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Failure rate after chronic rotator cuff repair is considerably high. Moreover, diabetes mellitus is known as a compromising factor of rotator cuff tear. The effect of Polydeoxyribonucleotide (PDRN) and polynucleotide (PN) on tendon healing and fatty infiltration is unclear as tissue regeneration activator in diabetic state. Therefore, a diabetic rat model with chronic rotator cuff tear was made for mechanical, histologic and blood tests. In the animal study using a diabetic rat cuff repair model, the administration of PDRN and PN increased the load to failure of repaired cuffs and improved tendon healing and decreased fatty infiltration. Also, the plasma levels of vascular endothelial growth factor and fibroblast growth factor were elevated in PDRN and PN administrated groups. We concluded that PDRN and PN appear to boost tendon recovery and reduce the presence of fatty infiltration following cuff repair in diabetic state. Also, PN showed a later onset and a longer duration than PDRN associated with the mean plasma growth factors., (© 2024. The Author(s).)

Background: The epidermal barrier acts as a defense against external agents as well as helps to maintain body homeostasis. Polynucleotides (PN), exogenous DNA fragments, promote wound repair through their stimulatory and anti-inflammatory effects. Recent findings indicate a synergistic effect of PN and hyaluronic acid (HA) combinations in regulating inflammation and promoting cell proliferation. This study aims to elucidate the effects of PN and HA on repairing the epidermal barrier following its disruption by tape stripping (TS) in a mouse model., Materials and Methods: After disrupting the epidermal barrier using TS, a formulation containing PN (14 mg/mL) and HA (6 mg/mL) was applied. Trans-epidermal water loss (TEWL) was measured at 0, 3, 6, 24, 48, and 72 h. Mice were euthanized after the final application at 72 h, and tissue samples were analyzed for epidermal/dermal thickness, neutrophil infiltration, and filaggrin expression., Results: We observed a significant reduction in TEWL in the PN+HA group compared to that in the control group (20.8 ± 0.5 vs. 43.7 ± 0.5 g/m 2 h at 72 h, p < 0.05), indicating an improvement in barrier function. Histological evaluation showed decreased epidermal and dermal thickening in the PN+HA group compared to that in the control group (epidermal: 29.4 ± 2.2 vs. 57.9 ± 3.5 μm; dermal: 464.8 ± 25.9 vs. 825.9 ± 44.8 μm, both p < 0.05). Additionally, neutrophil infiltration in the dermis was significantly reduced, and filaggrin protein levels were significantly higher in the PN+HA group compared to those in the control group (4.8 ± 0.4 vs. 21.1 ± 3.3 for neutrophils; 0.84 ± 0.04 vs. 0.42 ± 0.03 for filaggrin, both p < 0.05)., Conclusion: These results suggest that PN+HA may be an effective therapeutic strategy for repairing skin barrier damage., (© 2024 The Author(s). Skin Research and Technology published by John Wiley & Sons Ltd.)

Knee osteoarthritis (OA), an age-related degenerative disease characterized by severe pain and disability, is treated using polynucleotides (PNs) and hyaluronic acid (HA). The intra-articular (IA) injection of HA has been studied extensively in both animal models and in humans; however, the efficacy and mechanisms of action remain unclear. In addition, there has been a paucity of research regarding the use of PN alone or in combination with HA in OA. To investigate the effect of the combined injection of PN and HA in vivo, pathological and behavioral changes were assessed in an OA model. Anterior cruciate ligament transection and medial meniscectomy were performed in Sprague-Dawley rats to create the OA animal model. The locomotor activity improved following PNHA injection, while the OARSI grade improved in the medial tibia and femur. In mild OA, TNFα levels decreased histologically in the PN, HA, and PNHA groups but only the PNHA group showed behavioral improvement in terms of distance. In conclusion, PNHA exhibited anti-inflammatory effects during OA progression and improved locomotor activity regardless of the OARSI grade.

Osteoarthritis (OA) is characterized by degeneration of the joint cartilage, inflammation, and a change in the chondrocyte phenotype. Inflammation also promotes cell hypertrophy in human articular chondrocytes (HC-a) by activating the NF-κB pathway. Chondrocyte hypertrophy and inflammation promote extracellular matrix degradation (ECM). Chondrocytes depend on Smad signaling to control and regulate cell hypertrophy as well as to maintain the ECM. The involvement of these two pathways is crucial for preserving the homeostasis of articular cartilage. In recent years, Polynucleotides Highly Purified Technology (PN-HPT) has emerged as a promising area of research for the treatment of OA. PN-HPT involves the use of polynucleotide-based agents with controlled natural origins and high purification levels. In this study, we focused on evaluating the efficacy of a specific polynucleotide sodium agent, known as CONJURAN, which is derived from fish sperm. Polynucleotides (PN), which are physiologically present in the matrix and function as water-soluble nucleic acids with a gel-like property, have been used to treat patients with OA. However, the specific mechanisms underlying the effect remain unclear. Therefore, we investigated the effect of PN in an OA cell model in which HC-a cells were stimulated with interleukin-1β (IL-1β) with or without PN treatment. The CCK-8 assay was used to assess the cytotoxic effects of PN. Furthermore, the enzyme-linked immunosorbent assay was utilized to detect MMP13 levels, and the nitric oxide assay was utilized to determine the effect of PN on inflammation. The anti-inflammatory effects of PN and related mechanisms were investigated using quantitative PCR, Western blot analysis, and immunofluorescence to examine and analyze relative markers. PN inhibited IL-1β induced destruction of genes and proteins by downregulating the expression of MMP3, MMP13, iNOS, and COX-2 while increasing the expression of aggrecan (ACAN) and collagen II (COL2A1). This study demonstrates, for the first time, that PN exerted anti-inflammatory effects by partially inhibiting the NF-κB pathway and increasing the Smad2/3 pathway. Based on our findings, PN can potentially serve as a treatment for OA.

Background: After surgical repair of chronic rotator cuff tears, healing of the repaired tendons often fails and is accompanied by high-level fatty degeneration. Our purpose was to explore the effects of polydeoxyribonucleotide (PDRN) and polynucleotide (PN) on tendon healing and the reversal of fatty degeneration in a chronic rotator cuff tear model using a rat infraspinatus., Methods: Sixty rats were randomly assigned to the following three groups (20 rats per group: 12 for histological evaluation and 8 for mechanical testing): saline + repair (SR), PDRN + repair (PR), and PN + repair (PNR). The right shoulder was used for experimental intervention, and the left served as a control. Four weeks after detaching the infraspinatus, the torn tendon was repaired. Saline, PDRN, and PN were applied to the repair sites. Histological evaluation was performed 3 and 6 weeks after repair and biomechanical analysis was performed at 6 weeks., Results: Three weeks after repair, the PR and PNR groups had more CD168-stained cells than the SR group. The PR group showed a larger cross-sectional area (CSA) of muscle fibers than the SR and PNR groups. Six weeks after repair, the PR and PNR groups showed more adipose cells, less CD68-stained cells, and more parallel tendon collagen fibers than the SR group. The PR group had more CD 68-stained cells than the PNR group. The PR group showed a larger CSA than the SR group. The mean load-to-failure values of the PR and PNR groups were higher than that of the SR group, although these differences were not significant., Conclusion: PDRN and PN may improve tendon healing and decrease fatty degeneration after cuff repair., (© 2021. The Korean Tissue Engineering and Regenerative Medicine Society.)

