Unraveling inflammatory responses using systems genetics and gene-environment interactions in macrophages.

Many common diseases have an important inflammatory component mediated in part by macrophages. Here we used a systems genetics strategy to examine the role of common genetic variation in macrophage responses to inflammatory stimuli. We examined genome-wide transcript levels in macrophages from 92 strains of the Hybrid Mouse Diversity Panel. We exposed macrophages to control media, bacterial lipopolysaccharide (LPS), or oxidized phospholipids. We performed association mapping under each condition and identified several thousand expression quantitative trait loci (eQTL), gene-by-environment interactions, and eQTL "hot spots" that specifically control LPS responses. We used siRNA knockdown of candidate genes to validate an eQTL hot spot in chromosome 8 and identified the gene 2310061C15Rik as a regulator of inflammatory responses in macrophages. We have created a public database where the data presented here can be used as a resource for understanding many common inflammatory traits that are modeled in the mouse and for the dissection of regulatory relationships between genes.

Bridging the splicing gap in human genetics with long-read RNA sequencing: finding the protein isoform drivers of disease.

Aberrant splicing underlies many human diseases, including cancer, cardiovascular diseases and neurological disorders. Genome-wide mapping of splicing quantitative trait loci (sQTLs) has shown that genetic regulation of alternative splicing is widespread. However, identification of the corresponding isoform or protein products associated with disease-associated sQTLs is challenging with short-read RNA-seq, which cannot precisely characterize full-length transcript isoforms. Furthermore, contemporary sQTL interpretation often relies on reference transcript annotations, which are incomplete. Solutions to these issues may be found through integration of newly emerging long-read sequencing technologies. Long-read sequencing offers the capability to sequence full-length mRNA transcripts and, in some cases, to link sQTLs to transcript isoforms containing disease-relevant protein alterations. Here, we provide an overview of sQTL mapping approaches, the use of long-read sequencing to characterize sQTL effects on isoforms, the linkage of RNA isoforms to protein-level functions and comment on future directions in the field. Based on recent progress, long-read RNA sequencing promises to be part of the human disease genetics toolkit to discover and treat protein isoforms causing rare and complex diseases.

Genetic analysis of osteoblast activity identifies Zbtb40 as a regulator of osteoblast activity and bone mass.

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

Dissecting the Genetics of Osteoporosis using Systems Approaches.

Osteoporosis is a condition characterized by low bone mineral density (BMD) and an increased risk of fracture. Traits contributing to osteoporotic fracture are highly heritable, indicating that a comprehensive understanding of bone requires a thorough understanding of the genetic basis of bone traits. Towards this goal, genome-wide association studies (GWASs) have identified over 500 loci associated with bone traits. However, few of the responsible genes have been identified, and little is known of how these genes work together to influence systems-level bone function. In this review, we describe how systems genetics approaches can be used to fill these knowledge gaps.

The RNA demethylase FTO is required for maintenance of bone mass and functions to protect osteoblasts from genotoxic damage.

The fat mass and obesity-associated gene (FTO) encodes an m6A RNA demethylase that controls mRNA processing and has been linked to both obesity and bone mineral density in humans by genome-wide association studies. To examine the role of FTO in bone, we characterized the phenotype of mice lacking Fto globally (FtoKO ) or selectively in osteoblasts (FtoOcKO ). Both mouse models developed age-related reductions in bone volume in both the trabecular and cortical compartments. RNA profiling in osteoblasts following acute disruption of Fto revealed changes in transcripts of Hspa1a and other genes in the DNA repair pathway containing consensus m6A motifs required for demethylation by FtoFto KO osteoblasts were more susceptible to genotoxic agents (UV and H2O2) and exhibited increased rates of apoptosis. Importantly, forced expression of Hspa1a or inhibition of NF-κB signaling normalized the DNA damage and apoptotic rates in Fto KO osteoblasts. Furthermore, increased metabolic stress induced in mice by feeding a high-fat diet induced greater DNA damage in osteoblast of FtoOc KO mice compared to controls. These data suggest that FTO functions intrinsically in osteoblasts through Hspa1a-NF-κB signaling to enhance the stability of mRNA of proteins that function to protect cells from genotoxic damage.

Mouse genome-wide association and systems genetics identifies Lhfp as a regulator of bone mass.

