosinophilia is induced by IL-5, produced by the immune response to parasites.

Biological sex differences affect the course of HIV infection, with untreated women having lower viral loads compared to their male counterparts but, for a given viral load, women have a higher rate of progression to AIDS. However, the vast majority of data on viral evolution, a process that is clearly impacted by host immunity and could be impacted by sex differences, has been derived from men. We conducted an intensive analysis of HIV-1 gag and env-gp120 evolution taken over the first 6-11 years of infection from 8 Women's Interagency HIV Study (WIHS) participants who had not received combination antiretroviral therapy (ART). This was compared to similar data previously collected from men, with both groups infected with HIV-1 subtype B. Early virus populations in men and women were generally homogenous with no differences in diversity between sexes. No differences in ensuing nucleotide substitution rates were found between the female and male cohorts studied herein. As previously reported for men, time to peak diversity in env-gp120 in women was positively associated with time to CD4+ cell count below 200 (P = 0.017), and the number of predicted N-linked glycosylation sites generally increased over time, followed by a plateau or decline, with the majority of changes localized to the V1-V2 region. These findings strongly suggest that the sex differences in HIV-1 disease progression attributed to immune system composition and sensitivities are not revealed by, nor do they impact, global patterns of viral evolution, the latter of which proceeds similarly in women and men.

Transplant tolerance induced in adult animals is mediated by alloantigen-specific CD4 + CD25 + T cells, yet in many models, proliferation of CD4 + T cells from hosts tolerant to specific-alloantigen in vitro is not impaired. To identify changes that may diagnose tolerance, changes in the patterns of proliferation of CD4 + , CD4 + CD25 + , and CD4 + CD25 - T cells from DA rats tolerant to Piebald Virol Glaxo rat strain (PVG) cardiac allografts and from naïve DA rats were examined. Proliferation of CD4 + T cells from both naïve and tolerant hosts was similar to both PVG and Lewis stimulator cells. In mixed lymphocyte culture to PVG, proliferation of naïve CD4 + CD25 - T cells was greater than naïve CD4 + T cells. In contrast, proliferation of CD4 + CD25 - T cells from tolerant hosts to specific-donor PVG was not greater than CD4 + T cells, whereas their response to Lewis and self-DA was greater than CD4 + T cells. Paradoxically, CD4 + CD25 + T cells from tolerant hosts did not proliferate to PVG, but did to Lewis, whereas naïve CD4 + CD25 + T cells proliferate to both PVG and Lewis but not to self-DA. CD4 + CD25 + T cells from tolerant, but not naïve hosts, expressed receptors for interferon (IFN)-γ and IL-5 and these cytokines promoted their proliferation to specific-alloantigen PVG but not to Lewis or self-DA. We identified several differences in the patterns of proliferation to specific-donor alloantigen between cells from tolerant and naïve hosts. Most relevant is that CD4 + CD25 + T cells from tolerant hosts failed to proliferate or suppress to specific donor in the absence of either IFN-γ or IL-5. The proliferation to third-party and self of each cell population from tolerant and naïve hosts was similar and not affected by IFN-γ or IL-5. Our findings suggest CD4 + CD25 + T cells that mediate transplant tolerance depend on IFN-γ or IL-5 from alloactivated Th1 and Th2 cells.

Age-related health decline has been attributed to the accumulation of senescent cells recognized in vivo by p16(Ink4a) expression. The pharmacological elimination of p16(Ink4a)-positive cells from the tissues of mice was shown to extend a healthy lifespan. Here, we describe a population of mesenchymal cells isolated from mice that are highly p16(INK4a)-positive are proficient in proliferation but lack other properties of cellular senescence. These data, along with earlier reports on p16(Ink4a)-positive macrophages, indicate that p16(Ink4a)-positive and senescent cell populations only partially intersect, therefore, extending the list of potential cellular targets for anti- aging therapies.

Constitutive p16 Ink4a expression, along with senescence-associated β-galactosidase (SAβG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16 Ink4a -positive cell killing to the eradication of accumulated SCs. However, detection of p16 Ink4a /SAβG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16 Ink4a and SAβG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16 Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16 Ink4a /SAβG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16 Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16 Ink4a -positive cells may not be solely attributed to SCs but also to non-senescent p16 Ink4a /SAβG-positive macrophages.

CD4 + T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4 + CD25 + FOXP3 + Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg. This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4 + , especially CD4 + CD25 + T cells. CD4 + T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4 + T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4 + CD25 + T cells' response to rIL-5 and expression of IL-5Rα was also assessed. rIL-5 was sufficient to promote transplant tolerance mediating CD4 + T cells' survival in culture with specific-donor alloantigen. Tolerant CD4 + T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4 + T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4 + CD25 + T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4 + CD25 + T cells expressed IL-5Rα. This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4 + CD25 + T cells that mediate transplant tolerance., (Copyright © 2017 Elsevier B.V. All rights reserved.)

