John J. Callanan

Targeted suppression of claudin-5 decreases cerebral oedema and improves cognitive outcome following traumatic brain injury

Traumatic brain injury is the leading cause of death in children and young adults globally. Malignant cerebral oedema has a major role in the pathophysiology that evolves after severe traumatic brain injury. Added to this is the significant morbidity and mortality from cerebral oedema associated with acute stroke, hypoxic ischemic coma, neurological cancers and brain infection. Therapeutic strategies to prevent cerebral oedema are limited and, if brain swelling persists, the risks of permanent brain damage or mortality are greatly exacerbated. Here we show that a temporary and size-selective modulation of the blood-brain barrier allows enhanced movement of water from the brain to the blood and significantly impacts on brain swelling. We also show cognitive improvement in mice with focal cerebral oedema following administration in these animals of short interfering RNA directed against claudin-5. These observations may have profound consequences for early intervention in cases of traumatic brain injury, or indeed any neurological condition where cerebral oedema is the hallmark pathology.

The postpartum endometrial inflammatory response: a normal physiological event with potential implications for bovine fertility

After calving, the bovine endometrium undergoes marked morphological and functional changes that are necessary for subsequent re-breeding. Regulation and integration of these key events are largely uncharacterised. Here, endometrial swabs and biopsies were taken at 15, 30 and 60 days postpartum (DPP) from 13 healthy primiparous cows, 10 of which subsequently conceived, with a view to characterising innate and inflammatory gene expression profiles. Endometrial biopsies exhibited severe inflammation (>75 leukocytes per high-power field) at 15 DPP, which had begun to resolve by 30 DPP and had completely resolved by 60 DPP. The severe inflammation at 15 DPP coincided with uterine infection in all cows and a significant increase (P < 0.05) in the expression of all of 16 genes investigated, including CD45, IL8, IL6, IL1, TNF, TAP, SAA3 and HP at 15 DPP, relative to 60 DPP. All of these parameters had begun to return to normal physiological levels at 30 DPP. Systemically, serum protein concentrations of IL-8 were elevated at 15 DPP compared with 60 DPP (78 pgmL(-1)vs 48 pgmL(-1); P = 0.02). These results indicate that endometrial inflammation, leukocyte infiltration and increased expression of pro-inflammatory, antimicrobial and acute-phase protein genes are expected features of the postpartum period, critical to bacterial clearance and uterine involution.

Endometrial epithelial cells are potent producers of tracheal antimicrobial peptide and serum amyloid A3 gene expression in response to E. coli stimulation

Endometrial epithelial cells play a critical role in mediating inflammatory mechanisms key to bacterial clearance and tissue re-modelling postpartum. This study characterised innate immune gene expression by bovine endometrial epithelial cells from three animals in response to Escherichia coli, a common cause of bovine uterine disease. Expression of key innate immune genes, encoding Toll-like receptor 4 (TLR4), the transcription factor NFkB1, the chemokine interleukin 8 (IL8), inflammatory cytokines (interleukins IL1β, IL6; tumour necrosis factor, TNF), β-defensins (lingual antimicrobial peptides LAP, tracheal antimicrobial peptide TAP) and acute phase proteins (haptoglobin, HP; serum amyloid A, SAA3) was examined in endometrial epithelial cells stimulated with E. coli for 6 and 24h using qRT-PCR. Expression of all genes was increased significantly (P<0.05) 6h post-stimulation. Expression of IL1b, TNF and SAA3 genes was increased by 121-, 357- and 721-fold, respectively (P<0.05). Twenty four hours post-stimulation, IL1b, IL6, IL8, TNF and LAP gene expression was decreased compared to 6h, whereas TAP and SAA3 expression was further increased to 209- and 3452-fold (P<0.05). E. coli driven expression of immune effector genes demonstrates potent immune, antimicrobial and regulatory capacity of endometrial epithelial cells to respond to this pathogen.

Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3- and viral-induced expression of type I interferons

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression.

