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Tibetan kefir grain as the starter of milk fermentation has been applied as functional food with many bioactive characteristics. In this study, the milk whey product (TKG-MW) was obtained through the milk fermentation of Tibetan kefir grain containing the dominant Lactobacillus, Acetobacter, and Bacillus after 3 and 6 days of cultivation. Antioxidant, anti-inflammatory, and melanogenesis inhibition capacities under TKG-MW treatment were analyzed. Results revealed that the antioxidation of TKG-MW at 6 days of fermentation was higher than that at 3 days of fermentation according to the DPPH and ABTS+ radical scavenging analysis. However, the anti-inflammation of TKG-MW was only observed at 6 days of fermentation by using lipopolysaccharide-stimulated RAW 264.7 macrophages. The inhibition of mushroom tyrosinase activity by TKG-MW was demonstrated. The decrease of melanin content was verified using α-melanocyte-stimulating hormone-stimulated B16-F10 cell. The real-time quantitative reverse transcription polymerase chain reaction result indicated that the mRNA levels of Tyr, Trp-1, and Trp-2 of the B16 cell involved in melanin synthesis were down-regulated over a two-fold change by the TKG-MW treatment. Additionally, the protein expressions of Tyr, Trp-1, Trp-2, and Mitf of the B16 cell were reduced with the TKG-MW treatment. Organic acids, such as lactic acid, succinic acid, 3-phenyllactic acid, l-pyroglutamic acid, and malic acid, were identified by liquid chromatography-mass spectrometry in TKG-MW and were found to significantly inhibit tyrosinase activity. To the best of our knowledge, this work is the first to report melanogenesis suppression by TKG-MW. Results suggested that the fermentation product of TKG could be applied as a depigmenting agent in food and cosmetics., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In this study, we demonstrate the synthesis of carbonized nanogels (CNGs) from an amino acid (lysine hydrochloride) using a simple pyrolysis method, resulting in effective viral inhibition properties against infectious bronchitis virus (IBV). The viral inhibition of CNGs was studied using both in vitro (bovine ephemeral fever virus (BEFV) and pseudorabies virus (PRV)) and in ovo (IBV) models, which indicated that the CNGs were able to prevent virus attachment on the cell membrane and penetration into the cell. A very low concentration of 30 μg mL -1 was found to be effective (>98% inhibition) in IBV-infected chicken embryos. The hatching rate and pathology of IBV-infected chicken embryos were greatly improved in the presence of CNGs. CNGs with distinctive virus-neutralizing activities show great potential as a virostatic agent to prevent the spread of avian viruses and to alleviate the pathology of infected avian species.

Infection with influenza virus is a major public health problem, causing serious illness and death each year. Emergence of drug-resistant influenza virus strains limits the effectiveness of drug treatment. Importantly, a dual H275Y/I223R mutation detected in the pandemic influenza A 2009 virus strain results in multidrug resistance to current neuraminidase (NA) drugs. Therefore, discovery of new agents for treating multiple drug-resistant (MDR) influenza virus infections is important. Here, we propose a parallel screening strategy that simultaneously screens wild-type (WT) and MDR NAs, and identifies inhibitors matching the subsite characteristics of both NA-binding sites. These may maintain their potency when drug-resistant mutations arise. Initially, we analyzed the subsite of the dual H275Y/I223R NA mutant. Analysis of the site-moiety maps of NA protein structures show that the mutant subsite has a relatively small volume and is highly polar compared with the WT subsite. Moreover, the mutant subsite has a high preference for forming hydrogen-bonding interactions with polar moieties. These changes may drive multidrug resistance. Using this strategy, we identified a new inhibitor, Remazol Brilliant Blue R (RB19, an anthraquinone dye), which inhibited WT NA and MDR NA with IC(50) values of 3.4 and 4.5 µM, respectively. RB19 comprises a rigid core scaffold and a flexible chain with a large polar moiety. The former interacts with highly conserved residues, decreasing the probability of resistance. The latter forms van der Waals contacts with the WT subsite and yields hydrogen bonds with the mutant subsite by switching the orientation of its flexible side chain. Both scaffolds of RB19 are good starting points for lead optimization. The results reveal a parallel screening strategy for identifying resistance mechanisms and discovering anti-resistance neuraminidase inhibitors. We believe that this strategy may be applied to other diseases with high mutation rates, such as cancer and human immunodeficiency virus type 1.