Reconstruction of chronic ulcers is often hampered by lack of local tissues and poor general conditions. Conservative approaches with debridement and advanced medications, such as polyurethane foam, stand as mainstays. However, the healing process is often slow, thus increasing the risk for infection or other complications. In such cases, porcine dermis (PD) and polynucleotides-added hyaluronic acid (PAHA) were previously reported to accelerate healing. The aim of the study was to compare the efficacy of PD, PAHA and polyurethane foam in chronic ulcers. Thirty patients were randomly divided into 3 groups: group 1 was treated with advanced medications, group 2 with PD, group 3 with PAHA. Standardised photographs and biopsies were taken before treatment and at 30-day follow-up. Photographs were processed to calculate the wound area. Specimens were stained with Haematoxylin/Eosin, Masson trichrome, and immunohistochemically for CD34, alpha-Smooth Muscle Actin (α-SMA), Collagen types I and III, Ki67. The re-epithelialized area was larger in patients treated with PD and PAHA compared with those treated with polyurethane foam (P < .05 and P < .01, respectively). Specimens from patients treated with PD and PAHA showed a higher number of myofibroblasts (α-SMA+, P < .01), neo-angiogenesis (CD34+, P < .01), proliferating dermal cells (Ki67+, P < .01), proliferating keratinocytes (Ki67+, P < .01) and collagen type 1 deposition (P < .05). No difference was found between PD and PAHA. PD and PAHA proved to be more effective than polyurethane foam in the treatment of chronic ulcers. These approaches are a versatile and reliable option to address such cases., (© 2020 Medicalhelplines.com Inc (3M) and John Wiley & Sons Ltd.)

HA (Hyaluronic acid) filler, the most commonly used dermal filler, causes several side effects. HA-PN (Hyaluronic acid-Polynucleotide), a new composite filler, has excellent biocompatibility and induces tissue regeneration. In this study, we compare the efficacies and safety profiles of these fillers. The characteristics of HA and HA-PN fillers were compared using scanning electron microscopy and rheometry. No morphological difference was noted between the fillers. However, the latter had higher viscosity and elasticity values. The HA-PN filler induced higher cell migration than the HA filler in a wound healing assay. It was also found to stimulate better collagen synthesis in human and mouse fibroblasts. The HA and HA-PN fillers were injected into SKH1 hairless mice to determine changes in their volume for up to 24 weeks. Increased cell migration and collagen synthesis were observed in mice injected with the HA-PN complex filler. Although the safety and durability of the HA and HA-PN fillers were similar, the latter induced a lower transient receptor potential vanilloid 4 expression and caused less stimulation upon injection. In conclusion, HA-PN complex fillers can stimulate fibroblast growth and facilitate volume growth and skin regeneration.

The DNA polymeric molecules polydeoxynucleotide (PDRN) and polynucleotide (PN) can be used as new alternative treatment for osteoarthritis (OA); however, the underlying mechanisms are not fully understood. In this study, we investigated the effect of PDRN and PN on gene-expression profiles in a cell model of OA using transcriptome analysis. Under hypoxic conditions, human chondrosarcoma cells were stressed for 24 h in the presence of interleukin (IL)-1β and subsequently treated with PDRN, PN, or hyaluronic acid (HA) for another 24 h, followed by transcriptome analysis. The results of the transcriptome study comprising differentially expressed genes were analyzed using the Database of Annotation Visualization and Integrated Discovery program, which yielded Kyoto Encyclopedia of Genes and Genomes pathways. Toll-like receptor (TLR)- and nucleotide-binding oligomerization domain-like receptor (NLR)-signaling pathways were related between the IL-1β group and the group treated with DNA polymeric molecules. The genes involved in the TLR- and NLR-signaling pathways were validated using real-time quantitative polymerase chain reaction and western blot. Among these genes, IL-6, IL-1β, IL-8, and chemokine (C-C motif) ligand 3 were dramatically upregulated in the IL-1β group, but significantly downregulated in the group treated with DNA polymeric molecules. Specifically, PN treatment resulted in a greater decrease in the expression of these genes as compared with PDRN treatment. Both PDRN and PN treatments were involved in the anti-inflammatory response associated with OA progression, with PN treatment exhibiting additional anti-inflammatory properties relative to PDRN treatment. These results provide insight into potential therapeutic approaches involving PDRN and PN treatment of OA.

In keratitis, keratocytes play a vital role by releasing inflammatory cytokines and expressing intercellular cell adhesion molecule-1(ICAM-1). GC31 is a peptide derived from thrombomodulin, an endogenous protein with potential anti-inflammation properties. We evaluated the protective effect of GC31 in LPS- or poly(I:C)-induced corneal fibroblasts. Cultured keratocytes were treated with either LPS or poly(I:C); The mRNA and protein expressions of IL-6, IL-8, MCP-1, and IFN-γ were determined by real-time RT-PCR and ELISA. The expression level of ICAM-1 was estimated by real-time RT-PCR, immunofluorescence, and western blot. The underlying pathways were investigated by detecting NF-κB p65 translocation and phosphorylation of IκBα, p65, p38, JNK, and ERK. The MTS assay was used to measure cell viability of keratocytes after GC31 incubation. The elevation of IL-6, IL-8, MCP-1, and IFN-γ expression induced by LPS or poly(I:C) was significantly inhibited by GC31 in a dose-dependent manner at both mRNA and protein levels. GC31 also reduced the expression of ICAM-1 in keratocytes after LPS or poly(I:C) stimulation. LPS or poly(I:C) induced p65 translocation and phosphorylation of IκBα, p65, p38, and JNK were suppressed by GC31.GC31 is not only an effective inhibitor of LPS-induced inflammatory response, but it also inhibits poly(I:C)-induced release of inflammatory cytokines and ICAM-1 expression by blocking the NF-κB and MAPK (p38 and JNK) pathways. This suggested that GC31 may exert a protective effect in attenuating corneal inflammation by suppressing the immune response of the fibroblasts., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Impaired learning and cognitive function often occurs during systemic infection or inflammation. Although activation of the innate immune system has been linked to the behavioral and cognitive effects that are associated with infection, the underlying mechanisms remain poorly understood. Here we mimicked viral immune activation with poly(I:C), a synthetic analog of double-stranded RNA, and longitudinally imaged postsynaptic dendritic spines of layer V pyramidal neurons in the mouse primary motor cortex using two-photon microscopy. We found that peripheral immune activation caused dendritic spine loss, impairments in learning-dependent dendritic spine formation and deficits in multiple learning tasks in mice. These observed synaptic alterations in the cortex were mediated by peripheral-monocyte-derived cells and did not require microglial function in the central nervous system. Furthermore, activation of CX3CR1 high Ly6C low monocytes impaired motor learning and learning-related dendritic spine plasticity through tumor necrosis factor (TNF)-α-dependent mechanisms. Taken together, our results highlight CX3CR1 high monocytes and TNF-α as potential therapeutic targets for preventing infection-induced cognitive dysfunction.

