Platelet,Not Endothelial,P-Selectin Expression Contributes to Generation of Immunity in Cutaneous Contact Hypersensitivity

Leukocyte extravasation is a prerequisite for host defense and autoimmunity alike. Detailed understanding of the tightly controlled and overlapping sequences of leukocyte extravasation might aid development of novel therapeutic strategies. Leukocyte extravasation is initiated by interaction of selectins with appropriate carbohydrate ligands. Lack of P-selectin expression leads to decreased contact hypersensitivity responses. Yet, it remains unclear if this is due to inhibition of leukocyte extravasation to the skin or due to interference with initial immune activation in lymph nodes. In line with previous data, we here report a decreased contact hypersensitivity response, induced by 2,4,-dinitrofluorobenzene (DNFB), in P-selectin-deficient mice. Eliciting an immune reaction towards DNFB in wild-type mice, followed by adoptive transfer to P-selectin-deficient mice, had no impact on inflammatory response in recipients. This was significantly reduced in wild-type recipient mice adoptively transferred with DNFB immunity generated in P-selectin-deficient mice. To investigate if platelet or endothelial P-selectin was involved, mice solely lacking platelet P-selectin expression generated by bone marrow transplantation were used. Adoptive transfer of immunity from wild-type mice reconstituted with P-selectin-deficient bone marrow led to a decrease of inflammatory response. Comparing this decrease to the one observed using P-selectin-deficient mice, no differences were observed. Our observations indicate that platelet, not endothelial, P-selectin contributes to generation of immunity in DNFB-induced contact hypersensitivity.Infiltration of leukocytes is a hallmark of inflammation. To reach the affected tissues, leukocytes must leave the bloodstream. This process of leukocyte extravasation requires several distinct steps, occurring in sequence. Leukocyte extravasation is initiated by short-lived interactions of adhesion molecules from the selectin family with appropriate carbohydrate scaffolds displayed by several glycoproteins, leading to tethering and rolling of leukocytes along the endothelial lining of the vasculature. Of the numerous adhesion molecules, endothelial P-selectin, along with E-selectin and vascular cell adhesion molecule (VCAM)-1, initiates and sustains rolling interactions of leukocytes in the skin microvasculature.In addition to a direct interaction of leukocytes with the endothelium lining the vasculature, platelets are becoming more and more recognized to initiate leukocyte rolling in several organs, including peripheral lymph nodes,¹ atherosclerotic lesions² and skin.³ In detail, platelets may influence leukocyte rolling in numerous ways: First, formation of platelet-leukocyte aggregates in the bloodstream leads to activation of bound leukocytes,² leading to an increased avidity of leukocyte integrins,^4–6 which then may allow rolling interactions through binding of leukocyte VLA-4 to endothelial VCAM-1.⁷ Second, rolling of activated platelets along the vasculature^2,3,8,9 allows a deposition of platelet-derived pro-inflammatory substances directly to the endothelial cells, which in turn activates endothelial cells, leading to an increased expression/secretion of adhesion molecules and cytokines.^10–13 Third, formation of platelet-leukocyte aggregates increases leukocyte rolling in lymph nodes⁹ and the skin.³ In addition, this platelet-mediated leukocyte rolling has been shown to functionally relevant in lymph nodes, lung and the skin. Impaired extravasation of naïve T lymphocytes into the lymph nodes, and thus diminished generation of immunity, in L-selectin-deficient mice,¹⁴ can be restored by infusion of activated human platelets. Interestingly, infusion of activated platelets had no impact on generation of immunity in wild-type mice.¹⁵ Likewise, pulmonary leukocyte recruitment in platelet-depleted mice was abolished in a model of ovalbumin-induced asthma.¹⁶ Furthermore, in a model of chronically induced contact dermatitis, infusion of wild-type, but not P-selectin-deficient, platelets restored the inflammatory response in mice rendered thrombocytopenic.¹⁷The latter observation is in line with previous findings, showing an impairment of cutaneous inflammation induced by application of haptens, such as oxazolone, in P-selectin-deficient mice.¹⁸ However, while the work by Tamagawa-Mineoka and colleagues¹⁷ has clearly shown a dependency of leukocyte extravasation on platelet P-selectin expression to the skin, the role of P-selectin in the generation of immunity toward cutaneous applied haptens remained to be elucidated. We therefore investigated the role of P-selectin in the generation of immunity in the model of 2,4,-dinitrofluorobenzene (DNFB)-induced cutaneous hypersensitivity.     

Antibodies to Gliomedin Cause Peripheral Demyelinating Neuropathy and the Dismantling of the Nodes of Ranvier

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP.Guillain-Barré syndrome (GBS) is a group of inflammatory neuropathies that affect peripheral nerves. In Europe, acute inflammatory demyelinating polyneuropathy (AIDP) is the most common form of GBS. Autopsy and biopsy studies indicated that both humoral and cellular immune reaction against Schwann cell or axonal antigens are implicated in GBS etiology.¹ Early investigations have found that conduction defects closely correlate with myelin retraction and macrophage invasion in many patients.^{2, 3, 4, 5} Some GBS cases also involve acute demyelination without immune cell invasion and are primarily humorally mediated.^{6, 7} In particular, deposition of complement on the abaxonal surface of the Schwann cells has been shown during the early stage of GBS^{8, 9, 10} and in experimental allergic neuritis (EAN).¹¹ In a recent study, we demonstrated that nodes of Ranvier and paranodes are the targets of the immune attack in GBS and in chronic inflammatory demyelinating polyneuropathy (CIDP).¹² Notably, cell adhesion molecules (CAMs) at nodes or paranodes (gliomedin, neurofascin, and contactin) were recognized by IgG antibodies in patients with GBS or CIDP.^{12, 13} Autoantibodies against neurofascin and gliomedin were also detected in a rat model of AIDP and correlated with important conduction defects.¹⁴ This finding suggested that antibodies to nodal CAMs may participate to the pathogenesis of AIDP and CIDP. However, the exact mechanisms by which these humoral factors mediate demyelination and conduction defects are still elusive.Several CAMs are implicated in node formation and are responsible for the enrichment of voltage-gated sodium (Nav) channels at the nodes of Ranvier.¹⁵ At peripheral, nodes gliomedin and NrCAM are secreted into the nodal gap lumen and interact with neurofascin-186 (NF186) expressed at nodal axolemma.^{16, 17, 18, 19} This interaction is crucial for Nav channel aggregation at nodes.^{19, 20, 21} In addition, the paranodal axoglial junctions are made by the association of contactin and contactin-associated protein (Caspr) with neurofascin-155 (NF155), a variant expressed in glia.²² This adhesive junction forms a barrier to the lateral diffusion of nodal channels.^{19, 21, 23} In a rat model of AIDP, we found that the loss of NF186 and gliomedin at nodes preceded paranodal demyelination and the diffusion of Nav channels in demyelinated segments.¹⁴ This finding indicated that antibodies to nodal CAMs may participate to conduction defects by dismantling axoglial attachment at nodes and paranodes.We investigated whether immunity toward gliomedin and NF186 can trigger peripheral neuropathies and be responsible for demyelination in GBS patients. We found that immunization against gliomedin induced a biphasic condition associated with conduction loss and demyelination. Passive transfer of antibodies to gliomedin exacerbated the clinical signs of EAN and resulted in the disorganization of the nodes of Ranvier. Altogether, these results demonstrate that humoral immune response directed against nodal CAMs participates in conduction abnormalities in peripheral nerves and in the etiology of GBS and CIDP.     