Epidemiology supports a causal link between air pollutant exposure and childhood asthma, but the mechanisms are unknown. We have previously reported that ozone exposure can alter the anatomic distribution of CD25+ lymphocytes in airways of allergen-sensitized infant rhesus monkeys. Here, we hypothesized that ozone may also affect eosinophil trafficking to allergen-sensitized infant airways. To test this hypothesis, we measured blood, lavage, and airway mucosa eosinophils in 3-month old monkeys following cyclical ozone and house dust mite (HDM) aerosol exposures. We also determined if eotaxin family members (CCL11, CCL24, CCL26) are associated with eosinophil location in response to exposures. In lavage, eosinophil numbers increased in animals exposed to ozone and/or HDM. Ozone+HDM animals showed significantly increased CCL24 and CCL26 protein in lavage, but the concentration of CCL11, CCL24, and CCL26 was independent of eosinophil number for all exposure groups. In airway mucosa, eosinophils increased with exposure to HDM alone; comparatively, ozone and ozone+HDM resulted in reduced eosinophils. CCL26 mRNA and immunofluorescence staining increased in airway mucosa of HDM alone animals and correlated with eosinophil volume. In ozone+HDM animal groups, CCL24 mRNA and immunofluorescence increased along with CCR3 mRNA, but did not correlate with airway mucosa eosinophils. Cumulatively, our data indicate that ozone exposure results in a profile of airway eosinophil migration that is distinct from HDM mediated pathways. CCL24 was found to be induced only by combined ozone and HDM exposure, however expression was not associated with the presence of eosinophils within the airway mucosa., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a small subset of non small cell lung cancer (NSCLC) harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR inhibitors, suggesting that other factors are implicated in survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) function as transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been discovered in a number of cancers. In this study, we found that a small molecule, reactivation of p53 and induction of tumor cell apoptosis (RITA), showed anti-cancer activity against gefitinib-resistant H1650 cells through a p53-independent pathway. Stat3 suppression by RITA attracted our attention to investigate the role of Stat3 in sustaining survival of H1650 cells. Pharmacological and genetic approaches were employed to down-regulate Stat3 in H1650 cells. WP1066, a known Stat3 inhibitor, was shown to exhibit inhibitory effect on the growth of H1650 cells. Meanwhile, apoptosis activation by siRNA-mediated down-regulation of Stat3 in H1650 cells provides more direct evidence for the involvement of Stat3 in viability maintenance of H1650 cells. Moreover, as a novel identified Stat3 inhibitor, RITA increased doxorubicin sensitivity of H1650 cells in vitro and in vivo, suggesting that doxorubicin accompanied with Stat3 inhibitors may be considered as an alternative strategy to treat NSCLC patients who have inherent resistance to doxorubicin. Overall, our observations reveal that targeting Stat3 may be an effective treatment for certain NSCLC cells with oncogenic addition to Stat3., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have documented that exposure of rhesus monkeys to house dust mite aeroallergen during postnatal development resulted in significant recruitment of eosinophils into the airway mucosa (Clin Exp Allergy 33:1686-1694, 2003). Because eosinophils were not uniformly distributed throughout the five conducting airway generations examined, we speculated that trafficking within anatomic microenvironments of the lung is mediated by differential chemokine expression. To address this question, we used quantitative real-time RT-PCR to evaluate the related eosinophilic chemokines, eotaxin (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26) within isolated airways of infant monkey lung. Overall, chemokine mRNA expression levels in house dust mite-exposed airways were as follows: eotaxin-3 > eotaxin > eotaxin-2. Immunofluorescence staining for eotaxin-3 and CC chemokine receptor 3 (CCR3) showed positive cells within epithelium and peripherally located nerve fiber bundles of the airway wall. Epithelial volume of eotaxin-3 within the trachea correlated with epithelial volume of major basic protein. CCR3+ and MHC Class II+ dendritic cells, but not eosinophils or mast cells, co-localized within eotaxin-3+ nerve fiber bundles. We conclude that localized expression of eotaxin-3 plays an important role in the recruitment of diverse CCR3+ cell populations to different anatomic microenvironments within the infant airway in response to chronic allergen exposure.

