Local assessment of WC1+ γδ T lymphocyte subset in the different types of lesions associated with bovine paratuberculosis.

The local expression of WC1+ γδ T lymphocytes subset has been evaluated by immunohistochemical methods at the different types of lesions present in cows naturally infected with Mycobacterium avium subsp. paratuberculosis (Map) and in non-infected control animals. Infected cattle were either in the latent/subclinical (focal lesions) or clinical (diffuse paucibacillary and multibacillary forms) stage of paratuberculosis. To assess the cell distribution, a differential cell count was carried out at the lamina propria, gut-associated lymphoid tissue and submucosa. A significant increase in the number of WC1+ γδ T cells was observed in all the infected animals, regardless of the type of lesion. Cows with focal lesions showed higher number of labeled cells than those with diffuse forms, where no differences were found between the two types. This increase in the number of positively immunolabelled lymphocytes in infected animals was seen in the lamina propria, with higher values in those with focal lesions. While in the lymphoid tissue no differences in the numbers were observed, in animals with focal lesions, WC1+ γδ T cells tended to be located at the periphery of the granulomas. These findings suggest a proinflammatory action of WC1+ γδ T lymphocytes in bovine paratuberculosis, which might play an important role in the containment of the Map-infection in the focal granulomas located in the lymphoid tissue, helping to prevent the progression toward diffuse forms responsible for the clinical signs.

Assessment of a white matter reference region for 11C-UCB-J PET quantification.

11C-UCB-J is a positron emission tomography (PET) radioligand that has been used in humans for synaptic vesicle glycoprotein 2A (SV2A) imaging and as a potential synaptic density marker. The centrum semiovale (CS) is a proposed reference region for noninvasive quantification of 11C-UCB-J, due to negligible concentrations of SV2A in this region in baboon brain assessed by in vitro methods. However, in displacement scans with SV2A-specific drug levetiracetam in humans, a decrease in 11C-UCB-J concentration was observed in the CS, consistent with some degree of specific binding. The current study aims to validate the CS as a reference region by (1) optimizing CS region of interest (ROI) to minimize spill-in from gray matter with high radioactivity concentrations; (2) investigating convergence of CS ROI values using ordered subset expectation maximization (OS-EM) reconstruction, and (3) comparing baseline CS volume of distribution (VT) to nondisplaceable uptake in gray matter, VND. Improving ROI definition and increasing OS-EM iterations during reconstruction decreased the difference between CS VT and VND. However, even with these corrections, CS VT overestimated VND by ∼35-40%. These measures showed significant correlation, suggesting that, though biased, the CS may be a useful estimate of nondisplaceable uptake, allowing for noninvasive quantification for SV2A PET.

Application of an S-layer protein as a self-aggregating tag for cost-effective separation of recombinant human and yeast D-amino acid oxidases in the aqueous two-phase system.

OBJECTIVE: To evaluate whether the surface layer (S-layer) protein of Lactobacillus brevis serves as a self-aggregating protein tag for cost-effective separation of human and yeast D-amino acid oxidases (hDAAO and yDAAO) expressed in E. coli. RESULTS: In aqueous two-phase (PEG-phosphate) system, the S-layer:DAAO fusion proteins (shDAAO and syDAAO) were separated at the interface with a recovery of 82 ± 10.6% for shDAAO and 95 ± 1.9% for syDAAO. Some shDAAO proteins were separated as precipitates with a recovery of 41 ± 0.5% in phosphate (9%, w/w) using PEG 3000 and PEG 4000 (16%, w/w), while some syDAAO proteins were also isolated as precipitates with a recovery of 75 ± 17.5% in phosphate (9%, w/w) using PEG 4000 and PEG 8000 (16%, w/w). CONCLUSIONS: The S-layer of L. brevis was applied to a self-assembled protein tag to enable cost-effective separation of human and yeast D-amino acid oxidases expressed in E. coli cells. Because of the self-assembling properties of S-layer proteins, human and yeast D-amino acid oxidases fused with S-layer proteins could be easily separated by aggregates at the interface and/or in a few conditions by precipitates to the bottom of the PEG-phosphate aqueous system.

Chaihu-Shugan-San and absorbed meranzin hydrate induce anti-atherosclerosis and behavioral improvements in high-fat diet ApoE-/- mice via anti-inflammatory and BDNF-TrkB pathway.

