Broad-spectrum antibiotics alter the microbiome, increase intestinal fxr, and decrease hepatic steatosis in zebrafish short bowel syndrome.

Short bowel syndrome (SBS) is associated with changes in the intestinal microbiome and marked local and systemic inflammation. There is also a late complication of SBS, intestinal failure associated liver disease (IFALD) in which hepatic steatosis progresses to cirrhosis. Most patients with SBS arrive at massive intestinal resection after a contaminating intraabdominal catastrophe and have a history of exposure to broad-spectrum antibiotics. We therefore investigated whether the administration of broad-spectrum antibiotics in conjunction with SBS in zebrafish (ZF) would replicate these systemic effects observed in humans to identify potentially druggable targets to aid in the management of SBS and resulting IFALD. In zebrafish with SBS, broad-spectrum antibiotics altered the microbiome, decreased inflammation, and reduced the development of hepatic steatosis. After two weeks of broad-spectrum antibiotics, these fish exhibited decreased alpha diversity, with less variation in microbial community composition between SBS and sham fish. Additionally, administration of broad-spectrum antibiotics was associated with decreased expression of intestinal toll-like receptor 4 (tlr4), increased expression of the intestinal gene encoding the Farnesoid X receptor (fxr), decreased expression of downstream hepatic cyp7a1, and decreased development of hepatic steatosis. SBS in zebrafish reproducibly results in increased epithelial surface area as occurs in human patients who demonstrate intestinal adaptation, but antibiotic administration in zebrafish with SBS reduced these gains with increased cell death in the intervillus pocket that contains stem/progenitor cells. These alternate states in SBS zebrafish might direct the development of future human therapies.NEW & NOTEWORTHY In a zebrafish model that replicates a common clinical scenario, systemic effects of the administration of broad-spectrum antibiotics in a zebrafish model of SBS identified two alternate states that led to the establishment of fat accumulation in the liver or its absence. Broad-spectrum antibiotics given to zebrafish with SBS over 2 wk altered the intestinal microbiome, decreased intestinal and hepatic inflammation, and decreased hepatic steatosis.

Grading TESI: Crypt and villus formation in tissue-engineered small intestine alters with stem/progenitor cell source.

The small intestine has a remarkable ability to enhance its absorptive and digestive surface area through the formation of villi, a process known as villification. We sought to learn whether developing mouse and human tissue-engineered small intestine (TESI) followed known developmental biology routes to villification, such as Sonic hedgehog (SHH)/Indian hedgehog (IHH) and bone morphogenetic protein 4 (BMP4)/forkhead box F1 (FOXF1) signaling to identify targets to enhance the development of TESI. After generating TESI from prenatal and postnatal stem cell sources, we evaluated the effect of cell source derivation on villification with a grading scheme to approximate developmental stage. χ2 analysis compared the prevalence of TESI grade from each stem cell source. RNAscope probes detected genes known to direct villification and the development of the crypt-villus axis in mouse and human development. These were compared in TESI derived from various pluripotent and progenitor cell donor cell types as well as native human fetal and postnatal tissues. Prenatal and pluripotent cell sources form mature villus and crypt-like structures more frequently than postnatal donor sources, and there are alternate routes to villus formation. Human TESI recapitulates epithelial to mesenchymal crosstalk of several genes identified in development, with fetal and pluripotent donor-derived TESI arriving at villus formation following described developmental patterns. However, postnatal TESI is much less likely to form complete villus-crypt patterns and demonstrates alternate SHH/IHH and BMP4/FOXF1 signaling patterns. Grading TESI and other cellular constructs may assist discoveries to support future human therapies.NEW & NOTEWORTHY The small intestine can enhance its absorptive and digestive surface area through a process known as villification. Tissue-engineered small intestine achieves mature villification at varying levels of success between differing sources. We have developed a consistent grading schema of morphology and characterized it across multiple developmental pathways, allowing objective comparison between differing constructs and sources.

Pelvic pain in patients with complex mullerian anomalies including Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH), obstructed hemi-vagina ipsilateral renal anomaly (OHVIRA), and complex cloaca.

Caring for patients with congenital pelvic anomalies can be challenging in many ways but one crucial aspect is providing longitudinal into adulthood. Newborns with urinary, intestinal or vaginal obstruction require urgent operations to relieve obstruction followed by multiple reconstructive procedures involving the perineum. Openings are created in the pelvic floor musculature that did not exist in development. Adolescence presents further challenges for these postoperative patients while other diagnoses present for the first time in the peri-pubertal teenage years. Young adults can have new symptoms when they become sexually active and are faced with reproductive decisions. During all of these time periods, optimization of function is of paramount importance and patients who are suffering are not able to participate in school, sports or work. This study evaluates the prevalence of pelvic pain in newborns and adolescents with complex congenital pelvic anomalies, associated factors and possible treatment options.

