Large Extracellular Vesicles Can be Characterised by Multiplex Labelling Using Imaging Flow Cytometry.

Extracellular vesicles (EVs) are heterogeneous in size (30 nm-10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. METHODS: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. RESULTS: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. CONCLUSIONS: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.

Determination of ribosomal DNA copy number and comparison among strains of Coccidioides.

The use of PCR-based assays to detect fungi and diagnose fungal infections as well as to monitor fungal organ burden with diseases such as coccidioidomycosis is becoming more common. The target of these assays is frequently one or more of the ribosomal DNA (rDNA) gene subunits. The multicopy nature of this gene affords greater sensitivity over single-copy genes. However, there are few studies reporting the precise number of copies of the rDNA gene per genome in pathogenic fungi. Quantitative PCR was used to determine the number of copies of rDNA as well as CTS1, a single-copy gene, in samples of Coccidioides genomic DNA by the absolute quantification method. Variability of rDNA genome copy number was determined using 13 different Coccidioides isolates and was found to vary between 20 and 146 copies per genome. This suggests that detection of rDNA will likely afford an increased sensitivity of at least 20-fold over single-copy genes. However, estimation of the number of organisms present by quantification of the rDNA cannot be made prior to knowledge of each isolate's rDNA copy number because of the strain variation.

Coccidioides species determination: does sequence analysis agree with restriction fragment length polymorphism?

Fifteen Coccidioides isolates were previously examined for genetic diversity using restriction fragment length polymorphism (RFLP); two fragment patterns were observed. Two isolates demonstrated one banding pattern (designated RFLP group I), while the remaining 13 isolates demonstrated a second pattern (designated RFLP group II). Recently, molecular studies supported the division of the genera Coccidioides into two species: Coccidioides posadasii and Coccidioides immitis. It has been assumed that the species division corresponds to the RFLP grouping. We tested this hypothesis by amplifying the ribosomal DNA internal transcribed spacer region as well as the dioxygenase, serine proteinase, and urease genes from 13 isolates previously examined by RFLP and then sequencing the PCR products. The appropriate species for each isolate was assigned using phylogenetically informative sites. The RFLP grouping agreed with the Coccidioides species assignment for all but one isolate, which may represent a hybrid. In addition, polymorphic sites among the four genes examined were in agreement for species assignment such that analysis of a single gene may be sufficient for species assignment.

Monocyte-derived dendritic cells from chronic myeloid leukaemia have abnormal maturation and cytoskeletal function that is associated with defective localisation and signalling by normal ABL1 protein.

OBJECTIVES: Mature dendritic cells (DCs) may be derived from the BCR/ABL1 expressing monocytes in chronic myeloid leukaemia. These cells have potential therapeutic applications, but are recognised to have defective function. In normal DCs, activation and maturation depend on ABL1 dependent signals. We therefore tested the hypothesis that in the DCs of chronic myeloid leukaemia, the presence of the BCR/ABL1 molecule disrupts normal ABL1 signal pathways, and contributes to the observed functional defects of the cells. METHODS: We employed in vitro culture of clinical samples, combining microscopic and biochemical techniques with a phosphoproteomic approach to compare and characterise DCs from normal individuals and chronic myeloid leukaemia patients. RESULTS AND CONCLUSIONS: We identified an altered intracellular localisation for ABL1 within DCs derived from the monocytes of chronic myeloid leukaemia. The protein was found in the perinuclear region co-distributed with the adapter-protein CRKL and the BCR/ABL1 protein. This altered distribution was associated with defective generation of ABL1-dependent maturation signals, and a dislocation of ABL1 from the F-actin cytoskeleton. We suggest that abnormal ABL1-dependent signals contribute to the recognised functional defects affecting chronic myeloid leukaemia DCs.

Demonstration of Coccidioides immitis and Coccidioides posadasii DNA in soil samples collected from Dinosaur National Monument, Utah.

Soil samples were collected in 2006 from Dinosaur National Monument (DNM), Utah, the site of an outbreak of coccidioidomycosis in 2001. DNA was isolated from two soil samples, and polymerase chain reaction (PCR) amplified Coccidioides DNA present in both samples. Ribosomal RNA genes and internal transcribed spacer (ITS) region PCR products were sequenced. Single-nucleotide polymorphisms indicated that the DNA from sample SS06RH was that of Coccidioides immitis, while the DNA from sample SS06UM was C. posadasii. This is the first report to directly demonstrate Coccidioides in soils from DNM and the first to report the presence of both C. immitis and C. posadasii in the same geographic location.

