Toxicity of a novel antifungal agent (ATB1651 gel) in Yucatan minipigs (Sus scrofa) following 4 weeks of daily dermal administration.

ATB1651 gel is an antifungal drug candidate that enhances antifungal activity through substitution of several aryl rings, alkyl chains, and methyl groups. To ensure safety of use of ATB1651 gel, assessment of its potentially toxic side effects is necessary. In this study, we examined the repeated-dose toxicity of ATB1651 gel to Yucatan minipigs (Sus scrofa) in accordance with the Good Laboratory Practice guidelines. Five doses of ATB1651 gel (0%, 0.2%, 0.5%, 1.0%, 3.0%) were administered dermally to the left and right flanks of 38 minipigs daily for 4 weeks. Mortality, clinical symptoms, dermal scores, body weights, and physiological, biochemical, pathological, and toxicokinetic analyses were performed after the treatment period. No systemic toxicological damage was observed in either male or female minipigs regardless of dose; however, dermal application of ATB1651 gel caused some skin alterations at the application sites. Specifically, erythema and eschar formation, edema, and scabs or raise spots were observed at the application site(s) in males in the 3.0% ATB1651 gel treatment group and in females at ATB1651 gel concentrations ≥ 1.0%, with dermal scores ranging from grade 1 to 2. Additionally, histopathological assay indicated infiltration of different types of inflammatory cells and the presence of pustule/crust at the application site(s) in both males and females at ATB1651 gel concentrations ≥ 0.5%. However, these changes were reversible after a 2-week recovery period and were considered a local irritation effect of ATB1651 gel. The no-observed-adverse-effect level of ATB1651 gel was 3.0% with regard to topical and systemic toxicity in both male and female minipigs. Collectively, our results imply that ATB1651 gel is a safe candidate for clinical development as an antifungal drug with a wide therapeutic window.

Establishment of an experimental model of ovalbumin-induced atopic dermatitis in canines.

Introduction: A reliable standard model is required to evaluate the efficacy of new drugs for companion animals, especially dogs. Canine atopic dermatitis (cAD), also known as allergic inflammatory skin disease, is a common condition. Currently, the house dust mite animal model is used in the research of cAD; however, this model exhibits significant individual variation and is difficult to standardize. In this study, we used ovalbumin as an antigen to sensitize and stimulate dogs, thereby establishing a stable model mimicking the T-helper 2 (Th2) response seen in cAD. Our objective was to create a cAD model that could be employed to evaluate the efficacy of novel drugs and mimic the Th2 dominant allergic response observed in the pathogenesis of atopic dermatitis of dogs. Methods: In this study, six beagles were used. Normal saline was applied to two animals, and ovalbumin to four, on their dorsal skin. Results: The ovalbumin-treated groups exhibited clinical cAD symptoms, such as pruritus and erythema. Moreover, plasma levels of the cAD markers immunoglobulin E and CCL17 chemokine were higher in the ovalbumin-treated group than in the vehicle control group. The skin thickness of the epidermis was significantly increased in the ovalbumin-treated group, with infiltration of inflammatory cells observed in the thickened dermis region. In conclusion, treatment of canine skin with an optimal concentration of ovalbumin induced typical cAD-like symptoms, and histological and molecular analyses confirmed an enhanced Th2-related immune response. Conclusion: Therefore, we successfully established a suitable Th2-dominant response mimicking cAD, which will facilitate targeted research of atopic dermatitis in dogs.

A Comparison Between GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;TBM Pig Kidneys Transplanted in Nonhuman Primates.

Because there is a shortage of donor kidneys, researchers are exploring the possibility of using genetically modified pig kidneys for transplantation. Approaches involving knockout of carbohydrate genes or knockin of protective proteins have been attempted to determine the best gene modifications. In this study, we utilized GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;thrombomodulin (TBM) pigs for transplantation in nonhuman primates (NHPs). The NHPs survived for 4 weeks after kidney transplantation (4 WAT) from the GalT-/-;hCD39;hCD55 pig and for 6 WAT from the GalT-/-;hCD39;hCD46;hCD55;TBM pig. However, messenger RNA (mRNA) sequencing and immunohistochemistry analysis revealed that the 6 WAT kidney exhibited more severe apoptosis, inflammation, loss of renal function, and renal fibrosis than the 4 WAT kidney. These results indicate that additional knockin of complement regulator (hCD46) and coagulation regulator (TBM) is not enough to prevent renal damage, suggesting that improved immune suppression is needed for more prolonged survival.

