Cortical Processing of Multimodal Sensory Learning in Human Neonates.

Following birth, infants must immediately process and rapidly adapt to the array of unknown sensory experiences associated with their new ex-utero environment. However, although it is known that unimodal stimuli induce activity in the corresponding primary sensory cortices of the newborn brain, it is unclear how multimodal stimuli are processed and integrated across modalities. The latter is essential for learning and understanding environmental contingencies through encoding relationships between sensory experiences; and ultimately likely subserves development of life-long skills such as speech and language. Here, for the first time, we map the intracerebral processing which underlies auditory-sensorimotor classical conditioning in a group of 13 neonates (median gestational age at birth: 38 weeks + 4 days, range: 32 weeks + 2 days to 41 weeks + 6 days; median postmenstrual age at scan: 40 weeks + 5 days, range: 38 weeks + 3 days to 42 weeks + 1 days) with blood-oxygen-level-dependent (BOLD) functional magnetic resonance imaging (MRI) and magnetic resonance (MR) compatible robotics. We demonstrate that classical conditioning can induce crossmodal changes within putative unimodal sensory cortex even in the absence of its archetypal substrate. Our results also suggest that multimodal learning is associated with network wide activity within the conditioned neural system. These findings suggest that in early life, external multimodal sensory stimulation and integration shapes activity in the developing cortex and may influence its associated functional network architecture.

Lyme disease risk not amplified in a species-poor vertebrate community: similar Borrelia burgdorferi tick infection prevalence and OspC genotype frequencies.

The effect of biodiversity declines on human health is currently debated, but empirical assessments are lacking. Lyme disease provides a model system to assess relationships between biodiversity and human disease because the etiologic agent, Borrelia burgdorferi, is transmitted in the United States by the generalist black-legged tick (Ixodes scapularis) among a wide range of mammalian and avian hosts. The 'dilution effect' hypothesis predicts that species-poor host communities dominated by white-footed mice (Peromyscus leucopus) will pose the greatest human risk because P. leucopus infects the largest numbers of ticks, resulting in higher human exposure to infected I. scapularis ticks. P. leucopus-dominated communities are also expected to maintain a higher frequency of those B. burgdorferi outer surface protein C (ospC) genotypes that this host species more efficiently transmits ('multiple niche polymorphism' hypothesis). Because some of these genotypes are human invasive, an additive increase in human disease risk is expected in species-poor settings. We assessed these theoretical predictions by comparing I. scapularis nymphal infection prevalence, density of infected nymphs and B. burgdorferi genotype diversity at sites on Block Island, RI, where P. leucopus dominates the mammalian host community, to species-diverse sites in northeastern Connecticut. We found no support for the dilution effect hypothesis; B. burgdorferi nymphal infection prevalence was similar between island and mainland and the density of B. burgdorferi infected nymphs was higher on the mainland, contrary to what is predicted by the dilution effect hypothesis. Evidence for the multiple niche polymorphism hypothesis was mixed: there was lower ospC genotype diversity at island than mainland sites, but no overrepresentation of genotypes with higher fitness in P. leucopus or that are more invasive in humans. We conclude that other mechanisms explain similar nymphal infection prevalence in both communities and that high ospC genotype diversity can be maintained in both species-poor and species-rich communities.

Impact of molecular mutations on treatment response to DNMT inhibitors in myelodysplasia and related neoplasms.

We hypothesized that specific molecular mutations are important biomarkers for response to DNA methyltransferase inhibitors (DNMT inhibitors) and may have prognostic value in patients with myelodysplastic syndromes (MDS). Mutational analysis was performed in 92 patients with MDS and related disorders who received 5-azacytidine (n=55), decitabine (n=26) or both (n=11). Mutational status was correlated with overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) by univariate and multivariate analysis. Risk stratification models were created. TET2, DNMT3A, IDH1/IDH2, ASXL1, CBL, RAS and SF3B1 mutations were found in 18, 9, 8, 26, 3, 2 and 13% of patients, respectively. In multivariate analysis, TET2(MUT) and/or DNMT3A(MUT) (P=0.03), platelets > or = 100 × 10(9)/l (P=0.007) and WBC<3.0 × 10(9)/l (P=0.03) were independent predictors of better response. TET2(MUT) and/or DNMT3A(MUT) (P=0.04) status was also independently prognostic for improved PFS, as were good or intermediate cytogenetic risk (P<0.0001), age<60 (P=0.0001), treatment with both 5-azacytidine and decitabine (P=0.02) and hemoglobin > or = 10 g/dl (P=0.01). Better OS was associated with ASXL1(WT) (P=0.008) and SF3B1(MUT) (P=0.01), and, similar to PFS, cytogenetic risk (P=0.0002), age (P=0.02) and hemoglobin (P=0.04). These data support the role of molecular mutations as predictive biomarkers for response and survival in MDS patients treated with DNMT inhibitors.

Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies.

Polycomb repressive complex 2 (PRC2) is involved in trimethylation of histone H3 lysine 27 (H3K27), chromatin condensation and transcriptional repression. The silencing function of PRC2 complex is mostly attributed to its intrinsic activity for methylating H3K27. Unlike in B-cell lymphomas, enhancer of zeste homolog 2 (EZH2) mutations in myeloid malignancies are inactivating/hypomorphic. When we assessed the mutational status in myeloid malignancies (N=469 cases examined), we found EZH2 and EED/SUZ12 mutations in 8% and 3.3% of cases, respectively. In addition to mutant cases, reduced EZH2 expression was also found in 78% cases with hemizygous deletion (-7/del7q cases involving EZH2 locus) and 41% of cases with diploid chromosome 7, most interestingly cases with spliceosomal mutations (U2AF1/SRSF2 mutations; 63% of cases). EZH2 mutations were characterized by decreased H3K27 trimethylation and increased chromatin relaxation at specific gene loci accompanied by higher transcriptional activity. One of the major downstream target is HOX gene family, involved in the regulation of stem cell self-renewal. HOXA9 was found to be overexpressed in cases with decreased EZH2 expression either by EZH2/spliceosomal mutations or because of -7/del7q. In summary, our results suggest that loss of gene repression through a variety of mutations resulting in reduced H3K27 trimethylation may contribute to leukemogenesis.

CBL mutation-related patterns of phosphorylation and sensitivity to tyrosine kinase inhibitors.

Recurrent homozygous CBL-inactivating mutations in myeloid malignancies decrease ubiquitin ligase activity that inactivates SRC family kinases (SFK) and receptor tyrosine kinases (RTK). However, the most important SFK and RTK affected by these mutations, and hence, the most important therapeutic targets, have not been clearly characterized. We compared SFK and RTK pathway activity and inhibitors in acute myeloid leukemia cell lines containing homozygous R420Q mutation (GDM-1), heterozygous deletion (MOLM13) and wild-type (WT) CBL (THP1, U937). As expected with CBL loss, GDM-1 displayed high KIT expression and granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity. Ectopic expression of WT CBL decreased GDM-1 proliferation but not cell lines with WT CBL. GDM-1, but not the other cell lines, was highly sensitive to growth inhibition by dasatinib (dual SFK and RTK inhibitor, LD50 50 nM); there was less or no selective inhibition of GDM-1 growth by sunitinib (RTK inhibitor), imatinib (ABL, KIT inhibitor), or PP2 (SFK inhibitor). Phosphoprotein analysis identified phosphorylation targets uniquely inhibited by dasatinib treatment of GDM-1, including a number of proteins in the KIT and GM-CSF receptor pathways (for example, KIT Tyr721, STAT3 Tyr705). In conclusion, the promiscuous effects of CBL loss on SFK and RTK signaling appear to be best targeted by dual SFK and RTK inhibition.

Double-negative regulatory T cells induce allotolerance when expanded after allogeneic haematopoietic stem cell transplantation.

Double-negative (DN) regulatory T cells (Tregs) are specialized T lymphocytes involved in the down-modulation of immune responses, resulting in allotolerance after allogeneic haematopoietic stem cell transplantation (HSCT). Most of the properties of DN Tregs were identified in murine models, including the unique ability to suppress alloreactive syngeneic effector T cells in an antigen-specific manner via Fas/Fas-ligand interactions. We investigated the behaviour of DN Tregs following human allogeneic HSCT with regard to occurrence of graft-versus-host disease (GvHD) and restoration of T-cell receptor repertoire in a cohort of 40 patients. The frequency of DN Tregs and CD4/CD8 TCR repertoire was measured serially and at the time of diagnosis of GvHD by flow cytometry. Analysis demonstrated a positive correlation between degree of alloreactivity, as measured by grade of GvHD, and the number of variable beta chain (Vbeta) family expansions in both T-cell populations. We also found that a deficiency of DN Tregs was associated with an increased number of Vbeta family expansions, and most importantly, with the occurrence of GvHD. All individuals who demonstrated more than 1% DN Tregs did not develop GvHD, providing evidence that DN Tregs participate in peripheral tolerance to prevent GvHD when expanded after allogeneic HSCT.