Immunological characterization of bronchoalveolar lavage fluid in patients with acute pulmonary coccidioidomycosis.

BACKGROUND: The specific cellular immunological characteristics of bronchoalveolar lavage (BAL) fluid in acute pulmonary coccidioidomycosis have not been defined. METHODS: BAL fluid from patients living in a coccidioidomycosis-endemic region of Arizona who were undergoing bronchoscopy because of pulmonary infiltrates was analyzed. Mononuclear cells from BAL fluid and peripheral blood mononuclear cells (PBMCs) were incubated with the coccidioidal antigen T27K in vitro, and cellular immunological assays were performed. RESULTS: Forty-six patients were studied. Twelve received a diagnosis of acute pulmonary coccidioidomycosis, 17 received other diagnoses, and 17 had no diagnosis established. There was an increased proportion of polyfunctional CD8(+) T cells after antigen stimulation from subjects with coccidioidomycosis as compared to those with another diagnosis (P = .025). In cells collected from BAL fluid and in PBMCs, the concentrations of interferon γ, tumor necrosis factor α, and interleukin 17 (IL-17) were all significantly increased in samples from those with acute pulmonary coccidioidomycosis, compared with the other 2 groups (for all, P<.05). CONCLUSIONS: When incubated in vitro with a coccidioidal antigen preparation, cells from both BAL fluid and peripheral blood obtained from patients with pulmonary coccidioidomycosis demonstrated specific cellular immune responses, including expression of IL-17.

Serum (1->3)-ß-D-glucan measurement in coccidioidomycosis.

The serum (1→3)-ß-d-glucan assay has emerged as an important diagnostic test for invasive fungal disease. The utility of this assay in coccidioidomycosis has not been previously studied. Using a cutoff value of ≥80 pg/ml, we found the sensitivity (43.9%), specificity (91.1%), positive predictive value (81.8%), and negative predictive value (64.1%) to be similar to those of the assay in diagnosing other invasive mycoses.

Lesbian mothers and their children: the third wave.

Research regarding lesbian mothers and their children has gone through a transformation in the last forty years. The first wave of research examined lesbians who had become parents while in heterosexual relationships. The second wave examined women who became parents within the context of lesbian relationships. Both of these waves focused on family functioning and child outcome, using heterosexual-headed families as comparison groups. The third wave of research, which is now underway, is focusing on the unique challenges faced by these families, and how lesbian mothers are creating and raising their families on their own terms. This article explores the research as it has evolved over the years and the direction in which it is headed.

Early treatment with fluconazole may abrogate the development of IgG antibodies in coccidioidomycosis.

BACKGROUND: We have observed a number of patients who fail to develop coccidioidal complement fixing (CF) antibody (immunoglobulin [IgG]) after the initiation of early antifungal therapy. Although this is the first description of this phenomenon in mycology, a precedent for the abrogation of the immune response has been observed in other conditions, including primary syphilis and primary Lyme disease. METHODS: We conducted a retrospective case-control study to determine any patient-specific risk factors associated with this observation. Additionally, in vitro analysis of the coccidioidal CF (IgG) antigen (Cts1) was performed after Coccidioides was grown under escalating fluconazole concentrations. RESULTS: Seventeen patients persistently positive for coccidioidal IgM antibodies without developing an IgG response (cases) were compared with 64 consecutive patients who did develop coccidioidal CF (IgG) antibodies (controls). Early treatment with antifungals (within 2 weeks of symptom onset) was associated with an abrogation of IgG antibody production (P < .001). With immunodiffusion testing, control serum demonstrated a lack of IgG seroreactivity when Coccidioides posadasii grown in the presence of escalating fluconazole doses (0.5-128 µg/mL) was used as the antigen; however, control serum remained seroreactive for the presence of IgM. The coccidioidal IgG antigen (Cts1) was shown to be diminished when cultures were grown in the presence of fluconazole, lending further in vitro plausibility to our findings. CONCLUSIONS: The abrogation of an IgG response in patients treated early in the course of coccidioidal infection may complicate serodiagnosis and epidemiologic studies, and further study to determine the potential clinical implications should be performed.

Polyfunctional T lymphocytes are in the peripheral blood of donors naturally immune to coccidioidomycosis and are not induced by dendritic cells.

