Abnormalities of B cell subsets in patients with systemic lupus erythematosus

The prototypic autoimmune disease, SLE, is known to be associated with polyclonal B cell hyperreactivity. Developing an understanding of the complex nature of human B cell differentiation, largely through the application of multiparameter flow cytometry to an analysis of circulating B cells has permitted an assessment of whether specific stages of B cell maturation are affected by the tendency for polyclonal B cell activation. Moreover, the analysis of perturbations of the specific stages of B cell maturation has generated new information on whether abnormalities in B cell differentiation are primarily involved in autoimmune disease immunopathology or, rather, are secondary to the inflammatory environment characteristic of subjects with this autoimmune disease. Multivariant analysis has begun to document abnormalities in B cell maturation that are primarily associated with lupus, or, alternatively related to disease duration, disease activity and concomitant medication. Together, these analyses have provided new insights on the role of B cell over-reactivity in SLE.     

Changes over time in frontotemporal activation during a working memory task in patients with schizophrenia

Studies on working memory (WM) dysfunction in schizophrenia have reported several functionally aberrant brain areas including prefrontal and temporal cortex. Longitudinal studies have shown changes in prefrontal activation during treatment. We used event-related functional magnetic resonance imaging and a parametric verbal WM task to investigate cerebral function during WM performance in healthy subjects and medicated patients with schizophrenia with an acute symptom exacerbation. Patients were scanned twice: within the first week after admission to the hospital and after 7-8 weeks of a multimodal treatment including atypical antipsychotic agents. There were no differences in activation of lateral prefrontal regions in patients relative to healthy controls neither at baseline nor after 7-8 weeks. Controls showed relatively more activation in parietal, striatal and cerebellar regions. In patients with schizophrenia, frontotemporal function was bilaterally enhanced after 7-8 weeks. This activation change was associated with improved accuracy in a verbal WM task, improved verbal WM-span and symptom reduction as measured by the BPRS global score and the BPRS factor for thought disturbance. Although we could not replicate findings of functional hypofrontality in the patients with schizophrenia, frontotemporal activation changed with treatment and was associated with verbal WM performance and significant reduction of psychopathology.     

Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by Fluorescence In Situ Hybridization