Autoregulated paracellular clearance of amyloid-β across the blood-brain barrier

Size-selective and passive paracellular diffusion of amyloid-β across tight junctions of the blood-brain barrier in Alzheimer’s disease. The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and protecting neural tissue from damaging blood-borne agents. The barrier is characterized by endothelial tight junctions that limit passive paracellular diffusion of polar solutes and macromolecules from blood to brain. Decreased brain clearance of the neurotoxic amyloid-β (Aβ) peptide is a central event in the pathogenesis of Alzheimer’s disease (AD). Whereas transport of Aβ across the BBB can occur via transcellular endothelial receptors, the paracellular movement of Aβ has not been described. We show that soluble human Aβ(1–40) monomers can diffuse across the paracellular pathway of the BBB in tandem with a decrease in the tight junction proteins claudin-5 and occludin in the cerebral vascular endothelium. In a murine model of AD (Tg2576), plasma Aβ(1–40) levels were significantly increased, brain Aβ(1–40) levels were decreased, and cognitive function was enhanced when both claudin-5 and occludin were suppressed. Furthermore, Aβ can cause a transient down-regulation of claudin-5 and occludin, allowing for its own paracellular clearance across the BBB. Our results show, for the first time, the involvement of the paracellular pathway in autoregulated Aβ movement across the BBB and identify both claudin-5 and occludin as potential therapeutic targets for AD. These findings also indicate that controlled modulation of tight junction components at the BBB can enhance the clearance of Aβ from the brain.

Endogenous Oils Derived From Human Adipocytes Are Potent Adjuvants That Promote IL-1α–Dependent Inflammation

Obesity is characterized by chronic inflammation associated with neutrophil and M1 macrophage infiltration into white adipose tissue. However, the mechanisms underlying this process remain largely unknown. Based on the ability of oil-based adjuvants to induce immune responses, we hypothesized that endogenous oils derived from necrotic adipocytes may function as an immunological “danger signal.” Here we show that endogenous oils of human origin are potent adjuvants, enhancing antibody responses to a level comparable to Freund’s incomplete adjuvant. The endogenous oils were capable of promoting interleukin (IL)-1α–dependent recruitment of neutrophils and M1-like macrophages, while simultaneously diminishing M2-like macrophages. We found that endogenous oils from subcutaneous and omental adipocytes, and from healthy and unhealthy obese individuals, promoted comparable inflammatory responses. Furthermore, we also confirmed that white adipocytes in visceral fat of metabolically unhealthy obese (MUO) individuals are significantly larger than those in metabolically healthy obese individuals. Since adipocyte size is positively correlated with adipocyte death, we propose that endogenous oils have a higher propensity to be released from hypertrophied visceral fat in MUO individuals and that this is the key factor in driving inflammation. In summary, this study shows that adipocytes contain a potent oil adjuvant which drives IL-1α–dependent proinflammatory responses in vivo.

Mechanism of cell death mediated by a BF2-chelated tetraaryl-azadipyrromethene photodynamic therapeutic: Dissection of the apoptotic pathway in vitro and in vivo

Photodynamic therapy (PDT) is an established treatment modality for cancer. ADPM06 is an emerging non‐porphyrin PDT agent which has been specifically designed for therapeutic application. Recently, we have demonstrated that ADPM06‐PDT is well tolerated in vivo and elicits impressive complete response rates in various models of cancer when a short drug‐light interval is applied. Herein, the mechanism of action of ADPM06‐PDT in vitro and in vivo is outlined. Using a drug and light combination that reduces the clonogenicity of MDA‐MB‐231 cells by >90%, we detected a well‐orchestrated apoptotic response accompanied by the activation of various caspases in vitro. The generation of reactive oxygen species (ROS) upon photosensitizer irradiation was found to be the key instigator in the observed apoptotic response, with the endoplasmic reticulum (ER) found to be the intracellular site of initial PDT damage, as determined by induction of a rapid ER stress response post‐PDT. PDT‐induced apoptosis was also found to be independent of p53 tumor suppressor status. A robust therapeutic response in vivo was demonstrated, with a substantial reduction in tumor proliferation observed, as well as a rapid induction of apoptosis and initiation of ER stress, mirroring numerous aspects of the mechanism of action of ADPM06 in vitro. Finally, using a combination of 18F‐labeled 3′‐deoxy‐3′‐fluorothymidine (18F‐FLT) nuclear and optical imaging, a considerable decrease in tumor proliferation over 24‐hr in two models of human cancer was observed. Taken together, this data clearly establishes ADPM06 as an exciting novel PDT agent with significant potential for further translational development.

