Design and Functional Characterization of HIV-1 Envelope Protein-Coupled T Helper Liposomes.

Functionalization of experimental HIV-1 virus-like particle vaccines with heterologous T helper epitopes (T helper VLPs) can modulate the humoral immune response via intrastructural help (ISH). Current advances in the conjugation of native-like HIV-1 envelope trimers (Env) onto liposomes and encapsulation of peptide epitopes into these nanoparticles renders this GMP-scalable liposomal platform a feasible alternative to VLP-based vaccines. In this study, we designed and analyzed customizable Env-conjugated T helper liposomes. First, we passively encapsulated T helper peptides into a well-characterized liposome formulation displaying a dense array of Env trimers on the surface. We confirmed the closed pre-fusion state of the coupled Env trimers by immunogold staining with conformation-specific antibodies. These peptide-loaded Env-liposome conjugates efficiently activated Env-specific B cells, which further induced proliferation of CD4+ T cells by presentation of liposome-derived peptides on MHC-II molecules. The peptide encapsulation process was then quantitatively improved by an electrostatically driven approach using an overall anionic lipid formulation. We demonstrated that peptides delivered by liposomes were presented by DCs in secondary lymphoid organs after intramuscular immunization of mice. UFO (uncleaved prefusion optimized) Env trimers were covalently coupled to peptide-loaded anionic liposomes by His-tag/NTA(Ni) interactions and EDC/Sulfo-NHS crosslinking. EM imaging revealed a moderately dense array of well-folded Env trimers on the liposomal surface. The conformation was verified by liposomal surface FACS. Furthermore, anionic Env-coupled T helper liposomes effectively induced Env-specific B cell activation and proliferation in a comparable range to T helper VLPs. Taken together, we demonstrated that T helper VLPs can be substituted with customizable and GMP-scalable liposomal nanoparticles as a perspective for future preclinical and clinical HIV vaccine applications. The functional nanoparticle characterization assays shown in this study can be applied to other systems of synthetic nanoparticles delivering antigens derived from various pathogens.

Caspase-Cleaved Keratin 18 Measurements Identified Ongoing Liver Injury after Bariatric Surgery.

Bariatric surgery has emerged as an effective treatment option in morbidly obese patients with non-alcoholic fatty liver disease (NAFLD). However, worsening or new onset of non-alcoholic steatohepatitis (NASH) and fibrosis have been observed. Caspase-cleaved keratin 18 (ccK18) has been established as a marker of hepatocyte apoptosis, a key event in NASH development. Thus, ccK18 measurements might be feasible to monitor bariatric surgery patients. Clinical data and laboratory parameters were collected from 39 patients undergoing laparoscopic Roux-en-Y gastric bypass at six timepoints, prior to surgery until one year after the procedure. ccK18 levels were measured and a high-throughput analysis of serum adipokines and cytokines was carried out. Half of the cohort's patients (20/39) presented with ccK18 levels indicative of progressed liver disease. 21% had a NAFLD-fibrosis score greater than 0.676, suggesting significant fibrosis. One year after surgery, a mean weight loss of 36.87% was achieved. Six and twelve months after surgery, ccK18 fragments were significantly reduced compared to preoperative levels (p < 0.001). Yet nine patients did not show a decline in ccK18 levels ≥ 10% within one year postoperatively, which was considered a response to treatment. While no significant differences in laboratory parameters or ccK18 could be observed, they presented with a greater expression of leptin and fibrinogen before surgery. Consecutive ccK18 measurements monitored the resolution of NAFLD and identified non-responders to bariatric surgery with ongoing liver injury. Further studies are needed to elicit the pathological mechanisms in non-responders and study the potential of adipokines as prognostic markers.

Progranulin serum levels and gene expression in subcutaneous vs visceral adipose tissue of severely obese patients undergoing bariatric surgery.