Human S100A7 (psoriasin) is highly expressed in psoriasis and other inflammatory diseases; however, the function of S100A7 in wound repair remains largely unknown. Here we demonstrated that skin injury increased the expression of S100A7. Damaged cells from wounded skin induced the expression of S100A7 via the activation of Toll-like receptor 3 (TLR3) followed by the activation of p38 MAPK. S100A7, in turn, acted on keratinocytes to induce the expression of terminal differentiation marker gene loricrin through the activation of p38 MAPK and caspase-1. The differentiation of keratinocytes induced by S100A7 resulted in skin stratification, thus efficiently promoting wound closure. Taken together, our results demonstrate that the activation of TLR3 accelerates wound closure via the induction of S100A7 to induce keratinocyte differentiation. These findings also provide new insights into the development of different forms of treatment with skin wounds.

The common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial superinfections, resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential for 1,8-cineole to treat primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates poly(I:C)-induced activity of the antiviral transcription factor interferon regulatory factor 3 (IRF3), while simultaneously reducing proinflammatory nuclear factor (NF)-κB activity in human cell lines, inferior turbinate stem cells (ITSCs) and in ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared with poly(I:C) alone, whereas NF-κB activity was reduced. Accordingly, 1,8-cineole- and poly(I:C) treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared with the poly(I:C) treatment approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with lipopolysaccharide (LPS) and 1,8-cineole compared with the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on proinflammatory NF-κB signalling, and may thus broaden its field of application., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Speckled 110 kDa (Sp110) plays an important role in infectious diseases, as revealed by studies in humans. However, little is known regarding porcine Sp110. To elucidate its potential role in porcine resistance to viral diseases, here, the complete coding sequence of porcine Sp110 gene and its 26 alternatively spliced isoforms were isolated using reverse transcription (RT)-polymerase chain reaction (PCR), and another seven splicing patterns were obtained using a minigene construct. Subcellular distribution of 11 representative isoforms was characterized in PK-15 cells transiently transfected with their respective GFP fusion constructs, and only isoforms (R and V) bearing all functional domains were localized in nucleus, indicating all the other isoforms lose normal functions of Sp110 owing to alternative splicing. Real-time quantitative PCR and competitive RT-PCR showed that both isoforms R and V had similar tissue expression profile, half-life and response to poly(I:C), a synthetic analog of viral double-stranded RNA, while the longer one (isoform R) was transcribed at a higher level. The results indicated that porcine Sp110 has a role in viral infection and that isoform R is the dominant active form. Overall the data provide potential resource for molecular breeding of pig resistant to diseases and contributes to breeding pigs resistant to viral infection.

Nucleotides (NTs), such as adenosine triphosphate (ATP) and guanosine triphosphate (GTP), are signaling and bioenergy molecules to mediate a range of cellular pathways. We recently reported their significant endosomolytic activity. To evaluate whether polymeric NTs keep endosomolytic and bioenergetic functions of NTs in drug delivery and cell survival, NTs were polymerized by a coupling reaction to form polynucleotides (pNTs: pATP and pGTP) with their molecular weights around 500kDa. The cellular toxicity, indicated by IC50, of pNT was as low as that of corresponding monomeric NT. pNTs were degraded by an intracellular enzyme, alkaline phosphatase. Introduction of pNTs in a polycation-gene complex (polyplex) enhanced the extent of gene expression in cancerous, non-cancerous, and stem cells, up to 1500-fold higher than that of pNT-free polyplex. In addition, cells stored in a pATP solution resulted in a significantly higher survival rate (e.g., up to 20% increase) when exposed to low temperatures than pATP-free solution. The presence of pNT in polyplexes prevented the reduction of transfection efficiency induced by a low temperature. The findings in this study suggest that endosomolytic and bioenergetic pNTs serve as a non-toxic gene carrier component and protect cells from a cold shock or energy depletion., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Notch signaling plays an important role in regulation of innate immune responses and trophoblast function during pregnancy. To identify the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligands (DLL (Delta-like protein)-1/3/4), Jagged 1/2) and Notch-induced transcription factor Hes1 were assessed during preterm labor. Preterm labor was initiated on gestation day 14.5 by intrauterine (IU) injection of peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C). Notch1, Notch2, Notch4, DLL-1 and nuclear localization of Hes1 were significantly elevated in uterus and placenta during PGN+poly(I:C)-induced preterm labor. Ex vivo, Gamma secretase inhibitor (GSI) (inhibitor of Notch receptor processing) significantly diminished the PGN+poly(I:C)-induced secretion of M1- and M2-associated cytokines in decidual macrophages, and of proinflammatory cytokines (IFN-γ, TNF-α and IL-6) and chemokines (MIP-1β) in decidual and placental cells. Conversely, angiogenesis factors including Notch ligands Jagged 1/2 and DLL-4 and VEGF were significantly reduced in uterus and placenta during PGN+poly(I:C)-induced preterm labor. In vivo GSI treatment prevents PGN+poly(I:C)-induced preterm delivery by 55.5% and increased the number of live fetuses in-utero significantly compared to respective controls 48 hrs after injections. In summary, Notch signaling is activated during PGN+poly(I:C)-induced preterm labor, resulting in upregulation of pro-inflammatory responses, and its inhibition improves in-utero survival of live fetuses.

