Determination of the molecular subtypes of diffuse large B-cell lymphomas using immunohistochemistry: a case series from the Inselspital, Bern, and a critical appraisal of this determination in Switzerland

The two major subtypes of diffuse large B-cell lymphoma (DLBCL) (germinal centre B-cell - like (GCB-DLBCL) and activated B-cell - like (ABC-DLBCL)) are defined by means of gene expression profiling (GEP). Patients with GCB-DLBCL survive longer with the current standard regimen R-CHOP than patients with ABC-DLBCL. As GEP is not part of the current routine diagnostic work-up, efforts have been made to find a substitute than involves immunohistochemistry (IHC). Various algorithms achieved this with 80-90% accuracy. However, conflicting results on the appropriateness of IHC have been reported. Because it is likely that the molecular subtypes will play a role in future clinical practice, we assessed the determination of the molecular DLBCL subtypes by means of IHC at our University Hospital, and some aspects of this determination elsewhere in Switzerland. The most frequently used Hans algorithm includes three antibodies (against CD10, bcl-6 and MUM1). From records of the routine diagnostic work-up, we identified 51 of 172 (29.7%) newly diagnosed and treated DLBCL cases from 2005 until 2010 with an assigned DLBCL subtype. DLBCL subtype information was expanded by means of tissue microarray analysis. The outcome for patients with the GCB subtype was significantly better compared with those with the non-GC subtype, independent of the age-adjusted International Prognostic Index. We found a lack of standardisation in the subtype determination by means of IHC in Switzerland and significant problems of reproducibility. We conclude that the Hans algorithm performs well in our hands and that awareness of this important matter is increasing. However, outside clinical trials, vigorous efforts to standardise IHC determination are needed as DLBCL subtype-specific therapies emerge.

In search of epigenetic marks in testes and sperm cells of differentially fed boars

In search of transmittable epigenetic marks we investigated gene expression in testes and sperm cells of differentially fed F0 boars from a three generation pig feeding experiment that showed phenotypic differences in the F2 generation. RNA samples from 8 testes of boars that received either a diet enriched in methylating micronutrients or a control diet were analyzed by microarray analysis. We found moderate differential expression between testes of differentially fed boars with a high FDR of 0.82 indicating that most of the differentially expressed genes were false positives. Nevertheless, we performed a pathway analysis and found disparate pathway maps of development_A2B receptor: action via G-protein alpha s, cell adhesion_Tight junctions and cell adhesion_Endothelial cell contacts by junctional mechanisms which show inconclusive relation to epigenetic inheritance. Four RNA samples from sperm cells of these differentially fed boars were analyzed by RNA-Seq methodology. We found no differential gene expression in sperm cells of the two groups (adjusted P-value>0.05). Nevertheless, we also explored gene expression in sperm by a pathway analysis showing that genes were enriched for the pathway maps of bacterial infections in cystic fibrosis (CF) airways, glycolysis and gluconeogenesis p.3 and cell cycle_Initiation of mitosis. Again, these pathway maps are miscellaneous without an obvious relationship to epigenetic inheritance. It is concluded that the methylating micronutrients moderately if at all affects RNA expression in testes of differentially fed boars. Furthermore, gene expression in sperm cells is not significantly affected by extensive supplementation of methylating micronutrients and thus RNA molecules could not be established as the epigenetic mark in this feeding experiment.

Decrease in pneumococcal co-colonization following vaccination with the seven-valent pneumococcal conjugate vaccine.

Understanding the epidemiology of pneumococcal co-colonization is important for monitoring vaccine effectiveness and the occurrence of horizontal gene transfer between pneumococcal strains. In this study we aimed to evaluate the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal co-colonization among Portuguese children. Nasopharyngeal samples from children up to 6 years old yielding a pneumococcal culture were clustered into three groups: pre-vaccine era (n = 173), unvaccinated children of the vaccine era (n = 169), and fully vaccinated children (4 doses; n = 150). Co-colonization, serotype identification, and relative serotype abundance were detected by analysis of DNA of the total bacterial growth of the primary culture plate using the plyNCR-RFLP method and a molecular serotyping microarray-based strategy. The plyNCR-RFLP method detected an overall co-colonization rate of 20.1%. Microarray analysis confirmed the plyNCR-RFLP results. Vaccination status was the only factor found to be significantly associated with co-colonization: co-colonization rates were significantly lower (p = 0.004; Fisher's exact test) among fully vaccinated children (8.0%) than among children from the pre-PCV7 era (17.3%) or unvaccinated children of the PCV7 era (18.3%). In the PCV7 era there were significantly less non-vaccine type (NVT) co-colonization events than would be expected based on the NVT distribution observed in the pre-PCV7 era (p = 0.024). In conclusion, vaccination with PCV7 resulted in a lower co-colonization rate due to an asymmetric distribution between NVTs found in single and co-colonized samples. We propose that some NVTs prevalent in the PCV7 era are more competitive than others, hampering their co-existence in the same niche. This result may have important implications since a decrease in co-colonization events is expected to translate in decreased opportunities for horizontal gene transfer, hindering pneumococcal evolution events such as acquisition of antibiotic resistance determinants or capsular switch. This might represent a novel potential benefit of conjugate vaccines.

Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling.

Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.

Strain typing and antimicrobial susceptibility of methicillin-resistant coagulase-positive staphylococcal species in dogs and people associated with dogs in Thailand

AIMS This study was to investigate and to characterize methicillin-resistant coagulase-positive staphylococci (MRCoPS) harboring in dogs and people associated with dogs in Thailand. METHODS AND RESULTS Staphylococci were collected from 100 dogs, 100 dog owners, 200 small animal veterinarians and 100 people without pet association. Species of MRCoPS were identified phenotypically and genotypically. Molecular characteristics were determined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and SCCmec typing, and antimicrobial susceptibility was assayed by broth microdilution and by microarray analysis for resistance genes. Methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSSc) and methicillin-resistant Staphylococcus aureus (MRSA) were isolated from dogs (45, 17 and 1%, respectively), veterinarians (8, 2 and 1·5%, respectively) and dog owners (3, 2 and 0%, respectively). Seventeen sequence types (STs) were identified among 83 MRSP isolates which specifically carried SCCmec V, II-III, ΨSCCmec57395 and three uncharacterized SCCmec types. MRSP ST 45, 68 and novel STs including 169, 178, 181 and 183 were shared among canine and human isolates. Most of MRSA ST398 and MRSSc carried SCCmec type V. The MRCoPS commonly displayed multiple resistances to tested antimicrobials and carried various resistance genes. CONCLUSION Variety of MRCoPS, especially new MRSP clones, distributed in dogs and people in Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY The existence of MRCoPS circulating between dogs and humans in Thailand provides indirect evidence of interspecies transmission and represents a potential public health hazard.

Transcriptional profiles of cytokine/chemokine factors of immune cell-homing to the parasitic lesions: a comprehensive one-year course study in the liver of E. multilocularis-infected mice.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

Commensal bacteria protect against food allergen sensitization.

Environmentally induced alterations in the commensal microbiota have been implicated in the increasing prevalence of food allergy. We show here that sensitization to a food allergen is increased in mice that have been treated with antibiotics or are devoid of a commensal microbiota. By selectively colonizing gnotobiotic mice, we demonstrate that the allergy-protective capacity is conferred by a Clostridia-containing microbiota. Microarray analysis of intestinal epithelial cells from gnotobiotic mice revealed a previously unidentified mechanism by which Clostridia regulate innate lymphoid cell function and intestinal epithelial permeability to protect against allergen sensitization. Our findings will inform the development of novel approaches to prevent or treat food allergy based on modulating the composition of the intestinal microbiota.

Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: evidence for involvement of splicing regulatory proteins.

Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.

Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations. Results Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI. Conclusions In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.

Identification of Sleep-Modulated Pathways Involved in Neuroprotection from Stroke.

