Treatment of hepatitis C virus infection with direct-acting antivirals plus ribavirin eliminates viral RNA from peripheral blood mononuclear cells and reduces virologic relapse in diverse hepatic parenchymal changes.

Elimination of hepatitis C virus (HCV) may fail, leading to a non-response outcome because of inappropriate testing for viral RNA in peripheral blood mononuclear cells (PBMCs). Sequelae of HCV genotype 4 therapy with sofosbuvir and daclatasvir ± ribavirin were assessed in our study at the 12th week after end of treatment (EOT) by screening for viral genomic RNA in serum and PBMCs with correlation to hepatic parenchymal changes. We recruited 102 out of 2165 patients who had received sofosbuvir/daclatasvir, either alone (n = 1573) or together with ribavirin (n = 592). Subjects were classified into three groups based on testing by single-step reverse transcription PCR: group I, HCV negative in both serum and PBMCs (n = 25); group II, HCV positive in PBMCs only (n = 52); and group III, HCV positive in both serum and PBMCs (n = 25). Groups I and II (n = 77) were selected out of 2102 (every 27th subject), while group III (n = 25) were selected from every second or third serologic relapse (n = 63). The pre-sampling population (n = 2165) showed sustained virologic response (SVR) in 33.21%; serologic relapse in 2.91%; HCV RNA only in PBMCs (66.79%) compared to serologic relapses and potential cure (P < 0.0001); higher serologic (38 out of 63, P = 0.03210) and cellular (36 out of 52, P = 0.0002) relapses in dual therapy than in triple therapy. The post-sampling population (n = 102) showed more HCV relapses in dual (50 out of 60) than in triple (27 out of 42) therapy (P = 0.0351); increased HCV antisense RNA strand in relapses compared to positive-sense strands alone (P < 0.001); and significant SVR events in undetectable (15 out of 31) compared to early (10 out of 55, P = 0.0058) and cirrhotic liver tissue changes (0 out of 16, P = 0.0006). In summary, HCV treatment with sofosbuvir/daclatasvir is followed by higher rates of serologic and intracellular viral RNA relapse than treatment with sofosbuvir/daclatasvir plus ribavirin. Cellular and serum viral RNA relapses are accompanied by HCV-induced hepatic pathology. An increased SVR with no detectable liver tissue changes was observed after triple therapy due to elimination of HCV RNA from PBMCs.

Creatine Supply Attenuates Ischemia-Reperfusion Injury in Lung Transplantation in Rats.

Ischemia-reperfusion injury (IRI) is one of the factors limiting the success of lung transplantation (LTx). IRI increases death risk after transplantation through innate immune system activation and inflammation induction. Some studies have shown that creatine (Cr) protects tissues from ischemic damage by its antioxidant action. We evaluated the effects of Cr supplementation on IRI after unilateral LTx in rats. Sixty-four rats were divided into four groups: water + 90 min of ischemia; Cr + 90 min of ischemia; water + 180 min of ischemia; and Cr + 180 min of ischemia. Donor animals received oral Cr supplementation (0.5 g/kg/day) or vehicle (water) for five days prior to LTx. The left lung was exposed to cold ischemia for 90 or 180 min, followed by reperfusion for 2 h. We evaluated the ventilatory mechanics and inflammatory responses of the graft. Cr-treated animals showed a significant decrease in exhaled nitric oxide levels and inflammatory cells in blood, bronchoalveolar lavage fluid and lung tissue. Moreover, edema, cell proliferation and apoptosis in lung parenchyma were reduced in Cr groups. Finally, TLR-4, IL-6 and CINC-1 levels were lower in Cr-treated animals. We concluded that Cr caused a significant decrease in the majority of inflammation parameters evaluated and had a protective effect on the IRI after LTx in rats.

Copper accumulation and the effect of chelation treatment on cerebral amyloid angiopathy compared to parenchymal amyloid plaques.

