Quantitative imaging for development of companion diagnostics to drugs targeting HGF/MET.

The limited clinical success of anti-HGF/MET drugs can be attributed to the lack of predictive biomarkers that adequately select patients for treatment. We demonstrate here that quantitative digital imaging of formalin fixed paraffin embedded tissues stained by immunohistochemistry can be used to measure signals from weakly staining antibodies and provides new opportunities to develop assays for detection of MET receptor activity. To establish a biomarker panel of MET activation, we employed seven antibodies measuring protein expression in the HGF/MET pathway in 20 cases and up to 80 cores from 18 human cancer types. The antibodies bind to epitopes in the extra (EC)- and intracellular (IC) domains of MET (MET4EC, SP44_METIC, D1C2_METIC), to MET-pY1234/pY1235, a marker of MET kinase activation, as well as to HGF, pSFK or pMAPK. Expression of HGF was determined in tumour cells (T_HGF) as well as in stroma surrounding cancer (St_HGF). Remarkably, MET4EC correlated more strongly with pMET (r = 0.47) than SP44_METIC (r = 0.21) or D1C2_METIC (r = 0.08) across 18 cancer types. In addition, correlation coefficients of pMET and T_HGF (r = 0.38) and pMET and pSFK (r = 0.56) were high. Prediction models of MET activation reveal cancer-type specific differences in performance of MET4EC, SP44_METIC and anti-HGF antibodies. Thus, we conclude that assays to predict the response to HGF/MET inhibitors require a cancer-type specific antibody selection and should be developed in those cancer types in which they are employed clinically.

Impact of early cART in the gut during acute HIV infection.

Early after HIV infection there is substantial depletion of CD4+ T cells in the gastrointestinal (GI) tract lamina propria (LP), with associated epithelial barrier damage, leading to microbial translocation and systemic inflammation and immune activation. In this study, we analyzed these early events in the GI tract in a cohort of Thai acute HIV-infected patients and determined the effect of early combination antiretroviral treatment (cART). HIV-uninfected and chronically and acutely HIV-infected patients at different Fiebig stages (I-V) underwent colonic biopsies and then received cART. Immunohistochemistry and quantitative image analysis were performed on cross-sectional and longitudinal colon biopsy specimens (day 0 to week 96) to measure GI tract damage (infiltration of polymorphonuclear cells), inflammation (M×1, TNF-α), immune activation (Ki-67), and the CD4+ T cell population in the LP. The magnitude of GI tract damage, immune activation, and inflammation was significantly increased, with significantly depleted CD4+ T cells in the LP in all acutely infected groups prior to cART compared with HIV-uninfected control participants. While most patients treated during acute infection resolved GI tract inflammation and immune activation back to baseline levels after 24 weeks of cART, most acutely infected participants did not restore their CD4+ T cells after 96 weeks of cART.

Serum-based tracking of de novo initiated liver cancer progression reveals early immunoregulation and response to therapy.

BACKGROUND & AIMS: Liver inflammatory diseases associated with cancer promoting somatic oncogene mutations are increasing in frequency. Preclinical cancer models that allow for the study of early tumor progression are often protracted, which limits the experimental study parameters due to time and expense. Here we report a robust inexpensive approach using Sleeping Beauty transposition (SBT) delivery of oncogenes along with Gaussia Luciferase expression vector GLuc, to assess de novo liver tumor progression, as well as the detection of innate immune responses or responses induced by therapeutic intervention. METHODS: Tracking de novo liver tumor progression with GLuc was demonstrated in models of hepatocellular carcinoma (HCC) or adenoma (HCA) initiated by hydrodynamic delivery of SBT oncogenes. RESULTS: Rising serum luciferase levels correlated directly with increasing liver tumor burden and eventual morbidity. Early detection of hepatocyte apoptosis from mice with MET+CAT transfected hepatocytes was associated with a transient delay in HCC growth mediated by a CD8(+) T-cell response against transformed hepatocytes. Furthermore, mice that lack B cells or macrophages had an increase in TUNEL(+) hepatocytes following liver MET transfection demonstrating that these cells provide protection from MET-induced hepatocyte apoptosis. Treatment with IL-18+IL-12 of mice displaying established HCC decreased tumor burden which was associated with decreased levels of serum luciferase. CONCLUSIONS: Hydrodynamic delivery of the SBT vector GLuc to hepatocytes serves as a simple blood-based approach for real-time tracking of pathologically distinct types of liver cancer. This revealed tumor-induced immunologic responses and was beneficial in monitoring the efficacy of therapeutic interventions.

Suppressive oligodeoxynucleotides reduce lung cancer susceptibility in mice with silicosis.

