Paracrine SPARC signaling dysregulates alveolar epithelial barrier integrity and function in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease in which aging, environmental exposure(s) and genetic susceptibility have been implicated in disease pathogenesis, however, the causes and mechanisms of the progressive fibrotic cascade are still poorly understood. As epithelial-mesenchymal interactions are essential for normal wound healing, through human 2D and 3D in vitro studies, we tested the hypothesis that IPF fibroblasts (IPFFs) dysregulate alveolar epithelial homeostasis. Conditioned media from IPFFs exaggerated the wound-healing response of primary human Type II alveolar epithelial cells (AECs). Furthermore, AECs co-cultured with IPFFs exhibited irregular epithelialization compared with those co-cultured with control fibroblasts (NHLFs) or AECs alone, suggesting that epithelial homeostasis is dysregulated in IPF as a consequence of the abnormal secretory phenotype of IPFFs. Secretome analysis of IPFF conditioned media and functional studies identified the matricellular protein, SPARC, as a key mediator in the epithelial-mesenchymal paracrine signaling, with increased secretion of SPARC by IPFFs promoting persistent activation of alveolar epithelium via an integrin/focal adhesion/cellular-junction axis resulting in disruption of epithelial barrier integrity and increased macromolecular permeability. These findings suggest that in IPF fibroblast paracrine signaling promotes persistent alveolar epithelial activation, so preventing normal epithelial repair responses and restoration of tissue homeostasis. Furthermore, they identify SPARC-mediated paracrine signaling as a potential therapeutic target to promote the restoration of lung epithelial homoestasis in IPF patients.

Learning endometriosis phenotypes from patient-generated data.

Endometriosis is a systemic and chronic condition in women of childbearing age, yet a highly enigmatic disease with unresolved questions: there are no known biomarkers, nor established clinical stages. We here investigate the use of patient-generated health data and data-driven phenotyping to characterize endometriosis patient subtypes, based on their reported signs and symptoms. We aim at unsupervised learning of endometriosis phenotypes using self-tracking data from personal smartphones. We leverage data from an observational research study of over 4000 women with endometriosis that track their condition over more than 2 years. We extend a classical mixed-membership model to accommodate the idiosyncrasies of the data at hand, i.e., the multimodality and uncertainty of the self-tracked variables. The proposed method, by jointly modeling a wide range of observations (i.e., participant symptoms, quality of life, treatments), identifies clinically relevant endometriosis subtypes. Experiments show that our method is robust to different hyperparameter choices and the biases of self-tracking data (e.g., the wide variations in tracking frequency among participants). With this work, we show the promise of unsupervised learning of endometriosis subtypes from self-tracked data, as learned phenotypes align well with what is already known about the disease, but also suggest new clinically actionable findings. More generally, we argue that a continued research effort on unsupervised phenotyping methods with patient-generated health data via new mobile and digital technologies will have significant impact on the study of enigmatic diseases in particular, and health in general.

Metformin effectively treats Tsc1 deletion-caused kidney pathology by upregulating AMPK phosphorylation.

Tuberous sclerosis complex (TSC) is characterized by hamartomatous lesions in multiple organs, with most patients developing polycystic kidney disease and leading to a decline of renal function. TSC is caused by loss-of-function mutations in either Tsc1 or Tsc2 gene, but currently, there is no effective treatment for aberrant kidney growth in TSC patients. By generating a renal proximal tubule-specific Tsc1 gene-knockout (Tsc1 ptKO) mouse model, we observed that Tsc1 ptKO mice developed aberrantly enlarged kidneys primarily due to hypertrophy and proliferation of proximal tubule cells, along with some cystogenesis, interstitial inflammation, and fibrosis. Mechanistic studies revealed inhibition of AMP-activated protein kinase (AMPK) phosphorylation at Thr-172 and activation of Akt phosphorylation at Ser-473 and Thr-308. We therefore treated Tsc1 ptKO mice with the AMPK activator, metformin, by daily intraperitoneal injection. Our results indicated that metformin increased the AMPK phosphorylation, but decreased the Akt phosphorylation. These signaling modulations resulted in inhibition of proliferation and induction of apoptosis in the renal proximal tubule cells of Tsc1 ptKO mice. Importantly, metformin treatment effectively prevented aberrant kidney enlargement and cyst growth, inhibited inflammatory response, attenuated interstitial fibrosis, and protected renal function. The effects of metformin were further confirmed by in vitro experiments. In conclusion, this study indicates a potential therapeutic effect of metformin on Tsc1 deletion-induced kidney pathology, although currently metformin is primarily prescribed to treat patients with type 2 diabetes.

Dose-dependent skeletal deficits due to varied reductions in mechanical loading in rats.

