Higher CD4 Cell Research Articles

Abstract 2890: Trifluridine efficacy in cancer stem cell

Abstract Background: Cancer stem cells (CSC) have been involved in disease recurrence, metastasis, and therapeutic resistance, but effective anti-cancer agent for these cells is not still currently available. Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer who have been previously treated with fluoropyrimidine, oxaliplatin and irinotecan based chemotherapy etc. and improved overall survival. Here we showed the efficacy of FTD in CD44 and CD133 high expressing (CD44high/CD133high) cancer cells which has CSC like property. Method: CD44high /CD133high and CD44low /CD133low DLD-1 cells were separated by Fluorescence-Activated Cell Sorting (FACS) and subjected to sphere formation assay. Anti-proliferative and anti-sphere forming effects of FTD in CD44high /CD133high cells were measured. Then, 150 days FTD long exposed colorectal cell line's CSC markers CD44 and CD133 were measured by flow cytometry and its tumor initiating property in in vitro and in vivo were evaluated. Results: CD44 high /CD133 high DLD-1 cells formed much more sphere than CD44low /CD133low cells significantly. Compared to unsorted cells, CD44high /CD133high cells displayed 5-FU resistance (IC50 value of 5-FU for unsorted and CD44 high /CD133 high cells were 2.5 μM and 5.5 μM respectively) but not FTD resistance (IC50 value of FTD for unsorted and CD44 high /CD133 high cells were 8.9 μM and 10.7 μM, respectively) in in vitro proliferation assay. In addition, FTD treatment in a subtoxic concentration 1 μM for DLD-1 cells disrupted sphere formation. FTD-long exposed DLD-1 cell lines showed decreased CD44high /CD133high population compared to its parental cell line from 11.3% to 0.7%, and showed decreased sphere-forming efficacy. In addition, FTD-long exposed DLD-1 cell lines decreased tumor forming potential in in vivo. These results suggest that FTD is effective for CSC like cells and that long exposure to FTD might lead CSC depletion. Conclusion: FTD was proved to be effective for CSC like CD44high /CD133high cells. It suggests that FTD might be useful for CSC targeted chemotherapy in case of CD44 and CD133 are highly expressed subtype of tumors. Citation Format: Kenta Tsunekuni, Masamitsu Konno, Jun Koseki, Ayumu Asai, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Trifluridine efficacy in cancer stem cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2890.

Abstract 3812: A best in class anti-CD38 antibody with antitumor and immune-modulatory properties

Abstract Targeting CD38 in multiple myeloma has resulted in outstanding responses. CD38 is widely expressed on myeloma cells and other hematological malignancies. Not much is known about its expression on solid tumors and its role in the immune system. We have analysed a range of solid tumors for CD38 expression and distribution. To optimally target CD38, we have generated a novel antibody that is depleting CD38-high expressing cells, but also has immune modulatory properties. To dissect CD38 expression in solid tumors we exploited mRNA expression libraries, performed immune histochemistry (IHC) on tumor sections, and flow cytometry on patient tumor material. Bioinformatic analysis of the immune cell atlas revealed varying CD38 expression among all cancers analysed, and CD38 expression could be correlated with immune markers, e.g. Foxp3, PD-1/L1. IHC and flow cytometry confirmed CD38 expression across common cancer types, mostly confined to infiltrating lymphocyte and myeloid subsets. Expression on tumor cells was patient dependent. CD38 expression on immune cells was heterogenous and found on NK cells, T cells, suppressive myeloid cells, as well as regulatory T and B cells. Of note, high expression of CD38 was found to be correlated to a subset of exhausted T cells co-expressing PD-1 and other exhaustion markers. To target CD38 in solid tumors, we have screened a panel of CD38-binding antibodies. All antibodies have the potential to deplete CD38 positive tumor cells in vitro and in vivo. Additionally, their ability to influence effector T and NK cell activation has been evaluated. Among a panel of antibodies binding to distinct epitopes of CD38 and exerting unique functional properties, we have identified a fully human antibody, with strong capacity to deplete CD38-high cells in vitro and in vivo by varying killing mechanisms. This antibody was found to increase TCR-mediated signaling and proinflammatory cytokine secretion by human T cells, and further to enhance NK cell activation in vitro. Low dose injection to non-human primates resulted in increased expression of activation markers on both CD4 and CD8 T cells, while no T cell depletion was observed. Other selected antibodies comprise distinct modalities including strong to weak agonistic activity, differential killing properties, modulation of CD38 enzymatic activity, and offer a selection of candidates applicable for different treatment settings. In summary, we found heterogenous expression of CD38 in solid tumors, mostly confined to immune subsets. To target CD38, we present a potent anti-CD38 antibody with depleting effects on CD38-expressing cancer cells, as well as suppressive immune cells, and the capacity to increase the function of immune effector cells. This dual activity might allow to fully exploit the therapeutic potential of targeting CD38, not only in hematological malignancies but also in solid tumors. Citation Format: Nina Eissler, Simone Filosto, Jake Y. Henry, Michael F. Maguire, Kristina Witt, Andreas Lundqvist, Teresa Marafioti, Pascal Merchiers, Kevin Moulder, Beatriz Goyenechea, Haw Lu, Camilla Fairbairn, Sarah Windler, JD Aurellano, Omar Duramad, Dominic Smethurst, Sergio A. Quezada, Anne Goubier. A best in class anti-CD38 antibody with antitumor and immune-modulatory properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3812.