Pelargonium sidoides extract mediates nephrotoxicity through mitochondrial malfunction and cytoskeleton destabilization.

We investigated the cytotoxic effect of Pelargonium sidoides extract on Madin-Darby canine kidney (MDCK) cells. P. sidoides extract decreased the cell viability in a dose dependent manner (> 0.2%). The extract of P. sidoides decreased the mitochondrial action potential, increased the number of reactive oxygen species (ROS) inside the cell, and caused nicotinamide adenine dinucleotide hydride (NADH) to be released, all of which are signs of mitochondrial dysfunction. The results of unbiased mRNA sequencing showed that 0.3% P. sidoides extract upregulates the apoptosis-related gene (BBC3). This finding was supported by immunoblot analysis of apoptosis signal pathways, which included Bcl-2, Bax, cytochrome C (CytC), cleaved caspase 3 (CC3), cleaved caspase 7 (CC7), cleaved caspase 9 (CC9) and cleaved PARP (CP). It is interesting to note that the elevated levels of Bax, CytC, CC3, CC7, and CC9, as well as CP, were suppressed by N-acetyl-L-cysteine (NAC) pretreatment, which points to ROS-mediated apoptosis. The small GTPases, RhoA, and Rac1/cdc42-GTP-bound active form were all lowered when P. sidoides extract was used. Also, RhoA-related cytoskeleton signals (ROCK, p-LIMK1/2, p-cofilin) and Rac1/cdc42-related signals (N-WASP, WAVE-2) were inhibited by P. sidoides extract. NAC or RhoA/Rac1/cdc42 activator pretreatment reduced P. sidoides extract-induced actin destabilization. In this work, P. sidoides extract promotes apoptosis by causing mitochondrial dysfunction and cytoskeleton disassembly.

Structure based exploration of potential lead molecules against the extracellular cysteine protease (EcpA) of Staphylococcus epidermidis: a therapeutic halt.

Nosocomial infection caused by Staphylococcus epidermidis is one of the most widely spread diseases affecting the world's population. No strategies have been developed to overcome this infection and inhibit its spread in immunocompromised patients or patients with indwelling medical devices. EcpA is an extracellular cysteine protease protein involved in biofilm formation on medical devices. Thus, blocking this mechanism may be viable for developing a drug against S. epidermidis. The current research aimed to find new, potent inhibitors that could stop the S. epidermidis EcpA protein from functioning. This study attempted to identify the most promising drug candidates using structure-based virtual screening (SBVS) from libraries of natural ligands. The top-scored molecules were shortlisted based on their IC50 values and pharmacophore properties and further validated through density functional theory (DFT) studies. We found five inhibitors using virtual screening, and the results indicate that these drugs had the highest energy binding potential towards the EcpA targets when compared to the reference molecule E-64, a known cysteine protease inhibitor. In order to evaluate the binding conformational stability of protein-ligand complexes, molecular dynamics (MD) simulations were performed in triplicate for 100 ns, revealing the significant stability of anticipated molecules at the docked site. Furthermore, principal component analysis and binding free energy calculations were performed to understand the dynamics and stability of the complexes. The current study indicated that these compounds looked to be suitable novel inhibitors of the EcpA protein and pave the path for further discovery of novel inhibitors of EcpA.Communicated by Ramaswamy H. Sarma.

Less-Suture Vascular Anastomosis: Development of Alternative Protocols with Multifunctional Self-Wrapping, Transparent, Adhesive, and Elastic Biomaterials.

Blood vessel anastomosis by suture is a life-saving, yet time-consuming and labor-intensive operation. While suture-less alternatives utilizing clips or related devices are developed to address these shortcomings, suture anastomosis is still overwhelmingly used in most cases. In this study, practical "less-suture" strategies are proposed, rather than ideal "suture-less" methods, to reflect real-world clinical situations. In the case of rat artery (d = 0.64 mm) anastomosis, the less-suture anastomosis involves the application of thin, adhesive, transparent, and self-wrapping films to the site. This surprisingly reduces the number of stitches required from ten (without films) to four (with films), saving 27 min of operating time per vessel. Furthermore, the decreased number of stitches largely alleviates fibrosis-mediated wall-thickening. Thus, a less-suture strategy is particularly useful for anastomosis of multiple vessels in emergency conditions and small-diameter vessels.

