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Despite enormous progress in the understanding and treatment of disease during the 20th century, the amount of care individuals receive from health professionals is arguably less than in previous decades. Being in the presence of caring people who practised human caring has always been the bedrock of services to individuals who were ill. With the rise of scientific positivism in the mid-19th century, traditional ways of caring for sick people, not susceptible to scientific investigation and intervention, were either abandoned or discouraged. The spread of outcome-orientated health services has led to care being redefined as the provision of the finest form of treatment that is financially viable. The spectre of a service in which the human dimension of caring is either prescribed or seen as invalid gives cause for concern. This paper argues for urgent re-examination of what we understand by 'care'. [ABSTRACT FROM AUTHOR]

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations., (© 2024. The Author(s).)

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

Estrogen receptor positive (ER + ) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER + BCa cells and is a novel, tractable therapeutic target for the treatment of ER + BCa patients. Analyses of publicly available clinical datasets indicate that ER + BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER + BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER + BCa cells, implicating a growth dependency of ER + BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER + breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER + breast cancer signaling and growth that suggests ER + BCa patients may benefit from SEMA3C-targeted therapy.

Objectives: The effectiveness of mindfulness-based programs (MBPs) has been established in many randomized controlled trials. However, effect sizes are often modest, and there remains ample scope to improve their effectiveness. One approach to this challenge is to offer a "follow-on" course to people who have completed an MBP and are interested in further skill development. We developed and tested a new 8-week course for this purpose based on awareness of feeling tone ( vedanā ), an understudied aspect of mindfulness in many current MBPs, incorporating new developments in neuroscience and trauma sensitivity. We examined its effectiveness and the frequency and severity of unpleasant experience and harm., Methods: In an open trial, 83 participants, 78 of whom had previously taken part in an MBP (majority MBSR or MBCT), completed the program in nine groups. Participants completed questionnaires before and after and gave qualitative written feedback at completion., Results: Participants reported significantly reduced depression ( d  = 0.56), stress ( d  = 0.36), and anxiety ( d  = 0.53) and increased well-being ( d  = 0.54) and mindfulness ( d  = 0.65) with 38% meeting criteria for reliable change on anxiety and depression. As expected, about three-quarters of participants reported some unpleasant experiences associated with mindfulness practice during the course, but none reported harm. Five participants showed "reliable deterioration" (an increase) in either depression or anxiety, but four of these five also gave anonymous qualitative feedback describing benefits of the course., Conclusions: Findings support the added value of a follow-on course based on the exploration of feeling tone for participants who have a range of previous mindfulness experience., Supplementary Information: The online version contains supplementary material available at 10.1007/s12671-022-01929-0., Competing Interests: Conflict of InterestMark Williams, Chris Cullen, and Ruth Baer are affiliated with the Oxford Mindfulness Centre and receive occasional payments for training workshops and presentations related to mindfulness. Ruth Baer and Chris Cullen both have part-time employment with the Oxford Mindfulness Centre. Mark Williams and Ruth Baer also receive royalties for several books related to mindfulness. At the outset of the study, Laura Taylor, Emma Medlicott, Kath De Wilde, and Lucy Radley were affiliated with the Oxford Mindfulness Centre and funded by the Wellcome Trust on a strategic award exploring the role of mindfulness training in adolescence. They do not receive additional remuneration for training workshops or presentations related to mindfulness. Barbara Gerber, Mandy Johnson, Linda Kantor, Janine Kirby, and Matthew Watkin are affiliated to the Institute of Mindfulness South Africa and receive payment as mindfulness teachers as well as occasional payments for training workshops and presentations related to mindfulness. Melanie Fennell was a former member of The Department of Psychiatry at the University of Oxford and associated with the Oxford Mindfulness Centre. She receives occasional payments for training workshops and presentations related to mindfulness, and royalties from a book related to mindfulness. Helen Ma was formerly Director of the Hong Kong Mindfulness Centre and is associated with the Oxford Mindfulness Centre as their China Advisor. Ee-Lin Ong and Andy Phee are affiliated to the Oxford Mindfulness Centre as Teaching Partners, and receive payment as mindfulness teachers as well as occasional payments for training workshops and presentations related to mindfulness. Rebecca Crane is Director of the Bangor University Centre for Mindfulness Research and Practice, School of Psychology, and receives royalties from two books related to mindfulness. Danny Penman is an independent scholar and author and receives occasional payments for training workshops and presentations related to mindfulness. He also receives royalties for several books related to mindfulness. John Peacock and Martine Batchelor receive occasional payments for retreats, trainings, and presentations related to mindfulness and Buddhist psychology., (© The Author(s) 2022.)

