Higher CD4 Cell Research Articles

Association of CD34 Cell Dose with 5-Year Overall Survival after Peripheral Blood Allogeneic Hematopoietic Cell Transplantation in Adults with Hematologic Malignancies

Higher CD34 cell dose is associated with improved engraftment after peripheral blood allogeneic hematopoietic stem cell transplantation (alloHCT) but also may increase the risk of long-term complications, such as graft-versus-host disease (GVHD). Prior studies examining the relationship between CD34 cell dose and long-term survival outcomes have yielded conflicting results. In this study, we sought to clarify the prognostic impact of CD34 cell dose by examining a large contemporary cohort of patients undergoing alloHCT with a matched sibling peripheral blood stem cell (PBSC) donor. We retrospectively examined the impact of CD34 cell dose on overall survival (OS), neutrophil engraftment, platelet engraftment, treatment-related mortality, relapse, acute GVHD grade II-IV and III-IV, and chronic GVHD in 377 consecutive patients undergoing alloHCT with a PBSC graft source from a matched sibling donor at the University of Minnesota between 2002 and 2015. The patients were classified into 3 groups based on the tertile (T) of CD34 cell dose received: T1, <5×106 cells/kg; T2, 5 to 7.5×106 cells/kg; and T3, ≥7.5×106 cells/kg. Multivariable analysis demonstrated that high CD34 cell dose was associated with superior 5-year OS (hazard ratio [HR], 0.57; P=.01) and more rapid platelet engraftment (HR, 1.70; P < .01). Higher CD34 cell dose also was associated with improved absolute neutrophil count engraftment (T2: HR, 1.54; T3: HR, 1.52; P < .01). There was no association between CD34 cell dose and TRM or relapse at 5 years. Although higher CD34 cell dose was not associated with acute GVHD grade II-IV, it was associated with chronic GVHD (T2: HR, 1.68; T3: HR, 1.50; P=.04). Our data indicate that higher CD34 cell dose (>7.5×106/kg) is associated with superior OS at 5 years and improved engraftment but carries an increased risk of chronic GVHD. These data support a target CD34 cell dose goal of 7.5×106/kg for sibling PBSC graft donors.

Melatonin enhances cisplatin-induced cell death through inhibition of DERL1 in mesenchymal-like CD44high OSCC cells.

Melatonin is a hormone that is primarily produced in the pineal gland and is involved in wide range of biological functions. However, the impact of melatonin on chemotherapy-induced cell death remains to be elucidated in oral squamous cell carcinoma (OSCC) cells. The objective of this study was to clarify the role of melatonin in cisplatin-induced cytotoxicity in CD44high OSCC cells. CD44high OSCC cells were cultured on fibronectin-coated hydrogel. A lactate dehydrogenase cytotoxicity assay was performed to evaluate cisplatin-induced cell death. The effect of melatonin on cisplatin-induced cell death and Derlin-1 (DERL1) endoplasmic reticulum membrane protein expression was investigated. CD44high OSCC cells exhibited mesenchymal-like features when cultured on fibronectin-coated hydrogel. Mesenchymal-like CD44high OSCC cells demonstrated strong resistance to cisplatin-induced cell death compared with epithelial-like CD44high OSCC cells. DERL1mRNA and DERL1 protein expression levels were significantly higher in mesenchymal-like CD44high cells compared with epithelial-like CD44high cells. Cisplatin-induced cell death was significantly enhanced after DERL1siRNA knockdown, suggesting that DERL1 is involved in resistance to cisplatin-induced cell death. Melatonin significantly inhibited DERL1 expression and enhanced cisplatin-induced cell death in mesenchymal-like CD44high cells. miR-181c-5p expression was significantly upregulated in the presence of melatonin. Furthermore, melatonin-inhibited DERL1 expression was significantly recovered by miR-181c-5p inhibitor. In addition, melatoninenhanced cisplatin-induced cell death was attenuated by miR-181c-5p inhibitor. These results suggest that melatonin-induced miR-181c-5p enhances cisplatin-induced cell death through inhibition of DERL1 in mesenchymal-like CD44high cells. Melatonin plays a vital role in promoting cisplatin-induced cytotoxicity in mesenchymal-like CD44high OSCC cells.

