Upregulation of Xbp1 in NPY/AgRP neurons reverses diet-induced obesity and ameliorates leptin and insulin resistance.

The molecular mechanisms underlying neuronal leptin and insulin resistance in obesity and diabetes are not fully understood. In this study, we show that induction of the unfolded protein response transcription factor, spliced X-box binding protein 1 (Xbp1s), in Agouti-Related Peptide (AgRP) neurons alone, is sufficient to not only protect against but also significantly reverse diet-induced obesity (DIO) as well as improve leptin and insulin sensitivity, despite activation of endoplasmic reticulum stress. We also demonstrate that constitutive expression of Xbp1s in AgRP neurons contributes to improved insulin sensitivity and glucose tolerance. Together, our results identify critical molecular mechanisms linking ER stress in arcuate AgRP neurons to acute leptin and insulin resistance as well as liver glucose metabolism in DIO and diabetes.

GLP-1 increases preingestive satiation via hypothalamic circuits in mice and humans.

Glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs) are effective antiobesity drugs. However, the precise central mechanisms of GLP-1RAs remain elusive. We administered GLP-1RAs to patients with obesity and observed a heightened sense of preingestive satiation. Analysis of human and mouse brain samples pinpointed GLP-1 receptor (GLP-1R) neurons in the dorsomedial hypothalamus (DMH) as candidates for encoding preingestive satiation. Optogenetic manipulation of DMHGLP-1R neurons caused satiation. Calcium imaging demonstrated that these neurons are actively involved in encoding preingestive satiation. GLP-1RA administration increased the activity of DMHGLP-1R neurons selectively during eating behavior. We further identified that an intricate interplay between DMHGLP-1R neurons and neuropeptide Y/agouti-related peptide neurons of the arcuate nucleus (ARCNPY/AgRP neurons) occurs to regulate food intake. Our findings reveal a hypothalamic mechanism through which GLP-1RAs control preingestive satiation, offering previously unexplored neural targets for obesity and metabolic diseases.

GLP-1-directed NMDA receptor antagonism for obesity treatment.

The N-methyl-D-aspartate (NMDA) receptor is a glutamate-activated cation channel that is critical to many processes in the brain. Genome-wide association studies suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity are important for body weight homeostasis1. Here we report the engineering and preclinical development of a bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycaemia and dyslipidaemia in rodent models of metabolic disease. GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects neuroplasticity in the hypothalamus and brainstem. Importantly, targeting of MK-801 to GLP-1 receptor-expressing brain regions circumvents adverse physiological and behavioural effects associated with MK-801 monotherapy. In summary, our approach demonstrates the feasibility of using peptide-mediated targeting to achieve cell-specific ionotropic receptor modulation and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for safe and effective obesity treatment.

Minimizing the risk of Clostridioides difficile infection as an early complication of autologous stem cell transplantation.

This quality improvement project aimed to reduce institutional incidence of Clostridioides difficile infection (CDI) following autologous stem cell transplantation. CDI incidence per transplant was .17 in a baseline period and .09 following the implementation of postdischarge ultraviolet room cleaning (χ2 = 2.11, p = .15).

Exercise-induced hypothalamic neuroplasticity: Implications for energy and glucose metabolism.

BACKGROUND: Neuroplasticity refers to the brain's ability to undergo functional and structural changes in response to diverse challenges. Converging evidence supports the notion that exercise serves as a metabolic challenge, triggering the release of multiple factors both in the periphery and within the brain. These factors actively contribute to plasticity in the brain, and in turn, regulate energy and glucose metabolism. SCOPE OF REVIEW: The primary focus of this review is to explore the impact of exercise-induced plasticity in the brain on metabolic homeostasis, with an emphasis on the role of the hypothalamus in this process. Additionally, the review provides an overview of various factors induced by exercise that contribute to energy balance and glucose metabolism. Notably, these factors exert their effects, at least in part, through actions within the hypothalamus and more broadly in the central nervous system. MAJOR CONCLUSIONS: Exercise elicits both transient and sustained changes in metabolism, accompanied by changes in neural activity within specific brain regions. Importantly, the contribution of exercise-induced plasticity and the underlying mechanisms by which neuroplasticity influences the effects of exercise are not well understood. Recent work has begun to overcome this gap in knowledge by examining the complex interactions of exercise-induced factors which alter neural circuit properties to influence metabolism.

Prophylactic Pretransplant Ganciclovir to Reduce Cytomegalovirus Infection After Hematopoietic Stem Cell Transplantation.

OBJECTIVE/BACKGROUND: Cytomegalovirus (CMV) reactivation remains a serious complication after allogeneic hematopoietic cell transplantation (HCT) occurring in approximately 60-70% of CMV-seropositive HCT recipients. CMV reactivation leads to adverse outcomes including end-organ damage, graft-versus-host disease, and graft failure. METHODS: Ganciclovir was administered pretransplant at 5 mg/kg twice daily intravenously from the start of conditioning to Day T-2 to CMV-seropositive patients receiving their first allogeneic HCT. CMV DNA was monitored weekly until at least Day 100 posttransplant. RESULTS: A total of 109 consecutive patients were treated, median age 57 (range 20-73) years. Of these, 36 (33%) patients had a CMV reactivation within the first 105 days posttransplant with a median time of reactivation of 52.5 (range 36-104) days posttransplant. The cumulative incidence of CMV reactivation at Day 105 posttransplant was 33.1% (95% confidence interval: 24.4-42.0). One patient developed CMV disease. CONCLUSION: The use of pretransplant ganciclovir was associated with low incidence of CMV reactivation and disease. These data suggest that pretransplant ganciclovir with preemptive therapy for viral reactivation may be a useful strategy to reduce CMV reactivation. Future prospective trials are needed to compare strategies for CMV prophylaxis.