Background: Ultraviolet (UV) irradiation can result in premature skin aging (photoaging) which is characterized by decreased expression of collagen and increased expression of matrix metalloproteinases (MMPs). Double-stranded RNAs (dsRNAs) can be generated at various conditions including virally infected cells or UV-damaged skin cells. Recent studies have shown that a synthetic dsRNA, polyinosinic-polycytidylic acid (poly(I:C)), can reduce procollagen expression in human skin fibroblasts. However, little is known about the effect of poly(I:C) on the expression of MMPs in skin fibroblasts and its underlying mechanisms., Objective: We examined the effect of poly(I:C) on MMP-1, -2, and -3 expressions in human skin fibroblasts. Then, we further explored the underlying signaling pathways involved in the processes., Methods: Human skin fibroblasts were treated with poly(I:C) for the indicated times in the presence or the absence of various chemical inhibitors or small interfering RNAs (siRNAs) at the indicated concentrations. Protein and mRNA levels of various target molecules were examined by Western blotting and quantitative real-time PCR, respectively., Results: Poly(I:C) induced MMP-1, -2, and -3 expressions, which were dependent on TLR3. Poly(I:C) also induced activations of the mitogen-activated protein kinases (MAPKs), the nuclear factor-kappaB (NF-κB) and the interferon regulatory factor 3 (IRF3) pathways. By using specific inhibitors, we found that poly(I:C)-induced expressions of MMP-1, -2, and -3 were differentially regulated by these signaling pathways. In particular, we found that the inhibition of IRF3 signaling pathways attenuated poly(I:C)-induced expressions of all the three MMPs., Conclusion: Our data show that the expressions of MMP-1, -2, and -3 are induced by poly(I:C) through various signaling pathways in human skin fibroblasts and suggest that TLR3 and/or IRF3 may be good targets for regulating the expressions of MMP-1, -2, and -3 induced by dsRNAs., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The olfactory epithelium (OE) of mice deficient in cystic fibrosis transmembrane conductance regulator (CFTR) exhibits ion transport deficiencies reported in human CF airways, as well as progressive neuronal loss, suggesting defects in olfactory neuron homeostasis. Microvillar cells, a specialized OE cell-subtype, have been implicated in maintaining tissue homeostasis. These cells are endowed with a PLCβ2/IP3 R3/TRPC6 signal transduction pathway modulating release of neuropeptide Y (NPY), which stimulates OE stem cell activity. It is unknown, however, whether microvillar cells also mediate the deficits observed in CFTR-null mice. Here we show that Cftr mRNA in mouse OE is exclusively localized in microvillar cells and CFTR immunofluorescence is coassociated with the scaffolding protein NHERF-1 and PLCβ2 in microvilli. In CFTR-null mice, PLCβ2 was undetectable, NHERF-1 mislocalized, and IP3 R3 more intensely stained, along with increased levels of NPY, suggesting profound alteration of the PLCβ2/IP3 R3 signaling pathway. In addition, basal olfactory neuron homeostasis was altered, shown by increased progenitor cell proliferation, differentiation, and apoptosis and by reduced regenerative capacity following methimazole-induced neurodegeneration. The importance of CFTR in microvillar cells was further underscored by decreased thickness of the OE mucus layer and increased numbers of immune cells within this tissue in CFTR-KO mice. Finally, we observed enhanced immune responses to an acute viral-like infection, as well as hyper-responsiveness to chemical and physical stimuli applied intranasally. Taken together, these data strengthen the notion that microvillar cells in the OE play a key role in maintaining tissue homeostasis and identify several mechanisms underlying this regulation through the multiple functions of CFTR., (© 2014 Wiley Periodicals, Inc.)

Based on previously reported antiproliferative activity screening, four most promising disubstituted 2-phenylbenzothiazole hydrochlorides were chosen for detailed study. Water solubility, as well as liphophilicity/hydrophilicity balance of organic core were modified by conversion to mesylate salts. For purpose of structure/activity studies their structures were determined by X-ray structure analysis. Detailed analysis of interactions of new compounds with double stranded (ds-) DNA/RNA by UV/Vis and CD titrations, thermal melting and viscometry experiments revealed that most of studied compounds intercalate into ds-RNA but bind into minor groove of AT-DNA, and agglomerate along GC-DNA. Furthermore, compounds also interact with ss-RNA, but only amino-imidazolinyl 2-phenylbenzothiazole, 4b displayed well defined orientation and dominant binding mode (by induced CD signals) with poly A and poly G. Besides, in vitro investigations revealed moderate to high antiproliferative activity of benzothiazoles against seven human cancer cell lines, while in some cases (HTC 116, SW620, MIA PaCa-2) high correlation between the type of the amidino group and cytotoxic activity was observed., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

In the present study, we examined the liver protein profiles of the large yellow croaker (Pseudosciaena crocea) exposed to polyriboinosinic:polyribocytidylic acid [poly(I:C)], a viral mimic, using the differential proteomic approach. Sixteen altered protein spots were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry or matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry, including eight upregulated proteins and eight downregulated proteins. These altered host proteins were classified into six categories based on their biological function: cellular process, metabolic process, biological regulation, binding, and catabolic process, highlighting the fact that response to poly(I:C) induction in fish seems to be complex and diverse. Moreover, four corresponding genes of the differentially expressed proteins were validated by relative quantitative real-time PCR. Western blot analysis further demonstrated the changes in protein abundance of natural killer enhancing factor and peroxiredoxin 6. These results will be helpful in furthering our understanding of the changes of physiological processes in liver of fish during virus infection.

Chemokines are small, secreted cytokine peptides known principally for their ability to induce migration and activation of leukocyte populations and regulate the immune response mechanisms. The cobia (Rachycentron canadum), a marine finfish species, has a great potential for net cage aquaculture in the South China Sea. We isolated and characterized a CC chemokine cDNA from cobia-designated RcCC2. Its cDNA is 783 bp in length and encodes a putative protein of 110 amino acids. Homology and phylogenetic analysis revealed that the RcCC2 gene, which contains four conserved cysteine residues, shares a high degree of similarity with other known CC chemokine sequences and is closest to the CCL19/21 clade. The mRNA of RcCC2 is expressed constitutively in all tested tissues, including gill, liver, muscle, spleen, kidney, head kidney, skin, brain, stomach, intestine and heart, but not blood, with the highest level of expression in gill and liver. The reverse transcription quantitative polymerase chain reaction was used to examine the expression of the RcCC2 gene in immune-related tissues, including head kidney, spleen and liver, following intraperitoneal injection of the viral mimic polyriboinosinic polyribocytidylic acid, formalin-killed Vibrio carchariae (bacterial vaccine) and phosphate-buffered saline as a control. RcCC2 gene expression was up-regulated differentially in head kidney, spleen and liver during 12 h after challenge. These results indicate that the RcCC2 gene is inducible and is involved in immune responses, suggesting RcCC2 has an important role in the early stage of viral and bacterial infections.

Schizophrenia is associated with increased risk for multiple metabolic abnormalities, including altered glucose homeostasis, type-2 diabetes, obesity, and cardiovascular disease. Some of the metabolic alterations can already exist in psychosis-prone subjects prior to the onset of chronic schizophrenic disease and pharmacotherapy, indicating that they may have a developmental origin. In the present study, we tested the hypothesis that metabolic alterations pertinent to schizophrenic disease can be primed by an environmental risk factor associated with the disorder, namely prenatal exposure to immune challenge. We used a well-established mouse model of prenatal immune challenge induced by maternal gestational treatment with poly(I:C) (="polyriboinosinic-polyribocytidilic acid"), an analog of double-stranded RNA that stimulates a cytokine-associated viral-like acute phase response. Metabolic effects were studied using high-resolution computed tomography and fully automated indirect calorimetry system, along with an oral glucose tolerance test and plasma cytokine and corticosterone measurements. We found that prenatal immune activation caused altered glycemic regulation and abnormal ingestive behavior in periadolescence and led to an adult onset of excess visceral and subcutaneous fat deposition. These effects were accompanied by age-dependent changes in peripheral secretion of proinflammatory (interleukin [IL]-6 and tumor necrosis factor [TNF]-α) and T cell-related (IL-2 and interferon [IFN]-γ) cytokines and by increased release of the stress hormone corticosterone in periadolescence. Our findings show that schizophrenia-relevant metabolic and physiological abnormalities can be primed by prenatal viral-like immune activation, but at the same time, our study emphasizes that this environmental insult is unlikely to precipitate the full spectrum of metabolic and immunological changes pertinent to chronic schizophrenic disease.