Immune infiltrates in patients with localised high-risk soft tissue sarcoma treated with neoadjuvant chemotherapy without or with regional hyperthermia: A translational research program of the EORTC 62961-ESHO 95 randomised clinical trial

BackgroundThe EORTC 62961-ESHO 95 randomised trial showed improved long-term survival of patients with high-risk soft-tissue sarcoma by adding regional hyperthermia to neoadjuvant chemotherapy. We hypothesised that immune infiltrate of patients treated with neoadjuvant therapy associate with clinical outcome. MethodsTumour infiltrating lymphocytes (TILs) and CD8, FOXP3, PD-1, and PD-L1 were evaluated in sequential biopsies of patients after four cycles of therapy. ResultsFrom a subgroup of 109 patients who had been randomised between July 1997 and November 2006 to neoadjuvant chemotherapy (53 patients) or neoadjuvant chemotherapy with regional hyperthermia (56 patients), 137 biopsies were obtained. TILs increased in paired second biopsies independent of treatment allocation (p < 0.001). FOXP3 regulatory T cells decreased (p = 0.002), and PD-L1 expression of tumours became undetectable. In the multivariate analysis, post-treatment high TILs correlated to LPFS (HR: 0.34; 95% CI 0.15–0.75; p = 0.008) and DFS (HR: 0.38; 95% CI 0.17–0.82; p = 0.015). In comparing post-treatment immune infiltrate between treatment arms, tumour response was associated with neoadjuvant chemotherapy with regional hyperthermia (p = 0.013) and high TILs (p = 0.064). High CD8 cell infiltration was associated with improved LPFS (HR: 0.27; 95% CI 0.09–0.79; Log-rank p = 0.011) and DFS (HR: 0.25; 95% CI 0.09–0.73; Log-rank p = 0.006). Improved survival at 10 years was associated with immune infiltrate after neoadjuvant chemotherapy with regional hyperthermia. ConclusionPreoperative therapy re-programs a non-inflamed tumour at baseline into an inflamed tumour. The post-treatment immune infiltrate became predictive for clinical outcomes. The combination with regional hyperthermia primes the tumour microenvironment, enabling enhanced anti-tumour immune activity in high-risk soft tissue sarcomas. Trial registrationClinicalTrials.gov, NCT00003052.

NGF Expression and Elevation in Hip Osteoarthritis Patients with Pain and Central Sensitization.

Osteoarthritis (OA) is a chronic degenerative musculoskeletal disease that causes articular cartilage degeneration and chronic pain. Research into OA animal models suggests that elevated NGF levels in the synovium contribute to pain and central sensitization (CS). However, it is unclear whether synovial NGF contributes to CS in patients with OA. We investigated the association between synovial NGF expression and clinical assessments of pain and CS in hip OA (hOA) patients. We also aimed to identify which cells in the synovium of hOA patients express NGF. Sixty-six patients who received total hip replacement and a diagnosis of hOA were enrolled. We measured NGF mRNA expression in synovial samples obtained from 50 patients using qPCR and analyzed the correlation of NGF expression with the CS inventory (CSI) score and Japanese Orthopaedic Association (JOA) score, a clinical scoring system for OA. To identify the synovial cells expressing NGF, we analyzed NGF mRNA expression in CD14+ and CD14- cells, which represent macrophage-rich and fibroblast-rich fractions, respectively, extracted from 8 patients. To further identify which macrophage subtypes express NGF, we examined NGF mRNA expression in CD14high and CD14low cells sorted from 8 patients. Synovial NGF mRNA expression was negatively correlated with JOA score but positively correlated with CSI score (JOA pain, r = −0.337, P = 0.017; CSI score, r = 0.358, P = 0.011). Significantly greater levels of NGF were observed in CD14- cells compared to CD14+ cells (P = 0.036) and in CD14high cells compared to CD14low cells (P = 0.008). In conclusion, synovial NGF expression is correlated with the degree of pain and CS in hOA patients. NGF is predominantly expressed in synovial fibroblasts. Further, CD14high synovial macrophages expressed higher levels of NGF. Our results may provide a novel NGF-targeted therapeutic strategy for hOA pain.

Correlation between CD163 expression and resting pain in patients with hip osteoarthritis: Possible contribution of CD163+ monocytes/macrophages to pain pathogenesis.