High CD44 expression mediates p62-associated NFE2L2/NRF2 activation in breast cancer stem cell-like cells: Implications for cancer stem cell resistance

Cluster of differentiation 44 (CD44) is the most common cancer stem cell (CSC) marker and high CD44 expression has been associated with anticancer drug resistance, tumor recurrence, and metastasis. In this study, we aimed to investigate the molecular mechanism by which CD44 and nuclear factor erythroid 2-like 2 (NFE2L2; NRF2), a key regulator of antioxidant genes, are linked to CSC resistance using CD44high breast CSC-like cells. NRF2 expression was higher in CD44high cell populations isolated from doxorubicin-resistant MCF7 (ADR), as well as MCF7, MDA-MB231, and A549 cells, than in corresponding CD44low cells. High NRF2 expression in the CD44highCD24low CSC population (ADR44P) established from ADR cells depended on standard isoform of CD44. Silencing of CD44 or overexpression of CD44 resulted in the reduction or elevation of NRF2, respectively, and treatment with hyaluronic acid, a CD44 ligand, augmented NRF2 activation. As functional implications, NRF2 silencing rendered ADR44P cells to retain higher levels of reactive oxygen species and to be sensitive to anticancer drug toxicity. Moreover, NRF2-silenced ADR44P cells displayed tumor growth retardation and reduced colony/sphere formation and invasion capacity. In line with these, CD44 significantly colocalized with NRF2 in breast tumor clinical samples. The molecular mechanism of CD44-mediated NRF2 activation was found to involve high p62 expression. CD44 elevation led to an increase in p62, and inhibition of p62 resulted in NRF2 suppression in ADR44P. Collectively, our results showed that high CD44 led to p62-associated NRF2 activation in CD44high breast CSC-like cells. NRF2 activation contributed to the aggressive phenotype, tumor growth, and anticancer drug resistance of CD44high CSCs. Therefore, the CD44-NRF2 axis might be a promising therapeutic target for the control of stress resistance and survival of CD44high CSC population within breast tumors.

Capillary morphogenesis gene 2 maintains gastric cancer stem-like cell phenotype by activating a Wnt/\u03b2-catenin pathway

A growing body of evidence shows that the development and progression of gastric cancer (GC) is mainly associated to the presence of gastric cancer stem-like cells (GCSLCs). However, it is unclear how GCSLC population is maintained. This study aimed to explore the role of capillary morphogenesis gene 2 (CMG2) in GCSLC maintenance and the relevance to GC progression. We found that CMG2 was highly expressed in GC tissues and the expression levels were associated with the invasion depth and lymph node metastasis of GC, and inversely correlated with the survival of GC patients. Sorted CMG2High GC cells preferentially clustered in CD44High stem-like cell population, which expressed high levels of stemness-related genes with increased capabilities of self-renewal and tumorigenicity. Depletion of CMG2 gene resulted in reduction of GCSLC population with attenuated stemness and decrease of invasive and metastatic capabilities with subdued epithelial–mesenchymal transition phenotype in GC cells. Mechanistically, CMG2 interacted with LRP6 in GCSLCs to activate a Wnt/β-catenin pathway. Thus, our results demonstrate that CMG2 promotes GC progression by maintaining GCSLCs and can serve as a new prognostic indicator and a target for human GC therapy.

Interleukin-8 favors pro-inflammatory activity of human monocytes/macrophages

Interleukin-8 (IL-8, CXCL8) belongs to major chemokines to stimulate migration of neutrophils and monocytes/macrophages (Mc/Mphs) into the inflammation sites. We studied the direct effects of IL-8 on the functionality of human Mc/Mphs in vitro. CD14-positive cells were isolated from human peripheral blood mononuclear cells (PBMCs) by positive magnetic separation and were further cultured with or without lipopolysaccharide (LPS, 1.0 μg/ml) for 24 h. We showed that upon LPS activation of Mc/Mphs, IL-8 reduced markedly both the percentages and median fluorescence intensity (MFI) of CD16 (FcγRIII)-positive cells among CD14high cells, as well as in cells that reduced the expression of СD14 during their culturing. IL-8 was also found to be capable of reducing the expression of СD124 (IL-4 receptor subunit alpha, IL-4RA), with concomitant enhancement of the expression of both CD119 (interferon-gamma receptor 1) and CD197 (CCR7) in Mph cells. In addition, IL-8 up-regulated production of IL-6 and IL-1β [but not tumor necrosis factor-α (TNF-α) and IL-10] by activated Mc/Mphs. Our results suggest the ability for IL-8 to directly favor pro-inflammatory M1-type Mph activity.

CD44ICD promotes breast cancer stemness via PFKFB4-mediated glucose metabolism.

