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Introduction Measles, mumps, and rubella (MMR) and varicella (VAR) vaccines are the two vaccines administered in large recruit training sites (RTS) that require a single-use syringe to be prefilled with the diluent (ie water) before vaccine reconstitution. Since there are no preservatives in either MMR or VAR vaccines, it is critical to maintain the diluent sterile to ensure the sterility of the reconstituted vaccine. The Department of Defense/Defense Health Agency has instructions on reconstitution of lyophilized vaccines and guidelines for their storage. Vaccine manufacturers provide instructions on how to properly store the diluent. However, there is no clear guidance or standard operating procedures regarding the best practice for preparation and storage of the syringes prefilled with diluent. Various RTS across all four services have their respective routines to best fit their vaccination requirements. Currently, there are no available data on the sterility status of the diluent prepared using these various routines before they are used to reconstitute vaccines. Materials and Methods We investigated the sterility of the diluent (ie water) in prefilled syringes prepared using routines practiced at various RTS. Diluent was drawn up into single syringes and was kept under various conditions (4 °C or room temperature for overnight up to 24 hours) used by various RTS. At indicated time, diluent was injected into sterile vials and the sterility of the diluent was determined by monitoring the presence/growth of bacteria (including aerobic bacteria, mycoplasma, and an obligate intracellular bacterium, Coxiella burnetii), fungi, and viruses for up to 21 days after inoculation into proper and specific culture media. Both traditional cell culture and molecular assays were used to demonstrate the presence or absence of contamination that may compromise the sterility of the diluent. Results Our results demonstrate that the diluent, after being drawn up to fill the syringe, maintains sterility after storage for overnight up to 24 hours at room temperature or 4 °C with or without recapping the syringes, suggesting that current vaccine reconstitution routines practiced at large military RTS are safe. Conclusions Our results demonstrate that in spite of variations in current practices used in various RTS, the diluent in the prefilled syringe tested from each site maintains its sterility and was determined to be safe for use in military health system-wide vaccine reconstitution.

We previously reported the efficacy of prime-boost vaccination using three tetravalent (T) dengue vaccines, DNA (TDNA), purified inactivated vaccine (TPIV), and live attenuated vaccine (TLAV). We demonstrated that the TPIV/TLAV prime-boost vaccination yielded the highest and most durable neutralizing antibodies and 100% protection to all 4 serotypes of dengue virus in rhesus macaques. This study compares gene transcription, T and B cell responses elicited by these prime-boost combinations in rhesus macaques. This study shows that the TLAV vaccine increased the expression of the innate immune genes, DDX58 and TLR7, IL1A, IL1B, TNF, CXCL8, CXCL10, IRF1, IRF7, and IFNB, more robustly as compared to TDNA and TPIV vaccines. Overall, two doses of TDNA and one dose of TLAV efficiently elicited a T cell IFNγ response to PrM/E with a comparable magnitude. Compared to TDNA vaccine, the TLAV vaccine elicited additional IFNγ response to C, NS1, NS3, and NS5. The TPIV vaccine alone produced poor IFNγ response; however, the TLAV significantly boosted its IFNγ response. The T cell response repertoire associated with TPIV/TLAV prime-boost was to both the structural C/PrM/E and NS proteins, and the T cells were multifunctional as the CD4+ T cells produced IFNγ, TNF α, and IL2 and the CD8+ cells produced TNF α and IFNγ. Opposite to the pattern of CMI, the TPIV vaccine alone elicited the highest BMem compared to the other two vaccines, which continuously remained as the highest after boosting. In summary, the TDNA and TLAV vaccines elicited a strong T cell response whereas the TPIV vaccine elicited a superior BMem. The T cell response of the TPIV vaccine was significantly boosted by the TLAV vaccine. The elevated T cell response may have provided T cell help for a sustained antibody response for TPIV/TLAV vaccines, which is required for a protective immunity against a live virus challenge.

