KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine.

Urinary extracellular vesicles (EVs) are a promising source of biomarkers, which can be obtained in a non-invasive manner. However, the yield of EVs from urine samples may be insufficient for various analyses due to the entrapment of EVs by the Tamm-Horsfall protein (THP) meshwork. Here, we developed a simple dilution protocol to increase the urinary EV yield by disrupting the interaction between THP filaments and EVs with the help of alkaline pH and lowered ionic concentration. The integrity of the EVs and THP was assessed by electron microscopy. The effect of the protocol on the EV yield was quantified against an undiluted control by western blotting of four EV markers, nanoparticle tracking analysis and measuring of the RNA/miRNA concentration of the EV samples. The average EV yield from the dilution protocol was 2-7 fold the yield from the undiluted control i.e. increased by 130-624% as measured by western blotting and NTA. The yield increased most from samples with a high THP to EV ratio. The morphology and size range of the EVs were unaltered by the protocol. However, RNA/miRNA yields were the same as from the undiluted control and THP filaments could still be detected in EV samples. The dilution protocol, that we named KeepEX, provides a simple and efficient way to prevent loss of EVs thus increasing their yield from urine. Since KeepEX does not require individual adjustment of sample pH nor extra centrifugation steps, it could be used on its own or in combination with other EV purification protocols to improve EV isolation particularly from small urine volumes.

Seroreactivity for spotted fever rickettsiae and co-infections with other tick-borne agents among habitants in central and southern Sweden.

Patients seeking medical care with erythema migrans or flu-like symptoms after suspected or observed tick bite in the southeast of Sweden and previously investigated for Borrelia spp. and/or Anaplasma sp. were retrospectively examined for serological evidence of rickettsial infection (Study 1). Twenty of 206 patients had IgG and/or IgM antibodies to Rickettsia spp. equal to or higher than the cut-off titre of 1:64. Seven of these 20 patients showed seroconversion indicative of recent or current infection and 13 patients had titres compatible with past infection, of which five patients were judged as probable infection. Of 19 patients with medical records, 11 were positive for Borrelia spp. as well, and for Anaplasma sp., one was judged as positive. Five of the 19 patients had antibodies against all three pathogens. Erythema migrans or rash was observed at all combinations of seroreactivity, with symptoms including fever, muscle pain, headache and respiratory problems. The results were compared by screening an additional 159 patients (Study 2) primarily sampled for the analysis of Borrelia spp. or Mycoplasma pneumoniae. Sixteen of these patients were seroreactive for Rickettsia spp., of which five were judged as recent or current infection. Symptoms of arthritis, fever, cough and rash were predominant. In 80 blood donors without clinical symptoms, approximately 1 % were seroreactive for Rickettsia spp., interpreted as past infection. The study shows that both single and co-infections do occur, which illustrate the complexity in the clinical picture and a need for further studies to fully understand how these patients should best be treated.

Inclusion body myositis in Alzheimer's disease.

BACKGROUND: The prevalence of Alzheimer's disease is increasing. Could findings of similar deposits in brain and muscle tissue explain this increase? The purpose of this report is to illustrate that Alzheimer's disease and inclusion body myositis may share a common aetiology. RESULTS: We present a case where Alzheimer's disease and inclusion body myositis coexist in the same patient. Amyloid-beta deposition and the presence of phosphorylated tau protein have been noted in brain tissue and in muscle biopsy from patients with these disorders. METHODS: Electrophysiological methods are needed for proper diagnosis of this brain and muscle disorder. Recent data on deposit structures in both conditions may indicate an environmental aetiology for Alzheimer's disease and inclusion body myositis. CONCLUSION: By combining electrophysiological methods with muscle biopsy in cases of Alzheimer's disease, the possible aetiological connection between simultaneous affection of both muscle and brain in this condition can be established.

Association of various point mutations as risk factors for synthetic graft thrombosis in African American hemodialysis patients.

Single-nucleotide polymorphisms in the genes that code for coagulation factor V (factor V (Leiden)) and II (prothrombin G20210A), as well as the methyltetrahydrofolate reductase (MTHFR) have been implicated in the majority of cases of hereditary thrombophilia. In our hemodialysis patient population, comprised mainly of African Americans, we have seen a high incidence of early vascular access thrombosis without any identifiable predisposing factors. In some instances, there is recurrent access thrombosis that may be related to an inherited condition. In this study we evaluated the presence of these gene mutations and their association with increased risk of recurrent vascular access thrombosis in our hemodialysis patient population. Our results show that factor V Leiden and factor II prothrombin point mutations did not appear to increase the risk of vascular access thrombosis in African Americans with end-stage renal disease on hemodialysis. Patients heterozygous for the MTHFR gene, also did not show a significant increased risk for synthetic graft thrombosis.

Metallothionein gene expression in peripheral lymphocytes from cadmium-exposed workers.

Metallothionein (MT) plays an important role in the detoxification of cadmium. To investigate the usefulness of MT gene expression in peripheral blood lymphocytes (PBLs) as a biomarker of cadmium exposure and susceptibility, reverse transcriptase-polymerase chain reaction was used to measure the MT gene expression in PBLs from cadmium-exposed workers. Both basal and induced MT expressions were found to increase with increased blood cadmium (BCd) and urinary cadmium (UCd) levels. Both basal and induced MT expression levels were significantly correlated with the logarithm of BCd and the logarithm of UCd levels. The dose-response relationship between internal dose of cadmium and MT expression suggested the validity of MT expression in PBLs as a biomarker of cadmium exposure. In vitro induced MT expression level in PBLs was found to be inversely related to the level of renal dysfunction indicator, urinary N-acetyl-beta-D-glucosaminidase (UNAG). The latter finding indicates that MT expression in PBLs may be a useful biomarker of susceptibility to renal toxicity of cadmium.

