Differentiation of glomerular, tubular, and normal proteinuria: determinations of urinary excretion of β2-microglobulin, albumin, and total protein

A low molecular weight beta(2)-globulin (beta(2)-microglobulin), albumin, and total protein were measured in concentrated 24-hr urine specimens from 20 healthy subjects and 30 patients with clinical proteinuria of glomerular or tubular type. Classification of proteinuria was made on the basis of clinical diagnosis and size distribution of urinary proteins after gel chromatography. The molecular radii (Stokes' radii) of beta(2)-microglobulin and albumin, estimated by gel chromatography, were 15 A and 35 A.The average 24-hr urinary excretion in healthy subjects was 0.12 mg for beta(2)-microglobulin, 10 mg for albumin, and 80 mg for total protein. The patients with renal glomerular disorders had normal or only somewhat increased excretion of beta(2)-microglobulin, despite considerably increased excretion of albumin and total protein. Most of the patients with tubular dysfunction excreted large amounts of beta(2)-microglobulin, although they excreted normal or only slightly increased amounts of albumin and only moderately increased quantities of total protein. Consequently, the ratio or urinary albumin/urinary beta(2)-microglobulin was high in glomerular proteinuria (1100: 14,200), intermediate in normal proteinuria (33: 163), and low in tubular proteinuria (1.0: 13.3). Determinations of urinary clearances of beta(2)-microglobulin and albumin in four healthy subjects and 11 patients indicated that increased excretions of the two proteins were associated with increased clearances. The results suggest that quantitative determinations of urinary beta(2)-microglobulin and urinary albumin may be useful for detecting disorders of the renal handling of plasma proteins. The findings also seem to suggest a selective tubular reabsorption of the two proteins.Estimates on sera revealed a close correlation between serum levels of beta(2)-microglobulin and creatinine and also a greatly raised serum concentration of beta(2)-microglobulin after bilateral nephrectomy.     

A stop codon mutation in SCN9A causes lack of pain sensation

The general lack of pain experience is a rare occurrence in humans, and the molecular causes for this phenotype are not well understood. Here we have studied a Canadian family from Newfoundland with members who exhibit a congenital inability to experience pain. We have mapped the locus to a 13.7 Mb region on chromosome 2q (2q24.3-2q31.1). Screening of candidate genes in this region identified a protein-truncating mutation in SCN9A, which encodes for the voltage-gated sodium channel Na(v)1.7. The mutation is a C-A transversion at nucleotide 984 transforming the codon for tyrosine 328 to a stop codon. The predicted product lacks all pore-forming regions of Na(v)1.7. Indeed, expression of this altered gene in a cell line did not produce functional responses, nor did it cause compensatory effects on endogenous voltage-gated sodium currents when expressed in ND7/23 cells. Because a homozygous knockout of Na(v)1.7 in mice has been shown to be lethal, we explored why a deficiency of Na(v)1.7 is non-lethal in humans. Expression studies in monkey, human, mouse and rat tissue indicated species-differences in the Na(v)1.7 expression profile. Whereas in rodents the channel was strongly expressed in hypothalamic nuclei, only weak mRNA levels were detected in this area in primates. Furthermore, primate pituitary and adrenal glands were devoid of signal, whereas these two glands were mRNA-positive in rodents. This species difference may explain the non-lethality of the observed mutation in humans. Our data further establish Na(v)1.7 as a critical element of peripheral nociception in humans.     