The Glucagon-Like Peptide-1 Receptor Agonist Exendin 4 Has a Protective Role in Ischemic Injury of Lean and Steatotic Liver by Inhibiting Cell Death and Stimulating Lipolysis

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase – all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.The incidence of obesity and fatty liver disease is increasing worldwide. Non alcoholic fatty liver disease (NAFLD) includes a spectrum of liver abnormalities ranging from simple steatosis with preserved synthetic function to end-stage liver disease requiring transplantation.^{1, 2} The cause of hepatic dysfunction related to steatosis remains incompletely defined.³ However, it is known that a steatotic liver has increased susceptibility to ischemic insults, such as those induced during liver resections and liver surgery,^{4, 5, 6} heart failure,⁷ and shock.⁸ In addition, steatotic livers are known to weather the ischemic insult of transplantation poorly,⁹ resulting in increased rates of primary nonfunction and initial graft dysfunction.^{10, 11} As such, fatty livers are routinely turned down for transplantation and this impacts transplant wait list morbidity and mortality.¹² Thus, liver steatosis contributes to the public health burden and methods to mollify the adverse effects of liver steatosis are relevant across a large spectrum of hepatic diseases.The inability of a steatotic liver to withstand ischemic insult is directly related to increased post ischemic cell death, which can occur through necrosis and apoptosis. The fundamental connection between intracellular fat and poor hepatic cell survival¹³ is incompletely understood. However, it has been suggested that methods that decrease intracellular fat reverse this susceptibility and the use of glucagon-like peptide-1 (GLP-1) analogues is one such approach. GLP-1 is secreted from the L cells of the small intestine and its cognate receptor (GLP-1R) is present in several organs, such as the pancreas, brain, heart, kidney, and liver. Although it is well known for its incretin action,¹⁴ it also has pleotropic effects.^{15, 16, 17, 18, 19} In the liver we have shown that GLP-1 or its homologue Exendin 4 (Ex4) acts directly on steatotic hepatocytes to decrease their lipid content.^{20, 21} In addition, a cytoprotective action of Ex4 with improvement in cell survival has also been reported.²² Thus, we hypothesize that anti-steatotic effects of Ex4 in hepatocytes and cytoprotective effects in other organs make it a rational target for investigation in steatotic livers undergoing ischemia reperfusion injury (IRI), a common clinical scenario in people with NAFLD. In this study, we explore the role of Ex4 in protecting against necrosis and apoptosis, the two forms of cell death encountered in hepatic IRI, and we provide evidence to show that Ex4 stimulates lipolysis with a short course of treatment. To our knowledge, this is the first study showing a direct and rapid action of Ex4 in acutely reversing the vulnerability of a steatotic liver to ischemic insults, supporting the investigation of Ex4 as a potential therapeutic agent for treatment of people with NAFLD undergoing ischemic injury and at the time of procurement of a fatty liver for transplantation.     

Cytoprotective Mitochondrial Chaperone TRAP-1 As a Novel Molecular Target in Localized and Metastatic Prostate Cancer

Molecular chaperones of the heat shock protein-90 (Hsp90) family promote cell survival, but the molecular requirements of this pathway in tumor progression are not understood. Here, we show that a mitochondria-localized Hsp90 chaperone, tumor necrosis factor receptor-associated protein-1 (TRAP-1), is abundantly and ubiquitously expressed in human high-grade prostatic intraepithelial neoplasia, Gleason grades 3 through 5 prostatic adenocarcinomas, and metastatic prostate cancer, but largely undetectable in normal prostate or benign prostatic hyperplasia in vivo. Prostate lesions formed in genetic models of the disease, including the transgenic adenocarcinoma of the mouse prostate and mice carrying prostate-specific deletion of the phosphatase tensin homolog tumor suppressor (Pten^pc−/−), also exhibit high levels of TRAP-1. Expression of TRAP-1 in nontransformed prostatic epithelial BPH-1 cells inhibited cell death, whereas silencing of TRAP-1 in androgen-independent PC3 or DU145 prostate cancer cells by small interfering RNA enhanced apoptosis. Targeting TRAP-1 with a novel class of mitochondria-directed Hsp90 inhibitors, ie, Gamitrinibs, caused rapid and complete killing of androgen-dependent or -independent prostate cancer, but not BPH-1 cells, whereas reintroduction of TRAP-1 in BPH-1 cells conferred sensitivity to Gamitrinib-induced cell death. These data identify TRAP-1 as a novel mitochondrial survival factor differentially expressed in localized and metastatic prostate cancer compared with normal prostate. Targeting this pathway with Gamitrinibs could be explored as novel molecular therapy in patients with advanced prostate cancer.Apart from skin tumors, prostate cancer is the most commonly diagnosed malignancy in men in the United States.¹ Despite progress in early diagnosis,² and prolongation of patient survival,³ the disease still carries significant morbidity and mortality, with its advanced and metastatic phase claiming over 30,000 deaths per year in the United States alone. Similar to the genetic heterogeneity of most epithelial malignancies, prostate cancer progresses through a stepwise acquisition of multiple molecular changes,⁴ of which insensitivity to androgen deprivation,⁵ emergence of an ‘osteomimetic’ phenotype responsible for metastatic tropism to the bone,⁶ and deregulated cell proliferation and cell survival,⁷ are pivotal traits.In this context, advanced prostate cancer is almost invariably associated with a heightened anti-apoptotic threshold,⁴ which may contribute to disease progression and resistance to therapy. This process often involves aberrant resistance to mitochondrial cell death,⁸ with reduced organelle permeability to solutes, and attenuated release of mitochondrial apoptogenic proteins in the cytosol.⁹ The regulators of such ‘mitochondrial permeability transition’ normally triggered by cell death stimuli are still largely elusive, but knockout data in mice have identified pro-apoptotic Bcl-2 family proteins and the mitochondrial matrix immunophilin, cyclophilin D, as pivotal effectors of this process, controlling the integrity of the mitochondrial outer membrane,⁸ and the opening a permeability transition pore,^10,11 respectively.Recent data have shown that molecular chaperones of the heat shock protein-90 (Hsp90) family,¹² may function as novel regulators of mitochondrial permeability transition,¹³ especially in tumor cells.¹⁴ Accordingly, Hsp90, and its ortholog, tumor necrosis factor receptor-associated protein-1 (TRAP-1) are abundantly localized to mitochondria of tumor, but not most normal cells, and antagonize cyclophilin D-dependent pore-forming function, potentially via a protein (re)folding mechanism.¹⁴ Consistent with a general role of Hsp90 as a drug target in prostate cancer,¹⁵ this mitochondria-compartmentalized cytoprotective pathway could provide a novel therapeutic target to enhance tumor cell apoptosis.¹⁴In the current study, we demonstrate that TRAP-1 is dramatically expressed in all lesions that comprise the entire natural history of human prostate cancer, as well as genetic disease models in rodents, but undetectable in the normal prostate. Importantly, we show that Gamitrinibs, a novel class of small molecule Hsp90 antagonists selectively engineered to target the pool of these chaperones in mitochondria,¹⁶ cause sudden prostate cancer cell death without affecting nontransformed prostatic epithelium.     

Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions

Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.Pulmonary arterial (PA) neointimal lesions are observed in patients with PA hypertension that is idiopathic (IPAH) or associated with other medical conditions. These vascular abnormalities cause narrowing and even obliteration of the vessel lumen and contribute to the progressive increase in pulmonary vascular resistance that can lead to right ventricular failure (reviewed in Ref. ¹). Only a few murine or rodent models recapitulate this pathological feature, eg, mice exposed to ovalbumin or aspergillus² or to schistosomiasis,³ rats treated with the vascular endothelial receptor blocker Sugen 5416, exposed to chronic hypoxia and recovered in room air,⁴ mice that overexpress IL-6 and are subjected to chronic hypoxia,⁵ or mice that overexpress S100A4.⁶ The latter mice, when over 1 year of age, can on rare occasions “spontaneously” develop severe neointimal lesions.⁶ However, these lesions are observed consistently following infection with murine gammaherpesvirus-68 (MHV-68).⁷ S100A4 is also known as metastasin-1 (mts-1), and is a member of the calcium binding family of proteins that clusters on chromosome 1 and that has been related to cancer and inflammation.⁸The S100A4-overexpressing mouse infected with MHV-68 is relevant to clinical PAH. Increased immunoreactivity for S100A4 is observed in vascular lesions in patients with advanced PAH,⁶ and human immunodeficiency virus (HIV) and HHV-8/Kaposi''s sarcoma virus, the human homologue of MHV-68, have been implicated in clinical PAH. Specifically, the viral protein for HHV-8 has been detected in neointimal and plexiform lesions in lung tissues from some,⁹ albeit not all,¹⁰ series of PAH patients.Pulmonary vascular neointimal and plexiform lesions in S100A4 mice are associated with fragmentation of elastic laminae and with heightened activity of a serine elastase.⁷ Fragmentation of PA elastin has been observed in PAs of PAH patients,¹¹ and heightened activity of a serine elastase has been identified in the PA in a variety of experimental forms of PAH^12–15 and in cultured PA smooth muscle cells (SMC).^16–19 Moreover, inhibition of this elastase can attenuate or prevent^12–14 and even reverse¹⁵ experimental pulmonary vascular disease in rodents. In all rodent models where elastase inhibitors were used, the pulmonary vascular lesions were characterized by loss or increased muscularization of normally nonmuscular peripheral arteries at the alveolar wall and duct level, and medial hypertrophy of proximal muscular arteries. Neointimal lesion formation, however, was not present. Elastase inhibition should, however, attenuate these lesions since proliferation and migration of SMC in the neointima are likely the consequences of elastase-mediated release of growth factors from the extracellular matrix^16,17 and activation of growth factor receptors.²⁰We therefore hypothesized, and subsequently demonstrated in this study, that in the S100A4 overexpressing versus C57 mouse, heightened susceptibility of elastin to fragmentation, coupled to elevated serine elastase activity following M1-MHV-68 infection, can contribute to the development of neointimal lesions. We identified the elastase involved as neutrophil elastase (NE) produced by PA SMC, suggesting that it is the endogenous vascular elastase previously related to PAH in other experimental models.^12,16,18,19 Moreover, we showed that NE is produced in significantly greater amounts by cultured murine S100A4 versus C57 PA SMC, and by human PA SMC from IPAH versus control lungs, and we localized NE to neointimal and plexiform lesions in human lung specimens.     

Murine Cerebral Malaria Is Associated with a Vasospasm-Like Microcirculatory Dysfunction,and Survival upon Rescue Treatment Is Markedly Increased by Nimodipine

Brain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM.Cerebral malaria (CM) caused by Plasmodium falciparum claims the lives of nearly 1 million children every year.¹ Despite antimalarial treatment, 10% to 20% of patients die, and one in every four survivors develops neurological sequelae,^2,3 therefore adjunctive therapies are urgently needed. A number of clinical trials addressing potential adjunctive therapies for CM showed no proven benefits and some interventions were even deleterious,⁴ stressing the need for a better understanding of CM pathogenesis to develop effective therapies.An unresolved issue of CM pathogenesis regards the role of brain hemodynamic perturbations and ischemia. Sequestration of parasitized red blood cells (pRBCs) containing mature forms of the parasite in the brain microvasculature is a characteristic postmortem finding in human CM cases⁵ and together with rosetting⁶ and reduced RBC deformability⁷ may result in the obstruction of blood flow potentially leading to ischemia and hypoxia. In vivo studies of the microcirculation in human CM support this mechanism, with direct observation of retinal microvasculature showing impaired perfusion, retinal whitening, vascular occlusion, and ischemia.⁸ Accordingly, microvascular obstruction observed in the rectal mucosa of CM patients was proportional to the severity of the disease.⁹ In addition, hypovolemia, shock and intracranial hypertension, commonly associated with poor outcomes in CM,⁴ reduce tissue perfusion, and tissue hypoxia is one of the likely explanations for the acidosis frequently observed in severe malaria.^7,10 Ischemic damage has also been shown in children with CM and was associated with severe neurological sequelae.¹¹ On the other hand, transcranial Doppler sonography studies showed normal or even increased cerebral blood flow (CBF) velocities^12–15 in large arteries during CM, which associated with microcirculatory obstruction has been suggested to increase cerebral blood volume leading to intracranial hypertension.¹⁶ Alternatively, collateral flow has been proposed as a mechanism to reconcile the findings of normal or increased CBF velocities and impaired perfusion,¹⁷ an interpretation supported by findings of hyperdynamic flow in capillaries adjacent to obstructed vessels.⁹ Interventions that improve cerebral perfusion have been proposed to be beneficial in CM.^8,18The murine model of CM by Plasmodium berghei ANKA (PbA) shares many features with the human pathology,¹⁹ including the presence of multiple brain microhemorrhages and vascular obstruction, although the nature of the sequestered cell (leukocytes) differs. In murine CM, magnetic resonance imaging (MRI) and spectroscopy studies showed the presence of brain edema, decreased CBF, and ischemia.^20,21 Lack of resolution in MRI, however, precludes detailed studies of the microcirculation, which is a major target and player in CM pathogenesis. A few studies have addressed the in vivo microcirculatory changes in murine models of severe malaria,^22–24 however in sites other than the brain (cremaster muscle or skin). In the present work, we used for the first time brain intravital microscopy to follow the dynamic changes in the pial microcirculation during the course of PbA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow, and eventually vascular collapse, events similar to postsubarachnoid hemorrhage (SAH) vasospasm.²⁵ We also show that nimodipine, a calcium channel blocker used to treat post-SAH vasospasm,^25,26 markedly increased survival when given off-label to mice with CM as adjunctive therapy to artemether.     

PTEN,PIK3CA,p-AKT,and p-p70S6K Status: Association with Trastuzumab Response and Survival in Patients with HER2-Positive Metastatic Breast Cancer

Phosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies.Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% to 25% of invasive breast cancers. Patients with HER2-overexpressing tumors experience a shorter time to relapse and shorter overall survival than patients with tumors of normal HER2 levels.^1,2 HER2 overexpression can lead to activation of many downstream signaling molecules, including Ras-Gap, Src, phosphoinositide 3-kinase (PI3K)/AKT, and many other signaling events.^3,4 Trastuzumab (Herceptin; Genentech, CA), a humanized monoclonal antibody that directly targets the extracellular domain of HER2, has a remarkable therapeutic efficacy in treating patients with HER2-expressing metastatic breast cancer (MBC)⁵ and patients with HER2-positive early-stage disease in adjuvant settings.^6,7 Trastuzumab treatment, when combined with chemotherapy, resulted in a significant improvement in patients'' response rate, time to progression, and duration of response.⁸ The underlying mechanisms of trastuzumab''s antitumor activities include, but are not limited to, inducing antibody-dependent cellular cytotoxicity,⁹ inhibiting HER2 extracellular domain cleavage,¹⁰ activating phosphatase and tensin homolog (PTEN),¹¹ and inhibiting PI3K/AKT survival signaling.¹² However, the overall response rate to trastuzumab is low, and almost half of patients with HER2-positive breast cancer exhibit an initial resistance to trastuzumab-based therapy.^11,13 Despite the large amounts of preclinical and clinical data, the causes of trastuzumab resistance are still poorly understood.¹⁴The PI3K pathway, downstream of HER2, plays a central role in regulating a number of cellular processes, such as apoptosis, migration, angiogenesis, cell proliferation, and glucose metabolism, and it is involved in trastuzumab resistance.^15,16 PI3K phosphorylates phosphatidylinositols on the cell membrane, generating phosphatidylinositol-3,4,5-trisphosphate (PIP3) from phosphatidylinositol-4,5-bisphosphate (PIP2). Then, at the cell membrane, PIP3 recruits protein kinases and activates protein kinase B (PKB)/AKT.¹⁷ In breast cancer cells, HER2 overexpression can lead to activation of the PI3K/AKT pathway.¹⁸ The activation of AKT and its downstream signaling have been demonstrated to inhibit cell cycle arrest and block trastuzumab-mediated apoptosis.¹² AKT phosphorylation and AKT kinase activities were found to be increased in trastuzumab-resistant cells, derived from BT474 HER2-overexpressing breast cancer cells, when compared with parental cells.¹⁹ These data provide insight into the trastuzumab-resistance mechanism of PI3K/AKT signaling.¹⁵Aberrations in the components of the PI3K pathway have been reported in most solid tumors, including breast cancer.¹⁶ PTEN is a tumor suppressor that dephosphorylates the D3 position of PIP3 and inhibits the PI3K/AKT pathway.²⁰ Loss of PTEN function as a result of mutation, deletion, or promoter methylation has been reported in nearly 50% of breast cancers.¹¹ In addition, the gene encoding one of the PI3K catalytic subunits, p110α (PIK3CA), has been found to be mutated in about 25% of breast cancers.^21,22,23 Most of the reported mutations are localized to hotspots in exons 9 and 20 of the PIK3CA gene, which result in increased PI3K pathway signaling.^22,24 We previously discovered that PTEN activation is a novel mechanism of trastuzumab antitumor function, and PTEN loss confers trastuzumab resistance in HER2-overexpressing breast cancer cells.¹¹ PTEN loss significantly predicted poor response to trastuzumab-based therapy in a small cohort of HER2-positive patients with MBC.¹¹ Later, it was reported that both low PTEN levels and PI3K-activating PIK3CA mutations contribute to trastuzumab resistance in HER2-overexpressing breast cancer.^25,26 PTEN loss or PIK3CA mutations, which indicate activation of the PI3K pathway, are considered as markers for poor response to trastuzumab in patients with HER2-overexpressing breast cancer.²⁵ On the other hand, some studies found no correlation between PTEN expression and trastuzumab response or survival in patients with HER2-positive breast cancer.^27,28 These contradictory findings prompted us to further investigate the association between PTEN status and clinical outcomes in a large cohort of patients with MBC who were treated with trastuzumab-based therapy. We tested the hypothesis that a comprehensive assessment of PI3K pathway activation status provides biomarkers that can identify patients who may not benefit from trastuzumab-based therapy.     

Biological sample collections from minors for genetic research: a systematic review of guidelines and position papers

Stored tissue samples are an important resource for epidemiological genetic research. Genetic research on biological material from minors can yield valuable information on the development and genesis of early-onset genetic disorders and the early interaction of environmental and genetic factors. The use of such tissue raises some specific ethical and governance questions, which are not completely covered by the discussion on biological materials from adults. We have retrieved 29 guidelines and position papers pertaining to the storage and use of biological tissue samples for genetic research, originating from 27 different organizations. Five documents have an international scope, three have an European scope and 21 have a national scope. We discovered that 11 of these documents did not contain a section on biological materials from minors. The content of the remaining 18 documents was categorized according to four themes: consent, principles of non-therapeutic research on vulnerable populations, ethics committee approval and difference between anonymous and identifiable samples. We found out that these themes are not consistently mentioned by each document, but that documents discussing the same themes were mostly in agreement with their recommendations. However, a systematic reflection on the ethical and policy issues arising from the participation of minors in biobank research is missing.Stored tissue sample collections for genetic research exist in different forms. Some of these collections provide a resource for potentially unlimited genetic research, and gather samples and data from specific populations. An example is the ‘UK biobank''.¹ Other collections are stored for research on a specific disease. Collections that were originally gathered for different purposes, for example blood spot cards for newborn screening, could be reused for genetic research.²Genetic research on biological material from minors and the associated medical records can yield valuable information on the development and genesis of early-onset genetic disorders and the early interaction of environmental and genetic factors. For example, Rasmussen³ describes the incorporation of DNA sample collections into the ‘National Birth Defects Prevention Study'' in the United States to identify the risk factors for birth defects. Studies such as the ‘Avon Longitudinal Study of Parents and Children'' in Bristol (children of the nineties) use genetic, phenotypic and environmental information of 14 000 babies from their conception onwards to study the interaction between these data.⁴An extensive ethical literature exists on the collection, storage and use of biological samples for genetic research. The overwhelming majority of these documents discuss issues of privacy, confidentiality, commercialization and consent.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, research on pediatric data raises specific ethical questions with regard to consent and privacy. For example, who should give consent to the inclusion of tissue and data from children? Is the general requirement that non-therapeutic research can only be done with children if it involves no more than minimal risk, applicable to biobank research? We shall review whether and how guidelines and policy documents discuss children in the context of storing biological samples and DNA for non-therapeutic research.     

Interaction of MCM7 and RACK1 for Activation of MCM7 and Cell Growth

MCM7 is one of the pivotal DNA replication licensing factors in controlling DNA synthesis and cell entry into S phase. Its expression and DNA copy number are some of the most predictive factors for the growth and behavior of human malignancies. In this study, we identified that MCM7 interacts with the receptor for activated protein kinase C 1 (RACK1), a protein kinase C (PKC) adaptor, in vivo and in vitro. The RACK1 binding motif in MCM7 is located at the amino acid 221-248. Knocking down RACK1 significantly reduced MCM7 chromatin association, DNA synthesis, and cell cycle entry into S phase. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate dramatically decreased MCM7 DNA replication licensing and induced cell growth arrest. Activation of PKC induced redistribution of RACK1 from nucleus to cytoplasm and decreased RACK1-chromatin association. The MCM7 mutant that does not bind RACK1 has no DNA replication licensing or oncogenic transformation activity. As a result, this study demonstrates a novel signaling mechanism that critically controls DNA synthesis and cell cycle progression.Miniature chromosome maintenance (MCM) proteins were initially identified from autonomously replicating sequence in Saccharomyces cerevisiae. Mutations of some of these proteins, such as MCM7 or MCM3 result in loss of the large chunk of yeast chromosomes in yeast. MCM7 cDNA encodes a 543-amino acid protein and is ubiquitously expressed in all tissues. A large body of studies indicate that MCM7 is a critical component of DNA replication licensing complex in the yeast and xenopus.^1–4 Some studies suggest that MCM4, MCM6, and MCM7 complex contains DNA helicase activity.^5,6 DNA replication licensing complex is multimeric and phase specific. In yeast, DNA replication licensing proteins, such as MCM2-7 and several replication origin binding proteins, such as Cdc6, germinin, and Cdt1, form DNA replication licensing complex in G1 phase to enable DNA replication and to promote cell cycle entry into S phase. Initial implication of MCM7 involvement in human malignancies came from positive immunostaining of MCM7 in several human malignancies, including endometrial carcinoma,⁷ melanoma,⁸ esophageal adenocarcinoma,⁹ colorectal adenocarcinoma,¹⁰ oral squamous cell carcinoma,¹¹ glioblastoma,¹² and thyroid cancer.¹³ The first study addressing the oncogenic role of MCM7 in prostate cancer came from genome analysis of prostate cancer by performing a genome wide copy number analysis using biotin-labeled genome DNA on Affymetrix U95av2 chip.¹⁴ The DNA copy number of MCM7 was found to increase severalfold accompanied with a concomitant increase of MCM7 mRNA level. Subsequent validation analyses suggest that either copy number and/or protein level increase of MCM7 are associated with prostate cancer relapse and metastasis. Amplification of MCM7 was also found in esophageal carcinoma.⁹ The magnitude of MCM7 amplification correlates with the expression of MCM7, tumor grades, and the aggressiveness of esophageal cancer.⁹ It is presumed that amplification of MCM7 is the driving force of MCM7 overexpression in primary human malignancies. MCM7 is probably the primary target of Rb, the tumor suppressor that controls cell entry into S phase.¹⁵ There is growing evidence that other signaling pathways also regulate MCM7 activity.Receptor for activated protein kinase C 1 (RACK1), was initially identified as an adaptor of several protein kinase C (PKC) isoforms.¹⁶ The binding of RACK1 and PKC anchor PKC to its substrate to initiate second messenger signaling. It is suggested, according to recent studies that RACK1 interacts with a variety of other signaling molecules, including ras-GTPase activating protein,¹⁷ dynamin-1,¹⁸ src,¹⁹ integrins,²⁰ PTPμ,²¹ phosphodiesterase,²² hypoxia induced factor-1,²³ and so forth, that play an important role in several physiological processes, including, growth, hypoxia response, migration, adhesion, and cell differentiation. RACK1 only binds PKC activated by diacylglycerol or phorbol ester, but not quiescent PKC. In this study, we showed that RACK1 binds with MCM7 N-terminus. The MCM7/RACK1 interaction appears essential for DNA replication activity of MCM7.     