Bone mineral density (BMD) is a strong predictor of osteoporotic fracture. It is also one of the most heritable disease-associated quantitative traits. As a result, there has been considerable effort focused on dissecting its genetic basis. Here, we performed a genome-wide association study (GWAS) in a panel of inbred strains to identify associations influencing BMD. This analysis identified a significant (P = 3.1 x 10-12) BMD locus on Chromosome 3@52.5 Mbp that replicated in two separate inbred strain panels and overlapped a BMD quantitative trait locus (QTL) previously identified in a F2 intercross. The association mapped to a 300 Kbp region containing four genes; Gm2447, Gm20750, Cog6, and Lhfp. Further analysis found that Lipoma HMGIC Fusion Partner (Lhfp) was highly expressed in bone and osteoblasts. Furthermore, its expression was regulated by a local expression QTL (eQTL), which overlapped the BMD association. A co-expression network analysis revealed that Lhfp was strongly connected to genes involved in osteoblast differentiation. To directly evaluate its role in bone, Lhfp deficient mice (Lhfp-/-) were created using CRISPR/Cas9. Consistent with genetic and network predictions, bone marrow stromal cells (BMSCs) from Lhfp-/- mice displayed increased osteogenic differentiation. Lhfp-/- mice also had elevated BMD due to increased cortical bone mass. Lastly, we identified SNPs in human LHFP that were associated (P = 1.2 x 10-5) with heel BMD. In conclusion, we used GWAS and systems genetics to identify Lhfp as a regulator of osteoblast activity and bone mass.

A novel voluntary weightlifting model in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway.

Our understanding of the molecular mechanisms underlying adaptations to resistance exercise remains elusive despite the significant biological and clinical relevance. We developed a novel voluntary mouse weightlifting model, which elicits squat-like activities against adjustable load during feeding, to investigate the resistance exercise-induced contractile and metabolic adaptations. RNAseq analysis revealed that a single bout of weightlifting induced significant transcriptome responses of genes that function in posttranslational modification, metabolism, and muscle differentiation in recruited skeletal muscles, which were confirmed by increased expression of fibroblast growth factor-inducible 14 (Fn14), Down syndrome critical region 1 (Dscr1) and Nuclear receptor subfamily 4, group A, member 3 (Nr4a3) genes. Long-term (8 weeks) voluntary weightlifting training resulted in significantly increases of muscle mass, protein synthesis (puromycin incorporation in SUnSET assay) and mTOR pathway protein expression (raptor, 4e-bp-1, and p70S6K proteins) along with enhanced muscle power (specific torque and contraction speed), but not endurance capacity, mitochondrial biogenesis, and fiber type transformation. Importantly, weightlifting training profound improved whole-body glucose clearance and skeletal muscle insulin sensitivity along with enhanced autophagy (increased LC3 and LC3-II/I ratio, and decreased p62/Sqstm1). These data suggest that resistance training in mice promotes muscle adaptation and insulin sensitivity with simultaneous enhancement of autophagy and mTOR pathway.

Using "-omics" Data to Inform Genome-wide Association Studies (GWASs) in the Osteoporosis Field.

PURPOSE OF REVIEW: Osteoporosis constitutes a major societal health problem. Genome-wide association studies (GWASs) have identified over 1100 loci influencing bone mineral density (BMD); however, few of the causal genes have been identified. Here, we review approaches that use "-omics" data and genetic- and systems genetics-based analytical strategies to facilitate causal gene discovery. RECENT FINDINGS: The bone field is beginning to adopt approaches that are commonplace in other disease disciplines. The slower progress has been due in part to the lack of large-scale "omics" data on bone and bone cells. This is however changing, and approaches such as eQTL colocalization, transcriptome-wide association studies (TWASs), network, and integrative approaches are beginning to provide significant insight into the genes responsible for BMD GWAS associations. The use of "-omics" data to inform BMD GWASs has increased in recent years, leading to the identification of novel regulators of BMD in humans. The ultimate goal will be to use this information to develop more effective therapies to treat and ultimately prevent osteoporosis.

Genetic variability affects the response of skeletal muscle to disuse.

