Epithelial Integrity Is Maintained by a Matriptase-Dependent Proteolytic Pathway

A pericellular proteolytic pathway initiated by the transmembrane serine protease matriptase plays a critical role in the terminal differentiation of epidermal tissues. Matriptase is constitutively expressed in multiple other epithelia, suggesting a putative role of this membrane serine protease in general epithelial homeostasis. Here we generated mice with conditional deletion of the St14 gene, encoding matriptase, and show that matriptase indeed is essential for the maintenance of multiple types of epithelia in the mouse. Thus, embryonic or postnatal ablation of St14 in epithelial tissues of diverse origin and function caused severe organ dysfunction, which was often associated with increased permeability, loss of tight junction function, mislocation of tight junction-associated proteins, and generalized epithelial demise. The study reveals that the homeostasis of multiple simple and stratified epithelia is matriptase-dependent, and provides an important animal model for the exploration of this membrane serine protease in a range of physiological and pathological processes.The trypsin-like serine proteases are a particularly large group of proteolytic enzymes (approximately 200 in mammals) that reside in the pericellular space or on the cell surface and regulate multiple cellular processes in the context of embryonic development, tissue homeostasis, tissue repair, immunity, fertility, and more.^1,2 Specific catalytic functions include activation of hormones, growth factors, and cytokines, activation of signaling receptors, regulation of ion fluxes, and regulation of paracellular permeability.^{3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18}The surface of metazoan organs is lined by specialized epithelia that are endowed with multiple general and organ-specific functions. These include the maintenance of ion gradients, macromolecular transport, absorption, secretion of lipids, mucins, hormones and growth factors, and exclusion of pathogens. In its most simple form, an epithelium is a single layer of keratin-expressing cells attached to a basement membrane. Cells maintain close contact with each other through four types of specialized cell junctions termed desmosomes, gap junctions, adherens junctions, and tight junctions. These cell junctions are composed of an organized assembly of numerous integral and peripheral membrane proteins that are stabilized by anchorage to the cytoskeleton. The polarity of an epithelial sheath and prevention of paracellular diffusion is conferred by the tight junctions, which form a continuous belt encircling the apical end of the lateral membrane.¹⁹ However, how epithelial tight junctions are established and maintained is not fully understood. In particular, the specific contribution of extracellular serine proteases and their cognate inhibitors to epithelial tight junction formation, maintenance, and regulation of barrier function is unclear.^{3,4,5,6,20,21}Matriptase is a trypsin-like transmembrane serine protease encoded by the ST14 gene that belongs to the recently emerged type II transmembrane serine protease family. Orthologs of matriptase have been found in all vertebrate species analyzed to date, and homologs of the type II transmembrane serine proteases are present in bees and fruit flies.²² Mouse reverse genetics and clinical examination of humans homozygous for mutations in the ST14 gene have linked matriptase to terminal epidermal differentiation processes, including lipid extrusion, profilaggrin processing, and cornified envelope formation.^23,24,25 However, matriptase is also constitutively expressed in simple, stratified, transitional, and pseudo-stratified epithelia throughout the body that do not undergo cornification, secrete specialized lipids, or express profilaggrin. Moreover, this pattern of matriptase expression in epithelial cells is highly conserved between mice and humans.^26,27 These observations all suggest a wider role of the matriptase proteolytic cascade in epithelial physiology. Indeed, when we generated mice with a conditional St14 allele and performed tissue-specific, embryonic-onset or postnatal, acute St14 gene ablation, we found that loss of matriptase from mouse epithelia caused severe organ dysfunction, which was often associated with increased paracellular permeability, loss of tight junction function, mislocation of tight junction-associated proteins, and generalized epithelial demise. The study reveals a global role of matriptase in the maintenance of epithelial homeostasis and provides a useful animal model for the further exploration of matriptase in multiple physiological and pathophysiological processes.     

HIF1A Overexpression Is Associated with Poor Prognosis in a Cohort of 731 Colorectal Cancers

Tissue hypoxia commonly occurs in tumors. Hypoxia- inducible factor (HIF)-1 and HIF-2, which are essential mediators of cellular response to hypoxia, regulate gene expression for tumor angiogenesis, glucose metabolism, and resistance to oxidative stress. Their key regulatory subunits, HIF1A (HIF-1α) and endothelial PAS domain protein 1 (EPAS1; HIF-2α), are overexpressed and associated with patient prognosis in a variety of cancers. However, prognostic or molecular features of colon cancer with HIF expression remain uncertain. Among 731 colorectal cancers in two prospective cohort studies, 142 (19%) tumors showed HIF1A overexpression, and 322 (46%) showed EPAS1 overexpression by immunohistochemistry. HIF1A overexpression was significantly associated with higher colorectal cancer-specific mortality in Kaplan-Meier analysis (log-rank test, P < 0.0001), univariate Cox regression (hazard ratio = 1.84; 95% confidence interval, 1.37 to 2.47; P < 0.0001) and multivariate analysis (adjusted hazard ratio = 1.72; 95% confidence interval, 1.26 to 2.36; P = 0.0007) that adjusted for clinical and tumoral features, including microsatellite instability, TP53 (p53), PTGS2 (cyclooxygenase-2), CpG island methylator phenotype, and KRAS, BRAF, PIK3CA, and LINE-1 methylation. In contrast, EPAS1 expression was not significantly associated with patient survival. In addition, HIF1A expression was independently associated with PTGS2 expression (P = 0.0035), CpG island methylator phenotype-high (P = 0.013), and LINE-1 hypomethylation (P = 0.017). EPAS1 expression was inversely associated with high tumor grade (P = 0.0017) and obesity (body mass index ≥ 30 kg/m²) (P = 0.039). In conclusion, HIF1A expression is independently associated with poor prognosis in colorectal cancer, suggesting HIF1A as a biomarker with potentially important therapeutic implications.Tissue hypoxia commonly occurs in tumor, and adaptation to tissue hypoxia appears to be one of important characteristics of malignant cells.^1,2 Hypoxia-inducible factor (HIF)-1 and HIF-2 play a key role in cellular adaptation to hypoxia and regulate the expression of genes responsible for glucose metabolism, angiogenesis, and cell survival.^1,2,3 Thus, HIF and related pathways are potential therapeutic targets.^4,5 Cellular HIF levels are regulated not only by the oxygen-dependent pathway (eg, VHL and prolyl hydroxylase, EGLN) but also by the oxygen-independent pathway (eg, glycogen synthase kinase 3, the phosphatidylinositol 3-kinase pathway, the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway).^6,7 HIF and hypoxia signaling influence a wide variety of pathways including those related to vascular endothelial growth factor (VEGF), cyclins, and MTOR.^1,2 Thus, cellular HIF levels may modify responsiveness to drugs targeting those pathways or hypoxia signaling, and it is of particular interest to examine HIF expression in human cancers. Key regulatory subunits of HIF, HIF1A (the official symbol for HIF-1α), and endothelial PAS domain protein 1 (EPAS1; the official symbol for HIF-2α) are differentially overexpressed^8,9 and have distinct functions in human cancers.^9,10,11 HIF1A expression leads to increased tumor growth and metastasis in some studies,^12,13,14,15 whereas HIF1A inhibits tumor growth by cell cycle arrest or apoptosis induction in other studies.^16,17,18,19 Similar paradoxical effects of EPAS1 have also been reported; EPAS1 appears to promote cancer development and progression in neuroblastoma and renal carcinoma,^20,21,22 whereas it appears to inhibit tumor growth in other cancers including colon cancer.^23,24,25Previous data on HIF1A, EPAS1, and clinical outcome in colorectal cancer have been inconclusive. A study of 90 rectal cancer patients showed poor prognosis associated with HIF1A but not with EPAS1.²⁶ In contrast, another study of 87 colorectal cancer patients reported poor prognosis associated with EPAS1 but not with HIF1A.²⁷ Among studies assessing only HIF1A, some reported its independent prognostic effect^28,29 whereas others did not.^30,31 However, all previous studies^{26,27,28,29,30,31} were limited by small sample sizes (N <136). Considering the increasing importance of the HIF pathway as a potential target for cancer treatment,^1,2,6 the assessment of HIF1A and EPAS1 expression and clinical outcome using a large number of colorectal cancers is needed.We therefore examined prognostic effects of HIF1A and EPAS1 expression among 731 colorectal cancer patients identified in two prospective cohort studies. Moreover, because we concurrently assessed other important molecular events including mutations in KRAS, BRAF, and PIK3CA, LINE-1 hypomethylation, microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP), we could evaluate the effect of HIF1A or EPAS1 expression after controlling for these potential confounders.     

Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation

The attenuated S. typhimurium SL3261 (aroA) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-A^d/I-E^d antigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80⁺ and CD11b⁺ spleen cells.Salmonella infections still pose a serious health hazard worldwide, affecting both humans and animals. Salmonella typhi, the agent of human typhoid fever, is not pathogenic for common laboratory animals. Therefore, natural resistance and acquired immunity to Salmonella are studied mainly in the mouse model by using host-adapted salmonellae which cause systemic infections believed to mimic the human disease.In mice, early bacterial growth in the reticuloendothelial system (RES) is controlled by the innate resistance Nramp (Ity) gene, which is expressed in macrophages (22). In lethal infections, salmonellae rapidly reach large numbers in the tissues and death occurs presumably by endotoxin poisoning when bacterial counts reach levels of ca. 10⁸ CFU per organ (30). In sublethal infections, survival requires a host response that suppresses the exponential growth of the organisms in the RES towards the end of the first week, resulting in a plateau phase (17, 25). The establishment of the plateau phase does not require functional T cells. In fact, nude (T-cell-deficient) mice and mice depleted of T cells by administration of anti-CD4 and anti-CD8 antibodies can still suppress Salmonella growth in infected tissues (17). A bone marrow-dependent influx of radiation-sensitive cells is required for the plateau phase and for the formation of granulomas rich in mononuclear cells (17, 32). Most of the salmonellae in the spleens and livers of the infected animals are localized within the phagocytes present in the focal lesions (38). Tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and nitric oxide (NO) derivatives appear to be required for the suppression of salmonella growth in the RES (27, 28, 32, 36, 37, 48). TNF-α is needed for the recruitment of mononuclear cells in the tissues and for granuloma formation (32); IFN-γ can activate macrophages to kill salmonellae in vitro (20).The establishment of the plateau phase coincides with the development of hypersusceptibility to the toxic and lethal effects of bacterial lipopolysaccharide (LPS) (29, 33). We have previously shown that mice immunized with a live attenuated aromatic-dependent Salmonella vaccine strain show transient hypersusceptibility to LPS, which can be prevented by treatment with anti-TNF-α antibodies (29). The role of other cytokines in this phenomenon is not known.Interleukin-12 (IL-12) is a 70-kDa heterodimeric cytokine produced by macrophages, B cells, polymorphonuclear leukocytes, and dendritic cells in response to a variety of stimuli including products of bacterial origin (5, 10). IL-12 mediates resistance to intracellular organisms including Listeria, Toxoplasma, Candida, Leishmania, Mycobacterium tuberculosis, and Brucella abortus (8, 13, 18, 23, 39, 46, 50). IL-12 is generally believed to mediate host resistance by inducing IFN-γ production by NK and T cells as well as by contributing to the establishment of protective Th1 antigen-specific responses (5, 6, 9, 10, 12, 13, 24, 34, 39, 43, 47).Evidence for IL-12 induction in salmonellosis has been provided. IL-12 and IL-12-specific mRNA have been detected in vivo and in vitro in response to Salmonella. Elicited peritoneal mouse macrophages stimulated with Salmonella dublin express elevated levels of IL-12 p40-specific mRNA (4, 7). Oral infection with virulent or live attenuated S. dublin induces early (6 and 20 h postinfection) production of IL-12-specific mRNA in Peyer’s patches and mesenteric lymph nodes (3); biologically active IL-12 in lymph node homogenates has been documented 36 h after S. dublin infection (21). We and others previously reported that in vivo IL-12 neutralization reduces the ability of the host to suppress the growth of virulent salmonellae in the tissues and impairs IFN-γ production (21, 31). A recent report indicates that a mutation in the IL-12 receptors renders humans more susceptible to salmonellosis (11). Nevertheless, the mechanisms by which IL-12 mediates host resistance to Salmonella are still unclear.In the present study, we attempted to clarify the mechanisms by which IL-12 contributes to host resistance in mice infected with Salmonella. We investigated the role of IL-12 in survival, granuloma formation, and macrophage activation in mice infected with an attenuated Salmonella strain that normally causes very mild infections in BALB/c mice. We also investigated the involvement of IL-12 in the toxic and lethal effects of high bacterial loads in the tissues as well as in the expression of hypersusceptibility to LPS normally seen in mice infected with salmonellae. We also wished to clarify the involvement of IFN-γ in IL-12-mediated resistance to salmonellosis.     

Neuroprotective Effect of Oligodendrocyte Precursor Cell Transplantation in a Long-Term Model of Periventricular Leukomalacia

Perinatal white matter injury, or periventricular leukomalacia (PVL), is the most common cause of brain injury in premature infants and is the leading cause of cerebral palsy. Despite increasing numbers of surviving extreme premature infants and associated long-term neurological morbidity, our understanding and treatment of PVL remains incomplete. Inflammation- or ischemia/hypoxia-based rodent models, although immensely valuable, are largely restricted to reproducing short-term features of up to 3 weeks after injury. Given the long-term sequelae of PVL, there is a need for subchronic models that will enable testing of putative neuroprotective therapies. Here, we report long term characterization of a neonatal inflammation-induced rat model of PVL. We show bilateral ventriculomegaly, inflammation, reactive astrogliosis, injury to pre-oligodendrocytes, and neuronal loss 8 weeks after injury. We demonstrate neuroprotective effects of oligodendrocyte precursor cell transplantation. Our findings present a subchronic model of PVL and highlight the tissue protective effects of oligodendrocyte precursor cell transplants that demonstrate the potential of cell-based therapy for PVL.Premature, low-birth weight infants are commonly diagnosed with perinatal white matter damage, which leads to long-term neurological deficits including cerebral palsy.^1,2,3,4 Congenital encephalomyelitis was described almost 150 years ago, a disease in newborn children characterized by pale softened zones of degeneration within the deep white matter surrounding lateral ventricles and is now often referred to as periventricular leukomalacia (PVL).⁵ The white matter damage that occurs in preterm infants has more recently been shown to be also accompanied by significant cerebral-cortex and deep-gray matter abnormalities leading to neurodegeneration and altered neurobehavioral performance.⁶PVL is characterized by selective oligodendrocyte precursor cell (OPC) loss resulting in delayed or disrupted myelination, white matter atrophy and ventriculomegaly, neuronal loss, and cyst and scar formation. Extreme premature birth (approximately 23 to 32 weeks) accompanied by inflammation or infection corresponds to the period when immature dividing and differentiating oligodendrocytes predominate in the cerebral white matter.^7,8,9,10 PVL has a complex etiology. The two most important determinants are cerebral hypoperfusion and maternal intrauterine infection. Periventricular white matter is susceptible to hypoperfusion due to the comparative immaturity of the periventricular vasculature of the preterm infant.^11,12 Early oligodendrocyte lineage cells are vulnerable to the consequences of hypoperfusion and subsequent microglial and astrocytic activation on account of amplified glutamate receptor-mediated responses and lack of efficient antioxidant protection.^{13,14,15,16,17,18} Maternal infection, recently implicated as a causative factor in the pathogenesis of PVL,^{19,20,21,22,23} is believed to initiate an inflammatory/cytokine cascade that results in the release of early-response pro-inflammatory cytokines such as tumor necrosis factor–α (TNF-α), interleukin−1 β (IL-1β) and interleukin−6 (IL-6), and causes damage to immature oligodendrocytes.²⁴ TNF-α appears to have a particularly important role in PVL pathogenesis with in vitro evidence suggesting direct damage to OPCs,^25,26 while IL-1β and IL-6 modulate injury indirectly.^27,28Although cells of the oligodendrocyte lineage are regarded as the primary target in the pathogenesis of PVL, there is increasing evidence that neonatal white matter damage is accompanied by gray matter abnormalities, including neuronal loss, impaired axonal guidance, and altered synaptogenesis.^6,29 Premature newborns affected by PVL often have smaller cerebral cortex and deep gray matter volumes, reduced cortical neurons, and alterations in the orientation of central white matter fiber tracts.^6,30,31,32 Together, these data suggest that developing neurons, like immature oligodendrocyte lineage cells, are also vulnerable to injury caused by inflammatory response or hypoxia.Aspects of PVL have been modeled by ischemia/hypoxia and inflammation-mediated rodent models. Animal models of hypoxia-ischemia have clearly shown that following brain injury there is reduced myelination, enlarged ventricles, loss of neurons and damage to axons and dendrites and altered neurobehavioral performances.^{33,34,35,36,37,38,39} Experimentally induced inflammation has been used in a number of studies to model PVL pathology and test potential treatments.^23,40,41,42 Administration of the endotoxin lipopolysaccharide (LPS), a potent inducer of innate immune response and inflammation,^43,44 either intracerebrally during early neonatal period, intrauterine or peritoneally to a pregnant mother results in inflammation and hypomyelination.^20,22,23,38 Moreover, in animal models of LPS-induced PVL neuronal loss and a reduction in neurite length in the parietal cortex has been observed.^42,45 In vitro evidence suggests LPS is not directly toxic to OPCs, but causes injury through the activation of Toll-like receptor 4-positive microglia, which is a source of pro-inflammatory cytokines, nitric oxide, and free radicals.^44,45,46 Furthermore, it has been shown that LPS-induced inflammation increases the susceptibility of white matter to injury in response to otherwise “harmless” subthreshold hypoxic-ischemic insult.⁴⁷ Current models of PVL are typically short term, with studies using either hypoxia-ischemia or inflammation rarely extending analysis beyond 14 days. There is therefore a need to evaluate the longer-term, subchronic consequences of LPS injury and specifically address whether hypomyelination and neuronal injury are self-limiting or indeed spontaneously repair.Cellular therapeutic strategies are predicated on cell replacement and/or tissue protection independent of specific cellular differentiation. Progenitor cells including OPCs have previously been used for cellular replacement of damaged or lost cells in a variety of CNS injury models where damage to myelin and neuronal loss occur.^{48,49,50,51,52,53} Furthermore, there is accumulating evidence that implicates progenitor cell mediated neuroprotection through a variety of mechanisms including graft derived neurotrophic support independent of directed differentiation.^54,55 Moreover, there is evidence to suggest that OPCs secrete factors that are capable of supporting neuronal survival,^{56,57,58,59,60,61} thus suggesting they may be a potential therapeutic source for replacing lost or damaged cells and protecting healthy tissue following neonatal brain injury. In this study we have examined the subchronic effects of LPS induced injury and then examined the putative neuroprotective effects of OPC transplantation.     

