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.

Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.

Type 1 diabetes is an autoimmune disease in which the insulin-producing pancreatic beta cells are destroyed at an early age by an immune process that involves both CD4 and CD8 T lymphocytes. The identification of autoantigens in diabetes is very important for the design of antigen-specific immunotherapy. By screening a pancreatic islet cDNA library, we have identified the autoantigen recognized by highly pathogenic CD8 T cells in the non-obese diabetic mouse, one of the best animal models for human diabetes. This is the first identification, to our knowledge, of a CD8 T-cell epitope in an autoimmune disease. The peptide recognized by the cells is in the same region of the insulin B chain as the epitope recognized by previously isolated pathogenic CD4 T cells. This has very important implications for the potential use of insulin in preventative therapy.

Antigen presentation: TAP dances with ATP.

Assembly of antigen-presenting complexes between class I MHC molecules and peptide requires formation of a complex between the 'ABC' peptide transporter, TAP, and newly synthesized class I molecules. Recent studies have provided new insights into the role of ATP in peptide binding, transport and release.

Replacement of the macronuclear ribosomal RNA genes of a mutant Tetrahymena using electroporation.

The macronucleus of the ciliate Tetrahymena contains approx. 10(4) ribosomal RNA gene molecules (rDNA) in the form of linear, autonomously replicating palindromes. Previous studies have shown that macronuclear rDNA molecules derived from wild-type (wt) inbred strain C3 out-replicate those derived from wt inbred strain B, in macronuclei initially heterozygous for both, leading to the complete loss of the B rDNA. However, rmm-1, a cis-acting laboratory-induced mutation obtained previously by mutagenesis of inbred strain C3, causes the mutant rmm-1 rDNA to be completely out-replicated by B rDNA. These findings suggest the following hierarchy of replication potential: wt C3 greater than wt B greater than C3-rmm-1. We used electroporation to test whether cells containing only rmm-1 macronuclear rDNA are favorable recipients for transformation with either wt B or C3 donor rDNA molecules. The donor rDNA molecules carried the selectable marker Pmr (paromomycin resistance) located in the coding region of the 17S rRNA. Transformants were obtained, at a frequency greater than 1 in 10(5), by electroporation under a wide range of electrical discharge parameters. The fraction of cells surviving electroporation varied between 2 and greater than 95% in successful experiments. Replacement ('transplacement') of the recipient rDNA was observed, consistent with the prediction that B and C3 rDNA should out-replicate rmm-1 rDNA. These findings are also consistent with the previous conclusion that the differential replication determinants reside in the 5'-nontranscribed spacer of the rDNA.

Characteristics of and risk factors for compensated occupational injury and disease claims in dairy farmers: a case-control study.

Research indicates that dairy farmers have an elevated risk of work-related adverse health outcomes. This case-control study evaluated the characteristics of and risk factors for compensated occupational injury and disease claims among Finnish dairy farmers. The cases consisted of 19 farm couples in which both spouses had a history of multiple claims. There were 283 claims in total, a rate of 26.6 claims per 100 person-years. The controls consisted of 12 couples in which neither spouse had compensated or rejected claims during their work history as insured farmers. A combined mail/telephone survey charted potential risk factors for compensated claims. These claims frequently involved work tasks and causes related to animal husbandry. Cattle were the most common cause for injuries in general and for serious injuries in particular. Gender differences in farm work and claims were observed. Using logistic regression analyses, we identified personal and work-related risk factors including long work history, small-scale dairy farm operation, and conventional stanchion barn for dairy cattle. Outdated working conditions, while not statistically significant, were positively associated with claims as well. Declined current work ability and musculoskeletal or respiratory conditions were significantly associated with claims where each of these outcomes may contribute to the other. Identified factors could be used to select subgroups of dairy farmers with either elevated or reduced risk of claims. Prevention of adverse health outcomes could be most effective when targeted to farmers at highest risk of occupational injury and disease.