BACKGROUND: Progranulin represents an adipokine putatively mediating insulin resistance and inflammation. Data in humans are sparse, and the source of circulating progranulin in obesity is unknown. OBJECTIVES: Serum progranulin concentrations and subcutaneous (sc) as well as visceral (vis) adipose tissue (AT) progranulin expression were quantified in a large cohort of patients with obesity undergoing bariatric surgery (BS) (n = 153) or a low-calorie diet (LCD) (n = 121). COHORTS AND METHODS: Paired serum and AT mRNA samples were obtained from patients with severe obesity undergoing BS (ROBS cohort; Research in Obesity and Bariatric Surgery). Serum progranulin was measured by ELISA in both cohorts, and AT mRNA expression was analysed by quantitative real-time PCR in bariatric patients. RESULTS: There was no gender-specific effect in serum progranulin or AT progranulin expression. Importantly, circulating progranulin was independent from adipose tissue gene expression in paired samples. sc AT progranulin expression was higher than in vis AT (P = 0.027), and there was a positive correlation between sc AT and vis AT gene expression (P < 0.001; r = +0.34). Serum progranulin strongly and rapidly increased after BS within 3 days and remained elevated up to 12 months. Serum progranulin was strongly correlated with serum CTRP-3 levels. CONCLUSIONS: The present study provides detailed progranulin gene expression data in sc and vis AT in a large, prospective and observational cohort of patients with severe obesity. Serum progranulin concentrations are not predicted by sc or vis AT progranulin gene expression. Thus, AT seems not to be the main source of circulating progranulin levels in obesity.

Regulation of natriuretic peptides postprandially in vivo and of their receptors in adipocytes by fatty acids in vitro.

BACKGROUND AND AIM: Natriuretic peptides (NPs) and their receptors gain attention regarding adipocyte function. It was the aim to investigate the expression of natriuretic peptide receptors NPR-A, NPR-B and NPR-C during adipocyte differentiation (AD), upon stimulation with fatty acids (FA), and in murine and human adipose tissue depots (AT) of patients undergoing bariatric surgery (n = 44). PATIENTS, MATERIAL AND METHODS: The postprandial regulation of NT-proANP and NT-proBNP levels was measured by ELISA and was studied in two cohorts of healthy individuals undergoing an oral glucose tolerance test (OGTT) (n = 100) and an oral lipid tolerance test (OLTT) (n = 100). Adipocyte mRNA expression was investigated by quantitative real-time PCR. RESULTS: During AD, an early expression pattern could be described for NPR-C, a bimodal expression for NPR-B and a late expression pattern for NPR-A. NPR-A and NPR-B expression was high in epididymal and subcutaneous AT but low in peri-renal AT of mice. NPR-C showed a differential expression profile. FA stimulation caused a significant and differential regulation of NPRs in adipocytes. Serum NT-proANP and NT-proBNP concentrations did not change during OGTT, whereas NT-proANP significantly declined during OLTT. Basal NT-proANP and NT-proBNP concentrations were positively correlated with each other and with FGF-19 and FGF-21 levels. CONCLUSION: Adipocytes and AT show a characteristic expression of NPRs. FA are able to regulate NPR expression differentially. There is a postprandial and negative regulation of serum NT-proANP concentrations after OLTT and of NPR-A after FA stimulation. Both effects could represent a novel hypothetical negative feedback mechanism on adipocyte lipolysis.

Noncanonical NF-κB activation by the oncoprotein Tio occurs through a nonconserved TRAF3-binding motif.

Members of the nuclear factor κB (NF-κB) family of transcription factors regulate many cellular functions. Activation of NF-κB signaling is commonly classified as occurring through canonical or noncanonical pathways. Most NF-κB-inducing stimuli, including the viral oncoprotein Tio, lead to a concerted activation of both NF-κB pathways; however, extensive crosstalk at multiple levels between these signaling cascades restricts the ability to discriminate between the canonical and the noncanonical effects. We showed that noncanonical NF-κB activation by Tio depends on a distinct sequence motif that directly recruits tumor necrosis factor receptor-associated factor 3 (TRAF3). Through its TRAF3-binding motif, Tio triggered a ubiquitin-independent depletion of TRAF3 from the cytosol, which prevented TRAF3 from inhibiting signaling through the noncanonical NF-κB cascade. Furthermore, the Tio-TRAF3 interaction did not affect components of the canonical NF-κB signaling pathway or the expression of target genes; thus, Tio induced noncanonical NF-κB independently of crosstalk with the canonical pathway. Together, these data identify a distinct molecular mechanism of noncanonical NF-κB activation that should enable studies into the particular functions of this pathway.

Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip.

Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis.

Species restriction of Herpesvirus saimiri and Herpesvirus ateles: human lymphocyte transformation correlates with distinct signaling properties of viral oncoproteins.

The potential of Herpesvirus saimiri (HVS) subgroups A, B and C and Herpesvirus ateles (HVA) to transform primary T cells to permanent growth in vitro is restricted by the primate host species and by viral variability represented by distinct viral oncoproteins. We now addressed the relation between the transforming potential of the different viruses and the signaling pathways activated by transiently expressed oncoproteins. Marmoset lymphocytes were transformed by all HVS subgroups as well as HVA, while transformation of human cells was restricted to HVS-C and, unexpectedly, HVA. NF-κB and Src-family kinase (SFK) activity was required for survival of all transformed lymphocytes. Accordingly, NF-κB was induced by oncoproteins of all viruses. In contrast, SFK-related signaling was detectable only for oncoproteins of HVS-C and HVA. Thus, the restricted transformation of human lymphocytes likely correlates with the specific SFK targeting by these oncoproteins. These results will enable further studies into novel SFK effector mechanisms relevant for T-cell proliferation.