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.Human intestinal spirochetosis (HIS) is a histologically defined condition of the human distal intestinal tract characterized by helical microorganisms attached at one end to the surface epithelium of the colonic mucosa. This association forms a so-called “false brush border” (13). While certain Brachyspira spp. are recognized as the causative agents of swine dysentery and porcine intestinal spirochetosis (3, 11), their clinical significance and pathogenic potential for humans remain unclear. Various studies have reported on the association of these bacteria with intestinal disorders such as chronic watery diarrhea (8, 12) and on clinical improvement following antimicrobial therapy (29). In contrast, others have suggested that intestinal spirochetes are harmless commensals in humans (7). The prevalence of HIS ranges from 1.2% (23) to >40% (17, 34), depending on presumable patient risk factors, such as origin from developing countries, immunodeficiency, or homosexuality. Recently, Peruzzi et al. (30) discovered a prevalence of 12% in a selected population, indicating that HIS is an important differential diagnosis for patients with chronic gastrointestinal disorders and risk factors.Two intestinal spirochetes have been identified in humans so far: Brachyspira aalborgi (15) and Brachyspira pilosicoli (36). Both species require selective media, and B. aalborgi is an extremely slow growing, fastidious microorganism that requires anaerobic incubation for as long as 4 weeks (4, 34). For this reason, HIS is primarily diagnosed histopathologically. The fuzzy basophilic fringe, 4 to 7 μm thick, on the epithelial layer of the colonic mucosa is visible in hematoxylin-and-eosin (HE)-stained histological sections and is considered pathognomonic for HIS. Tissue morphology usually remains unaltered, and no inflammatory reaction is observed (20).However, diagnosis of HIS on the basis of HE staining requires experienced laboratory personnel and accurate interpretation, and silver staining is often needed to confirm the diagnosis (10). Therefore, a significant portion of cases may be missed, especially since B. pilosicoli might also colonize the epithelium without the characteristic end-on attachment, impeding identification by light microscopy at low magnification (24). Furthermore, histopathology does not provide information about the identity of the microorganisms, thereby precluding epidemiological studies. More importantly, the inability to identify the organism also hampers accurate therapy, since the intestinal spirochetes are suspected to differ in virulence, and therefore some cases of HIS may require antibiotic therapy more urgently than others (5, 29).The genus Brachyspira currently comprises seven established species and several proposed species. Among some Brachyspira species, the high level of 16S rRNA gene conservation precludes interspecies differentiation by 16S rRNA gene methods and necessitates further molecular analyses. However, all known species isolated from humans can be identified and differentiated via their 16S rRNA genes. In line with the genetic variation discovered in Brachyspira species, such as Brachyspira hyodysenteriae (2) and Brachyspira innocens (9), recent molecular studies have also identified human Brachyspira strains genetically distinct from B. aalborgi and B. pilosicoli. This heterogeneity was confirmed by sequencing of 16S rRNA (14, 21) or NADH oxidase (25) genes, fluorescence in situ hybridization (FISH) (16, 17), or multilocus enzyme electrophoresis (33). Pettersson et al. (31) analyzed biopsy samples from two adults by 16S rRNA gene sequencing and consequently proposed to divide the B. aalborgi lineage into three phylogenetic clusters, including the type strain, B. aalborgi 513A, in the first cluster.The extent of intraspecies genetic variation in human intestinal spirochetes is unclear and difficult to estimate, because few complete 16S rRNA gene sequences are available. Further epidemiologic and phylogenetic investigations are needed to elucidate spirochete genetic diversity and to facilitate the evaluation of the molecular diagnostic tools that are presently available.FISH is a microscopic method that allows simultaneous visualization and identification of microorganisms. Jensen and colleagues (3, 16, 17) designed several genus- or species-specific oligonucleotide probes targeting the 16S or 23S rRNA of Brachyspira spp. and applied them successfully to porcine and human intestinal biopsy specimens. However, no genus-specific 16S rRNA-directed probe for diagnostic use targeting all Brachyspira spp. known so far has been developed.In the present study, intestinal biopsy specimens from five patients with possible HIS were analyzed histopathologically and by culture, FISH, PCR amplification, and 16S rRNA gene sequencing. Biopsy specimens from a healthy control group were analyzed retrospectively by histopathology and FISH. The purpose was (i) to acquire further information about the phylogenetic structure of the Brachyspira spp. associated with HIS, (ii) to design a FISH probe covering all Brachyspira spp. based on the currently available sequence data, and (iii) to evaluate FISH as a fast and robust diagnostic screening tool for HIS.     

Distinct brain networks in recognition memory share a defined region in the precuneus

Current models of recognition memory performance postulate that there are two fundamentally distinct retrieval processes, i.e. recollection and familiarity. This view has been challenged and little is known from human research about the functional connectivity of the brain areas involved in these processes. In our study we used a Remember‐Know procedure to assess the functional connectivity of brain regions under recognition memory in 30 healthy adults. Using functional magnetic resonance imaging, we analysed the blood oxygen level‐dependent responses during correct Remember, correct Know, correct Rejection and missed responses of the subjects during recognition of non‐emotional nouns. One activation cluster was found in the left precuneus associated with both recollection and familiarity answers. To acquire information about the way in which activity in one brain region modulates activity in another brain region in response to the active task, we performed a psychophysiological interaction analysis with the left precuneus as a seed region. This analysis revealed functionally distinct networks of brain areas underlying recollection and familiarity. Furthermore, we discuss the differential involvement of the hippocampus in a recollection network as compared with a familiarity network. In summary, our results further strengthen the assumptions of a dual‐process view of recognition memory [e.g. H. Eichenbaum et al. (2007) Annual Review of Neuroscience, 30, 123–152; A.P. Yonelinas (2001) Philosophical Transactions of the Royal Society London B Biological Sciences, 356, 1363–1374] and add empirical findings about the functional interconnectivity of brain regions supporting either recollection or familiarity.     