Expression of CD163, a scavenger receptor specifically expressed by monocytes and macrophages, is elevated in the synovial tissue of patients with knee osteoarthritis (OA) compared with healthy controls. However, the association between CD163 expression in the synovium and pain in OA patients is unclear. We investigated the correlation between synovial CD163 expression and resting and active pain levels in patients with hip osteoarthritis (HOA). To investigate the possible contribution of CD163+ subsets to pain pathogenesis, we compared pain-related cytokine expression and M1/M2 macrophage marker expression in CD163+ and CD163- cells. We performed flow cytometric analysis to study the CD163+ cell population. We also examined pain-related cytokine expression and M1/M2 macrophage marker expression on CD163+ CD14high and CD163+ CD14low cells using cell sorting. Synovial CD163 expression significantly correlated with resting pain levels (p = 0.006; R = 0.321), but not active pain levels (p = 0.155; R = 0.169). Expression of the M1 macrophage marker CD80 was significantly higher in CD163+ than CD163- cells (p = 0.010), as was the expression of M2 macrophage markers CD206 and IL10 (CD206, p = 0.014; IL10, p = 0.005), and TNFA and IL1B (TNFA, p = 0.002; IL1B, p = 0.001). TNFA expression was significantly higher in CD163+ CD14low than CD163+ CD14high cells, while IL1B, IL10, and CD206 expression were comparable among these subsets. Our findings suggest that CD163 expression is associated with higher resting pain scores. As TNF-α plays a role in the pain process, CD163+ CD14low cells expressing TNFA may be a potent contributor to the pathogenesis of resting pain in HOA.

Surface Phenotype Changes and Increased Response to Oxidative Stress in CD4+CD25high T Cells.

Conversion of CD4+CD25+FOXP3+ T regulatory cells (Tregs) from the immature (CD45RA+) to mature (CD45RO+) phenotype has been shown during development and allergic reactions. The relative frequencies of these Treg phenotypes and their responses to oxidative stress during development and allergic inflammation were analysed in samples from paediatric and adult subjects. The FOXP3lowCD45RA+ population was dominant in early childhood, while the percentage of FOXP3highCD45RO+ cells began increasing in the first year of life. These phenotypic changes were observed in subjects with and without asthma. Further, there was a significant increase in phosphorylated ERK1/2 (pERK1/2) protein in hydrogen peroxide (H2O2)-treated CD4+CD25high cells in adults with asthma compared with those without asthma. Increased pERK1/2 levels corresponded with increased Ca2+ response to T cell receptor stimulation. mRNA expression of peroxiredoxins declined in Tregs from adults with asthma. Finally, CD4+CD25high cells from paediatric subjects were more sensitive to oxidative stress than those from adults in vitro. The differential Treg sensitivity to oxidative stress observed in children and adults was likely dependent on phenotypic CD45 isoform switching. Increased sensitivity of Treg cells from adults with asthma to H2O2 resulted from a reduction of peroxiredoxin-2, -3, -4 and increased pERK1/2 via impaired Ca2+ response in these cells.

The opioid crisis is driving mortality among under-served people living with HIV in British Columbia, Canada

IntroductionUniversal provision of effective antiretroviral medication has been essential to reduce mortality, increase longevity, and reduce onward transmission of HIV. This study aims to illuminate persistent threats to the health and longevity of under-served PLWH in British Columbia (BC), Canada.MethodsBetween 2007 and 2010, 1000 PLWH across BC were enrolled in the Longitudinal Investigation into Supportive and Ancillary health services (LISA) study and completed a cross-sectional survey on their HIV-care experiences and healthcare engagement. The sample generally reflects an under-served population of PLWH. A linkage to the provincial Vital Statistics registry is used in this analysis in order to examine overall mortality and cause-specific mortality trends; probability of death was modeled using logistic regression for participants with ongoing clinical monitoring (n = 910).ResultsBy June 2017, 208 (20.8%) participants had died. The majority of deaths 57 (27.4%) were attributed to drug-related complications or overdoses, 39 (18.8%) were attributed to HIV-related complications, and 36 (17.3%) to non-AIDS-defining malignancies. We observed elevated odds of death among PLWH who smoked tobacco (aOR: 2.11, 95% CI: 1.38, 3.23), were older (aOR: 1.06 per one-year increase, 95% CI: 1.04, 1.08), indicated heavy alcohol consumption (aOR: 1.57, 95% CI: 1.11, 2.22), and reported unstable housing (aOR: 1.96, 95% CI: 1.37, 2.80); while higher CD4 cell count was protective (aOR: 0.87 per 100-unit increase, 95% CI: 0.79, 0.94) as was male gender), though non-significant (aOR: 0.73, 95% CI: 0.49, 1.07).ConclusionsOverdose is - the leading cause of mortality among a cohort of under-served PLWH in BC, Canada. Public health efforts to end the HIV epidemic and support the health and well-being of PLWH are being thwarted by persistent health inequities and the enormous and persistent risks facing people who use drugs. Integrated low-barrier primary care is essential for supporting under-served PLWH, and safe drug supply is needed to support PLWH who use drugs.

