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.

Acute lymphoblastic leukaemia cells produce large extracellular vesicles containing organelles and an active cytoskeleton.

Extracellular vesicles have been described in non-paracrine cellular interactions in cancer. We report a similar phenomenon in B-cell precursor (BCP) acute lymphoblastic leukaemia (ALL). Using advanced microscopy and high throughput screening, we further characterise a subset of large vesicles (LEVs) identified in cell lines, murine models of human BCP-ALL and clinical samples. Primary ALL blasts and cell lines released heterogeneous anucleate vesicles <6 micron into extracellular fluids. Larger LEVs were enclosed in continuous membranes, contained intact organelles and demonstrated an organised cytoskeleton. An excess of circulating CD19-positive LEVs were observed in diagnostic samples and isolated from mice engrafted with BCP-ALL primary cells. LEVs exhibited dynamic shape change in vitro and were internalised by other leukaemic cell lines leading to phenotypic transformation analogous to the cell of origin. In patient-derived xenografts, LEVs were released by primary ALL cells into extracellular spaces and internalised by murine mesenchymal cells in vivo. Collectively these data highlight the heterogeneity but accessibility of LEVs in clinical samples and their potential to provide a unique insight into the biology of the cell of origin and to their development as novel biomarkers to aid diagnosis and improve therapeutic outcomes.

Cytokine Profiles from Antigen-Stimulated Whole-Blood Samples among Patients with Pulmonary or Nonmeningeal Disseminated Coccidioidomycosis.

The outcome of coccidioidomycosis depends on a robust specific cellular immune response. A T-helper type 1 (Th1) cellular immune response has been previously associated with resolution of clinical illness. However, the precise elements of this response and whether cytokines not involved with the Th1 response play a role in coccidioidomycosis are not known. Whole-blood samples were obtained from subjects with active coccidioidomycosis and controls and incubated for 18 h with T27K, a coccidioidal antigen preparation. The supernatant was then assayed for gamma interferon (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, IL-10, and IL-17A. A total of 43 subjects, 16 with acute pneumonia, 9 with pulmonary sequelae of nodules and cavities, and 18 with nonmeningeal disseminated coccidioidomycosis, were studied. Compared to concentrations in healthy immune and nonimmune donors, the median concentration of IL-17A was significantly higher in those with active coccidioidomycosis (for both, P < 0.01). In addition, IL-6 concentrations were higher while IL-2 and IFN-γ concentrations were significantly lower in those with nonmeningeal disseminated disease diagnosed within 12 months than in those with acute pneumonia (for all, P < 0.05). The cytokine profile among patients with active coccidioidomycosis is distinct in that IL-17A is persistently present. In addition, those with nonmeningeal disseminated disease have an increased inflammatory cytokine response and diminished Th1 responses that modulate over time.

Coccidioidomycosis and other systemic mycoses of marine mammals stranding along the central California, USA coast: 1998-2012.

A wide range of systemic mycoses have been reported from captive and wild marine mammals from North America. Examples include regionally endemic pathogens such as Coccidioides and Blastomyces spp., and novel pathogens like Cryptococcus gattii, which appear may have been introduced to North America by humans. Stranding and necropsy data were analyzed from three marine mammal stranding and response facilities on the central California coast to assess the prevalence, host demographics, and lesion distribution of systemic mycoses affecting locally endemic marine mammals. Between 1 January 1998 and 30 June 2012, >7,000 stranded marine mammals were necropsied at the three facilities. Necropsy and histopathology records were reviewed to identify cases of locally invasive or systemic mycoses and determine the nature and distribution of fungal lesions. Forty-one animals (0.6%) exhibited cytological, culture- or histologically confirmed locally invasive or systemic mycoses: 36 had coccidioidomycosis, two had zygomycosis, two had cryptococcosis, and one was systemically infected with Scedosporium apiospermum (an Ascomycota). Infected animals included 18 California sea lions (Zalophus californianus), 20 southern sea otters (Enhydra lutris nereis), two Pacific harbor seals (Phoca vitulina richardsi), one Dall's porpoise (Phocoenoides dalli), and one northern elephant seal (Mirounga angustirostris). Coccidioidomycosis was reported from 15 sea lions, 20 sea otters, and one harbor seal, confirming that Coccidioides spp. is the most common pathogen causing systemic mycosis in marine mammals stranding along the central California coast. We also report the first confirmation of C. gattii infection in a wild marine mammal from California and the first report of coccidioidomycosis in a wild harbor seal. Awareness of these pathogenic fungi during clinical care and postmortem examination is an important part of marine mammal population health surveillance and human health protection. Temporal-spatial overlap may be observed for pathogenic mycoses infecting coastal marine mammals and adjacent human populations.