CD4 + T cells that transfer alloantigen-specific transplant tolerance are short lived in culture unless stimulated with specific-donor alloantigen and lymphocyte derived cytokines. Here, we examined if IFN-γ maintained survival of tolerance transferring CD4 + T cells. Alloantigen-specific transplant tolerance was induced in DA rats with heterotopic adult PVG heart allografts by a short course of immunosuppression and these grafts functioned for >100days with no further immunosuppression. In previous studies, we found the CD4 + T cells from tolerant rats that transfer tolerance to an irradiated DA host grafted with a PVG heart, lose their tolerance transferring ability after 3days of culture, either with or without donor alloantigen, and effect rejection of specific-donor grafts. If cultures with specific-donor alloantigen are supplemented by supernatant from ConA activated lymphocytes the tolerance transferring cells survive, suggesting these cells depend on cytokines for their survival. In this study, we found addition of rIFN-γ to MLC with specific-donor alloantigen maintained the capacity of tolerant CD4 + T cells to transfer alloantigen-specific tolerance and their ability to suppress PVG allograft rejection mediated by co-administered naïve CD4 + T cells. IFN-γ suppressed the in vitro proliferation of tolerant CD4 + T cells. Tolerant CD4 + CD25 + T cells did not proliferate in MLC to PVG stimulator cells with no cytokine added, but did when IFN-γ was present. IFN-γ did not alter proliferation of tolerant CD4 + CD25 + T cells to third-party Lewis. Tolerant CD4 + CD25 + T cells' expression of IFN-γ receptor (IFNGR) was maintained in culture when IFN-γ was present. This study suggested that IFN-γ maintained tolerance mediating alloantigen-specific CD4 + CD25 + T cells., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Caseinolytic peptidases (ClpPs) regulate diverse aspects of cellular physiology in bacteria. Some species have multiple ClpPs, including the opportunistic pathogen Pseudomonas aeruginosa , in which there is an archetypical isoform, ClpP1, and a second isoform, ClpP2, about which little is known. Here, we use phenotypic assays to investigate the biological roles of ClpP1 and ClpP2 and biochemical assays to characterize purified ClpP1, ClpP2, ClpX, and ClpA. Interestingly, ClpP1 and ClpP2 have distinct intracellular roles for motility, pigment production, iron scavenging, and biofilm formation. Of particular interest, ClpP2, but not ClpP1, is required for microcolony organization, where multicellular organized structures first form on the pathway to biofilm production. We found that purified ClpP1 with ClpX or ClpA was enzymatically active, yet to our surprise, ClpP2 was inactive and not fully assembled in vitro ; attempts to assist ClpP2 assembly and activation by mixing with the other Clp components failed to turn on ClpP2, as did solution conditions that have helped activate other ClpPs in vitro We postulate that the active form of ClpP2 has yet to be discovered, and we present several potential models to explain its activation as well as the unique role ClpP2 plays in the development of the clinically important biofilms in P. aeruginosa IMPORTANCE Pseudomonas aeruginosa is responsible for severe infections of immunocompromised patients. Our work demonstrates that two different isoforms of the Clp peptidase, ClpP1 and ClpP2, control distinct aspects of cellular physiology for this organism. In particular, we identify ClpP2 as being necessary for microcolony organization. Pure active forms of ClpP1 and either ClpX or ClpA were characterized as assembled and active, and ClpP2 was incompletely assembled and inactive. By establishing both the unique biological roles of ClpP1 and ClpP2 and their initial biochemical assemblies, we have set the stage for important future work on the structure, function, and biological targets of Clp proteolytic enzymes in this important opportunistic pathogen., (Copyright © 2017 American Society for Microbiology.)

In the 1970s, the capacity of T cells to inhibit immunity and those from transplant tolerant hosts to transfer alloantigen-specific suppression to lymphopenic recipients was described. CD4 T suppressor cells that ex vivo reverted to effector cells were described in the 1980s. Their antigen-specific suppressor function could be preserved by stimulation by specific donor alloantigen and cytokines from activated lymphocytes. This led to the finding that alloantigen-specific T suppressor cells express IL-2 receptor (CD25) and that IL-2 in part promotes their survival.Whether these alloantigen-specific CD4CD25FOXP3 regulatory T (Treg) cells are progeny of thymic-derived CD4CD25FOXP3 Treg (tTreg) cells or are induced from peripheral effector CD4CD25FOXP3T cells (iTreg) cells is still debated.In vitro studies of antigen specific Treg has been difficult as they die in the absence of cytokines produced by immune activated cells. The antigen-specific CD4CD25 T cells that control rejection in tolerant hosts differ from the naive tTreg. Thymic-derived Treg cells are not antigen-specific, and very high ratios are required to suppress rejection. Thymic-derived Treg cells can be expanded ex vivo and are currently being tested in trials to control allograft rejection and graft versus host disease.Thymic-derived Treg cells with specific receptors for alloantigen are activated by either IL-2 or IL-4 but rapidly become dependent on other cytokines, respectively, IFN-γ or IL-12 if activated by IL-2, or IL-5 if activated by IL-4. The Th1 and Th2 pathways for activation of tTreg produce more potent antigen-specific Treg that only suppress specific donor rejection. After 25 years, much remains unknown about antigen-specific CD4CD25FOXP3 Treg-mediating transplant tolerance.