Silicosis is an inflammatory lung disease induced by the inhalation of silica-containing dust particles. There is conflicting data on whether patients with silicosis are more susceptible to lung cancer induced by cigarette smoke. To examine this issue experimentally, a model was developed in which one of the most abundant and potent carcinogens present in cigarette smoke [4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)] was administered to mice at the peak of silica-induced pulmonary inflammation. Results show that the incidence of lung tumors in silicotic mice treated with NNK was significantly increased compared with mice exposed to silica or NNK alone. Synthetic oligonucleotides (ODN) containing repetitive TTAGGG motifs can block pathologic inflammation. We therefore examined whether treatment with these suppressive (Sup) ODN could block silica-induced pulmonary inflammation and thereby reduce susceptibility to lung cancer. Results show that Sup (but not control) ODN inhibit pulmonary fibrosis and other inflammatory manifestations of chronic silicosis. Of greater import, Sup ODN reduced lung tumor incidence and multiplicity in silicotic mice exposed to NNK. These findings establish an experimental model for examining the role of silicotic inflammation in cancer susceptibility and demonstrate that Sup ODN represent a novel therapy for chronic silicosis.

C/EBPγ suppresses senescence and inflammatory gene expression by heterodimerizing with C/EBPß.

C/EBPß is an important regulator of oncogene-induced senescence (OIS). Here, we show that C/EBPγ, a heterodimeric partner of C/EBPß whose biological functions are not well understood, inhibits cellular senescence. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferated poorly, entered senescence prematurely, and expressed a proinflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPß. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPß. Covalently linked C/EBPß homodimers (ß∼ß) inhibited the proliferation and tumorigenicity of Ras(V12)-transformed NIH 3T3 cells, activated SASP gene expression, and recruited the CBP coactivator in a Ras-dependent manner, whereas Î³âˆ¼ß heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg(-/-) MEFs. C/EBPß depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPß homodimers in Cebpg(-/-) MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg(-/-) bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPß through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes.

Reduced inflammation and lymphoid tissue immunopathology in rhesus macaques receiving anti-tumor necrosis factor treatment during primary simian immunodeficiency virus infection.

BACKGROUND: Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections induce robust, generalized inflammatory responses that begin during acute infection and lead to pathological systemic immune activation, fibrotic damage of lymphoid tissues, and CD4⁺ T-cell loss, pathogenic processes that contribute to disease progression. METHODS: To better understand the contribution of tumor necrosis factor (TNF), a key regulator of acute inflammation, to lentiviral pathogenesis, rhesus macaques newly infected with SIVmac239 were treated for 12 weeks in a pilot study with adalimumab (Humira), a human anti-TNF monoclonal antibody. RESULTS: Adalimumab did not affect plasma SIV RNA levels or measures of T-cell immune activation (CD38 or Ki67) in peripheral blood or lymph node T cells. However, compared with untreated rhesus macaques, adalimumab-treated rhesus macaques showed attenuated expression of proinflammatory genes, decreased infiltration of polymorphonuclear cells into the T-cell zone of lymphoid tissues, and weaker antiinflammatory regulatory responses to SIV infection (ie, fewer presumed alternatively activated [ie, CD163⁺] macrophages, interleukin 10-producing cells, and transforming growth factor ß-producing cells), along with reduced lymphoid tissue fibrosis and better preservation of CD4⁺ T cells. CONCLUSIONS: While HIV/SIV replication drives pathogenesis, these data emphasize the contribution of the inflammatory response to lentiviral infection to overall pathogenesis, and they suggest that early modulation of the inflammatory response may help attenuate disease progression.

Molecular and organismal changes in offspring of male mice treated with chemical stressors.

Both gene methylation changes and genetic instability have been noted in offspring of male rodents exposed to radiation or chemicals, but few specific gene targets have been established. Previously, we identified the gene for ribosomal RNA, rDNA, as showing methylation change in sperm of mice treated with the preconceptional carcinogen, chromium(III) chloride. rDNA is a critical cell growth regulator. Here, we investigated the effects of paternal treatments on rDNA in offspring tissue. A total of 93 litters and 758 offspring were obtained, permitting rigorous mixed-effects models statistical analysis of the results. We show that the offspring of male mice treated with Cr(III) presented increased methylation in a promoter sequence of the rDNA gene, specifically in lung. Furthermore polymorphic variants of the multi-copy rDNA genes displayed altered frequencies indicative of structural changes, as a function of both tissue type and paternal treatments. Organismal effects also occurred: some groups of offspring of male mice treated with either Cr(III) or its vehicle, acidic saline, compared with those of untreated mice, had altered average body and liver weights and levels of serum glucose and leptin. Males treated directly with Cr(III) or acidic saline presented serum hormone changes consistent with a stress response. These results establish for the first time epigenetic and genetic instability effects in a gene of central physiological importance, in offspring of male mice exposed preconceptionally to chemicals, possibly related to a stress response in these males.