Antibody (Ab)-dependent enhancement (ADE) is a hypothesized mechanism of increased disease severity during secondary dengue virus (DENV) infection. This study investigates Ab-dependent cell cytotoxicity (ADCC) in counteracting ADE. In our system, DENV and DENV-immune sera were added to peripheral blood mononuclear cells (PBMCs), and ADE and NK cell activation were simultaneously monitored. ADE was detected in monocytes and a concurrent activation of NK cells was observed. Activated NK cells expressed IFN-γ and CD107a. IFN-γ was detected at 24 hours (24 h) followed by a rapid decline; CD107a expression peaked at 48 h and persisted for >7 days. Optimal activation of NK cells required the presence of enhancement serum together with ADE-affected monocytes and soluble factors, suggesting the coexistence of the counteractive ADCC Abs, in the same ADE-serum, capable of strongly promoting NK cell activation. The function of NK cells against ADE was demonstrated using a depletion assay. NK cell-depleted PBMCs had increased ADE as compared to whole PBMCs. Conversely, adding activated NK cells back into the NK-depleted-PBMCs or to purified monocytes decreased ADE. Blocking IFN-γ expression also increased ADE. The study suggests that under ADE conditions, NK cells can be activated by ADCC Abs and can control the magnitude of ADE.

Highlights • Assay that assesses influenza antibodies capable of NK cell degranulation. • Description of NK cell degranulation titer determination by CD107a expression. • Positive correlation between influenza HAI titers and NK cell degranulation titers. • Transchromosomic bovine intravenous immunoglobulin therapy has high NK cell titer., This study describes an antibody-dependent NK cell degranulation assay, as a biomarker to assess antibody-dependent cellular cytotoxicity (ADCC) response in influenza plasma and for antibody therapies against influenza infection. The concentration of neutralizing antibodies (NAbs) against the hemagglutinin receptor of influenza viruses is a current determinant in protection against infection, particularly following receipt of the seasonal influenza vaccine. However, this is a limited assessment of protection, because: (i) NAb titers that incur full protection vary; and (ii) NAb titers do not account for the entire breadth of antibody responses against viral infection. Previous reports have indicated that antibodies that prime ADCC play a vital role in controlling influenza infections, and thus should be quantified for assessing protection against influenza. This report demonstrates a non-radioactive assay that assesses NK cell activation as a marker of ADCC, in which NK cells interact with opsonized viral antigen expressed on the surface of infected Raji target cells resulting in effector cell degranulation (surrogate CD107a expression). A positive correlation was determined between HAI titers and sustained NK cell activation, although NK cell activation was seen in plasma samples with HAI titers below 40 and varied amongst samples with high HAI titers. Furthermore, sustained NK cell degranulation was determined for influenza-vaccinated transchromosomic bovine intravenous immunoglobulin, indicating the potential utility of this therapy for influenza treatment. We conclude that this assay is reproducible and relevant.

AIM To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT). METHODS In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR. RESULTS Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai, Zeb and Twist family of EMT transcription factors. CONCLUSION Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

Several studies published to date report associations between human leukocyte antigen (HLA) alleles and different types of Kaposi’s Sarcoma (KS). However, there is little concordance between the HLA alleles identified and the populations studied. To test whether HLA alleles associate with KS in a Cameroonian case–control study, we performed high-resolution HLA typing in KSHV seropositive individuals. Among HIV-positive individuals, carriers of HLA-B*14:01 were at a significantly higher risk of AIDS-KS (p = 0.033). For HIV-negative patients, a gene-wise comparison of allele frequencies identified the HLA-B (p = 0.008) and -DQA1 (p = 0.002) loci as possible risk factors for endemic KS. Our study provides additional understanding of genetic determinants of KS and their implications in disease pathogenesis. Further validation of these findings is needed to define the functional relevance of these associations.