Biochemical values in persons older than 82 years of age: report from a population-based study of twins

According to international standards, reference values for biochemical tests should be obtained in disease-free subjects and for therapies that may influence measurement values. For elderly persons, especially the very old, such a requirement is difficult to meet, since few of these individuals are free of ongoing or former diseases. The present study of a population-based Swedish twin sample comprises 535 individuals (186 males, 349 females) who were at least 82 years of age at blood sampling. Survival over a 6-year period is used as a reference of overall health. Baseline test values for subjects surviving 6 years are compared with results for persons who died in the follow-up period. Results, including mean and median values, standard deviations and ranges, were given for both genders in the total sample. Cases outside the 5th and 95th percentiles were then excluded, as were those who died during the follow-up period. In a third step, cases with disorders and therapies known to influence measurement values were also excluded. The risk of dying during the observation period was calculated across measurements from the lowest and highest 10th and 20th percentiles. The specific impact of proximity to death on the biochemical values, in addition to age effects, was analysed by comparing co-twins discordant for survival over 7 years. Increased mortality was indicated for subjects of both genders with high serum levels of urea, urate, gamma-GT, free thyroxin and plasma homocysteine. In females, low serum values for albumin and total cholesterol were associated with increased mortality. A lower cholesterol level was found 4 years before death, while a lower HDL-cholesterol level was associated with an increased risk of death across the entire 7-year period.     

Lyme neuroborreliosis in Swedish children—PCR as a complementary diagnostic method for detection of Borrelia burgdorferi sensu lato in cerebrospinal fluid

European Journal of Clinical Microbiology & Infectious Diseases - The aim of this study was to evaluate polymerase chain reaction (PCR) as a diagnostic method for the detection of Borrelia...     

Ion channel screening technology

Ion channels are at present the third biggest target class in drug discovery. Primary research is continually uncovering potential new ion channel targets in indications such as cancer, diabetes and respiratory diseases, as well as the more established fields of pain, cardiovascular disease, and neurological disorders. Despite the physiological significance and therapeutic relevance in a wide variety of biological systems, ion channels still remain under exploited as drug targets. This is to a large extent resulting from the historical lack of screening technologies to provide the throughput and quality of data required to support medicinal chemistry. Although technical challenges still lie ahead, this historic bottleneck in ion channel drug discovery is now being overcome by novel technologies that can be integrated into lead generation stages of ion channel drug discovery to allow the development of novel therapeutic agents. This review describes the variety of technologies available for ion channel screening and discusses the opportunities these technologies provide. The challenges that remain to be addressed are highlighted.     

High-dose aspirin is required to influence plasma fibrin network structure in patients with type 1 diabetes

OBJECTIVE

Patients with type 1 diabetes form a less permeable fibrin network, which could contribute to their increased risk of cardiovascular disease (CVD). Low-dose aspirin treatment is the standard in the management of CVD; however, the effect seems reduced in patients with diabetes. We investigated the effects of low- and high-dose aspirin treatment on fibrin network formation in patients with type 1 diabetes (primary aim) and the possible interaction between the treatment effects of aspirin on fibrin network permeability and glycemic control in these patients (secondary aim).

RESEARCH DESIGN AND METHODS

Forty-eight patients (24 subjects with good [HbA_1c <7.4%] and 24 subjects with poor [HbA_1c >8.4%] glycemic control) were randomly assigned to treatment with 75 or 320 mg/day aspirin during 4 weeks in a crossover fashion. A 4-week washout period separated the treatment periods. The plasma fibrin network was assessed by determination of the permeability coefficient (K_s).

RESULTS

Treatment with 75 mg aspirin did not influence fibrin network permeability (K_s). However, K_s increased significantly during treatment with 320 mg aspirin (P = 0.004), and a significant treatment effect was seen compared with treatment with 75 mg aspirin (P = 0.009). The increase in K_s during high-dose aspirin treatment was significant in patients with poor glycemic control (P = 0.02), whereas K_s only tended to increase in patients with good glycemic control (P = 0.06).