The shifting picture of HIV treatment, comorbidity and substance use among US MSM living with HIV.

People living with HIV (PLWH) have increased risk of chronic disease and poor mental health. We aimed to explore HIV disease indicators, comorbidity, and risk behavior of recent antiretroviral therapy (ART) initiators to inform current needs of PLWH. Men who have sex with men (MSM) in the Multicenter AIDS Cohort Study (MACS) who initiated ART between 2010 and 2018 (recent initiators) were compared with age-, race- and geographic location-matched men who initiated ART during 2000-2009 (early initiators). Measures of HIV disease, behavior, comorbidity and mental health were collected prospectively every 6months using standardized forms. Recent initiators had higher current CD4 (median CD4 451 vs. 307 cells/μL, P<0.0001) and nadir CD4 (451 vs. 300 cells/μL, P<0.0001) than earlier initiators. The proportion achieving viral suppression within a year of starting ART was significantly higher in recent compared with earlier initiators (92% vs. 74%, P<0.0001). Median [interquartile range (IQR)] time from HIV diagnosis to ART initiation was 5.4(1.7-23.1)months in recent initiators. Comorbidity prevalence was high in recent initiators, including obesity (24%), hypertension (25%) and kidney disease (15%). Substance use continues to be common, including cigarette use (40%), daily alcohol use (88%) and marijuana use (46%). Improvements in getting individuals onto ART at an early stage have led to substantially higher CD4 cell counts at initiation. However, the high burden of comorbidity, substance use and poor mental health affecting MSM living with HIV in the US underscore ongoing challenges and our need to adapt and coordinate care.

The antioxidant MnTBAP does not effectively downregulate CD4 expression in T cells in vivo

The antioxidant MnTBAP was previously shown to down-regulate the surface expression of CD4 molecule in T cells. This observation obviously holds great potential impact in a number of pathological human conditions, including autoimmunity. Three different single doses of MnTBAP reduced the frequency of CD4high cells. However, the median florescent intensity (MFI) was not different. Initiation of in vivo pharmacotherapy or vehicle control was performed inC57BL/6 mice that were actively immunized for experimental autoimmune encephalomyelitis (EAE). In contrast to published reports, the mean frequency of CD4high cells, and the median fluorescent intensity (MFI) of CD4 was similar in both treatment groups. 25-day survival following active immunization among the MnTBAP treated animals compared to vehicle controls was16.6 ± 6.9 days vs 23.6 ± 2.7 days; (P value <0.05). We conclude that MnTBAP (Sack and Herzog, 2009 (Sack and Herzog, 2009)) does not effectively downregulate CD4 expression in T cells in vivo, probably due to extensive mechanism that distinguishes it from an in vitro model (Harding, 1993 (Harding, 1993)) possesses toxic properties that may limit its clinic use in possible doses that could deliver the immunomodulation through down regulation of CD4 expression, and (Saizawa et al., 1987 (Saizawa et al., 1987)) has limited availability in specific tissues, including the CNS.