The full-length complementary DNA (cDNA) sequences encoding cd8α and cd8β molecules were sequenced and characterized from mandarin fish Siniperca chuatsi. Conserved motifs and residues were found to be present in derived peptides of the Cd8 molecules. For example, WXR motif, DXGXYXC motif, and four cysteine residues were present in the extracellular region of the Cd8 protein. Threonine, serine and proline residues involved in multiple O-linked glycosylation events were located in the membrane proximal hinge region. The common CPH motif in the cytoplasmic tail was detected similar to other teleost Cd8 molecules. Different from those in mammals, S. chuatsi Cd8 sequences have many extra cysteine residues (C149 in Cd8α sequence and C46, C51 and C158 in Cd8β sequence), which also exist in other teleost Cd8 molecules. Real-time polymerase chain reaction (RT-PCR) and Western blot analyses revealed that the thymus had the highest expression of cd8 messenger (m)RNA and protein. After stimulated with phytohaemagglutinin, polyriboinsine-polyribocyaidylic acid and concanavalin A (ConA), the expression level of cd8 mRNA increased significantly in head-kidney lymphocytes at 4 and 8 h, but decreased to normal level at 12 h. Similarly, stimulation with ConA in vivo also led to an increase in the cd8 mRNA level in the spleen. Immunohistochemistry analysis demonstrated that Cd8α-positive cells can be detected in the thymus, spleen and intestine by using polyclonal anti-Cd8α antibody., (© 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.)

Prenatal exposure to maternal infection may be associated with the development of neurodevelopmental disorders as well as increased susceptibility to the development of schizophrenia. Prenatal administration of polyriboinosinic-polyribocytidilic-acid, mimicking RNA virus exposure, has been shown to induce schizophrenia-like behavioral, neurochemical and neuorophysiological abnormalities in rodent offspring. In the present study PIC prenatal administration at gestation day 15 was associated with alterations in the acoustic-startle-response/prepulse-inhibition [ASR/PPI] and the HPA-axis stress response in rat offspring on day 90. We show that pretreatment with dehydroepiandrosterone (DHEA) reverses PIC-related ASR/PPI disruption in female rats and normalizes HPA-axis stress response in a united group of male and female rats. Further research in both animal and human studies is recommended in order to confirm these preliminary findings and their application to the understanding and management of schizophrenia and related conditions., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Cancer vaccine-based immunotherapy is emerging as a novel therapeutic strategy for cancer treatment. However, its antitumor effect remains unsatisfied due to the poor immunogenicity of tumor antigens (Ags). Although polyriboinosinic: polyribocytidylic acid (PIC), a TLR3 agonist, has been reported as a promising adjuvant for cancer vaccines, its immunopotency may be limited by insufficient cellular penetration. In the present study, we incorporated PIC into DOTAP cationic liposome to generate PIC-DOTAP Liposome Complex (PDLC) nanoparticles. The results showed that PDLC was more potent than DOTAP or PIC to enhance vaccine-induced tumor-specific cytotoxic T lymphocyte (CTL) response and IFN-γ production. Moreover, two doses of PDLC vaccines remarkably suppressed tumor growth in mice, which involved the participance of CD8(+) T cells and depended on the presence of Ag. The superior antitumor effect of PDLC vaccines could be attributable to enhanced maturation of mouse bone-marrow dendritic cells (BMDCs) and increased production of type I IFNs. More importantly, PDLC strengthened the TLR3 signaling in BMDCs by enhancing the interaction of PIC with TLR3 and augmenting downstream IRF-3 phosphorylation, as well as elevating IRF-3/IRF-7 mRNA transcription. Taking together, the complex of PIC and DOTAP liposomes enhanced PIC uptake and consequential TLR3 signaling in BMDCs, which in turn promoted DC maturation and type I IFN production, thereby augmenting the antitumor effect of cancer vaccines., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Maternal viral infection during pregnancy is associated with an increase in the incidence of psychiatric disorders with presumed neurodevelopmental origin, including autism spectrum disorders and schizophrenia. The enhanced risk for developing mental illness appears to be caused by deleterious effects of innate immune response-associated factors on the development of the central nervous system, which predispose the offspring to pathological behaviors in adolescence and adulthood. To identify the immune response-associated soluble factors that may affect central nervous system development, we examined the effect of innate immune response activation by polyriboinosinic-polyribocytidylic acid (poly(I:C)), a synthetic analogue of viral double-stranded RNA, on the expression levels of pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors in fetal and postnatal mouse brain 6 h and 24 h after treatment., Methods: C57BL/6J pregnant mice (gestational day 16) or newborn mice (postnatal day 4) received a single intraperitoneal injection of the synthetic analogue of viral double-stranded RNA poly(I:C) (20 mg/kg). Thirty-two immune response-associated soluble factors, including pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors, were assayed 6 h and 24 h after poly(I:C) injection using multiplexed bead-based immunoassay (Milliplex Map) and processed in a Luminex 100 IS instrument., Results: Maternal exposure to poly(I:C) at gestational day 16 induced a significant increase in cytokines interleukin (IL)-1β, IL-7 and IL-13; chemokines monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, interferon gamma-induced protein (IP)-10 and monokine induced by IFN-gamma (MIG); and in the colony stimulating factor vascular endothelial growth factor (VEGF) in the fetal brain. IL-1β showed the highest concentration levels in fetal brains and was the only cytokine significantly up-regulated 24 h after maternal poly(I:C) injection, suggesting that IL-1β may have a deleterious impact on central nervous system development. In contrast, poly(I:C) treatment of postnatal day 4 pups induced a pronounced rise in chemokines and colony stimulating factors in their brains instead of the pro-inflammatory cytokine IL-1β., Conclusions: This study identified a significant increase in the concentration levels of the cytokines IL-1β and IL-13, the chemokine MCP-1 and the colony stimulating factor VEGF in the developing central nervous system during activation of an innate immune response, suggesting that these factors are mediators of the noxious effects of maternal immune activation on central nervous system development, with potential long-lasting effects on animal behavior.

