Inhibitory Effect of Alloferons in Combination with Human Lymphocytes on Human Herpesvirus 1 (HHV-1) Replication In Vitro

Over the past two decades there has been intense study of compounds from vertebrates, microorganisms, plants, mushrooms, marine sponges, worms, etc. as well as insects in terms of their antiviral activity. Insects produce a variety of biologically active peptides. One of them is alloferon. The in vitro and in vivo experiments demonstrate that synthetic alloferon has an immunomodulatory properties. It was reported that alloferon and its analogues (alloferon I and II) have antimicrobial properties, as well. The aim of this study was to evaluate in vitro the effect of alloferon I and II, either alone or in combination with human lymphocytes, on human herpesvirus type 1 (HHV-1) McIntyre strain replication. On the base of results we can conclude that alloferon I and II inhibit the replication of HHV-1 McIntyre strain in HEp-2 cells. Enhanced antiviral activity was observed when infected cells were treated with alloferons and unstimulated or phytohemagglutinin PHA-stimulated lymphocytes simultaneously. After application of alloferons and PHA-stimulated lymphocytes to the HHV-1 infected HEp-2 culture, the mean HHV-1 titer reduction for alloferon and II, when used at the highest dose—400 µg/mL, were 3.69 and 3.27 log₁₀/TCID_50/mL, respectively.     

Formulation of Controlled-Release Baclofen Matrix Tablets II: Influence of Some Hydrophobic Excipients on the Release Rate and In Vitro Evaluation

The aim of this study was to investigate the influence of polymer level and type of some hydrophobic polymers, including hydrogenated castor oil (HCO); Eudragit RS100 (E-RS100); Eudragit L100 (E-L100), and some fillers namely mannitol [soluble filler], Dibasic calcium phosphate dihydrate (Emcompress) and anhydrous dibasic calcium phosphate [insoluble fillers] on the release rate and mechanism of baclofen from matrix tablets prepared by a hot-melt granulation process (wax tablets) and wet granulation process (E-RS100 and E-L100 tablets). Statistically significant differences were found among the drug release profile from different classes of polymeric matrices. Higher polymeric content (40%) in the matrix decreased the release rate of drug because of increased tortuosity and decreased porosity. At lower polymeric level (20%), the rate and extent of drug release was elevated. HCO was found to cause the strongest retardation of drug. On the other hand, replacement of Emcompress or anhydrous dibasic calcium phosphate for mannitol significantly retarded the release rate of baclofen, except for E-L100 (pH-dependent polymer). Emcompress surface alkalinity and in-situ increase in pH of the matrix microenvironment enhanced the dissolution and erosion of these matrix tablets. The release kinetics was found to be governed by the type and content of the excipients (polymer or filler). The prepared tablets showed no significant change in drug release rate when stored at ambient room conditions for 6 months.     

In Vitro Stimulation by Parathion of Porcine Pancreatic Carboxylesterase Activity toward Triacetin

In order to elucidate the relationship between bitter taste and chemical structure in peptides, various kinds of model bitter peptides containing arginine, proline and phenylalanine were synthesized, and the contribution of the individual amino acids to the bitter taste was made clear. It was confirmed that, in order to strengthen the bitterness in di- and tripeptides, the hydrophobic amino acid needs to be located at the C-terminal and, conversely, the basic amino acid should be located at the N-terminal Furthermore, a strong bitter taste was observed when arginine was contiguous to proline such as Arg-Pro, Gly-Arg-Pro and Arg-Pro-Gly. A synergistic effect for bitter taste was observed in the peptides whose structure is (Arg)_l-(Pro)_m-(Phe)_n (l=1, 2; m, n = 1 ~ 3) by increasing the number of amino acids. Among them, the octapeptide (Arg-Arg-Pro-Pro-Pro-Phe-Phe-Phe) possessed an extremely bitter taste with its threshold value of 0.002 mm and was found to be the most bitter among the peptides.     

CD36 Mediates the In Vitro Inhibitory Effects of Thrombospondin-1 on Endothelial Cells

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase–CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.     

