High efficiency ex vivo cloning of antigen-specific human effector T cells.

While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype.

Melanomas of unknown primary have a mutation profile consistent with cutaneous sun-exposed melanoma.

Melanoma of unknown primary (MUP) is an uncommon phenomenon whereby patients present with metastatic disease without an evident primary site. To determine their likely site of origin, we combined exome sequencing from 33 MUPs to assess the total rate of somatic mutations and degree of UV mutagenesis. An independent cohort of 91 archival MUPs was also screened for 46 hot spot mutations highly prevalent in melanoma including BRAF, NRAS, KIT, GNAQ, and GNA11. Results showed that the majority of MUPs exhibited high somatic mutation rates, high ratios of C>T/G>A transitions, and a high rate of BRAF (45 of 101, 45%) and NRAS (32 of 101, 32%) mutations, collectively indicating a mutation profile consistent with cutaneous sun-exposed melanomas. These data suggest that a significant proportion of MUPs arise from regressed or unrecognized primary cutaneous melanomas or arise de novo in lymph nodes from nevus cells that have migrated from the skin.

Open release of male mosquitoes infected with a wolbachia biopesticide: field performance and infection containment.

BACKGROUND: Lymphatic filariasis (LF) is a globally significant disease, with 1.3 billion persons in 83 countries at risk. A coordinated effort of administering annual macrofilaricidal prophylactics to the entire at-risk population has succeeded in impacting and eliminating LF transmission in multiple regions. However, some areas in the South Pacific are predicted to persist as transmission sites, due in part to the biology of the mosquito vector, which has led to a call for additional tools to augment drug treatments. Autocidal strategies against mosquitoes are resurging in the effort against invasive mosquitoes and vector borne disease, with examples that include field trials of genetically modified mosquitoes and Wolbachia population replacement. However, critical questions must be addressed in anticipation of full field trials, including assessments of field competitiveness of transfected males and the risk of unintended population replacement. METHODOLOGY/PRINCIPAL FINDINGS: We report the outcome of field experiments testing a strategy that employs Wolbachia as a biopesticide. The strategy is based upon Wolbachia-induced conditional sterility, known as cytoplasmic incompatibility, and the repeated release of incompatible males to suppress a population. A criticism of the Wolbachia biopesticide approach is that unintended female release or horizontal Wolbachia transmission can result in population replacement instead of suppression. We present the outcome of laboratory and field experiments assessing the competitiveness of transfected males and their ability to transmit Wolbachia via horizontal transmission. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that Wolbachia-transfected Aedes polynesiensis males are competitive under field conditions during a thirty-week open release period, as indicated by mark, release, recapture and brood-hatch failure among females at the release site. Experiments demonstrate the males to be 'dead end hosts' for Wolbachia and that methods were adequate to prevent population replacement at the field site. The findings encourage the continued development and extension of a Wolbachia autocidal approach to additional medically important mosquito species.

A high-throughput panel for identifying clinically relevant mutation profiles in melanoma.

Success with molecular-based targeted drugs in the treatment of cancer has ignited extensive research efforts within the field of personalized therapeutics. However, successful application of such therapies is dependent on the presence or absence of mutations within the patient's tumor that can confer clinical efficacy or drug resistance. Building on these findings, we developed a high-throughput mutation panel for the identification of frequently occurring and clinically relevant mutations in melanoma. An extensive literature search and interrogation of the Catalogue of Somatic Mutations in Cancer database identified more than 1,000 melanoma mutations. Applying a filtering strategy to focus on mutations amenable to the development of targeted drugs, we initially screened 120 known mutations in 271 samples using the Sequenom MassARRAY system. A total of 252 mutations were detected in 17 genes, the highest frequency occurred in BRAF (n = 154, 57%), NRAS (n = 55, 20%), CDK4 (n = 8, 3%), PTK2B (n = 7, 2.5%), and ERBB4 (n = 5, 2%). Based on this initial discovery screen, a total of 46 assays interrogating 39 mutations in 20 genes were designed to develop a melanoma-specific panel. These assays were distributed in multiplexes over 8 wells using strict assay design parameters optimized for sensitive mutation detection. The final melanoma-specific mutation panel is a cost effective, sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular-based therapeutics for the treatment of melanoma. When used in a clinical research setting, the panel may rapidly and accurately identify potentially effective treatment strategies using novel or existing molecularly targeted drugs.