Background: Accumulation of immune cell populations and their cytokine products within tracheobronchial airways contributes to the pathogenesis of allergic asthma. It has been postulated that peripheral regions of the lung play a more significant role than proximal airways with regard to inflammatory events and airflow obstruction., Objective: To determine whether immune cell populations and associated cytokines are uniformly distributed throughout the conducting airway tree in a non-human primate model of allergic asthma., Methods: We used a stereologic approach with a stratified sampling scheme to measure the volume density of immune cells within the epithelium and interstitium of trachea and 4-5 intrapulmonary airway generations from house dust mite (HDM) (Dermatophagoides farinae)-challenged adult monkeys. In conjunction with immune cell distribution profiles, mRNA levels for 21 cytokines/chemokines and three chemokine receptors were evaluated at four different airway generations from microdissected lungs., Results: In HDM-challenged monkeys, the volume of CD1a+ dendritic cells, CD4+ T helper lymphocytes, CD25+ cells, IgE+ cells, eosinophils, and proliferating cells were significantly increased within airways. All five immune cell types accumulated within airways in unique patterns of distribution, suggesting compartmentalized responses with regard to trafficking. Although cytokine mRNA levels were elevated throughout the conducting airway tree of HDM-challenged animals, the distal airways (terminal and respiratory bronchioles) exhibited the most pronounced up-regulation., Conclusion: These findings demonstrate that key effector immune cell populations and cytokines associated with asthma differentially accumulate within distinct regions and compartments of tracheobronchial airways from allergen-challenged primates.

Background: In addition to the common ABO phenotypes, numerous phenotypes with a weak expression of the A or B antigens on RBCs have been found. This study describes the molecular genetic analysis of the Ael and the A3 phenotypes., Study Design and Methods: The seven-exon regions of the ABO genes of Ael and A3B individuals were amplified by PCR and cloned, and the sequences of the exons and their adjacent splice sites were analyzed. Samples from 30 randomly selected A1 individuals were also assessed., Results: The A gene with wild-type coding sequence was demonstrated in the Ael propositus, but all the six unrelated Taiwanese people with the Ael or AelB phenotype were shown to possess an A allele with the G-->A mutation at the +5 position of intron 6 (IVS6+5G-->A). RT-PCR analysis showed that the complete A transcript structure was absent in the Ael RNA samples. The A3B individual possessed an A gene with an 838C-->T missense mutation., Conclusion: The results suggest an association of the Ael*IVS6+5G-->A allele with the Ael phenotype in Taiwanese people. The mechanism defining how the Ael*IVS6+5G-->A allele leads to the Ael phenotype awaits elucidation.