CD44 is a single-pass cell surface glycoprotein that is distinguished as the first molecule used to identify cancer stem cells in solid tumors based on its expression. In this regard, the CD44high cell population demonstrates not only the ability to regenerate a heterogeneous tumor, but also the ability to self-regenerate when transplanted into immune-deficient mice. However, the exact role of CD44 in cancer stem cells remains unclear in part because CD44 exists in various isoforms due to alternative splicing.Methods: Gain- and loss-of-function methods in different models were used to investigate the effects of CD44 on breast cancer stemness. Cancer stemness was analyzed by detecting SOX2, OCT4 and NANOG expression, ALDH activity, side population (SP) and sphere formation. Glucose consumption, lactate secretion and reactive oxygen species (ROS) levels were detected to assess glycolysis. Western blot, immunohistochemical staining, ELISA and TCGA dataset analysis were performed to determine the association of CD44ICD and PFKFB4 with clinical cases. A PFKFB4 inhibitor, 5MPN, was used in a xenograft model to inhibit breast cancer development.Results: In this report, we found that the shortest CD44 isoform (CD44s) inhibits breast cancer stemness, whereas the cleaved product of CD44 (CD44ICD) promotes breast cancer stemness. Furthermore, CD44ICD interacts with CREB and binds to the promoter region of PFKFB4, thereby regulating PFKFB4 transcription and expression. The resultant PFKFB4 expression facilitates the glycolysis pathway (vis-à-vis oxidative phosphorylation) and promotes stemness of breast cancer. In addition, we found that CD44ICD and PFKFB4 expressions are generally up-regulated in the tumor portion of breast cancer patient samples. Most importantly, we found that 5MPN (a selective inhibitor of PFKFB4) suppresses CD44ICD-induced tumor development.Conclusion: CD44ICD promotes breast cancer stemness via PFKFB4-mediated glycolysis, and therapies that target PFKFB4 (e.g., 5MPN therapy) may lead to improved outcomes for cancer patients.

Impact of intraoperative blood salvage on monocyte subsets alteration and intracellular tumor necrosis factor-α production.

Venous: venous patient blood (n = 20). Native: mixed salvaged native blood (n = 20). Filtered: mixed salvaged leukocyte filtered blood (n = 20). Irradiated: mixed salvaged irradiated blood (n = 20). The frequency of the surface receptors CD14, HLA-DR, and intracellular tumor necrosis factor (TNF)-α on peripheral blood mononuclear cells was analyzed by fluorescence-activated cell sorting analysis. The frequency of unstimulated CD14low and CD14high cells as well as unstimulated HLA-DR and TNF-α positive monocytes was comparable between venous and filtered salvaged blood. However, native and irradiated salvaged blood increased compared with venous (p < 0.05) and filtered salvaged blood (p < 0.05) for unstimulated CD14low cells, HLA-DR, and TNF-α positive monocytes. Stimulated intracellular TNF-α positive monocytes were decreased in native, filtered, and irradiated salvaged blood compared with venous blood (p < 0.05). Processing perioperative salvaged blood with leukofiltration minimizes the influence on monocytes activation compared with native and irradiated salvaged blood. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 815-821, 2018.

Chicken IL-17A is expressed in αβ and γδ T cell subsets and binds to a receptor present on macrophages, and T cells

IL-17A as important cytokine in host defense has been analysed intensively and various homologous have been identified. To further gain insight into the functional properties of chicken (gg) IL-17A its expression profile was analysed by intracellular cytokine staining. In splenocytes and peripheral blood mononuclear cells gg IL-17A was detected in subsets of CD4+ T cells and γδ T cells. In contrast the gg IL-17A producing populations in intestinal intraepithelial lymphocytes were characterized as either CD3+CD25+ cells or γδ T cells. Furthermore, using FLAG tagged gg IL-17A, binding to its receptor was demonstrated on the macrophage cell line HD11. In peripheral blood IL-17A binding activity was found on αβ and γδ T cell subsets, monocytes and a distinct population of CD25high cells. Treatment of HD11 cells with gg IL-17A induced IL-6 mRNA expression and nitric oxide production. These results demonstrate the presence of a αβ T helper17 cell subset and IL-17 producing γδ T cells in the chicken.

Abstract 2886: CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is highly invasive and resistant to therapies, where treatments often result in high rates of failure and disease recurrence. A subpopulation of tumor-initiating cancer stem cells (CSC) is thought to be responsible for metastatic invasion and drug resistance in many types of cancer, including HNSCC. CD44 is a known CSC marker in HNSCC but its role in maintaining CSC populations is not well understood. We previously reported that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) inhibition of bone morphogenetic protein (BMP) signaling is essential for maintaining a CD44-high CSC-like population in HNSCC. In this study, we sought to determine how CD44, a receptor for hyaluronic acid (HA) that constitutes a major component of the tumor stroma, is involved in the regulation of SMURF1 and the maintenance of a HNSCC CSC phenotype. CSC-like cells were enriched from HNSCC cell lines as CD44-high cells. CD44 signaling was stimulated by exogenous HA treatment and inhibited by CD44 knockout (KO) using an inducible CRISPR/Cas9 system. We then assessed changes in SMURF1 protein level, BMP signaling, transwell migration, Matrigel colony formation, and invasion through three-dimensional organotypic culture (OTC) following CD44 modulation. CD44-high cells were found to have increased extracellular HA production. Treatment with exogenous HA reduced BMP signaling, as determined by a reduction in phospho-SMAD1/5/8 levels, and increased transwell migration of CD44-high cells. CD44-KO reduced SMURF1 protein expression and inhibited Matrigel colony formation of an invasive and recurrent HNSCC-derived CD44-high cells but only partially reduced colony formation of a less-invasive cell line derived from a primary HNSCC. Knockout of CD44 expression showed a slight reduction in transwell migration of invasive cells. CD44-high cells also recapitulated an invasive and CSC-like growth pattern in OTC assays. In contrast, CD44-KO inhibited OTC invasion and the epithelial-to-mesenchymal transition (EMT) phenotype, resulting in increased apical epithelial growth and differentiation. CD44 reconstitution appeared to restore the invasive and EMT phenotype. Based on our current findings, CD44 may be crucial for maintaining an anchorage-independent and invasive phenotype of HNSCC but plays a minor role in carcinoma cell migration. Citation Format: Ali Khammanivong, Raj Gopalakrishnan, Erin B. Dickerson. CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2017-2886

