The strategic combination of trastuzumab emtansine with oncolytic rhabdoviruses leads to therapeutic synergy.

We have demonstrated that microtubule destabilizing agents (MDAs) can sensitize tumors to oncolytic vesicular stomatitis virus (VSVΔ51) in various preclinical models of cancer. The clinically approved T-DM1 (Kadcyla®) is an antibody-drug conjugate consisting of HER2-targeting trastuzumab linked to the potent MDA and maytansine derivative DM1. We reveal that combining T-DM1 with VSVΔ51 leads to increased viral spread and tumor killing in trastuzumab-binding, VSVΔ51-resistant cancer cells. In vivo, co-treatment of VSVΔ51 and T-DM1 increased overall survival in HER2-overexpressing, but trastuzumab-refractory, JIMT1 human breast cancer xenografts compared to monotherapies. Furthermore, viral spread in cultured HER2+ human ovarian cancer patient-derived ascites samples was enhanced by the combination of VSVΔ51 and T-DM1. Our data using the clinically approved Kadcyla® in combination with VSVΔ51 demonstrates proof of concept that targeted delivery of a viral-sensitizing molecule using an antibody-drug conjugate can enhance oncolytic virus activity and provides rationale for translation of this approach.

Dechlorane Plus increases adipogenesis in 3T3-L1 and human primary preadipocytes independent of peroxisome proliferator-activated receptor γ transcriptional activity.

BACKGROUND/OBJECTIVES: Polybrominated diphenyl ethers (PBDEs) are chemicals that were added to consumer products to reduce flammability but were deemed toxic and bioaccumulative and were phased out of commerce. Flame retardants (FRs) such as Dechlorane Plus (DP) were introduced as replacements. DP is being produced in high volumes and is detected in the environment, human milk, and human serum. Although human exposure to DP is evident, little is known about its potential effects on human health. We and others have shown that some FRs are potential obesogens, i.e., promote adipogenesis. However, the effects of DP on adipogenesis are not known. METHODS: Murine 3T3-L1 and human primary subcutaneous (Sc) and omental (Om) preadipocytes were differentiated in the presence of DP (0.001-10 µM) and adipogenic effects were measured. Further, the ability of DP to activate the adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) was also assessed. RESULTS: We show that treatment of murine preadipocytes with DP significantly (p < 0.05) increased lipid accumulation (2.5-fold) and the mRNA expression of adipogenic markers: fatty acid binding protein 4 (Fabp4), lipoprotein lipase (Lpl), perilipin (Plin), adipsin, and adiponectin. DP also significantly (p < 0.05) increased the protein levels of selected mature adipocyte markers. We further show using luciferase reporter assays that DP increased PPARγ transcriptional activity by threefold (p < 0.05). When the PPARγ agonist was replaced by DP in the human preadipocyte differentiation cocktail, DP significantly (p < 0.05) increased the mRNA levels of adipogenic markers, PPARγ, FABP4, and PLIN in human Sc as well as Om cultures. Finally, PPARγ antagonist studies revealed that DP-mediated upregulation of adipogenic markers Fabp4 and Lpl did not occur via PPARγ activation. CONCLUSION: The current study shows that DP can induce adipogenesis of murine and human preadipocytes. We show that, although DP can directly activate PPARγ, its adipogenic effects may be mediated via other pathways.

Elevated Carbohydrate Response Element-Binding Protein Beta (ChREBPß) and Thioredoxin Interacting Protein (TXNIP) Levels in Human Adipocytes Differentiated in High Glucose Concentrations.

OBJECTIVES: Obesity and type 2 diabetes often coexist. The effect of hyperglycemia on adipose tissue is, therefore, of interest. Although studies have shown that high glucose (HG) concentrations do not inhibit adipocyte differentiation, the resulting adipocyte phenotype has not been investigated. In particular, the levels of the glucose-responsive transcription factor carbohydrate-responsive response element binding protein (ChREBP) isoforms have not been assessed. METHODS: Human preadipocytes were differentiated into adipocytes in either normal glucose (NG) or HG conditions. RNA and protein analyses were used to measure the expression of ChREBP isoforms, thioredoxin interacting protein (TXNIP) and lipogenic genes. Insulin-stimulated glucose uptake was measured. RESULTS: HG- vs. NG-differentiated adipocytes expressed more ChREBPß and more TXNIP at the mRNA and protein levels. There was no change in lipogenic gene expression. HG- vs. NG-differentiated adipocytes displayed an inhibition of insulin-stimulated glucose uptake. CONCLUSIONS: HG-differentiated human adipocytes have distinct molecular differences and are insulin resistant. More studies are warranted to investigate potential mechanisms linking changes in ChREBPß and TXNIP to insulin responsiveness.

