Lysophosphatidic acid (LPA) acts on LPA2 receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA2 attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA2 alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA2 in intestinal epithelial cells (IECs) under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG) human LPA2; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA2 colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA2 compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA2 overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA2 alone can lead to intestinal dysplasia.
Lysophosphatidic acid (LPA) acts on LPA2 receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA2 attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA2 alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA2 in intestinal epithelial cells (IECs) under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG) human LPA2; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA2 colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA2 compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA2 overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA2 alone can lead to intestinal dysplasia.
Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20 days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8 days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. Statement of Significance Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.
Purpose of Review: Growing evidence supports the critical role of transcriptional mechanisms in promoting the spatial and temporal progression of bone healing. In this review, we evaluate and discuss new transcriptional and post-transcriptional regulatory mechanisms of secondary bone repair, along with emerging evidence for epigenetic regulation of fracture healing. Recent Findings: Using the candidate gene approach has identified new roles for several transcription factors in mediating the reactive, reparative, and remodeling phases of fracture repair. Further characterization of the different epigenetic controls of fracture healing and fracture-driven transcriptome changes between young and aged fracture has identified key biological pathways that may yield therapeutic targets. Furthermore, exogenously delivered microRNA to post-transcriptionally control gene expression is quickly becoming an area with great therapeutic potential. Summary: Activation of specific transcriptional networks can promote the proper progression of secondary bone healing. Targeting these key factors using small molecules or through microRNA may yield effective therapies to enhance and possibly accelerate fracture healing.
Huntington's disease (HD) is characterized by a progressive loss of neurons in the striatum and cerebral cortex and is caused by a CAG repeat expansion in the gene encoding huntingtin. Mice with the mutation inserted into their own huntingtin gene (knock-in mice) are, genetically, the best models of the human disease. Here we show for the first time that knock-in mice with 94 CAG repeats develop a robust and early motor phenotype at 2 months of age, characterized by increased rearing at night. This initial increase in repetitive movements was followed by decreased locomotion at 4 and 6 months, despite a normal life span. The decrease in striatal enkephalin mRNA that is known to occur at 4 months was not present at 2 months, when increased rearing was observed. Both the hyperactive and hypoactive phases of motor dysfunction prededed the detection of nuclear microaggregates of mutated huntingtin in striatal neurons. Nuclear microaggregates, defined as small huntingtin-positive punctas detected by light microscopy, were very rare at 4 months but became widely distributed in striatal neurons at 6 months. Nuclear inclusions did not appear until 18 months. When present, nuclear microaggregates predominated in the striosomal compartment of the striatum, providing a possible explanation for the different neuronal vulnerability of striatal compartments observed in humans. The early motor phenotype observed in the knock-in mouse is reminiscent of repetitive movements often observed in early HD and provides a novel opportunity to assess the ability of therapies to prevent the initial effects of the mutation in vivo.
Influenza vaccines with broad cross-protection are urgently needed to prevent an emerging influenza pandemic. A fusion protein of the Toll-like receptor (TLR) 5-agonist domains from flagellin and multiple repeats of the conserved extracellular domain of the influenza matrix protein 2 (M2e) was constructed, purified and evaluated as such a vaccine. A painless vaccination method suitable for possible self-administration using coated microneedle arrays was investigated for skin-targeted delivery of the fusion protein in a mouse model. The results demonstrate that microneedle immunization induced strong humoral as well as mucosal antibody responses and conferred complete protection against homo- and heterosubtypic lethal virus challenges. Protective efficacy with microneedles was found to be significantly better than that seen with conventional intramuscular injection, and comparable to that observed with intranasal immunization. Because of its advantages for administration, safety and storage, microneedle delivery of M2e-flagellin fusion protein is a promising approach for an easy-to-administer universal influenza vaccine.