Reduced skeletal loading leads to marked bone loss. Animal models of hindlimb suspension are widely used to assess alterations in skeleton during the course of complete unloading. More recently, the effects of partial unloading on the musculoskeletal system have been interrogated in mice and rats, revealing dose-dependent effects of partial weight bearing (PWB) on the skeleton and skeletal muscle. Here, we extended these studies to determine the structural and functional skeletal alterations in 14-week-old male Wister rats exposed to 20%, 40%, 70%, or 100% of body weight for 1, 2, or 4 weeks (n = 11-12/group). Using in vivo pQCT, we found that trabecular bone density at the proximal tibia declined in proportion to the degree of unloading and continued progressively with time, without evidence of a plateau by 4 weeks. Ex vivo measurements of trabecular microarchitecture in the distal femur by microcomputed tomography revealed deficits in bone volume fraction, 2 and 4 weeks after unloading. Histologic analyses of trabecular bone in the distal femur revealed the decreased osteoblast number and mineralizing surface in unloaded rats. Three-point bending of the femoral diaphysis indicated modest or no reductions in femoral stiffness and estimated modulus due to PWB. Our results suggest that this rat model of PWB leads to trabecular bone deterioration that is progressive and generally proportional to the degree of PWB, with minimal effects on cortical bone.

A bivalent vaccine confers immunogenicity and protection against Shigella flexneri and enterotoxigenic Escherichia coli infections in mice.

Vaccine studies for Shigella flexneri and enterotoxigenic Escherichia coli have been impaired by the lack of optimal animal models. We used two murine models to show that a S. flexneri 2a bivalent vaccine (CVD 1208S-122) expressing enterotoxigenic Escherichia coli colonization factor antigen-I (CFA/I) and the binding subunits A2 and B of heat labile-enterotoxin (LTb) is immunogenic and protects against weight loss and diarrhea. These findings document the immunogenicity and pre-clinical efficacy effects of CVD 1208S-122 vaccine and suggest that further work can help elucidate relevant immune responses and ultimately its clinical efficacy in humans.

PINK1 phosphorylates ubiquitin predominantly in astrocytes.

Loss-of-function mutations in PINK1 are causally linked to recessively inherited Parkinson's disease (PD), with marked loss of dopaminergic neurons in the substantia nigra that are required for normal movement. PINK1 is a nuclear-encoded mitochondrial-targeted kinase that phosphorylates a conserved serine at amino acid 65 (pS65) in ubiquitin as well as Parkin, another gene with loss-of-function mutations linked to recessive parkinsonism. The steady-state levels of PINK1 protein are very low, even in cells that express PINK1, because PINK1 is normally targeted for degradation after mitochondrial import by a process that is dependent upon mitochondrial membrane potential. Dissipation of the mitochondrial membrane potential with ionophores, such as CCCP and valinomycin, causes the accumulation of PINK1 on the outer mitochondrial membrane, a marked increase of pS65-ubiquitin and the recruitment of Parkin, which targets dysfunctional mitochondria for degradation by autophagy. While the high penetrance of PINK1 mutations establish its critical function for maintaining neurons, the activity of PINK1 in primary neurons has been difficult to detect. Mounting evidence implicates non-neuronal cells, including astrocytes and microglia, in the pathogenesis of both idiopathic and inherited PD. Herein we used both western analysis and immunofluorescence of pS65-ubiquitin to directly compare the activity of PINK1 in primary neurons, astrocytes, microglia, and oligodendrocyte progenitor cells cultured from the brains of wild-type (WT) and PINK1 knockout (KO) rat pups. Our findings that PINK1-dependent ubiquitin phosphorylation is predominantly in astrocytes supports increased priority for research on the function of PINK1 in astrocytes and the contribution of astrocyte dysfunction to PD pathogenesis.

Disparate Tuberculosis Disease Development in Macaque Species Is Associated With Innate Immunity.

While tuberculosis continues to afflict mankind, the immunological mechanisms underlying TB disease development are still incompletely understood. Advanced preclinical models for TB research include both rhesus and cynomolgus macaques (Macaca mulatta and Macaca fascicularis, respectively), with rhesus typically being more susceptible to acute progressive TB disease than cynomolgus macaques. To determine which immune mechanisms are responsible for this dissimilar disease development, we profiled a broad range of innate and adaptive responses, both local and peripheral, following experimental pulmonary Mycobacterium tuberculosis (Mtb) infection of both species. While T-cell and antibody responses appeared indistinguishable, we identified anti-inflammatory skewing of peripheral monocytes in rhesus and a more prominent local pro-inflammatory cytokine release profile in cynomolgus macaques associated with divergent TB disease outcome. Importantly, these differences were detectable both before and early after infection. This work shows that inflammatory and innate immune status prior to and at early stages after infection, critically affects outcome of TB infection.

miR-124/VAMP3 is a novel therapeutic target for mitigation of surgical trauma-induced microglial activation.