Poor CD4/CD8 ratio recovery in HBcAb-positive HIV patients with worse immune status is associated with significantly higher CD8 cell numbers

Low CD4+ cell count in patients with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) coinfection during combination antiretroviral therapy (cART) has been described; however, notably few studies have investigated coinfected patients positive for antibodies to the HBV c antigen (HBcAb). An observational retrospective study enrolling 190 patients was conducted by grouping patients with respect to HBV status and recording CD4+ T cell counts and percentages (CD4%), CD8+ T cell counts and percentages (CD8%), and the CD4+ to CD8+ T cell ratio (CD4/CD8) at the time of HIV diagnosis, at the start of treatment and at months 1, 2, 3, 4, 5, 6, 12, and 24 after beginning cART. One hundred and twenty patients (63.2%) were negative for previous HBV infection, while 70 (36.8%) were HBcAb-positive. A significant increase in the CD4/CD8 ratio was recorded in HIV monoinfected subjects compared to HBV coinfected patients from months 4 to 12 from the beginning of cART (p value = 0.02 at month 4, p value = 0.005 at month 5, p value = 0.006 at month 6, and p value = 0.008 at month 12). A significant increase in the absolute count of CD8+ T lymphocytes was described from months 2 to 24 from the start of cART in the subgroup of HBV coinfected patients with an AIDS event at the onset of HIV infection. The presence of HBcAb was observed to be associated with reduced CD4/CD8 ratio growth and a significantly higher proportion of subjects with CD4/CD8 < 0.45 in the HIV/HBV coinfected group. A significant increase in the CD8 T cell count was shown up to 24 months after the initiation of effective cART in the subgroup of patients with the worst immune status.

Translation and cross-cultural adaptation of WHOQOL-HIV Bref among people living with HIV/AIDS in Pakistan

BackgroundReliable Health-Related Quality of Life (HRQoL) assessment will be useful in identifying health issues and in identifying health care actions. Due to the lack of a psychometrically valid tool in Urdu, we aim to translate and examine the psychometric and cross-cultural adaptation of WHOQOL HIV Bref among people living with HIV/AIDS (PLWHA) in Pakistan.MethodsThe standard forward-backwards translation technique was used to convert English version of the WHOQOL HIV Bref into Urdu. After cognitive debriefing, final Urdu version of instrument was developed. Based on the principle of at least 5 subjects for each item, a sample of 182 patients was used using a universal random sampling technique from the Pakistan Institute of Medical Sciences, Islamabad. The Cronbach’s alpha and intra-class correlation coefficients (ICC) were estimated to assess internal validity and reliability of the translated version. Exploratory factor analysis was carried out to determine the factor structure and independent associations between the instrument domains and CD-4T-cell count were assessed using multivariable linear regressionResultsHigh Cronbach alpha 0.93 was found for all WHOQOL HIV Bref facets. The test–retest reliability demonstrated a statistically significant ICC ranged from 0.88 to 0.98 (p < 0.001). In known group validity, lower CD-4 lymphocytes count was significantly related to poor scores for all six domains (p < 0.001). Similarly, symptomatic subjects had significantly lower scores compared to asymptomatic subjects on the physical, psychological, social relationship and independence domains (p < 0.05). Statistically significant positive correlation of all six domains of instrument with CD4 cells count (p < 0.001), exhibiting patients with higher CD-4 cells will have higher mean scores of all domains. Factor analysis revealed 5 domains, including physical health, psychological health, social relationship, environmental, and spiritual health. Multivariable linear regression analysis reported; only physical, psychological health and environment health domains were found significantly associated with higher CD-4 lymphocytes count (Beta = 0.121, p < 0.001, Beta = 0.103, p = 0.002, and Beta = 0.032, p = 0.032).ConclusionFindings suggested that the Urdu version of WHOQOL HIV Bref is a psychometrically valid and culturally well-adapted HRQoL measurement tool for PLWHA in Pakistan.

Gender difference in mortality among pulmonary tuberculosis HIV co-infected adults aged 15-49 years in Kenya.