The comorbidity of coronary heart disease (CHD) and depression in patients is extremely prevalent, with a rate from 20 to 50%, while depression-like behaviors exist in a larger percentage of patients. Therefore, the study of comorbidities is particularly urgent. Chaihu-Shugan-San (CSS), a classical traditional Chinese medicine formula, has been used to treat CHD with depression for hundreds of years. However, the mechanism of its action on comorbidities remains unclear. Here, we focused on the behavioral changes in ApoE-/- mice fed a high-fat diet (HFD) and elucidated the mechanism of CSS and its main absorbed component, meranzin hydrate (MH), as an anti-atherosclerosis and anti-depression agent. In the present study, mice were fed an HFD for 16 weeks and were intragastrically administered high and low doses of CSS and MH. Depressive-like behaviors were evaluated by the sucrose preference test, the open-field test, the light-dark test and the tail-suspension test, after which atherosclerotic plaques, plasma lipids, inflammatory cytokine levels and the expression of BDNF/TrkB were measured. We demonstrated that the atherosclerosis model group exhibited significant depressant behaviors. Moreover, CSS inhibited depressive-like behavioral changes, reduced atherosclerotic plaque areas, plasma total cholesterol, triglycerides, LDL-cholesterol levels and inflammatory cytokines including TNF-α, IL-1ß, and IL-6 in plasma and hippocampi, increased the protein and mRNA expression of BDNF and TrkB in the aorta and the hippocampus. Interestingly, MH, the main component in CSS that is absorbed in the plasma and hippocampus, exerted effects similar to those of CSS. In addition, MH increased the protein and mRNA expression of BDNF and TrkB in human umbilical vein endothelial cells (HUVECs) induced by LPS. Collectively, these results suggest that MH represents the CSS decoction, induces anti-atherosclerosis effects and improves depression-like behaviors in HFD-fed ApoE-/- mice. Moreover, the regulation of proinflammatory factors and BDNF-TrkB signaling are possibly involved in this process. Our findings indicate that MH is a potential phytochemical compound for the prevention of the comorbidity of atherosclerosis and depression.

Safety assessment, biological effects, and mechanisms of Myrica rubra fruit extract for anti-melanogenesis, anti-oxidation, and free radical scavenging abilities on melanoma cells.

OBJECTIVE: Currently, the cosmetic and medical industries are paying considerable attention to solve or prevent skin damage or diseases, such as hyperpigmentation and oxidation and free radical damage. In this study, the effective compounds in Myrica rubra fruit were extracted and studied the biological effects of these M. rubra fruit extracts. METHODS: In this study, we extracted M. rubra fruit using solutions with various ratios of water to ethanol (100:0, 50:50, 5:95) and studied the anti-melanogenesis, anti-oxidation and radical scavenging effects of these M. rubra fruit extracts on two melanoma cell lines: mouse melanoma (B16-F0) and human melanoma (A2058). The cytotoxicity, melanin synthesis, mushroom and cellular tyrosinase activities, enzyme kinetics, melanogenesis-related gene expression, melanogenesis-related protein secretion, radical DPPH scavenging activity and ROS inhibition after treatment with M. rubra fruit extracts were determined. RESULTS: The results showed that the water extract of M. rubra fruit was less cytotoxic to the melanoma cell lines, effectively inhibited melanin synthesis and tyrosinase activity and down-regulated the gene expression and protein secretion of MITF and TRP-1. In addition, the M. rubra fruit extracts also showed the abilities to scavenge DPPH free radicals and suppress ROS production. Finally, the effective compounds in the water extract were Myricetin-O-deoxyhexoside, Quercetin-O-deoxyhexoside, and Kaempferol-O-hexoside determined by LC/MS/MS assay. CONCLUSION: Overall, the water extract of M. rubra fruit is a safe and effective melanin inhibitor and anti-oxidant and can be applied widely in the fields of cosmetics and medicine.

Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer.

Chimeric antigen receptor (CAR) T cells represent a novel targeted approach to overcome both quantitative and qualitative shortfalls of the host immune system relating to the detection and subsequent destruction of tumors. The identification of antigens expressed specifically on the surface of tumor cells is a critical first step in the ability to utilize CAR T cells for the treatment of cancer. The 5T4 is a tumor-associated antigen which is expressed on the cell surface of most solid tumors including ovarian cancer. Matched blood and tumor samples were collected from 12 patients with ovarian cancer; all tumors were positive for 5T4 expression by immunohistochemistry. Patient T cells were effectively transduced with 2 different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with matched autologous tumor disaggregates resulted in antigen-specific secretion of IFN-gamma. Furthermore, assessment of the efficacy of anti-5T4 CAR T cells in a mouse model resulted in therapeutic benefit against established ovarian tumors. These results demonstrate proof of principle that 5T4 is an attractive target for immune intervention in ovarian cancer and that patient T cells engineered to express a 5T4-specific CAR can recognize and respond physiologically to autologous tumor cells.