3'UTR elements inhibit Ras-induced C/EBPß post-translational activation and senescence in tumour cells.

C/EBPß is an auto-repressed protein that becomes post-translationally activated by Ras-MEK-ERK signalling. C/EBPß is required for oncogene-induced senescence (OIS) of primary fibroblasts, but also displays pro-oncogenic functions in many tumour cells. Here, we show that C/EBPß activation by H-Ras(V12) is suppressed in immortalized/transformed cells, but not in primary cells, by its 3' untranslated region (3'UTR). 3'UTR sequences inhibited Ras-induced cytostatic activity of C/EBPß, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3'UTR suppressed induction of senescence-associated C/EBPß target genes, while promoting expression of genes linked to cancers and TGFß signalling. An AU-rich element (ARE) and its cognate RNA-binding protein, HuR, were required for 3'UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPß translation controls de-repression by Ras signalling. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPß activation and OIS to proceed. Our findings reveal a novel mechanism whereby non-coding mRNA sequences selectively regulate C/EBPß activity and suppress its anti-oncogenic functions.

Residual NADPH oxidase and survival in chronic granulomatous disease.

BACKGROUND: Failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs) is the major defect in chronic granulomatous disease, causing recurrent infections and granulomatous complications. Chronic granulomatous disease is caused by missense, nonsense, frameshift, splice, or deletion mutations in the genes for p22(phox), p40(phox), p47(phox), p67(phox) (autosomal chronic granulomatous disease), or gp91(phox) (X-linked chronic granulomatous disease), which result in variable production of neutrophil-derived ROIs. We hypothesized that residual ROI production might be linked to survival in patients with chronic granulomatous disease. METHODS: We assessed the risks of illness and death among 287 patients with chronic granulomatous disease from 244 kindreds. Residual ROI production was measured with the use of superoxide-dependent ferricytochrome c reduction and flow cytometry with dihydrorhodamine oxidation assays. Expression of NADPH oxidase component protein was detected by means of immunoblotting, and the affected genes were sequenced to identify causal mutations. RESULTS: Survival of patients with chronic granulomatous disease was strongly associated with residual ROI production as a continuous variable, independently of the specific gene affected. Patients with mutations in p47(phox) and most missense mutations in gp91(phox) (with the exception of missense mutations in the nucleotide-binding and heme-binding domains) had more residual ROI production than patients with nonsense, frameshift, splice, or deletion mutations in gp91(phox). After adolescence, mortality curves diverged according to the extent of residual ROI production. CONCLUSIONS: Patients with chronic granulomatous disease and modest residual production of ROI have significantly less severe illness and a greater likelihood of long-term survival than patients with little residual ROI production. The production of residual ROI is predicted by the specific NADPH oxidase mutation, regardless of the specific gene affected, and it is a predictor of survival in patients with chronic granulomatous disease. (Funded by the National Institutes of Health.).

Serum S100A6 concentration predicts peritoneal tumor burden in mice with epithelial ovarian cancer and is associated with advanced stage in patients.

BACKGROUND: Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival. METHODOLOGY AND PRINCIPAL FINDINGS: Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. CONCLUSIONS: S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers.

An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

BACKGROUND: Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. METHODOLOGY/PRINCIPAL FINDINGS: Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately -1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from -388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5' leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5'-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. CONCLUSIONS/SIGNIFICANCE: The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5' leader sequence of the primary rRNA transcript.

MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance criteria based on comparability and consistency in the four parameters of the model. The assay is precise, accurate and robust and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability.

Immunity to murine prostatic tumors: continuous provision of T-cell help prevents CD8 T-cell tolerance and activates tumor-infiltrating dendritic cells.

We reported previously that tumor-specific CD8(+) T cells (TcR-I) become tolerant in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. In this study, we show that CD4(+) TcR transgenic (TcR-II) T cells transferred into TRAMP mice became activated in lymph nodes, trafficked to the prostate, and initially functioned as T(H)1 cells. Although a single cotransfer of TcR-II cells delayed TcR-I cell tolerization, repeated transfer of TcR-II cells was required to prevent TcR-I cell tolerization and significantly slowed progression of TRAMP prostate tumors. After transfer of TcR-II cells, dendritic cells within the tumor expressed higher levels of costimulatory molecules and displayed an enhanced ability to stimulate proliferation of naive T cells. Blockade of CD40-CD40L interactions during TcR-II transfer resulted in a profound reduction in dendritic cell stimulatory capacity and a partial loss of TcR-I effector functions and tumor immunity. These data show that sustained provision of activated tumor-specific CD4(+) T cells alters the immunosuppressive tumor microenvironment, ultimately leading to the control of tumor growth. These findings will assist in the design of more effective immunotherapeutic approaches for cancer.