Wnt signaling inhibition by monensin results in a period of Hippo pathway activation during intestinal adaptation in zebrafish.

Intestinal adaptation (IA) is a critical response to increase epithelial surface area after intestinal loss. Short bowel syndrome (SBS) may follow massive intestinal resection in human patients, particularly without adequate IA. We previously validated a model in zebrafish (ZF) that recapitulates key SBS pathophysiological features. Previous RNA sequencing in this model identified upregulation of genes in the Wnt and Hippo pathways. We therefore sought to identify the timeline of increasing cell proliferation and considered the signaling that might underpin the epithelial remodeling of IA in SBS. SBS was created in a ZF model as previously reported and compared with sham fish with and without exposure to monensin, an ionophore known to inhibit canonical Wnt signaling. Rescue of the monensin effects was attempted with a glycogen synthase kinase 3 inhibitor that activates wnt signaling, CHIR-99021. A timeline was constructed to identify peak cellular proliferation, and the Wnt and Hippo pathways were evaluated. Peak stem cell proliferation and morphological changes of adaptation were identified at 7 days. Wnt inhibition diminished IA at 2 wk and resulted in activation of genes of the Wnt/ß-catenin and Yes-associated protein (YAP)/Hippo pathway. Increased cytoplasmic YAP was observed in monensin-treated SBS fish. Genes of the WASP-interacting protein (WIP) pathway were elevated during Wnt blockade. In conclusion, cellular proliferation and morphological changes accompany SBS even in attempted Wnt blockade. Wnt/ß-catenin, YAP/Hippo pathway, and WIP pathway genes increase during early Wnt blockade. Further understanding of the effects of Wnt and YAP pathway signaling in proliferating stem cells might enrich our knowledge of targets to assist IA. NEW & NOTEWORTHY Intestinal adaptation is a critical response to increase epithelial surface area after large intestinal losses. Inhibition of Wnt/ß-catenin signaling impairs intestinal adaptation in a zebrafish model of short bowel syndrome. There is a subsequent upregulation in genes of the Yes-associated protein/Hippo and WIP pathway. These may be targets for future human therapies, as patients are salvaged by the compensation of increased intestinal epithelial surface area through successful intestinal adaptation.

Short bowel syndrome results in increased gene expression associated with proliferation, inflammation, bile acid synthesis and immune system activation: RNA sequencing a zebrafish SBS model.

BACKGROUND: Much of the morbidity associated with short bowel syndrome (SBS) is attributed to effects of decreased enteral nutrition and administration of total parenteral nutrition (TPN). We hypothesized that acute SBS alone has significant effects on gene expression beyond epithelial proliferation, and tested this in a zebrafish SBS model. METHODS: In a model of SBS in zebrafish (laparotomy, proximal stoma, distal ligation, n = 29) or sham (laparotomy alone, n = 28) surgery, RNA-Seq was performed after 2 weeks. The proximal intestine was harvested and RNA isolated. The three samples from each group with the highest amount of RNA were spiked with external RNA controls consortium (ERCC) controls, sequenced and aligned to reference genome with gene ontology (GO) enrichment analysis performed. Gene expression of ctnnb1, ccnb1, ccnd1, cyp7a1a, dkk3, ifng1-2, igf2a, il1b, lef1, nos2b, saa1, stat3, tnfa and wnt5a were confirmed to be elevated in SBS by RT-qPCR. RESULTS: RNA-seq analysis identified 1346 significantly upregulated genes and 678 significantly downregulated genes in SBS zebrafish intestine compared to sham with Ingenuity analysis. The upregulated genes were involved in cell proliferation, acute phase response signaling, innate and adaptive immunity, bile acid regulation, production of nitric oxide and reactive oxygen species, cellular barrier and coagulation. The downregulated genes were involved in folate synthesis, gluconeogenesis, glycogenolysis, fatty-acid oxidation and activation and drug and steroid metabolism. RT-qPCR confirmed gene expression differences from RNA-Sequencing. CONCLUSION: Changes of gene expression after 2 weeks of SBS indicate complex and extensive alterations of multiple pathways, some previously implicated as effects of TPN. The systemic sequelae of SBS alone are significant and indicate multiple targets for investigating future therapies.