Accumulation of fibrillar amyloid ß-protein (Aß) in parenchymal plaques and in blood vessels of the brain, the latter condition known as cerebral amyloid angiopathy (CAA), are hallmark pathologies of Alzheimer's disease (AD) and related disorders. Cerebral amyloid deposits have been reported to accumulate various metals, most notably copper and zinc. Here we show that, in human AD, copper is preferentially accumulated in amyloid-containing brain blood vessels compared to parenchymal amyloid plaques. In light of this observation, we evaluated the effects of reducing copper levels in Tg2576 mice, a transgenic model of AD amyloid pathologies. The copper chelator, tetrathiomolybdate (TTM), was administered to twelve month old Tg2576 mice for a period of five months. Copper chelation treatment significantly reduced both CAA and parenchymal plaque load in Tg2576 mice. Further, copper chelation reduced parenchymal plaque copper content but had no effect on CAA copper levels in this model. These findings indicate that copper is associated with both CAA deposits and parenchymal amyloid plaques in humans, but less in Tg2576 mice. TTM only reduces copper levels in plaques in Tg2576 mice. Reducing copper levels in the brain may beneficially lower amyloid pathologies associated with AD.

Kaempferol Treatment after Traumatic Brain Injury during Early Development Mitigates Brain Parenchymal Microstructure and Neural Functional Connectivity Deterioration at Adolescence.

Targeting mitochondrial ion homeostasis using Kaempferol, a mitochondrial Ca2+ uniporter channel activator, improves energy metabolism and behavior soon after a traumatic brain injury (TBI) in developing rats. Because of broad TBI pathophysiology and brain mitochondrial heterogeneity, Kaempferol-mediated early-stage behavioral and brain metabolic benefits may accrue from diverse sources within the brain. We hypothesized that Kaempferol influences TBI outcome by differentially impacting the neural, vascular, and synaptic/axonal compartments. After TBI at early development (P31), functional magnetic resonance imaging and diffusion tensor imaging (DTI) were applied to determine imaging outcomes at adolescence (2 months post-injury). Vehicle and Kaempferol treatments were made at 1, 24, and 48 h post-TBI, and their effects were assessed at adolescence. A significant increase in neural connectivity was observed after Kaempferol treatment as assessed by the spatial extent and strength of the somatosensory cortical and hippocampal resting-state functional connectivity (RSFC) networks. However, no significant RSFC changes were observed in the thalamus. DTI measures of fractional anisotropy (FA) and apparent diffusion coefficient, representing synaptic/axonal and microstructural integrity, showed significant improvements after Kaempferol treatment, with highest changes in the frontal and parietal cortices and hippocampus. Kaempferol treatment also increased corpus callosal FA, indicating measurable improvement in the interhemispheric structural connectivity. TBI prognosis was significantly altered at adolescence by early Kaempferol treatment, with improved neural connectivity, neurovascular coupling, and parenchymal microstructure in select brain regions. However, Kaempferol failed to improve vasomotive function across the whole brain, as measured by cerebrovascular reactivity. The differential effects of Kaempferol treatment on various brain functional compartments support diverse cellular-level mitochondrial functional outcomes in vivo.

The softness of tumour-cell-derived microparticles regulates their drug-delivery efficiency.

Extracellular microparticles (MPs) can function as drug-delivery vehicles for anticancer drugs. Here, we show that the softness of MPs derived from tumour-repopulating cells (TRCs) isolated from three-dimensional fibrin gels enhances the MPs' drug-delivery efficiency. We found that, compared with MPs derived from tumour cells cultured in conventional tissue-culture plastic, TRC-derived MPs intravenously injected in tumour-xenograft-bearing mice showed enhanced accumulation in tumour tissues, enhanced blood-vessel crossing and penetration into tumour parenchyma, and preferential uptake by highly tumorigenic TRCs. We also show that the cytoskeleton-related protein cytospin-A plays a critical role in the regulation of TRC-derived MP softness. The modulation of the mechanical properties of TRC-derived MPs could aid the efficiency of delivery of anticancer drugs.

Effects of Eucalyptol in respiratory system mechanics on acute lung injury after exposure to short-term cigarette smoke.

