CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

BACKGROUND: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. METHODS: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). RESULTS: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. CONCLUSIONS: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth.

Background: Though the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. Methods: To characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven Braf V600E /Pten -/- /Cxcr2 -/- and NRas Q61R /INK4a -/- /Cxcr2 -/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in Braf V600E /Pten -/- and NRas Q61R /INK4a -/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). Results: Genetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1 , a key tumor suppressive transcription factor, was the only gene significantly induced with a log 2 fold-change greater than 2 in these three different melanoma models. Conclusions: Here, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

Liver Transplantation: Recipients' Evaluation of Life From the Perspective of Living Donors.

AIM: Liver transplantation affects not only recipients and living donors' lives, but also the nature and quality of their relationship. Moreover, the ways in which recipients of liver transplant experience life and views of living donors on how recipients experience life may differ. These differences may account for relational changes. It is also important to understand how recipients and their living donors' views differ if the aim is to devise psychoeducational programs for recipients and living donors. Therefore, the present study examined the recipients' experience of life after a diagnosis of end-stage liver failure (ESLF) and transplantation surgery from donors' perspective. METHODS: The sample consisted of 16 living donors who donated a part of their liver to a patient with ESLF. Thematic analysis was undertaken in parallel with interviews during which an interview guide was followed. FINDINGS: Donors felt that recipients evaluated life after the diagnosis of ESLF and transplantation surgery in terms of limitations, mixed relationships, emotional changes, and improvement in life. CONCLUSION: Experience of social limitations, negative emotions, and the feeling that one is supported by others could be interpreted in terms of existing psychological theory. Some ways of adjusting that have not been reported before within the context of ESLF extended the literature. These included others being frightened of being infected by ESLF and being insensitive, experience of positive emotions, and ways of improving. Overall, compared with findings of previous qualitative work among recipients, our findings suggest that donors' evaluation of recipients' lives converge with that of recipients.

Predictors of onset of depression and anxiety in the year after diagnosis of breast cancer.

BACKGROUND: Depression and anxiety are common after diagnosis of breast cancer. We examined to what extent these are recurrences of previous disorder and, controlling for this, whether shame, self-blame and low social support after diagnosis predicted onset of depression and anxiety subsequently. METHOD: Women with primary breast cancer who had been treated surgically self-reported shame, self-blame, social support and emotional distress post-operatively. Psychiatric interview 12 months later identified those with adult lifetime episodes of major depression (MD) or generalized anxiety disorder (GAD) before diagnosis and onset over the subsequent year. Statistical analysis examined predictors of each disorder in that year. RESULTS: Of the patients, two-thirds with episodes of MD and 40% with episodes of GAD during the year after diagnosis were experiencing recurrence of previous disorder. Although low social support, self-blame and shame were each associated with both MD and GAD after diagnosis, they did not mediate the relationship of disorder after diagnosis with previous disorder. Low social support, but not shame or self-blame, predicted recurrence after controlling for previous disorder. CONCLUSIONS: Anxiety and depression during the first year after diagnosis of breast cancer are often the recurrence of previous disorder. In predicting disorder following diagnosis, self-blame and shame are merely markers of previous disorder. Low social support is an independent predictor and therefore may have a causal role.

A comparison of treatment outcomes for moxifloxacin versus ofloxacin/metronidazole for first-line treatment of uncomplicated non-gonococcal pelvic inflammatory disease.

Large randomized controlled trials support the efficacy of moxifloxacin for the treatment of uncomplicated pelvic inflammatory disease (PID). This study compares the clinical outcome and tolerability of treatment with moxifloxacin 400 mg once a day or ofloxacin 400 mg plus metronidazole 400 mg both twice daily in patients diagnosed with PID. A retrospective case-notes review was performed on patients diagnosed clinically with PID before and after local guidelines were changed to recommend moxifloxacin as first-line treatment for uncomplicated PID. Before the guidelines changed, 114/134 (85%) patients received the recommended first-line therapy versus 206/257 (80%) after the change, P = 0.3. There was no difference in the clinical outcomes between the two groups; significant improvement/resolved 77% versus 70%; marginal improvement 3% versus 11%; no change/worse 20% versus 18%, P = 0.14. Moxifloxacin is confirmed to be an effective alternative to ofloxacin/metronidazole for the treatment of PID in a large urban genitourinary clinic setting.

