An antibody that targets cell-surface glucose-regulated protein-78 inhibits expression of inflammatory cytokines and plasminogen activator inhibitors by macrophages.

Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α2 -macroglobulin (α2 M) and tissue-type plasminogen activator (tPA). In macrophages, α2 M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α2 M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.

Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability.

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

Enzymatically Inactive Tissue-Type Plasminogen Activator Reverses Disease Progression in the Dextran Sulfate Sodium Mouse Model of Inflammatory Bowel Disease.

Enzymatically inactive tissue-type plasminogen activator (EI-tPA) does not activate fibrinolysis, but interacts with the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1) in macrophages to block innate immune system responses mediated by toll-like receptors. Herein, we examined the ability of EI-tPA to treat colitis in mice, induced by dextran sulfate sodium. In two separate studies, designed to generate colitis of differing severity, a single dose of EI-tPA administered after inflammation established significantly improved disease parameters. EI-tPA-treated mice demonstrated improved weight gain. Stools improved in character and became hemoccult negative. Abdominal tenderness decreased. Colon shortening significantly decreased in EI-tPA-treated mice, suggesting attenuation of irreversible tissue damage and remodeling. Furthermore, histopathologic evidence of disease decreased in the distal 25% of the colon in EI-tPA-treated mice. EI-tPA did not decrease the number of CD45-positive leukocytes or F4/80-positive macrophage-like cells detected in extracts of colons from dextran sulfate sodium-treated mice as assessed by flow cytometry. However, multiple colon cell types expressed the NMDA-R, suggesting the ability of diverse cells, including CD3-positive cells, CD103-positive cells, Ly6G-positive cells, and epithelial cell adhesion molecule-positive epithelial cells to respond to EI-tPA. Mesenchymal cells that line intestinal crypts and provide barrier function expressed LRP1, thereby representing another potential target for EI-tPA. These results demonstrate that the NMDA-R/LRP1 receptor system may be a target for drug development in diseases characterized by tissue damage and chronic inflammation.

Clinicopathological Profile and Outcome of Childhood Urticaria Vasculitis: An Observational Study.

Background Urticaria is a type III hypersensitivity reaction usually triggered by an infection, medication, or food item. It usually subsides within 24 hours without any residual lesion and does not have any systemic manifestation. Urticaria vasculitis (UV) is a clinicopathological condition defined by the presence of an urticarial lesion lasting for >24 hours or recurrent episodes of urticaria associated with histopathological features of leukocytoclastic vasculitis. Methods This retrospective study was conducted in a tertiary care teaching institute in Eastern India over a period of 2 and ½ years. Children presenting with urticaria lesions for a duration of > 24 hours that did not subside either spontaneously or with anti-histamines were admitted for further workup and management. Results During the study period (July 2015 to December 2017), a total of 20 children with urticaria needed admission for symptom control and further workup. There were 16 boys and 4 girls. The mean (SD) age of presentation was 6.5 years (±2.4). Besides urticaria in all, pain abdomen was present in 13 (65%) and fever in 6 (30%) children. Only one had arthritis. Skin biopsy performed in these children was suggestive of leukocytoclastic vasculitis. One child was ANA (anti-nuclear antibody) positive with low C3. All except three children required systemic steroid for symptom control along with other medications (anti-histamines). None had suffered any complication or relapse.  Conclusions Urticaria vasculitis (most common cause being idiopathic) remains underdiagnosed because of the need of confirmation by biopsy, which might not always be attempted in every case. Though anti-histamines remain the main stay of treatment, adding short course oral steroid shortens the course once infection is ruled out.

NTHL1 in genomic integrity, aging and cancer.

Efficient DNA repair is essential to maintain genomic integrity. An average of 30,000 base lesions per cell are removed daily by the DNA glycosylases of the base excision repair machinery. With the advent of whole genome sequencing, many germline mutations in these DNA glycosylases have been identified and associated with various diseases, including cancer. In this graphical review, we discuss the function of the NTHL1 DNA glycosylase and how genomic mutations and altered function of this protein contributes to cancer and aging. We highlight its role in a rare tumor syndrome, NTHL1-associated polyposis (NAP), and summarize various other polymorphisms in NTHL1 that can induce early hallmarks of cancer, including genomic instability and cellular transformation.

Tissue-type plasminogen activator selectively inhibits multiple toll-like receptors in CSF-1-differentiated macrophages.