OBJECTIVE: To examine whether genetic variability plays a role in skeletal muscle response to disuse. METHODS: We examined skeletal muscle response to disuse in five different strains of mice: CAST/EiJ, NOD/ShiLtJ, NZO/HILtJ, 129S1/SvImJ and A/J. Mice had one limb immobilized by a cast for three weeks. RESULTS: Response to immobilization was dependent on the strain of mice. Skeletal muscle mass/body weight was decreased by immobilization in all strains except 1291/SvImJ. Immobilization decreased absolute skeletal muscle mass in quadriceps and gastrocnemius in NOD/ShiltJ and NZO/HILtJ mice. Three weeks of immobilization resulted in an increase in quadriceps levels of atrogenes in CAST/EiJ. Immobilization resulted in an increase in quadriceps and gastrocnemius levels of Myh4 in CAST/EiJ. A similar trend was observed for Myh7 in gastrocnemius muscle. Immobilization resulted in a decrease of the p-p70S6K1/total p706SK1 ratio in quadriceps of NOD/ShiLtJ mice and the gastrocnemius of A/J mice. Immobilization did not affect the p-4EBP1/total 4EBP1 ratio in quadriceps of any of the strains examined. However, the p-4EBP1/total 4EBP1 ratio in gastrocnemius was greater in immobilized, relative to control, limbs in CAST/EiJ mice. CONCLUSION: Genetic variability affects the response of skeletal muscle to disuse.

MicroRNA-30b Is Both Necessary and Sufficient for Interleukin-21 Receptor-Mediated Angiogenesis in Experimental Peripheral Arterial Disease.

The interleukin-21 receptor (IL-21R) can be upregulated in endothelial cells (EC) from ischemic muscles in mice following hind-limb ischemia (HLI), an experimental peripheral arterial disease (PAD) model, blocking this ligand-receptor pathway-impaired STAT3 activation, angiogenesis, and perfusion recovery. We sought to identify mRNA and microRNA transcripts that were differentially regulated following HLI, based on the ischemic muscle having intact, or reduced, IL-21/IL21R signaling. In this comparison, 200 mRNAs were differentially expressed but only six microRNA (miR)/miR clusters (and among these only miR-30b) were upregulated in EC isolated from ischemic muscle. Next, myoglobin-overexpressing transgenic (MgTG) C57BL/6 mice examined following HLI and IL-21 overexpression displayed greater angiogenesis, better perfusion recovery, and less tissue necrosis, with increased miR-30b expression. In EC cultured under hypoxia serum starvation, knock-down of miR-30b reduced, while overexpression of miR-30b increased IL-21-mediated EC survival and angiogenesis. In Il21r-/- mice following HLI, miR-30b overexpression vs. control improved perfusion recovery, with a reduction of suppressor of cytokine signaling 3, a miR-30b target and negative regulator of STAT3. Together, miR-30b appears both necessary and sufficient for IL21/IL-21R-mediated angiogenesis and may present a new therapeutic option to treat PAD if the IL21R is not available for activation.

Non-Invasive Identification of Nutrient Components in Grain.

Digital farming is a modern agricultural concept that aims to maximize the crop yield while simultaneously minimizing the environmental impact of farming. Successful implementation of digital farming requires development of sensors to detect and identify diseases and abiotic stresses in plants, as well as to probe the nutrient content of seeds and identify plant varieties. Experimental evidence of the suitability of Raman spectroscopy (RS) for confirmatory diagnostics of plant diseases was previously provided by our team and other research groups. In this study, we investigate the potential use of RS as a label-free, non-invasive and non-destructive analytical technique for the fast and accurate identification of nutrient components in the grains from 15 different rice genotypes. We demonstrate that spectroscopic analysis of intact rice seeds provides the accurate rice variety identification in ~86% of samples. These results suggest that RS can be used for fully automated, fast and accurate identification of seeds nutrient components.

Antiangiogenic VEGF165b Regulates Macrophage Polarization via S100A8/S100A9 in Peripheral Artery Disease.