Zika virus (ZIKV) recently caused a pandemic complicated by Guillain-Barre syndrome (GBS) and birth defects. ZIKV is structurally similar to the dengue viruses (DENV) and in vitro studies suggest antibody dependent enhancement occurs in ZIKV infections preceded by DENV; however, the clinical significance of this remains unclear. We undertook a PRISMA-adherent systematic review of all current human and non-human primate (NHP) data to determine if prior infection with DENV, compared to DENV-naïve hosts, is associated with a greater risk of ZIKV clinical complications or greater ZIKV peak viremia in vivo. We identified 1146 studies in MEDLINE, EMBASE and the grey literature, of which five studies were eligible. One human study indicated no increase in the risk of GBS in ZIKV infections with prior DENV exposure. Two additional human studies showed a small increase in ZIKV viremia in those with prior DENV exposure; however, this was not statistically significant nor was it associated with an increase in clinical severity or adverse pregnancy outcomes. While no meta-analysis was possible using human data, a pooled analysis of the two NHP studies leveraging extended data provided only weak evidence of a 0.39 log10 GE/mL rise in ZIKV viremia in DENV experienced rhesus macaques compared to those with no DENV exposure (p = 0.22). Using a customized quality grading criteria, we further show that no existing published human studies have offered high quality measurement of both acute ZIKV and antecedent DENV infections. In conclusion, limited human and NHP studies indicate a small and non-statistically significant increase in ZIKV viremia in DENV-experienced versus DENV-naïve hosts; however, there is no evidence that even a possible small increase in ZIKV viremia would correlate with a change in ZIKV clinical phenotype. More data derived from larger sample sizes and improved sero-assays are needed to resolve this question, which has major relevance for clinical prognosis and vaccine design., Author summary Zika virus (ZIKV) is a mosquito borne virus that recently caused a large epidemic with some cases complicated by ascending paralysis (Guillain-Barre syndrome) and birth defects. One major concern is that such complications may be more common in those who have had previous infection with the closely related mosquito-borne dengue virus (DENV) which also circulates in the tropics. Here, we undertook a thorough, structured review of all human and high-order animal literature to synthesize the current evidence about whether ZIKV outcomes are worse in those with previous DENV exposure. We further undertook a pooled analysis across the two major non-human primate studies to improve statistical power. We summarize that, in humans and in non-human primates, prior DENV exposure may lead to a small increase in ZIKV viral load during infection. However, we do not show that any possible increase in ZIKV viral replication is associated with a higher rate of Zika complications or Zika clinical severity. We further graded the quality of these published literature and indicate that substantial improvements in the immunological measurement of ZIKV and DENV exposure in humans are needed to answer these and other pertinent questions about these two epidemic pathogens.

The known human tumor viruses include the DNA viruses Epstein-Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV), Merkel cell polyomavirus (MCPyV), human papillomavirus (HPV), and hepatitis B virus (BV). RNA tumor viruses include human T-cell lymphotrophic virus type 1 (HTLV-1) and hepatitis C virus (HCV). The serological identification of antigens/antibodies in serum is a rapidly progressing field with utility for both scientists and clinicians. Serology is useful for conducting seroepidemiology studies and to inform on the pathogenesis and host immune response to a particular viral agent. Clinically, serology is useful for diagnosing current or past infection and for aiding in clinical management decisions. Serology is useful for screening blood donations for infectious agents and for monitoring the outcome of vaccination against these viruses. Serodiagnosis of human tumor viruses has improved in recent years with increased specificity and sensitivity of the assays, as well as reductions in cost and the ability to assess multiple antibody/antigens in single assays. Serodiagnosis of tumor viruses plays an important role in our understanding of the prevalence and transmission of these viruses and ultimately in the ability to develop treatments/preventions for these globally important diseases.

Lung cancer is a major public health problem causing more deaths than any other cancer. A better understanding of the biology of this disease and improvements in treatment are greatly needed. Increasing evidence supports the concept that a rare and specialized population of cancer cells, so-called cancer-initiating cells with stem cell-like characteristics, is responsible for tumor growth, maintenance, and recurrence. Cancer-initiating cells also exhibit characteristics that render them resistant to both radiation and chemotherapy, and therefore they are believed to play a role in treatment failure. This has led to the hypothesis that traditional therapies that indiscriminately kill tumor cells will not be as effective as therapies that selectively target cancer-initiating cells. Investigating putative cancer-initiating cells in lung cancer will greatly benefit the understanding of the origins of this disease and may lead to novel approaches to therapy by suggesting markers for use in either further isolating this population for study or for selectively targeting these cells. This review will discuss (1) lung cancer, (2) stem cells, and the role of cancer-initiating cells in tumorigenesis; (3) markers and functional characteristics associated with lung cancer-initiating cells; and (4) the potential to selectively target this subpopulation of tumor cells.

The study assessed antibody-dependent NK cell degranulation, a biomarker relevant to antibody-dependent cell cytotoxicity (ADCC), to analyze dengue immune sera. We first determined binding intensity of patient sera to the surface of DENV-infected cells and examined the types of antigens expressed on infected cells. Antigens from pre-membrane (PreM) and envelope (E), but not from NS proteins were detected on the surface of infected cells. After adding NK cells to infected target cells previously treated with patient sera, rapid NK cell degranulation was observed. Non-neutralizing patient sera generated comparable NK cell degranulation as that of neutralizing sera, suggesting ADCC may be a protective mechanism apart from Ab neutralization. The level of NK cell degranulation varied dramatically among human individuals and was associated with the level of CD16 expression on NK cells, informing on the complexity of ADCC among human population.