Murine gammaherpesvirus 68 (γHV68) infection of mice provides a tractable small-animal model system for assessing the requirements for the establishment and maintenance of gammaherpesvirus latency within the lymphoid compartment. The M2 gene product of γHV68 is a latency-associated antigen with no discernible homology to any known proteins. Here we focus on the requirement for the M2 gene in splenic B-cell latency. Our analyses showed the following. (i) Low-dose (100 PFU) inoculation administered via the intranasal route resulted in a failure to establish splenic B-cell latency at day 16 postinfection. (ii) Increasing the inoculation dose to 4 × 10 5 PFU administered via the intranasal route partially restored the establishment of B-cell latency at day 16, but no virus reactivation was detected upon explant into tissue cultures. (iii) Although previous data failed to detect a phenotype of the M2 mutant upon high-dose intraperitoneal inoculation, decreasing the inoculation dose to 100 PFU administered intraperitoneally revealed a splenic B-cell latency phenotype at day 16 that was very similar to the phenotype observed upon high-dose intranasal inoculation. (iv) After low-dose intraperitoneal inoculation, fractionated B-cell populations showed that the M2 mutant virus was able to establish latency in surface immunoglobulin B-negative (sIgD - ) B cells; by 6 months postinfection, equivalent frequencies of M2 mutant and marker rescue viral genome-positive sIgD - B cells were detected. (v) Like the marker rescue virus, the M2 mutant virus also established latency in splenic naive B cells upon low-dose intraperitoneal inoculation, but there was a significant lag in the decay of this latently infected reservoir compared to that seen with the marker rescue virus. (vi) After low-dose intranasal inoculation, by day 42 postinfection, latency was observed in the spleen, although at a frequency significantly lower than that in the marker rescue virus-infected mice; by 3 months postinfection, nearly equivalent levels of viral genome-positive cells were observed in the spleens of marker rescue virus- and M2 mutant virus-infected mice, and these cells were exclusively sIgD - B cells. Taken together, these data convincingly demonstrate a role for the M2 gene product in reactivation from splenic B cells and also suggest that disruption of the M2 gene leads to dose- and route-specific defects in the efficient establishment of splenic B-cell latency.
The brainstem locus coeruleus (LC) supplies norepinephrine to the forebrain and degenerates in Alzheimer’s disease (AD). Loss of LC neurons is correlated with increased severity of other AD hallmarks, including -amyloid (A) plaques, tau neurofibrillary tangles, and cognitive deficits, suggesting that it contributes to the disease progression. Lesions of the LC in amyloid-based transgenic mouse models of AD exacerbate A pathology, neuroinflammation, and cognitive deficits, but it is unknown how the loss of LC neurons affects tau-mediated pathology or behavioral abnormalities. Here we investigate the impact of LC degeneration in a mouse model of tauopathy by lesioning the LC of male and female P301S tau transgenic mice with the neurotoxin N-(2-chloroethyl)-N-ethyl-bromobenzylamine (DSP-4) starting at 2 months of age. By 6 months, deficits in hippocampal-dependent spatial (Morris water maze) and associative (contextual fear conditioning) memory were observed in lesioned P301S mice while performance remained intact in all other genotype and treatment groups, indicating that tau and LC degeneration act synergistically to impair cognition. By 10 months, the hippocampal neuroinflammation and neurodegeneration typically observed in unlesioned P301S mice were exacerbated by DSP-4, and mortality was also accelerated. These DSP-4-induced changes were accompanied by only a mild aggravation of tau pathology, suggesting that increased tau burden cannot fully account for the effects of LC degeneration. Combined, these experiments demonstrate that loss of LC noradrenergic neurons exacerbates multiple phenotypes caused by pathogenic tau, and provides complementary data to highlight the dual role LC degeneration has on both tau and A pathologies in AD.
Secretory IgA (SIgA) directed against gut resident bacteria enables the mammalian mucosal immune system to establish homeostasis with the commensal gut microbiota after weaning. Germinal centers (GCs) in Peyer’s patches (PPs) are the principal inductive sites where naive B cells specific for bacterial antigens encounter their cognate antigens and receive T-cell help driving their differentiation into IgA-producing plasma cells. We investigated the role of antigen sampling by intestinal M cells in initiating the SIgA response to gut bacteria by developing mice in which receptor activator of nuclear factor-jB ligand (RANKL)-dependent M-cell differentiation was abrogated by conditional deletion of Tnfrsf11a in the intestinal epithelium. Mice without intestinal M cells had profound delays in PP GC maturation and emergence of lamina propria IgA plasma cells, resulting in diminished levels of fecal SIgA that persisted into adulthood. We conclude that M-cell-mediated sampling of commensal bacteria is a required initial step for the efficient induction of intestinal SIgA.
In this report, we present a new strategy for targeting chemotherapeutics to tumors, based on targeting extracellular DNA. A gemcitabine prodrug was synthesized, termed H-gemcitabine, which is composed of Hoechst conjugated to gemcitabine. H-gemcitabine has low toxicity because it is membrane-impermeable; however, it still has high tumor efficacy because of its ability to target gemcitabine to E-DNA in tumors. We demonstrate here that H-gemcitabine has a wider therapeutic window than free gemcitabine.