Exudate macrophages attenuate lung injury by the release of IL-1 receptor antagonist in gram-negative pneumonia.

RATIONALE: Exudate macrophages are key players in host defense toward invading pathogens. Their antiinflammatory and epithelial-protective potential in gram-negative pneumonia, however, remains elusive. OBJECTIVES: We investigated whether exudate macrophages contributed to preservation of alveolar epithelial barrier integrity and analyzed the molecular pathways involved. METHODS: We evaluated the antiinflammatory and epithelial-protective effects of exudate macrophages in a model of LPS- and Klebsiella pneumoniae-induced lung injury comparing wild-type and CC-chemokine receptor 2 (CCR2)-deficient mice with defective lung macrophage recruitment and in in vitro studies using primary alveolar epithelial cells. MEASUREMENTS AND MAIN RESULTS: CCR2(-/-) mice exhibited enhanced alveolar epithelial cell apoptosis and lung leakage on intratracheal LPS treatment, which could be attributed to lack of exudate macrophage recruitment from the circulating pool as demonstrated in a model of wild-type/CCR2(-/-) bone-marrow chimeric mice. Among various antiinflammatory and proliferative mediators analyzed, the endogenous counterpart of resident macrophage-expressed IL-1ß, IL-1 receptor antagonist (IL-1ra), was highly up-regulated in flow-sorted exudate macrophages in LPS-treated wild-type mice. LPS/IL-1ß-induced impairment of alveolar epithelial cell integrity was antagonized by IL-1ra in vitro. Finally, intratracheal substitution of IL-1ra or intravenous adoptive transfer of IL-1ra(+/+) but not IL-1ra(-/-) blood mononuclear cells attenuated alveolar inflammation, epithelial apoptosis, and loss of barrier function in LPS-challenged or K. pneumoniae-infected CCR2(-/-) mice and enhanced survival after K. pneumoniae infection. CONCLUSIONS: We conclude that recruited lung macrophages attenuate IL-1ß-mediated acute lung injury in gram-negative pneumonia by release of IL-1ra.

Activation of noncanonical NF-kappaB signaling by the oncoprotein Tio.

NF-kappaB transcription factors are key regulators of cellular proliferation and frequently contribute to oncogenesis. The herpesviral oncoprotein Tio, which promotes growth transformation of human T cells in a recombinant herpesvirus saimiri background, potently induces canonical NF-kappaB signaling through membrane recruitment of the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, we show that, in addition to Tio-TRAF6 interaction, the Tio-induced canonical NF-kappaB signal requires the presence of the regulatory subunit of the inhibitor of kappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO), and the activity of its key kinase, IKKbeta, to up-regulate expression of endogenous cellular inhibitor of apoptosis 2 (cIAP2) and interleukin 8 (IL-8) proteins. Dependent on TRAF6 and NEMO, Tio enhances the expression of the noncanonical NF-kappaB proteins, p100 and RelB. Independent of TRAF6 and NEMO, Tio mediates stabilization of the noncanonical kinase, NF-kappaB-inducing kinase (NIK). Concomitantly, Tio induces efficient processing of the p100 precursor molecule to its active form, p52, as well as DNA binding of nuclear p52 and RelB. In human T cells transformed by infection with a Tio-recombinant virus, sustained expression of p100, RelB, and cIAP2 depends on IKKbeta activity, yet processing to p52 remains largely unaffected by IKKbeta inhibition. However, long term inhibition of IKKbeta disrupts the continuous growth of the transformed cells and induces cell death. Hence, the Tio oncoprotein triggers noncanonical NF-kappaB signaling through NEMO-dependent up-regulation of p100 precursor and RelB, as well as through NEMO-independent generation of p52 effector.

NF-kappaB activation by the viral oncoprotein StpC enhances IFN-gamma production in T cells.