The clinical and pathologic heterogeneity of human breast cancer has long been recognized. Now, molecular profiling has enriched our understanding of breast cancer heterogeneity and yielded new prognostic and predictive information. Despite recent therapeutic advances, including the HER2-specific agent, trastuzumab, locoregional and systemic disease recurrence remain an ever-present threat to the health and well being of breast cancer survivors. By definition, disease recurrence originates from residual treatment-resistant cells, which regenerate at least the initial breast cancer phenotype. The discovery of the normal breast stem cell has reignited interest in the identity and properties of breast cancer stem-like cells and the relationship of these cells to the repopulating ability of treatment-resistant cells. The cancer stem cell model of breast cancer development contrasts with the clonal evolution model, whereas the mixed model draws on features of both. Although the origin and identity of breast cancer stem-like cells is contentious, treatment-resistant cells survive and propagate only because aberrant and potentially druggable signaling pathways are recruited. As a means to increase the rates of breast cancer cure, several approaches to specific targeting of the treatment-resistant cell population exist and include methods for addressing the problem of radioresistance in particular. Refereed/Peer-reviewed

Tumour heterogeneity has several important consequences including: (i) making their classification by morphological and genetic analysis more difficult because of the diversity within single tumours and the common majority of cells as the bulk of a tumour will dominate this classification whether or not these cells are critical for diagnosis or treatment, (ii) treatments may fail to eradicate tumours simply by failing to eliminate one of the cell subtypes within the tumour and (iii) differing abilities of the cell subtypes for dissemination and metastasis. Recently, a rare subpopulation of cells within tumours has been described with the ability to initiate and sustain tumour growth, to resist traditional therapies and to allow for secondary tumour dissemination. These cells are termed tumour-initiating cells (TICs). Understanding tumour heterogeneity will be critical for advancing treatments for cancer that target TIC subpopulations of cells in a tumour able to resist traditional treatments and eliminate them before metastatic disease occurs. It follows that the TICs will be the most important cellular components in the tumour target. Therefore, knowledge of the molecular mechanism(s) of resistance of TICs to treatment and overcoming this problem will be essential in order to develop effective drug strategies for cancer therapy.

Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the study of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.

Thiophene and phenanthrene-9,10-quinone react in acidic solution to produce the deep blue extended quinone (Z,Z)-2,5-di(9-oxo-9,10-dihydro-10-phenanthrylidene)-2,5-dihydrothiophene the structure of which is confirmed by X-ray crystallography. Other aromatic 1,2-diketones give rise to related products.

Phenanthrene-9,10-quinone and P 4 S 10 react with phenols on being heated to give oxathiins, e.g., 9 the dioxin 22 , ethers, e.g., 14 arylphenanthrenols, e.g., 10 and furanols, e.g., 17 in addition to sulfides, e.g., 26 and disulfides, e.g., 25 . Under similar conditions benzil gives thiophene derivatives, e.g., 2 . Most of the reactions appear to proceed via thione intermediates but some of the products are also formed when P 4 S 10 is absent.

Thiophene reacts with aliphatic ketones in aqueous 75% v/v sulfuric acid to give condensation products containing up to five thiophene units. Ketones bearing phenyl substituents yield mono- and di-styrylthiophenes. All the reactions occur at the C-2 and -5 positions of thiophene. 2,5-Bis(α-phenylstyryl)thiophene undergoes photocyclisation to give a dinaphthothiophene.

The reaction of benzil with phenol at 180°C in the presence of SnCl4·5H2O produces a benzofuran, a benzofuranol, a benzodifuran, and a benzofuranone. Anhydrous tin(IV) chloride also gives a benzofuranofuranone. Other phenols and their methyl ethers yield related products. The good yields of the benzo- and naphtho-furanones make the method an attractive alternative to the benzilic acid route to such compounds.

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.