Background: Regional variations in hospital billing for total joint arthroplasty (TJA) have been reported. It is not clear whether differences exist in hospital charges for TJA based on hospital profit status., Methods: Data from the Centers for Medicare and Medicaid Services on Medicare Severity-Diagnosis Related Groups (MS-DRGs) 469 (TJA with comorbidity) and 470 (TJA without comorbidity) for fiscal year 2011 were analyzed. Differences in hospital charges and payments were investigated based on hospital profit status (nonprofit, government, and proprietary). Generalized estimating equations determined differences in charges and reimbursement between hospital types controlling for census region, MS-DRG, and number of discharges., Results: Significant differences in billing between institutions existed with median average hospital charges for nonprofit, government, and proprietary institutions being $70,514.30, $73,540.99, and $113,203.77 (P < .0001), respectively, for DRG 469 and $45,363.95, $44,956.57, and $62,715.39 (P < .0001), respectively, for DRG 470. Median average Centers for Medicare and Medicaid Services payments for nonprofit, government, and proprietary institutions for DRG 469 were $22,334.34, $21,346.65, and $21,281.30 (P = .017), respectively, and $14,461.95, $14,466.04, and $13,733.62 (P < .0001), respectively, for DRG 470. Multivariate analyses indicate that nonprofit hospitals charge 5% more (P = .021) and receive 3% less (P = .011) reimbursement than government hospitals. Proprietary hospitals charge 34% more (P < .0001) and receive 7% less (P < .0001) reimbursement than government hospitals., Conclusion: Significant differences in hospital charges based on institution profit status were found, with proprietary institutions charging significantly more than nonprofit and government institutions. However, proprietary institutions had the lowest median average reimbursement., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The scope of this review focuses on recent applications in preclinical and clinical magnetic resonance imaging (MRI) toward accomplishing the goals of early detection and responses to therapy in animal models of Alzheimer's disease (AD). Driven by the outstanding efforts of the Alzheimer's Disease Neuroimaging Initiative (ADNI), a truly invaluable resource, the initial use of MRI in AD imaging has been to assess changes in brain anatomy, specifically assessing brain shrinkage and regional changes in white matter tractography using diffusion tensor imaging. However, advances in MRI have led to multiple efforts toward imaging amyloid beta plaques first without and then with the use of MRI contrast agents. These technological advancements have met with limited success and are not yet appropriate for the clinic. Recent developments in molecular imaging inclusive of high-power liposomal-based MRI contrast agents as well as fluorine 19 ((19)F) MRI and manganese enhanced MRI have begun to propel promising advances toward not only plaque imaging but also using MRI to detect perturbations in subcellular processes occurring within the neuron. This review concludes with a discussion about the necessity for the development of novel preclinical models of AD that better recapitulate human AD for the imaging to truly be meaningful and for substantive progress to be made toward understanding and effectively treating AD. Furthermore, the continued support of outstanding programs such as ADNI as well as the development of novel molecular imaging agents and MRI fast scanning sequences will also be requisite to effectively translate preclinical findings to the clinic., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-gal(pH6)), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-gal(pH6)-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-gal(pH6)-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-gal(pH6)-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-gal(pH6)-positive cells and reconsideration of potential cellular target for anti-aging treatment., Competing Interests: statement A.P., O.B.C. and A.V.G. are co-founders and shareholders of Everon Biosciences.

There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-passage patient tumor-derived tissue into basement membrane extract, ensuring a low proportion of cell death to anoikis and growth complementation by coculture with patient-derived cancer-associated fibroblasts (CAF). A range of solid tumors proved amenable to growth and pharmacologic testing in this 3D assay. A study of 30 early-stage non-small cell lung cancer (NSCLC) specimens revealed high levels of de novo resistance to a large range of standard-of-care agents, while histone deacetylase (HDAC) inhibitors and their combination with antineoplastic drugs displayed high levels of efficacy. Increased resistance was seen in the presence of patient-derived CAFs for many agents, highlighting the utility of the assay for tumor microenvironment-educated drug testing. Standard-of-care agents showed similar responses in the 3D ex vivo and patient-matched in vivo models validating the 3D-Tumor Growth Assay (3D-TGA) as a high-throughput screen for close-to-patient tumors using significantly reduced animal numbers. Mol Cancer Ther; 15(4); 753-63. ©2016 AACR., (©2016 American Association for Cancer Research.)