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior.

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen.

BACKGROUND: Corneal stromal cells (keratocytes) are responsible for developing and maintaining normal corneal structure and transparency, and for repairing the tissue after injury. Corneal keratocytes reside between highly aligned collagen lamellae in vivo. In addition to growth factors and other soluble biochemical factors, feedback from the extracellular matrix (ECM) itself has been shown to modulate corneal keratocyte behavior. METHODS: In this study, we fabricate aligned collagen substrates using a microfluidics approach and assess their impact on corneal keratocyte morphology, cytoskeletal organization, and patterning after stimulation with platelet derived growth factor (PDGF) or transforming growth factor beta 1 (TGFß). We also use time-lapse imaging to visualize the dynamic interactions between cells and fibrillar collagen during wound repopulation following an in vitro freeze injury. RESULTS: Significant co-alignment between keratocytes and aligned collagen fibrils was detected, and the degree of cell/ECM co-alignment further increased in the presence of PDGF or TGFß. Freeze injury produced an area of cell death without disrupting the collagen. High magnification, time-lapse differential interference contrast (DIC) imaging allowed cell movement and subcellular interactions with the underlying collagen fibrils to be directly visualized. CONCLUSIONS: With continued development, this experimental model could be an important tool for accessing how the integration of multiple biophysical and biochemical signals regulate corneal keratocyte differentiation.

Assessment of Corneal Stromal Remodeling and Regeneration after Photorefractive Keratectomy.

This study utilizes high resolution multi-dimensional imaging to identify temporal and spatial changes in cell/extracellular matrix (ECM) patterning mediating cell migration, fibrosis, remodeling and regeneration during wound healing. Photorefractive keratectomy (PRK) was performed on rabbits. In some cases, 5([4,6-dichlorotriazin-2yl]-amino)fluorescein (DTAF) was applied immediately after surgery to differentiate native vs. cell-secreted collagen. Corneas were assessed 3-180 days postoperatively using in vivo confocal microscopy, and cell/ECM patterning was evaluated in situ using multiphoton and second harmonic generation (SHG) imaging. 7 days post-PRK, migrating fibroblasts below the ablation site were co-aligned with the stromal lamellae. At day 21, randomly patterned myofibroblasts developed on top of the ablation site; whereas cells underneath were elongated, co-aligned with collagen, and lacked stress fibers. Over time, fibrotic tissue was remodeled into more transparent stromal lamellae. By day 180, stromal thickness was almost completely restored. Stromal regrowth occurred primarily below the ablation interface, and was characterized by co-localization of gaps in DTAF labeling with elongated cells and SHG collagen signaling. Punctate F-actin labeling was detected along cells co-aligned with DTAF and non-DTAF labeled collagen, suggesting cell-ECM interactions. Overall, collagen lamellae appear to provide a template for fibroblast patterning during wound healing that mediates stromal repopulation, regeneration and remodeling.

Temporal and spatial analysis of stromal cell and extracellular matrix patterning following lamellar keratectomy.

Extracellular matrix (ECM) supplies both physical and chemical signals to keratocytes which can impact their differentiation to fibroblasts and/or myofibroblasts. It also provides a substrate through which they migrate during wound repair. We have previously shown that following transcorneal freeze injury (FI), migrating corneal fibroblasts align parallel to the stromal lamellae during wound repopulation. In this study, we compare cell and ECM patterning both within and on top of the stroma at different time points following lamellar keratectomy (LK) in the rabbit. Twelve rabbits received LK in one eye. Rabbits were monitored using in vivo confocal microscopy at 3, 7, 21 and 60 days after injury. A subset of animals was sacrificed at each time point to further investigate cell and matrix patterning. Tissue was fixed and labeled in situ with Alexa Fluor 488 phalloidin (for F-actin), and imaged using multiphoton fluorescence and second harmonic generation (SHG) imaging (for collagen). Immediately following LK, cell death occurred in the corneal stroma directly beneath the injury. At 7 and 21 days after LK, analysis of fluorescence (F-actin) and SHG results (collagen) indicated that fibroblasts were co-aligned with the collagen lamellae within this region. In contrast, stromal cells accumulating on top of the stromal wound bed were randomly arranged, contained more prominent stress fibers, and expressed alpha smooth muscle actin (α-SMA) and fibronectin. At 60 days, cells and matrix in this region had become co-aligned into lamellar-like structures; cells were elongated but did not express stress fibers. Corneal haze measured using in vivo confocal microscopy peaked at 21 days after LK, and was significantly reduced by 60 days. Cell morphology and patterning observed in vivo was similar to that observed in situ. Our results suggest that the topography and alignment of the collagen lamellae direct fibroblast patterning during repopulation of the native stroma after LK injury in the rabbit. In contrast, stromal cells accumulating on top of the stromal wound bed initially align randomly and produce a fibrotic ECM. Remarkably, over time, these cells appear to remodel the ECM to produce a lamellar structure that is similar to the native corneal stroma.