Activating transcription factor 3 (ATF3) participates in cellular processes to adapt to various extra- and intra-cellular changes including the modulation of immunity to prevent uncontrolled immune responses to pathogens. In teleost fishes, the involvement of ATF3 in immune response has not been documented. In this study, the putative Atlantic cod (Gadus morhua) ATF3 transcript was identified by performing rapid amplification of cDNA ends (RACE) based on unknown expressed sequence tags (ESTs) that are potentially inducible by polyriboinosinic polyribocytidylic acid (pIC, a synthetic double-stranded RNA viral mimic) in Atlantic cod. ATF3-like ESTs were the most abundant unknown transcript (i.e. lacking significant BLAST hits) generated from a previously constructed cDNA library enriched for pIC inducible transcripts in Atlantic cod spleen. The full-length cDNA of cod ATF3 consists of 2329 nucleotides with an open reading frame (ORF) of 735 bp encoding 244 amino acids. The deduced amino acid sequence of Atlantic cod ATF3 shares over 45% identity with its putative orthologs from other vertebrates. In addition, the presence of a conserved basic region leucine zipper (bZIP) domain in the deduced Atlantic cod ATF3-like protein further supports its identity as an ATF3 homolog. In the spleen of Atlantic cod challenged with intraperitoneal (IP) injections of pIC, the time-course transcript expression of ATF3 was studied using quantitative reverse transcription-polymerase chain reaction (QPCR). At 6 h following the pIC injection, the relative expression level of ATF3 mRNA was significantly up-regulated in comparison to a pre-injected control (61.9-fold) and its time-matched saline-injected control (97.3-fold). At 24 h following the pIC injection, the mRNA expression level of cod ATF3 had subsided and was no longer significantly different from its pre-injected control, but significantly higher (1.88-fold) than its time-matched saline-injected control. Collectively, these results suggest that ATF3 may be involved in the modulation of innate anti-viral response in Atlantic cod., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Epidemiological studies indicate that among other early life challenges, maternal infection with influenza during pregnancy increased the risk of developing schizophrenia in the child. One morphological manifestation of schizophrenia is hippocampal atrophy. In the hippocampus, playing a key role in learning and memory formation, new granule cell neurons are produced throughout life from resident precursor cells. We hypothesize that individuals exposed to a maternal anti-viral immune response would presumably enter life with a challenged neural precursor cell pool and might later be susceptible to psychiatric pathologies due to reduced adult neurogenesis. We used the injection of double-stranded RNA (polyriboinosinicpolyribocytidylic acid - PolyI:C) in pregnant C57Bl/6 and nestin-GFP reporter mice to induce a maternal viral-like infection and schizophrenia-like behavior in the offspring. In the progeny we found impairments in the open field test and in sensorimotor gating as measured by pre-pulse inhibition of the startle response. The behavioral deficits were accompanied by reduced baseline adult hippocampal neurogenesis. Telomerase activity in neural precursor cells was reduced from birth on and telomere shortening was found in the same cell type in adult life. When we subjected the progeny of viral-like infected dams to voluntary exercise, a known stimulus of adult hippocampal neurogenesis, we could rescue the phenotype in behavior, adult neurogenesis, and cellular senescence. In summary, maternal viral-like immune response reduced telomerase activity and resulted in telomere shortening in neural precursor cells. Further we demonstrate that beneficial behavioral and cellular effects induced by exercise can be studied in a rodent model of schizophrenia., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.

It has been suggested that maternal immune activation increases the risk of psychiatric disorders such as schizophrenia in offspring. There are many reports about hippocampal structural pathology in schizophrenia. Antipsychotic drug administration in adolescence prevented postpubertal hippocampal structural pathology in the maternal immune activation animal model. These findings suggest the possibility that maternal immune activation induces hippocampal dysfunction in juvenile offspring. To test this hypothesis, we investigated hippocampal function in juvenile offspring of maternal immune activation model rat. A synthetic double-stranded RNA polyriboinosinic-polyribocytidilic acid (Poly I:C; 4 mg/kg/day, I.P.) was injected to pregnant rats on gestation days 15 and 17, in order to cause immune activation by stimulating Toll-like receptor 3. Hippocampal synaptic function and morphology in their juvenile offspring (postnatal days 28-31) were compared to those in vehicle-injected control offspring. Field responses were recorded in the hippocampal CA1 region by stimulating commissural/Schaffer collaterals. Pre-synaptic fiber volley amplitudes (mV) and field excitatory post-synaptic potential slopes (mV/ms) were significantly lower in treated offspring. In addition, short-term synaptic plasticity, namely, the paired-pulse facilitation ratio, was significantly higher and long-term synaptic plasticity (long-term potentiation) was significantly impaired in treated offspring. Furthermore, major pre-synaptic protein (synaptophysin) expressions were decreased, but not major post-synaptic proteins (GluR1, GluR2/3, and NR1), in hippocampal CA1 of treated offspring, whereas neuronal loss was not detected in the hippocampal CA1-CA3 regions. These results indicate that maternal immune activation leads to synaptic dysfunction without neuronal loss in the hippocampus of juvenile offspring, and this may be one of the early stages of schizophrenia pathologies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified. The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.

Background: Immunostimulatory oligodeoxynucleotides (CpG-ODN) usually contain phosphorothioate (PS) backbones for nucleotide protection, which may result in some nonspecific side-effects like prolongation of coagulation time., Objective: The aim of the present study was to investigate the immunomodulatory potential of DNA molecules without PS backbones. Thus, we designed phosphorodiester (PO) molecules with a dumbbell-like covalently-closed structure (dSLIM-30L1)., Methods: We analyzed their effects on peripheral blood mononuclear cells (PBMC) from spontaneous high and low immunoglobulin (Ig)E producer (allergic and nonallergic donors) in comparison with linear CpG-ODN (lin-30L1) with PS backbones, using enzyme-linked immunosorbent assay and flow cytometry., Results: We observed a decrease of spontaneous IgE levels in PBMC from high IgE producer of approximately 27% with both dSLIM-30L1 and lin-30L1. In addition, both molecules enhanced the production of IgA, IgM and IgG1/IgG2, but with a slightly different pattern. Both molecules stimulated the secretion of the T(H)1-like cytokines interleukin (IL)-2, interferon-gamma and IL-12p40 and the pro-inflammatory cytokine IL-6. The immunomodulatory potential of dSLIM-30L1 and lin-30L1 was also effective in PBMC from nonallergic donors, as was confirmed for IL-2, IL-12p40, IgG1/IgG2 and IgM., Conclusion: Our data show an inhibition of IgE production but also enhancement of the inflammatory cytokine response in PBMC from allergic and nonallergic donors by covalently-closed PO-based dSLIM-30L1 with a pattern similar to that of linear PS-based lin-30L1, while avoiding PS-modifications and thus PS-mediated side-effects. Whether such molecules are useful for the treatment of allergic diseases will need further clarification by appropriate in vivo studies.

In this study, the contribution of type I interferons (IFNs) to protection against infection with enterovirus 71 (EV71) was investigated using a murine model where the virus was administrated to neonatal Institute of Cancer Research (ICR) mice by either the intraperitoneal (i.p.) or the oral route. In i.p. inoculated mice, post-infection treatment of dexamethasone (5 mg kg(-1) at 2 or 3 days after infection) exacerbated clinical symptoms and increased the tissue viral titre. In contrast, polyriboinosinic : polyribocytidylic acid [poly(I : C); 10 or 100 microg per mouse at 12 h before infection], a potent IFN inducer, improved the survival rate and decreased the tissue viral titres after EV71 challenge, which correlated with an increase in serum IFN-alpha concentration, the percentage of dendritic cells, their expression of major histocompatibility complex class II molecule and IFN-alpha in spleen. Treatment with a neutralizing antibody for type I IFNs (10(4) neutralizing units per mouse, 6 h before and 12 h after infection) resulted in frequent deaths and higher tissue viral load in infected mice compared with control mice. In contrast, an early administration of recombinant mouse IFN-alphaA (10(4) U per mouse for 3 days starting at 0, 1 or 3 days after infection) protected the mice against EV71 infection. In vitro analysis of virus-induced death in three human cell lines showed that human type I IFNs exerted a direct protective effect on EV71. It was concluded that type I IFNs play an important role in controlling EV71 infection and replication.