人集落刺激因子-1(CSF-1)对常见呼吸道病毒的抑制作用

本文观察集落刺激因子-1(CSF-1)及其诱生剂:内毒素(LPS)、胞壁二肽(MDP)和干扰素(IFN-α)对下列病毒所致细胞病变的抑制,包括不同型别腺病毒5株,单疱病毒Ⅰ型和Ⅱ型,流感病毒A_3型,鼻病毒,ECHO11型各1株,在人胚肺传代细胞株上病变抑制的结果如下:对HSV-1、HSV-2、腺病毒6、11、22型,流感A_3型,鼻病毒。(CSF-1)和IFN-α,一样有明显抑毒效果,LPS MDP联合使用对以上病毒有明显增强抑毒作用。CSF-1和IFN-α的抑毒作用能分别被CSF-1和IFN-α的抗体所解除。 CSF-1在人胚肺传代细胞和人胚皮肤肌肉传代细胞上对VSV的抑毒效果在人胚肺细胞中效果比人胚皮肤肌肉细胞更明显。LPS10ng/ml作用48小时比作用24小时效果更强。LPS MDP和IFN-α对二种细胞都有同样高效的抑毒作用。     

Recombinant Human Albumin in Cell Culture: Evaluation of Growth-Promoting Potential for NRK and SCC-9 Cells In Vitro

Serum-derived albumin has for a long time been used in cell culture media, but the exact role of albumin and/or impurities bound to albumin has not been precisely defined. In this study, recombinant human albumin was evaluated for its growth-promoting activity on two cell lines, NRK and SCC-9. For NRK cells, the recombinant human albumin was found to exert an inhibitory effect. The fact that fatty acid free HSA was also inhibitory while HSA fraction V was stimulatory suggested a role for fatty acids or some other bound moieties in growth stimulation by HSA fraction V. Addition of oleic acid, cholesterol, phosphatidylcholine, phosphatidylserine or a combination of these lipids, however, did not significantly improve the growth stimulating activity of either fatty acid free HSA or the recombinant human albumin. For SCC-9 cells, both recombinant human albumin and fatty acid free HSA showed slight stimulation (although they were not as active as HSA fraction V), suggesting that in some cell systems, the albumin molecule per se may promote cell growth and survival. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibitory Activity of Honeysuckle Extracts against Influenza A Virus In Vitro and In Vivo

Virologica Sinica - Honeysuckle has been used in the treatment of influenza virus infection for thousands of years in China. However, its main active components and the functional mechanisms remain...     

The Effect of Moisture on the Flowability of Pharmaceutical Excipients

The effect of moisture content on flowability of six pharmaceutical powders (microcrystalline cellulose (MCC), hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), corn starch, and potato starch) was investigated. Powder flowability was measured using established static techniques and emerging dynamic avalanche behavior measurements. Static techniques did not provide enough resolution to clearly identify changes in flowability due to increasing powder moisture content. Avalanche time and its standard deviation showed that flowability of MCC, CMC, PVP, and potato starch decreased after a critical moisture content, flowability of corn starch increased and flowability did not significantly change for HPMC. The moisture decreased flowability by forming stronger interparticle liquid bridges and increased flowability by acting as a lubricant. The dynamic density of the celluloses and PVP decreased linearly with increasing moisture content as the particles swelled with water. The starches also swelled and decreased in dynamic density, but only after a moisture content corresponding to monolayer coverage of water around the particles was reached. As flowability and dynamic density change with moisture content, to ensure consistent production of high-quality tablets, the moisture content of the powders must be measured and controlled.     

Potential DPP IV Inhibitory Peptides from Dry-Cured Pork Loins after Hydrolysis: An In Vitro and In Silico Study

Peptidyl peptidase IV (DPP-IV) is a pharmacotherapeutic target in type 2 diabetes, and inhibitors of this enzyme are an important class of drugs for the treatment of type 2 diabetes. In the present study, peptides (<7 kDa) isolated from dry-cured pork loins after pepsin and pancreatin hydrolysis were identified by mass spectrometry and tested as potential inhibitors of DPP-IV by the in silico method. Two peptides, namely WTIAVPGPPHS from myomesin (water-soluble fraction, A = 0.9091) and FKRPPL from troponin (salt-soluble fraction, A = 0.8333), were selected as the most promising inhibitors of DPP-IV. Both peptides were subjected to ADMET analysis. Fragments of these peptides showed promising drug-likeness properties as well as favorable absorption, distribution, metabolism, excretion, and toxicity functions, suggesting that they are novel leads in the development of DPP-IV inhibitors from food.     

