Urinary extracellular vesicles as a source of protein-based biomarkers in feline chronic kidney disease and hypertension.

OBJECTIVES: To validate a methodology for isolating feline urinary extracellular vesicles and characterise the urinary extracellular vesicle population and proteome in cats with normal renal function and cats with normotensive or hypertensive chronic kidney disease. METHODS: Feline urinary extracellular vesicles were isolated using three different methods (precipitation alone, precipitation followed by size exclusion chromatography and ultrafiltration followed by size exclusion chromatography, which were compared via transmission electron microscopy and nanoparticle tracking analysis. Cats with normal renal function (n=9), normotensive chronic kidney disease (n=10) and hypertensive chronic kidney disease (n=9) were identified and urinary extracellular vesicles isolated from patient urine samples via ultrafiltration followed by size exclusion chromatography. Extracellular vesicle size and concentration were determined using nanoparticle tracking analysis, and subsequently underwent proteomic analysis using liquid chromatography with tandem mass spectrometry to identify differences in protein expression between categories. RESULTS: Urinary extracellular vesicle preparations contained particles of the expected size and morphology, and those obtained by ultrafiltration + size exclusion chromatography had a significantly higher purity (highest particle: protein ratio). The urinary extracellular vesicle proteomes contained extracellular vesicle markers and proteins originating from all nephron segments. Urinary extracellular vesicle concentration and size were unaffected by renal disease or hypertension. There were no differentially expressed proteins detected when comparing urinary extracellular vesicles derived from cats in the healthy category with the combined chronic kidney disease category, but five differentially expressed proteins were identified between the normotensive chronic kidney disease and hypertensive chronic kidney disease categories. CLINICAL SIGNIFICANCE: Feline urinary extracellular vesicles can be successfully isolated from stored urine samples. Differentially expressed urinary extracellular vesicle proteins were discovered in cats with hypertensive chronic kidney disease, and warrant further investigation into their utility as biomarkers or therapeutic targets.

Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation.

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αß and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1-6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12-US21; a genetic arrangement, which is suggestive of an 'accordion' expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.

A rapid flow cytometric assay to detect CD4+ and CD8+ T-helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporin therapy.

BACKGROUND: The immune response in atopic dermatitis (AD) is thought to be driven by T-helper (Th) 2 cytokines. Using flow cytometry, higher frequencies of peripheral blood CD4+ and CD8+ T cells producing interleukin (IL)-4 and correspondingly lower frequencies of CD4+ T cells producing interferon (IFN)-gamma have been found in patients with AD compared with healthy controls. It would be of interest to know whether other Th1 and Th2 cytokines such as IL-5, IL-13 and tumour necrosis factor (TNF)-alpha are similarly skewed in patients with AD and whether this immune skewing, detected via a simple blood assay, can be correlated with other clinical measurements or treatments in AD. OBJECTIVES: To use a rapid (4-h) flow cytometric assay to study a wide range of Th1 and Th2 cytokine patterns in peripheral blood lymphocytes from patients with AD, comparing them with non-atopic healthy controls. To correlate cytokine patterns with the degree of eosinophilia observed and in the case of one patient with severe disease, to observe the effect of cyclosporin therapy on peripheral blood cytokine patterns. METHODS: Peripheral blood from eight patients with AD and 23 healthy controls was examined for the frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4, IL-5, IL-13, IFN-gamma and TNF-alpha using flow cytometry. RESULTS: Significantly higher frequencies of CD4+/IL-4+ (P < 0.005) and CD4+/IL-13+ (P < 0.0001) and lower frequencies of CD4+/IFN-gamma+ (P < 0.002) and CD8+/TNF-alpha+ (P < 0.05) T lymphocytes were found in patients with AD compared with controls. There were significant positive correlations with the increased percentages of CD4+/IL-4+ and CD4+/IL-13+ T lymphocytes and the degree of eosinophilia observed (P < 0.05, P < 0.001) and a negative correlation between the percentage of CD4+/IFN-gamma+ T lymphocytes and eosinophilia (P < 0.05). In one patient examined before and 8 days after cyclosporin therapy, 50% or greater reductions were observed in percentages of peripheral blood CD8+/IL-5+, CD8+/IL-13+, CD4+/IL-4+ and CD4+/IL-5+ T lymphocytes following cyclosporin therapy. A smaller reduction of 15% after cyclosporin therapy was found in percentages of CD4+/IL-13+ T cells. CONCLUSIONS: These data strongly support a Th2 predominance in the peripheral blood of AD. The results suggest that administration of cyclosporin therapy in patients with AD may help to restore the Th2 cytokine imbalance seen in these patients.

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis.