Tissue Factor-Deficiency and Protease Activated Receptor-1-Deficiency Reduce Inflammation Elicited by Diet-Induced Steatohepatitis in Mice

Altered hepatic lipid homeostasis, hepatocellular injury, and inflammation are features of nonalcoholic steatohepatitis, which contributes significantly to liver-related morbidity and mortality in the Western population. A collection of inflammatory mediators have been implicated in the pathogenesis of steatohepatitis in mouse models. However, the pathways essential for coordination and amplification of hepatic inflammation and injury caused by steatosis are not completely understood. We tested the hypothesis that tissue factor (TF)-dependent thrombin generation and the thrombin receptor protease activated receptor-1 (PAR-1) contribute to liver inflammation induced by steatosis in mice. Wild-type C57Bl/6J mice fed a diet deficient in methionine and choline for 2 weeks manifested steatohepatitis characterized by increased serum alanine aminotransferase activity, macrovesicular hepatic steatosis, hepatic inflammatory gene expression, and lobular inflammation. Steatohepatitis progression was associated with thrombin generation and hepatic fibrin deposition. Coagulation cascade activation was significantly reduced in low TF mice, which express 1% of normal TF levels. Hepatic triglyceride accumulation was not affected in low TF mice or PAR-1-deficient mice. In contrast, biomarkers of hepatocellular injury, inflammatory gene induction, and hepatic accumulation of macrophages and neutrophils were greatly reduced by TF-deficiency and PAR-1-deficiency. The results suggest that TF-dependent thrombin generation and activation of PAR-1 amplify hepatic inflammation and injury during the pathogenesis of steatohepatitis.Non-alcoholic fatty liver disease (NAFLD) is increasingly appreciated as a hepatic feature of the metabolic syndrome. NAFLD may occur in 25% of the Western population and altered hepatic function increases the risk for developing diseases including diabetes and atherosclerosis.^1,2 The progression of simple hepatic steatosis to the more severe nonalcoholic steatohepatitis (NASH) contributes significantly to liver-related morbidity and mortality.³ Requisite histological features of NASH include macrovesicular hepatic steatosis, evidence of hepatocellular injury, and lobular inflammation.⁴ In a subset of patients with chronic steatohepatitis, stellate cell activation coordinates a fibrogenic response causing fibrosis and cirrhosis.⁵ Of importance, the mechanisms required for the progression of hepatic inflammation during steatohepatitis are not completely understood.Animal models used to define mechanisms of steatohepatitis have used genetic and dietary modification to induce various features of the disease.² In particular, feeding mice a diet deficient in methionine and choline (MCD diet) is an established model to study the progression of steatohepatitis and has been extensively used to study mechanisms of hepatic inflammation and fibrosis. Rodents fed an MCD diet for 2 weeks manifest a defect in hepatic β oxidation resulting in accumulation of triglyceride and the induction of steatohepatitis.^2,6,7 Prolonged feeding (>4 weeks) of the MCD diet activates hepatic stellate cells and increases collagen expression and deposition in the liver. Utilization of the MCD diet model has revealed the contribution of hepatic triglyceride,⁸ various inflammatory mediators,^9,10 nuclear receptors,^11,12 and signaling pathways¹³ in the manifestation of steatohepatitis.An important physiological process disrupted by chronic liver disease is blood coagulation. Several studies have indicated that the progression of liver disease is associated with altered blood coagulation.¹⁴ For example, steatosis in patients with the metabolic syndrome is associated with a shift in the balance of procoagulant and antifibrinolytic factors favoring coagulation.^15–17 This links the progression of NAFLD with increased risk of thrombotic complications associated with vascular disease and the metabolic syndrome. However, it is not clear whether the altered coagulation impacts progression of the liver pathology in patients with NAFLD or NASH.The coagulation cascade is initiated by tissue factor (TF), the transmembrane receptor for coagulation factor VIIa.¹⁸ TF is expressed by the normal liver,¹⁹ albeit at much lower levels compared with other tissues (eg, lung, heart).²⁰ Of importance, potent inducers of TF expression such as bacterial lipopolysaccharide and pro-inflammatory cytokines (eg, tumor necrosis factor [TNF]α, monocyte chemoattractant protein [MCP]-1) are linked to the pathogenesis of NAFLD and NASH in humans and animal models.^21–24 TF-dependent coagulation cascade activation leads to generation of the serine protease thrombin, which cleaves circulating fibrinogen to form fibrin. Thrombin also elicits intracellular signaling by activating the G-protein coupled receptor protease activated receptor-1 (PAR-1).²⁵ This TF–PAR-1 pathway has been shown to increase inflammation in other models of tissue injury.^26–29 However, the contribution of both TF and PAR-1 to coagulation and inflammation during steatohepatitis has not been determined.To this end, we characterized the procoagulant response associated with steatohepatitis induced in mice by a MCD diet. Furthermore, we used mice expressing 1% of normal TF levels (ie, low TF mice³⁰ and PAR-1-deficient mice³¹ to test the hypothesis that TF-dependent thrombin generation contributes to the pathogenesis of murine steatohepatitis by activating PAR-1.     

Differential Effects of Myocilin and Optineurin,Two Glaucoma Genes,on Neurite Outgrowth

Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOC_WT-EGFP, pMYOC_P370L-EGFP, pMYOC_1-367-EGFP, pOPTN_WT-EGFP, and pOPTN_E50K-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTN_WT-EGFP and pOPTN_E50K-EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.Glaucoma, one of the leading causes of irreversible blindness worldwide, is characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. Primary open-angle glaucoma (POAG), the most common form of glaucoma, is frequently associated with increased intraocular pressure (IOP). The IOP is controlled by a balance between the production and outflow of the aqueous humor contained in the anterior chamber. The trabecular meshwork (TM), a specialized eye tissue neighboring the cornea, is the major site for regulation of the aqueous humor outflow.¹Recent studies have revealed that POAG is genetically heterogeneous, caused by several susceptibility genes and environmental factors.² To date, a total of 14 chromosomal loci have been mapped and designated as GLC1A to GLC1N.^3,4 Three candidate genes have been identified that include myocilin as the GLC1A,^5,6 optineurin as the GLC1E,^7,8 and WDR36 as the GLC1G⁹ gene.Myocilin is the first identified gene for both juvenile- and adult-onset POAG.⁶ More than 70 myocilin mutations have been found in a number of families.¹⁰ Patients with glaucoma with myocilin mutation tend to have high IOP.^11,12 Among the myocilin mutations, the Gln368Stop (Q368X) mutation is the most frequent¹⁰ and the Pro370Leu (P370L) mutation is responsible for one of the most severe glaucoma phenotypes.^13–15The human myocilin gene encodes a protein of 504 amino acids, containing a nonmuscle myosin-like domain near the amino (N)-terminus and an olfactomedin-like domain at the carboxyl (C)-terminus.¹⁶ Myocilin interacts with itself and a number of other proteins, mainly through the leucine zipper motif and a coiled-coil region in the myosin-like domain.^17–19 The wild-type myocilin is a secreted protein. Mutants with mutations in the olfatomedin-like domain, however, are not secreted. They are retained in the cells, aggregating to cause endoplasmic reticulum stress.^20–22Optineurin, identified in 2002,⁸ is a gene that links principally to normal tension glaucoma, a subtype of POAG.²³ Optineurin mutations were noted to vary with ethnic background.^24,25 The Glu50Lys (E50K) mutation, found in Caucasian and Hispanic populations,²⁵ seems to be associated with a more progressive and severe disease in patients with normal tension glaucoma.²⁶The human optineurin gene codes for a 577-amino acid protein that contains multiple coiled-coil domains and a C-terminal zinc finger.^27,28 The optineurin protein from different species has high amino acid homology.²⁸ The amino acid 50 glutamic acid residue is conserved in mice, rats, chickens, and cows.²³ Optineurin is expressed in many tissues including the brain and the retina.^27,28Despite intense interest, the roles of myocilin and optineurin in cellular functions still remain largely undefined. Because glaucoma is a group of diseases known as progressive optic neuropathies, we undertook the current study to investigate the effects of myocilin and optineurin on neurite outgrowth. We demonstrated that overexpression of wild-type myocilin or P370L and Q368X mutants caused an inhibition of neurite outgrowth, whereas forced expressed wild-type or E50K optineurin did not result in any changes in neuronal rat pheochromocytoma PC12²⁹ and RGC5³⁰ cells.     

Organic Cation Transporter 2 Mediates Cisplatin-Induced Oto- and Nephrotoxicity and Is a Target for Protective Interventions

The use of the effective antineoplastic agent cisplatin is limited by its serious side effects, such as oto- and nephrotoxicity. Ototoxicity is a problem of special importance in children, because deafness hampers their language and psychosocial development. Recently, organic cation transporters (OCTs) were identified in vitro as cellular uptake mechanisms for cisplatin. In the present study, we investigated in an in vivo model the role of OCTs in the development of cisplatin oto- and nephrotoxicity. The functional effects of cisplatin treatment on kidney (24 hours excretion of glucose, water, and protein) and hearing (auditory brainstem response) were studied in wild-type and OCT1/2 double-knockout (KO) mice. No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice. Comedication of wild-type mice with cisplatin and the organic cation cimetidine protected from ototoxicity and partly from nephrotoxicity. For the first time we showed that OCT2 is expressed in hair cells of the cochlea. Furthermore, cisplatin-sensitive cell lines from pediatric tumors showed no expression of mRNA for OCTs, indicating the feasibility of therapeutic approaches aimed to reduce cisplatin toxicities by competing OCT2-mediated cisplatin uptake in renal proximal tubular and cochlear hair cells. These findings are very important to establish chemotherapeutical protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.Cisplatin, one of the most effective and potent anticancer drugs, is used in the treatment of a wide variety of both pediatric and adult malignancies.¹ When combined with bleomycin and etoposide, cisplatin is considered to be curative treatment for testicular cancer.² However, the chemotherapeutic use of cisplatin is limited by serious side effects, such as nephrotoxicity and ototoxicity, sometimes requiring a reduction in dose or discontinuation of treatment. Even though nephrotoxicity can be managed with some success by concomitant use of i.v. hydration, it is still a major factor that limits the administration and efficacy of cisplatin in cancer therapy.³ Ototoxicity remains an unresolved clinical problem, especially in infants and younger children, where it leads to a considerable risk of delayed language development because of impaired perception of higher frequency consonant sounds that is of great importance in the presence of background noise. This may have devastating consequences for a young child''s social and educational development.⁴The molecular mechanisms of cellular cisplatin toxicity have been investigated intensively.² The ototoxic effects of cisplatin include loss of outer hair cells, blebbing of outer and inner hair cells, degeneration of the stria vascularis, and a decrease in the number of spiral ganglion cells.⁵ In the kidney, cisplatin highly accumulates in cells of the terminal proximal tubule and of the distal nephron, where it causes either apoptosis or necrosis, depending on exposure time and concentration.² Recently, some attention has been paid to the role of specific cellular uptake processes in cisplatin toxicity. The copper transporter (Ctr)1 seems to be involved in cisplatin accumulation in tumor cells⁶ as well as in the renal epithelial cells.⁷ However, the cisplatin uptake into renal tubular cells is also mediated by organic cation transporter (OCT)2 and the function of OCT2 is critically involved in the development of toxicity.^8,9 In humans, OCT2 (hOCT2) is highly expressed at the basolateral side of all three segments of the proximal tubule.¹⁰ It represents a subtype of three OCTs that belong to the SLC22 transporter family. OCTs are highly expressed in intestine, liver, and/or kidney and play a pivotal role in drug absorption and excretion.¹¹ They are polyspecific, electrogenic uniporters that may operate in both directions. Because the membrane potential provides part of the driving force, they preferably mediate organic cation uptake into cells at normal membrane potential.¹² OCT2 has been proposed as target for protective therapeutic interventions accompanying cisplatin treatment. In vitro studies showed that administration of cisplatin together with a second substrate of organic cation transporters decreases cellular cisplatin accumulation protecting renal cells from cisplatin toxicity.^8,9In the present study, we demonstrated in an in vivo model that OCT2 plays a pivotal role in the development of cisplatin induced oto- and nephrotoxicity. For the first time we showed that cisplatin ototoxicity is linked to the expression of OCT2 in hair cells of the cochlea. Furthermore, the feasibility of a therapeutic approach aimed to reduce cisplatin toxicities has been demonstrated in vivo. These findings are very important to establish chemotherapeutic protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.     

Genetic testing in asymptomatic minors Background considerations towards ESHG Recommendations

Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors, the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This paper has served as a background document in preparation of the development of the policy recommendations of the Public and Professional Committee of the European Society of Human Genetics. This background paper first discusses some general considerations with regard to the provision of genetic tests to minors. It discusses the concept of best interests, participation of minors in health-care decisions, parents'' responsibilities to share genetic information, the role of clinical genetics and the health-care system in communication within the family. Second, it discusses, respectively, the presymptomatic and predictive genetic testing for adult-onset disorders, childhood-onset disorders and carrier testing.Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors,^{1, 2} the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This background paper was preceded by an in-depth research on the topic by Eurogentest.³ Eurogentest (http://www.eurogentest.org aims to develop the necessary infrastructure, tools, resources, guidelines and procedures that will structure, harmonize and improve the overall quality of all the EU genetic services at the molecular, cytogenetic, biochemical and clinical level.⁴ Attention has also been paid to the provision of appropriate counselling related to genetic testing, the education of patients and professionals, as well as to the ethical, legal and social issues surrounding testing. The focus of the ethics unit of Eurogentest was oriented towards the study of the ethical issues related to genetic testing in minors. This work was the starting point for this background paper, which has been prepared and supported by different types of evidence. First, research has been performed on the existing recommendations regarding predictive genetic testing in minors¹ and carrier testing,² with the intention of identifying areas of agreement and disagreement. Second, the literature on medico–ethical and medico–legal aspects of predictive genetic testing in minors,⁵ carrier testing,^{6, 7} the position of minors⁸ and patient rights⁹ was studied. Third, a systematic literature review was performed to gather information regarding the attitudes of the different stakeholders (minors, health-care professionals, parents and relatives of the affected individuals) towards genetic testing in asymptomatic minors.^{10, 11} Fourth, the attitudes of European clinical geneticists regarding genetic testing in asymptomatic minors were gathered.^{12, 13, 14}In 2007, contacts were made with the Public and Professional Policy Committee of the European Society of Human Genetics with the aim of developing policy recommendations on the issue. On the basis of a decision of the PPPC meeting during the ESHG conference in Nice (June 2007), an ad hoc committee, consisting of Pascal Borry (Eurogentest), Kris Dierickx (Eurogentest), Angus Clarke, Gerry Evers-Kiebooms (PPPC) and Martina Cornel (PPPC), was created. This ad hoc committee met on 15 November 2007 to discuss a first draft of a background paper and recommendations that were prepared by Pascal Borry under the supervision of Kris Dierickx. A revised version was discussed during a PPPC meeting in Amsterdam (April 2008) and Barcelona (June 2008). In order not to repeat issues that have been discussed elsewhere, reference will often be made to the above-referenced publications.     