CONCLUSIONS

A high dose of aspirin is required to influence fibrin network permeability in patients with type 1 diabetes. The observed lack of effect with low-dose aspirin may contribute to aspirin treatment failure in diabetes.Diabetes is associated with increased platelet activation (1), elevated plasma fibrinogen levels (2), and impaired fibrinolysis (3), factors that may contribute to the elevated risk of cardiovascular disease (CVD) in these patients. Increased platelet activation in patients with diabetes is reflected by elevated levels of platelet microparticles, which are small circulating procoagulant vesicles shed from the platelet membrane upon activation (4). Low-dose aspirin therapy is one of the cornerstones in the management of CVD; however, the preventive effect seems reduced in patients with diabetes (5).Aspirin inhibits platelet function by irreversibly acetylating a serine residue in cyclooxygenase-1, thereby grossly reducing the production of the platelet-activating and vasoconstrictive compound thromboxane A2. This is the most accepted effect of aspirin in terms of cardiovascular protection. However, aspirin also may influence coagulation through effects on thrombin generation, factor XIII activation, and fibrin network formation (6). The fibrin network is an important part of the arterial thrombus, and its structure may influence the predisposition to atherothrombotic events (7). During thrombin activation, fibrinopeptides are released from fibrinogen, which polymerize and, in the presence of factor XIII, form a cross-linked fibrin network. The structure of the fibrin network is influenced by the environment in which it is formed and affects the fibrinolytic rate (8). A tighter and less permeable fibrin network, which is less susceptible to fibrinolysis, is formed in patients with manifest CVD or conditions associated with increased risk of atherothrombotic complications (7–10). In previous studies, we have shown that patients with type 1 diabetes have reduced fibrin network permeability and that improved metabolic control is associated with increased fibrin network permeability (11,12). The altered fibrin network in patients with type 1 diabetes may in part be attributed to increased fibrinogen glycation, as shown in studies on fibrinogen purified from diabetic patients (13,14). Treatment with aspirin increases fibrin network permeability in nondiabetic subjects, possibly through acetylation of lysine residues on plasma fibrinogen (15–17). However, the effect of aspirin on fibrin network permeability in patients with diabetes is unclear. Possible competition between acetylation and glycation on lysine residues in the fibrinogen molecule might contribute to the reduced preventive effect of aspirin in the management of CVD in patients with diabetes, and higher doses of aspirin might be required in these patients.The primary aim of the current study was to investigate the effects of low- and high-dose aspirin treatment on fibrin network permeability in patients with type 1 diabetes. The secondary aim was to investigate the possible interaction between the treatment effects of aspirin on fibrin network permeability and glycemic control in these patients. Because platelet microparticles may influence the fibrin formation (18,19), and because aspirin has well-known effects on platelet function, we also measured plasma concentrations of platelet microparticles.     

Prevalence and Diversity of Borrelia Species in Ticks That Have Bitten Humans in Sweden