Design, synthesis, computational prediction, and biological evaluation of ester soft drugs as inhibitors of dihydrofolate reductase from Pneumocystis carinii.

A series of lipophilic soft drugs structurally related to the nonclassical dihydrofolate reductase (DHFR) inhibitors trimetrexate and piritrexim have been designed, synthesized, and evaluated in DHFR assays, with special emphasis on the inhibition of P. carinii DHFR. The best inhibitors, encompassing an ester bond in the bridge connecting the two aromatic systems, were approximately 10 times less potent than trimetrexate and piritrexim. The metabolites were designed to be poor inhibitors. Furthermore, molecular dynamics simulations of three ligands in complex with DHFR from Pneumocystis carinii and from the human enzyme were conducted in order to better understand the factors determining the selectivity. A correct ranking of the relative inhibition of DHFR was achieved utilizing the linear interaction energy method. The soft drugs are intended for local administration. One representative ester was selected for a pharmacokinetic study in rats where it was found to undergo fast metabolic degradation to the predicted inactive metabolites.

Breast mass in a 55-year-old woman.

A 55-year-old white woman presented with a one-month history of a palpable breast mass. A mammogram was performed six months prior to her presentation and was read negative. However, the mammogram done two weeks prior to her clinical visit was suspicious for malignancy. Physical examination revealed a 1.5 cm to 2 cm palpable right breast mass. She underwent excision of the mass. Gross examination of the mass revealed a 2.5 cm well defined, ovoid-shaped, firm tan white tumor with a scalloped border, irregular extension into the surrounding tissue, and a granular cut surface. Largest tumor diameter was 2.5 cm. Microscopic examination of the tumor revealed small tumor cells with a solid, alveolar, and classic "single file" arrangement. There is also focal expansion and filling of the acini by similar small cells.

Computational predictions of binding affinities to dihydrofolate reductase: synthesis and biological evaluation of methotrexate analogues.

The relative binding affinities to human dihydrofolate reductase of four new potential antifolates, containing ester linkages between the two aromatic systems, were estimated by free energy perturbation simulations. The ester analogue, predicted to exhibit the highest binding affinity to human dihydrofolate reductase, and a reference ester (more structurally related to methotrexate) were synthesized. As deduced from the measured IC(50) values, the calculated ranking of the ligands was correct although a greater difference in affinity was indicated by the experimental measurements. Among the new antifolates the most potent inhibitor exhibited a similar pharmacokinetic profile to methotrexate but lacked activity in a complex antiarthritic model in rat in vivo.

Lead concentrations in elderly urban people related to blood pressure and mental performance: results from a population-based study.

BACKGROUND: The Kungsholmen project is a longitudinal study of ageing and dementia conducted in Stockholm in 1987. In a 1994-96 follow-up, 804 subjects had their blood samples analyzed for lead. METHODS: Lead concentration in blood in an elderly population aged 75+ (mean age of 88.4 years) was studied in relation to age, blood pressure (BP), body mass index (BMI), cognitive function measured with Mini-Mental State Examination (MMSE), gender, and smoking. RESULTS: The mean blood lead level (n = 762) was 3.7 microg/dL (0.18 micromol/L) whole blood with a standard deviation of 2.3, (0.11). There was a contribution of gender with men having higher blood lead levels than women (beta = -0.20; P = 0.000001) but not of smoking habits (beta = 0.07; P = 0.08) when these variables were entered into a multiple regression model with lead as the dependent variable (R = 0.22; P < 0.000001). Different multiple regression models were tested with lead as the dependent variable. No relation was found between lead concentrations and age, BMI, systolic BP, diastolic BP, or MMSE. Systolic and diastolic BP were correlated to BMI (R = 0.10; P = 0.01 and R = 0.22; P = 0.000 001, respectively). CONCLUSIONS: In this elderly population from a specified area of Stockholm it is unlikely that lead exposure affects BP or cognition. However, high lead levels in blood may reflect earlier occupational exposure or life style factors.

Toxicological aspects of metallothionein.

Metallothionein (MT) is expressed to a certain extent in almost all mammalian tissues. The biological significance of MT is related to its various forms MT-1, MT-2, MT-3 and MT4. For MT-1 several isoforms of the protein exist and it is likely that these isoforms are related to various functions involved in developmental processes occurring at various stages of gestation. Toxicokinetics and biochemistry of essential and toxic metals such as cadmium, zinc, mercury and copper in organs e.g. kidney, CNS, are often related to metallothionein. It is debated whether there is a relation or not for other metals e.g. selenium and bismuth. For the toxicokinetics of cadmium, MT plays an important role. By expanding techniques from experimental toxicology and biochemistry to include molecular biology methods, more specific and relevant studies can be performed of the actual role and biological function of MT. The present paper on toxicological aspects of metallothionein, presents an overview and evaluation of present knowledge concerning differences among organs and within organs of the expression of MT and how this affects tissue sensitivity to toxicity.