Coccidioidomycosis is a fungal infection endemic in the southwestern United States that is increasing in incidence. While cellular immunity correlates with protection from clinical illness, the precise elements of that response are undefined. Using the coccidioidal antigen preparation T27K and multiparametric flow cytometry, the in vitro frequency of polyfunctional T lymphocytes in the peripheral blood of naturally immune healthy donors and those who were nonimmune was determined. Polyfunctional CD4 lymphocytes, defined as producing intracellular interleukin 2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha simultaneously, had a frequency of 137 per 400,000 events among peripheral blood mononuclear cells (PBMC) of immune donors compared to 11 per 400,000 PBMC from nonimmune donors (P = 0.03). When monocyte-derived mature dendritic cells pulsed with T27K (mDC(T27K)) were used for antigen presentation, the frequency of polyfunctional CD4 T lymphocytes did not significantly increase for either group, although mDC(T27K) did significantly increase the concentrations of IL-2 and IFN-gamma released by PBMC from nonimmune donors (P = 0.02). After in vitro stimulation with T27K, polyfunctional CD4 and CD8 lymphocytes of PBMC from immune donors had a mixture of low- and high-expression CCR7 cells, suggesting both effector and central memory, compared with predominantly high-expression CCR7 cells when PBMC were incubated with the mitogen phytohemagglutinin (P = 0.03). These data demonstrate the presence of polyfunctional T lymphocytes in the peripheral blood of individuals with coccidioidal immunity and suggest a model for the in vitro testing of vaccine candidates for coccidioidomycosis.

Ribosome-associated nucleophosmin 1: increased expression and shuttling activity distinguishes prognostic subtypes in chronic lymphocytic leukaemia.

Two distinct groups of chronic lymphocytic leukaemia (CLL) are distinguished by the presence or absence of somatic hypermutation of the immunoglobulin heavy-chain gene. CLL without somatic hypermutation has an adverse outcome, but the precise biological differences that underlie this more aggressive clinical-course are unclear. Using a proteomic approach, we found that the two prognostic forms of CLL were consistently distinguished according to their protein expression pattern. The most important difference observed related to the different expression of nucleophosmin 1 between the two forms of CLL. This different expression was not related to apoptosis, proliferation or gene mutation. However, co-immunoprecipitation experiments identified an association between nucleophosmin 1 and ribosomal proteins. Using immunocytofluorescence, nucleophosmin 1 expression was identified in the nucleoli and nucleoplasm of all cells, but in a proportion of cells, nucleophosmin had been transferred from the nucleoplasm to the cytoplasm. Both the fluorescent intensity, and the frequency of cytoplasmic nucleophosmin 1 expression, was higher in CLL without somatic hypermutation. We propose therefore, that nucleophosmin 1, in association with ribosomal proteins, undergoes nucleo-cytoplasmic shuttling in CLL. This process is most prominent in un-mutated CLL and may signify altered protein biosynthesis.

Molecular cloning, characterization and expression analysis of two beta-N-acetylhexosaminidase homologs of Coccidioides posadasii.

Two full-length cDNAs were isolated from Coccidioides posadasii that encode two deduced proteins (CpHEX1 and CpHEX2) with homology to the glycosyl hydrolase 20 family of beta-N-acetylhexosaminidases. CpHEX1 consists of 595 amino acids, has a predicted molecular mass of 68 kDa and shares the highest identity with the N-acetylhexosaminidase (NAGA) of Aspergillus nidulans, while CpHEX2 consists of 603 amino acids, has a predicted molecular mass of 68.5 kDa and shares the highest identity with NAG1 from Paracoccidioides brasiliensis. CpHEX1 and CpHEX2 share only 23% identity and have dissimilar homologies showing more identity with other fungal beta-N-acetylhexosaminidases than with each other. Phylogenetic analysis of selected beta-N-acetylhexosaminidases placed CpHEX1 in a cluster with the orthologs from A. nidulans, Aspergillus oryzae, Penicillium chrysogenum and Candida albicans, while CpHEX2 grouped with the orthologs from P. brasiliensis and the Trichoderma spp. beta-N-acetylhexosaminidase activity and transcripts encoding CpHEX1 and CpHEX2 were detected in vitro during the spherule-endospore (SE) phase. Expression of the Cphex1 transcript exhibited a temporal increase that correlated with beta-N-acetylhexosaminidase activity, while the Cphex2 transcript remained relatively constant. The addition of N-acetylglucosamine to the cultures increased beta-N-acetylhexosaminidase activity and the expression of the Cphex1 transcript. A native beta-N-acetylhexosaminidase enzyme was purified from in vitro SE phase and identified as CpHEX1 by mass spectrometric analysis. Both the CpHEX1 and CpHEX2 cDNAs were expressed as recombinant fusion proteins and purified under denaturing conditions to apparent homogeneity but they lacked enzymatic activity.

