Sclerostin and Dickkopf-1 in post-menopausal renal allograft recipients.

BACKGROUND: Age, pre-existing renal osteodistrophy, impaired renal function, and chronic use of immunosuppressive drugs are the main factors involved in the onset and development of bone metabolism disturbances and skeletal alterations occurring after renal transplantation. However, at the state of the art, no reports have analyzed the additional post-menopausal physiological mechanisms associated with the onset and development of bone complications in renal transplant recipients. METHODS: We measured by means of molecular strategies (enzyme-linked immunoassay, chemiluminescence) the serum levels of Sclerostin and Dickkopf-1 (DKK1), two major antagonists of the Wnt/ß-catenin pathway, and several bone resorption/formation biomarkers (N-terminal procollagen type 1, bone-specific alkaline phosphatase, and serum C-terminal telopeptides of type I collagen) in 19 post-menopausal kidney transplant patients and 12 post-menopausal chronic kidney disease patients (CKD group) matched for age and renal function. RESULTS: Our results showed that the levels of both Wnt antagonists were similar in the two study groups (P=.15 and .96, respectively). Additionally, no correlation was found between Sclerostin and DKK1 serum levels in all patients included in the study (R2=0.03, P=.2). After statistical analysis, we found no differences in the bone resorption/formation biomarkers between renal transplant and CKD patients. Multivariate analysis showed that Sclerostin levels were significantly positively correlated with serum phosphorus levels (P=.008) and inversely correlated with renal function (P=.026). Surprisingly, no significant correlation was found between all the analyzed demographic and clinical parameters and DKK1. CONCLUSIONS: Our study demonstrated for the first time that renal transplantation per se and immunosuppressive treatments do not represent additional factors contributing to bone metabolic/biochemical alterations in post-menopausal women. However, our results emphasized that a better preservation of the graft function could significantly slow down the progression of bone metabolic deregulations and prevent clinical bone complications.

Pharmacogenomics: a new paradigm to personalize treatments in nephrology patients.

Although notable progress has been made in the therapeutic management of patients with chronic kidney disease in both conservative and renal replacement treatments (dialysis and transplantation), the occurrence of medication-related problems (lack of efficacy, adverse drug reactions) still represents a key clinical issue. Recent evidence suggests that adverse drug reactions are major causes of death and hospital admission in Europe and the United States. The reasons for these conditions are represented by environmental/non-genetic and genetic factors responsible for the great inter-patient variability in drugs metabolism, disposition and therapeutic targets. Over the years several genetic settings have been linked, using pharmacogenetic approaches, to the effects and toxicity of many agents used in clinical nephrology. However, these strategies, analysing single gene or candidate pathways, do not represent the gold standard, being the overall pharmacological effects of medications and not typically monogenic traits. Therefore, to identify multi-genetic influence on drug response, researchers and clinicians from different fields of medicine and pharmacology have started to perform pharmacogenomic studies employing innovative whole genomic high-throughput technologies. However, to date, only few pharmacogenomics reports have been published in nephrology underlying the need to enhance the number of projects and to increase the research budget for this important research field. In the future we would expect that, applying the knowledge about an individual's inherited response to drugs, nephrologists will be able to prescribe medications based on each person's genetic make-up, to monitor carefully the efficacy/toxicity of a given drug and to modify the dosage or number of medications to obtain predefined clinical outcomes.

Emotional symptoms, quality of life and cytokine profile in hemodialysis patients.