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8- T cells, IL-2 and interferon-gamma (IFN-gamma) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8+ T cells IL-2 was optimally induced after 10 h and IFN-gamma after 6 h. The levels of IL-2 and IFN-gamma in CD8+ and CD8- T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2+ (range 9.7-41.3) and CD3/IFN-gamma+ cells (10.1-44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3+/CD8+ peripheral blood T cells expressing IFN-gamma and an increased percentage of CD3+/CD8- T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.

The road to Cairo.

PIP: The final statement made by DAWN to the 3rd final preparatory meeting for the United Nations Conference on Population and Development scheduled for September, 1994, in Cairo was given in full. DAWN's recommendations were briefly stated in 10 points. The women's group suggested that 1) structural adjustment assurances be made that investments in social programs would not be reduced; 2) equity and poverty adjustment and job creation be included in the development model; 3) population policy promote the well-being of people and reproductive rights of women; 4) comprehensive, high quality health services for women be provided responsibly by governments; 5) women's rights be recognized as human rights; 6) health services be reorganized to incorporate a wide range of services, including safe abortion; 7) safe, legal abortion be recognized as a necessary right; 8) gender and empowerment programs receive financial support; 9) accountability be required and women's groups' representation be included; and 10) resources be restructured to incorporate the aforementioned recommendations. The closing statement stressed the need to use a broad context and not to separate population issues from other issues of development. Women's issues are inseparably linked with women's rights, women's empowerment, and provision of comprehensive health services. Equity is a central development issue. The impact of development models on women must be considered. Development models and women's rights are both topics that need to be addressed concurrently at the conference. The Vatican's attempt to separate the issues and focus exclusively on women's rights only in religiously defined terms is blatantly political and an exercise of power domination of women. Many women's groups from a broad base of support have lobbied this forthcoming proceeding for a broader framework of women's rights within the document. Respect for women's issues will come from greater involvement of women in monitoring policies and implementation of programs.^ieng

Structural adjustment: cure or curse? Implications for Caribbean development.

PIP: Applying a gender analysis to structural adjustment policies reveals why gender analysis should be viewed as a prerequisite which allows planners and development practitioners to understand the hidden biases in their policies. Structural adjustment policies seek to reduce consumption and stimulate production which is often export-oriented and fueled by foreign investment. The reallocation of governmental resources to such schemes has led to the virtual abandonment of the development goals of the 1970s. Gender analysis bridges the gap between macroeconomic analysis and micro-level policies and exposes the detrimental impact of structural adjustment policies at the household level. Gender analysis also reveals the importance of the dual role of women and how cuts in social services (reducing consumption) make women's lives harder while increases in production exploit women's cheap labor. Gender analysis exposes the dichotomies of private/public lives and exposes contradictions in policies which are derived without such analysis. Planners must recognize the essential links between women's productive and reproductive roles and avoid increasing burdens for women and, thus, undermining the effectiveness of economic and social development policies. In applying a gender perspective to analysis, planners can take advantage of the analytical tools which have been developed for the purpose. Planners can also recognize that those who use services are in the best position to determine priorities. This requires a consideration of whether it is possible to meet strategic gender needs simultaneously with meeting practical needs. The most useful purpose of gender analysis may simply be to question the fundamental principles of the development process itself in order to design projects to place more strategic power in the hands of those who will use the services.^ieng

Feminist issues in development.

PIP: The United Nations Decade for the Advancement of Women, from 1975 to 1985, leaves a legacy of a deeper understanding of the issues, and the emergence of new networks with the experience and commitment to work for further changes. However, the role and status of women did not improve. There is a new commitment to struggle for the ending of all oppression, injustice and violence of all kinds at all levels. Feminism is a consciousness of all forms of women's oppression and a commitment to work against them. Feminist critiques illuminate the larger structures that oppress both women and men. New development theories embracing feminism are necessary to understand how patriarchy and economic systems propogate oppression. The production-oriented approach to rural development is flawed n failing to address women's lack of access to land, credit, training and new technologies. Overwhelming household tasks, cultural norms, and traditional attitudes limit women's involvement in training programs and other development activities. The basic needs approach to rural development provides access to vital services to meet a family's basic needs for nutrition, housing and clothing, and allows people's participation in decision making. However, women have little actual role in decision making so their needs, concerns and perspectives are not taken into account. Women are treated as instruments to achieve goals without appreciating their perspective. Project-based approached emphasize short term goals rather than laying the foundation for longterm changes. Few projects address structural issues or empower women. Projects must include education to increase personal growth and self reliance. Development planning can be enormously enhanced by taking gender differences into account and recognizing that people, specially poor women, can promote their own devleopment. Longterm strategies that challenge existing structures, address the existing economic order, and, most of all, recognize women's voices are needed.^ieng