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The chronic lymphocytic leukemia (CLL) microenvironment has been receiving an increasing amount of attention, but there is currently limited data surrounding how the microenvironment affects initial development of CLL. We determined that the spleen is the initial site of CLL growth through monitoring of transgenic Eμ-TCL1 mice that develop CLL. Subsequently, we isolated stromal cells from the spleens of Eμ-TCL1 mice (EMST cells) that induce CLL cell division

Atherosclerotic disease of the coronary and carotid arteries is the primary global cause of significant mortality and morbidity. The chronic occlusive diseases have changed the epidemiological landscape of health problems both in developed and the developing countries. Despite the enormous benefit of advanced revascularization techniques, use of statins, and successful attempts of targeting modifiable risk factors, like smoking and exercise in the last four decades, there is still a definite “residual risk” in the population, as evidenced by many prevalent and new cases every year. Here, we highlight the burden of the atherosclerotic diseases and provide substantial clinical evidence of the residual risks in these diseases despite advanced management settings, with emphasis on strokes and cardiovascular risks. We critically discussed the concepts and potential underlying mechanisms of the evolving atherosclerotic plaques in the coronary and carotid arteries. This has changed our understanding of the plaque biology, the progression of unstable vs stable plaques, and the evolution of plaque prior to the occurrence of a major adverse atherothrombotic event. This has been facilitated using intravascular ultrasound, optical coherence tomography, and near-infrared spectroscopy in the clinical settings to achieve surrogate end points. These techniques are now providing exquisite information on plaque size, composition, lipid volume, fibrous cap thickness and other features that were previously not possible with conventional angiography.

Background:Myocardial infarction (MI) triggers myelopoiesis, resulting in heightened production of neutrophils. However, the mechanisms that sustain their production and recruitment to the injured heart are unclear.Methods:Using a mouse model of the permanent ligation of the left anterior descending artery and flow cytometry, we first characterized the temporal and spatial effects of MI on different myeloid cell types. We next performed global transcriptome analysis of different cardiac cell types within the infarct to identify the drivers of the acute inflammatory response and the underlying signaling pathways. Using a combination of genetic and pharmacological strategies, we identified the sequelae of events that led to MI-induced myelopoiesis. Cardiac function was assessed by echocardiography. The association of early indexes of neutrophilia with major adverse cardiovascular events was studied in a cohort of patients with acute MI.Results:Induction of MI results in rapid recruitment of neutrophils to the infarct, where they release specific alarmins, S100A8 and S100A9. These alarmins bind to the Toll-like receptor 4 and prime the nod-like receptor family pyrin domain-containing 3 inflammasome in naïve neutrophils and promote interleukin-1β secretion. The released interleukin-1β interacts with its receptor (interleukin 1 receptor type 1) on hematopoietic stem and progenitor cells in the bone marrow and stimulates granulopoiesis in a cell-autonomous manner. Genetic or pharmacological strategies aimed at disruption of S100A8/A9 and their downstream signaling cascade suppress MI-induced granulopoiesis and improve cardiac function. Furthermore, in patients with acute coronary syndrome, higher neutrophil count on admission and after revascularization correlates positively with major adverse cardiovascular disease outcomes.Conclusions:Our study provides novel evidence for the primary role of neutrophil-derived alarmins (S100A8/A9) in dictating the nature of the ensuing inflammatory response after myocardial injury. Therapeutic strategies aimed at disruption of S100A8/A9 signaling or their downstream mediators (eg, nod-like receptor family pyrin domain-containing 3 inflammasome, interleukin-1β) in neutrophils suppress granulopoiesis and may improve cardiac function in patients with acute coronary syndrome.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor that induces apoptosis in cancer cells. However, the physiological function of Par-4 remains unknown. Here we show that conventional Par-4 knockout (Par-4-/-) mice and adipocyte-specific Par-4 knockout (AKO) mice, but not hepatocyte-specific Par-4 knockout mice, are obese with standard chow diet. Par-4-/- and AKO mice exhibit increased absorption and storage of fat in adipocytes. Mechanistically, Par-4 loss is associated with mdm2 downregulation and activation of p53. We identified complement factor c3 as a p53-regulated gene linked to fat storage in adipocytes. Par-4 re-expression in adipocytes or c3 deletion reversed the obese mouse phenotype. Moreover, obese human subjects showed lower expression of Par-4 relative to lean subjects, and in longitudinal studies, low baseline Par-4 levels denoted an increased risk of developing obesity later in life. These findings indicate that Par-4 suppresses p53 and its target c3 to regulate obesity.