SettingKenya, 2012–2015ObjectiveTo explore whether there is a gender difference in all-cause mortality among smear positive pulmonary tuberculosis (PTB)/ HIV co-infected patients treated for tuberculosis (TB) between 2012 and 2015 in Kenya.DesignRetrospective cohort of 9,026 smear-positive patients aged 15–49 years. All-cause mortality during TB treatment was the outcome of interest. Time to start of antiretroviral therapy (ART) initiation was considered as a proxy for CD4 cell count. Those who took long to start of ART were assumed to have high CD4 cell count.ResultsOf the 9,026 observations analysed, 4,567(51%) and 4,459(49%) were women and men, respectively. Overall, out of the 9,026 patients, 8,154 (90%) had their treatment outcome as cured, the mean age in years (SD) was 33.3(7.5) and the mean body mass index (SD) was 18.2(3.4). Men were older (30% men’ vs 17% women in those ≥40 years, p = <0.001) and had a lower BMI <18.5 (55.3% men vs 50.6% women, p = <0.001). Men tested later for HIV: 29% (1,317/4,567) of women HIV tested more than 3 months prior to TB treatment, as compared to 20% (912/4,459) men (p<0.001). Mortality was higher in men 11% (471/4,459) compared to women 9% (401/4,567, p = 0.004). There was a 17% reduction in the risk of death among women (adjusted HR 0.83; 95% CI 0.72–0.96; p = 0.013). Survival varied by age-groups, with women having significantly better survival than men, in the age-groups 40 years and over (log-rank p = 0.006).ConclusionWomen with sputum positive PTB/HIV co-infection have a significantly lower risk of all-cause mortality during TB treatment compared to men. Men were older, had lower BMI and tested later for HIV than women.

Issue Highlights - November 2020.

Issue Highlights - November 2020.

TGF-β1 induces amoeboid-to-mesenchymal transition of CD44high oral squamous cell carcinoma cells via miR-422a downregulation through ERK activation and Cofilin-1 phosphorylation.

The objective of this study was to clarify the molecular mechanism of amoeboid-to-mesenchymal transition (AMT) of CD44high oral squamous cell carcinoma (OSCC) cells. Morphology and expression of mesenchymal genes were investigated in CD44high OSCC cells (CD44high OM-1 cells) cultured on laminin-coated soft silicone gel. Additionally, microarray analysis was performed to investigate microRNA (miRNA) expression inhibited by transforming growth factor-β1 (TGF-β1) in CD44high OM-1 cells. When CD44high OM-1 cells were cultured on 2.0-kPa laminin-coated silicone gel, the cells exhibited an amoeboid-like round morphology. Cofilin-1 expression was found in the nucleus and cytoplasm of amoeboid-like CD44high OM-1 cells. The invasive capacity was significantly reduced after Cofilin-1 knockdown. Additionally, Cofilin-1 knockdown cells had an irregularly extended shape. Phosphorylated Cofilin-1 was significantly upregulated by TGF-β1. Additionally, TGF-β1 enhanced N-cadherin and Snail mRNA expression and induced a spindle-shaped morphology. ERK1/2 phosphorylation was induced by TGF-β1. Microarray analysis revealed that miR-422a exhibited the greatest downregulation (fold change: 0.22) in the presence of TGF-β1. Importantly, TGF-β1-inhibited miR-422a expression was recovered by the ERK inhibitor or ERK1/2 knockdown. Additionally, miR-422a inhibitor-transfected CD44high OM-1 cells exhibited high N-cadherin and Snail mRNA expression. Furthermore, Cofilin-1 knockdown and miR-422a inhibition induced a spindle cell morphology. Cofilin-1 is involved in the invasive ability of CD44high OSCC cells. TGF-β1 contributes to AMT by downregulation of miR-422a via ERK activation and Cofilin-1 phosphorylation. Our findings suggest that miR-422a and Cofilin-1 play major roles in the maintenance of amoeboid-like CD44high cells.

Functional inhibition of regulatory CD4+CD25+T cells in peripheral blood of patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune bullous disease affecting the skin and mucous membranes. Its pathogenic mechanism is still not fully understood. Regulatory T cells (Tregs) have been reported to play a significant role in regulating immune homeostasis in autoimmune disorders, such as PV. To investigate the potential role of Tregs in the immunopathogenesis of PV. We enrolled 15 patients with PV and 15 healthy controls (HCs). Peripheral blood samples were collected from all participants before treatment. This was followed by flow cytometric, real-time reverse transcription PCR, and in vitro inhibition-based functional assays to explore the immunopathogenesis of Tregs in PV. Our results showed no statistically significant differences in total CD4+CD25+ cells and CD4+CD25high cells. In addition, expression levels of FOXP3 mRNA and the corresponding FOXP3 protein remained unchanged in the patients with PV and the HCs. However, the in vitro suppressive activity of CD4+CD25+ T cells was impaired in patients with PV compared with HCs. Our observations suggest that inhibition of suppressive activity of Treg cells may be involved in the pathogenesis of PV.