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.

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.

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.

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.

Detection of coccidioidal antibodies in serum of a small rodent community in Baja California, Mexico.

Coccidioidomycosis (Valley Fever) represents a serious threat to inhabitants of endemic areas of North America. Despite successful clinical isolations of the fungal etiological agent, Coccidioides spp., the screening of environmental samples has had low effectiveness, mainly because of the poor characterization of Coccidioides ecological niche. We explored Valle de las Palmas, Baja California, Mexico, a highly endemic area near the U.S.-Mexico border, where we previously detected Coccidioides via culture-independent molecular methods. By testing the serum from 40-trapped rodents with ELISA, we detected antibodies against Coccidioides in two species: Peromyscus maniculatus and Neotoma lepida. This study comprises the first report of wild rodent serum tested for coccidioidal antibodies, and sets the basis to analyze this pathogen in its natural environment and explore its potential ecological niche.

Lymphocytes from chronic lymphocytic leukaemia undergo ABL1-linked amoeboid motility and homotypic interaction as an early adaptive change to ex vivo culture.

BACKGROUND: Those stimuli that together promote the survival, differentiation and proliferation of the abnormal B-lymphocytes of chronic lymphocytic leukaemia (CLL) are encountered within tissues, where together they form the growth-supporting microenvironment. Different tissue-culture systems promote the survival of the neoplastic lymphocytes from CLL, partly replicating the in vivo tissue environment of the disorder. In the present study, we focussed on the initial adaptive changes to the tissue culture environment focussing particularly on migratory behaviour and cellular interactions. METHODS: A high-density CLL culture system was employed to test CLL cell-responses using a range of microscopic techniques and flow cytometric analyses, supported by mathematical measures of cell shape-change and by biochemical techniques. The study focussed on the evaluation of changes to the F-actin cytoskeleton and cell behaviour and on ABL1 signalling processes. RESULTS: We showed that the earliest functional response by the neoplastic lymphocytes was a rapid shape-change caused through rearrangement of the F-actin cytoskeleton that resulted in amoeboid motility and promoted frequent homotypic interaction between cells. This initial response was functionally distinct from the elongated motility that was induced by chemokine stimulation, and which also characterised heterotypic interactions between CLL lymphocytes and accessory cells at later culture periods. ABL1 is highly expressed in CLL lymphocytes and supports their survival, it is also recognised however to have a major role in the control of the F-actin cytoskeleton. We found that the cytoplasmic fraction of ABL1 became co-localised with F-actin structures of the CLL lymphocytes and that the ABL1 substrate CRKL became phosphorylated during initial shape-change. The ABL-inhibitor imatinib mesylate prevented amoeboid movement and markedly reduced homotypic interactions, causing cells to acquire a globular shape to rearrange F-actin to a microvillus form that closely resembled that of CLL cells isolated directly from circulation. CONCLUSION: We suggest that ABL1-induced amoeboid motility and homotypic interaction represent a distinctive early response to the tissue environment by CLL lymphocytes. This response is separate from that induced by chemokine or during heterotypic cell-contact, and may play a role in the initial entry and interactions of CLL lymphocytes in tissues.

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.

Case report: Abortion and disseminated infection by Coccidioides posadasii in an alpaca (Vicugna pacos) fetus in Southern California.

Coccidioidomycosis is a fungal disease affecting humans and other mammals caused by the soil-dwelling fungi Coccidioides immitis and C. posadasii. Abortion due to Coccidioides spp. infection is rare in domestic animals and transplacental transmission is considered uncommon in women. This report describes a case of placental-fetal infection and abortion in an alpaca with disseminated C. posadasii infection. PCR amplification and DNA sequencing were used to confirm the etiology, C. posadasii, in fetal tissues.

Challenges with using estimates when calculating ART need among adults in South Africa.

BACKGROUND: The Foundation for Professional Development (FPD) collects information annually on HIV/AIDS service provision and estimates service needs in the City of Tshwane Metropolitan Municipality (CTMM). METHODS: Antiretroviral therapy (ART) data from the Department of Health and Statistics South Africa (SSA) mid-year population estimates were used to approximate the ART need among adults in the CTMM. RESULTS: According to SSA data, ART need decreased dramatically from 2010 to 2011 and was lower than the number of adults receiving ART. Although the noted difference was probably due to changes in the calculations by SSA, no detailed or confirmed explanation could be offered. CONCLUSIONS: We provide a constructive contribution to the discussion about the use of model-derived estimates of ART need.

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.

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.

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.