Recent structural analysis of the peptide-MHC complex reveals that an antigenic peptide binds to MHC in only one conformation and that side chains anchoring in the binding pocket would not contact TCR. The identification of all the MHC-anchoring residues on an antigenic peptide is a prerequisite to understand how a given peptide interacts with the TCR. In a combination of binding analysis and model simulation, model peptide lambda repressor cl 16-26 was shown to bind to I-Ek through four anchor residues (Leu18, IIe21, Glu23 and Lys26), a pattern found in many I-Ek-binding peptides. TCR reactivity analysis clearly indicates a great variation in the interaction with cl 16-26 by T cells generated from different strains of I-Ek-bearing mice. Most of the T cell generated from A/J mice reacted with the central regions of cl 16-26, while there is a great diversity on the recognition of cl 16-26 by T cells from C3H and B10.BR mice. Despite the diverse interactions with antigenic peptide by these T cells, most TCR-E-k contacts are limited to the central region of the I-Ek beta-chain. T cells recognizing only the N-terminal part of cl 16-26 were found to contact I-Ek at nearly the same residues as T cells interacting with the C-terminal of cl 16-26. TCR-I-Ek recognition was apparently independent of TCR-cl 16-26 contact. The discordant TCR-peptide and TCR-MHC interaction may represent a unique feature of TCR recognition.

Alternative T cell receptor (TcR) gene usage between mice of different Mls alleles has been demonstrated in a number of T cell responses. A clear illustration of a flexible TcR V beta usage in the same strain of mice remains to be established. Using a model system in which I-Ek-restricted T cells recognizing lambda repressor cI protein (cI) 12-26 and pigeon cytochrome c (pcc) 81-104 predominantly use V beta 3 in B10.A and B10.BR mice, and V beta 1 in Mls-2a-bearing A/J and C3H mice, we have first demonstrated that the hierarchy of TcR V beta usage can not be inferred from one strain of mice to the other. The presumed flexibility of V beta 3 to V beta 1 did not exist in B10.BR mice in the given responses. Instead, a switch of dominant TcR from V beta 1/V beta 3 to V beta 8 was identified in C3H and B10.BR mice. In contrast, there was an absolute rigidity in TcR repertoire usage in some mouse strains such as A/J. The lack of flexibility was not due to slow generating kinetics of replacing T cells; since A/J mice treated with staphylococcal enterotoxin A from birth on still responded poorly to cI 12-26 and pcc 81-104. Therefore, whether TcR V beta usage in a T cell response would be flexible or rigid is highly dependent on each strain of mice. However, even the plasticity seen in B10.BR mice is very limited and further tolerance of the V beta 8+ population results in non-responsiveness toward the given antigens.

Kinetic data on potassium release from and killing of Candida albicans by the four polyene antibiotics amphotericin B, amphotericin B methyl ester hydrochloride, nystatin, and nystatin methyl ester hydrochloride are presented. The nystatins were relatively more effective than the amphotericins in causing potassium release rather than killing. These data suggest that the aqueous channels or pores formed by the polyene antibiotics are not central to the lethal action of the drugs.

The presence of free fatty acids in liposome model membranes sensitizes these membranes to the action of the imidazole antifungals, clotrimazole, micronazole, and sulconazole. Unsaturation of the fatty acids is an important variable; the effect of linoleic and oleic acids is much greater than that of stearic acid. The imidazoles differ somewhat in action, with clotrimazole potency greatest both on membranes with and without fatty acids. Sulconazole has very little activity on membranes without fatty acids even at the highest concentrations tested. The data are discussed with reference to the susceptibility of various cells to the imidazoles and the specificity of imidazole action. A modification of the enzymatic method generally used for assay of marker glucose with liposome systems is also presented.

Syrian hamsters are exquisitely sensitive to clindamycin; as little as 1 mg/kg of clindamycin given systemically causes a fatal colitis. Clindamycin and erythromycin were applied topically daily to the shaved backs of Syrian hamsters in a hydroalcoholic vehicle. A daily dose of 0.1 mg of clindamycin was lethal to more than half the hamsters and 1 mg to all the animals. The antibiotic-associated toxin from Clostridium difficile was present in their cecal material. Based on body surface areas and estimated usual volumes applied, the lethal dose in hamsters is not dissimilar to that given humans for acne. Oral tetracycline therapy protected the animals from clindamycin toxicity, but the animals died three days after stopping tetracycline if topical clindamycin applications were continued.