Eucalyptol is a compound that has demonstrated antioxidant, anti-inflammatory and bronchodilator effects, but there are no investigations about the effects of this constituent on the respiratory system mechanics in relation to acute lung injury caused by short-term cigarette smoke (CS) exposure. In view of the above, this work investigated the effects of Eucalyptol on the mechanics of the respiratory system of mice in short-term CS exposure. For this, we used data from respiratory mechanics in vivo, and histopathology and lung parenchymal morphometry analysis in vitro. The experiments were performed on C57black/6 mice divided into 5 groups. One group exposed to ambient air (AA + T), and another to cigarette smoke (CS + T) for 5 consecutive days and treated with 1% Tween 80 solution. The other groups were exposed to cigarette smoke for 5 consecutive days, and treated with Eucalyptol at doses of 30 mg/kg (CS + E30), 100 mg/kg (CS + E100), 300 mg/kg (CS + E300). Our results demonstrated significant changes in all variables of respiratory mechanics and lung parenchyma morphometry analyzed for the AA + T group compared to the CS + T group, confirming the establishment of the lesion induced by exposure to cigarette smoke. We also observed that mice treated with Eucalyptol orally at a dose of 300 mg/kg (CS + E300) showed improvement in all variables compared to the group exposed to cigarette smoke and treated with 1% Tween 80 (CS + T) demonstrating the effectiveness of Eucalyptol in preventing lung injury induced by exposure to CS. In conclusion, our results demonstrated that the Eucalyptol was able to prevent the acute lung injury in mice submitted to short-term cigarette smoke exposure.

Intranasal instillation of distilled water, hypertonic saline and sodium bicarbonate promotes redox imbalance and acute lung inflammation in adult mice.

Bronchial obstruction, caused by retained secretions, is often treated by the administration of mucoactive agents including distilled water, saline, hypertonic saline, and sodium bicarbonate. However, the inflammatory effect of these solutions on the lungs remains unclear. This study evaluated the instillation effects of different solutions on oxidative stress and lung inflammatory response in C57BL/6 mice. Fifty C57BL/6 mice were divided into 5 groups: control (CG); distilled water (DWG), hypertonic saline (HSG), saline (SG) and sodium bicarbonate (SBG). CG was exposed to ambient air while DWG, HSG, SG and SBG had 50 µl of respective solutions administered intranasally for 5 consecutive days. Twenty-four hours after the last intranasal instillation, all animals were euthanized for subsequent analysis. All solutions promoted increased recruitment of inflammatory cells to the lung compared to controls. Superoxide dismutase activity was lower in HSG compared to all other groups; catalase activity was reduced in SG, while it increased in SBG and DWG compared to CG. Finally, there was an increase in the inflammatory markers TNF-α, CCL2 and IFN-γ in DWG compared to CG, SG and HSG. In conclusions, the intranasal instillation of different solutions promotes redox imbalance and inflammation on lungs of adult mice.

Multisite Injection of Bioengineered Hepatic Units from Collagen Hydrogel and Neonatal Liver Cells in Parenchyma Improves Liver Cirrhosis.

Intrahepatic transplantation of hepatocytes/progenitor cells remodels or repopulates livers resulting in improved hepatic function, which represents a promising treatment option for liver diseases, but poor engraftment of transplanted liver cells in livers has been the major obstacle for exerting therapeutic efficacy on liver diseases. We herein demonstrated great potential of multisite injection of bioengineered hepatic units generating from collagen hydrogel and neonatal liver cells (MSI) in promoting engraftment, survival of liver cells, and the therapeutic potential of liver diseases in livers. The MSI allowed transplantation of large number of liver cells into parenchyma in one time. Superiorly, the MSI promoted the engraftment and survival of liver cells, and generated large, dispersively distributed tissue masses whose sizes were positively correlated with the number of injection sites in livers. Consistently, the MSI improved liver cirrhosis as evidenced by the reduction of collagen matrix deposit, improved liver architecture, and hepatic function in the MSI group compared to that of the nontreated control. These data indicate that the MSI represents a novel, improved bioengineered strategy that can exert therapeutic efficacy on liver diseases.

Characterization of changes in intrahepatic immune cell populations during HCV treatment with sofosbuvir and ribavirin.