Clonality of the stem cell compartment during evolution of myelodysplastic syndromes and other bone marrow failure syndromes.

Clonal hematopoiesis, observed in certain forms of marrow failure including aplastic anemia (AA), may be due to stem cell depletion. Alternatively, oligoclonality may be a result of recruitment of a preexisting defective clone, such as in paroxysmal nocturnal hemoglobinuria (PNH) or myelodysplastic syndromes (MDS). In PNH, exogenous permissive factors may be required for dominance of the abnormal clone, while in MDS, stem cells undergo transformation steps leading to a growth advantage. Stem or multipotent progenitor cell involvement in PNH is evidenced by long-term persistence of a clonal defect and its presence in all blood cells. In MDS, some clonal aberrations may have a 'founder-effect' and additional defects are secondary. Metaphase cytogenetics measures the proportion of clonal cells within dividing progenitor but not mature cells. Owing to low resolution, lesions can be found in only approximately 50% of MDS patients. This shortcoming may be overcome by application of newer technologies such as comparative genomic hybridization and SNP array-based karyotyping (SNP-A). SNP-A facilitates identification of cryptic lesions in bone marrow failure patients with normal or abnormal cytogenetics and allows for detection of loss of heterozygosity as a result of uniparental disomy, a lesion frequently found in MDS.

Large granular lymphocyte (LGL)-like clonal expansions in paroxysmal nocturnal hemoglobinuria (PNH) patients.

In paroxysmal nocturnal hemoglobinuria (PNH), clonal expansion of glycosylphosphatidylinositol-anchored proteins (GPI-AP)-deficient cells leads to a syndrome characterized by hemolytic anemia, marrow failure, and venous thrombosis. PNH is closely related to aplastic anemia and may share its immune pathophysiology. In vivo expansion of dominant T-cell clones can reflect an antigen-driven immune response but may also represent autonomous proliferation, such as in large granular lymphocytic (LGL)-leukemia. T-cell clonality can be assessed by a combination of T-cell receptor (TCR) flow cytometry and complementarity-determining-region-3 (CDR3) molecular analysis. We studied 24 PNH patients for evidence of in vivo dominant T-cell responses by flow cytometry; TCR-Vbeta-specific expansions were identified in all patients. In four cases, extreme expansions of one Vbeta-subset of CD8+/CD28-/CD56+ (effector) phenotype mimicked subclinical LGL-disease. The monoclonality of these expansions was inferred from unique CDR3-size peak distributions and sequencing of dominant clonotypes. We conclude that the molecular analysis of TCR-beta chain may demonstrate clonal LGL-like expansions at unexpected frequency in PNH patients. Our observations blur the classical boundaries between different bone marrow failure syndromes such as AA, PNH, and LGL, and support the hypothesis that in PNH, the mutant clone may expand as a result of an immune-escape from antigen-driven lymphocyte attack on hematopoietic progenitors.

Improving prognosis following a first variceal haemorrhage over four decades.

BACKGROUND: Variceal bleeding is a frequent cause of death in patients with cirrhosis and portal hypertension. Over the past 40 years a number of new techniques have been introduced to control active variceal haemorrhage. Many randomised controlled trials were performed to evaluate these new therapies. While most have demonstrated efficacy in controlling haemorrhage few showed improved survival. AIM: The aim of this study was to investigate whether the prognosis for cirrhotic patients following a first variceal haemorrhage has improved over the past four decades. PATIENTS AND METHODS: A total of 1475 patients included in the control or untreated arms of randomised controlled prophylactic trials for the primary prevention of variceal haemorrhage between 1960 and 2000. Twenty eight eligible randomised controlled studies were included. Over the 40 year period of observation there was a reduction in bleeding related mortality over time from approximately 65% to approximately 40% (p=0.024). CONCLUSION: This study suggests that there has been a significant reduction in bleeding related mortality in patients with cirrhosis and portal hypertension over the past 40 years.

Six-month outcomes for patients who switched to olanzapine treatment.