Thyroid-Stimulating Hormone-Stimulated Human Adipocytes Express Thymic Stromal Lymphopoietin.

When recombinant human (rh) thyroid-stimulating hormone (TSH) is administered to thyroid cancer survivors, an acute extra-thyroidal effect raises pro-inflammatory cytokines and activates platelets. Thymic stromal lymphopoietin (TSLP) is a cytokine recently implicated in platelet activation. Our aim was to measure platelet microparticle levels after rhTSH stimulation in vivo, and to investigate TSLP expression in TSH-stimulated human adipocytes in culture. Blood samples for total and platelet microparticle analysis were obtained from thyroid cancer survivors before (day 1) and after rhTSH administration (day 5). Adipocytes, differentiated from stromal preadipocytes isolated from adipose tissue from surgical patients, were stimulated with TSH. TSLP mRNA expression, protein expression, and protein release into the adipocyte medium were measured. The level of platelet microparticles in thyroid cancer patients rose 5-fold after rhTSH stimulation. TSH upregulated TSLP mRNA expression in adipocytes in culture through a pathway that was inhibited by 66% by H89, a protein kinase A inhibitor. TSLP protein expression rose in response to TSH, and TSH-stimulated TSLP release into the medium was completely blocked by dexamethasone. In conclusion, TSLP is a novel TSH-responsive adipokine. Future studies will be needed to address the potential role of adipocyte-derived TSLP and whether it is linked to TSH-dependent platelet activation.

The anti-adipogenic effect of peripheral blood mononuclear cells is absent with PCSK9 loss-of-function variants.

OBJECTIVE: To determine the effect of (1) an oral fat load and (2) pro-protein convertase subtilisin/kexin type (PCSK) 9 loss-of-function (LOF) variant status on the ability of peripheral blood mononuclear cells (PBMC) to inhibit human adipogenesis. METHODS: PBMC from subjects with one or more PCSK9 LOF variants versus non-variant controls were compared in the fasting state and after an oral fat load. RESULTS: Fasting triglyceride (TG) levels were lower in the LOF variant versus non-variant group but rose to the same level after the oral fat load. Conditioned medium from PBMC was obtained in fasting (PBMC-CM-F) and 4-h postprandial (PBMC-CM-PP) states. PBMC-CM-PP from non-variant controls inhibited adipogenesis of human preadipocytes more than did PBMC-CM-F. In contrast, PBMC-CM-F or -PP from PCSK9 LOF variant subjects had no effect on adipogenesis. After the oral fat load, PBMC from PCSK9 LOF variant subjects showed significant increases in mRNA levels of interleukin-1ß, tumor necrosis factor-α, sterol regulatory element binding protein-1c, CD36, and monocyte chemoattractant protein-1 (MCP-1), only MCP-1 mRNA levels increased in PBMC from non-variant controls. CONCLUSIONS: The absence of anti-adipogenic action of PBMC from PCSK9 LOF variant subjects points to a novel role for PCSK9 in PBMC-adipose cell interactions.

Discovery of bicyclic pyrazoles as class III histone deacetylase SIRT1 and SIRT2 inhibitors.

A series of bicyclic pyrazole carboxamides was synthesized and tested for inhibitory activity against the class III deacetylase sirtuin enzymes. Moderate to low micromolar inhibitory activities were obtained against SIRT1 and SIRT2. These bicyclic pyrazole compounds represent a new class of sirtuin inhibitors with a preference for SIRT1 over SIRT2.

Providing care to people on social assistance: how dentists in Montreal, Canada, respond to organisational, biomedical, and financial challenges.

BACKGROUND: Dentists report facing difficulties and experiencing frustrations with people on social assistance, one of the social groups with the most dental needs. Scientists ignore how they deal with these difficulties and whether they are able to overcome them. Our objective was to understand how dentists deal with critical issues encountered with people on social assistance. METHODS: We conducted in-depth, semi-structured interviews with 33 dentists practicing in Montreal, Canada. The interview guides included questions on dentists' experiences with people on social assistance and potential strategies developed for this group of people. Analyses consisted of interview debriefing, transcript coding, and data interpretation. RESULTS: Dentists described strategies to resolve three critical issues: missed appointments (organisational issue); difficulty in performing non-covered treatments (biomedical issue); and low government fees (financial issue). With respect to missed appointments, dentists developed strategies to maximise attendance, such as motivating their patients, and to minimise the impact of non-attendance, like booking two people at the same time. With respect to biomedical and financial issues, dentists did not find any satisfactory solutions and considered that it was the government's duty to resolve them. Overall, dentists seem reluctant to exclude people on social assistance but develop solutions that may discriminate against them. CONCLUSIONS: The efforts and failures experienced by dentists with people on social assistance should encourage us to rethink how dental services are provided and financed.