Differing transcriptional responses to pulsed or continuous estradiol exposure in human umbilical vein endothelial cells.

This study used human umbilical vein endothelial cells (HUVECs) that were treated with 17beta-estradiol for 5 days as 1h pulse or 24h continuous treatment at concentrations such that the 24h exposure (concentration x time) was identical in both conditions. Cell proliferation was studied and gene expression profiling was carried out using the Affymetrix GeneChip microarray analysis. Changes in morphology and apoptosis in HUVECs were examined with electron microscopy. Time-course studies of expression of genes vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were performed by quantitative PCR. We observed that cell proliferation was significantly decreased over days 3-5 with pulsed estradiol treatment relative to constant exposure. Microarray results showed that after 5 days, 801 genes differed (P<0.05) between continuous versus pulsed estradiol treatment. Functional analysis showed a significant number of genes to be associated with apoptosis and cell cycle pathways. We did not find any evidence of apoptosis from flow cytometry or electron microscopy examination. Our study highlights a large number of significantly different molecular responses to estradiol depending upon the mode of administration of estradiol. Significant changes were observed in genes involved in apoptosis and proliferation including VEGF, IGF receptors, and tumor protein p53.

Ishikawa cells exhibit differential gene expression profiles in response to oestradiol or 4-hydroxytamoxifen.

In this study, the oestrogen agonist/antagonist action of 4-hydroxytamoxifen (OHT; 1 x 10(-6) M) and 17beta-oestradiol (E(2); 1 x 10(-8) M) were assessed on the oestrogen receptor (ER)-positive epithelial cell line (Ishikawa) with respect to cell proliferation, and to gene and protein expression. qRT-PCR and western blotting confirmed that Ishikawa cells expressed both ER isoforms and that there was no change in transcript levels in response to either ligand. Gene expression profiles, using oligonucleotide arrays representing approxiamtely 19,000 human genes, showed that the expression of 716 and 534 genes were changed differentially by treatment with either OHT or E(2) respectively, at the 24-h time point, with modulation of 46 genes common to both ligands, whereas 335 (OHT) and 240 (E(2)) genes showed expression changes unique to ligand, with 13 common alterations at 48 h. Both OHT and E(2) had demonstrable oestrogen agonist actions on Ishikawa cells, exemplified by increased proliferation and expression of known oestrogen-responsive genes, such as creatine kinase B and by the induction of alkaline phosphatase activity. Additionally, the data indicate that the two oestrogen agonists generated not only common gene expression changes but also unique ligand-specific profiles, raising the intriguing possibility that tamoxifen has E(2)-independent effects on the uterine epithelium.

Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii Cu,Zn superoxide dismutase identified by proteomic analysis of the coccidioidal T27K vaccine.

Previous studies have demonstrated that the coccidioidal T27K vaccine preparation is protective in mice against respiratory challenge using Coccidioides posadasii (C. posadasii) arthroconidia. Proteomic methods have been employed to define the molecular components within the vaccine. This method has led to the identification of novel and previously uncharacterized coccidioidal proteins including a Cu,Zn superoxide dismutase. A two-dimensional gel of the T27K vaccine was run and spots were excised for mass spectrometric analysis. One peptide was obtained from the T27K gel that matched a TIGR C. posadasii 2.0 gene index tentative consensus sequence, TC1072, which is similar to fungal Cu,Zn superoxide dismutase. Activity assays performed with native PAGE gels of the T27K vaccine showed that the vaccine contains superoxide dismutase. The cDNA encoding the enzyme has been cloned and sequenced and expressed as a recombinant protein.

Coccidioides niches and habitat parameters in the southwestern United States: a matter of scale.