Assessment of Proliferative Potential of Odontogenic Keratocyst and Dentigerous Cyst using Podoplanin: An Immunohistochemical Study.

INTRODUCTION: Odontogenic cysts are commonly encountered lesions among head and neck pathologies. Odontogenic keratocyst (OKC) has unique features of recurrence and local aggressiveness. Podoplanin (PDP) is a lymphatic endothelial marker and is shown to be expressed in a variety of tissues. Hence, we planned to assess the significance of PDP in OKC and dentigerous cyst (DC). MATERIALS AND METHODS: The present study included assessment of immunoexpression of PDP in OKC and DC. Twenty specimens each of OKC and DC were included in the present study and were stained with D2-40 antibody. All the sections were analyzed and were categorized as negative staining, weakly positive staining, and strongly positive staining. All the results were analyzed by Statistical Package for the Social Sciences (SPSS) software. RESULTS: We detected PDP-positive staining in the cell membrane and cytoplasm of the cells of basal cell layer and supra-basal cell layers. In DC cases, we observed positive staining only in cases associated with inflammation. CONCLUSION: Podoplanin does play a significant role in enhancing the local invasive and neoplastic properties of OKC. CLINICAL SIGNIFICANCE: Podoplanin expression in OKC is potentially associated with moderate invasive nature of the neighboring structures.

Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection.

The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease.

In silico assessment of phosphorylation and O-ß-GlcNAcylation sites in human NPC1 protein critical for Ebola virus entry.

Ebola is a highly pathogenic enveloped virus responsible for deadly outbreaks of severe hemorrhagic fever. It enters human cells by binding a multifunctional cholesterol transporter Niemann-Pick C1 (NPC1) protein. Post translational modification (PTM) information for NPC1 is crucial to understand Ebola virus (EBOV) entry and action due to changes in phosphorylation or glycosylation at the binding site. It is difficult and costly to experimentally assess this type of interaction, so in silico strategy was employed. Identification of phosphorylation sites, including conserved residues that could be possible targets for 21 predicted kinases was followed by interplay study between phosphorylation and O-ß-GlcNAc modification of NPC1. Results revealed that only 4 out of 48 predicted phosphosites exhibited O-ß-GlcNAc activity. Predicted outcomes were integrated with residue conservation and 3D structural information. Three Yin Yang sites were located in the α-helix regions and were conserved in studied vertebrate and mammalian species. Only one modification site S425 was found in ß-turn region located near the N-terminus of NPC1 and was found to differ in pig, mouse, cobra and humans. The predictions suggest that Yin Yang sites may not be important for virus attachment to NPC1, whereas phosphosite 473 may be important for binding and hence entry of Ebola virus. This information could be useful in addressing further experimental studies and therapeutic strategies targeting PTM events in EBOV entry.

Effects of phosphodiesterase-5 inhibitor on ischemic kidney injury during nephron sparing surgery: quantitative assessment by NGAL and KIM-1.

PURPOSE: Interruption of renal blood flow is often necessary during nephron sparing surgery (NSS) and can induce renal injury. This study examines whether tadalafil, a phosphodiesterase-5 (PDE-5) inhibitor and well-known vasodilator, exerts nephroprotective effects in patients undergoing NSS. METHODS: This non-randomized study included 49 patients with enhancing solid renal mass. All patients were subjected to open NSS during which clamping the renal artery was performed. Twenty-two patients were pretreated with tadalafil 1 day prior NSS and 2 days following surgery. The other 27 patients underwent the same surgical procedure but did not receive tadalafil (controls). Urine samples were collected before surgery and following renal pedicle clamp removal. Urine levels of NGAL and KIM-1, two novel biomarkers for acute kidney injury (AKI), were determined. RESULTS: Clamping the renal artery induced kidney dysfunction as reflected by increases in urinary NGAL and KIM-1 in all participants. These increases in urinary NGAL and KIM-1 excretion were evident 1 h after renal ischemia and lasted for 72 and 24 h, respectively. Pretreatment with tadalafil reduced the absolute urinary excretion of KIM-1, but not of NGAL. Although the incidence of AKI was comparable between tadalafil-treated and untreated NSS subjects, the elevation in serum creatinine (SCr) was significantly attenuated in tadalafil-treated group as compared with NSS controls. CONCLUSIONS: Tadalafil exerts nephroprotective effects in AKI following NSS, as was evident by reduced urinary excretion of KIM-1 and attenuation of SCr elevation. Carefully controlled large clinical studies are needed before defining the role of PDE-5 inhibition therapy in these patients.