Members of the genus Borrelia are among the most common infectious agents causing tick-borne disease in humans worldwide. Here, we developed a Light Upon eXtension (LUX) real-time PCR assay that can detect and quantify Borrelia species in ticks that have fed on humans, and we applied the assay to 399 such ticks. Borrelia PCR-positive ticks were identified to species level by sequencing the products of conventional PCR performed using Borrelia group-specific primers. There was a 19% prevalence of Borrelia spp. in the detached ticks, and the number of spirochetes per Borrelia PCR-positive tick ranged from 2.0 × 10² to 4.9 × 10⁵, with a median of 7.8 × 10³ spirochetes. Adult ticks had a significantly larger number of spirochetes, with a median of 8.4 × 10⁴ compared to the median of nymphs of 4.4 × 10⁴. Adult ticks also exhibited a higher prevalence of Borrelia (33%) than nymphs (14%). Among the identified species, Borrelia afzelii was found to predominate (61%) and was followed by B. garinii (23%), B. valaisiana (13%), B. burgdorferi sensu stricto (1%), B. lusitaniae (1%), and B. miyamotoi-like (1%). Also, 3% of the ticks were coinfected with multiple strains of B. afzelii. Notably, this is the first report of B. lusitaniae being detected in ticks in Sweden. Our LUX real-time PCR assay proved to be more sensitive than a corresponding TaqMan assay. In conclusion, the novel LUX real-time PCR method is a rapid and sensitive tool for detection and quantification of Borrelia spp. in ticks.Lyme borreliosis (LB) is the most common tick-borne disease in humans in Europe (26), and it is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex. That group comprises the species B. burgdorferi sensu stricto, B. afzelii, and B. garinii, which are usually transmitted by the vector Ixodes ricinus. Furthermore, there have been reports of B. valaisiana, B. lusitaniae, and B. spielmanii being detected in samples of human skin and cerebrospinal fluid (5, 7, 30), which suggests that those three species can also give rise to LB. It is often hard to distinguish the clinical symptoms of LB from those of other diseases (10), and hence, it can be difficult to establish a correct diagnosis, especially if the patient is unable to recall having a tick bite.Today, diagnosis is based mainly on serological tests, although some PCR-based approaches, such as the TaqMan real-time PCR assay (3, 12), have been developed to detect Borrelia species in clinical samples. Even if real-time PCR is not yet considered to be a routine method in clinical practice, it can nonetheless provide valuable information about Borrelia infections, with regard to species type and the number of spirochetes present. Additional major advantages of PCR in this context are its simplicity, sensitivity, robustness, and speed. Other assays besides the TaqMan assay include a method based on SYBR green dye chemistry (37) and another using Light Upon eXtension (LUX) (Invitrogen Corporation). Compared to the SYBR green real-time PCR assay, the LUX assay offers the benefit of using a self-quenched primer with a hairpin loop structure, which makes it more specific; that is, it entails less unspecific binding and primer-dimer formation. Furthermore, the fluorophore is attached to the hairpin loop in the LUX setup, and thus, in contrast to the TaqMan assay, this PCR technique does not need an internal probe and is therefore a better choice if broader specificity is required. The LUX assay also has the capacity for melting curve analysis, which offers the possibility of discriminating between PCR products with different base pair compositions (23) and thereby revealing false-positive samples.Ixodes ricinus has been found in 23 of the 25 provinces in Sweden (9), but it is most common in the southern and central parts of the country and along the northeastern coast (14). Various investigators have described the prevalence and diversity of Borrelia in ticks collected in the field in Sweden (4, 8, 9, 14), and to date, five species of these bacteria have been recorded: B. afzelii, B. garinii, B. valaisiana, B. burgdorferi sensu stricto, and also one that is closely related to B. miyamotoi, which is known to be associated with relapsing fever. According to the cited studies, the prevalence of Borrelia spp. in Sweden varies between 3% and 23%. However, detection was not achieved by real-time PCR in those investigations, and thus, no attempts were made to quantify the Borrelia spirochetes in the ticks. To our knowledge, no quantification of Borrelia spirochetes in ticks detached from humans has ever been performed.Our aim was to study the prevalence of Borrelia and to quantify Borrelia cells in ticks that had fed on humans, and we developed a LUX real-time PCR assay for that purpose. In addition, we examined possible geographical differences in prevalence, and we also studied the temporal and spatial distribution of Borrelia species.     

Effects of lipid-lowering treatment on platelet reactivity and platelet-leukocyte aggregation in diabetic patients without and with chronic kidney disease: a randomized trial