Treatment of chronic hepatitis C virus (HCV) infection with direct-acting antivirals (DAAs) results in a sustained virologic response (SVR) in most patients. While highly efficacious, ~3%-5% of patients do not achieve SVR despite having virus that appears susceptible. It is unclear whether host factors contribute to treatment failures, although innate and adaptive immunity may play a role. Previous studies showed that after DAA treatment, the composition of intrahepatic immune cells does not normalize relative to healthy volunteers, even in cases where SVR is achieved. We used paired pre- and post-treatment liver biopsies from 13 patients treated with sofosbuvir and ribavirin, 4 of whom relapsed, to analyse intracellular immune changes during DAA treatment and explore correlations with inflammation and treatment outcome. We performed single marker immunohistochemistry followed by electronic image capture, manual annotation of parenchymal and non-parenchymal regions, and quantitative image analysis. The predominant cellular change during treatment was a decrease in CD8+ cellular density in both parenchymal and non-parenchymal regions. CD68+ Kupffer cell density correlated with hepatic inflammation (AST, ALT) pre-treatment, but did not change during treatment. CD4+ cellular density decreased in non-parenchymal regions and, intriguingly, was lower pre-treatment in subjects who eventually relapsed. Other cellular markers (CD56, CD20), as well as markers of apoptosis (TIA-1) and activated stellate cells, did not change significantly during treatment or differ by treatment outcome. The predominant intrahepatic cellular change during DAA treatment of chronic HCV infection is a reduction in CD8+ cellular density, but this did not correlate with treatment outcome.

Vesicular acetylcholine transport deficiency potentiates some inflammatory responses induced by diesel exhaust particles.

Endogenous acetylcholine (ACh), which depends of the levels of vesicular ACh transport (VAChT) to be released, is the central mediator of the cholinergic anti-inflammatory system. ACh controls the release of cytokine in different models of inflammation. Diesel exhaust particles (DEP) are one of the major environmental pollutants produced in large quantity by automotive engines in urban center. DEP bind the lung parenchyma and induce inflammation. We evaluated whether cholinergic dysfunction worsens DEP-induced lung inflammation. Male mice with decreased ACh release due to reduced expression of VAChT (VAChT-KD mice) were submitted to DEP exposure for 30 days (3 mg/mL of DEP, once a day, five days a week) or saline. Pulmonary function and inflammation as well as extracellular matrix fiber deposition were evaluated. Additionally, airway and nasal epithelial mucus production were quantified. We found that DEP instillation worsened lung function and increased lung inflammation. Higher levels of mononuclear cells were observed in the peripheral blood of both wild-type (WT) and VAChT-KD mice. Also, both wild-type (WT) and VAChT-KD mice showed an increase in macrophages in bronchoalveolar lavage fluid (BALF) as well as increased expression of IL-4, IL-6, IL-13, TNF-α, and NF-κB in lung cells. The collagen fiber content in alveolar septa was also increased in both genotypes. On the other hand, we observed that granulocytes were increased only in VAChT-KD peripheral blood. Likewise, increased BALF lymphocytes and neutrophils as well as increased elastic fibers in alveolar septa, airway neutral mucus, and nasal epithelia acid mucus were observed only in VAChT-KD mice. The cytokines IL-4 and TNF-α were also higher in VAChT-KD mice compared with WT mice. In conclusion, decreased ability to release ACh exacerbates some of the lung alterations induced by DEP in mice, suggesting that VAChT-KD animals are more vulnerable to the effects of DEP in the lung.