Colony-Stimulating Factor-1 Promotes Kidney Growth and Repair via Alteration of Macrophage Responses

Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.Macrophages are versatile cells that have been long recognized as immune effectors where their recruitment to sites of injury is a fundamental feature of inflammation. Although their role in host defense has been well documented, macrophages and their precursors are also important during embryogenesis, normal tissue maintenance, and postnatal organ repair.^1,2 Almost all developing organs contain a population of resident monocytes that infiltrate very early during organogenesis and persist throughout adult life.^3–6 In addition to their phagocytic capabilities during tissue remodeling-associated apoptosis,^5,7 fetal macrophages have many trophic effects that promote tissue and organ growth.^6,8,9Colony-stimulating factor (CSF)-1 controls the differentiation, proliferation, and survival of macrophages by binding to a high-affinity cell-surface tyrosine kinase receptor (CSF-1R), encoded by the c-fms proto-oncogene that is expressed on macrophages and their progenitors.⁶ CSF-1 is critical for both adult and embryonic macrophage development. This is manifested by multiple organ growth deficiencies observed in osteopetrotic (Csf1^op/Csf1^op) mice that have a spontaneous mutation in the csf-1 gene. These mice show growth restriction and developmental abnormalities of the bones, brain, and reproductive and endocrine organs,^10–13 a phenotype that can be rescued by injection of exogenous CSF-1 or insertion of a csf-1 transgene.^14–16In adult organs, there is considerable heterogeneity of monocytes and macrophages with distinct subsets defined by phenotype, function, and the differential expression of cell surface markers.^17–19 Subpopulations of macrophages directly contribute to wound healing and tissue repair, supporting the concept that some macrophage phenotypes can promote organ regeneration after a pro-inflammatory state of injury.²⁰ The concept of macrophage polarization states has emerged; the M1 “classically activated” pro-inflammatory cell type apparently opposed by an M2 “alternatively activated” immune regulatory macrophage.¹⁸ In general, these two states are thought to be analogous to the opposing T helper 1 and T helper 2 immune responses, although in both cases this model is probably too simplistic. Functionally, it is more likely that distinct subpopulations of macrophages may exist in the same tissue and play critical roles in both the injury and recovery phases of inflammatory scarring.²⁰Our previous study provided evidence that the addition of CSF-1 to a developing murine kidney promotes a growth and differentiation response that is accompanied by increased numbers of macrophages.³ Furthermore, with the use of expression profiling we demonstrated that fetal kidney, lung, and brain macrophages share a characteristic gene expression profile that includes the production of factors important in the suppression of inflammation and the promotion of proliferation.³ Embryonic macrophages appear to play a positive trophic role that may have parallel reparative functions in many adult tissues undergoing repair and cellular replacement.^1,20 A number of studies have suggested that infiltrating macrophages along with the trophic factors they release participate in tissue repair of the kidney,^20–22 brain,²³ skin,^24,25 lung,²⁶ liver,²⁷ heart,²⁸ gastrointestinal tract,^29,30 and skeletal muscle.^31,32 Indeed, the pleiotrophic roles for CSF-1 in reproduction, development of multiple organ systems, and maternal-fetal interactions during pregnancy by macrophage-mediated processes have also been well defined.^2,33,34To determine the physiological relevance of CSF-1 as a component of the mammalian growth regulatory axis, CSF-1 was administered to neonatal mice. We report that CSF-1 administration to newborn mice increased body weight and kidney weight and volume and was associated with increased numbers of macrophages. Our results also establish that CSF-1 injection into mice after ischemia-reperfusion (IR) injury promoted endogenous repair with characteristic rapid re-epithelialization of the damaged tubular epithelium, leading to functional recovery. Flow cytometric and gene expression analyses were used to delineate the macrophage profile present in the kidneys during the early and resolution phase of IR injury with and without CSF-1 therapy. We thus provide evidence that CSF-1 recruits macrophages to the reparative site and influences their phenotype, partly through an insulin-like growth factor (IGF)-1 signaling response. Therefore, macrophages under the stimulus of CSF-1 in an acute setting of renal disease markedly accelerate renal cell replacement and tissue remodeling while attenuating downstream interstitial extracellular matrix accumulation.     

Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage

Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.Recently, adult stem cells have been identified in several mature tissues, such as the adult intestine,¹ skin,² muscle,³ blood,⁴ and the nervous system^5–7 A stem cell is an undifferentiated cell that is defined by its ability to both self-renew and to produce mature progeny cells.⁸ Stem cells are classified based on their developmental potential as totipotent, pluripotent, oligopotent, and unipotent. Adult somatic stem cells were originally thought to be tissue specific and only able to give rise to progeny cells corresponding to their tissue of origin. Recent studies, however, have shown that adult mammalian stem cells are able to differentiate across tissue lineage boundaries,^9,10 although this “plasticity” of adult somatic stem cells remains controversial.Stem cell subpopulations (“side-population” (SP) cells) have been identified in many mammals, including humans, based on the ability of these cells to efflux the fluorescent dye Hoechst 33342.¹¹ Recent evidence suggests that the SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/Bcrp1.¹² Most recently, established malignant cell lines, which have been maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation.¹³The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding, and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely responsible for endometrial regeneration.¹⁴ Rare populations of human endometrial epithelial and stromal colony-forming cells¹⁵ and SP cells^16,17 have been identified. Although coexpression of CD146 and PDGFRβ isolates a population of mesenchymal stem like cells from human endometrium,¹⁸ specific stem cell markers of endometrium remain unclear. Recently, Gotte et al¹⁹ demonstrated that the adult stem cell marker Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells. Furthermore, they showed that telomerase and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue, compared with secretary endometrium, suggesting the concept of a stem cell origin of endometriosis and endometrial carcinoma.Recent evidence suggests that cancer stem-like cells exist in several malignant tumors, such as leukemia^20,21 breast cancer,²² and brain tumors,²³ and that these stem cells express surface markers similar to those expressed by normal stem cells in each tissue.^20,24Development of endometrial carcinoma is associated with a variety of genetic alterations. For example, increased expression and activity of telomerase^25,26 and frequent dysregulation of signaling pathways have been observed in endometrial carcinoma. Some of these pathways are important determinants of stem cell activity (Wnt-β-catenin and PTEN).^27–29 These suggest a stem cell contribution to endometrial carcinoma development.Recently, we isolated SP cells from the human endometrium. These SP cells showed long-term proliferating capacity in cultures and produced both gland and stromal-like cells. Additionally, they were able to function as progenitor cells.¹⁶ In this study, we isolated and characterized SP cells from human endometrial cancer cells and from rat endometrial cells expressing oncogenic [¹²Val] human K-Ras protein and demonstrated their cancer stem-like cell phenotypes.     

