Detection of Circulating Tumor Cell Subpopulations in Patients with Head and Neck Squamous Cell Carcinoma (HNSCC)

Background

Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples.

Methods

20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed.

Results

We were able to detect cells with epithelial properties like CK+/N-cadherin−/CD45− and CK+/CD133−/CD45− as well as cells with mesenchymal features such as N-cadherin+/CK−/CD45− and cells with both characteristics like N-cadherin+/CK+/CD45−. We also observed cells showing stem cell-like features like CD133+/CK−/CD45− and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45−. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1).

Conclusions

This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.     

Technical Performance Evaluation of the MyT4 Point of Care Technology for CD4+ T Cell Enumeration

Objective

Though absolute CD4+ T cell enumeration is the primary gateway to antiretroviral therapy initiation for HIV-positive patients in all developing countries, patient access to this critical diagnostic test is relatively poor. We technically evaluated the performance of a newly developed point-of-care CD4+ T cell technology, the MyT4, compared with conventional CD4+ T cell testing technologies.

Design

Over 250 HIV-positive patients were consecutively enrolled and their blood tested on the MyT4, BD FACSCalibur, and BD FACSCount.

Results

Compared with the BD FACSCount, the MyT4 had an r² of 0.7269 and a mean bias of −23.37 cells/µl. Compared with the BD FACSCalibur, the MyT4 had an r² of 0.5825 and a mean bias of −46.58 cells/µl. Kenya currently uses a CD4+ T cell test threshold of 350 cells/µl to determine patient eligibility for antiretroviral therapy. At this threshold, the MyT4 had a sensitivity of 95.3% (95% CI: 88.4–98.7%) and a specificity of 87.9% (95% CI: 82.3–92.3%) compared with the BD FACSCount and sensitivity and specificity of 88.2% (95% CI: 79.4–94.2%) and 84.2% (95% CI: 78.2–89.2%), respectively, compared with the BD FACSCalibur. Finally, the MyT4 had a coefficient of variation of 12.80% compared with 14.03% for the BD FACSCalibur.

Conclusions

We conclude that the MyT4 performed well at the current 350 cells/µl ART initiation eligibility threshold when used by lower cadres of health care facility staff in rural clinics compared to conventional CD4+ T cell technologies.     

Cross Talk with Hematopoietic Cells Regulates the Endothelial Progenitor Cell Differentiation of CD34 Positive Cells

Introduction

Despite the crucial role of endothelial progenitor cells (EPCs) in vascular regeneration, the specific interactions between EPCs and hematopoietic cells remain unclear.

Methods

In EPC colony forming assays, we first demonstrated that the formation of EPC colonies was drastically increased in the coculture of CD34⁺ and CD34⁻ cells, and determined the optimal concentrations of CD34⁺ cells and CD34⁻ cells for spindle-shaped EPC differentiation.

Results

Functionally, the coculture of CD34⁺ and CD34⁻ cells resulted in a significant enhancement of adhesion, tube formation, and migration capacity compared with culture of CD34⁺ cells alone. Furthermore, blood flow recovery and capillary formation were remarkably increased by the coculture of CD34⁺ and CD34⁻ cells in a murine hind-limb ischemia model. To elucidate further the role of hematopoietic cells in EPC differentiation, we isolated different populations of hematopoietic cells. T lymphocytes (CD3⁺) markedly accelerated the early EPC status of CD34⁺ cells, while macrophages (CD11b⁺) or megakaryocytes (CD41⁺) specifically promoted large EPC colonies.

Conclusion

Our results suggest that specific populations of hematopoietic cells play a role in the EPC differentiation of CD34⁺ cells, a finding that may aid in the development of a novel cell therapy strategy to overcome the quantitative and qualitative limitations of EPC therapy.     

Cortisol Patterns Are Associated with T Cell Activation in HIV

Objective

The level of T cell activation in untreated HIV disease is strongly and independently associated with risk of immunologic and clinical progression. The factors that influence the level of activation, however, are not fully defined. Since endogenous glucocorticoids are important in regulating inflammation, we sought to determine whether less optimal diurnal cortisol patterns are associated with greater T cell activation.

Methods

We studied 128 HIV-infected adults who were not on treatment and had a CD4⁺ T cell count above 250 cells/µl. We assessed T cell activation by CD38 expression using flow cytometry, and diurnal cortisol was assessed with salivary measurements.

Results

Lower waking cortisol levels correlated with greater T cell immune activation, measured by CD38 mean fluorescent intensity, on CD4⁺ T cells (r = −0.26, p = 0.006). Participants with lower waking cortisol also showed a trend toward greater activation on CD8⁺ T cells (r = −0.17, p = 0.08). A greater diurnal decline in cortisol, usually considered a healthy pattern, correlated with less CD4⁺ (r = 0.24, p = 0.018) and CD8⁺ (r = 0.24, p = 0.017) activation.