Breast milk HIV-1 transmission is currently the predominant contributor to pediatric HIV infections. Yet, only ~10% of breastfeeding infants born to untreated HIV-infected mothers become infected. This study assessed the protective capacity of natural HIV envelope-specific antibodies isolated from the milk of HIV-infected women in an infant rhesus monkey (RM), tier 2 SHIV oral challenge model. To mimic placental and milk maternal antibody transfer, infant RMs were i.v. infused and orally treated at the time of challenge with a single weakly neutralizing milk monoclonal antibody (mAb), a tri-mAb cocktail with weakly neutralizing and ADCC functionalities, or an anti-influenza control mAb. Of these groups, the fewest tri-mAb-treated infants had SHIV detectable in plasma or tissues (2/6, 5/6, and 7/8 animals infected in tri-mAb, single-mAb, and control-mAb groups, respectively). Tri-mAb-treated infants demonstrated significantly fewer plasma transmitted/founder variants and reduced peripheral CD4+ T cell proviral loads at 8 weeks post-challenge compared to control mAb-treated infants. Abortive infection was observed as detectable CD4+ T cell provirus in non-viremic control mAb- and single mAb-, but not in tri-mAb-treated animals. These results suggest that polyfunctional milk antibodies contribute to the natural inefficiency of HIV-1 transmission through breastfeeding and infant vaccinations eliciting non-neutralizing antibody responses could reduce postnatal HIV transmission.

Background: Fungal endocarditis is a rare and lethal cardiac infection which most commonly presents in immunocompromised patients or patients with other predisposing conditions. In a small subset of these patients, lesions present as mural masses and do not have any involvement with native valves or implanted devices. Here we present one such case which was diagnosed in the antemortem period in time to be managed with surgical resection., Case Presentation: A 70 year-old female patient who presented with multiple cerebral abscesses and was found on echocardiography to have a mass along the inferior wall of the left ventricle. She underwent surgical resection which revealed an Aspergillus vegetation along the left ventricle wall without any involvement of the cardiac valves. An intraoperative photograph was obtained and is presented in this case. The patient was started on antifungal therapy and expired on day 30 of treatment., Conclusions: Fungal endocarditis is a rare yet lethal disease. It can be difficult to detect and workup should be initiated immediately if there is any clinical suspicion. This is especially true in any patient with predisposing conditions or any patient who presents with undiagnosed, culture-negative fevers or evidence of embolic foci. Once diagnosis is made, early initiation of antifungal therapy coupled with aggressive surgical debridement is required for any significant chance of survival.

Despite the amenability of early-stage prostate cancer to surgery and radiation therapy, locally advanced and metastatic prostate cancer is clinically problematic. Chemical castration is often used as a first-line therapy for advanced disease, but progression to the castration-resistant prostate cancer phase occurs with dependable frequency, largely through mutations to the androgen receptor (AR), aberrant AR signaling, and AR-independent mechanisms, among other causes. Semaphorin 3C (SEMA3C) is a secreted signaling protein that is essential for cardiac and neuronal development and has been shown to be regulated by the AR, to drive epithelial-to-mesenchymal transition and stem features in prostate cells, to activate receptor tyrosine kinases, and to promote cancer progression. Given that SEMA3C is linked to several key aspects of prostate cancer progression, we set out to explore SEMA3C inhibition by small molecules as a prospective cancer therapy. A homology-based SEMA3C protein structure was created, and its interaction with the neuropilin (NRP)-1 receptor was modeled to guide the development of the corresponding disrupting compounds. Experimental screening of 146 in silico ‒identified molecules from the National Cancer Institute library led to the discovery of four promising candidates that effectively bind to SEMA3C, inhibit its association with NRP1, and attenuate prostate cancer growth. These findings provide proof of concept for the feasibility of inhibiting SEMA3C with small molecules as a therapeutic approach for prostate cancer.

Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition (EMT) and stem-like phenotypes in prostate cells. In the present study we show that ectopic expression of SEMA3C in RWPE-1 promotes the upregulation of EMT and stem markers, heightened sphere-formation, and cell plasticity. In addition, we show that SEMA3C promotes migration and invasion in vitro and cell dissemination in vivo.

The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is central to prostate cancer (PCa) progression. Ligand-activated AR engages androgen response elements (AREs) at androgen-responsive genes to drive the expression of gene batteries involved in cell proliferation and cell fate. Understanding the transcriptional targets of the AR has become critical in apprehending the mechanisms driving treatment-resistant stages of PCa. Although AR transcription regulation has been extensively studied, the signaling networks downstream of AR are incompletely described. Semaphorin 3C (SEMA3C) is a secreted signaling protein with roles in nervous system and cardiac development but can also drive cellular growth and invasive characteristics in multiple cancers including PCa. Despite numerous findings that implicate SEMA3C in cancer progression, regulatory mechanisms governing its expression remain largely unknown. Here we identify and characterize an androgen response element within the SEMA3C locus. Using the AR-positive LNCaP PCa cell line, we show that SEMA3C expression is driven by AR through this element and that AR-mediated expression of SEMA3C is dependent on the transcription factor GATA2. SEMA3C has been shown to promote cellular growth in certain cell types so implicit to our findings is the discovery of direct regulation of a growth factor by AR. We also show that FOXA1 is a negative regulator of SEMA3C. These findings identify SEMA3C as a novel target of AR, GATA2, and FOXA1 and expand our understanding of semaphorin signaling and cancer biology.

Heat shock protein 27 (Hsp27) is emerging as a promising therapeutic target for treatment of various cancers. Although the role of Hsp27 in protection from stress-induced intrinsic cell death has been relatively well studied, its role in Fas (death domain containing member of the tumor necrosis factor receptor superfamily)-induced apoptosis and cell proliferation remains underappreciated. Here, we show that Hsp27 silencing induces dual coordinated effects, resulting in inhibition of cell proliferation and sensitization of cells to Fas-induced apoptosis through regulation of PEA-15 (15-kDa phospho-enriched protein in astrocytes). We demonstrate that Hsp27 silencing suppresses proliferation by causing PEA-15 to bind and sequester extracellular signal-regulated kinase (ERK), resulting in reduced translocation of ERK to the nucleus. Concurrently, Hsp27 silencing promotes Fas-induced apoptosis by inducing PEA-15 to release Fas-associating protein with a novel death domain (FADD), thus allowing FADD to participate in death receptor signaling. Conversely, Hsp27 overexpression promotes cell proliferation and suppresses Fas-induced apoptosis. Furthermore, we show that Hsp27 regulation of PEA-15 activity occurs in an Akt-dependent manner. Significantly, Hsp27 silencing in a panel of phosphatase and tensin homolog on chromosome 10 (PTEN) wild-type or null cell lines, and in LNCaP cells that inducibly express PTEN, resulted in selective growth inhibition of PTEN-deficient cancer cells. These data identify a dual coordinated role of Hsp27 in cell proliferation and Fas-induced apoptosis via Akt and PEA-15, and indicate that improved clinical responses to Hsp27-targeted therapy may be achieved by stratifying patient populations based on tumor PTEN expression.

Two distinct biochemical signals are delivered by the CD95/Fas death receptor. The molecular basis for the differential mitochondrially independent (type I) and mitochondrially dependent (type II) Fas apoptosis pathways is unknown. By analyzing 24 Fas-sensitive tumor lines, we now demonstrate that expression/activity of the PTEN tumor suppressor strongly correlates with the distinct Fas signals. PTEN loss-of-function and gain-of-function studies demonstrate the ability to interconvert between type I and type II Fas pathways. Importantly, from analyses of Bcl-2 transgenic Pten(+/-) mice, Pten haploinsufficiency converts Fas-induced apoptosis from a Bcl-2-independent to a Bcl-2-sensitive response in primary thymocytes and activated T lymphocytes. We further show that PTEN influences Fas signaling, at least in part, by regulating PEA-15 phosphorylation and activity that, in turn, regulate the ability of Bcl-2 to suppress Fas-induced apoptosis. Thus, PTEN is a key molecular rheostat that determines whether a cell dies by a mitochondrially independent type I versus a mitochondrially dependent type II apoptotic pathway upon Fas stimulation.