BACKGROUND: Atherosclerotic occlusions decrease blood flow to the lower limbs, causing ischemia and tissue loss in patients with peripheral artery disease (PAD). No effective medical therapies are currently available to induce angiogenesis and promote perfusion recovery in patients with severe PAD. Clinical trials aimed at inducing vascular endothelial growth factor (VEGF)-A levels, a potent proangiogenic growth factor to induce angiogenesis, and perfusion recovery were not successful. Alternate splicing in the exon-8 of VEGF-A results in the formation of VEGFxxxa (VEGF165a) and VEGFxxxb (VEGF165b) isoforms with existing literature focusing on VEGF165b's role in inhibiting vascular endothelial growth factor receptor 2-dependent angiogenesis. However, we have recently shown that VEGF165b blocks VEGF-A-induced endothelial vascular endothelial growth factor receptor 1 (VEGFR1) activation in ischemic muscle to impair perfusion recovery. Because macrophage-secreted VEGF165b has been shown to decrease angiogenesis in peripheral artery disease, and macrophages were well known to play important roles in regulating ischemic muscle vascular remodeling, we examined the role of VEGF165b in regulating macrophage function in PAD. METHODS: Femoral artery ligation and resection were used as an in vivo preclinical PAD model, and hypoxia serum starvation was used as an in vitro model for PAD. Experiments including laser-Doppler perfusion imaging, adoptive cell transfer to ischemic muscle, immunoblot analysis, ELISAs, immunostainings, flow cytometry, quantitative polymerase chain reaction analysis, and RNA sequencing were performed to determine a role of VEGF165b in regulating macrophage phenotype and function in PAD. RESULTS: First, we found increased VEGF165b expression with increased M1-like macrophages in PAD versus non-PAD (controls) muscle biopsies. Next, using in vitro hypoxia serum starvation, in vivo pre clinical PAD models, and adoptive transfer of VEGF165b-expressing bone marrow-derived macrophages or VEGFR1+/- bone marrow-derived macrophages (M1-like phenotype), we demonstrate that VEGF165b inhibits VEGFR1 activation to induce an M1-like phenotype that impairs ischemic muscle neovascularization. Subsequently, we found S100A8/S100A9 as VEGFR1 downstream regulators of macrophage polarization by RNA-Seq analysis of hypoxia serum starvation-VEGFR1+/+ versus hypoxia serum starvation-VEGFR1+/- bone marrow-derived macrophages. CONCLUSIONS: In our current study, we demonstrate that increased VEGF165b expression in macrophages induces an antiangiogenic M1-like phenotype that directly impairs angiogenesis. VEGFR1 inhibition by VEGF165b results in S100A8/S100A9-mediated calcium influx to induce an M1-like phenotype that impairs ischemic muscle revascularization and perfusion recovery.

RNA-sequencing analysis of differential gene expression associated with arterial stiffness.

OBJECTIVES: Arterial stiffness is recognized as an important predictor of cardiovascular disease morbidity and mortality, independent of traditional cardiovascular disease risk factors. Given that arterial tissue is not easily accessible, most gene expression studies on arterial stiffness have been conducted on animals or on patients who have undergone by-pass surgeries. In order to obtain a deeper understanding of early changes of arterial stiffness, this study compared transcriptome profiles between healthy adults with higher and lower arterial stiffness. METHODS: The sample included 20 healthy female adults without cardiovascular disease. Arterial stiffness was measured by carotid-femoral pulse wave velocity, the "gold-standard" measure of central arterial stiffness. Peripheral blood samples collected to PAXgene™ RNA tubes were used for RNA sequencing (RNA-seq). The potential confounding effects of age, body mass index, and mean arterial pressure were controlled for in RNA-seq analysis. To validate RNA-seq results, quantitative real-time PCR (qRT-PCR) was performed for six selected genes. RESULTS: The findings demonstrated that genes including CAPN9, IL32, ERAP2, RAB6B, MYBPH, and miRNA626 were down-regulated, and that MOCS1 gene was up-regulated among the people with higher arterial stiffness. Real-time PCR showed that the changes of CAPN9, IL32, ERAP2, and RAB6B were in concordance with RNA-seq data, and confirmed the validity of the gene expression profiles obtained by RNA-seq analysis. CONCLUSIONS: Previous studies have suggested the potential roles of CAPN9, IL32, and ERAP2 in structural changes of the arterial wall through up-regulation of metalloproteinases. However, the current study showed that CAPN9, IL32, and ERAP2 were down-regulated in the individuals with higher arterial stiffness, compared with those with lower arterial stiffness. The unexpected directions of expression of these genes may indicate an effort to maintain vascular homeostasis during increased arterial stiffness among healthy individuals. Further studies are guaranteed to investigate the roles of CAPN9, IL32, and ERAP2 in regulating arterial stiffness in people with and without cardiovascular disease.