Knockdown of Osteopontin Reduces the Inflammatory Response and Subsequent Size of Postsurgical Adhesions in a Murine Model

Adhesions between organs after abdominal surgery remain a significant unresolved clinical problem, causing considerable postoperative morbidity. Osteopontin (OPN) is a cytokine up-regulated after cell injury and tissue repair. Our previous studies have shown that blocking OPN expression at sites of cutaneous wounding resulted in reduced granulation tissue and scarring. We hypothesize that it may be possible to similarly reduce inflammation-associated fibrosis that causes small-bowel adhesions after abdominal surgery. By using a mouse model, we deliver OPN antisense oligodeoxynucleotides via Pluronic gel to the surface of injured, juxtaposed small bowel and show a significant reduction of inflammatory cell influx to the developing adhesion and a dramatic reduction in the resulting adhesion size. A significant reduction in α-smooth muscle actin expression and collagen deposition within the mature adhesion is also demonstrated. We see no impact on mortality, and the healing of serosal injury to intact bowel appeared normal given the reduced inflammatory response. Our studies suggest that dampening OPN levels might be a potentially important target for anti-adhesion therapeutics.The peritoneum is an extensive and complex organ consisting of a layer of mesothelial cells lining the peritoneal cavity and all organs within it.¹ One of the main functions of the peritoneum is to allow friction-free movement between abdominal viscera and the peritoneal wall.² Any surgery that breaches the peritoneal lining causes injury to the peritoneum, which responds by raising inflammatory signals that attract innate immune cells in parallel with a wound repair response and subsequent fibrosis.^3–5 This almost invariably results in permanent peritoneal adhesion formation.⁶ The result can be tethering of adjacent small-bowel loops that may lead to abdominal pain⁷ and/or bowel obstruction,⁸ which is a significant cause of postoperative morbidity in clinical practice. Readmission rates secondary to adhesional complications are as high as 5% to 10% after abdominal surgery.^9,10 Adhesion prevention options in clinical practice are limited to either barrier methods¹¹ or flotation fluids,¹² which use the concept of keeping damaged peritoneal surfaces separated during their healing process; however, these options are of limited effectiveness.^13,14 Pathophysiological manipulation of the cascade events leading to fibrosis has been investigated,^15–18 but none has led to a clinically usable product. Herein, we investigate whether therapeutic strategies used to block scar formation after skin healing might also be effective during peritoneal repair. Microarray studies of wound tissues from wild-type mice versus PU.1 mice (lacking neutrophils, macrophages, and mast cells) reveal an inflammation-dependent gene, osteopontin (OPN), that is expressed by wound granulation tissue fibroblasts, coincident with a skin wound inflammatory response.^19,20 PU.1 mice heal skin wounds without the standard inflammatory cascade, which results in less fibrosis and scarring at the healed wound site.¹⁹ OPN acts both as a secreted chemokine-like protein and as part of an intracellular signaling complex.²¹ It plays key roles in several processes associated with tissue repair, including cell adhesion, migration, and survival.^21,22 Short-term local knockdown of OPN in cutaneous wounds leads to decreased granulation tissue and reduced scar formation.²³ In this study, we investigate whether these effects are transferable to peritoneal repair and also might block i.p. fibrosis.     

Non-invasive prenatal testing: ethical issues explored

This paper explores the ethical implications of introducing non-invasive prenatal diagnostic tests (NIPD tests) in prenatal screening for foetal abnormalities. NIPD tests are easy and safe and can be performed early in pregnancy. Precisely because of these features, it is feared that informed consent may become more difficult, that both testing and selective abortion will become ‘normalized'', and that there will be a trend towards accepting testing for minor abnormalities and non-medical traits as well. In our view, however, the real moral challenge of NIPD testing consists in the possibility of linking up a technique with these features (easy, safe and early) with new genomic technologies that allow prenatal diagnostic testing for a much broader range of abnormalities than is the case in current procedures. An increase in uptake and more selective abortions need not in itself be taken to signal a thoughtless acceptance of these procedures. However, combining this with considerably enlarging the scope of NIPD testing will indeed make informed consent more difficult and challenge the notion of prenatal screening as serving reproductive autonomy. If broad NIPD testing includes later-onset diseases, the ‘right not to know'' of the future child will become a new issue in the debate about prenatal screening. With regard to the controversial issue of selective abortion, it may make a morally relevant difference that after NIPD testing, abortion can be done early. A lower moral status may be attributed to the foetus at that moment, given the dominant opinion that the moral status of the foetus progressively increases with its development.Since the discovery of cell-free foetal DNA/RNA (cffDNA/RNA) in maternal plasma in 1997,¹ the possibility to use this cffDNA/RNA for non-invasive prenatal diagnosis (NIPD) has been investigated many times.^{2, 3, 4, 5, 6} cffDNA/RNA can be obtained from a maternal blood sample, as early as 4 weeks of gestation,⁷ but currently only reliably so from 7 weeks of gestation.⁴ This development holds the promise of NIPD testing early in pregnancy and without the small, but significant risk of foetal loss that the current invasive procedures of chorionic villus sampling (CVS) and amniocentesis (AP) carry. NIPD testing for the determination of a Y-signal for pregnancies at risk of X-linked disorders and for diagnosis of Rhesus factor status in RhD-negative women is now being translated into clinical practice.⁴ In many European countries, discussion about broader applications of NIPD testing can be expected in the coming years.^{8, 9} The feasibility of NIPD for trisomy 21, 13 and 18 has already been shown,² but large-scale independent studies are still needed. Sex-chromosomal abnormalities (eg, Turner syndrome (X0) and triple X syndrome (XXX)) could in principle be diagnosed by NIPD testing as well,⁴ if reliable quantitative tests become available in the future and the maternal ‘background'' can be excluded from testing. Even if accurate NIPD testing for the mentioned abnormalities becomes possible, the clinical utility of the test remains to be assessed. This includes balancing the benefits to the harms also with regard to its psychological, ethical, legal, social and economic implications.^{10, 11} The possible ethical implications of NIPD as a new approach to prenatal testing have so far been reviewed in a few publications.^{4, 8, 9, 12, 13, 14, 15, 16, 17} Apart from clear benefits related to avoiding the miscarriage risk of present invasive methods, important potential drawbacks have been mentioned as well. For one thing, proper counselling and informed consent is argued to become more challenging when offering NIPD testing. Moreover, there is a concern that the ease and safety of NIPD may lead to prenatal screening being increasingly conceived as a matter of course, both by those making the offer and by the women undergoing the test. Related to this is the concern that selective abortion of foetuses with minor abnormalities, the wrong sex or unwanted paternity, will become normalized.This paper aims to expand and refine these ethical evaluations and will add some new ethical perspectives with regard to possible implications of NIPD at present and in the future.In our view, it is not so much the fact that foetal material used for prenatal testing can be obtained early and non-invasively (allowing easy and safe testing) that would lead to moral challenges. Rather, it is the fact that a technology with these features would be open to being used for testing a potentially much broader range of abnormalities than those included in the presently used method of microscopic chromosome analysis (karyotyping).Although NIPD testing can also be applied in high genetic-risk families and for the management of pregnancy, the focus of this paper will primarily be on the application of NIPD testing in the screening context. The reason for this focus on prenatal screening is that in the near future, the question if, and if so, in what way NIPD testing is to be applied within prenatal screening strategies should be considered and discussed by policy makers, health care professionals and society at large.To avoid confusion, a preliminary remark is needed on terminology. In medicine, ‘screening'' is often used as referring to a kind of test for risk assessment or disease discovery. However, after the convention in normative and regulatory discourse, we will use ‘screening'' as referring to any systematic and unsolicited offer of predictive testing (using whatever types of test) involving individuals who themselves have no reason (yet) to seek medical help for the condition in question.¹⁸ In this broader sense, screening stands in contrast to ‘diagnosis'' as testing on indication.