Comparison of Graft Survival Between Full-Thickness and Lamellar Pig-to-Monkey Corneal Xenotransplantation from the Same Genetically Engineered Pig Model with Minimal Immunosuppression.

BACKGROUND: The graft survival rate of full-thickness corneal xenotransplantation (XTP) with minimal immunosuppression in genetically engineered pigs is unknown, whereas lamellar corneal XTP shows satisfactory results. We compared graft survival between full-thickness and lamellar transplantations in the same genetically engineered pig. METHODS: Six pig-to-monkey corneal transplantations were performed on 3 transgenic pigs. Two corneas harvested from 1 pig were transplanted into 2 monkeys using full-thickness and lamellar corneal xenotransplantation. The transgenic donor pigs used were α1,3-galactosyltransferase gene-knockout + membrane cofactor protein (GTKO+CD46) in one recipient and GTKO+CD46+ thrombomodulin (TBM) in the other. RESULTS: The graft survival time for GTKO+CD46 XTP was 28 days. With the addition of TBM, the survival differences between lamellar and full-thickness XTP were 98 days versus 14 days and >463 days (ongoing) versus 21 days, respectively. An excessive number of inflammatory cells was observed in failed grafts, but none were in the recipient's stromal bed. CONCLUSIONS: Unlike full-thickness corneal XTP, lamellar xenocorneal transplantation does not exhibit surgical complications, such as retrocorneal membrane or anterior synechia. The graft survival of lamellar XTP in this study was not as good as in our previous experiments, although the survival period was superior to that of full-thickness XTP. The difference in graft survival based on transgenic type is not definitive. Further studies using transgenic pigs and minimal immunosuppression need to focus on improving graft survival of lamellar XTP and using a larger sample size to determine the potential of full-thickness corneal XTP.

Current Status of Genetically Engineered Pig to Monkey Kidney Xenotransplantation in Korea.

BACKGROUND: In South Korea, pig-to-nonhuman primate trials of solid organs have only been performed recently, and the results are not sufficiently satisfactory to initiate clinical trials. Since November 2011, we have performed 30 kidney pig-to-nonhuman primate xenotransplantations at Konkuk University Hospital. METHODS: Donor αGal-knockout-based transgenic pigs were obtained from 3 institutes. The knock-in genes were CD39, CD46, CD55, CD73, and thrombomodulin, and 2-4 transgenic modifications with GTKO were done. The recipient animal was the cynomolgus monkey. We used the immunosuppressants anti-CD154, rituximab, anti-thymocyte globulin, tacrolimus, mycophenolate mofetil, and steroids. RESULTS: The mean survival duration of the recipients was 39 days. Except for a few cases for which survival durations were <2 days because of technical failure, 24 grafts survived for >7 days, with an average survival duration of 50 days. Long-term survival was observed 115 days after the removal of the contralateral kidney, which is currently the longest-recorded graft survival in Korea. We confirmed functioning grafts for the surviving transplanted kidneys after the second-look operation, and no signs of hyperacute rejection were observed. CONCLUSIONS: Although our survival results are relatively poor, they are the best-recorded results in South Korea, and the ongoing results are improving. With the support of government funds and the volunteering activities of clinical experts, we aim to further improve our experiments and contribute to the commencement of clinical trials of kidney xenotransplantation in Korea.

Antiviral Lipid Nanocarrier Loaded with Remdesivir Effective Against SARS-CoV-2 in vitro Model.

Introduction: The ongoing SARS-CoV-2 pandemic has affected public health, the economy, and society. This study reported a nanotechnology-based strategy to enhance the antiviral efficacy of the antiviral agent remdesivir (RDS). Results: We developed a nanosized spherical RDS-NLC in which the RDS was encapsulated in an amorphous form. The RDS-NLC significantly potentiated the antiviral efficacy of RDS against SARS-CoV-2 and its variants (alpha, beta, and delta). Our study revealed that NLC technology improved the antiviral effect of RDS against SARS-CoV-2 by enhancing the cellular uptake of RDS and reducing SARS-CoV-2 entry in cells. These improvements resulted in a 211% increase in the bioavailability of RDS. Conclusion: Thus, the application of NLC against SARS-CoV-2 may be a beneficial strategy to improve the antiviral effects of antiviral agents.

Temporal quantitative profiling of sialyllactoses and sialic acids after oral administration of sialyllactose to mini-pigs with osteoarthritis.