Abstract 4166: Role of CD44 as a regulator of adaptive plasticity in pancreatic cancer cells

Abstract Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) may show an initial objective response to chemotherapy that is usually of short duration. Our research goal is to understand the molecular mechanisms for this short duration response and to more fully define differences between chemosensitive and chemoresistant cells. Cancer cells that undergo an EMT and metastatic tumors show an increase in CD44s expression. In this study, we use CD44 Low and CD44 High isogenic cell models to identifying key molecular networks and potential targets that could benefit therapy. Experiments design: CFPAC1-CD44 Hi and Low cells were sorted by flowcytometry and single clone selection. Isogenic model systems were generated by overexpression CD44s in CF-Low cells. Western blot, MTT, migration and invasion assays had been used to characterize cell growth, invasiveness and gemcitabine sensitivity. Receptor Tyrosine Kinase arrays were used to identify the CD44 regulated molecular networks in these isogenic cell models treated with/without CD44 ligand hyaluronan acid (HA). Summary: CD44s high cells show an EMT phenotype (increase of vimentin and loss of E-cadherin) and increase invasion. Expression of CD44s in CD44 low cells was sufficient to drive EMT partially and drive invasion. By in vitro testing over expressing CD44s is not sufficient to decrease sensitivity to gemcitabine. By Receptor Tyrosine Kinase arrays, CD44 Hi clone cells show increase in IGF-1R phosphorylation. Preliminary data had shown that HA may stimulate ROR2, VEGFR1, and EphB2 phosphorylation in CD44 Hi clone cells. Conclusion: Our previous study suggested that CD44 could be a therapeutic target in Gemcitabine resistant PDAC cells. The current study will further identify the molecular targets that regulated by CD44 pathways. These newly identified targets could be hopefully used for the optimized multimodality therapy to sensitize the PDAC cells to therapy and to benefit the pancreatic cancer patients. Citation Format: Chen Chen, Shujie Zhao, Xiangru Zhao, James W. Freeman. Role of CD44 as a regulator of adaptive plasticity in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4166. doi:10.1158/1538-7445.AM2017-4166

Abstract 141: CD44 positive and sorafenib resistant hepatocellular carcinomas respond to the ATP-competitive mTOR inhibitor INK128

Abstract The PI3K/AKT/mTOR pathway is activated in about 50% of patients with hepatocellular carcinoma (HCC). Allosteric mTOR inhibitors, also known as rapalogs, fail to show any significant clinical utility. In an effort to identify new pathways and compounds to treat advanced HCC, we considered the ATP-competitive mTOR inhibitor INK128. ATP-competitive mTOR inhibitors attenuate both mTORC1 and mTORC2. We evaluated INK128 in sorafenib sensitive and insensitive HCC cell lines, CD44low and CD44high HCC and those cell lines with acquired sorafenib resistance. CD44 was significantly increased in Huh7 cells made resistant to sorafenib. Forced expression of CD44 enhanced cellular proliferation and migration, and rendered the cells more sensitive to the anti-proliferative effects of INK128. INK128 suppressed CD44 expression by blocking phosphorylation of eukaryotic translation initiation factor 4EBP1 in HCC cells while allosteric mTOR inhibitors do not. Moreover, INK128 exhibited potent anti-proliferative and anti-migration effects on the mesenchymal-like HCC cells, CD44high and sorafenib resistant HCC cells compared to the allosteric mTOR inhibitor everolimus. Combination studies of INK128 and sorafenib showed an additive to synergistic anti-proliferative effect in CD44high HCC cells. Our findings suggest that ATP-competitive mTOR inhibitors are effective in treating advanced HCC patients who express CD44, and are insensitive or resistant to sorafenib. It also highlights the potential use of CD44 as a biomarker of response in these patients. Citation Format: Mohamed Badawi, Jihye Kim, Dhruvitkumar Sutaria, Tasneem Motiwala, Ryan Reyes, Nissar Wani, Samson Jacob, Mitch Phelps, Thomas Schmittgen. CD44 positive and sorafenib resistant hepatocellular carcinomas respond to the ATP-competitive mTOR inhibitor INK128 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 141. doi:10.1158/1538-7445.AM2017-141

Glucose metabolism-targeted therapy and withaferin A are effective for epidermal growth factor receptor tyrosine kinase inhibitor-induced drug-tolerant persisters.