Preferences for partner notification method: variation in responses between respondents as index patients and contacts.

There have been very few studies focusing on what form of communication patients would find acceptable from a clinic. This study looks at the differences in preferences for various partner notification methods when the respondents were index patients compared with when they had to be contacted because a partner had a sexually transmitted infection (STI). There were 2544 respondents. When the clinic had to notify partners, respondents were more likely to report the method as good when a partner had an STI and they were being contacted compared with when the respondents had an infection and the partner was being contacted. The opposite was true for patient referral partner notification. Therefore, there are variations in the preferences of respondents for partner notification method, which depend on whether they see themselves as index patients or contacts.

Patient preferences for partner notification.

OBJECTIVE: To identify patient preferences for notification of sexual contacts when a sexually transmitted infection (STI) is diagnosed. METHODS: A questionnaire survey of 2544 patients attending three large genitourinary clinics at Derby, Birmingham, and Coventry in the United Kingdom. RESULTS: The median age of the respondents was 24 with 1474 (57.9%) women, 1835 (72.1%) white, 1826 (71.8%) single. The most favoured method of partner notification was patient referral, which was rated a "good" method by 65.8% when they had to be contacted because a sexual partner has an STI. Notifying contacts by letter as a method of provider partner notification is more acceptable than phoning, text messaging, or email. Respondents with access to mobile telephones, private emails, and private letters were more likely to rate a method of partner notification using that mode of communication as "good" compared to those without. With provider referral methods of partner notification respondents preferred to receive a letter, email, or text message asking them to contact the clinic rather than a letter, email or text message informing them that they may have an STI. CONCLUSION: Most respondents think that being informed directly by a partner is the best method of being notified of the risk of an STI. Some of the newer methods may not be acceptable to all but a significant minority of respondents prefer these methods of partner notification. The wording of letters, emails, or text messages when used for partner notification has an influence on the acceptability of the method and may influence success of the partner notification method. Services should be flexible enough to utilise the patients' preferred method of partner notification.

Assessment of signal transducer and activator of transcription 6 as a target of glucocorticoid action in human airway epithelial cells.

BACKGROUND: Activation of signal transducer and activator of transcription (STAT)6 by IL-4 and IL-13 is essential in many key epithelial responses in the asthmatic airway including expression of numerous chemokines, goblet cell differentiation and mucus production and expression of other allergic inflammatory genes. While these responses are all inhibited by glucocorticoids (GC) administered systemically or by inhalation, the inhibitory mechanisms are unknown. OBJECTIVE: To test the hypothesis that GC suppress allergic responses by blocking IL-4-induced STAT6 signalling in airway epithelial cells. METHODS: Western blotting and reporter gene assays were used to determine whether GC could inhibit STAT6 production, phosphorylation or nuclear translocation, or whether GC could affect STAT6 transcriptional activity in the BEAS-2B airway epithelial cell line. RESULTS: Our results showed that GC had no inhibitory effect on the total cellular or nuclear levels of STAT6 or phospho-STAT6. GC did not inhibit transcription from three different STAT6-driven reporter constructs, indicating that GC also did not inhibit STAT6 function. CONCLUSION: We conclude that airway epithelial STAT6 is not the central target of GC in allergic inflammation and that the inhibitory effect of GC on STAT6-mediated IL-4- and IL-13-induced responses is exerted by targeting pathways distinct from STAT6.

Is telephone follow-up as good as traditional clinic follow-up in achieving the proposed national outcome standards for chlamydia management?

A retrospective review of 400 cases of genital chlamydia managed with a traditional clinic follow-up was compared to 400 cases with a telephone follow-up appointment. We satisfactorily treated more patients with the telephone follow-up appointment than with a traditional clinic follow-up (204 [51%] vs 121 [30%]; P <0.0001). We also satisfactorily treated more partners with the telephone follow-up system than a traditional clinic appointment system (0.57 vs 0.45 contacts per case; P =0.0006). The introduction of the telephone follow-up appointment system in the clinic increased the number of patients and contacts of patients successfully managed for genital chlamydial infection. Our findings should lead to increased research and adoption of different methods of follow-up and help develop proper outcome standards.

Antiapoptotic function of NF-kappaB in T lymphocytes is influenced by their differentiation status: roles of Fas, c-FLIP, and Bcl-xL.

