Computer-supported detection of M-components and evaluation of immunoglobulins after capillary electrophoresis

BACKGROUND: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenstr?m macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. METHODS: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the gamma- and ss-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. RESULTS: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. CONCLUSIONS: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.     

Typing of genetic variants of alpha 1-antitrypsin from dried blood

The alpha 1-antitrypsin phenotype was determined from both plasma and dried blood in 35 children and adolescents who had or were suspected to have alpha 1-antitrypsin deficiency. A disc of paper with dried blood was eluted and analysed by flat bed electrofocusing on polyacrylamide gel. The gel was stained according to a silver-staining technique until the microheterogeneous bands appeared with adequate intensity. The alpha 1-antitrypsin patterns of plasma and dried blood were identical. The stability of the dried blood samples was 3 days at room temperature, up to 1 week at +4 degrees C, and up to 1 month at -20 degrees C. The alpha 1-antitrypsin screening method and the Pi-typing procedure described in this study may be combined in future screening studies using dried blood specimens obtained for the Guthrie test.     

High Proportions of Proinflammatory Bacteria on the Colonic Mucosa in a Young Patient with Ulcerative Colitis as Revealed by Cloning and Sequencing of 16S rRNA Genes

The pathogenesis of ulcerative colitis (UC) remains unknown. It is thought to be due to an abnormal and uncontrolled immune response to normally occurring constituents of the intestine. Microbial agents appear to be involved in the pathogenesis and intestinal bacteria seem to be an important factor in the development and chronicity. The aim of this study was to investigate the colonic microbiota of a patient with UC. The colonic tissues were taken during surgery from a 12-year-old girl suffering from UC. The microbiota on the colonic samples was studied by cloning and sequencing of amplified 16S rRNA genes. Compared with healthy subjects, alteration of the dominant bacterial group was observed in the UC patient. We found a high incidence of Enterobacteriaceae, Bacteroides fragilis, and the single phylotype of the Faecalibacterium prausnitzii-like “Butyrate-producing bacterium” L2-6. Furthermore, there was a substantial presence of Pseudomonas aeruginosa in the present case of UC. The high proportion of adverse proinflammatory species is striking in the present case compared with more normal situations. Even if those bacteria are not the cause of the UC, they most probably enhance the symptoms of the disease.     

Ileal pelvic pouch microbiota from two former ulcerative colitis patients, analysed by DNA-based methods, were unstable over time and showed the presence of Clostridium perfringens

OBJECTIVE: Ileal pouch anal anastomosis (IPAA) is the preferred method for restorative surgery in patients with ulcerative colitis who have to undergo proctocolectomy. The most common complication is pouchitis and several studies have pointed to the microbiota of the pouch as being a risk factor. The aim of this study was to follow the development of the bacterial microbiota in pouches during the first year. MATERIAL AND METHODS: Terminal restriction fragment length polymorphism (T-RFLP) combined with cloning and sequencing was used to identify the most predominant bacteria on the different sampling occasions. A total of 274 clones were grouped by T-RFLP and clones from each group were selected for sequencing and identified by comparison with known sequences. RESULTS: Differences in T-RFLP profiles and clone libraries were found between the patients, and also in changes apparent in each patient at different time-points. The main bacterial groups in the pouches resembled those of the normal colonic microbiota, with a predominance of the clostridia clusters XIVa and IV, Bacteroides and Enterobacteriaceae. Exceptions were clones with sequences resembling those of the Clostridium perfringens group, in both patients and on all sampling occasions, and the dominance of clones resembling Turicibacter in one of the patients at the time of pouch construction. CONCLUSIONS: The pouch microbiota showed similarities to the normal colon microbiota except for the presence of clones with sequences resembling those of the C. perfringens group and Turicibacter. The bacterial composition differed between the two patients and the microbiota changed with time, suggesting that the composition is not stable during the first year.     

Endotoxin- and d-galactosamine-induced liver injury improved by the administration of Lactobacillus, Bifidobacterium and blueberry