Conclusions

These data suggest that the hypothalamic-pituitary-adrenal (HPA) axis contributes to the regulation of T cell activation in HIV. This may represent an important pathway through which psychological states and the HPA axis influence progression of HIV.     

Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

Background

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.

Objective

The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.

Methods and Results

We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84^−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84^−/− mice. In vivo, Cd84^−/− mice exhibited unaltered hemostatic function and arterial thrombus formation.

Conclusion

These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.     

Jak3 Is Involved in Dendritic Cell Maturation and CCR7-Dependent Migration

Background

CCR7-mediated signalling is important for dendritic cell maturation and homing to the lymph nodes. We have previously demonstrated that Jak3 participates in the signalling pathway of CCR7 in T lymphocytes.

Methodology and Principal Findings

Here, we used Jak3^−/− mice to analyze the role of Jak3 in CCR7-mediated dendritic cells migration and function. First, we found no differences in the generation of DCs from Jak3^−/− bone marrow progenitors, when compared to wild type cells. However, phenotypic analysis of the bone marrow derived DCs obtained from Jak3^−/− mice showed reduced expression of co-stimulatory molecules compared to wild type (Jak3^+/+). In addition, when we analyzed the migration of Jak3^−/− and Jak3^+/+ mature DCs in response to CCL19 and CCL21 chemokines, we found that the absence of Jak3 results in impaired chemotactic responses both in vitro and in vivo. Moreover, lymphocyte proliferation and contact hypersensitivity experiments showed that DC-mediated T lymphocyte activation is reduced in the absence of Jak3.

Conclusion/Significance

Altogether, our data provide strong evidence that Jak3 is important for DC maturation, migration and function, through a CCR7-mediated signalling pathway.     

Targeting Serum Glucocorticoid-Regulated Kinase-1 in Squamous Cell Carcinoma of the Head and Neck: A Novel Modality of Local Control

Purpose

The inhibition of serum glucocorticoid-regulated kinase-1 (SGK-1) has been found to decrease growth of colon and prostate cancer cells. The purpose of this study is to evaluate the therapeutic effect of SGK-1 inhibition in head and neck squamous cell carcinoma (SCC).

Experimental Design

Human head and neck tumors (HTB41/43) were established in athymic mice. Growth rates between mice treated with vehicle (PBS) injection (group 1, n = 5), SGK-1 Inhibitor GSK 650394 (group 2, n = 6), systemic cisplatin (group 3, n = 6), and a combination of SGK-1 Inhibitor and cisplatin (group 4, n = 6) were compared using repeated measures one-way ANOVA with Newman-Keuls Multiple Comparison Test. Tumor cells were subsequently submitted to further analyses.

Results

At the end of the experiment mean tumor sizes were 122.33+/−105.86, 76.73+/−36.09, 94.52+/−75.92, and 25.76+/−14.89 mm² (mean +/− SD) for groups 1 to 4. Groups 2 and 3 showed decreased tumor growth compared to controls (p<0.001). Group 4 displayed even greater growth suppression (p<0.0001). Importantly, group 4 fared better than group 3 (p<0.001). CD44 expression was reduced in group 2 (p<0.05), and to an even greater extent in groups 3 and 4 (p<0.0025). A trend towards reduction of HER 2 expression was noted in group 4.

Conclusions

SGK-1 inhibition suppresses tumor growth, and in combination with systemic cisplatin exceeds the effect of cisplatin alone. Decreased expression of CD44 and HER 2 implies depletion of tumor stem cells, and less tumorigenicity. SGK-1 inhibition represents a potential modality of local control for palliation in advanced cases.     

Hematopoietic Stem/Progenitor Cell Proliferation and Differentiation Is Differentially Regulated by High-Density and Low-Density Lipoproteins in Mice

Rationale

Hematopoietic stem/progenitor cells (HSPC) are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation.

Objectives

We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells.

Methods and Results

HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr^−/−) mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB). In addition, an increased proportion of BM HSPC was in G₂M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP) increased in BM of LDLr^−/− mice. When BM Lin-Sca-1+cKit+ (i.e. “LSK”) cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin−) cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL) or reconstituted HDL (rHDL), the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro.

Conclusion

Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.     

Central Muscarinic Cholinergic Activation Alters Interaction between Splenic Dendritic Cell and CD4+CD25- T Cells in Experimental Colitis

Background

The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4⁺/CD25⁻T cell activation in the context of experimental colitis.

Methods

The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4⁺CD25⁻T cell co-culture were determined.

Results

McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4⁺CD25⁻T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy.

Conclusions

Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.     