Noninvasive and Nondestructive Detection of Cowpea Bruchid within Cowpea Seeds with a Hand-Held Raman Spectrometer.

Insect damage to crops is a serious issue, in particular when the pest dwells within its host. The cowpea bruchid ( Callosobruchus maculatus) is an herbivore of legumes including beans and peas. The bruchid lays its eggs on the seeds themselves; after hatching, the larvae burrow into and develop inside the seed, complicating detection and treatment. Left unchecked, two insects could destroy up to 50% of 1 ton of harvest cowpea ( Vigna unguiculata) after several months of storage. In this study, we investigated the possibility of using a hand-held Raman spectrometer to detect the pest during its development within intact cowpeas. Our results show that Raman spectroscopy can detect chemical signatures of bruchid larvae as well as their excrements inside the intact seeds. Additionally, using chemometric methods, we distinguished between healthy and infested seeds as well as among seeds hosting developmentally early or late-stage larvae with high accuracy. This study demonstrates Raman spectroscopy's efficacy in not only detection of pathogens and pests present on the surface of plant leaves and the grain but also inside the seeds. This Raman-based method may prove useful as a rapid means of screening crops for internal pests.

Nanoscale Structural Organization of Plant Epicuticular Wax Probed by Atomic Force Microscope Infrared Spectroscopy.

The cuticle covers external surfaces of plants, protecting them from biotic and abiotic stress factors. Epicuticular wax on the outer surface of the cuticle modifies reflectance and water loss from plant surfaces and has direct and indirect effects on photosynthesis. Variation in epicuticular wax accumulation, composition, and nanoscale structural organization impacts its biological function. Atomic force microscope infrared spectroscopy (AFM-IR) was utilized to investigate the internal and external surfaces of the cuticle of Sorghum bicolor, an important drought-tolerant cereal, forage, and high-biomass crop. AFM-IR revealed striking heterogeneity in chemical composition within and between the surfaces of the cuticle. The wax aggregate crystallinity and distribution of chemical functional groups across the surfaces was also probed and compared. These results, along with the noninvasive nondestructive nature of the method, suggest that AFM-IR can be used to investigate mechanisms of wax deposition and transport of charged molecules through the plant cuticle.

Raman spectroscopy as an early detection tool for rose rosette infection.

MAIN CONCLUSION: Hand-held Raman spectroscopy is a potential tool for a confirmatory, non-invasive, and non-destructive detection and identification of rose rosette disease. Using this spectroscopic approach, structural changes in roses that are associated with this viral infection can be revealed. The commercial rose shrub industry in the United States is one of the largest of its kind. All commercial rose varieties are susceptible to rose rosette disease (RRD), a deadly viral disease vectored by eriophyid mites. This disease is typically diagnosed visually and/or by PCR-based detection assays. The present work demonstrates that Raman spectroscopy can detect RRD in intact leaf tissue. It is shown that chemometric analysis can distinguish between spectra collected from symptomatic and asymptomatic tissue, as well as between healthy and asymptomatic tissue. This method will be useful as an initial screen for RRD prior to PCR analysis to help conserve reagents and save time.

A MicroRNA93-Interferon Regulatory Factor-9-Immunoresponsive Gene-1-Itaconic Acid Pathway Modulates M2-Like Macrophage Polarization to Revascularize Ischemic Muscle.