Rationale: There is incomplete knowledge of the impact of bone marrow cells on the gut microbiome and gut barrier function. Objective: We postulated that diabetes mellitus and systemic ACE2 (angiotensin-converting enzyme 2) deficiency would synergize to adversely impact both the microbiome and gut barrier function. Methods and Results: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from wild-type, ACE2 −/y , Akita (type 1 diabetes mellitus), and ACE2 −/y -Akita mice. Gut barrier integrity was assessed by immunofluorescence, and bone marrow cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2 −/y -Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells, but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2 −/y -Akita mice demonstrated a marked increase in peptidoglycan-producing bacteria. When compared with control cohorts treated with saline, intraperitoneal administration of myeloid angiogenic cells significantly decreased the microbiome gene expression associated with peptidoglycan biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of peptidoglycan and FABP-2 (intestinal fatty acid-binding protein 2) were observed in plasma of human subjects with type 1 diabetes mellitus (n=21) and type 2 diabetes mellitus (n=23) compared with nondiabetic controls (n=23). Using human retinal endothelial cells, we determined that peptidoglycan activates a noncanonical TLR-2 (Toll-like receptor 2) associated MyD88 (myeloid differentiation primary response protein 88)-ARNO (ADP-ribosylation factor nucleotide-binding site opener)-ARF6 (ADP-ribosylation factor 6) signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of peptidoglycan on the endothelium. Conclusions: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2 −/y -Akita mice can be favorably impacted by exogenous administration of myeloid angiogenic cells.

Platelets play a critical role in both the initiation and progression of atherosclerosis, and even more so in the ensuing atherothrombotic complications. Low-dose aspirin remains the mainstay of antiplatelet therapy in high-risk patients by reducing the risk of myocardial ischemia (MI), stroke, or death due to cardiovascular disease (CVD). However, antiplatelet therapies lose their efficacy in people with diabetes mellitus (DM), increasing the risk of future atherothrombotic events. The molecular mechanisms that promote platelet hyperactivity remain unclear but could be due to glycation-induced conformational changes of platelet membranes resulting in impaired aspirin entry, or less-efficient acetylation/compensatory increase in COX-2 expression in newborn platelets. Emerging evidence from our laboratory and elsewhere suggest that enhanced platelet turnover (thrombopoiesis), particularly the production of immature reticulated platelets from the bone marrow, could contribute to atherosclerotic complications. We have identified a major role for neutrophil-derived S100A8/A9, a damage-associated molecular pattern, in driving reticulated thrombopoiesis by directly interacting with its receptors on Kupffer cells in the liver. In this review, we discuss the role of hepatic inflammation in driving reticulated platelet production and suggest potential targets to control their production, improve efficacy of current antiplatelet therapies, and reduce the risk of atherothrombotic complications.

Chronic lymphocytic leukemia (CLL) patients progressively develop an immunosuppressive state. CLL patients have more plasma IL-10, an anti-inflammatory cytokine, than healthy controls. In vitro human CLL cells produce IL-10 in response to BCR cross-linking. We used the transgenic Eμ–T cell leukemia oncogene-1 (TCL1) mouse CLL model to study the role of IL-10 in CLL associated immunosuppression. Eμ-TCL mice spontaneously develop CLL because of a B cell–specific expression of the oncogene, TCL1. Eμ-TCL1 mouse CLL cells constitutively produce IL-10, which is further enhanced by BCR cross-linking, CLL-derived IL-10 did not directly affect survival of murine or human CLL cells in vitro. We tested the hypothesis that the CLL-derived IL-10 has a critical role in CLL disease in part by suppressing the host immune response to the CLL cells. In IL-10R−/− mice, wherein the host immune cells are unresponsive to IL-10–mediated suppressive effects, there was a significant reduction in CLL cell growth compared with wild type mice. IL-10 reduced the generation of effector CD4 and CD8 T cells. We also found that activation of BCR signaling regulated the production of IL-10 by both murine and human CLL cells. We identified the transcription factor, Sp1, as a novel regulator of IL-10 production by CLL cells and that it is regulated by BCR signaling via the Syk/MAPK pathway. Our results suggest that incorporation of IL-10 blocking agents may enhance current therapeutic regimens for CLL by potentiating host antitumor immune response.

