Involvement of the immune system in the pathogenesis of Crohn's disease. Expression of the T9 antigen on peripheral immunocytes correlates with the severity of the disease

Peripheral lymphocyte cells from patients suffering from Crohn's disease were analyzed for the expression of the "activation" antigens T9 and HLA-DR on their cell surface. It was found that high numbers of "activated" lymphocytes, the majority of which have proven to be T cells, could be detected in patients with active Crohn's disease, whereas in healthy controls and inactive disease only a small subfraction of lymphocytes was positive for these antigens. This difference was highly significant (p = 0.0001). Within the subpopulation of T9-positive cells the ratio between T4- and T8-positive cells is about 1.8 (compared with 2.0 in the total T-cell subset). All HLA-DR-positive, non-B and non-glass-adherent cells could be detected in the T9-positive cell fraction. The presence of T9 antigens was found to correlate with the grade of severity of the disease as assessed by a Crohn's disease activity index. The presence of high amounts of T cells exhibiting this antigen is not restricted to Crohn's disease but is thought to be of importance as a marker for the involvement of the immune system in other maladies as well. Nevertheless, the determination of T9 antigen is expected to provide objective data reflecting the severity of Crohn's disease.     

Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae.

Uptake of glucose, fructose, and the nonmetabolizable analog 6-deoxyglucose was measured in wild-type Saccharomyces cerevisiae and two mutant strains, one (hxk1 hxk2) lacking both hexokinase A(P-I) and B(P-II) but containing glucokinase (and hence able to grow on glucose but not fructose) and the other (hxk1 hxk2 glk) also lacking glucokinase (and not able to grow on glucose either). Uptake of the nonmetabolized substances (i.e., 6-deoxyglucose in all three strains, fructose in the two mutants, and glucose in the triple mutant) reached a plateau at or below the external concentration. The kinetic characteristics of uptake were determined from 5-sec incubations by plotting velocity (V) vs. velocity/substrate concentration (V/S) curves. According to such plots, in the wild-type strain uptake had two components, "high affinity uptake" with Km values of ca. 1 mM for glucose and 6 mM for fructose and "low affinity uptake" with Km values of ca. 20 and 50 mM, respectively. The double kinase mutant showed both components for glucose but only the high Km component for fructose, while the triple kinase mutant showed only high Km uptake for both glucose and fructose. Genetic analysis showed that only in strains lacking both hexokinases (hxk1 hxk2) was the low Km system for fructose absent. Low Km uptake was restored to the triple mutant by introduction of the cloned wild-type genes: HXK1 or HXK2, for fructose uptake, and HXK1, HXK2, or GLK1, for glucose uptake. A phosphoglucose isomerase mutant had both low and high Km uptake for glucose. These results indicate the presence of two types of uptake mechanism for glucose and fructose in yeast, the functioning of one of which, the low Km system, is influenced by the cognate kinases.     

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.New therapeutic strategies are needed to treat complex human diseases. Because disease phenotypes are often regulated by interwoven genetic networks, exploiting combination therapy to target multiple pathways, as opposed to only single ones, can enhance treatment efficacy (1). However, discovering effective combination therapies for human diseases is challenging with existing methods, due to the cost, effort, and labor required to construct and analyze each combination (2). For example, the National Cancer Institute tested ∼5,000 pairwise combinations of 100 cancer drugs against the NCI-60 panel in a study that took 2 y and cost about USD $4 million (3). Thus, there is a need for technological advances to accelerate the identification of effective combinatorial therapies. Here, we used our combinatorial genetics en masse (CombiGEM)-CRISPR platform to perform rapid pooled screening of pairwise genetic knockouts against genes coding for epigenetic regulators and then translated our screen hits into drug combinations against human ovarian cancer cells.CRISPR-Cas9 technology has been used for large-scale genetic perturbation screens with single-guide RNA (sgRNA) libraries for gene knockouts (4–7), repression, and activation (8, 9). Despite its simplicity for multiplexed genetic perturbations (10–12), new methods are needed to enable high-throughput CRISPR-Cas9–based screening with combinatorial sets of guide RNAs (gRNAs), which would be broadly useful for studying combinatorial gene functions in multigenic phenotypes and diseases. By using CombiGEM-based DNA assembly (13, 14), we developed a strategy for the simple and efficient assembly of barcoded combinatorial gRNA libraries. These libraries can be delivered into human cells by lentiviruses to create genetically ultradiverse cell populations harboring unique gRNA combinations that can be tracked via barcode sequencing in pooled assays. This strategy, termed CombiGEM-CRISPR, uses one-pot cloning steps to enable the assembly of combinatorial gRNA libraries, thus simplifying and accelerating the workflow toward systematic analysis of combinatorial gene functions.     

Benefits and Sustainability of a Learning Collaborative for Implementation of Treat‐to‐Target in Rheumatoid Arthritis: Results of a Cluster‐Randomized Controlled Phase II Clinical Trial

Objective

We conducted a 2‐phase randomized controlled trial of a learning collaborative to facilitate implementation of treat‐to‐target (T2T) to manage rheumatoid arthritis (RA). We found substantial improvement in implementation of T2T in phase I. Here, we report on a second 9 months (phase II), where we examined the maintenance of response in phase I and predictors of greater improvement in T2T adherence.

Methods

We recruited patients from 11 rheumatology sites and randomized them to either receive the learning collaborative during phase I or to a wait‐list control group that received the learning collaborative intervention during phase II. The outcome was change in T2T implementation score (0–100, where 100 = best) from pre‐ to postintervention. The T2T implementation score was defined as a percent of components documented in visit notes. Analyses examined the extent to which the phase‐I intervention teams sustained improvement in T2T, as well as predictors of T2T improvement.

Results

The analysis included 636 RA patients. At baseline, the mean T2T implementation score was 11% in phase I intervention sites and 13% in phase II sites. After the intervention, T2T implementation score improved to 57% in the phase I intervention sites and to 58% in the phase II sites. Intervention sites from phase I sustained the improvement during the phase II (52%). Predictors of greater T2T improvement included having only rheumatologist providers at the site, academic affiliation of the site, having fewer providers per site, and the rheumatologist provider being a trainee.

Conclusion

Improvement in T2T remained relatively stable over a postintervention period.

     

Osteoporosis knowledge,self-efficacy,and health beliefs among Chinese individuals with HIV

Summary

The worldwide uptake of FRAX is described.

Introduction

The aim of this report was to determine the usage of FRAX worldwide over a 1-year period from 1 May 2012.

Methods

The number of FRAX calculations from each country was assessed over a 1-year period and expressed as calculations per million of the population aged 50 years or more. Countries were colour coded according to usage to populate a world map.

Results

Over the index year, there were estimated to be 2,391,639 calculations sourced from 173 counties. Uptake was high in North America, the Antipodes and most countries of Europe; intermediate in Latin America and the Middle East; and very low in Africa and much of South East Asia.

Conclusions

It is expected that the comparative data will encourage the development of new FRAX models and the uptake of FRAX into assessment guidelines.