Lymphatic vessel assessment by podoplanin (D2-40) immunohistochemistry in breast cancer.

CONTEXT: Lymph node metastasis has an important bearing on the staging of breast cancer. Lymph node metastasis occurs by hematogenous and lymphatic spread. The hematogenous and lymphatic spread can be quantified by the blood vessel and lymphatic vessel density in the intra-tumoral and peri-tumoral zone by specific markers for blood vessels and lymph vessels. AIMS: In this study, we are trying to study the localization of podoplanin in lymph vessels of invasive breast carcinoma, to quantify lymphangiogenesis in tissue sections of invasive breast carcinomas by podoplanin immunohistochemistry (IHC) by D2-40 antibody, and compare it with blood microvessel count using CD-31 antibody and correlating clinicopathologic parameters with the results of IHC. MATERIALS AND METHODS: IHC for biomarkers D2-40 and CD-31 were performed on sections from 30 mastectomy specimens to assess blood vessel and lymphatic vessel density in intra-tumoral and peri-tumoral zone. The data were analyzed using Statistical Package for Social Sciences (SPSS) version 15.0 statistical analysis software. RESULTS: The results showed that both lymph vessel density and blood vessel density increased with the increase in lymph node ratio. Lymph node ratio is the ratio of positive lymph nodes to the total number of lymph nodes removed. CONCLUSION: Taking into account our small sample size, we conclude that a further large-sized study should be carried out to further prove the role of lymphatics in tumor dissemination. New therapeutic options can be developed targeting the lymphatic channels to arrest the lymphatic spread of the breast cancer.

Quantitative immunohistochemical assessment of blood and lymphatic microcirculation in cutaneous lichen planus lesions.

Latest advances have brought to light the hypothesis that angiogenesis and lymphangiogenesis are tightly connected to some chronic inflammatory diseases. The present study focuses on immunohistochemical assessment of the quantitative changes in the blood and lymphatic microcirculatory bed in common chronic dermatosis - cutaneous lichen planus. Double immunohistochemistry with CD34 and podoplanin antibodies was used to detect blood and lymphatic endothelium, while anti-human VEGF was used for the observation of a key angiogenesis and lymphangiogenesis inducer. Morphometric analysis was performed with QuickPhoto Micro image analysis software. Results confirmed statistically significant enlargement of both the blood and lymphatic microcirculatory beds. Compared to healthy skin, cutaneous lichen planus lesions revealed 1.6 times enlarged blood microcirculatory bed and 1.8 times enlarged lymphatic microcirculatory bed. Vascular endothelial growth factor (VEGF) expression in lesional skin was significantly higher in the epidermis (19.1 times increase) than in the dermis (10.3 times increase). These findings indicate a tight association of angiogenesis and lymphangiogenesis with the pathogenesis of cutaneous lichen planus.

Assessment of atherosclerosis in chronic granulomatous disease.

BACKGROUND: Patients with chronic granulomatous disease (CGD) experience immunodeficiency because of defects in the phagocyte NADPH oxidase and the concomitant reduction in reactive oxygen intermediates. This may result in a reduction in atherosclerotic injury. METHODS AND RESULTS: We prospectively assessed the prevalence of cardiovascular risk factors, biomarkers of inflammation and neutrophil activation, and the presence of magnetic resonance imaging and computed tomography quantified subclinical atherosclerosis in the carotid and coronary arteries of 41 patients with CGD and 25 healthy controls in the same age range. Univariable and multivariable associations among risk factors, inflammatory markers, and atherosclerosis burden were assessed. Patients with CGD had significant elevations in traditional risk factors and inflammatory markers compared with control subjects, including hypertension, high-sensitivity C-reactive protein, oxidized low-density lipoprotein, and low high-density lipoprotein. Despite this, patients with CGD had a 22% lower internal carotid artery wall volume compared with control subjects (361.3±76.4 mm(3) versus 463.5±104.7 mm(3); P<0.001). This difference was comparable in p47(phox)- and gp91(phox)-deficient subtypes of CGD and independent of risk factors in multivariate regression analysis. In contrast, the prevalence of coronary arterial calcification was similar between patients with CGD and control subjects (14.6%, CGD; 6.3%, controls; P=0.39). CONCLUSIONS: The observation by magnetic resonance imaging and computerized tomography of reduced carotid but not coronary artery atherosclerosis in patients with CGD despite the high prevalence of traditional risk factors raises questions about the role of NADPH oxidase in the pathogenesis of clinically significant atherosclerosis. Additional high-resolution studies in multiple vascular beds are required to address the therapeutic potential of NADPH oxidase inhibition in cardiovascular diseases. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01063309.