Risk factors for occupational injuries among full-time farmers in Finland.

The objectives of this study were to evaluate the frequency of and farm management-related risk factors for occupational injuries among full-time farmers. A computer-assisted telephone interview was conducted among randomly selected self-employed full-time farmers (n = 1182; 911 male and 271 female), The response rate was 86%. Two-thirds of the respondents raised dairy or beef cattle. Nearly 16% of the farmers had experienced one or more occupational injuries requiring medical consultation during the past 12 months; the total number of such injuries was 222. Injuries were more common among male (17 injuries/100 person-years) than female farmers (13 injuries/ 100 person-years). The injuries occurred most frequently in animal husbandry work (n = 97). Falling or slipping was the most common mechanism of injury. Poisson regression with a stepwise (forward) model selection procedure identified the following risk factors for occupational injuries: male gender, younger age, cooperation with other farmers, perceived high accident risk, and stress symptoms. The adjusted rate ratios for these risk factors ranged from 1.40 to 1.96. This study indicates that interventions are needed, particularly among male farmers in their early years of full-time farm operation. At this stage of life, heavy financial burden and stress while establishing and expanding production may contribute to injuries. To reduce stress and related injuries, we recommend guidance for farmers regarding the organization and management of farm work.

Risk factors and prevalence of declined work ability among dairy farmers.

Farm work, particularly with livestock, exposes farmers to injuries, occupational diseases, and disabling health conditions, which in many cases result in early retirement and loss of quality of life. The objectives of this study were to evaluate the prevalence of and risk factors for declined work ability among full-time dairy farmers. We conducted a postal survey using the standard Work Ability Index (WAI) questionnaire augmented with a form containing questions about working conditions and lifestyle factors that potentially affect work ability. We received 399 usable responses (245 female and 154 male; 41.5% response rate). The prevalence of declined work ability (poor or moderate WAI score) was 39% overall, 44% among females, and 32% among males. Older age, small herd size, lack of mental breaks from work, inadequate leisure, and non-use of alcohol were significantly associated with declined work ability. The odds ratios for these risk factors ranged from 2.04 to 4.67. Current injuries and diseases are part of the WAI questionnaire contributing to declined work ability. This study indicates that interventions are needed, particularly among older farmers and farmers with injuries or diseases, to restore their work ability. Life-long measures to maintain work ability are also important, preventing the steep decline in work ability currently occurring among older farmers. Based on this study, we recommend guidance addressing the identified risk factors, particularly the importance of organizing both farm and domestic work with adequate rest, leisure time, and mental breaks as counterbalance to the daily workload among livestock farmers.

Work Ability Index among Finnish dairy farmers.

Full-time farmers and particularly dairy farmers who plan to expand their production have voiced concerns about their physical and mental work ability in recent studies. The objectives of this study were to characterize the work ability of dairy farmers and to identify demographic groups at risk of disability and in greatest need of interventions to promote work ability. We conducted a postal survey using the Work Ability Index (WAI) questionnaire. The WAI of 399 dairy farmers (245 female and 154 male) was analyzed (response rate 41.5%). The mean WAI score was 36.0 among female and 39.0 among male respondents (scale: 7 = worst to 49 = best). The WAI decreased with age. The WAI was systematically better among males compared to females in all age groups, and the difference was greatest among those over 45 years of age. About one-fourth of females and one-tenth of males over 45 years of age were at an imminent risk of disability (poor WAI). The WAI of farmers in our study was similar to farmers in previous studies where entrepreneurs and salaried workers had better WAI compared to farmers. This study indicates that interventions are needed among older dairy farmers, particularly females, to help them improve their work ability. The first question (of seven) in the WAI questionnaire correlated well with the complete questionnaire-based WAI. The first question could be used in surveys as a condensed version of the WAI, if the same correlation is found in future studies. Based on this study, we recommend using the Work Ability Index questionnaire for assessing the health of those working in agriculture.

Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene.