Intestinal adaptation in proximal and distal segments: Two epithelial responses diverge after intestinal separation.

BACKGROUND: In short bowel syndrome, luminal factors influence adaptation in which the truncated intestine increases villus lengths and crypt depths to increase nutrient absorption. No study has evaluated the effect of adaptation within the distal intestine after intestinal separation. We evaluated multiple conditions, including Igf1r inhibition, in proximal and distal segments after intestinal resection to evaluate the epithelial effects of the absence of mechanoluminal stimulation. METHODS: Short bowel syndrome was created in adult male zebrafish by performing a proximal stoma with ligation of the distal intestine. These zebrafish with short bowel syndrome were compared to sham-operated zebrafish. Groups were treated with the Igf1r inhibitor NVP-AEW541, DMSO, a vehicle control, or water for 2 weeks. Proximal and distal intestine were analyzed by hematoxylin and eosin for villus epithelial circumference, inner epithelial perimeter, and circumference. We evaluated BrdU+ cells, including costaining for ß-catenin, and the microbiome was evaluated for changes. Reverse transcription quantitative polymerase chain reaction was performed for ß-catenin, CyclinD1, Sox9a, Sox9b, and c-Myc. RESULTS: Proximal intestine demonstrated significantly increased adaptation compared to sham-operated proximal intestine, whereas the distal intestine showed no adaptation in the absence of luminal flow. Addition of the Igf1r inhibitor resulted in decreased adaption in the distal intestine but an increase in distal proliferative cells and proximal ß-catenin expression. While some proximal proliferative cells in short bowel syndrome colocalized ß-catenin and BrdU, the distal proliferative cells did not co-stain for ß-catenin. Sox9a increased in the distal limb after division but not after inhibition with the Igf1r inhibitor. There was no difference in alpha diversity or species richness of the microbiome between all groups. CONCLUSION: Luminal flow in conjunction with short bowel syndrome significantly increases intestinal adaption within the proximal intestine in which proliferative cells contain ß-catenin. Addition of an Igf1r inhibitor decreases adaptation in both proximal and distal limbs while increasing distal proliferative cells that do not colocalize ß-catenin. Igf1r inhibition abrogates the increase in distal Sox9a expression that otherwise occurs in short bowel syndrome. Mechanoluminal flow is an important stimulus for intestinal adaptation.

Management of pediatric intramuscular venous malformations.

BACKGROUND: Intramuscular venous malformations (VMs) are rare, but can be highly symptomatic. There are few reports on outcomes, particularly pain, functional limitations, and muscle contractures. We aimed to compare results of medical management, sclerotherapy, and surgical resection. METHODS: We retrospectively reviewed 45 patients with an extremity or truncal intramuscular VM between June 2005 and June 2015 at a single institution. Outcomes were compared between treatment modalities with ANOVA and χ2 tests. RESULTS: Six patients (13%) were treated with medical management, 4 (9%) with surgical resection, 23 (51%) with sclerotherapy, and 12 (27%) with both surgery and sclerotherapy. Sclerotherapy alone decreased pain in 72%. Only 20% of patients presented with muscle contracture. For these patients, 33% resolved with sclerotherapy, physical therapy, and aspirin; 22% resolved with surgery, and 45% had persistent contracture. 40% of patients treated with sclerotherapy then surgery developed new muscle contractures, compared to 4% of sclerotherapy only patients and 0% of surgery only patients (p=0.04). CONCLUSIONS: Medical management, surgery and sclerotherapy are effective treatments for intramuscular VMs. Observation and supportive care can be a primary treatment for patients with minimal symptomatology and no functional limitations. Sclerotherapy is more effective for treating pain than contractures and when used alone, rarely causes a new muscle contracture.

Inhibition of Fgf signaling in short bowel syndrome increases weight loss and epithelial proliferation.