Sialyllactose (SL) is the most abundant acidic oligosaccharide in human breast milk and plays a primary role in various biological processes. Recently, SL has attracted attention as an excellent dietary supplement for arthritis because it is effective in cartilage protection and treatment. Despite the superior function of SL, there are few pharmacological studies of SL according to blood concentrations in arthritis models. In this study, we investigated quantitative changes in SL and sialic acids in the plasma obtained from mini-pigs with osteoarthritis throughout exogenous administration of SL using liquid chromatography-multiple reaction monitoring mass spectrometry. Plasma concentrations of SL and sialic acids in the SL-fed group showed a significant difference compared to the control group. Mini pigs were fed only Neu5Ac bound to SL, but the concentration patterns of the two types of sialic acid, Neu5Ac and Neu5Gc, were similar. In addition, the relative mRNA expression level of matrix metalloproteinases (MMPs), which is known as a critical factor in cartilage matrix degradation, was remarkably decreased in the synovial membrane of the SL-fed group. Consequently, the temporal quantitative profiling suggests that dietary SL can be metabolized and utilized in the body and may protect against cartilage degradation by suppressing MMP expression during osteoarthritis progression.

Biological response of nonhuman primates to controlled levels of acute blood loss.

Introduction: The global shortage of human blood for medical use has prompted the development of alternative blood sources. Nonhuman primates (NHPs) are commonly used owing to their physiological similarities to humans. The objective of the current study was to establish a controlled-blood-loss model in NHPs to explore their clinical and biological responses. Methods: Blood was sequentially withdrawn from 10 cynomolgus monkeys (10, 14, 18, 22, and 25% of the total blood volume); their vital signs were monitored, and blood parameters were serially analyzed. Humoral mediators in the blood were measured using flow cytometry and enzyme-linked immunosorbent assays. Results: In NHPs subjects to 25% blood loss and presenting with related clinical symptoms, the systolic blood pressure ratio on day 0 after bleeding was significantly lower than that of the animals from the other groups (median: 0.65 vs. 0.88, P = 0.0444). Red blood cell counts from day 0-14 and hematocrit levels from day 0-7 were markedly decreased relative to the baseline (P < 0.01). These parameters showed a direct correlation with the extent of blood loss. The levels of creatine phosphokinase, aspartate aminotransferase, and alanine aminotransferase exhibited increases in response to blood loss and had a stronger correlation with the hemoglobin ratio than the volume of blood loss. The levels of C3a and C4a, as well as interleukin (IL)-1α and IL-15, displayed a strong correlation, with no apparent association with blood loss. Conclusion: The findings of the present study showed that only NHPs with 25% blood loss exhibited clinical decompensation and significant systolic blood pressure reduction without fatalities, suggesting that this level of blood loss is suitable for evaluating blood transfusion efficacy or other treatments in NHP models. In addition, the ratio of hemoglobin may serve as a more dependable marker for predicting clinical status than the actual volume of blood loss. Thus, our study could serve as a basis for future xenotransfusion research and to predict biological responses to massive blood loss in humans where controlled experiments cannot be ethically performed.

Key factors to establish the ovalbumin-induced atopic dermatitis minipig model: age and body weight.

BACKGROUND: Given its similar structure and immune response to the human skin, porcine is a good model for dermal studies. Here, we sensitized ovalbumin (Ova) on minipig back skin for 2-4 weeks to induce chronic atopic dermatitis (AD). RESULTS: Gross observation, serum cytokine level, epidermal thickness, and epidermal integrity did not change after 4 weeks of Ova induction compared with the control, indicating AD modeling failure. Only the neutrophils in the blood and macrophages in bronchoalveolar lavage fluid changed slightly until 3 or 2 weeks after Ova sensitization, respectively. The successful and failed Ova-induced AD minipig models only differ in age and body weight of the minipigs. The minipigs, 12 months old with a 30-kg median weight, had a two-fold thicker dermis than minipigs 8-10 months old, with an 18.97-kg median weight, resulting in impaired Ova permeability and immune response. CONCLUSION: Age and body weight are key factors that should be considered when developing an Ova-induced AD minipig model.

Regenerative and anti-inflammatory effect of a novel bentonite complex on burn wounds.