Human Immunodeficiency Virus (HIV) Infection of Human Macrophages Is Increased by Dopamine : A Bridge between HIV-Associated Neurologic Disorders and Drug Abuse

The prevalence of human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) that result from HIV infection of the central nervous system is increasing. Macrophages, the primary target for HIV within the central nervous system, play a central role in HIV-induced neuropathogenesis. Drug abuse exacerbates HAND, but the mechanism(s) by which this increased neuropathology results in more severe forms of HAND in HIV-infected drug abusers is unclear. The addictive and reinforcing effects of many drugs of abuse, such as cocaine and methamphetamine, are mediated by increased extracellular dopamine in the brain. We propose a novel mechanism by which drugs of abuse intensify HIV neuropathogenesis through direct effects of the neurotransmitter dopamine on HIV infection of macrophages. We found that macrophages express dopamine receptors 1 and 2, and dopamine activates macrophages by increasing ERK 1 phosphorylation. Our results demonstrate for the first time that dopamine increases HIV replication in human macrophages and that the mechanism by which dopamine mediates this change is by increasing the total number of HIV-infected macrophages. This increase in HIV replication is mediated by activation of dopamine receptor 2. These findings suggest a common mechanism by which drugs of abuse enhance HIV replication in macrophages and indicate that the drug abuse-heightened levels of central nervous system dopamine could increase viral replication, thereby accelerating the development of HAND.Human immunodeficiency virus (HIV) enters the central nervous system (CNS) soon after initial infection,¹ resulting in ongoing inflammation and neurological damage that leads to the development of HIV-associated neurocognitive disorders (HAND) in as many as 50% of infected individuals.^2,3 The prevalence of these complications is increasing despite the advent of antiretroviral therapy, due to the longer lifespan of infected individuals on antiretroviral therapies⁴ and the poor ability of most antiretroviral drugs to penetrate the blood-brain barrier.^5,6 HIV is thought to enter the brain through the transmigration of infected monocytes across the blood-brain barrier.^7,8,9,10 Within the CNS, macrophages are the primary source of HIV and the virus spreads primarily through infection of brain macrophages and microglia.^11,12 Infected macrophages produce numerous factors that are neurotoxic and contribute to the neurological damage that occurs in HIV-infected individuals.^13,14,15 Thus, HIV infection and replication within CNS macrophages plays a central role in the development of HANDs.The incidence and severity of HAND are exacerbated by drugs of abuse, such as the psychostimulants cocaine and methamphetamine,^16,17,18,19 which have been shown to increase both HIV neuropathogenesis and viral replication.^{20,21,22,23,24} However, the mechanism(s) by which drugs of abuse enhance HIV-related neuropathologies are not well understood. Dopamine (DA), a neurotransmitter involved in the control of locomotion, cognition, positive reinforcement, and neuroendocrine secretion,²⁵ is central to the action of drugs of abuse. Psychostimulants such as cocaine and methamphetamine exert addictive and reinforcing effects through elevation of extracellular DA levels in the CNS.^{26,27,28,29,30} The use of both cocaine and methamphetamine generates extracellular CNS dopamine levels far higher than those found in the brains of non-drug-abusers.^{26,31,32,33,34,35,36}Dopamine acts through dopamine receptors, which are members of the G-protein coupled seven transmembrane domain family of receptors. Dopamine receptors (DRs) are divided into two subtypes designated D1-like DRs, comprised of dopamine receptor 1 (D1R) and D5R, and D2-like DRs, comprised of D2R, D3R, and D4R.²⁵ Classically, DRs have been studied on neurons, but DR expression has also been reported in several types of peripheral blood leukocytes, including T lymphocytes and monocytes.^37,38,39 Dopamine receptors have been shown to modulate the immune function of T lymphocytes.^25,40,41 A recent study showed D1R on human macrophages,²⁴ but the expression of other DRs and the functions of DRs in this cell type have not been well characterized.In studies with simian immunodeficiency virus-infected macaques, injection with or oral administration of L-DOPA, a DA precursor that crosses the blood-brain barrier, or selegiline, a blocker of DA breakdown by monoamine oxidase, resulted in increased levels of simian immunodeficiency virus in the CNS.^42,43 In addition, infected macaques exhibited an increased incidence of simian immunodeficiency virus encephalitis and induction of a spongiform polioencephalopathy in dopaminergic regions of the CNS.^42,43 These studies suggest that the enhanced extracellular DA elicited by use of drugs like cocaine and methamphetamine could be sufficient to increase HIV replication in the CNS and exacerbate HIV neuropathogenesis. Macrophages play a central role in the development of HIV-induced neuropathology. Thus, examination of DA modulation of HIV infection of macrophages, as well as the characterization of intracellular signaling pathways that are involved in the DA-mediated increase in HIV infectivity, are important to the identification of mechanisms by which drugs of abuse enhance the development of HAND.This report demonstrates that primary human monocyte-derived macrophages (MDMs) inoculated with HIV in the presence of DA exhibit increased levels of viral replication when compared with MDMs inoculated with HIV in the absence of DA. The DA-induced increase in viral replication correlated with an increase in the percentage of MDMs infected with HIV. HIV infection in the presence of the D2R agonist, quinpirole, increased viral replication similarly to DA, while infection in the presence of the D1R agonist, SKF 82958, did not alter HIV replication, suggesting that D2R is involved in the DA-mediated increase in HIV replication. The data also confirm that uninfected MDMs expressed both D1R and D2R on the cell surface and show that endogenous macrophage D2R was active by demonstrating that DA induced extracellular signal regulated kinase 1 (ERK 1) phosphorylation in macrophages through D2R. These results suggest that dopamine-induced increases in HIV replication in macrophages may be an important mechanism by which specific drugs of abuse, characterized by their ability to increase extracellular DA levels in the CNS, exacerbate the neuropathogenesis of HIV infection.     

Characterization of RAGE,HMGB1, and S100β in Inflammation-Induced Preterm Birth and Fetal Tissue Injury

Immune activation represents an adaptive reaction triggered by both noxious exogenous (microbes) and endogenous [high mobility group box-1 protein (HMGB1), S100 calcium binding proteins] inducers of inflammation. Cell stress or necrosis lead the release of HMGB1 and S100 proteins in the extracellular compartment where they act as damage-associated molecular pattern molecules (or alarmins) by engaging the receptor for advanced glycation end-products (RAGE). Although the biology of RAGE is dictated by the accumulation of damage-associated molecular pattern molecules at sites of tissue injury, the role of RAGE in mediating antenatal fetal injury remains unknown. First, we studied the relationships at birth between the intensity of human fetal inflammation and sRAGE (an endogenous RAGE antagonist), HMGB1, and S100β protein. We found significantly lower sRAGE in human fetuses that mounted robust inflammatory responses. HMGB1 levels correlated significantly with levels of interleukin-6 and S100β in fetal circulation. We then evaluated the levels and areas of tissue expression of RAGE, HMGB1, and S100β in specific organs of mouse fetuses on E16. Using an animal model of endotoxin-induced fetal damage and preterm birth, we determined that inflammation induces a significant change in expression of RAGE and HMGB1, but not S100β, at sites of tissue damage. Our findings indicate that RAGE and HMGB1 may be important mediators of cellular injury in fetuses delivered in the setting of inflammation-induced preterm birth.Conventional wisdom holds that the primary causes of the high neonatal morbidity and mortality attendant preterm birth are complications of immature organ systems.^1,2,3,4 However, a growing body of investigation suggests that the poor outcome observed in many preterm children is not entirely dependent on their gestational age at birth.^2,5,6 After correcting for gestational age, several risk factors remain significantly associated with an increased risk of cerebral palsy, such as intra-amniotic infection, histological chorioamnionitis, prolonged rupture of the membranes, and hypoxemic fetal growth restriction.^7,8,9 Therefore, particularities of the fetal innate immune response to infection appear to cause pathology unique to the premature fetus. This includes a heightened inflammatory and oxidative stress state that acts synergistically with microbial insult to induce cell damage and multisystem organ failure.^7,10,11,12The host’s response to microbial pathogens involves a series of carefully orchestrated mechanisms that include the newly described damage-associated molecular pattern molecules (DAMPs).^13,14 DAMPs, also known as “alarmins,”¹⁵ are a pleiotropic group of intracellular proteins that include among others the high-mobility group box-1 (HMGB1 or amphoterin) and S100β proteins.^13,16 When released into the extracellular compartment in excess as a result of cell activation or injury, DAMPs become “danger signals” that specifically activate the receptor of advanced glycation end-products (RAGE).^14,17 RAGE is a transmembrane receptor,¹⁸ a member of the immunoglobulin superfamily, and functions as a chief receptor for products of nonenzymatic glycoxidation (advanced glycation end-products, AGEs), HMGB1, and S100β proteins.¹⁴ In adult humans and animals, RAGE has been shown to be expressed on the cellular surface of cortical neurons and numerous endothelial, smooth muscle, inflammatory, and vascular cells positioned in vital organs such as the brain, lung, heart, liver, and bowel.^19,20,21,22 Binding of DAMPs to the RAGE extracellular domain results in sustained activation of nuclear factor (NF)-κB and recruitment of inflammatory cells (CD68- and Cd11c-positive mononuclear phagocyte), which in turn amplify the process of tissue damage.¹⁴ That RAGE and HMGB1 play a fundamental role in inflammation and oxidative stress-induced tissue injury is demonstrated by experiments in animal models where administration of quercetin (flavonoid with potent antioxidant properties and HMGB1 inhibitor)²³ or soluble RAGE (sRAGE, an extracellular truncated form of RAGE that acts as a decoy receptor) or antibodies or peptides targeted against RAGE or HMGB1 attenuate the lethal effects of endotoxin, acetaminophen and ischemia-reperfusion.^{24,25,26,27,28,29,30}Recently, we demonstrated that the S100A12-RAGE axis is actively engaged in modulating the intensity of the human intra-amniotic inflammatory response to infection.^31,32 We attributed a key role to the presence and activity of amniotic fluid (AF) sRAGE.³¹ In this study we sought to evaluate the role of RAGE, HMGB1, and S100β proteins as mediators of fetal organ injury in the context of infection and/or inflammation. Specifically, we have begun by assessing whether the intensity of the human maternal and fetal inflammation impacts on the fetal systemic levels of sRAGE (as marker of the RAGE system activation),³³ HMGB1, or S100β levels at birth. Given that sRAGE acts as a decoy for RAGE we anticipated that in the setting of a robust fetal inflammatory response the circulatory levels of sRAGE are low. We thought that this may be related to inhibition or dysfunction of the mechanisms responsible for synthesis of this decoy receptor in the setting of overwhelming cytokemia. Alternatively, low levels of total sRAGE may be related to successful removal/detoxification of AGEs known to be generated in high amounts in the context of increased metabolic and oxidative stress, such as that associated with fetal prematurity and infection.^34,35 To elucidate whether RAGE, HMGB1, and S100β are participants in the mechanisms underlying infection/inflammation induced fetal cell/organ damage, we turned to an animal model of preterm birth induced by maternal administration of endotoxin (lipopolysaccharide, LPS).³⁵ First, we evaluated the level of RAGE, HMGB1, and S100β tissue expression and regional distribution in specific organs of mouse fetuses at developmental stage E16. Furthermore, we aimed to determine whether altered expression of RAGE, HMGB1, or S100β co-exists with tissue inflammation and cellular damage in vital organs such as fetal brain and liver.     