We have used the bacterial beta-galactosidase gene (lacZ) as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer [Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834]. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic beta-galactosidase substrate fluorescein di-beta-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/Ab complex, was transfected with the NF-AT-lacZ construct. lacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacological agents, to the physiological peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concentrations of the stimulus increased the fraction of lacZ+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ+ cells remained within a remarkably narrow range. These results demonstrate that TCR-mediated activation can be readily measured in single T cells and strongly suggest that, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concentration of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.

Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens.

The alpha/beta T-cell receptor a complex ligand formed by the association of antigenic peptides with molecules of the major histocompatibility complex (MHC). The inherent limitations of the conventional T-cell activation assays used to detect these peptide/MHC ligands have, until now, hampered the development of expression cloning systems for T-cell antigens. To overcome these limitations, we have recently introduced a method for detecting ligand-induced activation of individual T cells. This assay, which makes use of a lacZ reporter construct, differs from conventional ligand-induced activation assays in that it allows the detection of single, activated T cells in large pools of resting cells. We applied the lacZ assay to the problem of screening expression libraries, which requires the ability to detect ligand-bearing antigen-presenting cells when they are present at very low frequency. We show here that ligand-expressing antigen-presenting cells can be detected at frequencies of 1:10(3)-10(4), a level of sensitivity compatible with the screening of cDNA libraries. Furthermore, by using as antigen-presenting cells COS-7 cells stably transfected with the murine Kb class I MHC molecule, we demonstrate that transiently expressed ovalbumin is efficiently processed and presented to an ovalbumin/Kb-specific T-cell hybridoma. lacZ expression is induced in a detectable number of cocultured T cells, even when the ovalbumin cDNA consists of only 1:10(4) of the total DNA used to transfect the COS cells. These results suggest that unknown T-cell antigens may be identified by screening cDNA libraries in MHC-expressing COS cells using lacZ-inducible T cells as indicators of peptide antigen expression.

Murine memory B cells are multi-isotype expressors.

Flow cytometric analyses of the surface immunoglobulins of murine memory B cells revealed the existence of populations expressing multiple isotypes, including an IgM+/IgG+ population that could be stimulated in vitro with antigen to secrete both IgM and IgG. Female BALB/c mice were immunized with R-phycoerythrin (RPE), a fluorescent photosynthetic accessory protein from red algae. Pooled splenocytes from these mice at different stages of immunization were stained with RPE as well as with allophycocyanin- and fluorescein-conjugated anti-isotype antibodies and analysed on a two-laser FACS. RPE-binding cell sub-populations were defined and selectively sorted to verify their phenotype and to demonstrate that the various subpopulations (IgM+/IgG+, IgM+/IgG-, IgM-/IgG+) had different isotype-secretion patterns when challenged with RPE in vitro. These results re-affirm the notion that a transcriptional processing mechanism may be responsible for the simultaneous expression of multiple isotypes in memory cells.

The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation.

In its attempt to evade cytotoxic T cell recognition, human cytomegalovirus encodes several genes that target MHC class I molecules at different points in their assembly pathway. We show here that the human cytomegalovirus US6 gene encodes a 22-kDa glycoprotein that binds the transporter-associated with antigen processing (TAP)/class I complex and inhibits translocation of peptide from the cytosol to the endoplasmic reticulum. Major histocompatibility complex class I molecules are therefore unable to load TAP-dependent peptides, resulting in the retention of MHC class I molecules in the endoplasmic reticulum, with a consequent reduction in class I at the cell surface. Interferon-gamma treatment of US6 transfected cells overcomes this inhibition of peptide translocation and restores class I at the cell surface to wild type levels. The functional consequence of TAP inhibition is that US6 transfected cells are unable to present endogenous antigen to cytotoxic T lymphocytes and are therefore resistant to cytotoxic T lymphocyte lysis.

Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP).

The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.

CD40 is a cellular receptor mediating mycobacterial heat shock protein 70 stimulation of CC-chemokines.

The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.