Background Diabetes mellitus (DM) is associated with hyperreactive platelets and increased platelet-leukocyte aggregation (PLA), but the impact of concomitant chronic kidney disease (CKD) has been much less studied. Lipid-lowering treatment (LLT) may have favorable effects on platelet activation and inflammation. The objective of this mechanistic study was to investigate the impact of CKD on platelet function and inflammatory parameters in patients with DM and the effects of LLT. Methods After a placebo run-in period, the effects of simvastatin alone (S) or simvastatin + ezetimibe (S + E) were compared in a randomized, double-blind, cross-over study on platelet reactivity, PLA formation and inflammatory parameters. Eighteen DM patients with estimated glomerular filtration rate (eGFR) 15-59 mL/min × 1.73 m(2) (CKD stages 3-4) (DM-CKD) and 21 DM patients with eGFR >75 mL/min (DM-only) were included. Results PLAs were elevated at baseline in DM-CKD compared with DM-only (P = 0.04). S + E reduced PLAs among total leukocytes and neutrophils in DM-CKD patients (P = 0.01 for both) but not in the DM-only group. Platelet reactivity did not differ between patient groups or with LLT. Plasma levels of sCD40L (P < 0.001), elastase (P < 0.01) and von Willebrand factor (VWF) (P < 0.001) were elevated in DM-CKD compared with DM-only. S + E reduced sCD40L in DM-CKD patients (P = 0.01), but LLT did not influence VWF or elastase. Conclusions DM patients with CKD stages 3-4 had increased PLA and inflammatory activity compared with DM patients with normal GFR. Simvastatin + ezetimbe decreased PLAs and plasma sCD40L in DM patients with concomitant CKD. Clinical Trial registration http://www.clinicaltrials.gov. Identifier NCT01035320.     

Association of biochemical values with morbidity in the elderly: a population-based Swedish study of persons aged 82 or more years

BACKGROUND: Various inter-dependent factors influence serum biochemical values. In the elderly, the impact of these factors may differ compared with younger age groups and therefore population-based studies among older people are needed. The specific morbidity in old age, including also various types of drug therapy, should be observed. METHODS: Various biochemical tests in 349 females and 186 males over 81 years of age were carried out and the associations of biochemical values with morbidity, drug therapy, anthropometry and gender were estimated. RESULTS: Biochemical serum values deviate in various diseases, characterized by increased frequency in the elderly, i.e. congestive heart failure, osteoporosis, hip fractures, depression and dementia. All of these diseases present a tendency to increased homocysteine, usually combined with low folate. Cases with intact cognitive function throughout the six years after sampling are characterized by low homocysteine, which is the opposite of what is found in dementia. Furthermore, congestive heart failure is associated with impaired creatinine clearance and increased urea and urate, and osteoporosis and hip fractures are characterized by low albumin and cholesterol. Increased values for urate and impaired creatinine clearance are found in coronary diseases. In gout, multiple biochemical changes take place. For cases with a history of diabetes, arterial hypertension, peptic ulcer and malignancy, few changes are found compared with the values of the total sample. Furosemide therapy is associated with the same pattern as congestive heart failure, and laxative treatment is characterized by low folate and high homocysteine values.     

Pediatric norovirus diarrhea in Nicaragua

Information about norovirus (NoV) infections in Central America is limited. Through a passive community and hospital pediatric diarrhea surveillance program, a total of 542 stool samples were collected between March 2005 and February 2006 in León, Nicaragua. NoV was detected in 12% (65/542) of the children; of these, 11% (45/409) were in the community and 15% (20/133) were in the hospital, with most strains (88%) belonging to genogroup II. NoV infections were age and gender associated, with children of <2 years of age (P < 0.05) and girls (P < 0.05) being most affected. Breast-feeding did not reduce the number of NoV infections. An important proportion (57%) of NoV-infected children were coinfected with diarrheagenic Escherichia coli. A significant proportion (18/31) of NoV-positive children with dehydration required intravenous rehydration. Nucleotide sequence analysis (38/65) of the N-terminal and shell region in the capsid gene revealed that at least six genotypes (GI.4, GII.2, GII.4, GII.7, GII.17, and a potentially novel cluster termed “GII.18-Nica”) circulated during the study period, with GII.4 virus being predominant (26/38). The majority (20/26) of those GII.4 strains shared high nucleotide homology (99%) with the globally emerging Hunter strain. The mean viral load was approximately 15-fold higher in children infected with GII.4 virus than in those infected with other G.II viruses, with the highest viral load observed for the group of children infected with GII.4 and requiring intravenous rehydration. This study, the first of its type from a Central American country, suggests that NoV is an important etiological agent of acute diarrhea among children of <2 years of age in Nicaragua.