BACKGROUND: Signaling by fibroblast growth factor is critical for epithelial proliferation, differentiation, and the development of many organs, including the intestine. Fibroblast growth factor 10 and fibroblast growth factor 2c are upregulated after massive bowel resection during intestinal adaptation. This pathway is conserved highly. We hypothesized that inhibition of fibroblast growth factor signaling would impair intestinal adaptation in the zebrafish model of short bowel syndrome and allow insight into the negative regulation of this pathway. METHODS: Short bowel syndrome equivalent to a high jejunostomy was generated in adult male hsp70:dnfgfr1-GFP zebrafish, wildtype fish exposed to tyrosine-kinase inhibitor, and wildtype fish in absence of tyrosine-kinase inhibitor. Heat shock in hsp70:dnfgfr1-GFP fish decreases fgf 1 expression. Parameters including weight, proliferation, and differentiation were evaluated after harvest in experimental and control groups. RESULTS: Although short bowel syndrome zebrafish lost more weight relative to sham zebrafish in both groups, heat shock fish with short bowel syndrome lost more weight compared with non-heat shock fish with short bowel syndrome. In the non-heat shock controls, the villus epithelial perimeter increased in short bowel syndrome compared with sham fish, but this did not occur in heat shock fish. Non-heat shock fish with short bowel syndrome fish had significantly increased Bromodeoxyuridine(+) proliferative cells per hemivillus compared with non-heat shock-sham, while heat shock-short bowel syndrome had a more substantial increase in Bromodeoxyuridine(+) cells compared with HS-sham. Non-heat shock-short bowel syndrome demonstrated a significantly increased percentage of Alcian blue(+) goblet cells per hemivillus compared with non-heat shock-sham, while the heat shock-short bowel syndrome demonstrated decreased Alcian blue(+) cells compared with non-heat shock-short bowel syndrome. In contrast, SU5402 inhibited epithelial proliferation while increasing weight loss. CONCLUSION: Inhibition of fibroblast growth factor-1 signaling in short bowel syndrome decreases epithelial adaptation, increases Bromodeoxyuridine-labeled cells at 2 weeks, and exacerbates weight loss while decreasing epithelial goblet cells.

Human and Murine Tissue-Engineered Colon Exhibit Diverse Neuronal Subtypes and Can Be Populated by Enteric Nervous System Progenitor Cells When Donor Colon Is Aganglionic.

PURPOSE: Tissue-engineered colon (TEC) might potentially replace absent or injured large intestine, but the enteric nervous system (ENS), a key component, has not been investigated. In various enteric neuropathic diseases in which the TEC is derived from aganglionic donor colon, the resulting construct might also be aganglionic, limiting tissue engineering applications in conditions such as Hirschsprung disease (HD). We hypothesized that TEC might contain a diverse population of enteric neuronal subtypes, and that aganglionic TEC can be populated by neurons and glia when supplemented with ENS progenitor cells in the form of neurospheres. MATERIALS AND METHODS: Human and murine organoid units (OU) and multicellular clusters containing epithelium and mesenchyme were isolated from both mouse and human donor tissues, including from normally innervated and aganglionic colon. The OU were seeded onto a biodegradable scaffold and implanted within a host mouse, resulting in the growth of TEC. Aganglionic murine and human OU were supplemented with cultured neurospheres to populate the absent ENS not provided by the OU to rescue the HD phenotype. RESULTS: TEC demonstrated abundant smooth muscle and clusters of neurons and glia beneath the epithelium and deeper within the mesenchyme. Motor and afferent neuronal subtypes were identified in TEC. Aganglionic OU formed TEC with absent neural elements, but neurons and glia were abundant when aganglionic OU were supplemented with ENS progenitor cells. CONCLUSION: Murine and human TEC contain key components of the ENS that were not previously identified, including glia, neurons, and fundamental neuronal subtypes. TEC derived from aganglionic colon can be populated with neurons and glia when supplemented with neurospheres. Combining tissue engineering and cellular replacement therapies represents a new strategy for treating enteric neuropathies, particularly HD.

Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine.

Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

A dynamic postoperative protocol provides efficient care for pediatric patients with non-ruptured appendicitis.

PURPOSE: Postoperative management of pediatric patients with non-ruptured appendicitis is highly variable and often includes an overnight stay in the hospital. We implemented a criteria-based postoperative protocol designed to eliminate postoperative antibiotics and facilitate timely discharge by utilizing the bedside nurse to evaluate for readiness for discharge. METHODS: We collected data on all patients with non-ruptured appendicitis at our institution following protocol implementation (May 1, 2012 to April 30, 2013) and compared them to a control group. RESULTS: 580 patients were treated for non-ruptured appendicitis (285 prior protocol, 295 new protocol). Following implementation of our protocol, there was an overall reduction in length of stay from 40.1 (SD 27.5) to 23.5 (SD 20.8)h, and total cost of care per patient also decreased from $5783 (SD $2501) to $4499 (SD $1983) (p<0.001). There was no change in hospital readmission rate (1.1% prior protocol, 1.4% new protocol) or postoperative abscess rate (0.8% prior protocol, 0.3% new protocol). CONCLUSION: Our new protocol reveals the value of eliminating postoperative antibiotics and leveraging the continuous availability of the bedside nurse in the determination of readiness for discharge.