Tissue-type plasminogen activator (tPA) is a major activator of fibrinolysis, which also attenuates the pro-inflammatory activity of lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The activity of tPA as an LPS response modifier is independent of its proteinase activity and instead, dependent on the N-methyl-D-aspartate Receptor (NMDA-R), which is expressed by BMDMs. The major Toll-like receptor (TLR) for LPS is TLR4. Herein, we show that enzymatically-inactive (EI) tPA blocks the response of mouse BMDMs to selective TLR2 and TLR9 agonists, rapidly reversing IκBα phosphorylation and inhibiting expression of TNFα, CCL2, interleukin-1ß, and interleukin-6. The activity of EI-tPA was replicated by activated α2-macroglobulin, which like EI-tPA, signals through an NMDA-R-dependent pathway. EI-tPA failed to inhibit cytokine expression by BMDMs in response to agonists that target the Pattern Recognition Receptors (PRRs), NOD1 and NOD2, providing evidence for specificity in the function of EI-tPA. Macrophages isolated from the peritoneal space (PMs), without adding eliciting agents, expressed decreased levels of cell-surface NMDA-R compared with BMDMs. These cells were unresponsive to EI-tPA in the presence of LPS. However, when PMs were treated with CSF-1, the abundance of cell-surface NMDA-R increased and the ability of EI-tPA to neutralize the response to LPS was established. We conclude that the anti-inflammatory activity of EI-tPA is selective for TLRs but not all PRRs. The ability of macrophages to respond to EI-tPA depends on the availability of cell surface NMDA-R, which may be macrophage differentiation-state dependent.

Adolescence benzo[a]pyrene treatment induces learning and memory impairment and anxiolytic like behavioral response altering neuronal morphology of hippocampus in adult male Wistar rats.

Exposure to benzo[a]pyrene (B[a]P), a prototype of polycyclic aromatic hydrocarbons (PAHs) easily cross blood brain barrier (BBB) and is associated with impaired learning and memory in adult rats. However, there is no symmetric study reported on association between B[a]P exposure during the early development and hippocampal dendritic architecture causing behavioral changes like learning and memory deficit of adult rats. We investigated a fourteen day consecutive B[a]P administration, intraperitonial (i.p.), with two different doses (0.1 and 1µM) during early adolescence at PND30-44 and learning behavior assessed between PND 45-60 in adult male rats. The anxiolytic like behavioural analysis was done by LDPT. Depressive like behaviour was estimated through sucrose preference and learning and memory by T-maze. After B[a]P administration oxidative stress markers like glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), reduced (GSH) and oxidized glutathione (GSSG) were evaluated. To parallel these behavioral and antioxidant level changes to alteration in dendritic morphology, Golgi-Cox staining was performed in the hippocampus. Our study showed anxiolytic like behavioral response with significant increase in time spent in light zone and significant (p < 0.05) decrease in preference for sucrose and a reduction in percentage of spontaneous responses in T-maze test in B[a]P administered group as compared to vehicle control. B[a]P exposed male rats showed significant increase in GST activity (p < 0.05) and concentration of GSSG with a decay in GSH, GPx and GR in both the groups as compared to control. B[a]P administered rats showed significant loss in total dendritic length and number (28%) with reduced spine density (18%) in both higher and lower doses. These results suggested that B[a]P administration can be associated with an increase ROS production showing altered antioxidant defence system through glutathione biosynthesis and causing profound alterations in dendritic length and spine density of hippocampal neurons leading towards learning and memory deficits in adult rats.

Novel Regulation of Integrin Trafficking by Rab11-FIP5 in Aggressive Prostate Cancer.

The laminin-binding integrins, α3ß1 and α6ß1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. ß1 integrins share the Rab11 recycling machinery, but the trafficking of α3ß1 and α6ß1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6ß1 trafficking. Rab11-FIP5 and its membrane-binding domain were required for α6ß1 recycling, without affecting the other laminin-binding integrin (i.e., α3ß1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6ß1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6ß1 recycling in cell-cell locations. Taken together, these data demonstrate that α6ß1 is distinct from α3ß1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location.Implications: Rab11-FIP5-dependent α6ß1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Mol Cancer Res; 16(8); 1319-31. ©2018 AACR.

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual ) of 3.25 min-1 , threefold faster than α3 integrin (1.0 min-1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min-1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min-1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6ß4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6ß1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc.

Hypertriglyceridemia Thalassemia Syndrome: Common Disease, Uncommon Association.