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 T-cell immune responses at mucosal sites. We have constructed recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing an HIV-1 group M consensus envelope (Env) either as a surface, intracellular, or secreted protein as an immunogen. rBCG containing HIV-1 env plasmids engineered for secretion induced optimal Env-specific T-cell gamma interferon enzyme-linked immunospot responses in murine spleen, female reproductive tract, and lungs. While rBCG-induced T-cell responses to HIV-1 envelope in spleen were lower than those induced by adenovirus prime/recombinant vaccinia virus (rAd-rVV) boost, rBCG induced comparable responses to rAd-rVV immunization in the female reproductive tract and lungs. T-cell responses induced by rBCG were primarily CD4(+), although rBCG alone did not induce anti-HIV-1 antibody. However, rBCG could prime for a protein boost by HIV-1 envelope protein. Thus, rBCG can serve as a vector for induction of anti-HIV-1 consensus Env cellular responses at mucosal sites.

Aims/hypothesis: Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake., Materials and Methods: Insulin sensitivity in Pten heterozygous (Pten(+/-)) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten(+/-) mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3beta (GSK3beta), a substrate of PKB/Akt, was determined by western immunoblotting., Results: Following i.p. insulin challenge, blood glucose levels in Pten(+/-) mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten(+/-) mice. Enhanced glucose uptake was observed both in Pten(+/-) myocytes and in skeletal muscle of Pten(+/-) mice by PET. PKB and GSK3beta phosphorylation was enhanced and prolonged in Pten(+/-) myocytes., Conclusions/interpretation: Pten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten(+/-) mice.

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 immune responses at mucosal sites. We have generated recombinant Mycobacterium smegmatis vectors that express the HIV-1 group M consensus envelope protein (Env) as a surface, intracellular, or secreted protein and have tested them in animals for induction of both anti-HIV-1 T-cell and antibody responses. Recombinant M. smegmatis engineered for expression of secreted protein induced optimal T-cell gamma interferon enzyme-linked immunospot assay responses to HIV-1 envelope in the spleen, female reproductive tract, and lungs. Unlike with the induction of T-cell responses, priming and boosting with recombinant M. smegmatis did not induce anti-HIV-1 envelope antibody responses, due primarily to insufficient protein expression of the insert. However, immunization with recombinant M. smegmatis expressing HIV-1 Env was able to prime for an HIV-1 Env protein boost for the induction of anti-HIV-1 antibody responses.

Background: Progression to a lethal androgen-independent (AI) stage of advanced prostate cancer is a critical clinical obstacle limiting patient survival. PTEN inactivation is frequently observed in advanced prostate cancer and correlates with a poor prognosis. However, the functional significance of PTEN inactivation in AI progression has not been demonstrated., Methods: PTEN expression was examined in benign, hormone naïve and AI human prostate cancer specimens, and in recurrent AI Shionogi tumors. The effect of antisense oligonucleotide (ASO)-mediated PTEN downregulation in AI progression of the Shionogi tumor model was determined., Results: Significantly reduced PTEN expression was observed in AI versus benign and hormone naïve prostate tumors. Seven of 14 AI Shionogi tumors exhibited marked downregulation or complete loss of PTEN. ASO-mediated PTEN inhibition reduced androgen-withdrawal induced regression of Shionogi tumors and accelerated AI progression., Conclusions: These data suggest that PTEN inactivation may play a role in progression to androgen independence., (Copyright 2006 Wiley-Liss, Inc.)