BACKGROUND: Mental disorders are frequent in hemodialysis (HD) patients. Depression and anxiety along with physical co-morbidity affect quality of life (QOL). Uremia is associated with inflammation and release of cytokines by lymphomonocytes. Inflammatory cytokines are relevant in depression. The aim of this study was to assess the psychological alterations and QOL in HD patients, and to correlate them with pattern of cytokine production. PATIENTS: 30 HD patients and 20 subjects with CKD Stage I-II K-DOQI. Psychometric tests were administered: 1) Hospital Anxiety and Depression Scale (HADS) composed of an anxiety subscale (HADS-A) and a depression subscale (HADS-D); 2) Kidney Disease Quality of Life (KDQOL) modified, including a cognitive function subscale (KDQOL-CF). Whole blood samples collected at beginning of HD session were diluted with RPMI/heparin and incubated for 24 h in presence of lipopolysaccharide (LPS). IL-1Gamma, IL-6, TNF-alpha and IL-10 were assayed on supernatants and results were normalized per number of lymphomonocytes (ng/106 cells). RESULTS: A depressive mood was more frequent in HD patients (50%) than controls (20%, p < 0.0001). No difference for anxiety (HD = 43%, controls = 45%) was observed. QOL score was significantly lower in HD than controls (p = 0.006) and correlated inversely with HADS total, HADS-A and HADS-D (p < 0.0001). Albumin, Kt/V and phosphate were comparable in patients with or without anxiety or depression. Cytokine production was significantly higher in HD patients than controls (IL-1beta p = 0.05; IL-6 p = 0.010; TNF-alpha p < 0.0001; IL-10, p = 0.0019). HD patients with the HADS-A positive for anxiety showed higher IL-6 production (p = 0.026), while IL-1beta levels were not associated with symptoms of depression. KDQOL-CF correlated inversely with levels of IL-6, TNF-alpha and IL-10. CONCLUSIONS: HD patients have symptoms of depression and anxiety that negatively affect QOL. These symptoms are independent of the efficiency of dialysis and nutritional status. On the contrary, IL-6 is linked to the presence of psychological discomfort in these patients.

Immunogenicity of a 23-valent pneumococcal polysaccharide vaccine in elderly residents of a long-term care facility

S. pneumoniae is a significant cause of community-acquired pneumonia in the elderly, and accounts for the majority of the pneumonia deaths among the elderly. We conducted this randomized double-blind study to evaluate the immune response to a 23-valent pneumococcal polysaccharide vaccine and the persistence of antibodies two years after the vaccination in an elderly population in Santiago, Chile. A total of 118 elderly nursing home residents received either the pneumococcal or a tetanus control vaccine. Serum samples were taken at enrolment, at two months, and at two years post-vaccination. Pre-vaccination anti-pneumococcal antibody geometric mean concentrations (GMC) were similar in both study groups, with increased levels of antibodies found only against serotype 14. The pneumococcal vaccine was highly immunogenic at 2 months, and titers remained high two years after the vaccination for the 10 serotypes studied in this elderly population. The results thus support the benefits of this pneumococcal vaccine in this elderly population who are at increased risk of invasive pneumococcal disease.

[Nasal carriage of Streptococcus pneumoniae in elderly subjects according to vaccination status]. / Portación nasal de Streptococcus pneumoniae en adulto mayor y su respuesta frente a la vacunación antineumocócica.

BACKGROUND: S pneumoniae is the main cause of community-acquired pneumonia in the elderly, group that concentrates 95% of deaths. AIM: To assess the prevalence of nasal carriage of S pneumoniae in institutionalized elderly patients. MATERIAL AND METHODS: One hundred eighteen institutionalized subjects aged over 60 years (65 males) were enrolled. Since they were also participating in a controlled protocol related to the immunogenicity of an anti-pneumococcal vaccine, our investigation was also blind and randomized. According to randomization, they received pneumococcal or tetanic vaccine. Nasal swab cultures were taken at the beginning of the trial and two months after vaccination. According to recommended methods, we identified S pneumoniae, the serotypes and their antimicrobial susceptibility. RESULTS: In the first nasal sample, 16% of subjects were positive for S pneumoniae. The second sample was positive in 12%. Of the 33 isolated serotypes, 9.1% demonstrated intermediate resistance to penicillin and 3.3% were resistant to chloramphenicol. CONCLUSIONS: The study demonstrated a greater percentage of colonized patients than in the general population. The isolated serotypes are the same that cause invasive diseases in this age group, according to data of the Institute of Public Health of Chile. There were no differences in the percentage of colonization between subjects vaccinated against S pneumoniae and control groups, after two months of follow up. Isolated strains had a low resistance to penicillin. High level resistance was not observed.