Activation of microglia and the subsequently elevated inflammatory cytokine release in the brain during surgery predispose individuals to cognitive dysfunction, also known as postoperative cognitive dysfunction (POCD). miR-124 is one of the most abundant microRNAs in the brain that regulates microglial function. Elucidating the role of miR-124 in microglial activation in the context of surgery may therefore promote understanding of as well as therapeutic development for post-surgical disorders involving microglial activation. The downstream targets of miR-124 were investigated using bioinformatic screening and dual-luciferase reporter assay validation, and vesicle-associated membrane protein 3 (VAMP3) was identified as a potential target. The kinetics of miR-124/VAMP3 expression was first examined in vitro in microglial cells (primary microglia and BV2 microglial cells) following lipopolysaccharide (LPS) stimulation. LPS induced a time-dependent decrease of miR-124 and upregulated the expression of VAMP3. Manipulating miR-124/VAMP3 expression by using miR-124 mimics or VAMP3-specific siRNA in LPS-stimulated BV2 microglial cells inhibited BV2 microglial activation-associated inflammatory cytokine release. To further examine the role of miR-124/VAMP3 in a surgical setting, we employed a rat surgical trauma model. Significant microglial activation and altered miR-124/VAMP3 expression were observed following surgical trauma. We also altered miR-124/VAMP3 expression in the rat surgical trauma model by administration of exogenous miR-124 and by using electroacupuncture, which is a clinically applicable treatment that modulates microglial function and minimizes postoperative disorders. We determined that electroacupuncture treatment specifically increases the expression of miR-124 in the hypothalamus and hippocampus. Increased miR-124 expression with a concomitant decrease in VAMP3 expression resulted in decreased inflammatory cytokine release related to microglial activation post-surgery. Our study indicates that miR-124/VAMP3 is involved in surgery-induced microglial activation and that targeting miR-124/VAMP3 could be a potential therapeutic strategy for postoperative disorders involving microglial activation.

Longitudinal time course of muscle impairments during partial weight-bearing in rats.

In the near future, space agencies plan to send the first crews for extended stays on the Moon and Mars, where gravity is significantly reduced compared to Earth (0.16×g and 0.38×g, respectively). However, the long-term effects of partial gravity have not yet been elucidated, and ensuring astronauts' health and performance is crucial to the success of these missions. Using a quadrupedal partial weight-bearing (PWB) model in rats that we designed, we investigated the longitudinal time course of muscle function at three different PWB levels. We demonstrated that both muscle mass and muscle function are significantly impaired in reduced weight-bearing environments as early as after 7 days of suspension. Moreover, we showed that muscular alterations are correlated to the PWB level and do not reach a plateau during a 1-month exposure to reduced weight-bearing, emphasizing the need for mitigating countermeasures for safe and successful extraterrestrial exploration.

Successful application of prime and pull strategy for a therapeutic HSV vaccine.

One promising approach for a herpes simplex virus vaccine uses a vaccine to prime and a chemoattractant to pull immune cells into the genital tract. We evaluated subunit vaccines (prime) and imiquimod (pull) in the guinea pig (gp) model of recurrent Herpes simplex virus type-2 (HSV-2). Following vaginal HSV-2 infection, gps were vaccinated with various combination of glycoproteins and adjuvant with or without subcutaneous or local applications of imiquimod after infection. Animals were examined daily for recurrent lesions and vaginal swabs collected for recurrent shedding. Although both the vaccines alone and imiquimod alone reduced recurrent HSV disease, the combination of local imiquimod and vaccine (Prime and Pull) was the most effective. In the first study, immunization with the trivalent vaccine alone or imiquimod alone decreased recurrent disease. However, the largest decrease was with the combination of vaccine and local imiquimod (P < 0.001 vs. placebo or vaccine alone). No effect on recurrent shedding was observed. In the second study, recurrent disease scores were similar in the PBS control group and the trivalent-immunized group treated with subcutaneous imiquimod however, significant reductions with glycoprotein vaccines and local imiquimod (p < 0.01 vs. placebo) were noted. The number of qPCR-positive recurrent swabs, ranged from 5 to 11% in the vaccinated+local imiquimod groups compared 29% in the PBS control group (P < 0.05). No recurrent swab samples from vaccinated groups were culture positive. We conclude that the strategy of prime (subunit HSV vaccine) and topical pull (intravaginal/topical imiquimod) decreased recurrent HSV more effectively than vaccine alone.