Chronic Lymphocytic Leukemia (CLL), the most prevalent adult leukemia, is characterized by the clonal expansion of CD5+ CD19+ B cells within lymphoid organs and accumulation of quiescent cells in the blood. Eμ-Tcl1 transgenic mice expressing the Tcl1 oncogene in a B cell-specific manner develop CLL and adoptive transfer of CLL cells from these mice induces CLL growth in C57BL/6 mice. These models typically cause splenomegaly as the CLL burden increases, and CLL cells localize mainly in the spleen within the first 10 days post-injection. We hypothesized that the splenic microenvironment is vital for CLL expansion. Accordingly, we found that splenectomy significantly delayed and decreased CD5+ CD19+ CLL cell burden. The role of the splenic microenvironment was further explored by isolating splenic stromal cells from Eμ-Tcl1 mice and establishing stable cell lines (EMST cells). In cell culture, CLL cells divide poorly and remain mostly quiescent. The EMST cells induced proliferation in a majority of mouse and human CLL cells without the addition of any cytokines or growth factors commonly used to accomplish in vitro proliferation of CLL cells. They do not express CD40L, an important stimulatory molecule for B cell proliferation. EMST cells lack conventional immune cell markers, but they express the cytokines and chemokines BAFF, CCL2, CXCL12, and Wnt4. CCL2 levels dramatically increased upon co-culture with CLL. EMST cells also enhance IL-10 secretion by the CLL cells and thus may enhance an immunosuppressive environment to prevent anti-CLL immunity from developing. Our studies support the hypothesis that the spleen is vital for the establishment of CLL and EMST cells produce specific factors that may play a role in CLL growth.

Ashwagandha, an ayurvedic medicinal plant, has been found to be a major contributor in treating medical ailments. Ayurveda is one of the world’s oldest traditional medical systems originated in India through various indigenous theories, beliefs and experiences. Often translated as science of “ayus” (life) and “ved” (knowledge), it emphasizes the use of herbs and their derivatives for prevention of disease, rejuvenation of body systems, and improving the quality of life. In this chapter, we will discuss some of the immuno-modulatory effects of withaferin A (WFA) as well as its growth-inhibitory effects on B cell lymphoma, leukemia and myelodysplastic syndrome.

Studies on the physical nature of the structural heterogeneity of chromatin in their native states are few. The eukaryotic chromatin as observed by dye staining studies is of heterogeneous intensity when observed by fluorescent stains, where less and more bright regions apparently correspond to euchromatin and heterochromatin respectively. These are also associated with differential gene expression where it is believed that euchromatin is transcriptionally more active due to increased flexibility. Unfixed squashed preparations of polytene chromosomes of Drosophila were stained with a dsDNA specific dye PicoGreen and fluorescence lifetimes as well as fluorescence anisotropy decay kinetics were measured. Here we report a positive correlation between fluorescence lifetimes and fluorescence intensities, and show that less bright regions corresponding to euchromatin have shorter lifetimes, whereas more bright regions corresponding to heterochromatin have longer lifetimes. We interpret this as less bright regions being more dynamic, a conclusion also supported by fluorescence anisotropy decay kinetics. We infer that the comparatively higher flexibility associated with euchromatin can be directly measured by fluorescence lifetimes and fluorescence anisotropy decay kinetics.

We report a study of dynamics with a dsDNA-specific dye called PicoGreen bound to plasmid DNA (3.4 kb), and show that at low dye/DNA phosphate ratios (1 : 100 and below), PicoGreen dynamics reflect the motional dynamics of dsDNA. We further evaluated the usefulness of this probe by measuring the time-resolved fluorescence dynamics of PicoGreen bound to dsDNA in the presence of cationic reagents that affect DNA dynamics [MgCl2 and polyethyleneimine (PEI)] and also with plasmid DNA in different topological states. Among these conditions, MgCl2, PEI and the supercoiled form of plasmid resulted in increases in the very short component (0.2–0.4 ns) of the rotational correlation time. Separately, HMGB1 protein enhanced DNA dynamics, as observed from the rotational correlation times of very short (0.2–0.4 ns) and short (2–4 ns) rotational correlation timescale components. By studying the effect of specific deletion mutants HMGB1-ΔA (deletion of 98 N-terminal amino acids) and HMGB1-ΔC (deletion of 30 C-terminal amino acids), we show that the acidic C-terminal tail is required for enhancement of DNA dynamics. We then discuss the possible mechanisms and implications of HMGB1-mediated flexibility of DNA in the context of formation of large nucleoprotein complexes.

Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the western world, is characterized by accumulation of clonally expanded CD5+CD19+ B lymphocytes in the peripheral blood and secondary lymphoid organs. CLL cell survival is dependent on B cell receptor signaling and cross-talk within the leukemia microenvironment. CLL presents with enlargement of the spleen, and lymph nodes due to accumulation of long-lived lymphocytes with impaired apoptotic mechanisms. The Eμ-Tcl1 mice, with a B cell specific overexpression of the oncogene, T cell Leukemia 1 (Tcl1), serve as an excellent model to study CLL as they develop a CLL like disease by 9–13 months of age. They exhibit splenomegaly, enlarged lymph nodes and a high peripheral blood white count. Adoptive transfer of primary CD5+CD19+ CLL cells from the Eμ-Tcl1 mice into syngeneic recipients leads to rapid disease development. Ultrasonography of recipient mice showed a dramatic enlargement of the spleen, which preceded detection of CLL cells in the blood. Unlike various genetic modifications, which can only delay the onset of CLL, splenectomy in these models, cures or significantly delays disease development from 30–35 days to 250–270 days. Interestingly, splenectomy did not delay CLL development as significantly in animals deficient for prostate apoptotic response-4 (Par-4) compared to C57BL/6 wild type mice. Par-4 is a known tumor suppressor protein that can be secreted and induce apoptosis selectively in cancer cells but not in normal cells. Par-4 appears to regulate a specific microenvironment required for CLL growth. Presently we are investigating the role of Par-4 in the microenvironment and the cell types that are critical for CLL growth within the splenic niche.

Although transcriptional activation by NF-κB is well appreciated, physiological importance of transcriptional repression by NF-κB in cancer has remained elusive. Here we show that an HDAC4-RelB-p52 complex maintains repressive chromatin around proapoptotic genes Bim and BMF and regulates multiple myeloma (MM) survival and growth. Disruption of RelB-HDAC4 complex by a HDAC4-mimetic polypeptide blocks MM growth. RelB-p52 also represses BMF translation by regulating miR-221 expression. While the NIK-dependent activation of RelB-p52 in MM has been reported, we show that regardless of the activation status of NIK and the oncogenic events that cause plasma cell malignancy, several genetically diverse MM cells including Bortezomib-resistant MM cells are addicted to RelB-p52 for survival. Importantly, RelB is constitutively phosphorylated in MM and ERK1 is a RelB kinase. Phospho-RelB remains largely nuclear and is essential for Bim repression. Thus, ERK1-dependent regulation of nuclear RelB is critical for MM survival and explains the NIK-independent role of RelB in MM.

We report a study of dynamics with a dsDNA-specific dye called PicoGreen bound to plasmid DNA (3.4 kb), and show that at low dye/DNA phosphate ratios (1 : 100 and below), PicoGreen dynamics reflect the motional dynamics of dsDNA. We further evaluated the usefulness of this probe by measuring the time-resolved fluorescence dynamics of PicoGreen bound to dsDNA in the presence of cationic reagents that affect DNA dynamics [MgCl2 and polyethyleneimine (PEI)] and also with plasmid DNA in different topological states. Among these conditions, MgCl2, PEI and the supercoiled form of plasmid resulted in increases in the very short component (0.2-0.4 ns) of the rotational correlation time. Separately, HMGB1 protein enhanced DNA dynamics, as observed from the rotational correlation times of very short (0.2-0.4 ns) and short (2-4 ns) rotational correlation timescale components. By studying the effect of specific deletion mutants HMGB1-DeltaA (deletion of 98 N-terminal amino acids) and HMGB1-DeltaC (deletion of 30 C-terminal amino acids), we show that the acidic C-terminal tail is required for enhancement of DNA dynamics. We then discuss the possible mechanisms and implications of HMGB1-mediated flexibility of DNA in the context of formation of large nucleoprotein complexes.