Monitoring temporal abundance and spatial distribution of Aedes polynesiensis using BG-Sentinel traps in neighboring habitats on Raiatea, Society Archipelago, French Polynesia.

Adult numbers and sizes of mosquitoes were monitored for 2 yr in neighboring habitats on the western coast of Raiatea (Society Archipelago) in anticipation of testing new vector control technologies. Aedes polynesiensis Marks females comprised the overwhelming majority (approximately 99%) of the three species of mosquitoes captured in Biogent Sentinel traps placed at fixed sites on three small satellite islands (motus) of the western lagoon and on the shoreline of Raiatea. Aedes polynesiensis males, Aedes aegypti (L.), and Culex quinquefasciatus Say rarely were collected. Numbers of Ae. polynesiensis females per collection differed among trapping dates and locations, with the majority of females captured on two motus, Horea and Toamaro. Shoreline and Horea females had significantly longer mean wing lengths than females from Tiano and Toamaro. Thus, wing lengths were influenced more by local developmental conditions than overall numbers of adults. Significantly more females were captured during the wet season than the dry season. Nonetheless, at least on the two highly productive motus, dry-season females had larger wing lengths than their wet season counterparts. Local weather patterns predicted about half the variation in mosquito numbers. Differences in vector abundance observed when comparing neighboring motus are likely because of differences in human activity and mosquito suppression.

Identification of TFG (TRK-fused gene) as a putative metastatic melanoma tumor suppressor gene.

High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation "hotspot" at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.

A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma.

So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families. Mutations in CDKN2A account for approximately 40% of familial cases, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds. Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma. We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF). Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant. Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case-control sample. Likewise, it was similarly associated in an independent case-control sample from the United Kingdom. In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both. The variant allele was also associated with increased naevus count and non-blue eye colour. Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.

Epidemiology of West Nile virus: a silent epiornitic in Northern Delaware in 2007 without associated human cases.

ABSTRACT. We performed a 2-year longitudinal study (2006-2007) of West Nile virus (WNV) infections in wild birds, mosquitoes, and sentinel chickens at 6 WNV-endemic sites in northern Delaware. We determined virus infection rates of Culex pipiens and other mosquito vectors as well as seroprevalence and antibody titers of amplifying hosts. Endemicity status varied widely among the 6 sites based on 3 criteria-mosquito infections, sentinel chicken seropositivity, and wild bird seropositivity. A highly endemic site would display at least 2 of the 3 criteria during each year of the study, while a site with just 1 positive criterion was considered to have low endemicity. Culex pipiens was the principal vector detected at 2 highly endemic sites in 2006 vs. 1 site in 2007. However, in 2006, we also found 2 other WNV-positive vector species as well as an unidentifiable Culex species at 1 highly endemic site, suggesting increased activity at the end of the 1st year of the study. Wild birds were early indicators of WNV at highly endemic sites in mid-July to early August of both 2006-2007. Mosquitoes were positive in mid- to late August, appearing concurrently with seroconverted sentinel chickens, with wild resident birds appearing approximately 4 wk prior to those indicators. Of birds tested with n > or = 9, Northern cardinals had the highest seropositivity rates (47%) followed by Carolina wrens (19%), house sparrows (13%), American robins (13%), tufted titmice (11%), and gray catbirds (9%). The overall seropositive rates in trapped birds increased from 5.0% in 2006 to 20.0% in 2007, while the geometric mean titers of all positive birds increased from 1:34 to 1:47 during the comparable periods. Based on these results, we suggest that an epiornitic in birds occurred in 2007, but that greatly reduced abundance of mosquito vectors caused by an extreme drought largely precluded human infection.

Life satisfaction, self-esteem, and perceived health status among elder Korean women: focus on living arrangements.