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.     

In Vivo and In Vitro Synthesis of Human Rhinovirus Type 2 Ribonucleic Acid

HeLa cells infected with human rhinovirus type 2 synthesize a mixture of single-and double-stranded ribonucleic acid (RNA). The RNA synthesized by the membrane-bound RNA polymerase complex in vitro is also a mixture of single- and double-stranded RNA, whereas the deoxycholate-treated RNA polymerase complex synthesized only double-stranded RNA. Although twice as much cell-associated viral RNA is synthesized in vivo at 34 C than at 37 C, there is no difference in the rate of RNA synthesized in vitro at 34 C and 37 C by the polymerase complex. The RNA polymerase complex, after treatment with deoxycholate, sediments as a broad peak with an average sedimentation value of 120S.     

Borrelia burgdorferi Downregulates ICAM-1 on Human Synovial Cells In Vitro

Lyme arthritis following infection with Borrelia burgdorferi (B. burgdorferi) is associated with the presence of bacteria in the joint, but the mechanism of persistent infection in the presence of specific antibodies and lymphocytes remains unknown. To investigate how an infection with B. burgdorferi might influence the local immune response in the joint, we examined the expression of cell adhesion molecules, human leucocyte antigens and inducible nitric oxide synthase (iNOS)-1 and -2 in human synovial cells after infection with B. burgdorferi in vitro. Synovial cells are known to influence the function of local immunologic effector cells and play a key role in the pannus formation of erosive arthritis. It has been shown previously that B. burgdorferi can persist in the cytosol of human synovial cells. The expression of the surface molecules ICAM-1, VCAM-1, HLA-class-I and -class-II and the cytosolic production of iNOS-1 and -2 in synovial cells was measured by flow cytometry for up to 5 days after infection with B. burgdorferi. A significant, lasting downregulation of surface ICAM-1 could be demonstrated on synovial cells, whereas no significant changes were seen in the expression of VCAM-1, HLA-class-I and -II, and of iNOS-1 and -2. To determine the biological significance of this downregulation an in vitro adhesion assay using peripheral blood mononuclear cells was developed. After infection with B. burgdorferi a significantly smaller number of mononuclear cells was adhering to the synovial cell monolayer. Adhesion of peripheral mononuclear cells was shown to be in part mediated by ICAM-1 by using a blocking mononuclear antibody against ICAM-1. Downregulation of ICAM-1 on synovial cells due to infection with B. burgdorferi might suppress the local immunosurveillance and might help the bacteria to persist in joint cells in vivo.     

Evaluation of the Potential In Vitro Antiproliferative Effects of Millimeter Waves at Some Therapeutic Frequencies on RPMI 7932 Human Skin Malignant Melanoma Cells

The potential antiproliferative effects of low power millimeter waves (MMWs) at 42.20 and 53.57 GHz on RPMI 7932 human skin melanoma cells were evaluated in vitro in order to ascertain if these two frequencies, comprised in the range of frequency used in millimeter wave therapy, would have a similar effect when applied in vivo to malignant melanoma tumours. Cells were exposed for 1 h exposure/day and to repeated exposure up to a total of four treatments. Plane wave incident power densities <1 mW/cm² were used in the MMWs-exposure experiments so that the radiations did not cause significant thermal effects. Numerical simulations of Petri dish reflectivity were made using the equations for the reflection coefficient of a multilayered system. Such analysis showed that the power densities transmitted into the aqueous samples were ≤0.3 mW/cm². Two very important and general biological endpoints were evaluated in order to study the response of melanoma cells to these radiations, i.e. cell proliferation and cell cycle. Herein, we show that neither cell doubling time nor the cell cycle of RPMI 7932 cells was affected by the frequency of the GHz radiation and duration of the exposure, in the conditions above reported.     