To determine habitat attributes and processes suitable for the growth of Coccidioides, soils were collected from sites in Arizona, California, and Utah where Coccidioides is known to have been present. Humans or animals or both have been infected by Coccidioides at all of the sites. Soil variables considered in the upper 20 cm of the soil profile included pH, electrical conductivity, salinity, selected anions, texture, mineralogy, vegetation types and density, and the overall geomorphologic and ecological settings. Thermometers were buried to determine the temperature range in the upper part of the soil where Coccidioides is often found. With the exception of temperature regimes and soil textures, it is striking that none of the other variables or group of variables that might be definitive are indicative of the presence of Coccidioides. Vegetation ranges from sparse to relatively thick cover in lower Sonoran deserts, Chaparral-upper Sonoran brush and grasslands, and Mediterranean savannas and forested foothills. No particular grass, shrub, or forb is definitive. Material classified as very fine sand and silt is abundant in all of the Coccidioides-bearing soils and may be their most common shared feature. Clays are not abundant (less than 10%). All of the examined soil locations are noteworthy as generally 50% of the individuals who were exposed to the dust or were excavating dirt at the sites were infected. Coccidioides has persisted in the soil at a site in Dinosaur National Monument, Utah for 37 years and at a Tucson, Arizona site for 41 years.

Characteristics of the protective subcellular coccidioidal T27K vaccine.

While the whole killed spherule vaccine, protective in mice and monkeys, did not prevent coccidioidal disease in humans, the 27K vaccine, a soluble derivative, retains protective activity in mice with little irritant action. Gel filtration and anion exchange fractions of thimerosal-inactivated spherules (T27K), when administered with alum adjuvant, also protect mice against lethal respiratory coccidioidal challenge. However, the superb protection afforded by T27K antigens is maintained for some 3 months, but may then diminish. This appears unrelated to the aging of the mice. Prolongation of the protective action may require addition of a different adjuvant or administration of booster doses of vaccine.

Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-alpha-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods.

The coccidioidal T27K vaccine is protective in mice against respiratory challenge with Coccidioides posadasii (C. posadasii) arthroconidia. The vaccine is a subcellular multicomponent preparation that has not been fully characterized. To identify potential protective antigens in the heterogeneous mixture, the vaccine has been separated by two-dimensional gel electrophoresis and then analyzed for seroreactive proteins using immunoblot analysis with pooled sera from patients with coccidioidomycosis. Two seroreactive spots of identical apparent molecular weight were identified and sequenced using tandem mass spectrometry. Three peptides were generated, two of which matched a tentative consensus sequence in the TIGR C. posadasii 2.0 gene index database that is similar to fungal 1,2-alpha-mannosidases. The 5' and 3' ends of the mannosidase cDNA were mapped using rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR), and a full-length cDNA was then obtained using reverse-transcription (RT) PCR. The cDNA was cloned and sequenced and expressed as a recombinant protein. The predicted protein consists of 519 amino acids, has a theoretical molecular weight and pI of 56,918 Da and 4.84, respectively, and is very similar (>60%) to other fungal 1,2-alpha-mannosidases. Class I 1,2-alpha-mannosidase enzyme activity was also detected in the T27K vaccine using the substrate, Man-alpha-1,2-Man-alpha-OCH(3) in a spectrophotometric assay.

Safety, antigenicity, and efficacy of a recombinant coccidioidomycosis vaccine in cynomolgus macaques (Macaca fascicularis).

The safety, immunogenicity and efficacy of recombinant Ag2/PRA106 + CSA chimeric fusion protein (CFP) vaccine in ISS/Montanide adjuvant-administered intramuscular (IM) was assessed in adult female cynomolgus macaques challenged with Coccidioides posadasii. Animals received three immunizations with either 5 microg CFP, 50-microg CFP, or adjuvant alone and were challenged 4 weeks following the final immunization. Although significant antibody response was produced in response to vaccination, there were no discernable adverse effects, suggesting that the vaccine was well tolerated. Upon intratracheal challenge, all animals showed evidence of disease. Two animals that received 5-microg doses of CFP were euthanatized prior to the study's end because of severe symptoms. Animals vaccinated with 50-microg doses of CFP showed evidence of enhanced sensitization compared to adjuvant controls and animals vaccinated with 5-microg doses of CFP. This was based on higher serum anti-CFP titers, enhanced secretion of interferon-gamma (IFN-gamma) from stimulated bronchoalveolar lavage mononuclear cells (BALMC), reduced pulmonary radiologic findings following intratracheal challenge, reduced terminal complement fixation titers, and reduced necropsy findings. Overall the vaccine was well tolerated, induced sensitization, and resulted in a protective response when given at the higher 50-microg dose. Additional experiments may be needed to optimize the vaccination and to confer greater protection against lethal challenge.