Apoptosis of Hippocampal Pyramidal Neurons Is Virus Independent in a Mouse Model of Acute Neurovirulent Picornavirus Infection

Many viruses, including picornaviruses, have the potential to infect the central nervous system (CNS) and stimulate a neuroinflammatory immune response, especially in infants and young children. Cognitive deficits associated with CNS picornavirus infection result from injury and death of neurons that may occur due to direct viral infection or during the immune responses to virus in the brain. Previous studies have concluded that apoptosis of hippocampal neurons during picornavirus infection is a cell-autonomous event triggered by direct neuronal infection. However, these studies assessed neuron death at time points late in infection and during infections that lead to either death of the host or persistent viral infection. In contrast, many neurovirulent picornavirus infections are acute and transient, with rapid clearance of virus from the host. We provide evidence of hippocampal pathology in mice acutely infected with the Theiler’s murine encephalomyelitis picornavirus. We found that CA1 pyramidal neurons exhibited several hallmarks of apoptotic death, including caspase-3 activation, DNA fragmentation, and chromatin condensation within 72 hours of infection. Critically, we also found that many of the CA1 pyramidal neurons undergoing apoptosis were not infected with virus, indicating that neuronal cell death during acute picornavirus infection of the CNS occurs in a non–cell-autonomous manner. These observations suggest that therapeutic strategies other than antiviral interventions may be useful for neuroprotection during acute CNS picornavirus infection.Many viruses maintain neurovirulent potential, even when the vast majority of infections are silent or subclinical.^1,2,3,4,5 For example, in the correct context, even the common cold virus is neurovirulent.^6,7 Picornaviruses are of particular concern due to ubiquitous distribution, widespread exposure, ease of transmission, and a propensity for neurovirulence.⁸ Non-polio picornaviruses, and especially neurovirulent enteroviruses, are emerging or re-emerging pathogens with the potential for global socio-economic impact.^{1,2,9,10,11,12,13} For example, enterovirus 71(EV71), first isolated from a child with encephalitis in California in 1969, has caused large epidemics with neurological consequences in Eastern Europe and Southeast Asia.¹² Of 130,000 people infected in Taiwan in 1998, 405 required hospitalization for severe central nervous system (CNS) disease.¹⁴ Because EV71 infection results in clinical manifestations predominantly in infants and young children,¹² the potential for long-term cognitive consequences is significant. Indeed, subsequent follow-up of those hospitalized patients that survived acute infection in the 1998 Taiwan outbreak revealed that many suffered long-term neurological deficits,¹⁵ including attention-deficit/hyperactivity disorder.¹⁶ These cognitive deficits were most pronounced in young children.¹⁵The indispensable and largely irreplaceable nature of neurons in the CNS requires that these cells maintain a flawless apoptotic balance. Viral infection of the CNS is known to alter this tightly regulated equilibrium, leading to neuron death.⁸ For example, acute CNS infection by members of the neurotropic picornavirus family, including Theiler’s murine encephalomyelitis virus (TMEV),^17,18 the coxsackieviruses,¹⁹ and encephalomyocarditis virus,^20,21 induces the death of hippocampal neurons in a range of hosts.⁸ Case reports indicate that infection with neurovirulent picornaviruses results in extensive hippocampal lesions and cognitive impairment in humans.^15,22,23 Likewise, in mice acutely infected with TMEV, we have observed damage to the CA1 hippocampal subfield and a consequent inability to form spatial memories.¹⁷ A previous study concluded that hippocampal neuronal apoptosis during TMEV infection was a cell-autonomous event triggered by direct neuron infection, and that cell death was a protective response that limited viral replication before the development of a humoral or cellular immune response.²⁴ However, this previous study only assessed neuron death at one time point (7 days postinfection [dpi]) and within a viral infection system that either killed the host by overwhelming viral encephalitis or led to persistent viral infection of the CNS. In contrast to fatal or chronic viral models, many common neurovirulent picornavirus infections are acute and transient, with clearance of virus from the host CNS and rapid disease resolution.²⁵ Based on this discrepancy, we used the Daniel’s strain of TMEV and an infection paradigm in mice of the H-2^b major histocompatibility complex haplotype that models an acute brain infection that is cleared from the host CNS within 14 to 21 dpi.²⁶ We exploited this model to evaluate CNS pathology in mice infected with a neurovirulent picornavirus and to elucidate the canonical mechanisms underlying neuronal apoptosis in the hippocampus of infected hosts.     

The Immunoconjugate “Icon” Targets Aberrantly Expressed Endothelial Tissue Factor Causing Regression of Endometriosis

Endometriosis is a major cause of chronic pain, infertility, medical and surgical interventions, and health care expenditures. Tissue factor (TF), the primary initiator of coagulation and a modulator of angiogenesis, is not normally expressed by the endothelium; however, prior studies have demonstrated that both blood vessels in solid tumors and choroidal tissue in macular degeneration express endothelial TF. The present study describes the anomalous expression of TF by endothelial cells in endometriotic lesions. The immunoconjugate molecule (Icon), which binds with high affinity and specificity to this aberrant endothelial TF, has been shown to induce a cytolytic immune response that eradicates tumor and choroidal blood vessels. Using an athymic mouse model of endometriosis, we now report that Icon largely destroys endometriotic implants by vascular disruption without apparent toxicity, reduced fertility, or subsequent teratogenic effects. Unlike antiangiogenic treatments that can only target developing angiogenesis, Icon eliminates pre-existing pathological vessels. Thus, Icon could serve as a novel, nontoxic, fertility-preserving, and effective treatment for endometriosis.Endometriosis is a gynecological disorder characterized by the presence of functional endometrial tissue outside of the uterus.¹ The disease affects up to 10% of all reproductive-age women and up to 50% of infertile women.^1,2,3 Endometriotic lesions are primarily located on the pelvic peritoneum and ovaries, but can also be found in the colon, pericardium, pleura, lung parenchyma, and brain.² Implants can cause pelvic adhesions, chronic pelvic pain, bowel obstruction, and infertility requiring repetitive, extensive, and expensive medical and surgical treatments.^1,4,5The etiology of the disease has been ascribed to retrograde menstruation, coelomic metaplasia, or both.^1,6,7,8,9 Although its pathogenesis involves a complex interplay of genetic, anatomical, environmental, and immunological factors, there is general agreement that it is associated with a local inflammatory response and that vascularization at the site of ectopic attachment of the lesions is a key determinant in its pathogenesis.^{1,10,11,12,13} Angiogenic agents such as vascular endothelial growth factor and the angiopoietins are likely mediators of endometriotic neovascularization.^1,3,11,12,13Tissue factor (TF), the transmembrane initiator of hemostasis, is not physiologically expressed by endothelial cells but plays a crucial role in embryonic and oncogenic angiogenesis.^14,15,16,17 Recent studies indicate that the type-2 proteinase activated receptor (PAR-2) is intimately involved in TF-mediated signaling and angiogenesis.^15,18,19 Tissue factor may initiate differential signaling pathways in physiological compared with pathological angiogenesis,²⁰ to wit, the physiological pathway is mediated by TF–thrombin–PAR-1 and the pathological pathway by TF/factor FVIIa/PAR-2 signaling.²⁰ These latter vessels display abnormal structure and function and are poorly associated with pericytes causing leakiness and edema.^21,22Our prior studies demonstrated that both TF and PAR-2 are up-regulated in endometria of women with endometriosis compared with unaffected women.²³In this report, we describe the anomalous endothelial expression of TF in ectopic endometrium derived from women with endometriosis. We posited that a novel chimeric immunoconjugate molecule (Icon) will specifically target endothelial TF in ectopic implants leading to their devascularization and atrophy. Icon is composed of a mutated low-coagulation–inducing factor VII (fVII) domain that binds to TF with high affinity and an IgG1 Fc (fVII/IgG1 Fc) effector domain that activates a natural killer cell cytolytic response against TF bearing endothelial cells.^24,25,26,27 This report confirms our hypothesis by demonstrating that Icon largely destroys pre-established human endometriotic lesions in an athymic mouse model without untoward systemic effects, altered fertility, or subsequent teratogenesis.     