Here we report the cloning of the rat 90K, a homolog of the mouse cyclophilin C-associated protein/mouse adherent macrophage and human 90K. The protein is constitutively expressed by FRTL-5 thyrocytes, and its levels are modulated by TSH, insulin/IGF-I, and gamma-interferon. Transfection of the cells with 90K cDNA or exposure to purified 90K resulted in a significant increase of the expression of major histocompatibility complex class I but not class II antigens. An increased expression of 90K was obtained after viral infection or introduction into the cells of fragments of viral, bacterial, or mammalian double-strand polynucleotides. The increase was sequence independent, not CpG mediated, and associated with the expression of molecules characterizing antigen-presenting-cell phenotype. The present data along with results from previous studies suggest that 90K plays an important role in the maintenance of an appropriate level of immune response.

In the case of viral infection, various viral proteins and genetic components are disseminated in the body. The former viral proteins may be captured by immature dendritic cells (DC) and the latter genetic components may stimulate the antigen-loading DC to maturate via specific Toll-like receptors (TLR), leading to the establishment of virus-specific cellular immunity; in particular, cytotoxic T lymphocytes (CTL) that control intracellular virions. Polyriboinosinic polyribocytidylic acid [poly(I:C)], which might reflect a natural genetic product from a variety of viruses during replication, has recently been identified as one of the critical stimuli for TLR3. Based on these observations, we speculated that stimulation of TLR3 with poly(I:C) might drive the direction of acquired/adaptive immunity to the cellular arm. Indeed, when BALB/c mice were immunized with purified recombinant HIV-1 envelope gp120 or influenza hemagglutinin (HA) protein together with poly(I:C), epitope-specific CD8(+) class I MHC molecule-restricted CTL were primed from naive CD8(+) T cells in vivo. In contrast, when the same proteins were immunized with lipopolysaccharide, a stimulant of TLR4, specific CTL were not primed at all. Moreover, we show here that immature DC could present processed antigen from captured purified protein in association with class I MHC molecules in the presence of poly(I:C), but not of LPS. These results indicate that we are able to manipulate the direction of acquired/adaptive effector immune responses using an appropriate stimuli and the findings presented in this paper will offer a new therapeutic strategy using poly(I:C) administration for priming antigen-specific CD8(+) CTL with purified viral protein in vivo.

Virus-induced secretion of proinflammatory chemokines (e.g., regulated on activation, normal T cells expressed and secreted [RANTES], interleukin [IL]-8) by airway epithelial cells helps to initiate antiviral responses and airway inflammation by enhancing inflammatory cell recruitment. To define mechanisms for virus-induced chemokine secretion, monolayers of nontransformed bronchial epithelial cells were transfected or incubated with polydeoxyinosinic-deoxycytidylic acid (synthetic double-stranded [ds] RNA), rhinovirus dsRNA, or single-stranded RNA (ssRNA), and the secretion of selected chemokines was determined. Transfection or incubation with dsRNA, but not ssRNA, significantly enhanced secretion of RANTES and IL-8, but not eotaxin or macrophage inflammatory protein-1alpha. Mechanistically, dsRNA induced and activated dsRNA-dependent protein kinase (PKR), and activated nuclear factor-kappaB and p38 mitogen-activated protein kinase. Furthermore, the PKR inhibitor 2-aminopurine significantly blocked dsRNA-induced RANTES and IL-8 secretion, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 suppressed dsRNA-induced IL-8, but not RANTES. These findings indicate that dsRNA selectively induce the secretion of chemokines such as IL-8 and RANTES, and implicate dsRNA-sensitive signaling proteins in this process. Moreover, these data suggest that this may be an important mechanism for the selective secretion of chemokines by viruses (e.g., rhinovirus, respiratory syncytial virus, influenza) that synthesize dsRNA during replication.

A series of novel N1 alkylated purine nucleic acids were polymerized either enzymatically or by automated synthesis to further establish the SAR requirements for HIV, RT, and HCMV activity. Out of the series, two constructs, 2'-O-methyl-1-allylinosinic acid phosphorothioate 33-mer (16) and an oligomer incorporating 1-propyl-6-thioinosinic acid residues (20), were found to be highly active under all three assay conditions. SAR studies indicate that sulfur incorporation, high molecular weight, and low steric bulk at N1 all can be important for activity.

HLA class II-restricted proliferative and cytotoxic T cell (CTL) responses to B cell chronic lymphocytic leukaemia (B-CLL) can be generated using autologous dendritic cells (DCs) pulsed with tumour cell lysate. In this study a number of different approaches were used to optimize further the in vitro system. First, the effects of a variety of maturation agents were studied. The addition of TNF-alpha, polyriboinosinic polyribocytidylic acid (Poly(I:C)) and LPS to autologous DCs resulted in the emergence of only a small percentage of CD83+ DCs, IFN-alpha having no demonstrable effect. Only the addition of Poly(I:C) to DCs resulted in modestly increased specific cytotoxicity to B-CLL targets, IFN-alpha and LPS having no effect. Secondly, T cells were pretreated with IL-15, prior to culturing with lysate-pulsed autologous DCs. A significant increase in T cell activation (P = 0.038), IFN-gamma secretion (P = 0.030) and specific cytotoxicity to B-CLL targets (P = 0.006) was demonstrated compared to untreated T cells. Thirdly, monocyte derived DCs electrofused with B-CLL B cells were compared with lysate-pulsed DCs. T cells stimulated by fused DCs generated higher levels of specific cytotoxicity to autologous B-CLL B cell targets than those stimulated by lysate pulsed DCs (P = 0.013). Blocking studies demonstrated inhibition of this cytotoxicity by both anti-CD4 (P = 0.062) and anti-CD8 monoclonal antibodies (P = 0.018), suggesting the generation of both HLA class I- and HLA class II-restricted CTL responses. In summary, in vitro B-CLL-specific T cell responses can be enhanced further by preincubating T cells with IL-15 and using autologous fused DC-B-CLL hybrids instead of autologous lysate-pulsed DCs. These preliminary data require confirmation with larger numbers of patients. Such an approach, however, may eventually provide effective immunotherapy for treatment of B-CLL.