The safety and efficacy of transcatheter clamshell occlusion of patent foramen ovale for relief of severe arterial desaturation and dyspnea in the upright position due to intracardiac shunting were examined in eight patients with excessive risk of surgical patent foramen ovale closure. All patients had successful reduction of intracardiac shunting with an immediate rise in oxygen saturation −95% by implantation of a clamshell device on the atrial septum. Despite two early incidents of device embolization, retrieval and immediate re-implantation, and one patient with nonsustained atrial and ventricular arrhythmias, there were no adverse clinical sequelae. In follow-up evaluation transcatheter clamshell closure of patent foramen ovale has provided persistent relief from shuntrelated arterial desaturation and symptomatology in all living patients. © 1995 Wiley-Liss, Inc.

Treatment of 1,2-di(thien-2-yl)ethanone with aqueous 75% (v/v) sulfuric acid gives a dimer in which the units are linked head-to-head via a thiophene–tetrahydrothiophene bond.

Breast cancer is a heterogeneous disease, composed of tumour cells with differing gene expressions and phenotypes. Very few antigens have been identified and a better understanding of tumour initiating-cells as targets for therapy is critically needed. Recently, a rare subpopulation of cells within tumours has been described with the ability to: (i) initiate and sustain tumour growth; (ii) resist traditional therapies and allow for secondary tumour dissemination; and (iii) display some of the characteristics of stem cells such as self-renewal. These cells are termed tumour-initiating cells or cancer stem cells, or alternatively, in the case of breast cancer, breast cancer stem cells. Previous studies have demonstrated that breast cancer stem cells can be enriched for in "tumoursphere" culture. Proteomics represents a novel way to investigate protein expression between cells. We hypothesise that characterisation of the proteome of the breast cancer line MCF-7 tumourspheres compared to adherent/differentiated cells identifies proteins of novel interest for further isolating or targeting breast cancer stem cells. We present evidence that: (i) the proteome of adherent cells is different to the proteome of cells grown in sphere medium from either early passage (passage 2) or late passage (passage 5) spheres; (ii) that spheres are enriched in expression of a variety of tumour-relevant proteins (including MUC1 and Galectin-3); and (iii) that targeting of one of these identified proteins (galectin-3) using an inhibitor (N-acetyllactosamine) decreases sphere formation/self-renewal of MCF-7 cancer stem cells in vitro and tumourigenicity in vivo. Hence, proteomic analysis of tumourspheres may find use in identifying novel targets for future therapy. The therapeutic targeting of breast cancer stem cells, a highly clinically relevant sub-population of tumour cells, has the potential to eliminate residual disease and may become an important component of a multi-modality treatment of cancer.

Cancer initiating cells (CICs) represent a unique cell population essential for the maintenance and growth of tumors. Most in vivo studies of CICs utilize human tumor xenografts in immunodeficient mice. These models provide limited information on the interaction of CICs with the host immune system and are of limited value in assessing therapies targeting CICs, especially immune-based therapies. To assess this, a syngeneic cancer model is needed. We examined the sphere-forming capacity of thirteen murine lung cancer cell lines and identified TC-1 and a metastatic subclone of Lewis lung carcinoma (HM-LLC) as cell lines that readily formed and maintained spheres over multiple passages. TC-1 tumorspheres were not enriched for expression of CD133 or CD44, putative CIC markers, nor did they demonstrate Hoechst 33342 side population staining or Aldefluor activity compared to adherent TC-1 cells. However, in tumorsphere culture, these cells exhibited self-renewal and long-term symmetric division capacity and expressed more Oct-4 compared to adherent cells. HM-LLC sphere-derived cells exhibited increased Oct-4, CD133, and CD44 expression, demonstrated a Hoechst 33342 side population and Aldefluor activity compared to adherent cells or a low metastatic subclone of LLC (LM-LLC). In syngeneic mice, HM-LLC sphere-derived cells required fewer cells to initiate tumorigenesis compared to adherent or LM-LLC cells. Similarly TC-1 sphere-derived cells were more tumorigenic than adherent cells in syngeneic mice. In contrast, in immunocompromised mice, less than 500 sphere or adherent TC-1 cells and less than 1,000 sphere or adherent LLC cells were required to initiate a tumor. We suggest that no single phenotypic marker can identify CICs in murine lung cancer cell lines. Tumorsphere culture may provide an alternative approach to identify and enrich for murine lung CICs. Furthermore, we propose that assessing tumorigenicity of murine lung CICs in syngeneic mice better models the interaction of CICs with the host immune system.