Enterococcus faecalis YM0831 suppresses sucrose-induced hyperglycemia in a silkworm model and in humans.

Hyperglycemia caused by excessive intake of sucrose leads to lifestyle-related diseases such as diabetes. Administration of a lactic acid bacterial strain to mice suppresses sucrose-induced hyperglycemia, but evidence for a similar effect in humans is lacking. Here we show that Enterococcus faecalis YM0831, identified using an in vivo screening system with silkworms, suppressed sucrose-induced hyperglycemia in humans. E. faecalis YM0831 also suppressed glucose-induced hyperglycemia in silkworms. E. faecalis YM0831 inhibited glucose uptake by the human intestinal epithelial cell line Caco-2. A transposon insertion mutant of E. faecalis YM0831, which showed decreased inhibitory activity against glucose uptake by Caco-2 cells, also exhibited decreased inhibitory activity against both sucrose-induced and glucose-induced hyperglycemia in silkworms. In human clinical trials, oral ingestion of E. faecalis YM0831 suppressed the increase in blood glucose in a sucrose tolerance test. These findings suggest that E. faecalis YM0831 inhibits intestinal glucose transport and suppresses sucrose-induced hyperglycemia in humans.

Murine obscurin and Obsl1 have functionally redundant roles in sarcolemmal integrity, sarcoplasmic reticulum organization, and muscle metabolism.

Biological roles of obscurin and its close homolog Obsl1 (obscurin-like 1) have been enigmatic. While obscurin is highly expressed in striated muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations have been linked to myopathies, whereas mutations in Obsl1 result in 3M-growth syndrome. To further study unique and redundant functions of these closely related proteins, we generated and characterized Obsl1 knockouts. Global Obsl1 knockouts are embryonically lethal. In contrast, skeletal muscle-specific Obsl1 knockouts show a benign phenotype similar to obscurin knockouts. Only deletion of both proteins and removal of their functional redundancy revealed their roles for sarcolemmal stability and sarcoplasmic reticulum organization. To gain unbiased insights into changes to the muscle proteome, we analyzed tibialis anterior and soleus muscles by mass spectrometry, uncovering additional changes to the muscle metabolism. Our analyses suggest that all obscurin protein family members play functions for muscle membrane systems.

Preliminary design of a new degradable medical device to prevent the formation and recurrence of intrauterine adhesions.

Intrauterine adhesions lead to partial or complete obliteration of the uterine cavity and have life-changing consequences for women. The leading cause of adhesions is believed to be loss of stroma resulting from trauma to the endometrium after surgery. Adhesions are formed when lost stroma is replaced by fibrous tissue that join the uterine walls. Few effective intrauterine anti-adhesion barriers for gynecological surgery exist. We designed a degradable anti-adhesion medical device prototype to prevent adhesion formation and recurrence and restore uterine morphology. We focused on ideal degradation time for complete uterine re-epithelialization for optimal anti-adhesion effect and clinical usability. We developed a triblock copolymer prototype [poly(lactide) combined with high molecular mass poly(ethylene oxide)]. Comparative pre-clinical studies demonstrated in vivo anti-adhesion efficacy. Ease of introduction and optimal deployment in a human uterus confirmed clinical usability. This article provides preliminary data to develop an intrauterine medical device and conduct a clinical trial.

Arid1b haploinsufficient mice reveal neuropsychiatric phenotypes and reversible causes of growth impairment.

Sequencing studies have implicated haploinsufficiency of ARID1B, a SWI/SNF chromatin-remodeling subunit, in short stature (Yu et al., 2015), autism spectrum disorder (O'Roak et al., 2012), intellectual disability (Deciphering Developmental Disorders Study, 2015), and corpus callosum agenesis (Halgren et al., 2012). In addition, ARID1B is the most common cause of Coffin-Siris syndrome, a developmental delay syndrome characterized by some of the above abnormalities (Santen et al., 2012; Tsurusaki et al., 2012; Wieczorek et al., 2013). We generated Arid1b heterozygous mice, which showed social behavior impairment, altered vocalization, anxiety-like behavior, neuroanatomical abnormalities, and growth impairment. In the brain, Arid1b haploinsufficiency resulted in changes in the expression of SWI/SNF-regulated genes implicated in neuropsychiatric disorders. A focus on reversible mechanisms identified Insulin-like growth factor (IGF1) deficiency with inadequate compensation by Growth hormone-releasing hormone (GHRH) and Growth hormone (GH), underappreciated findings in ARID1B patients. Therapeutically, GH supplementation was able to correct growth retardation and muscle weakness. This model functionally validates the involvement of ARID1B in human disorders, and allows mechanistic dissection of neurodevelopmental diseases linked to chromatin-remodeling.