How health professionals perceive and experience treating people on social assistance: a qualitative study among dentists in Montreal, Canada.

BACKGROUND: In Canada, the prevalence of oral diseases is very high among people on social assistance. Despite great need for dental treatment, many are reluctant to consult dental professionals, arguing that dentists do not welcome or value poor patients. The objective of this research was thus to better understand how dentists perceived and experienced treating people on social assistance. METHODS: This descriptive qualitative research was based on in-depth semi-structured interviews with 33 dentists practicing in Montreal, Canada. Generally organized in dentists' offices, the interviews lasted 60 to 120 minutes; they were digitally recorded and later transcribed verbatim. The interview transcripts were coded with NVivo software, and data was displayed in analytic matrices. Three members of the research team interpreted the data displayed and wrote the results of this study. RESULTS: Dentists express high levels of frustration with people on social assistance as a consequence of negative experiences that fall into 3 categories: 1) Organizational issues (people on social assistance ostensibly make the organization of appointments and scheduling difficult); 2) Biomedical issues (dentists feel unable to provide them with adequate treatment and fail to improve their oral health); 3) Financial issues (they are not lucrative patients). To explain their stance, dentists blame people on social assistance for neglecting themselves, and the health care system for not providing adequate coverage and fees. Despite dentists' willingness to treat all members of society, an accumulation of frustration leads to feelings of powerlessness and discouragement. CONCLUSIONS: The current situation is unacceptable; we urge public health planners and governmental health agencies to ally themselves with the dental profession in order to implement concrete solutions.

The role of interleukin 1ß in the anti-adipogenic action of macrophages on human preadipocytes.

When adipose tissue accumulates in obesity, the ability of preadipocytes to differentiate permits a hyperplastic expansion of functional adipocytes that preserves insulin sensitivity. Adipose infiltration by macrophages is associated with an adipogenic deficit and the appearance of inflamed, insulin-resistant hypertrophied adipocytes. Interleukin 1ß (IL1ß) has been reported to account for the anti-adipogenic action of macrophages in a mouse model. Using the THP-1 human macrophage cell line and human primary preadipocytes, our objective was to determine whether IL1ß was necessary for the ability of conditioned medium from THP-1 macrophages (THP-1-MacCM) to: i) stimulate human preadipocyte inhibitor of κB kinase ß (IKKß) and ii) inhibit human adipocyte differentiation. IL1ß is present in THP-1-MacCM, and THP-1-MacCM or IL1ß (500 pg/ml; its concentration in THP-1-MacCM) acutely stimulated IKKß phosphorylation and inhibitor of κB (IκB) degradation in preadipocytes. IL1ß was sufficient to inhibit adipogenesis on its own, and this was blocked by SC-514, an IKKß inhibitor, as has been reported for THP-1-MacCM. IκB degradation by IL1ß-immunodepleted THP-1-MacCM was attenuated, whereas IKKß phosphorylation and the inhibition of adipocyte differentiation were unchanged. Therefore, in contrast to what has been suggested for mouse cell models, IL1ß is not required for the ability of MacCM to inhibit adipogenesis in human cell models.

Life course experiences and lay diagnosis explain low-income parents' child dental decisions: a qualitative study.

OBJECTIVE: This study aimed to better understand low-income parents' child dental care decisions through a life course approach that captured parents' experiences within the social context of poverty. METHODS: We conducted 43 qualitative life history interviews with 10 parents, who were long-term social assistance recipients living in Montreal, Canada. Thematic analysis involved interview debriefing, transcript coding, theme identification and data interpretation. RESULTS: Our interviews identified two emergent themes: lay diagnosis and parental oral health management. Parents described a process of 'lay diagnosis' that consisted of examining their children's teeth and interpreting their children's oral signs and symptoms based on their observations. These lay diagnoses were also shaped by their own dental crises, care experiences and oral health knowledge gained across a life course of poverty and dental disadvantage. Parents' management strategies included monitoring and managing their children's oral health themselves or by seeking professional recourse. Parents' management strategies were influenced both by their lay diagnoses and their perceived ability to manage their children's oral health. Parents felt responsible for their children's dental care, empowered to manage their oral health and sometimes forgo dental visits for their children because of their own self-management life history. CONCLUSION: This original approach revealed insights that help to understand why low-income parents may underutilize free dental services. Further research should consider how dental programs can nurture parental empowerment and capitalize on parents' perceived ability to diagnose and manage their children's oral health.