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.

Representing a renewable source for cell replacement, neural stem cells have received substantial attention in recent years. The neurosphere assay represents a method to detect the presence of neural stem cells, however owing to a deficiency of specific and definitive markers to identify them, their quantification and the rate they expand is still indefinite. Here we propose a mathematical interpretation of the neurosphere assay allowing actual measurement of neural stem cell symmetric division frequency. The algorithm of the modeling demonstrates a direct correlation between the overall cell fold expansion over time measured in the sphere assay and the rate stem cells expand via symmetric division. The model offers a methodology to evaluate specifically the effect of diseases and treatments on neural stem cell activity and function. Not only providing new insights in the evaluation of the kinetic features of neural stem cells, our modeling further contemplates cancer biology as cancer stem-like cells have been suggested to maintain tumor growth as somatic stem cells maintain tissue homeostasis. Indeed, tumor stem cell's resistance to therapy makes these cells a necessary target for effective treatment. The neurosphere assay mathematical model presented here allows the assessment of the rate malignant stemlike cells expand via symmetric division and the evaluation of the effects of therapeutics on the self-renewal and proliferative activity of this clinically relevant population that drive tumor growth and recurrence. © 2011 Deleyrolle et al.

The reaction of benzil with phenol at 180°C in the presence of SnCl4·5H2O produces a benzofuran, a benzofuranol, a benzodifuran, and a benzofuranone. Anhydrous tin(IV) chloride also gives a benzofuranofuranone. Other phenols and their methyl ethers yield related products. The good yields of the benzo- and naphtho-furanones make the method an attractive alternative to the benzilic acid route to such compounds.

Ludwig Institute of Cancer Research National Breast Cancer Foundation Introduction: Metastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.Methods: We used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.Results: Most brain metastases were triple negative and basal-like. the brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. in particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.Conclusions: Deregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. the need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules. Univ Queensland, Clin Res Ctr, Brisbane, Qld 4029, Australia Queensland Inst Med Res, Brisbane, Qld 4006, Australia Universidade Federal de São Paulo, EPM, Dept Anat Patol, BR-04024000 São Paulo, Brazil Griffith Univ, Brisbane, Qld 4011, Australia Univ Queensland, Ctr Magnet Resonance, Brisbane, Qld 4072, Australia Eijkman Inst, Jakarta 10430, Indonesia Inst Nacl Canc, Dept Patol, BR-20230130 Rio de Janeiro, Brazil Lab Salomao & Zoppi, Dept Patol, BR-04104000 São Paulo, Brazil Charles Univ Prague, Fac Med, Dept Pathol, Plzen 30605, Czech Republic Univ Sydney, Inst Clin Pathol & Med Res, Sydney W Area Hlth Serv, Sydney, NSW 2145, Australia Univ Sydney, Westmead Millennium Inst, Sydney W Area Hlth Serv, Sydney, NSW 2145, Australia Peter MacCallum Canc Ctr, Dept Pathol, Melbourne, Vic 3002, Australia Univ Queensland, Queensland Brain Inst, Brisbane, Qld 4072, Australia Royal Brisbane & Womens Hosp, Brisbane, Qld 4029, Australia Universidade Federal de São Paulo, EPM, Dept Anat Patol, BR-04024000 São Paulo, Brazil Web of Science

Dendritic cells are powerful antigen-presenting cells that can generate primary cytolytic T lymphocyte responses against tumors. Consequently, there has been much interest in their application as antitumor vaccines. A number of dendritic cell-based vaccine trials targeting a variety of tumors have been conducted in different countries; however, the rate of clinical responses remains low. The majority of these studies have administered dendritic cells loaded with synthetic peptide epitopes or tumor lysates. Genetic modification of dendritic cells to express tumor antigens or immunostimulatory molecules through gene transfer or mRNA transfection offers a logical alternative with potential advantages over antigen loading in dendritic cells. In this chapter, we review the current and future prospects for genetically modified dendritic cell vaccines for cancer therapy.

The concept of cancer stem cells responsible for tumour origin, maintenance, and resistance to treatment has gained prominence in the field of breast cancer research. The therapeutic targeting of these cells has the potential to eliminate residual disease and may become an important component of a multimodality treatment. Recent improvements in immunotherapy targeting of tumour-associated antigens have advanced the prospect of targeting breast cancer stem cells, an approach that might lead to more meaningful clinical remissions. Here, we review the role of stem cells in the healthy breast, the role of breast cancer stem cells in disease, and the potential to target these cells.