In pathway‐targeted cancer drug therapies, the relatively rapid emergence of drug‐tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. In this study, we investigated the features of epidermal growth factor receptor–tyrosine kinase inhibitor‐induced DTPs and explored a new treatment strategy to overcome the emergence of these DTPs. We used two EGFR‐mutated lung adenocarcinoma cell lines, PC9 and II‐18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 months. We analyzed the mRNA expression of the stem cell‐related markers by quantitative RT‐PCR and the expression of the cellular senescence‐associated proteins. Then we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by glucose metabolism targeting therapies and a stem‐like cell targeting drug, withaferin A. Drug‐tolerant persisters were composed of at least two types of cells, one with the properties of cancer stem‐like cells (CSCs) and the other with the properties of therapy‐induced senescent (TIS) cells. The CD133high cell population had CSC properties and the CD133low cell population had TIS properties. The CD133low cell population containing TIS cells showed a senescence‐associated secretory phenotype that supported the emergence of the CD133high cell population containing CSCs. Glucose metabolism inhibitors effectively eliminated the CD133low cell population. Withaferin A effectively eliminated the CD133high cell population. The combination of phloretin and withaferin A effectively suppressed gefitinib‐resistant tumor growth.

Low HDL-cholesterol among HIV-1 infected and HIV-1 uninfected individuals in Nairobi, Kenya

BackgroundAntiretroviral treatment (ART) is associated with dyslipidemia yet little is known about the burden of dyslipidemia in the absence of ART in sub-Saharan Africa. We compared the prevalence and risk factors for dyslipidemia among HIV-infected ART-naïve adults and their uninfected partners in Nairobi, Kenya.MethodsNon-fasting total cholesterol (TC) and high density lipoprotein cholesterol (HDL) levels were measured by standard lipid spectrophotometry on thawed plasma samples obtained from HIV-infected participants and their uninfected partners. Dyslipidemia, defined by high TC (>200 mg/dl) or low HDL (<40 mg/dl) was compared between HIV-infected and uninfected men and women.ResultsAmong 196 participants, median age was 32 years [IQR: 23–41]. Median CD4 count among the HIV-infected was 393 cells/ μl (IQR: 57–729) and 90% had a viral load >1000 copies/ml. Mean TC and HDL were comparable for HIV-infected and uninfected participants. Prevalence of dyslipidemia was 83.8% vs 78.4% (p = 0.27). Among the HIV-infected, those with a viral load >1000 copies/ml were 1.5-fold more likely to have dyslipidemia compared to those with ≤1000 copies/ml (adjusted prevalence ratio [aPR] 1.5, 95% CI: 1.22–30.99, p = 0.02). BMI, age, gender, blood pressure and smoking were not significantly associated with dyslipidemia.ConclusionsAmong ART-naïve HIV-infected adults, high viral load and low CD4 cell count were independent predictors of dyslipidemia, underscoring the importance of early initiation of ART for viral suppression.

Inflammatory mediator ultra-low-molecular-weight hyaluronan triggers necrosis of B-precursor leukemia cells with high surface CD44 expression

Acute lymphoblastic leukemia (ALL) with mixed lineage leukemia (MLL) gene rearrangements (MLL+ALL) has a dismal prognosis and is characterized by high surface CD44 expression. Known that CD44 has the specific binding sites for a natural ligand hyaluronan (HA), we investigated biological effects of HA with different molecular sizes on MLL+ALL cell lines, and found that the addition of ultra-low-molecular-weight (ULMW)-HA strongly suppressed their thymidine uptakes. The MLL+ALL cell line lacking surface CD44 expression established by genome editing showed no suppression of thymidine uptake. Surface CD44-high B-precursor ALL cell lines other than MLL+, but not T-ALL cell lines, were also suppressed in their thymidine uptakes. The inhibition of thymidine uptakes was because of induction of cell death, but dead cells lacked features of apoptosis on cytospin smears and flow cytometric analysis. The cell death was neither blocked by pan-caspase inhibitor nor autophagy inhibitor, but was completely blocked by necrosis inhibitor necrostatin-1. Necrotic cell death was further supported by a marked release of a high-mobility protein group B1 and morphological changes on transmission electron microscopy. Elevation of intracellular reactive oxygen species production suggested a role for inducing this necrotic cell death. ULMW-HA-triggered cell death was similarly demonstrated in surface CD44-high primary B-precursor leukemia cells. Assuming that ULMW-HA is abundantly secreted at the site of infection and inflammation, this study sheds light on understanding the mechanism of a transient inflammation-associated remission of leukemia. Further, the CD44-targeting may become an effective approach in future for the treatment of refractory B-precursor ALL by its capability of predominantly eradicating CD44-high leukemia-initiating cells.