Effectiveness of antiepileptic drug combination therapy for partial-onset seizures based on mechanisms of action.

IMPORTANCE: To our knowledge, the current study is the first to describe antiepileptic drug (AED) combination therapy patterns according to their mechanism of action (MOA) in a real-world setting and to evaluate the differences in outcomes comparing different-MOA combination therapy with same-MOA combination therapy for patients with partial-onset seizure. OBJECTIVE: To compare treatment persistence and health care use with AED combinations categorized by MOA in patients with partial-onset seizures. DESIGN, SETTING, AND PARTICIPANTS: Using the Truven Health MarketScan Commercial Claims Database containing 96 million covered lives from July 1, 2004, through March 31, 2011, adults with concomitant use of 2 different AEDs and a recent partial-onset seizure diagnosis were selected. Antiepileptic drugs were categorized by MOA: sodium channel blockers (SC), gamma-aminobutyric acid analogs (G), synaptic vesicle protein 2A binding (SV2), and multiple mechanisms (M). Patients were assigned a combination category based on their concomitant AED use. MAIN OUTCOMES AND MEASURES: Treatment persistence was measured from the start of AED combination therapy until the end of the combination. Health care resource use was measured during the combination treatment duration. Multivariate analyses evaluated AED discontinuation risk and health care use according to MOA combinations. RESULTS: Distribution of 8615 selected patients by combination was 3.3% for G+G, 7.5% for G+SV2, 8.6% for G+M, 13.9% for SC+SC, 19.0% for G+SC, 21.5% for SC+M, and 26.3% for SC+SV2. The same-MOA (G+G and SC+SC) combinations had the shortest persistence (mean [SD], 344 [345] days and 513 [530] days, respectively) and greater hazard of discontinuation compared with different-MOA combinations. Patients with different-MOA G combinations had a significantly lower risk for inpatient admission (odds ratio, 0.716; 95% CI, 0.539-0.952; P = .02) compared with G+G combinations. Patients with different-MOA SC combinations had significantly lower risks for emergency department visits (odds ratio, 0.853; 95% CI, 0.742-0.980; P = .03) compared with SC+SC combinations. CONCLUSIONS AND RELEVANCE: The findings suggest that AED combinations with different MOAs have greater effectiveness as measured by treatment persistence and lower risks for hospitalization and emergency department visits. Further research is needed to more fully understand the role of the MOA in achieving optimal outcomes.

Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases.

Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 wk) treated ± ethanol in drinking water (20% wt/vol, 12 wk) ± orally gavaged GSH in methylcellulose vehicle (300 mg x kg(-1) x day(-1), during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) ± GSH (500 µM, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) ± orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia.

Characterization of surface layer proteins and its role in probiotic properties of three Lactobacillus strains.

The objective of this study was the characterization of the surface layer proteins (SLPs) and their functional role in the probiotic activity of Lactobacillus helveticus fb213, L. acidophilus fb116 and L. acidophilus fb214. SLPs were extracted and identified by SDS-PAGE, circular dichroism spectra and LC-MS analysis. The results revealed that the molecular masses of the three proteins were 49.7 kDa, 46.0 kDa and 44.6 kDa, respectively. The secondary structures and amino acid compositions of the three proteins were found to be similar. After removing SLPs, the survival of the three lactobacilli in simulated gastric and intestinal juices was reduced by 2-3log as compared with survival of the intact cells. And the adhesion ability of the three strains to HT-29 cells decreased by 61%, 65% and 92%, respectively. SLPs also inhibited the adhesion and invasion of Escherichia coli ATCC 43893 to HT-29 cells. These results suggest that SLPs are advantageous barriers for lactobacilli in the gastrointestinal tract, and these proteins help make it possible for lactobacilli to serve their probiotic functions.