Dendritic cells (DCs) are powerful antigen-presenting cells that process antigens and present peptide epitopes in the context of the major histocompatibility complex molecules to generate immune responses. DCs are being studied as potential anticancer vaccines because of their ability to present antigens to naïve T cells and to stimulate the expansion of antigen-specific T-cell populations. We investigated an antitumor vaccination using DCs modified by transfer of a nonsignaling neu oncogene, a homologue of human HER-2/neu, in a transgenic model of breast cancer. BALB-neuT mice develop breast cancers as a consequence of mammary gland-specific expression of an activated neu oncogene. We vaccinated BALB-neuT mice with bone marrow-derived DCs transduced with Ad.Neu, a recombinant adenovirus expressing a truncated neu oncoprotein. The vaccine stimulated the production of specific anti-neu antibodies, enhanced interferon-γ expression by T cells, and prevented or delayed the onset of mammary carcinomas in the mice. Over 65% of vaccinated mice remained tumor free at 28 weeks of age, whereas all of the mice in the control groups developed tumors. When challenged with a neu-expressing breast cancer cell line, vaccinated tumor-free animals had delayed tumor growth compared with controls. The antitumor effect of the vaccine was specific for expression of neu. Studies showed that CD4+ T cells were required in order to generate antitumor immunity. Importantly, the effectiveness of the vaccine was not diminished by preexisting immunity to adenovirus, whereas the protection afforded by vaccination that used direct injection of Ad.Neu was markedly reduced in mice with anti-adenovirus antibody titers. DCs modified by recombinant adenoviruses expressing tumor-associated antigens may provide an effective antitumor vaccination strategy.

Treatment of 2,2-di(3,4-dimethoxyphenyl)indane-1,3-dione with concentrated sulfuric acid gives 2,3,6,7-tetramethoxydibenzo[a,c]fluoren-13-one while under the same conditions 2,2-di(3,4-dimethoxyphenyl)acenaph-thenone produces 2,3,6,7-tetramethoxydibenzo[j,l]fluoranthene.

We report the unusual case of a young man with acute respiratory failure caused by a left atrial myxoma. The patient's rapid clinical deterioration and severe hypoxemia suggested the acute respiratory distress syndrome; however, unexpected physical examination findings and a markedly elevated pulmonary capillary wedge pressure implied cardiac abnormality. The use of echocardiography as a bedside tool allowed correct, rapid diagnosis.