Nutritional status and metabolic risk in HIV-infected university students: challenges in their monitoring and management

Objective: This study aims to describe challenges in the management of HIV-infected university students focusing on their nutritional status and metabolic risk.&#x0D; &#x0D; Methods: A cross-sectional, descriptive study design was used to assess the anthropometry, food intake and clinical status of a cohort of known HIV-infected registered students at a South African university.&#x0D; &#x0D; Results: Participants n = 63) had a mean CD4 cell count of 411 (SD = 219.9) cells/mm3, a mean body mass index (BMI) of 28.05 (SD = 7.9) kg/m2 and only half of the participants (n = 31) were on antiretroviral therapy (ART). A higher BMI (&gt; 25 kg/m2) was significantly (p &lt; 0.05; V = 0.32) associated with higher CD4 cell counts of &gt; 350 cells/mm3. Some 40% (n = 25) of students were at risk for metabolic complications based on their waist circumference and 11% (n = 7) had clinical signs of lipodystrophy. The ‘obese’ group consumed a mean energy intake of 24 kcal/kg bodyweight which was lower than the ‘overweight’ and normal weight groups.&#x0D; &#x0D; Conclusions: In total 51% of HIV-positive students in the sample presented with signs of metabolic complications. Side effects of ART can be prevented and/or treated by regular physical activity, adequate nutritional intake, monitoring of side effects and BMI, combined with optimal care and support.&#x0D; &#x0D; (Full text of the research articles are available online at www.medpharm.tandfonline.com/ojfp)&#x0D; &#x0D; S Afr Fam Pract 2017; DOI: 10.1080/20786190.2016.1248143

Predictors and incidence of sexually transmitted Hepatitis C virus infection in HIV positive men who have sex with men

BackgroundSexual transmission of Hepatitis C virus (HCV) in men who have sex with men (MSM) and its interaction with HIV status, sexually transmitted infections and sexual behaviour is poorly understood. We assessed the incidence and predictors of HCV infection in HIV positive MSM.MethodsThe electronic medical record and laboratory results from HIV positive MSM in care at a large urban public specialist HIV clinic embedded in a sexual health centre in Melbourne Australia were collected. Patients with two or more HCV antibody tests between January 2008 and March 2016 and with no record of injecting drug use were included. The HCV exposure intervals were the periods between a negative HCV test and the next HCV test. We compared HCV exposure intervals temporally associated with and without newly acquired syphilis or anorectal chlamydia. HCV exposure intervals were also categorised as being before or after HIV virological suppression and by most recent and nadir CD4 cell count.ResultsThirty seven new HCV infections were diagnosed in 822 HIV positive MSM with no history of injecting drug use over 3114 person years (PY) of follow-up. Mean age was 43.1 years (±12.5) and mean CD4 cell count nadir was 362 cells/uL (±186). The incidence of HCV infection in the study population was 1.19/100PY (0.99–1.38). The incidence in exposure periods temporally close to new syphilis infection was 4.72/100PY (3.35–6.08) and to new anorectal chlamydia infection was 1.37/100PY (0.81–1.93). The incidence in men without supressed viral load was 3.19/100PY (1.89–4.49). In the multivariate Cox regression analysis only younger age (aHR 0.67 (0.48–0.92)), exposure periods temporally associated to new syphilis infection (aHR 4.96 (2.46–9.99)) and higher CD4 cell count nadir (aHR 1.26 per 100 cells/uL (1.01–1.58)) were associated with increased risk of HCV infection. During the study period the incidence of syphilis increased dramatically but the incidence of HCV infection remained the same.ConclusionsIncidence of HCV infection is associated with syphilis but not anorectal chlamydia which suggests a biological rather than behavioural risk modification. Rising syphilis incidence may offset declines in HCV transmission through HCV treatment as prevention.

Cancer stem cell, cytokeratins and epithelial to mesenchymal transition markers expression in oral squamous cell carcinoma derived from ortothopic xenoimplantation of CD44high cells

Oral squamous cell carcinoma (OSCC) is the most prevalent neoplasia of oral cavity worldwide and prognosis remains unchanged in decades. Recently, different authors reported that head and neck squamous cell carcinomas have a subpopulation of tumor initiating cells that apparently correspond to cancer stem cells (CSC) and are also responsible for tumor growth and metastasis. The purpose of the present study was to investigate the microscopic and phenotypic characteristics of OSCC tumors induced after orthotopic xenoimplantation of SCC9WT cell line and CSC-enriched subpopulation isolated from SCC9 cell line based on high expression of the putative CSC marker CD44. Different numbers of FACS-sorted SCC9 CD44high and CD44low cells as well as SCC9WT (wild type) were transplanted into the tongue of BALB/C nude (NOD/SCID) mice to evaluate their tumorigenic potential. Sixty days post-induction, tumors were morphologically characterized and immunostained for CSC markers (CD44, Nanog and Bmi-1), epithelial-mesenchymal transition (Snail, Slug) and epithelial differentiating cell markers (cytokeratins 4, 13, 15, 17 and 19), as well as E-cadherin and β-catenin. The data presented here shows that SCC9 CD44high cells have higher ability to form tumors than SCC9 CD44low cells, even when significantly lower numbers of SCC9 CD44high cells were transplanted. Immunoassessment of tumors derived from SCC9 CD44high cells revealed high expression of cytokeratin CK19, β-catenin, E-cadherin and CD44, and negative or low expression of CK17, CK4, CK15, CK13, Nanog, Bmi-1, Snail and Slug. While tumors derived from SCC9WT showed high expression of CK17, CK19, CD44, Nanog, Bmi-1, Snail and Slug, and negative or low expression of CK4, CK15, CK13, β-catenin and E-cadherin. Thus, SCC9 CD44high cells were highly tumorigenic, capable of originating heterogeneous tumors and these tumors have a immunohistochemical profile different from those formed by the wild type cell line.