Kindlin-1 Is Required for RhoGTPase-Mediated Lamellipodia Formation in Keratinocytes

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with β1 integrin, α-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.Kindlins are a family of novel regulators of integrin signaling and cell-matrix adhesion,¹ which are causally linked to human genetic disorders. The family members, kindlin-1, kindlin-2, and kindlin-3 (also known as fermitin family homologs 1, -2, and -3), localize to integrin adhesion sites inside the cell and, together with talin, co-activate integrins to mediate outside-in signaling and to control cell behavior.^2,3,4 Kindlins are evolutionarily conserved multidomain proteins¹ that contain a hallmark C-terminal four point one band/ezrin/radixin/moesin (FERM) domain, but exhibit distinct tissue expression patterns: kindlin-1 is an epithelial-specific protein, kindlin-2 is widely expressed, and kindlin-3 is confined to the hematopoietic system.⁵Pivotal information on kindlin functions has been gained from investigation of their defects in human diseases or mouse models.^2,3,6,7,8 In particular, the biological relevance of kindlin-1 was underlined by its association with Kindler syndrome (KS), a genetic skin disorder caused by mutations in the FERMT1 (also known as KIND1) gene. Thus far, no human disease is known to be associated with genetic defects of kindlin-2,⁹ but mutations in the kindlin-3 gene FERMT3 were recently identified in rare leukocyte adhesion deficiency syndromes LAD-III and LAD-1/variant, with defective integrin activation in platelets, neutrophils, and lymphocytes.^{7,10,11,12,13}KS is an intriguing human disorder affecting the skin, oral and urogenital mucosa, and the intestine.^14,15 It has an evolving phenotype that is not well understood: the clinical features change with advancing age and encompass congenital skin blistering, progressive poikiloderma, mucosal fragility, ulcerative colitis, photosensitivity, and propensity to epithelial cancer.^14,16 Epithelial fragility and atrophy are clinical hallmarks,¹⁶ but despite rapid developments in understanding the genetic basis of KS, little is known about the molecular pathology and disease mechanisms underlying the clinical symptoms.Morphologically, KS skin resembles the skin of mice with a keratinocyte-restricted β1 integrin knock out,¹⁷ and the functional abnormalities of KS keratinocytes mirror those of perturbed integrin mediated processes.^6,17,18,19 This is in line with in vitro observations,^6,19 which suggest that kindlin-1 is required for keratinocyte proliferation, attachment to the extracellular matrix and motility. It may act via integrin activation and recruitment of specific molecules into the integrin-associated platforms, focal adhesions (FA), which allow force transmission. These processes require the integrity of the actin cytoskeleton and a fine-tuned regulation of its remodeling in response to different stimuli.²⁰ Depending on the stimulus and the signals it sets into motion, actin remodeling results in different structures. Such processes are governed by Rho GTPases, which induce either actin microfilaments that project filopodia, bundling of actin into fibers for efficient acto-myosin contraction during translocation of the cell body, or circumferential actin assembly and cell spreading.²¹ Numerous actin-binding and other associated proteins act in a coordinated manner to respond to upstream signals and to remodel the actin cytoskeleton. Since such functions are important for cell survival, many molecules act in a redundant manner, as shown by the fact that more than 50 FA proteins have been identified.^22,23,24,25Here, we disclosed three novel FERMT1 mutations and uncovered new physiological functions of the gene product, kindlin-1, by identifying novel ligands and showing that kindlin-1 modulates the cytoskeleton through Rho GTPase governed signaling processes in epithelial cells. These findings lay an essential basis for understanding the molecular pathology of KS and, consequently, for design of biologically valid therapeutic strategies for this incurable human disorder.     

Selective Cell Death of Hyperploid Neurons in Alzheimer’s Disease

Aneuploidy, an abnormal number of copies of a genomic region, might be a significant source for neuronal complexity, intercellular diversity, and evolution. Genomic instability associated with aneuploidy, however, can also lead to developmental abnormalities and decreased cellular fitness. Here we show that neurons with a more-than-diploid content of DNA are increased in preclinical stages of Alzheimer’s disease (AD) and are selectively affected by cell death during progression of the disease. Present findings show that neuronal hyperploidy in AD is associated with a decreased viability. Hyperploidy of neurons thus represents a direct molecular signature of cells prone to death in AD and indicates that a failure of neuronal differentiation is a critical pathogenetic event in AD.Understanding the mechanisms underlying generation of neuronal variability and complexity remains a basic challenge to neuroscience. Structural variation in the human genome is likely to be one important mechanism for neuronal diversity and brain disease.¹ The genetic profile of a cell can be permanently altered by chromosomal aneuploidy (i.e., an abnormal number of copies of a genomic region). A combination of multiple different forms of aneuploid cells due to loss or gain of whole chromosomes (mosaic aneuploidy) giving rise to cellular diversity at the genomic level have been described in neurons of the normal and diseased adult human brain.^{2,3,4,5,6,7,8,9,10,11,12}Cells in normal individuals have basically been assumed to contain identical euploid genomes. Still, earlier hypotheses suggested that a number of mammalian somatic tissues are populated by polyploid cells. Adult neurons of mammals were assumed to be postmitotic cells characterized to some extent by a polyploid chromosome complement. Testing this hypothesis in the past through histochemical methods, however, yielded controversial results through technical limitations.^13,14 However, with the recent development of molecular cytogenic techniques, aneuploid cells in the normal developing and mature brain have clearly been identified, indicating that the maintenance of aneuploid neurons in the adult CNS is a widespread, if not universal, property of organization.^{2,3,4,5,6,7,8,9,10,11,12,15}Recent studies of the embryonic brain have shown that approximately one-third of the dividing cells that give rise to the cerebral cortex have genetic variability, manifested as chromosome aneuploidy.^3,7,12 Neurons that constitute the adult brain arise from mitotic neural progenitor cells in the ventricular zone, a proliferating region where aneuploid cells appear to be generated through various chromosome segregation defects initially.^3,10 While a portion of these aneuploid cells apparently die during development,^3,16,17 aneuploid neurons have been identified in the mature brain in all areas assayed^{2,3,4,5,6,7,8,9,10,11,12,15} indicating that aneuploidy does not necessarily impair viability.¹⁸ Aneuploid neurons in the adult have been shown to make distant connections and express markers associated with neural activity, which indicates that these neurons can be integrated into brain circuitry.^4,12Contrary to this physiological consequences of “low-level” aneuploidy potentially contributing to neuronal diversity, aneuploidy above a critical threshold might be detrimental. Genomic instability and imbalances in gene dosage associated with aneuploidy can lead to developmental abnormalities, decreased cellular and organismal fitness, and increased susceptibility to disease.^19,20Aneuploid cells have typically been associated with pathophysiological conditions such as cancer,²¹ and most aneuploid syndromes present brain phenotypes and show a high vulnerability for psychiatric disorders.²² Mental impairment is a characteristic feature of all recognizable autosomal aneuploidy syndromes.Recent studies have shown an increased rate of aneuploid neurons in Alzheimer’s disease (AD), schizophrenia, autism, and ataxia-telangiectasia.^2,5,23,24,25 So far, however, direct evidence for a pathogenetic role for neuronal aneuploidy in these disorders is lacking. In particular, it remains unclear whether neuronal aneuploidy affects cellular viability, thus contributing directly to neurodegeneration and cell death.Here, we analyzed the fate of hyperploid neurons at the conversion from preclinical to mild AD and during further progression to severe stages of the disease. We can show that neurons with a more-than-diploid content of DNA are increased in preclinical stages of AD and are selectively affected by cell death during progression of the disease. Present findings show that neuronal hyperploidy in AD is associated with a decreased viability and directly linked to cell death.     