Immunization by the nasal route is an established method for the induction of mucosal and systemic humoral and cell-mediated antigen-specific responses. However, the effectiveness of nasal immunization is often hampered by the need for increased doses of antigen. Bioadhesives and absorption enhancers were investigated for their ability to enhance immune responses in mice after nasal immunization with model HIV-1 peptide and protein immunogens. Two additives, hydroxypropylmethylcellulose (HPMC) and capric acid, consistently enhanced antigen-specific serum IgG endpoint titers under conditions in which antigen dose was limiting. Nasal immunization of mice with 20 microg of an HIV-1 peptide immunogen plus cholera toxin (CT) as adjuvant induced serum antipeptide IgG titers of 1:9.5log2 after four immunizations while the addition of CA or HPMC to the vaccine formulation increased serum antipeptide IgG titers to 1:15.4log2 and 1:17.6log2, respectively. When 5 microg recombinant HIV-1 gp41 was used as the immunogen, the addition of CA or HPMC to the vaccine formulation increased serum anti-gp41 IgG titers to 1:11.6log2 and 1:8.8log2, respectively, compared to 1:5.2log2 after three nasal immunizations with 5 microg gp41 + CT alone. Thus, HPMC and capric acid may be useful additives that increase the immunogenicity of nasally administered vaccines and permit less antigen to be used with each immunization.

Rationally designed therapeutics that target the phosphatidylinositol 3'-kinase (PI3K) cell survival pathway are currently in preclinical and clinical development for cancer therapy. Drugs targeting the PI3K pathway aim to inhibit proliferation, promote apoptosis, and enhance chemosensitivity and radiosensitivity of cancer cells. The phosphatase and tensin homologue (PTEN) phosphatidylinositol 3'-phosphatase is a key negative regulator of the PI3K pathway. Inactivation of the PTEN tumor suppressor results in constitutive activation of the PI3K pathway and is found in approximately 50% of advanced prostate cancers, which correlates with a high Gleason score and poor prognosis. Inhibition of the PI3K pathway leads to apoptosis of prostate cancer cells; however, the precise mechanism by which this occurs is unknown. Here we report that apoptotic cell death of PTEN-deficient LNCaP and PC3 prostate cancer cells induced by the PI3K inhibitor LY294002 can be abrogated by disrupting Fas/Fas ligand (FasL) interactions with recombinant Fas:Fc fusion protein or FasL neutralizing antibody (Nok-1), or by expressing dominant-negative Fas-associated death domain. Furthermore, we find that apoptosis induced by expression of wild-type PTEN, driven by a tetracycline-inducible expression system in LNCaP cells, can be inhibited by blocking Fas/FasL interaction using Fas:Fc or Nok-1. These data show that apoptosis induced by blockade of the PI3K pathway in prostate tumor cells is mediated by an autocrine Fas/FasL apoptotic mechanism and the Fas apoptotic pathway is both necessary and sufficient to mediate apoptosis by PI3K inhibition.

The transition to flowering occurs at the shoot apex; however, most of the characterized genes that affect the timing of floral induction are expressed throughout the plant. To further our understanding of these genes and the flowering process, the vegetative molecular phenotypes of 16 Arabidopsis mutants associated with the major flowering initiation pathways were assayed using a 13,000 clone microarray under two different conditions that affect flowering. All mutants showed at least one change in gene expression other than the mutant flowering gene. Metabolism- and defence-related pathways were the areas with the most frequent gene expression changes detected in the mutants. Several genes such as EARLI1 were differentially expressed in a number of flowering mutants from different flowering pathways. Analysis of the promoter regions of genes differentially expressed identified common promoter elements, indicating some form of common regulation.

Induction of mucosal anti-human immunodeficiency virus type 1 (HIV-1) T-cell responses in males and females will be important for the development of a successful HIV-1 vaccine. An HIV-1 envelope peptide, DNA plasmid, and recombinant modified vaccinia virus Ankara (rMVA) expressing the H-2D(d)-restricted cytotoxic T lymphocyte P18 epitope were used as immunogens to test for their ability to prime and boost anti-HIV-1 T-cell responses at mucosal and systemic sites in BALB/c mice. We found of all prime-boost combinations tested, an HIV-1 Env peptide subunit mucosal prime followed by systemic (intradermal) boosting with rMVA yielded the maximal induction of gamma interferon (IFN-gamma) spot-forming cells in the female genital tract and colon. However, this mucosal prime-systemic rMVA boost regimen was minimally immunogenic for the induction of genital, colon, or lung anti-HIV-1 T-cell responses in male mice. We determined that a mucosal Env subunit immunization could optimally prime an rMVA boost in female but not male mice, as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tracts, colon, and lung. Defective mucosal priming in male mice could not be overcome by multiple mucosal immunizations. However, rMVA priming followed by an rMVA boost was the optimal prime-boost strategy for male mice as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tract and lung. Thus, prime-boost immunization strategies able to induce mucosal antigen-specific IFN-gamma responses were identified for male and female mice. Understanding the cellular and molecular basis of gender-determined immune responses will be important for optimizing induction of anti-HIV-1 mucosal immune responses in both males and females.