Study of Regulatory T-Cells in Patients with Gastric Malt Lymphoma: Influence on Treatment Response and Outcome

Purpose

FOXP3+ regulatory T cells (Treg) play an essential role in modulating host responses to tumors and infections. The role of these cells in the pathogenesis of MALT lymphomas remains unknown. The aims of the study were to quantify the number of infiltrating FOXP3+ and CD3+ cells in patients with gastric MALT lymphoma at diagnosis and to study kinetics of these cells and CD20+ tumor cells after treatment and during long-term follow-up.

Methods

FOXP3+, CD3+ and CD20+ cells were analyzed by immunohistochemistry and the number of cells was quantified using a micrometric ocular. Samples of 35 patients with gastric MALT lymphoma at diagnosis and after treatment were included. Diagnostic samples were compared to 19 cases of chronic gastritis and diffuse large B-cell lymphoma (DLBCL) of the stomach.

Results

The median number of FOXP3+ infiltrating cells was higher (27 cells/cm²) in gastric MALT patients than in DLBCL (10 cells; p = 0.162) but similar to chronic gastritis (20 cells; p = 0.605). No characteristic or specific distribution pattern of infiltrating FOXP3+ cells was found. Gastric MALT lymphoma patients responding to bacterial eradication therapy had higher number of FOXP3+ cells at study entry. Kinetics of both infiltrating FOXP3+ cells and tumor CD20+ cells were strongly dependent on the treatment administered.

Discussion

Gastric MALT lymphomas have a number of Treg cells more similar to chronic gastritis than to DLBCL. Patients with higher number of tumor infiltrating FOXP3+ cells at study entry seem to have better response to antibiotics. Kinetics of Treg and tumor cells are influenced by type of treatment.     

Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu,Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

Purpose

The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 ^−/−) mouse.

Methods

Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 ^−/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed.

Results

Corneal vital staining scores in the Sod1 ^−/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 ^−/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 ^−/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 ^−/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 ^−/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 ^−/− mice.

Conclusions

Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 ^−/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations.     

Dynamic Transcription of Long Non-Coding RNA Genes during CD4+ T Cell Development and Activation

Background

Recent evidence shows that long non-coding RNA (LncRNA) play important regulatory roles in many biology process, including cell development, activation and oncogenesis. However, the roles of these LncRNAs in the development and activation of CD4⁺ T cells, which is an important component of immune response, remain unknown.

Results

To predict the function of LncRNA in the development and activation of CD4⁺ T cells, first, we examined the expression profiles of LncRNAs and mRNAs in CD4⁻CD8⁻ (DN), CD4⁺CD8⁺ (DP), CD4⁺CD8⁻, and activated CD4⁺CD8⁻ T cells in a microarray analysis and verified these results by real time PCRs (qPCR). We found that the expression of hundreds of LncRNAs significantly changed in each process of developmental transition, including DN into DP, DP into CD4⁺CD8⁻, and CD4⁺CD8⁻ into activated CD4⁺ T cells. A Kendall distance analysis suggested that the expression of LncRNAs in DN, DP, CD4⁺CD8⁻ T cells and activated CD4⁺ T cells were correlated with the expression of mRNAs in these T cells. The Blat algorithm and GO analysis suggested that LncRNAs may exert important roles in the development and activation of CD4⁺ T cells. These roles included proliferation, homeostasis, maturation, activation, migration, apoptosis and calcium ion transportation.

Conclusion

The present study found that the expression profiles of LncRNAs in different stages of CD4⁺ T cells are distinguishable. LncRNAs are involved in the key biological process in CD4⁺ T cell development and activation.     

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Background

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.

Methodology/Principal Findings

After transplantation with CD34⁺CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2^−/−IL-2Rγc^−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19⁺CD27⁺ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.

Conclusion/Significance

This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.     

Period2 Deficiency Blunts Hypoxia-Induced Mobilization and Function of Endothelial Progenitor Cells

Background

In the clinic, variations in circadian rhythm are evident in patients with cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. In this study, we focused on the role of the circadian gene period2 (per2) in mobilization and function of endothelial progenitor cells (EPCs) in vitro and in vivo after myocardial infarction (MI) in mice.

Methods and Results

MI was produced by surgical ligation of the left anterior descending coronary artery in mice with and without per2 deficiency. Trans-thoracic echocardiography was used to evaluate cardiac function in mice. Per2^−/− mice with MI showed decreased cardiac function and increased infarct size. The number of CD34+ cells and capillary density were decreased in the myocardium of per2^−/− mice on immunohistochemistry. Flow cytometry revealed decreased number of circulating EPCs in per2^−/− mice after MI. In vitro, per2^−/− EPCs showed decreased migration and tube formation capacity under hypoxia. Western blot analysis revealed inhibited activation of extracellular signal-regulated kinase and Akt signaling in the bone marrow of per2^−/− mice and inhibited PI3K/Akt expression in per2^−/− EPCs under hypoxia.