The purpose of this study was to examine whether living arrangements significantly affect life satisfaction, self-esteem, and perceived health status of elder Korean women. A total of 121 women aged 65 to 89 was interviewed in an urban community senior center in Korea. The convenience sample was obtained by face-to-face interviews using a structured questionnaire. Life satisfaction, self-esteem, and perceived health status were strongly correlated with each other. Living arrangements significantly affected life satisfaction, self-esteem, and perceived health status. Women who live with their married son had the highest life satisfaction and self-esteem and perceived themselves to be healthier in comparison to their counterparts. Therefore, government programs need to be developed to assist children in caring for their parents to improve their overall well-being.

Dendritic cell immunotherapy for stage IV melanoma.

Active boosting of the antitumour immune response of patients with solid malignancies has been tested in a large number of trials. Isolated complete clinical responses have been reported, however, they have not been replicated in subsequent studies. We recently reported objective clinical responses to a dendritic cell/irradiated autologous tumour cell 'vaccine' in patients with distant metastatic (stage IV) melanoma. Here we describe our experience in a second cohort of patients with stage IV melanoma, using this dendritic cell-based immunotherapy in a cryopreserved format. Of 46 patients enrolled into the study, three had complete remission of all detectable disease, and a further three had partial clinical responses. These data confirm that dendritic cell-based immunotherapy has potential as a therapy in a limited number of patients with stage IV melanoma. To our knowledge, this is the first demonstration that cryopreserved dendritic cells can elicit complete clinical responses in patients with advanced cancer. Our observations support randomized controlled trials to validate the findings.

Stormwater ponds, constructed wetlands, and other best management practices as potential breeding sites for West Nile virus vectors in Delaware during 2004.

We performed longitudinal surveys of mosquito larval abundance (mean mosquito larvae per dip) in 87 stormwater ponds and constructed wetland in Delaware from June to September 2004. We analyzed selected water quality factors, water depth, types of vegetation, degree of shade, and level of insect predation in relation to mosquito abundance. The 2004 season was atypical, with most ponds remaining wet for the entire summer. In terms of West Nile virus (WNV) vectors, wetlands predominantly produce Aedes vexans, culex pipiens pipiens, and Culex restuans. Retention ponds generally produced the same species as wetlands, except that Cx. p. pipiens was more abundant than Cx. restuans in retention ponds. Aedes vexans and Culex salinarius were the most abundant species to Conservation Restoration Enhancement Program ponds. Sand filters uniquely produced high numbers of Cx. restuans, Cx. p. pipiens, and Aedes japonicus japonicus, a newly invasive vector species. Site that alternately dried and flooded, mostly detention ponds, forebays of retention ponds, and some wetlands often produced Ae. vexans, an occasional WNV bridge vector species. Overall, seasonal distribution of vectors was bimodal, with peaks occurring during early and late summer. Ponds with shallow sides and heavy shade generally produced an abundance of mosquitoes, unless insect predators were abundant. Bright, sunny ponds with steep sides and little vegetation generally produced the fewest mosquitoes. The associations among mosquito species and selected vegetation types are discussed.

Immunological characteristics correlating with clinical response to immunotherapy in patients with advanced metastatic melanoma.

Current treatment options for advanced metastatic melanoma are limited to experimental regimen that provide poor survival outcomes. Immunotherapy is a promising alternative and we recently reported a clinical trial in which 6 out of 19 patients enrolled had objective clinical responses to a fully autologous melanoma/dendritic cell vaccine. The mechanism of the vaccine is not well understood, but we hypothesized that general immunocompetence may be a determinant of clinical response. We therefore examined the immune status of an expanded series of 21 patients who displayed varying clinical responses to the melanoma/dendritic cell vaccine. Immunocompetence was assessed using in vitro assays of lymphocyte function: survival, proliferation and cytokine responses to mitogen stimulation as well as T-cell receptor zeta expression and lymphocyte subset analysis. Although lymphocytes from patients mostly performed comparably to age-matched and sex-matched controls, in some assays we identified significant differences between complete clinical responders and other patients, both before and following vaccination. Surprisingly, before vaccination, only lymphocytes from clinical responder patients showed impaired in vitro survival. Following vaccination, T lymphocyte survival improved and cells recovered their ability to produce the Th1-associated cytokines TNF and IFN-gamma in response to anti-CD3 stimulation in vitro. No increase in Th1 cytokine production was observed in lymphocytes from patients who experienced partial clinical responses or progressive disease. We conclude that, before vaccination, patients who go on to have complete responses have immune characteristics suggestive of high cell turnover and low Th1-associated cytokine production, and that these can be reversed with vaccination. These results have potential implications for future immunotherapeutic strategies.