BACKGROUND: D-galactosamine together with lipopolysaccharide can lead to a pronounced secretion by Kupffer cells of pro-inflammatory mediators, which have been shown to be early and important mediators of liver injury. Probiotics and dietary supplementation with fruit or vegetable extracts with high content of antioxidants, such as blueberry, could be beneficial in protecting against hepatotoxicity. AIMS: To investigate whether blueberry and probiotics could attenuate liver injury induced by D-galactosamine and lipopolysaccharide. SUBJECTS: Sprague-Dawley rats were used. METHODS: Six experimental groups: acute liver injury control and five groups of liver injury treated by blueberry alone or by each of the probiotics strains (Lactobacillus plantarum DSM 15313 and Bifidobacterium infantis DSM 15159) with and without blueberry. Samples were collected 24 h after induction for bacterial test, liver function test, short chain fatty acids, myeloperoxidase, cytokines, malondialdehyde and glutathione. RESULTS: Alanine aminotransferase levels decreased significantly in all groups compared to liver injury control and DSM 15313 groups. Bilirubin, liver TNF-alpha, myeloperoxidase and acetic acid in cecum content decreased significantly in all groups, while liver glutathione values increased significantly in all groups compared to liver injury control. Liver IL-1beta and bacterial translocation to the liver and mesenteric lymph nodes decreased significantly in all groups except B. infantis DSM 15159 group compared to the liver injury control. Enterobacteriaceae count in cecum decreased significantly in the groups with blueberry plus probiotics compared to the other groups. CONCLUSION: Blueberry and probiotics exert protective effects on acute liver injury. They reduce the hepatocytes injury, the inflammation and the pro-inflammatory cytokines, and improve the barrier functions and antioxidant activity.     

Adjuvant therapy for locally advanced renal cell cancer: A systematic review with meta-analysis

Background  

Many adjuvant trials have been undertaken in an attempt to reduce the risk of recurrence among patients who undergo surgical resection for locally advanced renal cancer. However, no clear benefit has been identified to date. This systematic review was conducted to examine the exact role of adjuvant therapy in renal cancer setting.     

Probiotic therapy to men with incipient arteriosclerosis initiates increased bacterial diversity in colon: A randomized controlled trial

ObjectiveThis study aimed to clarify the microbial change in the intestinal microbiota in patients, with cardiovascular disease, consuming a drink with high numbers of live Lactobacillus plantarum.MethodsSixteen males, with atherosclerotic plaque on the carotid wall, were randomly selected from a larger cohort and included in this double blind, placebo controlled study. Colonic biopsies, taken before and after four weeks of probiotic treatment, were analysed with Terminal Restriction Fragment Length Polymorphism, including digestion with MspI and HaeIII. Microbial diversity was calculated, short-chain fatty acids in faeces, and blood markers were analysed.ResultsConsumption of one probiotic strain of L. plantarum (DSM 9843) increased intestinal microbial diversity. The probiotic group had an increased diversity after consumption of the probiotic drink compared to the change in the placebo group when Shannon and Weaner diversity index (MspI and HaeIII, p = 0.026) and Simpson index of diversity (MspI, p = 0.044 and HaeIII, p = 0.026) were calculated. The fermentation pattern of short-chain fatty acids in faeces were unaffected for most acids, but the probiotic group had decreased concentration of isovaleric acid (p = 0.006) and valeric acid (p = 0.029). Viable count of lactobacilli increased in the probiotic group (p = 0.001), but no significant changes in blood markers were observed.ConclusionAdministration of a single-strain probiotic increases the bacterial diversity in the gut, and affects the concentration of some short-chain fatty acids. Consumption of the single strain L. plantarum DSM 9843 might be a strategy to favour a diverse intestinal microbiota, which is beneficial for the host.     

Simvastatin protects against cholestasis-induced liver injury

Background:

Bile duct obstruction is associated with hepatic accumulation of leukocytes and liver injury. The aim of this study was to evaluate the effect of simvastatin on cholestasis-induced liver inflammation and tissue damage.

Experimental approach:

C57BL/6 mice were treated with simvastatin (0.02 and 0.2 mg·kg⁻¹) and vehicle before and after undergoing bile duct ligation (BDL) for 12 h. Leukocyte recruitment and microvascular perfusion in the liver were analysed using intravital fluorescence microscopy. CXC chemokines in the liver were determined by enzyme-linked immunosorbent assay. Liver damage was monitored by measuring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Hepatic levels of myeloperoxidase (MPO) were also determined.

Key results:

Administration of 0.2 mg·kg⁻¹ simvastatin decreased ALT and AST by 87% and 83%, respectively, in BDL mice. This dose of simvastatin reduced hepatic formation of CXC chemokines by 37–82% and restored sinusoidal perfusion in cholestatic animals. Moreover, BDL-induced leukocyte adhesion in sinusoids and postsinusoidal venules, as well as MPO levels in the liver, was significantly reduced by simvastatin. Notably, administration of 0.2 mg·kg⁻¹ simvastatin 2 h after BDL induction also decreased cholestatic liver injury and inflammation.

Conclusions and implications:

These findings show that simvastatin protects against BDL-induced liver injury. The hepatoprotective effect of simvastatin is mediated, at least in part, by reduced formation of CXC chemokines and leukocyte recruitment. Thus, our novel data suggest that the use of statins may be an effective strategy to protect against the hepatic injury associated with obstructive jaundice.