Hypertriglyceridemia has been rarely described with thalassemia, an entity called hypertriglyceridemia-thalassemia syndrome. The authors describe a young infant diagnosed with thalassemia major with severe hypertriglyceridemia. The presence of severe hypertriglyceridemia in this child which rapidly resolved after transfusion, probably suggests a self limited mechanism which may not require therapy. Though hypertriglyceridemia has been reported with hemolytic anemias, the mechanism is unclear. This case illustrates that thalassemia may be associated with hypertriglyceridemia; once familial and secondary causes are ruled out, clinicians may wait for spontaneous resolution before considering specific therapy.

Neonatal exposure to benzo[a]pyrene induces oxidative stress causing altered hippocampal cytomorphometry and behavior during early adolescence period of male Wistar rats.

Environmental neurotoxicants like benzo[a]pyrene (B[a]P) have been well documented regarding their potential to induce oxidative stress. However, neonatal exposure to B[a]P and its subsequent effect on anti-oxidant defence system and hippocampal cytomorphometry leading to behavioral changes have not been fully elucidated. We investigated the effect of acute exposure of B[a]P on five days old male Wistar pups administered with single dose of B[a]P (0.2 µg/kg BW) through intracisternal mode. Control group was administered with vehicle i.e., DMSO and a separate group of rats without any treatment was taken as naive group. Behavioral analysis showed anxiolytic-like behavior with significant increase in time spent in open arm in elevated plus maze. Further, significant reduction in fall off time during rotarod test showing B[a]P induced locomotor hyperactivity and impaired motor co-ordination in adolescent rats. B[a]P induced behavioral changes were further associated with altered anti-oxidant defence system involving significant reduction in the total ATPase, Na(+) K(+) ATPase, Mg(2+) ATPase, GR and GPx activity with a significant elevation in the activity of catalase and GST as compared to naive and control groups. Cytomorphometry of hippocampus showed that the number of neurons and glia in B[a]P treated group were significantly reduced as compared to naive and control. Subsequent observation showed that the area and perimeter of hippocampus, hippocampal neurons and neuronal nucleus were significantly reduced in B[a]P treated group as compared to naive and control. The findings of the present study suggest that the alteration in hippocampal cytomorphometry and neuronal population associated with impaired antioxidant signaling and mood in B[a]P treated group could be an outcome of neuromorphological alteration leading to pyknotic cell death or impaired differential migration of neurons during early postnatal brain development.

Schwann Cells Increase Prostate and Pancreatic Tumor Cell Invasion Using Laminin Binding A6 Integrin.

Human pancreatic and prostate cancers metastasize along nerve axons during perineural invasion. The extracellular matrix laminin class of proteins is an abundant component of both myelinated and non-myelinated nerves. Analysis of human pancreatic and prostate tissue revealed both perineural and endoneural invasion with Schwann cells surrounded or disrupted by tumor, respectively. Tumor and nerve cell co-culture conditions were used to determine if myelinating or non-myelinating Schwann cell (S16 and S16Y, respectively) phenotype was equally likely to promote integrin-dependent cancer cell invasion and migration on laminin. Conditioned medium from S16 cells increased tumor cell (DU145, PC3, and CFPAC1) invasion into laminin approximately 1.3-2.0 fold compared to fetal bovine serum (FBS) treated cells. Integrin function (e.g., ITGA6p formation) increased up to 1.5 fold in prostate (DU145, PC3, RWPE-1) and pancreatic (CFPAC1) cells, and invasion was dependent on ITGA6p formation and ITGB1 as determined by function-blocking antibodies. In contrast, conditioned medium isolated from S16Y cells (non-myelinating phenotype) decreased constitutive levels of ITGA6p in the tumor cells by 50% compared to untreated cells and decreased ITGA6p formation 3.0 fold compared to S16 treated cells. Flow cytometry and western blot analysis revealed loss of ITGA6p formation as reversible and independent of overall loss of ITGA6 expression. These results suggest that the myelinating phenotype of Schwann cells within the tumor microenvironment increased integrin-dependent tumor invasion on laminin.

Unique C2V3 sequence in HIV-1 envelope obtained from broadly neutralizing plasma of a slow progressing patient conferred enhanced virus neutralization.

Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an env clone amplified from cross neutralizing plasma of an antiretroviral naïve chronically infected Indian patient (ID50 >600-fold higher compared to other autologous env clones). The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env) was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.