Clearance of p-cresol sulfate and β-2-microglobulin from dialysate by commercially available sorbent technology

Dialysate regeneration by sorbents is an alternative to conventional single-pass dialysis. Little is known about the capacity of sorbents to clear dialysate of “middle molecules” and protein-bound uremic toxins. We studied p-cresol sulfate (PCS) and β-2-microglobulin (β2M) removal from dialysate by a sorbent: 1. PCS (40 mg PCS dissolved in 4 L of fresh dialysate) was recirculated through a sorbent cartridge (SORB Technology, Inc.) for analysis of PCS removal. 2. Spent peritoneal dialysate was recirculated on the “blood” side of a high-flux dialyzer. On the “dialysate” side of the membrane, bicarbonate dialysate was recirculated through a sorbent cartridge. β2M was measured in both streams. Two results are of particular importance for the use of regenerated fluid in chronic dialysis: 1. PCS was virtually completely removed from the dialysate. On average, PCS concentration was reduced to 1.4% of the starting concentration after 60 minutes. PCS extraction across the sorbent was nearly complete at any time. 2. β2M was on average reduced to 14.3% of the starting concentration after 60 minutes. Postsorbent concentrations were consistently below the validated range of the test method. We conclude that PCS and β2M are efficiently removed from the dialysate by commercially available sorbent technology. Spent peritoneal dialysis fluid can be cleared of β2M when circulated against sorbent-regenerated dialysate using a high-flux membrane.

Prognostic impact of β-2-microglobulin expression in colorectal cancers stratified by mismatch repair status

β-2-microglobulin (B2M) is essential for antigen presentation, yet may also possess proto-oncogenic properties.

Factors other than the glomerular filtration rate that determine the serum beta-2-microglobulin level.

BACKGROUND β2-microglobulin has been increasingly investigated as a diagnostic marker of kidney function and a prognostic marker of adverse outcomes. To date, non-renal determinants of β2-microglobulin levels have not been well described. Non-renal determinants are important for the interpretation and appraisal of the diagnostic and prognostic value of any endogenous kidney function marker. METHODS This cross-sectional analysis was performed within the framework of the www.seniorlabor.ch study, which includes subjectively healthy individuals aged ≥ 60 years. Factors known or suspected to have a non-renal association with kidney function markers were investigated for a non-renal association with serum β2-microglobulin. As a marker of kidney function, the Berlin Initiative Study equation 2 for the estimation of the estimated glomerular filtration rate (eGFR(BIS2)) in the elderly was employed. RESULTS A total of 1302 participants (714 females and 588 males) were enrolled in the study. The use of a multivariate regression model adjusting for age, gender and kidney function (eGFR(BIS2)) revealed age, male gender, and C-reactive protein level to be positively associated with β2-microglobulin levels. In addition, there was an inverse non-renal relationship between systolic blood pressure, total cholesterol and current smoking status. No association with markers of diabetes mellitus, body stature, nutritional risk, thyroid function or calcium and phosphate levels was observed. CONCLUSIONS Serum β2-microglobulin levels in elderly subjects are related to several non-renal factors. These non-renal factors are not congruent to those known from other markers (i.e. cystatin C and creatinine) and remind of classical cardiovascular risk factors.

β2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis

Aging drives cognitive and regenerative impairments in the adult brain, increasing susceptibility to neurodegenerative disorders in healthy individuals. Experiments using heterochronic parabiosis, in which the circulatory systems of young and old animals are joined, indicate that circulating pro-aging factors in old blood drive aging phenotypes in the brain. Here we identify β2-microglobulin (B2M), a component of major histocompatibility complex class 1 (MHC I) molecules, as a circulating factor that negatively regulates cognitive and regenerative function in the adult hippocampus in an age-dependent manner. B2M is elevated in the blood of aging humans and mice, and it is increased within the hippocampus of aged mice and young heterochronic parabionts. Exogenous B2M injected systemically, or locally in the hippocampus, impairs hippocampal-dependent cognitive function and neurogenesis in young mice. The negative effects of B2M and heterochronic parabiosis are, in part, mitigated in the hippocampus of young transporter associated with antigen processing 1 (Tap1)-deficient mice with reduced cell surface expression of MHC I. The absence of endogenous B2M expression abrogates age-related cognitive decline and enhances neurogenesis in aged mice. Our data indicate that systemic B2M accumulation in aging blood promotes age-related cognitive dysfunction and impairs neurogenesis, in part via MHC I, suggesting that B2M may be targeted therapeutically in old age.

Detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler

BACKGROUND: Cystic fibrosis (CF) is associated with at least 1 pathogen point sequence variant on each CFTR allele. Some symptomatic patients, however, have only 1 detectable pathogen sequence variant and carry, on the other allele, a large deletion that is not detected by conventional screening methods. METHODS: For relative quantitative real-time PCR detection of large deletions in the CFTR gene, we designed DNA-specific primers for each exon of the gene and primers for a reference gene (beta2-microglobulin). For PCR we used a LightCycler system (Roche) and calculated the gene-dosage ratio of CFTR to beta2-microglobulin. We tested the method by screening all 27 exons in 3 healthy individuals and 2 patients with only 1 pathogen sequence variant. We then performed specific deletion screenings in 10 CF patients with known large deletions and a blinded analysis in which we screened 24 individuals for large deletions by testing 8 of 27 exons. RESULTS: None of the ratios for control samples were false positive (for deletions or duplications); moreover, for all samples from patients with known large deletions, the calculated ratios for deleted exons were close to 0.5. In addition, the results from the blinded analysis demonstrated that our method can also be used for the screening of single individuals. CONCLUSIONS: The LightCycler assay allows reliable and rapid screening for large deletions in the CFTR gene and detects the copy number of all 27 exons.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Does subjective burden of early breast cancer and its treatment affect immune measures during adjuvant therapy?

Psychosocial factors have been described as affecting cellular immune measures in healthy subjects. In patients with early breast cancer we explored bi-directional psycho-immune effects to determine whether subjective burden has an impact on immune measures, and vice versa. Patients (n = 239) operated for early breast cancer and randomized into International Breast Cancer Study Group (IBCSG) adjuvant clinical trials were assessed immediately before the beginning of adjuvant treatment (baseline) and 3 and 6 months thereafter, at the beginning of the corresponding treatment cycle. Cellular immune measures (leukocytes, lymphocytes, lymphocyte subset counts), markers of activation of the cellular immune system (beta2-microglobulin, soluble interleukin-2 receptor serum levels), and self-report subjective burden (global indicators of physical well-being, mood, coping effort) were assessed concurrently. The relationship between subjective burden and gradients of immune measures was investigated with regression analyses controlling for adjuvant treatment. There was a pattern of small negative associations between all variables assessing subjective burden before the beginning of adjuvant therapy with the gradients of the markers of activation of the cellular immune system and NK cell counts. In particular, better mood predicted a decline in the course of beta2-microglobulin and IL-2r at months 3 and 6. The gradient of beta2-microglobulin was associated with mood and coping effort at month 3. However, the effect sizes were very small. In conclusion, in this explorative investigation, there was an indication for subjective burden affecting and being affected by markers of activation of the cellular immune system during the first 3 and 6 months of adjuvant therapy. The question of clinical significance remains unanswered. These associations have to be investigated with refined assessment tools and schedules.

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells.

Bovine colostrogenesis is distinguished by the specific transfer of IgG1 from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E2 ) and progesterone (P4 ) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta-2-Microglobulin (β2M). We hypothesized that steroid hormone treatments (E2 and P4 ) of bovine mammary epithelial cells in vitro would induce up-regulation of IgG1 transcytosis candidate gene mRNA expression suggesting involvement in IgG1 transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up-regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG1 transcytosis in bovine mammary cells.