Conclusions

Per2 modulates EPC mobilization and function after MI, which is important to recovery after MI in mice.     

A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.

Objectives

We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.

Methods

Subjects were randomly assigned to receive either a nominal dose of 5×10⁹ CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.

Principal Findings

One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.

Conclusions

This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.

Trial Registration

Controlled-trials.comISRCTN91111837     

A T Cell-Inducing Influenza Vaccine for the Elderly: Safety and Immunogenicity of MVA-NP+M1 in Adults Aged over 50 Years

Background

Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults.

Methods

Thirty volunteers (aged 50–85) received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10⁸ plaque forming units (pfu). Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP) and matrix protein 1 (M1) was determined by interferon-gamma (IFN-γ) ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8⁺ T cells, T cell receptor (TCR) gene expression was evaluated using an unbiased molecular approach.

Results

Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4⁺ and CD8⁺ T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8⁺ T cells, which displayed a predominant CD27⁺CD45RO⁺CD57⁻CCR7⁻ phenotype both before and after vaccination.

Conclusions

MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00942071","term_id":"NCT00942071"}}NCT00942071     

Heterogeneity of Functional Properties of Clone 66 Murine Breast Cancer Cells Expressing Various Stem Cell Phenotypes

Introduction

Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous.

Methods

Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis.

Results

The proportion of cells expressing CD44^highCD24^low/neg, side population (SP) cells, ALDH1⁺, CD49f^high, CD133^high, and CD34^high differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1⁺, CD34^low, and CD49f^high suggested properties of transit amplifying cells. Colony formation was higher from ALDH1⁻ and non-SP cells than ALDH1⁺ and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than “non-stem” cells. Fewer SP cells were needed to form tumors than ALDH1⁺ cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined.

Conclusions

These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.     

Agonistic Anti-TIGIT Treatment Inhibits T Cell Responses in LDLr Deficient Mice without Affecting Atherosclerotic Lesion Development

Objective

Co-stimulatory and co-inhibitory molecules are mainly expressed on T cells and antigen presenting cells and strongly orchestrate adaptive immune responses. Whereas co-stimulatory molecules enhance immune responses, signaling via co-inhibitory molecules dampens the immune system, thereby showing great therapeutic potential to prevent cardiovascular diseases. Signaling via co-inhibitory T cell immunoglobulin and ITIM domain (TIGIT) directly inhibits T cell activation and proliferation, and therefore represents a novel therapeutic candidate to specifically dampen pro-atherogenic T cell reactivity. In the present study, we used an agonistic anti-TIGIT antibody to determine the effect of excessive TIGIT-signaling on atherosclerosis.

Methods and Results

TIGIT was upregulated on CD4⁺ T cells isolated from mice fed a Western-type diet in comparison with mice fed a chow diet. Agonistic anti-TIGIT suppressed T cell activation and proliferation both in vitro and in vivo. However, agonistic anti-TIGIT treatment of LDLr^−/− mice fed a Western-type diet for 4 or 8 weeks did not affect atherosclerotic lesion development in comparison with PBS and Armenian Hamster IgG treatment. Furthermore, elevated percentages of dendritic cells were observed in the blood and spleen of agonistic anti-TIGIT-treated mice. Additionally, these cells showed an increased activation status but decreased IL-10 production.

Conclusions

Despite the inhibition of splenic T cell responses, agonistic anti-TIGIT treatment does not affect initial atherosclerosis development, possibly due to increased activity of dendritic cells.     

In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

Background

The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro.

Methodology

Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture.

Principal Findings

The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line.

Conclusions

The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).     

Functional Glycosylation of Dystroglycan Is Crucial for Thymocyte Development in the Mouse

Background

Alpha-dystroglycan (α-DG) is a cell surface receptor providing a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton. During its biosynthesis, α-DG undergoes specific and unusual O-glycosylation crucial for its function as a high-affinity cellular receptor for ECM proteins.

Methodology/Principal Findings

We report that expression of functionally glycosylated α-DG during thymic development is tightly regulated in developing T cells and largely confined to CD4⁻CD8⁻ double negative (DN) thymocytes. Ablation of DG in T cells had no effect on proliferation, migration or effector function but did reduce the size of the thymus due to a significant loss in absolute numbers of thymocytes. While numbers of DN thymocytes appeared normal, a marked reduction in CD4⁺CD8⁺ double positive (DP) thymocytes occurred. In the periphery mature naïve T cells deficient in DG showed both normal proliferation in response to allogeneic cells and normal migration, effector and memory T cell function when tested in acute infection of mice with either lymphocytic choriomeningitis virus (LCMV) or influenza virus.

Conclusions/Significance

Our study demonstrates that DG function is modulated by glycosylation during T cell development in vivo and that DG is essential for normal development and differentiation of T cells.