Comparison of prognostic usefulness (three years) of computed tomographic angiography versus 64-slice computed tomographic calcium scanner in subjects without significant coronary artery disease

Coronary computerized tomographic angiography (CTA) has been used as a noninvasive method for ruling out high-grade stenoses. Even in the absence of such stenoses, analysis of coronary atherosclerosis may provide for important prognostic information, and this may be superior to exclusive coronary artery calcium scoring. We tested this hypothesis in patients undergoing CTA for clinical indications who had no stenoses requiring revascularization. From December 2004 to December 2006, 706 consecutive patients who underwent CTA but had no high-grade stenoses were included (58% men, mean age 59 ± 11 years). CTA and coronary artery calcium scoring (Agatston method) were performed using a 64-slice CT scanner with a gantry rotation time of 330 ms. CT angiograms were categorized as completely normal (group 1), showing minor plaque (group 2), or showing intermediate stenoses (group 3). Follow-up information was obtained in 670 patients (95%) over a mean of 3.2 years. There were 31 major adverse events (5%), namely 9 deaths (all noncoronary), 2 myocardial infarctions, 5 strokes, 13 coronary revascularization procedures (percutaneous or surgical > 6 months after CTA), and 2 peripheral percutaneous interventions. Coronary status as defined by CTA was predictive of major events after adjustment for age and gender. In group 1, the probability of event-free survival at 3 years was 100%; in group 2, it was 96%; and in group 3, it was 91%. Compared to group 1, the risk in group 2 was increased 2.3-fold, and in group 3, it was increased 5.6-fold after adjusting for age and gender. However, after addition of the coronary artery calcium score to the regression analysis, CT angiographic status no longer appeared to be predictive. In conclusion, the risk of a major adverse cardiovascular event or death increased in a graded manner with degree of coronary atherosclerosis as defined by CTA even in the absence of high-grade coronary stenoses. However, in the absence of high-grade stenoses, we were unable to demonstrate a superior prognostic value of CTA compared to coronary artery calcium.     

Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines

There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r² = 0.47, P = 0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r² = 0.56, P = 0.03 and r² = 0.62, P = 0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA.LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.     

Scintigraphy for risk stratification of iodine-induced thyrotoxicosis in patients receiving contrast agent for coronary angiography: a prospective study of patients with low thyrotropin

The risk of iodine-induced thyrotoxicosis in euthyroid patients receiving iodine-containing contrast agents is known to be low, but data on this risk in patients with latent hyperthyroidism are scarce. We investigated the role of thyroid scintigraphy using Tc-99m preceding the application of iodine-containing contrast material to estimate the risk of iodine-induced thyrotoxicosis in patients with low levels of TSH. In a prospective study on 91 patients, thyroid scintigraphy was performed before coronary angiography (CA). In patients with technetium thyroid uptake (TCTU) less than 1%, CA was done without prophylactic drugs (n = 56). Patients with TCTU greater than 1% were treated either with 900 mg of perchlorate or, depending on the autonomous volume, combined with 20 to 60 mg thiamazole. In the 56 patients with TCTU less than 1%, no case of iodine-induced hyperthyroidism occurred within 4 wk after CA. In the patients who received prophylactic drugs, two cases of mild thyrotoxicosis were observed. Our data suggest that in patients with low levels of TSH, the risk of hyperthyroidism after application of iodine-containing contrast media is negligible if TCTU is less than 1%. In these patients, CA can be performed without administration of prophylactic drugs.