Macrophages alter the differentiation-dependent decreases in fibronectin and collagen I/III protein levels in human preadipocytes.

Adipose tissue of obese individuals is characterized by increased fibrosis and macrophage infiltration. Extensive remodeling of the extracellular matrix (ECM) that occurs during adipogenesis can be influenced by macrophages, but it remains unclear how macrophage-secreted factors alter preadipocyte ECM protein expression under non-adipogenic versus adipogenic conditions. Confluent human subcutaneous abdominal preadipocytes were cultured for 14 days, with or without adipogenic inducers, in either control medium, medium conditioned by THP-1 monocytes (THP-1-MonCM), or medium conditioned by THP-1 macrophages (THP-1-MacCM). Under non-adipogenic conditions in THP-1-MacCM, collagen I/III and fibronectin protein levels rose by 40 and 70 %, respectively (p < 0.05, n = 3; compared to control non-adipogenic medium). When preadipocytes were exposed to adipogenic inducers in THP-1-MacCM, collagen I/III levels increased by 50 %, but those of fibronectin fell by 48 %, both compared to non-adipogenic THP-1-MacCM conditions. The rise in collagen I/III levels contrasts with the 51 % decrease in collagen I/III that occurs with induction of differentiation in control medium, whereas, the decrease in fibronectin is more modest, but consistent in THP-1-MacCM (48 %) and control medium (92 %). A similar effect on fibronectin levels occurred using medium conditioned by LPS-treated human monocyte-derived macrophages (MD-MacCM). Our data indicate macrophage-derived factors regulate levels of collagen I/III and fibronectin in preadipocytes under non-adipogenic and adipogenic conditions. Further studies are needed to determine if these changes in these ECM proteins contribute to the anti-adipogenic action of MacCM.

How do dentists perceive poverty and people on social assistance? A qualitative study conducted in Montreal, Canada.

Despite significant needs, people on social assistance are sometimes reluctant to consult dentists because of previous negative experience and communication barriers. They feel poorly understood by oral health professionals and sometimes complain of being stigmatized. It is thus important to know how dentists perceive poverty and this group of patients. The aim of this study was to understand how dentists perceive poverty and people on social assistance. To investigate this largely unexplored question, a qualitative study was conducted based on in-depth interviews with thirty-three dentists practicing in Montreal, Canada. Interviews were audiotaped and transcribed for qualitative analysis. The study revealed two perspectives on poverty: 1) the individualistic-deficit perspective and 2) the socio-lifecourse perspective. In the individualistic-deficit perspective, which predominated among these participants, dentists explained poverty by individual factors and emphasized individuals' negative attitudes toward work and lack of capabilities. Conversely, dentists with a socio-lifecourse perspective described poverty as a structural rather than an individual process. Acknowledging individuals' distress and powerlessness, these dentists expressed more empathy toward people on social assistance. The results suggest the individualistic-deficit perspective impedes the care relationship between dentists and poor patients as well as highlighting the need to better prepare dentists for addressing issues of poverty and social inequities in clinical practice.

Conserved molecular interactions within the HBO1 acetyltransferase complexes regulate cell proliferation.

Acetyltransferase complexes of the MYST family with distinct substrate specificities and functions maintain a conserved association with different ING tumor suppressor proteins. ING complexes containing the HBO1 acetylase are a major source of histone H3 and H4 acetylation in vivo and play critical roles in gene regulation and DNA replication. Here, our molecular dissection of HBO1/ING complexes unravels the protein domains required for their assembly and function. Multiple PHD finger domains present in different subunits bind the histone H3 N-terminal tail with a distinct specificity toward lysine 4 methylation status. We show that natively regulated association of the ING4/5 PHD domain with HBO1-JADE determines the growth inhibitory function of the complex, linked to its tumor suppressor activity. Functional genomic analyses indicate that the p53 pathway is a main target of the complex, at least in part through direct transcription regulation at the initiation site of p21/CDKN1A. These results demonstrate the importance of ING association with MYST acetyltransferases in controlling cell proliferation, a regulated link that accounts for the reported tumor suppressor activities of these complexes.

Macrophage-conditioned medium inhibits the activation of cyclin-dependent kinase 2 by adipogenic inducers in 3T3-L1 preadipocytes.