Extracellular matrix composition modulates PDAC parenchymal and stem cell plasticity and behavior through the secretome.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Its aggressiveness is driven by an intense fibrotic desmoplastic reaction in which the increasingly collagen I-rich extracellular matrix (ECM) and several cell types, including cancer stem cells (CSCs), create a tumor-supportive environment. However, how ECM composition regulates CSC dynamics and their relationship with the principle parenchymal tumor population to promote early invasive growth is not yet characterized. For this, we utilized a platform of 3D organotypic cultures composed of laminin-rich Matrigel, representative of an early tumor, plus increasing concentrations of collagen I to simulate malignant stroma progression. As ECM collagen I increases, CSCs progress from a rapidly growing, vascular phenotype to a slower growing, avascular phase, while maintaining their endothelial-like gene signatures. This transition is supported autocrinically by the CSCs and paracrinically by the parenchymal cells via their ECM-dependent secretomes. Indeed, when growing on an early tumor ECM, the CSCs are dedicated toward the preparation of a vascular niche by (a) activating their growth program, (b) secreting high levels of proangiogenic factors which stimulate both angiogenesis and vasculogenic mimicry, and (c) overexpressing VEGFR-2, which is activated by VEGF secreted by both the CSC and parenchymal cells. On Matrigel, the more differentiated parenchymal tumor cell population had reduced growth but a high invasive capacity. This concerted high local invasion of parenchymal cells into the CSC-derived vascular network suggests that a symbiotic relationship between the parenchymal cells and the CSCs underlies the initiation and maintenance of early PDAC infiltration and metastasis.

The TrkB agonist 7,8-dihydroxyflavone changes the structural dynamics of neocortical pyramidal neurons and improves object recognition in mice.

BDNF and its receptor TrkB have important roles in neurodevelopment, neural plasticity, learning, and memory. Alterations in TrkB expression have been described in different CNS disorders. Therefore, drugs interacting with TrkB, specially agonists, are promising therapeutic tools. Among them, the recently described 7,8-dihydroxyflavone (DHF), an orally bioactive compound, has been successfully tested in animal models of these diseases. Recent studies have shown the influence of this drug on the structure of pyramidal neurons, specifically on dendritic spine density. However, there is no information yet on how DHF may alter the structural dynamics of these neurons (i.e., real-time study of the addition/elimination of dendritic spines and axonal boutons). To gain knowledge on these effects of DHF, we have performed a real-time analysis of spine and axonal dynamics in pyramidal neurons of barrel cortex, using cranial windows and 2-photon microscopy during a chronic oral treatment with this drug. After confirming TrkB expression in these neurons, we found that DHF increased the gain rates of spines and axonal boutons, as well as improved object recognition memory. These results help to understand how the activation of the BDNF-TrkB system can improve basic behavioral tasks through changes in the structural dynamics of pyramidal neurons. Moreover, they highlight DHF as a promising therapeutic vector for certain brain disorders in which this system is altered.

Semi-Mechanistic Population Pharmacokinetic Modeling of L-Histidine Disposition and Brain Uptake in Wildtype and Pht1 Null Mice.

PURPOSE: To develop a semi-mechanistic population pharmacokinetic (PK) model to quantitate the disposition kinetics of L-histidine, a peptide-histidine transporter 1 (PHT1) substrate, in the plasma, cerebrospinal fluid and brain parenchyma of wildtype (WT) and Pht1 knockout (KO) mice. METHODS: L-[14C]Hisidine (L-His) was administrated to WT and KO mice via tail vein injection, after which plasma, cerebrospinal fluid (CSF) and brain parenchyma samples were collected. A PK model was developed using non-linear mixed effects modeling (NONMEM). The disposition of L-His between the plasma, brain, and CSF was described by a combination of PHT1-mediated uptake, CSF bulk flow and first-order micro-rate constants. RESULTS: The PK profile of L-His was best described by a four-compartment model. A more rapid uptake of L-His in brain parenchyma was observed in WT mice due to PHT1-mediated uptake, a process characterized by a Michaelis-Menten component (Vmax = 0.051 nmoL/min and Km = 34.94 µM). CONCLUSIONS: A semi-mechanistic population PK model was successfully developed, for the first time, to quantitatively characterize the disposition kinetics of L-His in brain under in vivo conditions. This model may prove a useful tool in predicting the uptake of L-His, and possibly other PHT1 peptide/mimetic substrates, for drug delivery to the brain.

Understanding and managing atherosclerotic renovascular disease: still a work in progress.