Does Stepwise Voltage Ramping Protect the Kidney from Injury During Extracorporeal Shockwave Lithotripsy? Results of a Prospective Randomized Trial

BACKGROUND Renal damage is more frequent with new-generation lithotripters. However, animal studies suggest that voltage ramping minimizes the risk of complications following extracorporeal shock wave lithotripsy (SWL). In the clinical setting, the optimal voltage strategy remains unclear. OBJECTIVE To evaluate whether stepwise voltage ramping can protect the kidney from damage during SWL. DESIGN, SETTING, AND PARTICIPANTS A total of 418 patients with solitary or multiple unilateral kidney stones were randomized to receive SWL using a Modulith SLX-F2 lithotripter with either stepwise voltage ramping (n=213) or a fixed maximal voltage (n=205). INTERVENTION SWL. OUTCOMES MEASUREMENTS AND STATISTICAL ANALYSIS The primary outcome was sonographic evidence of renal hematomas. Secondary outcomes included levels of urinary markers of renal damage, stone disintegration, stone-free rate, and rates of secondary interventions within 3 mo of SWL. Descriptive statistics were used to compare clinical outcomes between the two groups. A logistic regression model was generated to assess predictors of hematomas. RESULTS AND LIMITATIONS Significantly fewer hematomas occurred in the ramping group(12/213, 5.6%) than in the fixed group (27/205, 13%; p=0.008). There was some evidence that the fixed group had higher urinary β2-microglobulin levels after SWL compared to the ramping group (p=0.06). Urinary microalbumin levels, stone disintegration, stone-free rate, and rates of secondary interventions did not significantly differ between the groups. The logistic regression model showed a significantly higher risk of renal hematomas in older patients (odds ratio [OR] 1.03, 95% confidence interval [CI] 1.00-1.05; p=0.04). Stepwise voltage ramping was associated with a lower risk of hematomas (OR 0.39, 95% CI 0.19-0.80; p=0.01). The study was limited by the use of ultrasound to detect hematomas. CONCLUSIONS In this prospective randomized study, stepwise voltage ramping during SWL was associated with a lower risk of renal damage compared to a fixed maximal voltage without compromising treatment effectiveness. PATIENT SUMMARY Lithotripsy is a noninvasive technique for urinary stone disintegration using ultrasonic energy. In this study, two voltage strategies are compared. The results show that a progressive increase in voltage during lithotripsy decreases the risk of renal hematomas while maintaining excellent outcomes. TRIAL REGISTRATION ISRCTN95762080.

The cystatin C/creatinine ratio, a marker of glomerular filtration quality: associated factors, reference intervals, and prediction of morbidity and mortality in healthy seniors.

The ratio of cystatin C (cysC) to creatinine (crea) is regarded as a marker of glomerular filtration quality associated with cardiovascular morbidities. We sought to determine reference intervals for serum cysC-crea ratio in seniors. Furthermore, we sought to determine whether other low-molecular weight molecules exhibit a similar behavior in individuals with altered glomerular filtration quality. Finally, we investigated associations with adverse outcomes. A total of 1382 subjectively healthy Swiss volunteers aged 60 years or older were enrolled in the study. Reference intervals were calculated according to Clinical & Laboratory Standards Institute (CLSI) guideline EP28-A3c. After a baseline exam, a 4-year follow-up survey recorded information about overall morbidity and mortality. The cysC-crea ratio (mean 0.0124 ± 0.0026 mg/μmol) was significantly higher in women and increased progressively with age. Other associated factors were hemoglobin A1c, mean arterial pressure, and C-reactive protein (P < 0.05 for all). Participants exhibiting shrunken pore syndrome had significantly higher ratios of 3.5-66.5 kDa molecules (brain natriuretic peptide, parathyroid hormone, β2-microglobulin, cystatin C, retinol-binding protein, thyroid-stimulating hormone, α1-acid glycoprotein, lipase, amylase, prealbumin, and albumin) and creatinine. There was no such difference in the ratios of very low-molecular weight molecules (urea, uric acid) to creatinine or in the ratios of molecules larger than 66.5 kDa (transferrin, haptoglobin) to creatinine. The cysC-crea ratio was significantly predictive of mortality and subjective overall morbidity at follow-up in logistic regression models adjusting for several factors. The cysC-crea ratio exhibits age- and sex-specific reference intervals in seniors. In conclusion, the cysC-crea ratio may indicate the relative retention of biologically active low-molecular weight compounds and can independently predict the risk for overall mortality and morbidity in the elderly.