In Vitro Studies to Define the Cell-Surface and Intracellular Targets of Polyarginine-Conjugated Sodium Borocaptate as a Potential Delivery Agent for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) requires pharmaceutical innovations and molecular-based evidence of effectiveness to become a standard cancer therapeutic in the future. Recently, in Japan, 4-borono-L-phenylalanine (BPA) was approved as a boron agent for BNCT against head and neck (H&N) cancers. H&N cancer appears to be a suitable target for BPA-BNCT, because the expression levels of L-type amino acid transporter 1 (LAT1), one of the amino acid transporters responsible for BPA uptake, are elevated in most cases of H&N cancer. However, in other types of cancer including malignant brain tumors, LAT1 is not always highly expressed. To expand the possibility of BNCT for these cases, we previously developed poly-arginine peptide (polyR)-conjugated mercaptoundecahydrododecaborate (BSH). PolyR confers the cell membrane permeability and tumor selectivity of BSH. However, the molecular determinants for the properties are not fully understood. In this present study, we have identified the cluster of differentiation 44 (CD44) protein and translational machinery proteins as a major cell surface target and intracellular targets of BSH-polyR, respectively. CD44, also known as a stem cell-associated maker in various types of cancer, is required for the cellular uptake of polyR-conjugated molecules. We showed that BSH-polyR was predominantly delivered to a CD44High cell population of cancer cells. Once delivered, BSH-polyR interacted with the translational machinery components, including the initiation factors, termination factors, and poly(A)-biding protein (PABP). As a proof of principle, we performed BSH-polyR-based BNCT against glioma stem-like cells and revealed that BSH-polyR successfully induced BNCT-dependent cell death specifically in CD44High cells. Bioinformatics analysis indicated that BSH-polyR would be suitable for certain types of malignant tumors. Our results shed light on the biochemical properties of BSH-polyR, which may further contribute to the therapeutic optimization of BSH-BNCT in the future.

Reactive oxygen species levels differentiate CD34+ human progenitors based on CD38 expression.

Low reactive oxygen species (ROS) levels are well-established characteristics of mouse hematopoietic stem cells (HSCs). However, little is known about these levels in human HSCs. This study aimed at quantifying ROS levels in human CD34+ CD38low and CD34+ CD38high human progenitors from bone marrow, cord blood and cells mobilized for autologous HSC transplantation. A specifically devised multiparameter flow cytometry method was used to quantify ROS levels (H2 DCFDA staining) in sub-populations of primary cells. Results were confirmed by assessing gene expression level of the ROS scavenger GPX3, a key determinant of HSC self-renewal, in sorted CD34+ CD38low and CD34+ CD38high cells. CD34+ CD38low cells from bone marrow and cord blood displayed significantly lower levels of ROS than CD34+ CD38high cells and other leukocytes. Moreover, the correlation between ROS and GPX3 expression was verified in sorted CD34+ CD38low and CD34+ CD38high cells. These results confirm, in human, data previously reported in mice. Moreover, the flow cytometry assay we developed could allow for a more precise enumeration of repopulating primitive progenitors in the course of HSC transplantation.

Abstract B32: Impact of cancer-associated fibroblasts on head and neck cancer stem cells