Atherosclerotic renovascular disease (ARVD) is an unresolved therapeutic dilemma despite extensive pre-clinical and clinical studies. The pathophysiology of the disease has been widely studied, and many factors that may be involved in progressive renal injury and cardiovascular risk associated with ARVD have been identified. However, therapies and clinical trials have focused largely on attempts to resolve renal artery stenosis without considering the potential need to treat the renal parenchyma beyond the obstruction. The results of these trials show a staggering consistence: although nearly 100% of the patients undergoing renal angioplasty show a resolution of the vascular obstruction, they do not achieve significant improvements in renal function or blood pressure control compared with those patients receiving medical treatment alone. It seems that we may need to take a step back and reconsider the pathophysiology of the disease in order to develop more effective therapeutic strategies. This mini-review discusses potential therapeutic alternatives that focus on the renal parenchyma distal to the vascular obstruction and may provide additional tools to enhance current treatment of ARVD.

Parenchymal Pressure Inconsistency in Different Brain Areas After Kaolin Injection into the Subarachnoid Space of Neonatal Rats.

AIM: To describe the relationship between the parenchymal pressure changes and the development of hydrocephalus in kaolininjected neonatal rats according to cerebral regions and time intervals of developing hydrocephalus. MATERIAL AND METHODS: Neonatal rats aged 2 to 3 days were examined in 5 groups as kaolin frontal "K-F", kaolin parietal "KP", saline frontal "SF-F", saline parietal "SF-P" and control "C", based on the injected material and injection sites. All injections were performed into the cortical subarachnoid space of the right frontal and right parietal regions. The fifth group was injection free. On the 3 < sup > rd < /sup > , 7 < sup > th < /sup > , 15 < sup > th < /sup > , 30 < sup > th < /sup > and 60 < sup > th < /sup > days after injection, parenchymal pressures (PP) of 5-7 rats from each group were measured from different regions. RESULTS: We compared the control group with saline-injected and kaolin-injected groups and found statistically significant parenchymal pressure differences based on regional measurements. In the kaolin groups, the mean PP values were obviously higher than the saline-injected group. Within each kaolin-injected group, the pressure values were variable and inconsistent regarding the parenchymal regions. CONCLUSION: Hydrocephalus cannot be totally explained with existent "bulk-flow" or "hydrodynamic" theories. Although our experimental design was planned to develop hydrocephalus according to the bulk flow theory, our results were more compatible with the hydrodynamic theory. The present comments on the occurrence and pathogenesis of hydrocephalus are still open to debate and may require further comprehensive studies.

An in vitro study examining the duration between deep inspirations on the rate of renarrowing.

The factors altering the bronchodilatory response to a deep inspiration (DI) in asthma are important to decipher. In this in vitro study, we investigated the effect of changing the duration between DIs on the rate of force recovery post-DI in guinea pig bronchi. The airway smooth muscle (ASM) within the main bronchi were submitted to length oscillation that simulated tidal breathing in different contractile states during 2, 5, 10 or 30min prior to a larger length excursion that simulated a DI. The contractile states of ASM were determined by adding either methacholine or isoproterenol. Irrespective of the contractile state, the duration between DIs neither affected the measured force during length oscillation nor the bronchodilator effect of DI. Contrastingly, the rate of force recovery post-DI in contracted state increased as the duration between DIs decreased. Similar results were obtained with contracted parenchymal strips. These findings suggest that changing the duration between DIs may alter the rate of ASM force recovery post-DI and thereby affect the rate of renarrowing and the duration of the respiratory relief afforded by DI.

Identification of highly connected hub genes in the protective response program of human macrophages and microglia activated by alpha B-crystallin.

The glial stress protein alpha B-crystallin (HSPB5) is an endogenous agonist for Toll-like receptor 2 in CD14+ cells. Following systemic administration, HSPB5 acts as a potent inhibitor of neuroinflammation in animal models and reduces lesion development in multiple sclerosis patients. Here, we show that systemically administered HSPB5 rapidly crosses the blood-brain barrier, implicating microglia as additional targets for HSPB5 along with peripheral monocytes and macrophages. To compare key players in the HSPB5-induced protective response of human macrophages and microglia, we applied weighted gene co-expression network analysis on transcript expression data obtained 1 and 4 h after activation. This approach identified networks of genes that are co-expressed in all datasets, thus reducing the complexity of the nonsynchronous waves of transcripts that appear after activation by HSPB5. In both cell types, HSPB5 activates a network of highly connected genes that appear to be functionally equivalent and consistent with the therapeutic effects of HSPB5 in vivo, since both networks include factors that suppress apoptosis, the production of proinflammatory factors, and the development of adaptive immunity. Yet, hub genes at the core of the network in either cell type were strikingly different. They prominently feature the well-known tolerance-promoting programmed-death ligand 1 as a key player in the macrophage response to HSPB5, and the immune-regulatory enzyme cyclooxygenase-2 (COX-2) in that of microglia. This latter finding indicates that despite its reputation as a potential target for nonsteroidal anti-inflammatory drugs, microglial COX-2 plays a central role in the therapeutic effects of HSPB5 during neuroinflammation. GLIA 2017;65:460-473.