Viral infections have long been suspected to play a role in the pathogenesis of multiple sclerosis. In the present study, two different rodent models of experimental autoimmune encephalomyelitis (EAE) were used to demonstrate the ability of murine gammaherpesvirus-68 (gammaHV-68) to exacerbate development of neurological symptoms. SJL mice received UV-inactivated gammaHV-68 or intranasalgammaHV-68, followed by immunization against proteolipid-protein peptide 139-151. Infected mice became moribund within 10 days post-immunization, whereas mice exposed to UV-inactivated gammaHV-68 recovered. In the second model, Lewis rats were exposed to UV-inactivated gammaHV-68 or to gammaHV-68, followed by passive transfer of encephalitogenic T lymphocytes specific for myelin basic protein. Consistently, infected rats had higher clinical scores, and this result was observed during acute or latent gammaHV-68 infection. It is unlikely that this gammaHV-68-induced exacerbation was due to significant viral replication within the central nervous system since nested PCR, viral plaque assays, and infectious-centers assays demonstrated no detectable virus in spinal cords or brains of infected rodents undergoing EAE. Taken together, these studies demonstrate increased clinical symptoms of EAE in rodents infected by a gammaherpesvirus that has a limited ability to invade the central nervous system.

We evaluated a two-component transposon iAc/Ds system for generating a library of insertional mutants in rice. The constructs used have gene or enhancer trapping properties, plasmid rescue and T-DNA/Ds launching pad reporter facilities. Mutagenic iAc/Ds lines were produced by three methods: crossing iAc and Ds containing lines; co-transformation with iAc and Ds constructs; and super-transformation of iAc transgenic calli with Ds constructs. First and second generation screening populations, derived from crosses (F2 and F3) or double transformation (DtT1 and DtT2), were analysed for stable insertion lines containing Ds transposed to locations unlinked to iAc. The average frequencies of putative stable insertion (PSI) lines in the F2, DtT1, F3 and DtT2 populations were 6.61, 5.58, 11.47 and 7.05% respectively, with large variations in these frequencies in screening populations derived from different mutagenic lines. Further analyses indicated that 41, 33, 65 and 64% of the PSI lines, respectively, have Ds transposed to locations unlinked to the original Ds launching pad. Using the plasmid rescue system, sequences flanking Ds from 137 PSI lines were obtained. Sixty-eight of these lines had unique insertions in genomic regions, of which 18 were known sequences. Because the average frequency of proven stable insertion lines in any of our screening populations has been less than 5%, we suggest that additional features should be incorporated in this two-component iAc/Ds system to increase the screening efficiency, and to make it suitable for large-scale insertional mutagenesis and determination of gene function in rice.

Based on its genomic sequence and its pathogenesis, murine gammaherpesvirus-68 (gammaHV-68) has been established as a tractable model for the study of viral infections caused by the human gammaherpesviruses, Epstein-Barr virus or human herpesvirus-8. Despite significant advances, the mechanisms responsible for gammaHV-68-induced alterations in the protective host response, and the accompanying virus-induced leukocytosis, are not clear. In the present study, we questioned whether viral infection resulted in endogenous interleukin-10 (IL-10) production that might alter the host response. Infection of C57BL/6 mice resulted in increased IL-10 expression, demonstrating that gammaHV-68 could induce endogenous production of this cytokine. Infected C57BL/6 mice demonstrated the characteristic splenomegaly associated with this viral infection, however, we were surprised to discover that the splenomegaly was greater in syngeneic mice genetically deficient in IL-10 (IL-10-/-). These results strongly suggested that endogenously produced IL-10 might serve to limit leukocytosis in wild-type mice. Quantification of viral burden demonstrated a significant elevation in C57BL/6 versus IL-10-/- mice, with increases in virus being observed in both the macrophage and B-lymphocyte populations. The decreased viral load in syngeneic IL-10-/- mice correlated with an increased expression of endogenous IL-12, suggesting a mechanism of protection that was IL-12 dependent. Taken together, these studies demonstrate a surprising dichotomy for endogenous IL-10 production during gammaHV-68 infection. While the lack of IL-10 results in increased IL-12 expression and a lower viral burden, IL-10-/- mice also experience an increased leukocytosis.