BACKGROUND: Bentonite, a montmorillonite clay, has been used as a classical remedy strategy for a long time. Recently, bentonite has been used as a raw material in cosmetic products because of its antibacterial and antioxidant properties. However, the therapeutic effect of bentonite on burn injuries has not yet been identified. OBJECTIVES: The aim of this study was to explore the therapeutic effect of a novel bentonite complex, which was developed for medical use, on burn wounds and the anti-inflammatory function of bentonite clay in the complex in vitro. METHODS: A novel bentonite complex and bentonite clay were prepared for each in vivo and in vitro assay (C&L Biotech, Seoul, Korea). Burn wounds were induced on the dorsal skin of two Yucatan minipigs, and effects of tissue regeneration and anti-inflammation were assessed by histological and gene expression analysis. RESULTS: The bentonite complex improved skin regeneration in burn wounds by inducing collagen synthesis, cell proliferation and angiogenesis in vivo. Furthermore, expression of inflammatory cytokines was significantly inhibited by treatment of the bentonite complex. In vitro study for identification of anti-inflammatory effect showed that bentonite clay significantly regulated COX-2 signalling in both HacaT keratinocytes and 3D4/2 macrophage cell lines. CONCLUSIONS: This study identified the therapeutic effect of a novel bentonite complex in burn wounds by inducing skin regeneration and bentonite-mediated anti-inflammatory responses.

Evaluation of Spatial Distribution of Three Major Leptocorisa (Hemiptera: Alydidae) Pests Using MaxEnt Model.

We targeted three major Leptocorisa species (L. chinensis, L. acuta, and L. oratoria) and evaluated their potential distributions using MaxEnt. The results showed that most Asian countries and northern Australia would be suitable for at least one of these pest species, and climate change will expand their habitat northward. All of the developed models were evaluated to be excellent with AUC, TSS, and OR10%. Most of the recorded regions of the Leptocorisa species are consistent with the result of potential distributions predicted in this study. The results confirmed that the minimum temperature of the coldest month mainly influences the three Leptocorisa species distributions. The potential distributions of the three species cover major rice cultivation areas regardless of climate change, suggesting that it would be necessary to establish a sustainable control strategy for the pests.

Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages.

BACKGROUND: Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. RESULTS: As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, ß-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. CONCLUSIONS: We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions.

Sweep Sampling Comparison of Terrestrial Insect Communities Associated with Herbaceous Stratum in the Riparian Zone of the Miho River, Korea.

To investigate insect and plant community relationships in riparian zones, terrestrial insect communities were compared in plant communities in the riparian zone of the Miho River, Korea. The sweep netting method was used to sample insects in 50 m transects in three herbaceous plant communities. In 2020, each plant community-Chenopodium album, Beckmannia syzigachne, and Artemisia indica-was swept 100 times (50 sweeps × 2). In 2021, two communities had an additional 100 sweeps collected using 10 subsamples of 10 sweeps (excluding C. album communities). The surveyed dominant species or subdominant species of the insect community in each site preyed on the dominant plant species at the site. The Bray-Curtis similarity was significantly higher than the Sørensen similarity when comparing datasets across different years for the same plant species community. The predicted optimum sampling size to obtain approximately 80% of the total species estimated to be at each survey site, for effective quantitative collection of terrestrial insect herbivores in each plant community, was examined. Fifty sweeps were required for the A. indica community and 100 sweeps were required for the B. syzigachne community. The results of this study provide important data for riparian biodiversity conservation and future pest monitoring.

Formaldehyde exposure induces differentiation of regulatory T cells via the NFAT-mediated T cell receptor signalling pathway in Yucatan minipigs.

The use of minipigs (Sus scrofa) as a platform for toxicological and pharmacological research is well established. In the present study, we investigated the effect of formaldehyde (FA) exposure on helper T cell-mediated splenic immune responses in Yucatan minipigs. The minipigs were exposed to different inhaled concentrations of FA (0, 2.16, 4.62, or 10.48 mg/m3) for a period of 2 weeks. Immune responses elicited by exposure to FA were determined by assessing physiological parameters, mRNA expression, and cytokine production. Additionally, the distribution of helper T cells and regulatory T (Treg) cells and expression of NFAT families, which are well-known T cell receptor signalling proteins associated with regulatory T cell development, were evaluated. Exposure to FA suppressed the expression of genes associated with Th1 and Th2 cells in minipigs in a concentration-dependent manner. The subsequent production of cytokines also declined post-FA exposure. Furthermore, exposure to FA induced the differentiation of CD4+ Foxp3+ Treg cells with divergent expression levels of NFAT1 and NFAT2. These results indicated that exposure to FA increased the Treg cell population via the NFAT-mediated T cell receptor signalling pathway, leading to suppression of effector T cell activity with a decline in T cell-related cytokine production.