BACKGROUND: Currently, no therapies exist for treating and improving outcomes in patients with severe peripheral artery disease (PAD). MicroRNA93 (miR93) has been shown to favorably modulate angiogenesis and to reduce tissue loss in genetic PAD models. However, the cell-specific function, downstream mechanisms, or signaling involved in miR93-mediated ischemic muscle neovascularization is not clear. Macrophages were best known to modulate arteriogenic response in PAD, and the extent of arteriogenic response induced by macrophages is dependent on greater M2 to M1 activation/polarization state. In the present study, we identified a novel mechanism by which miR93 regulates macrophage polarization to promote angiogenesis and arteriogenesis to revascularize ischemic muscle in experimental PAD. METHODS: In vitro (macrophages, endothelial cells, skeletal muscle cells under normal and hypoxia serum starvation conditions) and in vivo experiments in preclinical PAD models (unilateral femoral artery ligation and resection) were conducted to examine the role of miR93-interferon regulatory factor-9-immunoresponsive gene-1 (IRG1)-itaconic acid pathway in macrophage polarization, angiogenesis, arteriogenesis, and perfusion recovery. RESULTS: In vivo, compared with wild-type controls, miR106b-93-25 cluster-deficient mice (miR106b-93-25-/-) showed decreased angiogenesis and arteriogenesis correlating with increased M1-like macrophages after experimental PAD. Intramuscular delivery of miR93 in miR106b-93-25-/- PAD mice increased angiogenesis, arteriogenesis, and the extent of perfusion, which correlated with more M2-like macrophages in the proximal and distal hind-limb muscles. In vitro, miR93 promotes and sustains M2-like polarization even under M1-like polarizing conditions (hypoxia serum starvation). Delivery of bone marrow-derived macrophages from miR106b-93-25-/- to wild-type ischemic muscle decreased angiogenesis, arteriogenesis, and perfusion, whereas transfer of wild-type macrophages to miR106b-93-25-/- had the opposite effect. Systematic analysis of top differentially upregulated genes from RNA sequencing between miR106b-93-25-/- and wild-type ischemic muscle showed that miR93 regulates IRG1 function to modulate itaconic acid production and macrophage polarization. The 3' untranslated region luciferase assays performed to determine whether IRG1 is a direct target of miR93 revealed that IRG1 is not an miR93 target but that interferon regulatory factor-9, which can regulate IRG1 expression, is an miR93 target. In vitro, increased expression of interferon regulatory factor-9 and IRG1 and itaconic acid treatment significantly decreased endothelial angiogenic potential. CONCLUSIONS: miR93 inhibits interferon regulatory factor-9 to decrease IRG1-itaconic acid production to induce M2-like polarization in ischemic muscle to enhance angiogenesis, arteriogenesis, and perfusion recovery in experimental PAD.

Detection and Identification of Plant Pathogens on Maize Kernels with a Hand-Held Raman Spectrometer.

Rapid detection and identification of crop pathogens is essential for improving crop yield. Typical pathogen assaying methods, such as polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA), are time-consuming and destructive to the sample. Raman spectroscopy (RS) is a noninvasive nondestructive analytical technique that provides insight on the chemical structure of the specimen. In this study, we demonstrate that using a hand-held Raman spectrometer, in combination with chemometric analyses, we can distinguish between healthy and diseased maize ( Zea mays) kernels, as well as between different diseases with 100% accuracy. Our analysis is portable and sample-agnostic, suggesting that it could be retooled for other crops and conducted autonomously.

Genomic Modifiers of Natural Killer Cells, Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection.

The MHC class I D(k) molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds D(k), are required to control viral spread. The extent of D(k)-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust D(k)-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.

A phase 1/2 trial of ublituximab, a novel anti-CD20 monoclonal antibody, in patients with B-cell non-Hodgkin lymphoma or chronic lymphocytic leukaemia previously exposed to rituximab.

This phase 1/2 study evaluated the safety, pharmacokinetic behavior and anti-tumour activity of ublituximab, a unique type I, chimeric, glycoengineered anti-CD20 monoclonal antibody, in rituximab-relapsed or -refractory patients with B-cell non-Hodgkin lymphoma (B-NHL) or chronic lymphocytic leukaemia (CLL). Induction therapy (doses of 450-1200 mg) consisted of 4 weekly infusions in cycle 1 for NHL and 3 weekly infusions in cycles 1 and 2 for CLL. Patients received ublituximab maintenance monthly during cycles 3-5, then once every 3 months for up to 2 years. Enrolled patients with B-NHL (n = 27) and CLL (n = 8) had a median of 3 prior therapies. No dose-limiting toxicities or unexpected adverse events (AEs) occurred. The most common AEs were infusion-related reactions (40%; grade 3/4, 0%); fatigue (37%; grade 3/4, 3%); pyrexia (29%; grade 3/4, 0%); and diarrhoea (26%; grade 3/4, 0%). Common haematological AEs were neutropenia (14%; grade 3/4, 14%) and anaemia (11%; grade 3/4, 6%). The overall response rate for evaluable patients (n = 31) was 45% (13% complete responses, 32% partial responses). Median duration of response and progression-free survival were 9·2 months and 7·7 months, respectively. Ublituximab was well-tolerated and efficacious in a heterogeneous and highly rituximab-pre-treated patient population.