Murine gammaherpesvirus-68 (gammaHV-68) induces a lymphocytosis in mice and establishes a latent infection of B lymphocytes following intranasal administration in anaesthetized animals. Because gammaHV-68 is a gammaherpesvirus, it has been used as a model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8) infections. In this study, we investigated the unlikely possibility that gammaHV-68 could survive the harsh gastrointestinal environment to efficiently infect intestinal epithelial cells, and then disseminate from mucosal sites to cause systemic disease. Surprisingly, oral administration, or gastric instillation which by-passed the oral cavity, readily caused a systemic lymphocytosis and established a latent infection in splenic leukocytes. The finding that gammaHV-68 could readily infect adult mice following gastric instillation strongly suggested that intestinal epithelial cells could be productively infected. Unlike the more routinely used method of intranasal inoculation, gammaHV-68 given intragastrically resulted in lytic virus, viral RNA and viral DNA being present in isolated intestinal epithelial cells. Furthermore, gammaHV-68 RNA and DNA, but not latent virus, could be detected in epithelial cells as long as 30 days post-infection, suggesting that some of these cells might be persistently infected. Taken together, these studies demonstrate that gammaHV-68 can survive passage through the gastrointestinal tract and infect intestinal epithelial cells. Following infection of gut epithelial cells, gammaHV-68 can disseminate from mucosal sites to induce a systemic lymphocytosis which is similar to the disease induced following intranasal inoculation.

Stromal cell-derived factor-1 (SDF-1), a C-X-C family chemokine, is a potent T lymphocyte chemoattractant. We investigated the effects of T cell activation on the chemotactic response to SDF-1. Anti-CD3 Ab stimulation of either Jurkat T cells or murine peripheral CD4+ T lymphocytes produced a dramatic inhibition of SDF-1-induced chemotaxis. In contrast, the SDF-1 responses of Jurkat clones with deficiencies in key TCR signaling components (Lck, CD45, and TCR-beta), were only marginally reduced by anti-CD3 stimulation. Similar to PMA treatment, which abolished both CXCR4 receptor expression and the chemotactic response of Jurkat cells to SDF-1, anti-CD3 Ab treatment reduced cell surface expression of CXCR4 to 65% of the control value, an effect that was blocked by protein kinase C inhibitors. Our data suggest that initial T cell activation events inhibit the response of Jurkat T cells to CXCR4 stimulation. In contrast, SDF-1 treatment resulted in a reduction of tyrosine phosphorylation of the TCR downstream effectors, ZAP-70, SLP-76, and LAT (linker for activation of T cells), suggesting that this chemokine potentially regulates the threshold for T cell activation.

The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPalpha, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-alpha. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPalpha expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPalpha-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPalpha in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPalpha, and there was evidence of association between PTPalpha and Src kinase(s). PTPalpha expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125(FAK). In addition, paxillin, a Src and/or pp125(FAK) substrate, displayed increased levels of tyrosine phosphorylation in PTPalpha-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPalpha-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPalpha may have a role in regulating cell-substratum adhesion.

Rat embryo fibroblasts transformed with HPV-16 E7 and the Ha-ras oncogene (ER clones) fall into two distinct groups based on their endogenous p53 status, wild-type or mutant. We have taken advantage of such clones in order to study the p53 target genes by the differential display method of RNA fingerprinting. We have identified a cDNA clone, clone 16, that recognises a large transcript on Northern blots. The clone 16 transcript is overexpressed in ER cell lines that express wild-type p53 compared with ER cell lines that express mutant p53. Similar to the waf1/p21 gene, which is transcriptionally activated in cells treated with ionizing radiation in a p53-dependent manner, the clone 16 transcript was also induced in response to cell irradiation. The sequence of clone 16 exhibits a high homology to two members of RAL retroviral-like elements.