Abstract B68: Role of CD44 in pancreatic cancer cell plasticity

Abstract Two subpopulations of pancreatic adenocarcinoma (PDAC) cells were separated from different cell lines on the basis of CD44 expression level. CD44 high and CD44 low expressing cells were compared for their invasive potential, epithelial/mesenchymal phenotype, in vitro response and in vivo development of resistance to gemcitabine. CD44 high cells showed a mesenchymal phenotype, formed tumor spheres, were more invasive and were less responsive to gemcitabine. In contrast, CD44 low cells had an epithelial phenotype, were weakly invasive, did not grow as tumor spheres and showed greater response to gemcitabine. CD44 high cells showed a predominant expression of CD44 standard isoform with lower levels of various CD44 variant isoforms. Interestingly, gemcitabine treatment of CD44 low cells induced a phenotypic switch, loss of ESRP1 expression with concurrent up regulation in expression of CD44 standard. In an orthotopic mouse model, tumors from CD44 high cells had a selective growth advantage and formed metastases more rapidly. Gemcitabine treatment continuously suppressed tumor growth in animals implanted with CD44 low cells during the 22 weeks of the experiment; whereas, tumors from CD44 high cells initially showed some response to gemcitabine but developed resistance by weeks 10-12. Knockdown of CD44 in CD44 high cells from two different PDAC cell lines, inhibited invasion and increased their response to gemcitabine similar to that seen for CD44 low cells. Collectively this study suggests that a subpopulation of CD44 high expressing PDAC cells with a mesenchymal-like phenotype tends to be highly invasive, more resistant to gemcitabine in vitro and to develop gemcitabine resistance in vivo. Thus, initial targeting CD44 or reversing the CD44 high phenotype may improve therapeutic response of PDAC. Citation Format: James W. Freeman, Shujie Zhao, Chen Chen, Anand Karnad.{Authors}. Role of CD44 in pancreatic cancer cell plasticity. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B68.

Abstract B30: Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a propensity for metastasis and drug resistance. To overcome this and improve the outcome of patients with PDAC, there is an urgent need to elucidate the molecular mechanisms of metastasis in which cancer stem cells (CSCs) may play a role. Prrx1, a homeobox transcription factor has been identified by us, as a key molecule involved in pancreatic development, regeneration and early carcinogenesis (Reichert et al. Genes and Development 2013). Furthermore, we have reported recently that Prrx1 isoform switching governs EMT-MET plasticity to regulate PDAC invasion and metastasis (Takano et al. Genes and Development 2016). Herein, we examined that the correlation between the two Prrx1 isoforms and putative CSC markers to investigate the role of Prrx1 on pancreatic CSC functions. Materials and Methods: Mouse pancreatic ductal cell lines were isolated and established from Pdx1Cre mice (normal pancreatic ductal cells), Pdx1Cre;KrasG12D/+ mice (PanIN cells), Pdx1Cre;KrasG12D/+;p53R175H/+ mice (primary and metastatic PDAC cells). Either Prrx1a and Prrx1b was overexpressed by lentiviral transduction in each cell line with controls. Prrx1 knockdown was done in primary PDAC cells and their matched liver metastatic cells by two isoform specific siRNAs. We immunophenotyped putative pancreatic CSCs using antibodies against CSC markers (CD133, CD44, CD24 and Epcam) by flow cytometry. These subpopulations of cells were evaluated in 3D cultures, invasion assays and single-pancreatosphere formation assays. Additionally, we evaluated the expression of pancreatic CSC markers in Prrx1a and Prrx1b overexpressing and knockdown cells by qRT-PCR and flow cytometry. Results: CD133High cells and CD44HighCD24High cells are expressed highly in metastatic PDAC cells compared to primary PDAC cells. CD133High cells form spheroid cysts and CD133Low cells form spindle-shaped cells in 3D culture. CD133High cells demonstrate more invasiveness and higher self-renewal capacity compared to CD133 Low cells. The percentage of CD133High cells, CD44HighCD24High cells and EpCAMHigh cells are increased significantly in Prrx1a overexpressing primary PDAC cells. However, these cells are decreased in Prrx1a knockdown metastatic cells. Contrary to this, these subpopulation of cells are significantly decreased in Prrx1b overexpressing primary PDAC cells, and increased in Prrx1b knockdown metastatic cells. These data suggest that Prrx1a might regulate pancreatic CSC functions in metastatic PDAC cells. Conclusion: Prrx1a may be associated with a subpopulation of pancreatic cancer cells that have features of cancer stem cells. This may influence metastatic properties of pancreatic cancer cells to the liver. Citation Format: Shigetsugu Takano, Maximilian Reichert, Hideyuki Yoshitomi, Basil Bakir, Koushik K. Das, Steffen Heeg, Shingo Kagawa, Hiroaki Shimizu, Masayuki Ohtsuka, Katsunori Furukawa, Masaru Miyazaki, Anil K. Rustgi.{Authors}. Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B30.