Urothelial overexpression of insulin-like growth factor-1 increases susceptibility to p-cresidine-induced bladder carcinogenesis in transgenic mice.

To establish a role for insulin-like growth factor-1 (IGF-1) in bladder cancer susceptibility, we tested the effect of p-cresidine, a potent bladder carcinogen, in transgenic (TG) mice with human IGF-1 expression in the bladder driven by the bovine keratin 5 promoter (referred to as BK5.IGF-1 TG mice). Indomethacin was also tested to determine if the cyclooxygenase (COX) pathway is a target for bladder cancer prevention in this model. Thirty-three female BK5.IGF-1 TG mice and 29 female nontransgenic littermates were randomized to the following treatments: (1) AIN-76A diet; (2) AIN-76A diet with 0.5% p-cresidine; or (3) AIN-76A diet with 0.5% p-cresidine + 0.00075% indomethacin. BK5.IGF-1 TG mice, with twofold greater total serum IGF-1 than nontransgenic mice, exhibited greatly increased susceptibility to p-cresidine-induced bladder tumors compared to nontransgenic mice. The most common type of bladder tumor in the BK5.IGF-1 TG mice was transitional cell carcinoma, which is the predominant type of bladder cancer observed in developed countries. Indomethacin inhibition of bladder tumor development in BK5.IGF-1 TG mice was not statistically significant. These results present further evidence for the role of IGF-1 in bladder cancer progression. In addition, these transgenic mice provide a useful model for studying the role of the IGF-1 pathway in bladder carcinogenesis and its prevention.

The effects of alternate polypurine tracts (PPTs) and mutations of sequences adjacent to the PPT on viral replication and cleavage specificity of the Rous sarcoma virus reverse transcriptase.

We previously reported that a mutant Rous sarcoma virus (RSV) with an alternate polypurine tract (PPT), DuckHepBFlipPPT, had unexpectedly high titers and that the PPT was miscleaved primarily at one position following a GA dinucleotide by the RNase H of reverse transcriptase (RT). This miscleavage resulted in a portion of the 3' end of the PPT (5'-ATGTA) being added to the end of U3 of the linear viral DNA. To better understand the RNase H cleavage by RSV RT, we made a number of mutations within the DuckHepBFlipPPT and in the sequences adjacent to the PPT. Deleting the entire ATGTA sequence from the DuckHepBFlipPPT increased the relative titer to wild-type levels, while point mutations within the ATGTA sequence reduced the relative titer but had minimal effects on the cleavage specificity. However, mutating a sequence 5' of ATGTA affected the relative titer of the virus and caused the RNase H of RSV RT to lose the ability to cleave the PPT specifically. In addition, although mutations in the conserved stretch of thymidine residues upstream of the PPT did not affect the relative titer or cleavage specificity, the mutation of some of the nucleotides immediately upstream of the PPT did affect the titer and cleavage specificity. Taken together, our studies show that the structure of the PPT in the context of the cognate RT, rather than a specific sequence, is important for the proper cleavage by RSV RT.

Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys.

BACKGROUND: Patients with Birt-Hogg-Dubé (BHD) syndrome harbor germline mutations in the BHD tumor suppressor gene that are associated with an increased risk for kidney cancer. BHD encodes folliculin, a protein that may interact with the energy- and nutrient-sensing 5'-AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling pathways. METHODS: We used recombineering methods to generate mice with a conditional BHD allele and introduced the cadherin 16 (KSP)-Cre transgene to target BHD inactivation to the kidney. Kidney cell proliferation was measured by BrdU incorporation and phospho-histone H3 staining. Kidney weight data were analyzed with Wilcoxon's rank-sum, Student's t, and Welch's t tests. Hematoxylin and eosin staining and immunoblot analysis and immunohistochemistry of cell cycle and signaling proteins were performed on mouse kidney cells and tissues. BHD knockout mice and kidney cells isolated from BHD knockout and control mice were treated with the mTOR inhibitor rapamycin. Mouse survival was evaluated by Kaplan-Meier analyses. All statistical tests were two-sided. RESULTS: BHD knockout mice developed enlarged polycystic kidneys and died from renal failure by 3 weeks of age. Targeted BHD knockout led to the activation of Raf-extracellular signal-regulated protein kinase (Erk)1/2 and Akt-mTOR pathways in the kidneys and increased expression of cell cycle proteins and cell proliferation. Rapamycin-treated BHD knockout mice had smaller kidneys than buffer-treated BHD knockout mice had (n = 4-6 mice per group, relative kidney/body weight ratios, mean = 4.64% vs 12.2%, difference = 7.6%, 95% confidence interval = 5.2% to 10.0%; P < .001) and longer median survival time (n = 4-5 mice per group, 41.5 vs 23 days; P = .0065 ). CONCLUSIONS: Homozygous loss of BHD may initiate renal tumorigenesis in the mouse. The conditional BHD knockout mouse may be a useful research model for dissecting multistep kidney carcinogenesis, and rapamycin may be considered as a potential treatment for Birt-Hogg-Dubé syndrome.