A population of HLA-DR+ immature cells accumulates in the blood dendritic cell compartment of patients with different types of cancer.

Dendritic cell (DC) defects are an important component of immunosuppression in cancer. Here, we assessed whether cancer could affect circulating DC populations and its correlation with tumor progression. The blood DC compartment was evaluated in 136 patients with breast cancer, prostate cancer, and malignant glioma. Phenotypic, quantitative, and functional analyses were performed at various stages of disease. Patients had significantly fewer circulating myeloid (CD11c+) and plasmacytoid (CD123+) DC, and a concurrent accumulation of CD11c(-)CD123(-) immature cells that expressed high levels of HLA-DR+ immature cells (DR(+)IC). Although DR(+)IC exhibited a limited expression of markers ascribed to mature hematopoietic lineages, expression of HLA-DR, CD40, and CD86 suggested a role as antigen-presenting cells. Nevertheless, DR(+)IC had reduced capacity to capture antigens and elicited poor proliferation and interferon-gamma secretion by T-lymphocytes. Importantly, increased numbers of DR(+)IC correlated with disease status. Patients with metastatic breast cancer showed a larger number of DR(+)IC in the circulation than patients with local/nodal disease. Similarly, in patients with fully resected glioma, the proportion of DR(+)IC in the blood increased when evaluation indicated tumor recurrence. Reduction of blood DC correlating with accumulation of a population of immature cells with poor immunologic function may be associated with increased immunodeficiency observed in cancer.

Nursing care quality initiative for care of hospitalized elders and their families.

This report describes a Nursing Care Quality Initiative (NCQI) through which a new model of care for hospitalized older adults and their families, the Family-Centered Geriatric Resource Nurse (FCGRN) model, was introduced. Eighteen units in 10 hospitals participated in the NCQI Project. Educational, clinical, and evaluation components of the NCQI Project are described.

Hospitalized elders: changes in functional and mental status.

This article describes outcomes of a new model of care for hospitalized elders and their families. Patient functional and cognitive status on admission and discharge were evaluated for changes as a result of an educational program for preparing family-centered geriatric resource nurses. Patients in the intervention group (n = 173) demonstrated significant improvements in outcome measures (functional and cognitive status) from admission to discharge. A subset (n = 50) was selected from the 173 subjects who comprised the intervention group; this subset was compared with control subjects (n = 44); no statistically significant differences were noted between the 2 groups. Suggestions for future research are presented.

Reinforcing organizationwide pressure ulcer reduction on high-risk geriatric inpatient units.

Skin care and pressure ulcer prevention programs abound, although their content varies and their outcomes are often difficult to quantify. This article describes 2 complementary programs, their quality improvement processes, and a variety of ways of measuring their success. The first program was broad in scope, emphasizing system-wide changes in administration and coordination of resources, while the second focused on nursing education on high-risk units. These 2 approaches could be adapted for use in any health care setting.

A family assessment tool for hospitalized elders.

This article describes the development of the FAMILY card, a family assessment instrument used in the Nursing Care Quality Initiative (NCQI) to assess the involvement and needs of family members of hospitalized elders. Through the NCQI project, particular attention has been paid to integrating both clinical expertise in geriatric nursing care and the principles of family-centered care into the nursing care provided by staff on the NCQI study units. Two case studies illustrating use of the SPICES geriatric assessment instrument and the newly developed FAMILY assessment card are included as exemplars.