Inducible protection from apoptosis in vivo controls the size of cell populations. An important question in this respect is how differentiation affects mechanisms of apoptosis regulation. Among mature T lymphocytes, the NF-kappaB/Rel transcription factors are coupled to receptors that control cell population sizes by concurrently regulating survival and multiplication. In the present study, we used a transgenic inhibitor of NF-kappaB/Rel signaling to investigate the role of this pathway in proliferation and death of mature T cells in vivo. The results indicate that NF-kappaB integrates two critical yet distinct molecular pathways preventing apoptosis affected by the death receptor Fas, coordinately regulating levels of FLIP and Bcl-x(L) in primary T cells. Surprisingly, NF-kappaB blockade preferentially impacted naive as compared to memory T cells. The Fas/FasL pathway was linked to these findings by evidence that the abnormalities imposed by NF-kappaB inhibition were ameliorated by Fas deficiency, particularly for the CD4(+) lineage. Moreover, levels of an inhibitor of Fas-mediated apoptosis, c-FLIP, were diminished in cells expressing the transgenic inhibitor. NF-kappaB was also linked to T cell survival in vivo by mediating induction of Bcl-x(L): restoration of Bcl-x(L) levels reversed the preferential deficit of naive T cells, differentially impacting the CD4 and CD8 subsets. These results show that promoting survival and effective multiplication are central roles for NF-kappaB in T lymphoid homeostasis in vivo, but this effect and its underlying mechanisms are influenced by the developmental state of the lymphocyte.

Inefficient ZAP-70 phosphorylation and decreased thymic selection in vivo result from inhibition of NF-kappaB/Rel.

Signaling from the TCR regulates T lymphoid survival, deletion by apoptosis, and selective clonal expansion. One set of signaling pathways activated during thymic selection leads to degradation of a cytosolic retention protein, the inhibitor of kappaB (IkappaB)alpha, followed by nuclear translocation of the NF-kappaB/Rel family of transcription factors. It has been found previously that NF-kappaB proteins mediate a pathway signaling the survival of mature T cells and protection of thymocytes against TNF-induced apoptosis. In contrast, we show in this study that a transgenic inhibitor of NF-kappaB/Rel signaling interferes with the negative selection of immature thymocytes by endogenous MHC ligands in vivo. Positive selection of the H-Y TCR also was diminished. This attenuation of thymic selection efficiency was associated with decreased ZAP-70 phosphorylation and TCR signaling of CD69 induction. These findings demonstrate that the NF-kappaB transcriptional pathway plays an important role in normal processes of clonal deletion and they indicate that the NF-kappaB/IkappaB axis can regulate the efficiency of TCR signaling.

Inhibition of NF-kappaB-dependent T cell activation abrogates acute allograft rejection.

Using a heterotopic model of transplantation, we investigated the role of T cell activation in vivo during allograft rejection in I-kappaB(DeltaN)-transgenic mice that express a transdominant inhibitor of NF-kappaB in T cells. Our results show indefinite prolongation of graft survival in the I-kappaB(DeltaN)-transgenic recipients. Interestingly, at the time of rejection of grafts in wild-type recipients, histology of grafts in the I-kappaB(DeltaN)-transgenic recipients showed moderate rejection; nevertheless, grafts in the I-kappaB(DeltaN) recipients survived >100 days. Analysis of acute phase cytokines, chemokine, chemokine receptors, and immune responses shows that the blockade of NF-kappaB activation in T cells inhibits up-regulation of many of these parameters. Interestingly, our data also suggest that the T cell component of the immune response exerted positive feedback regulation on the expression of multiple chemokines that are produced predominantly by non-T cells. In conclusion, our studies indicate NF-kappaB activation in T cells is necessary for acute allograft rejection.

NF-kappa B activation plays an important role in the IL-4-induced protection from apoptosis.