Potential of Cyclodextrin Complexation and Liposomes in Topical Delivery of Ketorolac: In Vitro and In Vivo Evaluation

The objective of this investigation was to evaluate the effect of delivery strategies such as cyclodextrin complexation and liposomes on the topical delivery of ketorolac acid (KTRA) and ketorolac tromethamine. Ketorolac acid–hydroxypropyl-β-cyclodextrin solid dispersions (KTRA-CD) were prepared by kneading method. The liposomes containing ketorolac tromethamine (KTRM) and KTRA-CD were prepared. The in vitro permeation of KTRM solution, KTRA solution, KTRA-CD, and liposomes containing KTRM or KTRA-CD through guinea pig skin was evaluated. The anti-inflammatory activity of the topically applied KTRA-CD gel (containing 1% w/w KTRA) was compared to that of orally delivered KTRM solution. The KTRA-CD demonstrated significantly higher transdermal transport of ketorolac as compared to all other systems whereas liposomes significantly reduced the transport of ketorolac. The anti-inflammatory activity of the topically applied KTRA-CD gel was similar to that of the orally administered KTRM. Thus, cyclodextrin complexation enabled effective transdermal delivery of the ketorolac.     

Potential Anti-proliferative and Immunomodulatory Effects of Marine Microalgal Exopolysaccharide on Various Human Cancer Cells and Lymphocytes In Vitro

Marine microalgal exopolysaccharides (EPSs) have drawn great attention due to their biotechnological potentials such as anti-viral, anti-oxidant, anti-lipidemic, anti-proliferative, and immunomodulatory activities, etc. In the present study, the EPS derived from microalgae Thraustochytriidae sp.-derived mutant GA was investigated for its anti-proliferation and immunomodulation. Anti-cancer efficacy of the microalgal EPS was examined for the alterations in cell proliferation and cell cycle-related gene expression that occur in three types of human cancer cell lines, BG-1 ovarian, MCF-7 breast, and SW-620 colon cancer cell lines, by its treatment. Alterations in immunoreactivity by the microalgal EPS were examined by measuring its influence on the growth of T and B lymphocytes and cytokine production of T cells. In cell viability assay, the microalgal EPS inhibited cancer cell growth at the lowest concentration of 10^?11 dilution and in a dose-responsive manner within the range of dilution of 10^?11~10^?3. In addition, the protein expression of cell cycle progression genes such as cyclin D1 and E in these cancer cell lines was significantly reduced by the microalgal EPS in a dose- and a time-dependant manner. In cell proliferation assay using T and B cells, the microalgal EPS induced B cell proliferation even at the lowest dilution of 10^?11, but not T cells. In cytokine assay, the microalgal EPS decreased the formation of IL-6 and INF-γ at 10^?3 dilution compared to the control and had no significant effects on TNF-α. Collectively, these findings suggest that the EPS derived from microalgae Thraustochytriidae sp. GA has an anti-proliferative activity against cancer cells and an immunomodulatory effect by having an influence on B cell proliferation and cytokine secretion of T cells.     

In Vitro Activity of Coumermycin A1

The in vitro activity of coumermycin A(1) was compared with that of novobiocin, ampicillin, and minocycline. Coumermycin was found to be the most active antibiotic of the four against Staphylococcus aureus. It was about 50 times more active than novobiocin or minocycline against the strains tested. Coumermycin also showed good activity against group A streptococci and pneumococci, moderate activity against Escherichia coli, indole-positive Proteus species, and Pseudomonas aeruginosa, and poor activity against Klebsiella-Enterobacter and enterococci. Against P. mirabilis, however, coumermycin activity was almost equal to that of ampicillin. The new antibiotic was further found to be greatly reduced in activity in the presence of plasma, but its minimal inhibitory concentration was not greatly affected by inoculum size. Coumermycin was found to be bacteriostatic in its action, and resistance to it developed slowly. Also, cross-resistance was present with novobiocin but absent with ampicillin or minocycline.     

Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO₂, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery.     

Release of sICAM-1 in Oocytes and In Vitro Fertilized Human Embryos

Background

During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G) by 48–72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection.

Methodology/Principal Findings

The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes.

Conclusions/Significance

The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques.     

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.