Macrophage infiltration into adipose tissue, associated with obesity, is thought to contribute to abnormal adipose tissue remodeling, low-grade inflammation, and insulin resistance. Medium conditioned by macrophages (MacCM) inhibits 3T3-L1 and human adipocyte differentiation, as well as early adipogenic cell cycle events including MCE and retinoblastoma protein (Rb) phosphorylation. Our objective was to determine if the inhibition of Rb phosphorylation was linked to changes in cell cycle-related proteins. We treated 3T3-L1 preadipocytes with adipogenic inducers for 24 h in control medium versus J774A.1-MacCM. The differentiation-induced mRNA and protein expression of cyclin A, an activator of cyclin-dependent kinase (cdk) 2 which phosphorylates Rb, was inhibited by 82% and 73%, respectively, by J774A.1-MacCM; adipogenic expression of Myc, a transcriptional regulator of cyclin A, was also suppressed significantly. Consistent with the reduction in cyclin A levels, the activation of cdk2 by adipogenic inducers was inhibited by 75% by J774A.1-MacCM. J774A.1-MacCM also lowered levels of cyclins D1 and D2. Inhibition studies demonstrated that platelet-derived growth factor, an anti-adipogenic factor found in J774A.1-MacCM, was not responsible for the inhibitory effect on differentiation. The anti-adipogenic effect of J774A.1-MacCM was resistant to proteinase K and heat treatment, and was present in a <3 kDa fraction. Our data indicate that J774A.1-MacCM interferes with the upregulation of cyclin A levels and cdk2 activity that are required for Rb phosphorylation and MCE in 3T3-L1 adipogenesis.

HBO1 HAT complexes target chromatin throughout gene coding regions via multiple PHD finger interactions with histone H3 tail.

The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity toward its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification crosstalk within a single protein complex.

IKKbeta and the anti-adipogenic effect of platelet-derived growth factor in human abdominal subcutaneous preadipocytes.

To clarify how anti-adipogenic factors act on preadipocytes to inhibit their differentiation, we compared preadipocyte signaling responses generated by platelet-derived growth factor (PDGF; anti-adipogenic) versus insulin (pro-adipogenic). PDGF, but not insulin, stimulated the phosphorylation of inhibitor of kappaB kinase beta (IKKbeta) in a time-dependent manner. This PDGF-dependent phosphorylation event was inhibited by 60% (P<0.05) when the cells were pretreated with wortmannin, indicating a requirement for the phosphatidylinositol (PI) 3-kinase/AKT pathway. IKKbeta phosphorylation by PDGF was neither accompanied by IkappaBalpha degradation nor NF-kappaB activation. PDGF inhibited human adipocyte differentiation, assessed by triacylglycerol accumulation (75% reduction; P<0.01) and by fatty acid synthase protein expression (60% reduction; P<0.05); these responses were no longer apparent in the presence of sc-514, a selective inhibitor of IKKbeta. Our data describe a novel PDGF response in human preadipocytes that involves the pro-inflammatory kinase IKKbeta and demonstrate that it is required for the inhibition of adipogenesis.

Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes.

This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n=3; p<0.001), and BrdU incorporation was impaired by 55% (n=3; p<0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27(kip1), a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n=3; p<0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBPbeta were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.

Facility-based maternal death reviews: effects on maternal mortality in a district hospital in Senegal.

OBJECTIVE: The improvement of obstetric services is one of the key components of the Safe Motherhood Programme. Reviewing maternal deaths and complications is one method that may make pregnancy safer, but there is no evidence about the effectiveness of this strategy. The objective of our before and after study is to assess the effect of facility-based maternal deaths reviews (MDR) on maternal mortality rates in a district hospital in Senegal that provides primary and referral maternity services. METHODS: We included all women who were admitted to the maternity unit for childbirth, or within 24 hours of delivery. We recorded maternal mortality during a 1-year baseline period from January to December 1997, and during a 3-year period from January 1998 to December 2000 after MDR had been implemented. Effects of MDR on organization of care were qualitatively evaluated. FINDINGS: The MDR strategy led to changes in organizational structure that improved life-saving interventions with a relatively large financial contribution from the community. Overall mortality significantly decreased from 0.83 (95% CI (confidence interval) = 0.60 -1.06) in baseline period to 0.41 (95% CI = 0.25 -0.56) per 100 women 3 years later. CONCLUSION: MDR had a marked effect on resources, management and maternal outcomes in this facility. However, given the design of our study and the local specific context, further research is needed to confirm the feasibility of MDR in other settings and to confirm the benefits of this approach for maternal health in developing countries.