Interferon-gamma (IFN-gamma) is an essential regulator of innate and adaptive immune responses and a hallmark of the Th1 T-cell subset. It is produced at high levels by human T lymphocytes upon transformation with Herpesvirus saimiri, which depends on the expression of the viral oncoproteins saimiri transformation-associated protein of subgroup C (StpC) and tyrosine kinase-interacting protein (Tip). Here, we show that IFN-gamma production was induced by Tip in Jurkat T cells. StpC by itself did not affect IFN-gamma expression, but enhanced the effect of Tip. Our results substantiated the findings that StpC induces NF-kappaB activation and demonstrated that other transcription factors, including NFAT, AP-1 and serum response element regulators, were not activated by StpC in unstimulated T cells. Studies using StpC mutants deficient in NF-kappaB activation, dominant negative IkappaBalpha and constitutively active IKK2, established the importance of NF-kappaB in StpC-mediated upregulation of IFN-gamma production. These observations suggest that NF-kappaB induction by StpC contributes to the Th1-like phenotype of virus-transformed human T cells.

Chest wall defect and chronic pleural infection: surgical treatment with thoracomyoplasty and open window thoracostomy.

We report a rare case of a 75-year-old hemiplegic man with a chronic pleural infection, a bronchopleural fistula, and a full-thickness defect of the chest. In one operation we performed open-window thoracostomy and pleural decortication as well as reconstruction of the chest defect and reclosure of the bronchopleural fistula with a latissimus dorsi muscle flap. The patient made a good recovery and was sent for rehabilitation in good condition. Surgical treatment was essential to control and ultimately halt the septic process. Use of a muscle transplant in a hemiplegic patient did not reduce mobility.

NFkappaB signaling is induced by the oncoprotein Tio through direct interaction with TRAF6.

The transcription factor NFkappaB is a major regulator of genes involved in inflammation and oncogenesis. NFkappaB is induced upon stimulation of cellular receptors coupled to different intracellular signaling molecules. Further downstream, TRAF6 links at least two receptor pathways to take control of IkappaB, the administrator of NFkappaB activity. Here we report on a strong NFkappaB activation by Tio, a unique herpesviral oncoprotein promoting transformation of human T cells in a Src-kinase-dependent manner. NFkappaB induction by Tio is independent of Src-kinase interaction and tyrosine phosphorylation of Tio. Mutation of a glutamic acid-rich motif at the N terminus of Tio, corresponding to a TRAF6 consensus binding motif, completely abrogated NFkappaB activation. Cotransfection of a dominant negative TRAF6 construct led to a decrease in NFkappaB activation. Furthermore, we provide evidence that TRAF6 directly binds to the Tio oncoprotein. Identification of TRAF6 as the direct target of Tio describes a novel mechanism for the constitutive activation of NFkappaB through an oncoprotein.

Tyrosine phosphorylation of the Tio oncoprotein is essential for transformation of primary human T cells.

Human T cells are transformed to antigen-independent permanent growth in vitro upon infection with herpesvirus saimiri subgroup C strains. The viral oncoproteins required for this process, StpC and Tip, could be replaced by Tio, the oncoprotein of herpesvirus ateles. Here we demonstrate that proliferation of lymphocytes transformed with Tio-recombinant herpesvirus saimiri required the activity of Src family kinases. Src kinases had previously been identified as interaction partners of Tio. This interaction was now shown to be independent of any of the four tyrosine residues of Tio but to be dependent on an SH3-binding motif. Mutations within this motif abrogated the transforming capabilities of Tio-recombinant herpesvirus saimiri. Furthermore, kinase interaction resulted in the phosphorylation of Tio on a single tyrosine residue at position 136. Mutation of this residue in the viral context revealed that this phosphorylation site, but none of the other tyrosine residues, was required for T-cell transformation. These data indicate that the interaction of Tio with a Src kinase is essential for both the initiation and the maintenance of T-cell transformation by recombinant herpesvirus saimiri. The requirement for the tyrosine phosphorylation site at position 136 suggests a role for Tio beyond simple deregulation of the kinase.

Herpesvirus ateles Tio can replace herpesvirus saimiri StpC and Tip oncoproteins in growth transformation of monkey and human T cells.

Herpesvirus saimiri group C strains are capable of transforming human and simian T-lymphocyte populations to permanent antigen-independent growth. Two viral oncoproteins, StpC and Tip, that are encoded by a single bicistronic mRNA, act in concert to mediate this phenotype. A closely related New World monkey herpesvirus, herpesvirus ateles, transcribes a single spliced mRNA at an equivalent genome locus. The encoded protein, Tio, has sequence homologies to both StpC and Tip. We inserted the tio sequence of herpesvirus ateles strain 73 into a recombinant herpesvirus saimiri C488 lacking its own stpC/tip oncogene. Simian as well as human T lymphocytes were growth transformed by the chimeric Tio-expressing viruses. Thus, a single herpesvirus protein appears to be responsible for the oncogenic effects of herpesvirus ateles.