IL-4 alone protects cells from apoptosis by insulin receptor substrate (IRS)-dependent and -independent mechanisms. However, in vivo cells are typically exposed to a number of signals at the same time. To determine the contribution of co-stimulatory signals to the regulation of apoptosis by IL-4, we first analyzed whether tumor necrosis factor (TNF)-alpha, which has been shown to inhibit the activation of IRS-1 by insulin, could modify IL-4 signaling and protection from apoptosis. We found that TNF-alpha cooperates with IL-4 in protecting 32D cells from factor withdrawal-induced apoptosis. This effect was independent of the expression of IRS-1, indicating that this cooperation is via an alternative anti-apoptotic pathway. Moreover, TNF-alpha had no effect on the activation of IRS-1 induced by IL-4. IL-4 enhanced TNF-alpha-induced activation of the transcription factor NF-kappaB. Interestingly, pharmacologic inhibition of NF-kappaB activation or protein synthesis resulted in the induction of cell death that could not be inhibited by IL-4, suggesting that IL-4 cooperates with NF-kappaB to signal protection from apoptosis. Supporting this hypothesis, IL-4 also increased NF-kappaB activation induced by anti-CD3 antibodies in primary T cells and protected them from apoptosis induced by receptor engagement. However, IL-4 was not able to inhibit apoptosis induced by anti-CD3 in T lymphocytes isolated from transgenic mice expressing a dominant-negative form of IkappaBalpha that prevents NF-kappaB activation. Thus, in addition to the previously identified IRS-1 pathway, IL-4-induced protection from apoptosis may also be mediated through cooperation with the NF-kappaB family of transcription factors.

IL-4 signaling, gene transcription regulation, and the control of effector T cells.

The central goal of our laboratory is to understand the regulation of lymphoid cells through molecular mechanisms of signal transduction and transcriptional control. A long-standing focus has been on changes that influence the effector function of mature lymphocytes. Work in the laboratory is oriented toward the identification of new regulatory mechanisms using cell lines and primary cells, and the validation of these in vitro findings in mouse models of immune responses and diseases. In this review, we summarize key insights into the regulation of T helper cell function during the phase of immunity where effector responses arise de novo. Particular interest has been centered on cytokine gene regulation as part of T cell differentiation into the Th1 and Th2 subsets. Information on IL-4 receptor signaling and the role of NF-kappaB transcription factors is reviewed. Our more recent work is designed to understand how regulation at the Th1/2 effector stages is related to the control of memory T cell survival, immune recall responses, and the role of these responses in immune-mediated disease.

Redirection of T cell effector function in vivo and enhanced collagen-induced arthritis mediated by an IL-2R beta/IL-4R alpha chimeric cytokine receptor transgene.

Chronic inflammatory autoimmune diseases such as diabetes, experimental autoimmune encephalomyelitis, and collagen-induced arthritis (CIA) are associated with type 1 (Th1, Tc1) T cell-dependent responses against autoantigens. Immune deviation toward type 2 (Th2, Tc2) response has been proposed as a potential means of gene therapy or immunomodulation to treat autoimmune diseases based on evidence that type 2 cytokines can prevent or alleviate these conditions. In this report we assessed the effects of elevated type 2 responses on CIA using transgenic mice expressing an IL-2R beta/IL-4R alpha chimeric cytokine receptor transgene specifically in T cells. In response to IL-2 binding, this chimeric receptor transduces IL-4-specific signals and dramatically enhances type 2 responses. In contrast to published reports of Th2-mediated protection, CIA was exacerbated in IL-2R beta/IL-4R alpha chimeric receptor transgenic mice, with increased disease incidence, severity, and earlier disease onset. The aggravated disease in transgenic mice was associated with an increase in type 2 cytokines (IL-4, IL-5, IL-10) and an increase in collagen-specific IgG1 levels. However, IFN-gamma production is not affected significantly in the induction phase of the disease. There is also an extensive eosinophilic infiltration in the arthritic joints of the transgenic animal, suggesting a direct contribution of type 2 response to joint inflammation. Taken together, our findings provide novel evidence that enhancement of a polyclonal type 2 response in immunocompetent hosts may exacerbate an autoimmune disease such as CIA, rather than serving a protective role. This finding raises significant caution with regard to the potential use of therapeutic approaches based on immune deviation toward type 2 responses.

NF-kappa B/Rel participation in the lymphokine-dependent proliferation of T lymphoid cells.