Elastases are proteinases capable of solubilizing fibrous elastin. They may belong to the class of serine proteinases, cysteine proteinases and metalloproteinases. Mammalian elastases occur mainly in the pancreas and the phagocytes. Among non-mammalian elastases there is a great variety of bacterial metallo and serine elastases. The elastolytic activity varies from one elastase to another and is usually not correlated with the catalytic efficiency of these proteinases. One may measure this activity using native or labelled elastins. With pure elastases one may use synthetic substrates. There is a large number of natural (proteins) and synthetic elastase inhibitors. Elastases play a pathologic role in pulmonary emphysema, cystic fibrosis, infections, inflammation and atherosclerosis.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

DNA and RNA have been shown for the first time to function as chiral photosensitizers in aqueous solution, to effect the enantiodifferentiating photoisomerization of (Z)-cyclooctene (1Z), giving the chiral (E)-isomer in enantiomeric excesses (ee's) of up to 15%. In order to elucidate the effect of nucleotide sequence, enantiodifferentiating photoisomerization of 1Z was also performed using oligo and homopolynucleotides as chiral sensitizer. The -18.8% ee was observed by using d(T)15.d(A)15 as sensitizer, whereas sensitization by the poly(U).poly(A) duplex gave only racemic (E)-cyclooctene. From these results, oligothymidine sequence is essential for efficient enantioselective photoisomerization of 1Z.

Interferon titers in the blood and brain of mice and their protection from the herpes virus were compared after the animal exposure to poly(G).poly(C) duplex, both native and modified with cis-diammine dichloroplatinum (II). It was shown that the duplex platination especially at the level of the poly(G) strand resulted in sharp rising of the interferon titers in the extracts of the animal brain and rearrangement of the types of interferon induced in the brain to predominance of gamma-interferon. The interferonogenesis indices correlated with the duplex protective activity against the herpes virus. It was concluded that the platinum binding could increase the membrane specificity of the duplex and stimulate its penetration through the hematoencephalic barrier. Possible structural changes in the duplex under the action of platinum (II) resulting in the observed effect are discussed.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.

Polynucleotides enhance T-helper (Th) cell-mediated humoral immune responses in naive resting Th cells, B cells, and antigen-presenting cells (APC) from unprimed mouse spleen. If polynucleotides augment Th cell functions independent of the activation stage of Th cells, then polynucleotides may cause hyperimmune responses. In this study we examined the effects of polynucleotides on effector-stage murine Th cell clones in vitro. The A.E7 clone (primed with pigeon cytochrome C, origin: B10.A mice) and CDC35 clone (primed with rabbit gamma-globulin, origin: DBA/2 mice) were used as representative type 1 (Th1) and type 2 (Th2) Th cells, respectively. Th clones were stimulated with antigen (Ag) in polynucleotide-supplemented or control cultures in the presence of syngeneic spleen cells (either CD4- or irradiated). The number of antibody (Ab)-secreting cells was counted to measure T-dependent Ab production. Production of interferon-gamma (IFNgamma) for the Th1 clone and interleukin-5 (IL-5) for the Th2 clone were measured. Without Ag stimulation, cytokine production and the number of Ab-secreting cells formed were very low and not altered by polynucleotides. With suboptimal Ag challenges provided by Ag-primed spleen cells, polynucleotides enhanced IFNgamma production by the Th1 clone, while they suppressed Th1 clone-mediated Ab production and IL-5 production by the Th2 clone. Polynucleotides did not alter Th2 clone-mediated Ab production. These actions of polynucleotides appeared to be dose-dependent. With optimal Ag challenges, polynucleotides did not affect our measures of Th cell activation. Polynucleotide action in vitro on effector-stage Th cell clones differed in each Th cell subset and depended on Ag concentration.

A paper summarises the results of author's and his collaborators studies on the mutagenic action of DNA and other natural and synthetic polynucleotides. These results were published with rare exception in Russian and Ukrainian scientific journals. Analogous results obtained by other authors of several countries are also discussed. It was proved that unlike conventional physical and chemical mutagens, polynucleotides selectively induce visible and lethal mutations preferentially in certain genes, different in case of treatment with different polynucleotides. Polynucleotides often induce mutation in several non-allelic genes in a single germ cell of the host (multimutational effect). Polynucleotides do not induce gross chromosome aberration.

Beside the direct inhibition of thrombin and its regulatory functions, many of the newer antithrombin agents produce several additional effects, unrelated to their anticoagulant actions. Synthetic peptide inhibitors are capable of producing fibrinolytic compromise by virtue of their actions on fibrinolytic enzymes such as t-PA, plasmin, urokinase and protein Ca. In addition, the low molecular weight arginine-containing peptides are also known to produce hemodynamic and hemostatic deficits. The designs of the ongoing clinical trials are largely empirical because of the non-availability of valid pharmacologic and toxicologic data on thrombin inhibitors. In contrast to heparin, none of the thrombin inhibitors produce endogenous release of tissue factor pathway inhibitor (TFPI) in the experimental and clinical settings. These observations suggest that beside the direct inhibition of thrombin, these agents also produce multiple additional effects that can significantly contribute to their pharmacologic and toxicologic profile.

Combinatorial libraries and protein engineering represent two new powerful tools in drug discovery and development. The application of a combinatorial ssDNA library to thrombin led to the discovery of a sequence-specific nucleotide-based thrombin inhibitor (thrombin aptamer). The thrombin aptamer has a novel tertiary structure revealed by NMR and shows potent rapid anticoagulation with a short half-life in vivo. It has been used successfully to replace heparin in a canine cardiopulmonary bypass model. Functional mapping of the surface residues of thrombin led to the generation of a modified thrombin with markedly diminished procoagulant properties while retaining its ability to activate protein C. This engineered thrombin functions as a protein C activator and demonstrates potent anticoagulation in vivo without prolongation of the bleeding time.

Actions of nucleotides on in vitro humoral immune responses were studied in mice fed a nucleotide-free diet, a nucleotide-free diet plus a mononucleotide-nucleoside mixture or a nucleotide-free diet plus yeast RNA (polynucleotides). Cultured spleen cells from mice fed a nucleotide-free diet produced fewer numbers of antibody-secreting cells in response to a T-dependent antigen, compared with those from controls fed nucleotide-supplemented diets. Immunoglobulin M concentrations in these supernatants were significantly lower in cultured cells from mice fed the nucleotide-free diet or the nucleotide-free diet plus the mononucleotide/nucleoside mixture compared with concentrations in cells from mice fed the nucleotide-free diet plus RNA. Concanavalin A-potentiated cytokine (interleukin-4 and interleukin-5) production by purified T helper cells was also lower in cultured cells from mice fed a nucleotide-free diet than in those from mice fed nucleotide-supplemented diets. In vivo supplementation with the mononucleotide/nucleoside mixture restored impaired in vitro antibody and concanavalin A-potentiated cytokine production in mice fed a nucleotide-free diet. However, addition of RNA to the culture enhanced antibody production in spleen cells from mice of all diet groups. Supplementing the culture with RNA did not enhance mitogen-potentiated cytokine production. This in vitro action of RNA was retained after the removal of oligonucleotides (molecular weight < 1000), but was reduced by modification of bases and cleavage of phosphodiester bonds of RNA. Thus the in vitro action of RNA is mainly attributed to polynucleotides, indicating their potential role in modulation of local humoral immune responses in the body.