The Metastasis-Associated Anterior Gradient 2 Protein Is Correlated with Poor Survival of Breast Cancer Patients

The secreted metastasis-inducing protein, human anterior gradient 2 (AGR2), has been independently reported to be associated with either a reduced or an increased survival of different groups of patients with breast cancer. We now aim to analyze the expression of AGR2 in a third completely independent group of patients using a specific AGR2 monoclonal antibody (mAb). Primary tumors from a group of 315 patients suffering from operable (stage I and II) breast cancer with 20-years follow-up were immunocytochemically stained with a specific mAb to AGR2 and associations with prognostic factors and patient survival were analyzed. The mAb specifically recognized AGR2 in Western blots, and positive staining for AGR2 was significantly associated with involved lymph nodes and staining for estrogen receptor α, progesterone receptor, and the metastasis-inducing proteins osteopontin, S100P, and S100A4. After 20 years of follow-up, only 26% of patients with AGR2-positive carcinomas survived compared with 96% of those with AGR2 negative carcinomas, with the highly significant difference in median survival times of 68 and >216 months, respectively (P < 0.0001). Cox’s multivariate regression analysis showed that staining for AGR2 was one of the most significant independent prognostic indicators, with a corrected relative risk of 9.4. The presence of AGR2 in the primary tumor is therefore a possible prognostic indicator of poor patient outcome in breast cancer.Anterior gradient 2 (AGR2) protein is a secreted protein first described in Xenopus laevis embryos, where it induces the formation of the forebrain and the mucus-secreting cement gland.¹ Human AGR2 is also found co-expressed with estrogen receptor α (ERα) in breast cancer cell lines² and its presence significantly correlates with ERα in breast carcinoma specimens.³ Subsequent studies have found elevated expression of AGR2 in adenocarcinomas of the esophagus, pancreas, prostate, and non-small cell lung cancer, showing that it is a widely overexpressed protein in human carcinomas.^{4,5,6,7,8,9,10} We have shown that human AGR2 is expressed at higher levels in malignant, rather than in benign breast tumors,¹¹ and that, when introduced in an expression vector into the benign, nonmetastatic rat mammary cell line, Rama 37,¹² it causes metastasis in syngeneic rats.¹¹ These results suggest that AGR2’s metastasis-inducing properties may contribute toward the malignant progression of some breast cancers. Certain molecules shown to induce metastasis in experimental breast cancer in rodents, for example, S100A4, S100P, and osteopontin (OPN),^13,14,15,16 provide a potential source for markers that may be useful as prognostic factors in predicting patient outcome in human breast cancer.Using a polyclonal antibody (PAb) to AGR2, a strong positive association was shown between AGR2 and ERα and between AGR2 and low histological grade in a retrospective cohort of 351 breast cancer patients treated by adjuvant hormonal therapy.¹⁷ In these patients, levels of AGR2 were also associated with a relatively poor outcome in patients with ERα-positive breast cancers after treatment with antiestrogen therapy.¹⁷ In contrast to these results, it has been reported that in another panel of 155 breast carcinoma patients treated with various adjuvant therapies, immunocytochemically detected AGR2 in the cancer cells was associated with significantly longer patient survival times, although there was still a significant correlation with the presence of ERα and lower tumor grade.¹⁸ To resolve these apparent conflicting associations of AGR2 protein with patient survival, the expression of immunocytochemically detectable AGR2 protein has now been determined in the primary tumors of an independent group of 315 patients suffering from operable breast cancer and treated by surgery alone with no accompanying adjuvant therapy. A new specific monoclonal antibody (mAb) to AGR2, which does not cross-react with the closely-related AGR3 protein,³ has been used to improve the accuracy of detection of AGR2. The results show that, in this large group of breast cancer patients, the presence of immunocytochemically detectable AGR2 protein in the primary tumor is strongly associated with a reduced survival of the corresponding patients.     

Activation of mTORC1 Signaling Pathway in AIDS-Related Lymphomas

Using immunohistochemistry with antibodies against the phosphoserine residues in both S6rp and 4E binding protein 1, we identified the activation of the mammalian target of rapamycin (mTORC)1 pathway in 29 cases of AIDS-related lymphoma. These cases represented a diverse spectrum of histological types of non-Hodgkin lymphoma (24 cases) and classic Hodgkin lymphoma (five cases). mTORC1 was also activated in the hyperplastic but not involuted follicles of HIV-associated lymphadenopathy in eight cases, supporting the notion that mTORC1 activation is a common feature of transformed lymphocytes irrespective of either their reactive or malignant phenotype. We also found that in B-cell lines that represent diffuse large B-cell lymphoma, Burkitt lymphoma, Epstein-Barr virus-infected lymphocytes, and human herpesvirus 8-positive primary effusion lymphoma, inhibitors of Syk, MEK, and, seemingly, phosphoinositide 3 kinases suppressed mTORC1 activation, in particular when these inhibitors were used in combination. These findings indicate that AIDS-related lymphoma and other histologically similar types of lymphomas that are derived from transformed B lymphocytes may display clinical responses to inhibitors that directly target mTORC1 or, possibly, upstream activators of the mTORC1 pathway.The incidence of lymphomas in HIV-positive patients is nearly 200 times higher than in those uninfected by the virus. Lymphomas accounts for an increasing percentage of AIDS-defining illness, particularly from the advent of highly active antiretroviral therapy therapy.^1,2 These AIDS-related lymphomas (ARLs) typically represent the proliferation of enlarged, transformed B lymphocytes, which usually fall into the category of diffuse large cell lymphoma (DLBCL), with morphology ranging from centroblasts to immunoblasts. Other histological types such as Burkitt lymphoma (BL), Hodgkin lymphoma, and T/null cell anaplastic large cell lymphoma are also overrepresented among ARLs.^1,2,3,4 The pathogenesis of ARL is poorly understood. It has been postulated that cell proliferation occurring in the setting of severe immunosuppression and driven by chronic antigenemia resulting at first in the polyclonal and ultimately in the monoclonal lymphoproliferation plays a key role in lymphomagenesis in HIV patients. In addition, cell infection by the Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) most likely contributes to the malignant cell phenotype in some subtypes of ARL, with the association of primary effusion lymphoma with HHV8 being essentially universal.^1,2,3,4 Regardless of the histological type of ARL, chemotherapy is typically ineffective and new treatment approaches are clearly needed to combat this group of lymphomas. In addition to ARL, HIV patients develop a benign reactive lymphadenopathy, particularly early after the infection as an overall ineffective response to the virus. This immune response is characterized by florid follicular hyperplasia that over time may lead to follicular involution and lymphocyte depletion.Mammalian target of rapamycin (mTOR) is a ubiquitously expressed serine/threonine kinase involved in key cellular functions including protein synthesis and proliferation.^5,6 mTOR associates with several proteins including either raptor or rictor to form the mTORC1 and mTORC2 complexes, respectively, with the signaling pathways activated by mTORC1 being much better characterized.^4,5,6,7 Accordingly, it is well established that mTORC1 activates p70S6 kinase 1 and inhibits 4E binding protein 1 (4E-BP1). In turn, p70S6 kinase 1 phosphorylates an S6 protein of the 40S ribosomal subunit (S6rp) at several sites including serines 235 and 236. The exact mechanisms of mTORC1 activation are less understood but both phosphoinositide 3 kinases (PI3K)/Akt^8,9,10 and extracellular regulated (ERK)/mitogen-activated kinase (MEK) kinases^11,12 signaling pathways have been found to activate mTORC1 with members of the insulin growth factor family providing the primary signal, at least in some instances. The highly potent and specific inhibitors from the rapamycin family can functionally inactivate mTORC1. In addition to being used as immunosuppressants, mTORC1 inhibitors are evaluated as therapeutic agents in various types of cancer,^5,6 with high efficacy already documented in renal cell carcinoma.¹³Syk is a protein tyrosine kinase expressed in B lymphocytes,¹⁴ monocytes/macrophages,¹⁵ mast cells,¹⁶ and other cell types. Syk has been found to be involved in signal transduction through several types of receptors including the antigen B-cell receptor¹⁷ and at least three different receptors for the Fc component of immunoglobulins G and E.^{14,15,16,18,19,20} A recent report suggests that Syk may be involved in mTORC1 activation in a follicular and possibly other types of B-cell lymphoma.²¹ Although inhibitors of either PI3K/Akt or MEK/ERK signaling pathways did fully inhibit mTORC1 activation in transformed B lymphocytes, at least when applied alone,²² these pathways have been found to contribute to mTORC1 stimulation in two types of T-cell lymphoma.^23,24In this study, we identified the activation of the mTORC1 pathway in all ARL cases examined, regardless of their specific histological classification and immunophenotype. mTORC1 was also activated in the hyperplastic follicles of the HIV-associated lymphadenopathy. Furthermore, we found that in the different types of transformed B-lymphocytes cell lines, inhibition of Syk, MEK, and, seemingly, PI3K resulted in suppression of the mTORC1 activation, in particular when the combination of the inhibitors was used. These findings indicate that ARL and histologically similar types of lymphoma may benefit from targeted therapy with inhibitors of mTORC1 or, possibly, its upstream activators.     

Long-Term Expression of Tissue-Inhibitor of Matrix Metalloproteinase-1 in the Murine Central Nervous System Does Not Alter the Morphological and Behavioral Phenotype but Alleviates the Course of Experimental Allergic Encephalomyelitis

Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related proteins that inhibit matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMPs 2, 3, and 4 are constitutively expressed at high levels, whereas TIMP1 can be induced by various stimuli. Here, we studied the effects of constitutive expression of TIMP1 in the CNS in transgenic mice. Transgene expression started prenatally and persisted throughout lifetime at high levels. Since MMP activity has been implicated in CNS development, in proper function of the adult CNS, and in inflammatory disorders, we investigated Timp1-induced CNS alterations. Despite sufficient MMP inhibition, high expressor transgenic mice had a normal phenotype. The absence of compensatory up-regulation of MMP genes in the CNS of Timp1 transgenic mice indicates that development, learning, and memory functions do not require the entire MMP arsenal. To elucidate the effects of strong Timp1 expression in CNS inflammation, we induced experimental allergic encephalomyelitis. We observed a Timp1 dose-dependent mitigation of both experimental allergic encephalomyelitis symptoms and histological lesions in the CNS of transgenic mice. All in all, our data demonstrate that (1) long-term CNS expression of TIMP1 with complete suppression of gelatinolytic activity does not interfere with physiological brain function and (2) TIMP1 might constitute a promising candidate for long-term therapeutic treatment of inflammatory CNS diseases such as multiple sclerosis.Tissue-inhibitor of matrix metalloproteinase-1 (TIMP1), a tightly regulated 28.5-kDa glycoprotein,^1,2 belongs to a family of multifunctional secreted proteins (TIMPs 1 to 4) that regulate the proteolytic activity of matrix metalloproteinases (MMPs). Together, MMPs and TIMPs control the pericellular environment, including the turnover of extracellular matrix proteins, bioavailability of growth factors and cytokines, and shedding of membrane receptors.³ In the central nervous system (CNS), expression of MMP and TIMP genes is highly orchestrated during development and normal brain function.^4,5,6 The spatio-temporal expression pattern of TIMP1 indicates a developmental role in the hippocampus and the cerebellum.^4,7,8,9 TIMP1 was also proposed as a candidate plasticity protein in learning and memory.^10,11 Several MMPs on the other hand are expressed in the developing cerebellum,⁴ and in migrating neural precursors,^12,13 and MMP activity was localized to the growth cone of cultured sympathetic neurons.^14,15,16 In the normal adult CNS, however, MMP expression is low.^17,18 Alterations of the MMP/TIMP ratio are tightly associated with pathological processes and clearly influence the course of various diseases.^12,17,18,19 In autoimmune diseases such as multiple sclerosis and the animal model experimental allergic encephalomyelitis (EAE), elevated activity of several MMPs was detected and suspected to influence the outcome of the disease.^{17,20,21,22,23} Accordingly, synthetic MMP inhibitors blocked the blood-brain-barrier disruption and improved the clinical condition of the animals,^23,24,25 and Timp1 deficient mice revealed increased leukocyte infiltration as well as enhanced spinal cord demyelination.²⁶ Due to serious side-effects, however, only few synthetic MMP-inhibitors have been introduced to human therapy.²⁷Since high levels of Timp1 were expressed by astrocytes around the inflammatory lesions in EAE¹⁷ and to further investigate the role of Timp1 in the CNS, we targeted its expression to astrocytes in a transgenic mouse model.     

Development of Arterial Blood Supply in Experimental Liver Metastases

In this study, we present a mechanism for the development of arterial blood supply in experimental liver metastases. To analyze the arterialization process of experimental liver metastases, we elucidated a few key questions regarding the blood supply of hepatic lobules in mice. The microvasculature of the mouse liver is characterized by numerous arterioportal anastomoses and arterial terminations at the base of the lobules. These terminations supply one hepatic microcirculatory subunit per lobule, which we call an arterial hepatic microcirculatory subunit (aHMS). The process of arterialization can be divided into the following steps: 1) distortion of the aHMS by metastasis; 2) initial fusion of the sinusoids of the aHMS at the tumor parenchyma interface; 3) fusion of the sinusoids located at the base of the aHMSs, which leads to the disruption of the vascular sphincter (burst pipe); 4) incorporation of the dilated artery and the fused sinusoids into the tumor; and 5) further development of the tumor vasculature (arterial tree) by proliferation, remodeling, and continuous incorporation of fused sinusoids at the tumor–parenchyma interface. This process leads to the inevitable arterialization of liver metastases above the 2000- to 2500-μm size, regardless of the origin and growth pattern of the tumor.It is widely accepted that hepatic metastases and tumors are predominantly supplied by arterial blood, a notion that serves as the basis for hepatic arterial chemotherapy and chemoembolization.^{1,2,3,4,5,6,7} The most cited article on this field dates back to the 1950s.¹ Since then numerous papers have been published using human and experimental materials and different methods such as corrosion casting, confocal and electron microscopy, angiography, radiolabeled microspheres, and in vivo microscopy, have been used to study the blood supply of liver metastases.^{2,3,4,5,6,7,8,9,10,11,12,13,14,15,16} A large proportion of these articles have confirmed the original observation of Breedis and Young,¹ but no mechanism for the development of the arterial blood supply in metastases has ever been presented.^2,3,4,5,6,7 On the other hand, numerous papers, including ours, have emphasized the contribution of the portal vein, either directly or through the sinusoids in the blood supply of hepatic metastases.^{8,9,10,11,12,13,14} This apparent contradiction might result from the observed continuity of the sinusoidal with the tumor vasculature and the presumption that blood flows in an “outside-in” direction from the sinusoids toward the tumor vasculature. Most of the studies dealing with the blood supply of metastases have neglected the importance of arterioportal anastomoses and other interspecies differences in the hepatic microcirculation, which could lead to seriously biased results. According to the observations of Yamamoto et al¹⁵ there are extensive arterioportal anastomoses throughout the vascular tree in rats, whereas a separate arterial and portal tree, without direct arterioportal communication, can be observed in hamster and human liver. Opinions about the presence of arterioportal anastomoses in mice are controversial^10,16; therefore, we have addressed this question first.The classic lobule can be divided into several conical hepatic microcirculatory subunits (HMSs) supplied by a single inlet portal venule. Hepatic arterioles terminate either on the inlet venules or directly on sinusoids. The number of these terminations within a lobule is species-dependent. The blood flow through the inlet venules and terminal arterioles is regulated by sphincters.¹⁷ The most detailed studies on microcirculation of the liver and vessel architecture of liver metastases were performed by corrosion casting. However, in these studies the livers were completely filled with uncolored resin, which made analyzing the three-dimensional organization of the deep interlobular vessels difficult.^10,14,15In the present study, we used a two color corrosion casting technique to analyze the blood supply in liver metastases of experimental tumors in mice. A special filling method was used to prevent the mixing of the “portal and arterial resin” upstream of the hepatic sinusoids. This technique enabled us to analyze separately the contribution of the two vascular systems to the blood supply of liver metastases and to establish the steps of the arterialization process.     

Dual Roles of CD40 on Microbial Containment and the Development of Immunopathology in Response to Persistent Fungal Infection in the Lung

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4⁺ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.CD40, a 48-kDa type I transmembrane protein and member of the tumor necrosis factor receptor family, is a well-described costimulatory molecule expressed on B cells, dendritic cells (DC), macrophages, basophils, and platelets as well as nonhematopoietic cells including fibroblasts, epithelial, and endothelial cells. The ligand for CD40, known as CD154 or CD40L, is a type II transmembrane protein member of the tumor necrosis factor (TNF) superfamily expressed primarily by activated T cells, B cells, and platelets.^1,2,3 CD40 can be induced on DC, monocytes, and macrophages under inflammatory conditions.^4,5 Signaling via the CD40/CD40L pathway exerts numerous biological effects including: i) increased cytokine expression (especially TNF-α and Th1 cytokines interleukin (IL)-12 and interferon (IFN)-α) and nitric oxide production; ii) upregulation of additional costimulatory molecules (CD80 and CD86) on antigen-presenting cells (APC); iii) enhanced cell survival (particularly of B and T cells, DC, and endothelial cells); iv) Ig isotype switching; and v) somatic hypermutation of Ig.^1,4,5The CD40/CD40L signaling pathway contributes to adaptive Th1 immune responses required to clear Leishmanisa spp.,^6,7,8 Trypanosoma spp.,^6,7,8,9 Shistosoma mansoini,¹⁰ and the fungi Candida albicans¹¹ and Pneumocystis spp.¹² The enhanced production of IFN-γ, TNF-α, and nitric oxide associated with CD40/CD40L signaling is thought to be responsible for this protective effect. However, other studies have suggest that CD40/CD40L signaling is not required for successful host defense against Listeria monocytogenes,^13,14 Toxoplasma gondi,¹⁵ lymphocytic choriomeningitis virus,^16,17 or the fungus Hisoplasma capsulatum.^18,19 In models of Mycobacterium spp. infection, CD40 appears dispensable for clearance of an i.v. infection,^20,21 but essential for clearing the organism in response to aerosolized infection in the lungs.^22,23 Thus, the role of CD40 in antimicrobial host defense varies and depends not only on the specific pathogen but also on the primary site of infection.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with AIDS, lymphoid or hematological malignancies, or patients receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation.^24,25 Infection in non-immunocompromised patients has been reported.^26,27,28 Murine models of cryptococcal infection in CBA/J or BALB/c mice demonstrate that development of a Th1 antigen-specific immune response characterized by IFN-γ production and classical activation of macrophages is required to eradicate the organism.^{29,30,31,32,33,34,35,36,37,38,39,40} In contrast, a model of persistent cryptococcal infection has been developed using C57BL/6 mice;^{41,42,43,44,45,46,47} this model reflects many features observed in humans diagnosed with allergic bronchopulmonary mycosis.⁴⁸ Specifically, these mice fail to clear the organism from the lung and develop characteristic Th2-mediated immunopathology including: i) tissue eosinophilia; ii) airway hyperreactivity, mucus production, and fibrosis; and iii) alternative macrophage activation associated with YM1 crystal deposition.The molecular mechanisms responsible for the immunopathologic response associated with persistent cryptococcal infection are not clearly defined. These features are abrogated in the absence of IL-4,⁴⁵ whereas more severe Th2-mediated lung injury occurs in the absence of IFN-γ.^29,41 TNF-α exerts a protective effect by enhancing IFN- γ production and the subsequent classical activation of lung macrophages.^31,35,49,50 Lymphocytes are critical mediators of this Th2 response as the pathological features of chronic cryptococcal infection are substantially diminished in CD4 T cell-depleted mice despite no change in fungal clearance.⁴²Although interactions between CD4 T cells and APC are critical determinants of T cell polarization in response to cryptococcal lung infection,^{49,51,52,53,54,55} the contribution of specific costimulatory molecules including the CD40/CD40L signaling pathway has not been fully elucidated. In vitro studies suggest that activation of the CD40/CD40L pathway in response to Cryptococcus promotes IFN-γ production by T cells and TNF-α, and nitric oxide (NO) production by monocytes.⁵⁶ In the absence of CD40L, primary pulmonary infection with a weakly virulent strain of C. neoformans was associated with impaired fungal clearance; however, measurements of immune function at the site of infection in the lung or evidence of systemic fungal dissemination were not evaluated.⁵⁷ The potential to target CD40 therapeutically is highlighted by studies showing that treatment of mice with disseminated or intracerebral cryptococcal infection with an agonist antibody to CD40 in combination with IL-2 improves survival.^58,59 In this study, we used gene-targeted CD40-deficient mice (on a C57BL/6 genetic background), a clinically relevant model, and assessments of immune function and histopathology in the lung to identify two unique roles for the CD40-signaling pathway in response to persistent cryptococcal lung infection.     