Response-based therapy for ruptured appendicitis reduces resource utilization.

PURPOSE: We examined the effectiveness of a postoperative ruptured appendicitis protocol that eliminated Pseudomonas coverage and based the duration of IV antibiotic treatment and length of hospital stay on the patient's clinical response. METHODS: In our new protocol, IV antibiotics were administered until the patient met discharge criteria: adequate oral intake, pain control with oral medications, and afebrile for 24h. We collected data on all patients with ruptured appendicitis at our institution following protocol implementation (May 1, 2012, to April 30, 2013) and compared them to a control group. RESULTS: 306 patients were treated (154 prior protocol, 152 new protocol). The new clinical response-based protocol led to a decrease in hospital stay from 134h (SD 66.1) to 94.5h (SD 61.7) (p<0.001) and total cost of care per patient also decreased from $13,610 (SD $6859) to $9870 (SD $5670) (p<0.001). CONCLUSION: Our clinical response-based protocol for pediatric patients with ruptured appendicitis decreased LOS, cost, and IV antibiotics use without significant changes in adverse events.

Complete VACTERL evaluation is needed in newborns with rectoperineal fistula.

PURPOSE: Given that a rectoperineal fistula is developmentally the most mature lesion in the spectrum of anorectal malformations, it is not clear whether it merits a complete VACTERL evaluation. We sought to determine if the same evaluation is required to rule out associated anomalies in newborns with rectoperineal fistula as those with more complex anorectal malformations. METHODS: We performed a retrospective review of the pediatric colorectal center database at our tertiary care children's hospital from 2000 to 2012. Patients with anorectal malformations were categorized as rectoperineal fistula or "other" using the Krickenbeck classification. Records were reviewed to identify associated anomalies. RESULTS: 308 patients (156 males) were treated at our institution during the time period (rectoperineal fistula=102). Thirty-five (34%) patients with a perineal fistula had at least one associated anomaly. The most common anomalies were cardiac lesions (29% excluding PFO and PDA), genitourinary (20.6%), and malformations of the spine (15.7%). The overall occurrence of anomalies was lower than the "other" group. CONCLUSION: Our review demonstrates that newborns with a rectoperineal fistula frequently have associated anomalies and should undergo an evaluation similar to more complex lesions. These findings illustrate the importance of a structured approach to the evaluation of even the most straightforward lesions.

Peroxisome proliferator-activated receptor-gamma activation suppresses HIV-1 replication in an animal model of encephalitis.

OBJECTIVE: Poor penetration of antiretroviral therapy across the blood-brain barrier poses an impediment on control of HIV-1 infection in brain macrophages. Peroxisome proliferator-activated receptor (PPAR)-gamma, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. As PPARgamma agonists are rapidly absorbed by oral administration and efficiently permeate the blood-brain barrier, we hypothesized that PPARgamma stimulation may suppress HIV-1 replication. DESIGN AND METHODS: We investigated the effect of PPARgamma ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages and in vivo using a murine model (immunodeficient mice reconstituted with human lymphocytes and intracerebrally inoculated with HIV-1 infected macrophages) of HIV-1 encephalitis. RESULTS: Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARgamma stimulation inhibited virus replication by modulating NF-kappaB activation in a receptor-dependent manner, leading to downregulation of HIV-1 long terminal repeat (LTR) promoter activity and suppression of HIV-1 replication. These effects were PPARgamma specific as PPARgamma silencing or addition of PPARgamma antagonist abolished effects of PPARgamma stimulation on HIV-1 LTR and virus replication. Using a murine model for HIV-1 encephalitis, we demonstrated that PPARgamma ligand suppressed HIV-1 replication in macrophages in brain tissue and reduced viremia by 50%. CONCLUSION: In vitro data delineated the novel mechanism by which PPARgamma activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARgamma agonists to reduce HIV-1 replication in lymphocytes and brain macrophages, thus offering a new therapeutic intervention in brain and systemic infection.

Activation of protein tyrosine kinases and matrix metalloproteinases causes blood-brain barrier injury: Novel mechanism for neurodegeneration associated with alcohol abuse.

Blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Activation of matrix metalloproteinases (MMPs) and alteration of basement membrane (BM) associated with BBB injury was documented in stroke patients. While chronic alcoholism is a risk factor for developing stroke, underlying mechanisms are not well understood. We hypothesized that ethanol (EtOH)-induced protein tyrosine kinase (PTK) signaling resulted a loss of BBB integrity via MMPs activation and degradation of BM component, collagen IV. Treatment of BMVEC with EtOH or acetaldehyde (AA) for 2-48 h increased MMP-1, -2 and -9 activities or decreased the levels of tissue inhibitors of MMPs (TIMP-1, -2) in a PTK-dependent manner without affecting protein tyrosine phosphatase activity. Enhanced PTK activity after EtOH exposure correlated with increased phosphorylated proteins of selective receptor and nonreceptor PTKs. Up-regulation of MMPs activities and protein contents paralleled a decrease in collagen IV content, and inhibitors of EtOH metabolism, MMP-2 and -9, or PTK reversed all these effects. Using human BMVEC assembled into BBB models, we found that EtOH/AA diminished barrier tightness, augmented permeability, and monocyte migration across the BBB via activation of PTKs and MMPs. These findings suggest that alcohol associated BBB injury could be mediated by MMPs via BM protein degradation and could serve as a comorbidity factor for neurological disorders like stroke or neuroinflammation. Furthermore, our preliminary experiments indicated that human astrocytes secreted high levels of MMP-1 and -9 following exposure to EtOH, suggesting the role of BM protein degradation and BBB compromise as a result of glial activation by ethanol. These results provide better understanding of multifaceted effects of alcohol on the brain and could help develop new therapeutic interventions.

Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood-brain barrier dysfunction.

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Oxidative stress is a major underlying cause of neurodegenerative and neuroinflammatory disorders and BBB injury associated with them. Using human BMVEC grown on porous membranes covered with basement membrane (BM) matrix (BBB models), we demonstrated that reactive oxygen species (ROS) augmented permeability and monocyte migration across BBB. ROS activated matrix metalloproteinases (MMP-1, -2, and -9) and decreased tissue inhibitors of MMPs (TIMP-1 and -2) in a protein tyrosine kinase (PTK)-dependent manner. Increase in MMPs and PTK activities paralleled degradation of BM protein and enhanced tyrosine phosphorylation of tight junction (TJ) protein. These effects and enhanced permeability/monocyte migration were prevented by inhibitors of MMPs, PTKs, or antioxidant suggesting that oxidative stress caused BBB injury via degradation of BM protein by activated MMPs and by PTK-mediated TJ protein phosphorylation. These findings point to new therapeutic interventions ameliorating BBB dysfunction in neurological disorders such as stroke or neuroinflammation.

HIV-1 gp120 compromises blood-brain barrier integrity and enhances monocyte migration across blood-brain barrier: implication for viral neuropathogenesis.

Human immunodeficiency virus-1 (HIV-1) encephalitis is characterized by brain infiltration of virus-infected monocytes and macrophages. Cellular products and viral proteins secreted by infected cells likely play an important role in blood-brain barrier (BBB) impairment and the development of HIV-1-associated dementia (HAD). We previously demonstrated that HIV-1 envelope glycoprotein gp120 induces toxicity and alters expression of tight junction proteins in human brain microvascular endothelial cells (HBMECs). Here, we delineate the mechanisms of gp120-induced BBB dysfunction. Human brain microvascular endothelial cells expressed HIV-1 co-receptors (CCR5 and CXCR4). Exposure of HBMECs to gp120 derived from macrophage (CCR5) or lymphocyte (CXCR4)-tropic viruses decreased BBB tightness, increased permeability, and enhanced monocyte migration across in vitro BBB models. Blood-brain barrier integrity was restored after gp120 removal. CCR5 antibodies and inhibitors of myosin light chain kinase or protein kinase C (PKC) blocked gp120-enhanced monocyte migration and permeability of BBB in vitro. Exposure of HBMECs to gp120 induced release of intracellular calcium ([Ca(2+)](i)) that was prevented by CCR5 antibody and partially blocked by CXCR4 antagonist. Human immunodeficiency virus-1 gp120 activated three PKC isoforms in HBMECs [PKC-alpha/betaII, PKC(pan)-betaII and PKC-zeta/lambda]. Furthermore, specific PKC inhibitors (acting at the ATP-binding and calcium release site) blocked gp120-induced PKC activation and prevented increase in BBB permeability, supporting the biologic significance of these results. Thus, gp120 can cause dysfunction of BBB via PKC pathways and receptor mediated [Ca(2+)](i) release leading to cytoskeletal alterations and increased monocyte migration.