The neuropathogenesis of feline immunodeficiency virus infection: Barriers to overcome

Feline immunodeficiency virus (FIV), like human immunodeficiency virus (HIV)-1, is a neurotropic lentivirus, and both natural and experimental infections are associated with neuropathology. FIV enters the brain early following experimental infection, most likely via the blood-brain and blood-cerebrospinal fluid barriers. The exact mechanism of entry, and the factors that influence this entry, are not fully understood. As FIV is a recognised model of HIV-1 infection, understanding such mechanisms is important, particularly as HIV enters the brain early in infection. Furthermore, the development of strategies to combat this central nervous system (CNS) infection requires an understanding of the interactions between the virus and the CNS. In this review the results of both in vitro and in vivo FIV studies are assessed in an attempt to elucidate the mechanisms of viral entry into the brain.

Pellino3 targets RIP1 and regulates the pro-apoptotic effects of TNF-α

Tumour necrosis factor-α (TNF) can activate NF-κB to induce pro-inflammatory genes but can also stimulate the caspase cascade to promote apoptosis. Here we show that deficiency of the ubiquitin E3 ligase, Pellino3, sensitizes cells to TNF-induced apoptosis without inhibiting the NF-κB pathway. Suppressed expression of Pellino3 leads to enhanced formation of the death-induced signalling complex, complex II, in response to TNF. We show that Pellino3 targets RIP1, in a TNF-dependent manner, to inhibit TNF-induced complex II formation and caspase 8-mediated cleavage of RIP1 in response to TNF/cycloheximide co-stimulation. Pellino3-deficient mice also show increased sensitivity to TNF-induced apoptosis and greatly increased lethality in response to TNF administration. These findings define Pellino3 as a novel regulator of TNF signalling and an important determining factor in dictating whether TNF induces cell survival or death.

Comparative proteomic analysis of lung tissue from guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) reveals a decrease in abundance of host proteins involved in cytoskeletal and cellular organization

UNLABELLED Leptospiral pulmonary haemorrhage syndrome (LPHS) is a particularly severe form of leptospirosis. LPHS is increasingly recognized in both humans and animals and is characterized by rapidly progressive intra-alveolar haemorrhage leading to high mortality. The pathogenic mechanisms of LPHS are poorly understood which hampers the application of effective treatment regimes. In this study a 2-D guinea pig proteome lung map was created and used to investigate the pathogenic mechanisms of LPHS. Comparison of lung proteomes from infected and non-infected guinea pigs via differential in-gel electrophoresis revealed highly significant differences in abundance of proteins contained in 130 spots. Acute phase proteins were the largest functional group amongst proteins with increased abundance in LPHS lung tissue, and likely reflect a local and/or systemic host response to infection. The observed decrease in abundance of proteins involved in cytoskeletal and cellular organization in LPHS lung tissue further suggests that infection with pathogenic Leptospira induces changes in the abundance of host proteins involved in cellular architecture and adhesion contributing to the dramatically increased alveolar septal wall permeability seen in LPHS. BIOLOGICAL SIGNIFICANCE The recent completion of the complete genome sequence of the guinea pig (Cavia porcellus) provides innovative opportunities to apply proteomic technologies to an important animal model of disease. In this study, the comparative proteomic analysis of lung tissue from experimentally infected guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) revealed a decrease in abundance of proteins involved in cellular architecture and adhesion, suggesting that loss or down-regulation of cytoskeletal and adhesion molecules plays an important role in the pathogenesis of LPHS. A publically available guinea pig lung proteome map was constructed to facilitate future pulmonary proteomics in this species.