Rous sarcoma virus (RSV) integration in vivo: a CA dinucleotide is not required in U3, and RSV linear DNA does not autointegrate.

The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3' end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5' end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5' end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo.

A covalent inhibitor targeting an intermediate conformation of the fusogenic subunit of the HIV-1 envelope complex.

Peptide inhibitors corresponding to sequences in the six helix bundle structure of the fusogenic portion (gp41) of the HIV envelope glycoprotein have been successfully implemented in preventing HIV entry. These peptides bind to regions in HIV gp41 transiently exposed during the fusion reaction. In an effort to improve upon these entry inhibitors, we have successfully designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to facilitate covalent attachment. Using a temperature-arrested state prime wash in vitro assay we show evidence for the trapping of a pre-six helix bundle fusion intermediate by a covalent reaction with the specific anti-HIV-1 peptide. This is the first demonstration of the trapping of an intermediate conformation of a viral envelope glycoprotein during the fusion process that occurs in live cells. The permanent specific attachment of the covalent inhibitor is projected to improve the pharmacokinetics of administration in vivo and thereby improve the long-term sustainability of peptide entry inhibitor therapy and help to expand its applicability beyond salvage therapy.

Replicating Ad-recombinants encoding non-myristoylated rather than wild-type HIV Nef elicit enhanced cellular immunity.

OBJECTIVE: To determine if immunization with non-myristoylated nef would elicit enhanced cellular immune responses resulting from improved presentation of Nef peptides by MHC-I on the cell surface, and enhanced T-cell help. DESIGN: The myristoylation site of HIV and SIV Nef is required for several Nef functions that modulate the immune response in an infected host, including downregulation of MHC-I, MHC-II, and CD4, and increased expression of the invariant chain on the cell surface. We constructed replication-competent Ad5- and Ad7-HIV recombinants encoding wild-type nef (nefWT) or a nef mutant (nefNM) lacking 19 amino-terminal amino acids, including the myristoylation site, and sequentially immunized chimpanzees mucosally, first with Ad5-HIVnef recombinants and subsequently with Ad7-HIVnef recombinants. METHODS: Peripheral blood lymphocytes were evaluated over the immunization course for Nef-specific cellular immune responses by interferon (IFN)-gamma ELISPOT and T-cell proliferation assays. Nef-specific CD4 and CD8 memory T cells that produced intracellular IFN-gamma, interleukin-2, and tumor necrosis factor (TNF)-alpha were assessed by flow cytometry. RESULTS: In comparison to immunization with Ad-HIVnefWT, Ad-HIVnefNM elicited statistically significant increases in numbers of IFN-gamma-secreting cells after the Ad7-HIVnefNM immunization and increased T-cell proliferative responses following both Ad5- and Ad7-HIVnefNM immunizations. Nef-specific CD4 and CD8 memory T-cell populations secreting TNF-alpha were also significantly increased in the Ad-HIVnefNM immunization group. CONCLUSIONS: The results support the hypothesis that immunization with Ad-recombinants encoding HIVnefNM rather than HIVnefWT elicits enhanced cellular immunity resulting from improved antigen presentation and greater T-cell help.

Alternate polypurine tracts affect rous sarcoma virus integration in vivo.

When the endogenous polypurine tract (PPT) of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z was replaced with alternate retroviral PPTs, the fraction of unintegrated viral DNA with the normal consensus ends significantly decreased and the retention of part of the PPT significantly increased. If the terminus of the U3 long terminal repeat (LTR) is aberrant, RSV integrase can correctly process and integrate the normal U5 LTR into the host genome. However, the canonical CA is not involved in joining the aberrant U3 LTR to the host DNA, generating either large duplications or deletions of the host sequences instead of the normal 5- or 6-bp duplication.