Portación nasal de Streptococcus pneumoniae en adulto mayor y su respuesta frente a la vacunación antineumocócica / Nasal carriage of Streptococcus pneumoniae in elderly subjects according to vaccination status

Background: S pneumoniae is the main cause of community-acquired pneumonia in the elderly, group that concentrates 95 percent of deaths. Aim: To assess the prevalence of nasal carriage of S pneumoniae in institutionalized elderly patients. Material and methods: One hundred eighteen institutionalized subjects aged over 60 years (65 males) were enrolled. Since they were also participating in a controlled protocol related to the immunogenicity of an anti-pneumococcal vaccine, our investigation was also blind and randomized. According to randomization, they received pneumococcal or tetanic vaccine. Nasal swab cultures were taken at the beginning of the trial and two months after vaccination. According to recommended methods, we identified S pneumoniae, the serotypes and their antimicrobial susceptibility. Results: In the first nasal sample, 16 percent of subjects were positive for S pneumoniae. The second sample was positive in 12 percent. Of the 33 isolated serotypes, 9.1 percent demonstrated intermediate resistance to penicillin and 3.3 percent were resistant to chloramphenicol. Conclusions: The study demonstrated a greater percentage of colonized patients than in the general population. The isolated serotypes are the same that cause invasive diseases in this age group, according to data of the Institute of Public Health of Chile. There were no differences in the percentage of colonization between subjects vaccinated against S pneumoniae and control groups, after two months of follow up. Isolated strains had a low resistance to penicillin. High level resistance was not observed.

Accuracy of the urinary albumin titrator stick 'Micral-Test' in kidney-disease patients.

BACKGROUND: A quick, accurate, and easy measurement of microalbuminuria can be useful for the management of many patients. The Micral-Test stick (Boehringer Mannheim, Germany) gives a semiquantitative estimation of urinary albumin concentration at discrete readings of 0, 10, 20, 50 or 100 mg/l. The purpose of this study was to test its accuracy. METHODS: From 46 non-diabetic patients, 491 urinary samples were analysed both with Micral-Test and with immunochemical nephelometry. RESULTS: Of 491 samples, 308 were from non-proteinuric patients (urinary albumin < 300 mg/day). In these patients the correlation coefficient of nephelometric values versus the stick readings was 0.79: 120/123 samples from non-microalbuminuric (< or = 30 mg/24 h) and 164/185 from microalbuminuric patients were correctly identified by the stick, giving an 89% sensitivity and a 98% specificity. The readings around the threshold for microalbuminuria (20 and 50 mg/l patches) were the most reliable ones. Analysis of the correct/uncorrect readings' ratio for every patch in the 245 samples in the 0-150 mg/l range shows a relative uniformity (chi2 = 8.5, P = 0.07), while analysis of the over/correct/underreadings for the 10, 20 and 50 patches, shows that incorrect responses tend to be underestimations for the 10 patch and overestimations for the 20 and 50 mg/l patches (chi2 = 10.5, P = 0.03). CONCLUSIONS: The Micral-Test stick is useful for screening, but less reliable for follow-up, unless considerable changes in albumin excretion are expected.

Albuminuria and transferrinuria in essential hypertension. Effects of antihypertensive therapy.