Role of Cell Source and Graft Composition in Haploidentical Transplantation Using Post-Transplant Cyclophosphamide

Introduction: Cell source and graft composition are known to have a prognostic role in allogeneic hematopoietic cell transplant (HCT). Currently, there are no data in the setting of haploidentical-HCT with post-transplant cyclophosphamide (PT-Cy).Patients and methods: One-hundred patients undergoing haplo-HCT with PT-Cy from Sept 2011 to Nov 2015 were included in this multicenter retrospective analysis.Bone marrow (BM) was used as graft source in 25 patients and peripheral blood stem cell (PBSC) in 75. The two groups (BM vs PBSC) were similar in terms of age, Disease Risk Index (DRI) and HCT-CI. The only differences were on disease type (lymphoid malignancies 76% vs 57%, p=0.02) and conditioning regimen (myeloablative: 100% versus 30%, p<0.01).Graft cellular subsets analysis was performed by means of flow cytometry (CD34, CD3, CD4, CD8, CD56, CD19, CD38, CD31, CD25, CD45RA, CD95, CD127, CCR4, CCR6, CCR7). Overall Survival (OS) and Progression Free Survival (PFS) were performed with Kaplan-Meier analysis. Acute GVHD, chronic GVHD, Non-Relapse Mortality (NRM) and Relapse Incidence/Progression of disease (RI/POD) were obtained with competing risk analysis.Results: For the whole cohort, neutrophil and platelet engraftment cumulative incidences at day +30 were 97% (95% CI: 91-99) and 64% (95% CI: 53-73), with no differences between BM or PBSC. Grade II-IV and grade III-IV aGVHD cumulative incidences at day +100 were 36% (95% CI: 26-48) and 10% (95%CI: 5-18), respectively. Acute GVHD cumulative incidence was significantly lower for BM grafts [16% (95% CI: 5-33) vs 43% (95% CI: 31-54), p=0.02], but no differences were observed regarding grade III-IV acute GVHD [8% (CI 95%: 1-24) vs 11% (CI 95%: 5-20), p=0.73]. Chronic GVHD cumulative incidence at 18 months was 23% (95%CI: 15-32) with no difference between BM or PBSC. With a median follow up of 16.5 months (range 0.8 - 40.6), the 18-months PFS and OS were 43% (95%CI: 32-54) and 63% (95% CI: 52-73), respectively. NRM and RI/POD at 18 months were 18% (95%CI: 11-26) and 38% (95%CI: 27-49).Multivariable analysis was performed using patient (age, gender, HCT-CI, DRI) and transplant characteristics (donor gender, donor-recipient relation, graft source). A DRI>1 was the only factor associated with higher RI/POD (HR 3.45, 95% CI: 1.4-8.2, p<0.01), lower PFS (HR 2.77, 95%CI: 1.4-5.3, p<0.01), and lower OS (HR 2.33, 95%CI: 1.1-4.9, p=0.02). Graft source did not influence survival outcomes.Graft cell composition analysis was performed separately for BM and PBSC cohorts. For the BM group, univariable analysis showed that CD4 graft count ≥20x10^6/kg was associated with a prolonged PFS [60% (95%CI: 28-81) vs 0%, p<0.01] (fig. 1), a trend toward a decreased NRM [0% vs 27% (95%CI: 5-56), p=0.05] and a prolonged OS [92%(95%CI: 57-99) vs 58% (95%CI:27-80), p=0.05]. Among the CD4 subsets analyzed (activated, central memory, effector, effector memory, memory stem cells, regulatory, Th17), naive T cells ≥8.5x10^6/kg (CD3+/CD8-/CD4+/CCR7+/CD45RA+/CD95-) and recent thymic emigrants ≥6.9x10^6/kg (median) (RTEs, CD4+/CD31+/CD45RA+) were the only phenotypes associated with a better PFS and OS (p=0.02 and p=0.04 respectively). For the PBSC group, CD8 graft count ≥ 75 x10^6/kg (median) was associated with a lower NRM [3% (95%CI: 0-12) vs 16% (95%CI:6-30), p=0.01]. No associations were found regarding graft cell composition and incidence of acute or chronic GVHD for both BM and PBSC groups.Conclusions: In our retrospective analysis, we did not observe significant differences in survival outcomes based on graft source. Patients receiving BM grafts developed fewer grade II-IV aGVHD, but grade III-IV aGVHD incidence was similar between the two groups.For the BM group, a higher CD4 count was predictive of better PFS, TRM and OS, as reported with standard GVHD prophylaxis and matched donors (Waller EK, J Clin Oncol 2014. 32: 2365-2372). Interestingly, of the CD4 population, only naive T cells and RTEs were associated with an improved PFS and OS. A protective effect of CD8 cell count was observed in the PBSC group. This is similar to the non PT-Cy setting where higher CD8 cells are associated with better survival outcomes (Reshef R, J Clin Oncol 2015). These data should be validated in a prospective analysis to guide the choice of cell source in the haplo PT-Cy setting. [Display omitted] DisclosuresCorradini:Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Servier: Honoraria; Gilead: Honoraria; Takeda: Consultancy.