Impact of NKT Cells and LFA-1 on Liver Regeneration under Subseptic Conditions.

BACKGROUND: Activation of the immune system in terms of subseptic conditions during liver regeneration is of paramount clinical importance. However, little is known about molecular mechanisms and their mediators that control hepatocyte proliferation. We sought to determine the functional role of immune cells, especially NKT cells, in response to partial hepatectomy (PH), and to uncover the impact of the integrin lymphocyte function-associated antigen-1 (LFA-1) on liver regeneration in a subseptic setting. METHODS: Wild-type (WT) and LFA-1-/- mice underwent a 2/3 PH and low-dose lipopolysaccharid (LPS) application. Hepatocyte proliferation, immune cell infiltration, and cytokine profile in the liver parenchyma were determined. RESULTS: Low-dose LPS application after PH results in a significant delay of liver regeneration between 48h and 72h, which is associated with a reduced number of CD3+ cells within the regenerating liver. In absence of LFA-1, an impaired regenerative capacity was observed under low-dose LPS application. Analysis of different leukocyte subpopulations showed less CD3+NK1.1+ NKT cells in the liver parenchyma of LFA-1-/- mice after PH and LPS application compared to WT controls, while CD3-NK1.1+ NK cells markedly increased. Concordantly with this observation, lower levels of NKT cell related cytokines IL-12 and IL-23 were expressed in the regenerating liver of LFA-1-/- mice, while the expression of NK cell-associated CCL5 and IL-10 was increased compared to WT mice. CONCLUSION: A subseptic situation negatively alters hepatocyte proliferation. Within this scenario, we suggest an important impact of NKT cells and postulate a critical function for LFA-1 during processes of liver regeneration.

Cigarette smoke and decreased oxygen tension inhibit pulmonary claudin-6 expression.

PURPOSE: Chronic obstructive pulmonary disease is a condition involving perturbed barrier integrity coincident with both emphysema and inflammation of the airways, and smoking is considered a major risk factor. Claudins (Cldns) stabilize barriers and contribute to tight junctions by preventing paracellular transport of extracellular fluid constituents. METHODS: To determine Cldn6 was differentially influenced by tobacco smoke, Cldn6 was evaluated in cells and tissues by q-PCR, immunoblotting, and immunohistochemistry following exposure. Cldn6 transcriptional regulation was also assessed using luciferase reporter constructs. RESULTS: Q-PCR and immunoblotting revealed that Cldn6 was decreased in alveolar type II-like epithelial cells (A549) and primary small airway epithelial cells when exposed to cigarette smoke extract (CSE). Cldn6 was also markedly decreased in the lungs of mice exposed to acute tobacco smoke delivered by a nose-only automated smoke machine compared to controls. Luciferase reporter assays incorporating 0.5-kb, 1.0-kb, or 2.0-kb of the Cldn6 promoter revealed decreased transcription of Cldn6 following exposure to CSE. Cldn6 transcriptional regulation was also assessed in hypoxic conditions due to low oxygen tension observed during smoking. Hypoxia and hypoxia inducible factor-1 alpha caused decreased transcription of the Cldn6 gene via interactions with putative response elements in the proximal promoter sequence. CONCLUSIONS: These data reveal that tight junctional proteins such as Cldn6 are differentially regulated by tobacco-smoke exposure and that Cldns are potentially targeted when epithelial cells respond to tobacco smoke. Further research may show that Cldns expressed in tight junctions between parenchymal cells contribute to impaired structural integrity of the lung coincident with smoking.