The objectives of this study were to evaluate the effects of an ACE inhibitor (fosinopril) and a calcium antagonist (amlodipine) on the urinary albumin and transferrin excretion and their relationship to the blood pressure in essential hypertension. Twenty-four never-treated patients (mean age, 46.4 +/- 8.9 years) with a diastolic blood pressure between 90 and 114 mm Hg and normal renal function, randomly received amlodipine or fosinopril and, if the diastolic blood pressure was not normalized, doxazosin was added to the therapy. Twenty-four-hour ambulatory blood pressure monitoring and 24-h urine collection for albumin and transferrin measurements were performed before and after 3 and 6 months of therapy. Diastolic blood pressure was normalized in 23 patients (96%). Before treatment, microalbuminuria was present in 50% of patients. In the amlodipine and fosinopril group, antihypertensive therapy significantly decreased blood pressure and, only in the fosinopril group, albuminuria. Transferrinuria did not change significantly in both groups. Fosinopril lowered albuminuria in all patients, whereas amlodipine only in half of patients. Albuminuria, but not transferrinuria, was significantly correlated to the ambulatory blood pressure. This correlation was more pronounced for systolic than for diastolic pressure. In essential hypertensive patients with normal renal function, a high prevalence of microalbuminuria can be observed. Albuminuria appears to correlate with ambulatory blood pressure, particularly with systolic pressure. Intrarenal hemodynamic changes seem to play a more important role than systemic blood pressure decrease in the reduction of albuminuria. Transferrinuria does not seem a useful marker to follow-up nondiabetic hypertensive patients with early signs of glomerular dysfunction.

Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica.

Since 1993, strains of Entamoeba histolytica sensu lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. histolytica, this sequence shows 1.7% nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts.

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verified with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram. The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and in their self-pollination progenies. While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points to in vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.

Glycated proteins in serum: effect of their relative proportions on their alkaline reducing activity in the fructosamine test.

The fructosamine test is considered clinically useful for assessing short-term integrated control of blood glucose, but there are few published data to support this hypothesis. We fractionated glycated and nonglycated proteins by affinity chromatography on phenylboronate columns and, with specific immunochemical methods, determined in the eluted fractions the following proteins, selected according to their biological half-lives and relative concentrations in serum: albumin, IgA, IgG, IgM, apolipoprotein B, haptoglobin, transferrin, alpha 1-antitrypsin, and alpha 2-macroglobulin. We found the following correlations between fructosamine (mmol/L) and, respectively, glycated albumin, IgG, and (albumin + IgG) (each in grams per liter): r = 0.901, 0.702, 0.878. IgM had the highest percentage of glycated molecules (range 11.1-37.5%, mean 22.4%), haptoglobin and alpha 1-antitrypsin the least. This result was almost independent of the proteins' molecular masses and fractional catabolic rate. Albumin evidently contributes most to results of the fructosamine test, confirming conclusions obtained in different ways by others.

Assessment of an automatic liquid chromatograph for foetal and abnormal haemoglobins.

The DIAMAT TM (Bio-Rad) analyser is a microprocessor-operated HPLC system using a silica-based weak cation exchange column with three phosphate buffers of increasing ionic strength as the step gradient mobile phase. A dual wavelength detector measures absorbance at 415 and 690 nm; each sample is completely processed in 8 minutes. The instrument effectively separates and quantifies HbF in a discrete peak. We have verified that the HbF assay is linear up to about 65% values. We calculated within-run imprecision for n = 20 in 3 different haemolysed blood samples; the results are shown below as percent of total haemoglobin: A) Mean = 1.06 SD = 0.048 CV% = 4.5 B) Mean = 1.90 SD = 0.041 CV% = 2.1 C) Mean = 8.93 SD = 0.047 CV% = 0.5 Between-run imprecision for similar samples (n = 18) was: D) Mean = 3.74 SD = 0.20 CV% = 5.5 E) Mean = 9.94 SD = 0.15 CV% = 1.5 Accuracy was assessed in different series by correlating Hb values (y) with those obtained by the alkali denaturation test (x). The regression line equation was y = 1.03 x - 0.33 (r = 0.999, n = 62). The DIAMAT TM instrument also reveals the presence of any HbS and calculates its peak area correctly, as we found by electrophoretic reassay in heterozygous subjects. We also noted the presence of other abnormal haemoglobins characterized by specific retention times.