STUDY OBJECTIVES Sleep deprivation (SDp) performed before stroke induces an ischemic tolerance state as observed in other forms of preconditioning. As the mechanisms underlying this effect are not well understood, we used DNA oligonucleotide microarray analysis to identify the genes and the gene-pathways underlying SDp preconditioning effects. DESIGN Gene expression was analyzed 3 days after stroke in 4 experimental groups: (i) SDp performed before focal cerebral ischemia (IS) induction; (ii) SDp performed before sham surgery; (iii) IS without SDp; and (iv) sham surgery without SDp. SDp was performed by gentle handling during the last 6 h of the light period, and ischemia was induced immediately after. SETTINGS Basic sleep research laboratory. MEASUREMENTS AND RESULTS Stroke induced a massive alteration in gene expression both in sleep deprived and non-sleep deprived animals. However, compared to animals that underwent ischemia alone, SDp induced a general reduction in transcriptional changes with a reduction in the upregulation of genes involved in cell cycle regulation and immune response. Moreover, an upregulation of a new neuroendocrine pathway which included melanin concentrating hormone, glycoprotein hormones-α-polypeptide and hypocretin was observed exclusively in rats sleep deprived before stroke. CONCLUSION Our data indicate that sleep deprivation before stroke reprogrammed the signaling response to injury. The inhibition of cell cycle regulation and inflammation are neuroprotective mechanisms reported also for other forms of preconditioning treatment, whereas the implication of the neuroendocrine function is novel and has never been described before. These results therefore provide new insights into neuroprotective mechanisms involved in ischemic tolerance mechanisms.

The Phosphoinositide 3-Kinase p110α Isoform Regulates Leukemia Inhibitory Factor Receptor Expression via c-Myc and miR-125b to Promote Cell Proliferation in Medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K) pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi)-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α) was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH) subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.

Detection of Chlamydia pneumoniae in a collection of captive snakes and response to treatment with marbofloxacin.

In a collection of 58 snakes comprising predominantly Eurasian vipers in Switzerland, five snakes died unexpectedly during hibernation from 2009 to 2012. In one snake, organisms resembling chlamydiae were detected by immunohistochemistry in multiple histiocytic granulomas. Real-time quantitative PCR and microarray analysis were used to determine the presence of Chlamydia pneumoniae in tissue samples and cloacal/choanal swabs from snakes in the collection; 8/53 (15.1%) of the remaining snakes were positive. Although one infected snake had suppurative periglossitis, infection with C. pneumoniae did not appear to be associated with specific clinical signs in snakes. Of seven snakes treated with 5 mg/kg marbofloxacin IM once daily, five became PCR negative for C. pneumoniae following treatment, whereas one animal remained positive and one snake was lost to follow-up.

Analysis of independent microarray datasets of renal biopsies identifies a robust transcript signature of acute allograft rejection

Transcriptomics could contribute significantly to the early and specific diagnosis of rejection episodes by defining 'molecular Banff' signatures. Recently, the description of pathogenesis-based transcript sets offered a new opportunity for objective and quantitative diagnosis. Generating high-quality transcript panels is thus critical to define high-performance diagnostic classifier. In this study, a comparative analysis was performed across four different microarray datasets of heterogeneous sample collections from two published clinical datasets and two own datasets including biopsies for clinical indication, and samples from nonhuman primates. We characterized a common transcriptional profile of 70 genes, defined as acute rejection transcript set (ARTS). ARTS expression is significantly up-regulated in all AR samples as compared with stable allografts or healthy kidneys, and strongly correlates with the severity of Banff AR types. Similarly, ARTS were tested as a classifier in a large collection of 143 independent biopsies recently published by the University of Alberta. Results demonstrate that the 'in silico' approach applied in this study is able to identify a robust and reliable molecular signature for AR, supporting a specific and sensitive molecular diagnostic approach for renal transplant monitoring.

CD47 protein expression in acute myeloid leukemia: A tissue microarray-based analysis

Binding of CD47 to signal regulatory protein alpha (SIRPα), an inhibitory receptor, negatively regulates phagocytosis. In acute myeloid leukemia (AML), CD47 is overexpressed on peripheral blasts and leukemia stem cells and inversely correlates with survival. Aim of the study was to investigate the correlation between CD47 protein expression by immunohistochemistry (IHC) in a bone marrow (BM) tissue microarray (TMA) and clinical outcome in AML patients. CD47 staining on BM leukemia blasts was scored semi-quantitatively and correlated with clinical parameters and known prognostic factors in AML. Low (scores 0-2) and high (score 3) CD47 protein expression were observed in 75% and 25% of AML patients. CD47 expression significantly correlated with percentage BM blast infiltration and peripheral blood blasts. Moreover, high CD47 expression was associated with nucleophosmin (NPM1) gene mutations. In contrast, CD47 expression did not significantly correlate with overall or progression free survival or response to therapy. In summary, a BM TMA permits rapid and reproducible semi-quantitative analysis of CD47 protein expression by IHC. While CD47 expression on circulating AML blasts has been shown to be a negative prognostic marker for a very defined population of AML patients with NK AML, CD47 expression on AML BM blasts is not.