Abstract Background: The tumor microenvironment (TME) is made up of epithelial cancer cells and mesenchymal stromal cells including tumor-infiltrating lymphocytes, cancer-associated fibroblasts (CAFs) and endothelial cells. Within the cancer cell population are cancer stem cells (CSC), a subpopulation of cells defined as having the capacity for self-renewal, tumorigenesis, and the ability to recapitulate a heterogeneous tumor. The interaction of the TME with cancer cells remains of interest. The focus of this study is to investigate the effect of patient-derived CAFs on head and neck CSC and tumor activity. Methods: Patient-derived head and neck cancer cell lines and patient-derived fibroblasts lines have previously been established by our group. Tumor cells were grown alone or in combination with patient-matched fibroblasts in a coculture chamber system, allowing for mixture of media but not cells. Scratch tests were performed and closure rates were compared between the two conditions (tumor cells alone and coculture). Tumor cells were then grown alone or in direct contact with matched fibroblasts for one week. Cancer cells were selected for based on epithelial cell adhesion molecule (EpCAM) status and in vitro tumor growth and stem cells percentages (defined as proportion of CD44-high and ALDH-positive cells) were calculated. Results: Patient-derived cancer cells cocultured with matched fibroblasts showed a significant increase in closure rates compared with tumor cells alone at three defined time points (p=0.03-0.002). Coculture conditions resulted in greater in vitro tumor growth after one week (6.4-fold vs. 3.7-fold increase) and an increased proportion of CD44-high tumor cells (9% vs. 5%). The effect of coculture on ALDH-positive percentages was variable. Conclusion: CAFs have previously been shown to potentiate tumorigenesis and metastases. We validated these findings with matched patient derived tumor cells and CAFs. We were then able to demonstrate that CAFs increase the proportion of head and neck CSC, suggesting the mechanism for CAF-associated increased tumor activity may be mediated through CSC. Further studies investigating the relationship between CAFs and CSC are warranted. Citation Format: Molly E. Heft Neal, John Henry J. Owen, Matthew E. Spector, Thomas E. Carey, Mark E.P. Prince, Steven B. Chinn. Impact of cancer-associated fibroblasts on head and neck cancer stem cells [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B32.

Salvia miltiorrhiza inhibits allospecific memory CD8+ T cell generation and allograft rejection in mice

Abstract It’s well known that memory T cells pose a threat to long-term allograft survival. The objective of this study is to suppress generation of donor-specific memory T cells via short-term treatment with a natural and non-cytotoxic herb. Previous studies have demonstrated that Chinese herb Salvia miltiorrhiza (Danshen) regulates immune responses. In this study, B6 mice were transplanted with BALB/c islets and treated with Danshen and/or low doses of rapamycin (0.25 mg/kg/day). To generate allospecific memory T cells, Thy1.1+ CD8+CD44low T cells were sorted by FACSAria and injected into B6 recipients one day before transplantation. Graft-infiltrating T cell proliferation was determined by BrdU staining while the memory cells were enumerated via FACS. We found that Danshen alone significantly extended islet allograft survival (MST = 28 vs. 13 days, n=9, P&amp;lt;0.05). Interestingly, administration of both Danshen and low doses of rapamycin further prolonged islet allograft survival (MST = 83 versus 28 days), with 44% of recipients achieving long-term islet graft survival (&amp;gt;100 days). Moreover, Danshen suppressed generation of memory CD8+ T cells compared to control two months after transplantation (Thy1.1+CD8+CD44high cell numbers: 1.3±0.2 vs. 2.5±0.3, ×104/LN, P&amp;lt;0.05 and 4.3±0.6 vs. 8.5±0.9, ×105/spleen, P&amp;lt;0.05) while Danshen plus rapamycin further reduced CD8+ memory T cells compared to Danshen alone (0.7±0.1 vs. 1.3±0.2/LN, P&amp;lt;0.05 and 2.6±0.4 vs. 4.3±0.6,/spleen, P&amp;lt;0.05). Finally, Danshen alone inhibited graft-infiltrating T cell proliferation at one week (52±4 vs. 35±3 %, P&amp;lt;0.05). Thus, Danshen inhibited donor-specific memory CD8+ T cell generation and promoted induction of long-term murine allograft survival.

Salinomycin Derivatives Kill Breast Cancer Stem Cells by Lysosomal Iron Targeting.

Salinomycin (1) exhibits a large spectrum of biological activities including the capacity to selectively eradicate cancer stem cells (CSC), making it and its derivatives promising candidates for the development of drug leads against CSC. It has been previously shown that salinomycin and its C20-propargylamine derivative (Ironomycin (2)) accumulate in lysosomes and sequester iron in this organelle. Herein, a library of salinomycin derivatives is reported, including products of C20-amination, C1-esterification, C9-oxidation, and C28-dehydration. The biological activity of these compounds is evaluated against transformed human mammary epithelial HMLER CD24low /CD44high cells, a well-established model of breast CSC, and HMLER CD24high /CD44low cells deprived of CSC properties. Unlike other structural alterations, derivative 4, which displays a cyclopropylamine at position C20, showed a strikingly low IC50 value of 23 nm against HMLER CD24low /CD44high cells. This study provides highly selective molecules to target the CSC niche, a potential interesting advance for drug development to prevent cancer resistance.