In vitro assessment of IL-4- or IL-13-mediated changes in the structural components of keratinocytes in mice and humans.

T helper type 2 (Th2) cytokines, IL-4 and IL-13, attenuate the expression of genes that regulate epidermal cellular structures and the barrier function at the terminal stage of keratinocyte differentiation. However, whether these Th2 cytokines act at earlier stages remains unknown. We investigated the roles of cytokines in expression levels of mRNAs and/or proteins in primary mouse keratinocytes and human keratinocyte HaCaT cells at earlier stages. We showed that IL-4 downregulated the expression levels of Krt1, Krt10, Dsg1, and Dsc1 via IL-4Rα- and signal transducer and activator of transcription factor 6 (STAT6)-dependent mechanisms in differentiating mouse keratinocytes at early stages. As the expression levels of keratin-1 and -10 in the keratinocytes transiently expressing an active form of STAT6 were not downregulated, STAT6 and other IL-4-induced molecules may synergistically regulate this expression. The restoration of the downregulated expression levels of Krt1 and Krt10 induced by IL-4 with the MEK (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase) inhibitor U0126 indicated the involvement of the p44/42 MAPK signaling pathway in the attenuated expression. IL-13 also downregulated the expression of the four genes. Furthermore, IL-4 or IL-13 caused the downregulation of these genes in HaCaT cells and promoted the fragmentation of cell sheets with mechanical stress. Our results showed that IL-4 or IL-13 acted on differentiating keratinocytes in vitro at early stages to attenuate the gene expression.

Large unstained cells: a potentially valuable parameter in the assessment of immune activation levels in HIV infection.

INTRODUCTION: Chronic immune activation is associated with the accelerated progression of HIV to AIDS; however, affordable markers reflecting this have not yet been determined. The percentage of large unstained cells (%LUCs) is a differential count parameter measured by certain routine hematology analyzers and reflects activated lymphocytes and peroxidase-negative cells. We hypothesized that the %LUCs would be increased in HIV infection and would correlate with markers of immune activation [i.e. CD38 expression on CD8+ T cells (%CD38onCD8) and lipopolysaccharide-binding protein (LBP)] and CD4 counts. METHODS: In this cross-sectional study, 78 HIV-infected, antiretroviral therapy-naïve adults and 52 uninfected controls were recruited. %CD38onCD8 and CD4 counts were determined by flow cytometry, LBP levels were assessed by immunoassay, and the %LUCs was tested on a Siemens ADVIA 2120. RESULTS: Significant differences were found between the HIV-infected and control groups for %LUCs (95% CI 2.3-2.7 vs. 1.8-2.2, respectively; p = 0.001), as well as for %CD38onCD8, LBP, and CD4 counts. Furthermore, %LUCs correlated directly with %CD38onCD8 and LBP and inversely with CD4 counts. CONCLUSION: The %LUCs was significantly increased in this untreated, asymptomatic, HIV-infected group and correlated with markers of immune activation and CD4 counts. Therefore, the %LUCs may be of value in identifying HIV-infected patients at risk of accelerated disease progression.

Assessment of ORAI1-mediated basal calcium influx in mammary epithelial cells.

BACKGROUND: The entry of calcium ions into mammary gland epithelial cells is one of the least well-understood processes in the transport of calcium into milk during lactation. The store-operated calcium entry channel ORAI1, has been suggested as a potential mechanism for the entry of Ca(2+) into mammary gland epithelial cells from the maternal blood supply during lactation. The down regulation of the canonical ORAI1 activator STIM1 during lactation suggests that other known ORAI activators such as STIM2 and SPCA2 may be important during lactation. RESULTS: Differentiation of HC11 mammary gland epithelial cells was associated with enhanced basal Ca(2+) influx. Silencing of Orai1 abolished this enhancement of Ca(2+) influx. Stim2 had a modest effect on Ca(2+) influx in this in vitro model of lactation, whereas Stim1 and Spca2 silencing had no effect. Despite pronounced increases in Spca2 mRNA during lactation there was no change in the generation of the alternative splice product generated by Mist1, which increases during lactation. CONCLUSIONS: These studies support the hypothesis that lactation is associated with a remodelling of Ca(2+) influx and this is associated with enhancement of basal Ca(2+) influx. This enhanced Ca(2+) influx appears to occur through the calcium channel Orai1.