OBJECTIVE: This study evaluated the outcomes of patients in a community mental health center who switched from treatment with another antipsychotic to olanzapine treatment. It also sought to determine whether simultaneous access to case management and psychosocial rehabilitation and olanzapine leads to enhanced functional improvement. METHODS: Six-month outcomes for a consecutive series of 104 patients who switched from a conventional antipsychotic medication to olanzapine were evaluated. Forty-nine patients in the same treatment program who continued to take conventional antipsychotics were also monitored as a reference group. Outcomes of the group receiving olanzapine were compared with their own baseline status and with outcomes of the reference group. RESULTS: At six months, patients in the olanzapine group demonstrated significant improvement over baseline across multiple measures of symptoms and psychosocial function. Compared with the reference group, the olanzapine group was more symptomatic at baseline and demonstrated significantly greater improvement at follow-up on the Brief Psychiatric Rating Scale and all subscales; Mini Psychiatric Rating Scale negative symptom, disorganization, anxiety, depression, and medication side effects items; and Clinical Global Improvement scale and Case Manager's Rating Scale-Plus illness factors. There was a trend toward superior improvement in psychosocial functioning among patients in the olanzapine group that achieved significance when patients in acute relapse at baseline were excluded. CONCLUSIONS: Olanzapine is effective in managing markedly to severely ill patients with psychotic disorders in a community mental health center. Simultaneous treatment with olanzapine, case management, and psychosocial rehabilitation leads to enhanced functional improvement among nonrelapsing patients.

Alpha satellite DNA variant-specific oligoprobes differing by a single base can distinguish chromosome 15 homologs.

The ability to distinguish homologous chromosomes is a powerful cytogenetic tool. However, traditional techniques can only distinguish extreme physical variants and are highly dependent on sample preparation. We have previously reported oligonucleotide probes, specific for human chromosome 17 alpha satellite DNA sequence variants, that distinguish cytogenetically normal homologous chromosomes by FISH. Here we report the development of similar oligoprobes, differing at a single nucleotide position, that not only distinguish homologous chromosomes 15 but can be used to follow the transmission of a chromosome from parents to their offspring. We also identified a novel array-size polymorphism in another family. The alphoid array of one chromosome is quite small and below the detection threshold for our oligoprobes, although it is detectable by conventional FISH probes. This size polymorphism provides an additional FISH-based method for distinguishing homologs. Most importantly, this work illustrates the potential applicability of the technique to the entire human chromosome complement.

Effectiveness of combining atypical antipsychotics and psychosocial rehabilitation in a community mental health center setting.

This article presents a consecutive case series of 70 patients treated with olanzapine, case management, and psychosocial rehabilitation in a community mental health setting. This group demonstrated highly significant improvement on all analyzed measures of symptoms and psychosocial function at 6-month follow-up. These findings suggest that results of efficacy studies of olanzapine will generalize to the community mental health setting. Furthermore, prescribing olanzapine in combination with case management and rehabilitation yields positive functional outcomes.

Strand displacement amplification and homogeneous real-time detection incorporated in a second-generation DNA probe system, BDProbeTecET.

BACKGROUND: Amplified DNA probes provide powerful tools for the detection of infectious diseases, cancer, and genetic diseases. Commercially available amplification systems suffer from low throughput and require decontamination schemes, significant hands-on time, and specially trained laboratory staff. Our objective was to develop a DNA probe system to overcome these limitations. METHODS: We developed a DNA probe system, the BDProbeTecTMET, based on simultaneous strand displacement amplification and real-time fluorescence detection. The system uses sealed microwells to minimize the release of amplicons to the environment. To avoid the need for specially trained labor, the system uses a simple workflow with predispensed reagent devices; a programmable, expandable-spacing pipettor; and the 96-microwell format. Amplification and detection time was 1 h, with potential throughput up to 564 patient results per shift. We tested 122 total patient specimens obtained from a family practice clinic with the BD ProbeTecET and the Abbott LCx(R) amplified system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. RESULTS: Based on reportable results, the BDProbeTecET results for both organisms were 100% sensitive and 100% specific relative to the LCx. CONCLUSIONS: The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C. trachomatis and N. gonorrhoeae and other organisms.

Plasma Cleaning and Its Applications for Electron Microscopy.

: The effectiveness of applying a high-frequency, low-energy, reactive gas plasma for the removal of hydrocarbon contamination from specimens and components for electron microscopy has been investigated with a variety of analytical techniques. Transmission electron microscopy (TEM) analysis of specimens that have been plasma cleaned shows an elimination of the carbonaceous contamination from the specimen. With extended cleaning times the removal of existing carbon contamination debris due to previously conducted microanalysis is shown. Following plasma cleaning, specimens may be examined in the electron microscope for several hours without exhibiting evidence of recontamination. The effectiveness of plasma cleaning is not limited to applications for TEM specimens. Scanning electron microscopy (SEM) specimens that have been plasma cleaned likewise show an elimination of carbonaceous contamination. Furthermore, other electron microscopy parts and accessories, such as aperture strips, specimen clamping rings, and Wehnelts, among others, can benefit from plasma cleaning.