Proliferative responses of lymphoid cells to IL-2 and IL-4 depend on activation of the cells, but the mechanism(s) by which activation enhances cellular competence to respond to cytokines is not fully understood. The NF-kappaB/Rel family represents one signal transduction pathway induced during such activation. We show in this study that inhibition of NF-kappaB through the expression of an IkappaBalpha (inhibitory protein that dissociates from NF-kappaB) mutant refractory to signal-induced degradation (IkappaBalpha(DeltaN)) interfered with the acquisition of competence to proliferate in response to IL-4 as well as IL-2. Thymocytes and T cells from IkappaBalpha(DeltaN) transgenic mice expressed normal levels of IL-2R subunits. However, transgenic cells exhibited a dramatic defect in Stat5A activation treatment with IL-2, and a similar defect was observed for IL-4-induced Stat5. In contrast, T lymphoid cells with inhibition of NF-kappaB showed normal insulin receptor substrate-2 phosphorylation and only a modest decrease in Stat6 activation and insulin receptor substrate-1 phosphorylation after IL-4 stimulation. These results indicate that the NF-kappaB/Rel/IkappaBalpha system can regulate cytokine receptor capacitation through effects on the induction of downstream signaling by the Stat transcription factor family.

Lymphokine-dependent proliferation of T-lymphoid cells: regulated responsiveness and role in vivo.

The discovery of lymphokines stemmed from their ability to promote T-lymphocyte proliferation in vitro. Even after 20 years of intensive investigation, crucial aspects remain to be clarified about the role of specific lymphokines in T-cell proliferation and the biochemical mechanisms by which they play these roles, particularly in vivo. The present review focuses on conventional populations of TCR(alpha)beta T cells. Older findings and new insights into the function of specific lymphokines in T-lymphocyte proliferation in vivo are summarized along with unanswered questions raised by these observations. Vital contributions of lymphokines to clonal proliferation arise from two processes: the protection of cells against apoptosis and the activation of cell cycling. Findings are underscored indicating that the activity of a particular lymphokine depends on the subset of T cells (CD4 vs. CD8; naive vs. memory) to which it binds, and that point to potential pitfalls of extrapolating from tissue culture-adapted models to the regulation of T cells in vivo. After summaries of signaling mechanisms related to the proliferative activity of lymphokines, recent findings are highlighted suggesting that such signaling is a regulated and plastic process rather than one fixed schema of action.

IL-4-dependent induction of BCL-2 and BCL-X(L)IN activated T lymphocytes through a STAT6- and pi 3-kinase-independent pathway.

Both B and T lymphocytes require ongoing signals to maintain their viability. The pleiotropic cytokine interleukin (IL-) 4 plays an important role in the maintenance of activated T cells, perhaps reflecting induction of the anti-apoptotic genes Bcl-2 and Bcl-X(L). However, it is not known which of the signalling pathways known to link the IL-4 receptor with transcription regulation are required, or if the levels of Bcl-2/X induction under such physiologic conditions are sufficient to account for the anti-apoptotic effects of IL-4. We report here that although blockade of pathways (PI 3-kinase and pp70 S6 kinase) recruited by the IRS-1/2 adaptor proteins inhibited the anti-apoptotic function of IL-4, Bcl-2/X induction were normal. These findings were recapitulated in primary and culture-adapted T cells whose Stat6 signalling pathway also was defective. These results demonstrate that both the Stat6 and PI 3-kinase pathways can be dispensable for Bcl-2/X induction by IL-4, thus suggesting the involvement of an additional signal transduction pathway. Moreover, the preservation of Bcl-2/X induction despite inhibition of the anti-apoptotic function of IL-4 indicates that this cytokine activates additional protective mechanisms.

The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis.

BACKGROUND: The Fas ligand/Fas receptor (FasL/Fas) system is an important mediator of apoptosis in the immune system where the juxtaposition of cells expressing the cell-surface ligand induces the apoptotic pathway in Fas-expressing lymphocytes. The FasL/Fas system has also been shown to be involved in apoptosis in epithelial tissues, including the involuting rodent prostate. FasL can be shed through the action of an hitherto unidentified metalloproteinase to yield soluble FasL (sFasL), although the biological activity of sFasL has been disputed. RESULTS: Here we report that the matrix metalloproteinase matrilysin can process recombinant and cell-associated FasL to sFasL, and that matrilysin-generated sFasL was effective at inducing apoptosis in a target epithelial cell population. In the involuting mouse prostate, FasL and matrilysin colocalized to the cell surface in a restricted population of epithelial cells. Mice deficient in matrilysin demonstrated a 67% reduction in the apoptotic index in the involuting prostate compared with wild-type animals, implicating matrilysin in this FasL-mediated process. CONCLUSIONS: The results show that a functional form of sFasL was generated by the action of the metalloproteinase matrilysin, and suggest that matrilysin cleavage of FasL is an important mediator of epithelial cell apoptosis.