Rat embryo fibroblast clones transformed with the human papillomavirus type 16 E7 gene and the H-ras oncogene (ER clones) fall into two groups on the basis of endogenous p53 genotype, wild type or mutant. We have compared these clones with the aim of indentifying physiological differences that could be attributed to p53 protein function. We show that all ER clones, regardless of p53 gene status, are tumorigenic and metastatic in severe combined immunodeficiency mice. We demonstrate that only the wild-type p53 protein expressed in ER clones is functional on the basis of its site-specific double-stranded DNA-binding activity and its ability to confer a G1 delay on cells following treatment with ionizing radiation. These data indicate that disruption of the p53 growth-regulatory pathway is not a prerequisite for the malignant conversion of rat embryo fibroblasts expressing the E7 gene and mutant ras. Differences in phenotype that were correlated with loss of p53 protein function included the following: serum-independent growth of ER clones in culture, decreased tumor doubling time in vivo, and increased radioresistance. In addition, we demonstrate the p53-dependent G1 checkpoint alone does not determine radiosensitivity.

Rat embryo fibroblasts transformed with the HPV-16 E7 gene and the activated c-H-ras gene fall into two distinct phenotypic classes. At high cell density, clones of one class form colonies in methylcellulose supplemented with low serum; at low cell density, these cells display responsiveness to mitogenic factors present in serum-free conditioned medium from rat embryo fibroblasts. In contrast, clones of the second class exhibit an absolute dependency on growth factors present in serum at all cell densities in the methylcellulose colony assay and fail to respond to conditioned medium. We find that the status of the endogenous p53 gene is tightly correlated with these two classes of clones. Clones of the first class contain missense mutations in the p53 gene and have lost the wild-type allele. Clones of the second class express wild-type p53 protein. The importance of mutant p53 expression in reducing the growth factor dependency of transformed clones was confirmed in a separate series of experiments in which rat embryo fibroblasts were transformed with three genes, E7 + ras + mutant p53. The growth behaviour of these triply transfected clones was similar to that of the E7 + ras clones expressing endogenous mutant p53. We demonstrate that the enhanced proliferation of E7 + ras clones expressing mutant p53 protein involves an autocrine mechanism.

A number of cellular and viral genes encode proteins that play a role in the establishment of normal cells in culture. In addition, these genes cooperate with activated ras genes to induce cellular transformation. We show that ras-dependent transformation of rat embryo fibroblasts is more efficient when two establishment genes are used together compared with one alone. Both quantitative and qualitative differences in the efficiency of transformation were detected. The number of transformed foci generated was greater than the sum of the foci obtained with ras and each of the establishment genes used separately. In addition, the foci had a distinct morphology. Synergism was seen between the HPV-16 E7 gene and certain mutant alleles of the cellular p53 gene as well as between E7 and c-myc.

The Friend erythroleukemia virus complex contains no cell-derived oncogene. Transformation by this virus may therefore involve mutations affecting cellular gene expression. We provide evidence that inactivating mutations of the cellular p53 gene are a common feature in Friend virus-induced malignancy, consistent with an antioncogene role for p53 in this disease. We have shown that frequent rearrangements of the p53 gene cause loss of expression or synthesis of truncated proteins, whereas overexpression of p53 protein is seen in other Friend cell lines. We now demonstrate that p53 expression in the latter cells is also abnormal, as a result of missense mutations in regions encoding highly conserved amino acids. Three of these aberrant alleles obtained from cells from different mice were cloned and found to function as dominant oncogenes in gene transfer assays, supporting the view that certain naturally occurring missense mutations in p53 confer a dominant negative phenotype on the encoded protein.

The principal feeding stimulants for the beetle Scolytus multistriatus Marsham from the twig bark of Ulmus americana L. have been identified as (+)-catechin-5-beta-D-xylopyranoside and lupeyl cerotate.