Loss-of-Function FERMT1 Mutations in Kindler Syndrome Implicate a Role for Fermitin Family Homolog-1 in Integrin Activation

Kindler syndrome is an autosomal recessive disorder characterized by skin atrophy and blistering. It results from loss-of-function mutations in the FERMT1 gene encoding the focal adhesion protein, fermitin family homolog-1. How and why deficiency of fermitin family homolog-1 results in skin atrophy and blistering are unclear. In this study, we investigated the epidermal basement membrane and keratinocyte biology abnormalities in Kindler syndrome. We identified altered distribution of several basement membrane proteins, including types IV, VII, and XVII collagens and laminin-332 in Kindler syndrome skin. In addition, reduced immunolabeling intensity of epidermal cell markers such as β1 and α6 integrins and cytokeratin 15 was noted. At the cellular level, there was loss of β4 integrin immunolocalization and random distribution of laminin-332 in Kindler syndrome keratinocytes. Of note, active β1 integrin was reduced but overexpression of fermitin family homolog-1 restored integrin activation and partially rescued the Kindler syndrome cellular phenotype. This study provides evidence that fermitin family homolog-1 is implicated in integrin activation and demonstrates that lack of this protein leads to pathological changes beyond focal adhesions, with disruption of several hemidesmosomal components and reduced expression of keratinocyte stem cell markers. These findings collectively provide novel data on the role of fermitin family homolog-1 in skin and further insight into the pathophysiology of Kindler syndrome.Kindler syndrome (KS; OMIM 173650) is a rare autosomal recessive disorder characterized by trauma-induced skin blistering, skin atrophy, and poikiloderma.¹ KS results from pathogenic mutations in the FERMT1 (formerly KIND1 or C20orf42) gene that encodes fermitin family homolog-1 (FFH1) (formerly kindlin-1 or kindlerin), an actin cytoskeleton and focal adhesion-associated molecule.^2,3 FFH1 is mainly expressed in basal keratinocytes.^3,4,5,6 In KS, however, there is often a complete absence of FFH1 protein expression, although some variability may occur.⁶ Thus far, 37 different loss-of-function FERMT1 mutations have been identified.^7,8,9 The mechanism by which these pathogenic FERMT1 mutations result in skin atrophy and blistering, however, remains unclear. Indeed, insight from studies on KS skin and FFH1 has been limited. Ultrastructural and immunohistochemical studies on KS skin have revealed a disrupted and reduplicated cutaneous basement membrane,^{4,10,11,12,13,14,15,16} and silencing of FFH1 in HaCaT cells results in reduced cell adhesion, proliferation, and spreading.¹⁷ In addition, cultured KS keratinocytes display decreased cell adhesion and proliferation and exhibit multiple cell polarities and undirected migration.⁵ Moreover, FFH1 is able to bind to the cytoplasmic tails of β1 and β3 integrins,^17,18 colocalizes with vinculin and paxillin at focal adhesions in keratinocytes,¹⁷ and associates biochemically with FFH2 and filamin binding LIM protein.⁶ Overall, these studies suggest a critical role for FFH1 in cell-extracellular matrix (ECM) interactions but do not fully explain the clinicopathological abnormalities present in KS.Cell-ECM interactions are principally mediated by integrins through integrin activation and cytoskeletal organization.^19,20 An important step in integrin activation is the binding of the FERM (four point one protein, ezrin, radixin, and moesin) domain of talin to the cytoplasmic tail of integrin.^{21,22,23,24,25,26,27} It is being increasingly recognized that other proteins may also regulate integrin activation. FFH2 (expressed predominantly in the heart) and FFH3 (expressed mainly in hematopoietic tissue) recently have been identified as essential regulators of integrin activation with deficiencies in these two focal adhesion proteins leading to cardiac malformation and platelet dysfunction, respectively.^{28,29,30,31,32,33,34,35,36} FFH1, 2, and 3 form part of a protein family that shares a high degree of sequence homology as well as a bipartite FERM domain interrupted by a pleckstrin homology domain.^3,37 Nevertheless, it remains to be determined whether FFH1 is involved in integrin activation in keratinocytes. In the current study, we have identified new pathological abnormalities involving epidermal stem cell markers, hemidesmosomal-associated proteins and integrin activation in KS. Our study also highlights a crucial role of FFH1 in integrin biology because induction of its overexpression in KS keratinocytes is able to restore integrin activation and partially rescue the cellular phenotype.     

Fenfluramine Disrupts the Mitral Valve Interstitial Cell Response to Serotonin

Serotonin (5HT) receptor signaling and 5HT-related agents, such as the anorexogen fenfluramine (Fen), have been associated with heart valve disease. We investigated the hypothesis that Fen may disrupt mitral valve interstitial cell (MVIC) homeostasis through its effects on mitogenesis and extracellular matrix biosynthesis. Normal and myxomatous mitral valves, both human and canine, were harvested, and primary MVIC cultures were established. 5HT caused increased phosphorylation of extracellular signal-related kinase in MVIC; Fen alone did not. However, Fen combined with 5HT increased the level of MVIC extracellular signal-related kinase, when compared with 5HT alone. In addition, MVIC mitogenesis per ³H-thymidine (³HTdR) demonstrated a 5HT dose-dependent increase, with no effect of Fen alone. In contrast, Fen combined with 5HT inhibited the MVIC ³HTdR response when compared with 5HT alone. Furthermore, fluoxetine, a 5HT transporter inhibitor, while having no effect alone, suppressed Fen-5HT ³HTdR inhibition when administered with Fen plus 5HT. Finally, MVIC incorporations of ³H-proline and ³H-glucosamine, measures of extracellular matrix collagen and glycosaminoglycan respectively, were increased with 5HT alone; however, Fen did not affect MVIC glycosaminoglycan or collagen either alone or in combination with 5HT. Taken together, the ratios of ³H-proline or ³H-glycosaminoglycan to ³HTdR in MVIC, normalized to 5HT alone, demonstrated a significant imbalance of extracellular matrix production versus proliferation in MVIC cultures with Fen plus 5HT exposure. This imbalance may explain in part the pathophysiology of Fen-related mitral valve disease.Serotonin (5HT) is a neurotransmitter that has been demonstrated to be associated with heart valve disease in both clinical settings^{1,2,3,4,5,6,7,8} and in experimental animals.^9,10,11,12 5HT-associated heart valve disease, affecting primarily the right-sided heart valves, was first noted with carcinoid tumors,⁶ which are chromaffin cell malignancies that affect the small intestine and produce serotonin and other catecholamines. Dopamine agonist administration has also been shown to be associated in rare cases with heart valve disease affecting either the mitral or aortic valves.^7,8 5HT administration to mice⁹ and rats^10,11 results in progressive heart valve disease, and transgenic mice that have the 5HT transporter (5HTT) gene deleted, resulting in delayed processing of 5HT, also develop heart valve disease that affects predominantly the mitral and aortic valves.¹² Interestingly, fenfluramine (Fen) has never been demonstrated to cause an experimental valvulopathy.In the mid-1990s, heart valve disease was shown to be associated with the use of Fen as a diet drug.^1,2,3,4,5 Fen has been reported to have 5HT receptor (5HTR) agonist activity in neuronal cells and 5HT-releasing activity from 5HTT.¹³ Fen-related heart valve disease was reported both with administration of Fen alone, or in combination with phentermine (Phen), a monamine oxidase inhibitor that was co-administered to sustain Fen’s effects.^1,2,3,4,5 Fen was withdrawn from human use by the U.S. Food and Drug Administration in 1997.¹⁴ The pathogenesis of Fen-induced heart valve disease is still incompletely understood. However, since Fen affects 5HT mechanisms and the pathology of the Fen valve lesions in some, but not all of the published cases^4,5 resembled the carcinoid syndrome valvulopathy,^4,5 it has been strongly suggested that a 5HT mechanism may be involved.^1,2,3,4,5 Prior studies^{15,16,17,18,19,20} explored the pathogenesis of Fen-associated heart valve disease, using a variety of model systems, and in general concluded that Fen was likely acting as a 5HTR agonist. Since cardiac valve anatomy, physiology, and pathophysiology are unique for each of the different cardiac valves, we sought to focus the present investigations on the mitral valve. Mitral valves were also the most frequently affected in the Fen cases reported in both of the largest human pathology series.^4,5Thus, the present study examined the mitral valve interstitial cell (MVIC) response to 5HT and Fen, to investigate why Fen may have caused mitral valve disease. Our working hypothesis is that Fen may disrupt MVIC homeostasis through its effects on mitogenesis and associated extracellular matrix (ECM) biosynthetic activity via mechanisms involving 5HTR signal transduction and off-target effects. We investigated this hypothesis with cell culture studies using both human and canine MVIC assessing the effects of 5HT and Fen on canine and human MVIC with endpoints assessing signal transduction, mitogenesis, and ECM biosynthesis.