DCs are specialized antigen presenting cells that generate immune responses by presenting antigen epitopes in the context of MHC. DCs also secrete cytokines that direct the immune response. Interleukin-12 (IL-12) is important for directing immune system toward a cytolytic T cell response. We studied the effect of vaccination with DCs modified to express the murine IL-12 gene using a recombinant adenovirus. DCs were generated from bone marrow of C57Bl/6 mice in media supplemented with GM-CSF. Recombinant adenoviral vectors, Ad.mIL-12 expressing the p35/p40 subunits of murine IL-12 and a control vector, Ad.null, were generated in 293 cells. DCs were infected with Ad.mIL-12 at various MOIs and IL-12 secretion was measured by ELISA. Splenocytes (Spc) from naive C57Bl/6 mice were mixed with irradiated DCs infected with Ad.mIL-12 (DCAd.mIL-12) or Ad.null (DCAd.null) and in the presence or absence of anti-CD4 or anti-CD8 antibodies. Spc proliferation was assessed by [3H]-thymidine incorporation and IFN-γ production was measured. Groups of C57Bl/6 or IFN-γ −/− knockout C57Bl/6 mice were injected weekly with 1×106 DCAd.mIL-12, DCAd.null or with unmodified DCs for 2 weeks. A week after the last vaccination, mice were injected with 1×105 MC38.KRas murine colon carcinoma cells expressing mutant human K-Ras (G12D). Some groups of DCAd.mIL-12 treated mice received injections of anti-CD4 or anti-CD8 antibodies beginning either two days before initiation of vaccine or before tumor challenge. Tumor and Spc were obtained from each group of mice 21 days after injection of tumor. Tissues were examined for CD4 and CD8 cells, and for IFN-γ expression by in situ hybridization. Spc were co-cultured with irradiated MC38.KRas for 5 days and IFN-γ and IL-4 concentrations were measured by ELISA. After infection with Ad.mIL-12 (MOI = 10 and 30), mIL-12 p70 1628 and 3826 pg/mL/105 cells was measured, respectively. Spc stimulated with DCAd.mIL-12 had 2-fold greater [3H]-thymidine incorporation than controls. Anti-CD4 antibody, but not anti-CD8-antibody abolished this stimulation. DCAd.mIL-12 co-cultured with Spc produced 10-fold higher concentrations of IFN-γ than Spc stimulated with 300 ng of recombinant murine IL-12. When mice were challenged with MC38.KRas cells, inhibition of tumor growth was seen in the DCAd.mIL-12 vaccinated group but not the controls. Sixty percent of DCAd.mIL-12 treated mice were free of tumor at 77 days, while less than 20% of mice in the control groups tumor-free. Infiltration of tumors by CD4+ cells and expression of IFN-γ was seen in DCAd.mIL-12 treated mice. When Spc from mice treated with DCAd.mIL-12, DCAd.null or unmodified DC were mixed with MC38.KRas in vitro, supernatant contained 120, 35, 0.7 ng/ml of IFN-γ and 17.3, 8.9, 42.3 pg/ml of IL-4, respectively. Protection by DCAd.mIL-12 was abolished by depletion of CD4+ cells, but not by depletion of CD8+ cells. DCAd.mIL-12 vaccination was ineffective at inhibiting tumor growth in IFN-γ −/− knockout mice. [Conclusion] DCs modified with Ad.mIL-12 successfully protected C57Bl/6 mice from challenge with MC38KRas tumor cells. CD4+ T cells and secretion of IFN-γ appear to be important for the effectiveness of DCAd.mIL-12 vaccination.

Adult patients (pts) with cyanotic congenital heart disease (CCHD) and elevation (↑) in pulmonary vascular resistance (PVR) may have a marked decrease in exercise capacity (EX) and quality of life due to intravascular volume overload, ventricular dysfunction and cyanosis-related morbidity. Concerns of increasing right-to-left shunting and cyanosis have limited characterization of the safety and effects of angiotensin converting enzyme inhibition (ACEI) in these pts. We evaluated ACEI in 7 symptomatic adults (ages 29–36 yrs) with CCHD and ↑PVR from 6/92 to 6/94. Intracardiac shunting was present at the ventricular level (single ventricle, double outlet right ventricle) in 3 pts, at the atrial septum (Ebstein's malformation, peripheral pulmonary stenoses) in 2 pts, and at both levels (AV canal defect) in 2 pts. All pts had marked exercise incapacity; 6/7 were candidates for heart-lung transplantation. Baseline serum hemoglobin (Hgb) oxygen saturation (O2sat) and 6 minute walking capacity (6 min) were compared before and after 6 weeks of ACEI. Baseline Hgb was ≥6 gm/dl (19.3 ± 1.7) and O2sat was ≤90% (79 ± 4) in all pts. ACEI caused no decrease in O2sat or increase in Hgb in any pt. When compared to baseline, EX increased ≥30% (6 min: 326 ± 129 vs 441 ± 160 yds) with ACEI in 5/5 pts (p = 0.01). All pts noted subjective functional improvement with ACEI; during treatment 3/6 pts removed themselves from consideration for organ transplantation and remain well. Our preliminary findings suggest that short-term ACEI is safe and may improve exercise capacity and quality of life in adult pts with CCHD and ↑PVR . Further investigation is required to elucidate the mechanisms of these benefits and to determine the long-term effects of ACEI in this cohort.

For the past three years, a Learjet has been making microphysical measurements in new cloud development on the flanks of multicellular storms in the eastern Transvaal area of South Africa. Data from an imaging probe and a forward scattering spectrometer have been averaged for each storm for all first cloud penetrations between −8° and −12°C. Clear images of drops of diameters greater than 300 μm are found in 40% of the 42 storms measured. Most of the observed drops are associated with the more “maritime” droplet spectra. Also, the appearance of coalescence around −10°C appears to be related to cloud base temperatures and buoyancies, rather than changes in air masses, suggesting that cloud thermodynamics may play a dominant role in determining cloud microphysics in the Nelspruit area.