Differential susceptibility to lipopolysaccharide affects the activation of toll-like-receptor 4 signaling in THP-1 cells and PMA-differentiated THP-1 cells.

Monocytes and macrophages that originate from common myeloid progenitors perform various crucial roles in the innate immune system. Stimulation with LPS combined with TLR4 drives the production of pro-inflammatory cytokines through MAPKs and NF-κB pathway in different cells. However, the difference in LPS susceptibility between monocytes and macrophages is poorly understood. In this study, we found that pro-inflammatory cytokines-IL-1ß, IL-6 and TNFα showed greater induction in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells than in THP-1 cells. To determine the difference in cytokine expression, the surface proteins such as TLR4-related proteins and intracellular adaptor proteins were more preserved in PMA-differentiated THP-1 cells than in THP-1 cells. MyD88 is a key molecule responsible for the difference in LPS susceptibility. Moreover, MAPKs and NF-κB pathway-related molecules showed higher levels of phosphorylation in PMA-differentiated THP-1 cells than in THP-1 cells. Upon MyD88 depletion, there was no difference in the phosphorylation of MAPK pathway-related molecules. Therefore, these results demonstrate that the difference in LPS susceptibility between THP-1 cells and PMA-differentiated THP-1 cells occur as a result of gap between the activated MAPKs and NF-κB pathways via changes in the expression of LPS-related receptors and MyD88.

Disruption of Membrane Integrity as a Molecular Initiating Event Determines the Toxicity of Polyhexamethylene Guanidine Phosphate Depending on the Routes of Exposure.

Polyhexamethylene guanidine phosphate (PHMG-P), a cationic biocide, is widely used in household products due to its strong bactericidal activity and low toxicity. However, it causes fatal lung damage when inhaled. In this study, we investigated why PHMG-P causes fatal lung injury when inhaled, and demonstrated that the disruption of membrane integrity through ionic interaction-a molecular initiating event of PHMG-P-determines toxicity. Mice were injected intravenously with 0.9 or 7.2 mg/kg PHMG-P (IV group), or instilled intratracheally with 0.9 mg/kg PHMG-P (ITI group); they were euthanatized at 4 h and on days 1 and 7 after treatment. Increased total BAL cell count and proinflammatory cytokine production, along with fibrotic changes in the lungs, were detected in the ITI group only. Levels of hepatic enzymes and hepatic serum amyloid A mRNA expression were markedly upregulated in the 7.2 mg/kg IV and ITI groups at 4 h or day 1 after treatment, but returned to baseline. No pathological findings were detected in the heart, liver, or kidneys. To simulate the IV injection, A549, THP-1, and HepG2 cells were treated with PHMG-P in cell culture media supplemented with different serum concentrations. Increased serum concentration was associated with an increase in cell viability. These results support the idea that direct contact between PHMG-P and cell membranes is necessary for PHMG-induced toxicity.

Therapeutic effect of intra-articular injected 3'-sialyllactose on a minipig model of rheumatoid arthritis induced by collagen.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease of joint, but there is no known cure. 3'-sialyllactose (3'-SL) is an oligosaccharide that is abundant in breast milk of mammals, and has anti-inflammatory properties. However, the efficacy of 3'-SL on RA remains unclear. The objective of the present study was to evaluate the therapeutic effect of 3'-SL after it was directly injected into the knee joint cavity of a RA minipig model. RESULTS: Minipig RA model was induced by intra-articular injection of bovine type II collagen emulsified with complete or incomplete Freund's adjuvant into left knee joint. In clinical assessment, lameness and swelling of the hindlimb and increased knee joint width were observed in all animals. After the onset of arthritis, 3'-SL (0, 2, 10, and 50 mg/kg) was directly administered to the left knee joint cavity once a week for 4 weeks. Compared to the vehicle control group, no significant difference in macroscopic observation of the synovial pathology or the expression of inflammation-related genes (IL-1ß, TNF-α, and COX2) in the synovial membrane of the knee joint was found. In microscopic observation, cell cloning of the articular cartilage was significantly reduced in proportion to the concentration of 3'-SL administered. CONCLUSIONS: Our results suggest that intra-articular injected 3'-SL had a therapeutic effect on collagen-induced arthritis at the cellular level with potential as a medication for RA.