Pig Cells Research Articles

The order of immunoglobulin light chain κ and λ usage in primary and secondary lymphoid tissues of germ-free and conventional piglets

In pigs (Sus scrofa), the initial immunoglobulin rearrangement of the κ light chain is replaced by λ before the heavy chains rearrange, and the light chains may rearrange even later. This study investigates whether these developmental differences are reflected in the usage of IGK and IGL genes. We found large differences between peripheral B cells and those developing in the bone marrow, and between B cells in germ-free piglets and conventional pigs. During early B cell development in the bone marrow, more 3′ V and 5′ J gene segments for both light chains are used. However, in the peripheral naive repertoire, more 5′ IGLV and 3′ IGLJ genes are used. A similar shift toward the use of more 5′ IGKV and 3′ IGKJ genes is observed later after antigen exposure in conventional pigs. The expression profile showed that most λ+ B cells are generated earlier, while κ+ B cells develop from late precursors that already contain the λ rearrangement. The initial λ rearrangement is retained in both λ+ and κ+ B lymphocytes, and multiple λ transcripts can be found in individual cells. The overall pool of the IGLV repertoire is therefore much larger and more diversified than for IGKV. The κ repertoire is further restricted to the preferential use of only two major IGKV genes, reflecting the limitation for only two consecutive rearrangements. Tracing of silenced λ transcripts in κ+ B cells further confirmed the unconventional mechanism of differential rearrangements in pigs. Our results underline the diversity of the immune system among mammals.

814 Differences in Human, Pig, and Rat Spinal Cord Stem Cells in Response to Inflammatory and Regenerative Factors In Vitro

INTRODUCTION: The translation of preclinical science to human applications, including regenerative strategies for spinal cord injury, has been poor. Historically, animal models, such as rats and pigs, have been used to model human spinal cord given the difficulty in obtaining live human spinal cord. METHODS: To mimic post-injury inflammation, primary-derived NSPCs from adult humans (n=8), rats (n=6), and pigs (n=5) were treated with interleukin-6 (IL-6), tumor necrosis factor- α (TNFα), or transforming growth factor- β (TGFβ). To direct regeneration, NSPCs were treated with retinoic acid (RA), platelet-derived growth factor (PDGFα), or bone morphogenic protein-4 (BMP4) to induce neurons, oligodendrocytes or astrocytes, respectively. NSPCs were treated for 7 or 14 days and characterized by immunocytochemistry (GFAP, β-iii tubulin, O4). RESULTS: IL-6, TNFα and TGFβ induced astrogenesis of rat NSPCs (3.9±0.7, 5.0±0.9, and 4.0±0.6 fold, respectively) and reduced neurogenesis (0.14±0.90, 0.07±0.04, 0.07±0.05 fold, respectively) after 7 days. Pig NSPCs increased astrogenesis (1.38±0.04, 1.26±0.05, and 1.45±0.04 fold, respectively) after 14 days. Human NSPCs had reduced astrogenesis (0.14±0.07, 0.6±0.2, and 0.12±0.07 fold, respectively) over 14 days, but generated more neurons with IL-6 and TGFβ treatments (1.23±0.05 and 1.34±0.04 fold, respectively). RA increased neurogenesis of human and rat NSPCs, PDGFα increased oligodendrocyte differentiation of rat NSPCs, and BMP4 increased astrogenesis of human and rat NSPCs at low (40ng/mL) and high (100ng/mL) concentrations, respectively. CONCLUSIONS: For the first time, we have compared primary-derived adult spinal cord NSPCs from humans, pigs, and rats and identified differences in their response to pathophysiological and regenerative factors which would impact successful translation of stem cell-based therapies.

SARS-CoV-2 VIRUS: ISOLATION, GROWTH, THERMOSTABILITY, INACTIVATION AND PASSAGES

Coronaviruses are a large family of RNA-containing viruses capable of infecting humans and a number of animal species. Several species of coronaviruses are now known to circulate in the population and generally cause moderate to severe upper respiratory tract and lung disease. A new coronavirus infection, COVID-19 (SARS-CoV-2), has been added to this list. The virus has spread rapidly around the world. Recently in Kazakhstan, this infection has been observed with pronounced pneumonia among the population. In developing the vaccine, we had to work with live SARS-CoV-2 virus. It should be noted that when working with live virus, we faced some issues concerning the biological and physicochemical properties of the virus, which have remained outside the attention of scientists of the world. In this connection, in these studies we studied some physicochemical and biological properties of Kazakh strain of SARS-CoV-2 virus isolated from a patient with new coronavirus infection COVID-19, such as thermal stability (persistence), virus inactivation by different concentrations of formaldehyde at different temperature and time regimes and virus passivation in cell culture. Studies were conducted to find additional biological models sensitive to the SARS-CoV-2 virus and used in vitro. Of the 11 cell culture species tested, monkey and pig cell cultures were found to be the most sensitive to the SARS-CoV-2 virus, in which the virus was actively reproducing. We studied the effect of storage conditions of SARS-CoV-2 nucleic acid samples as well as the effect of environmental conditions on the SARS-CoV-2 virus itself. The results of our studies showed that freezing at minus 70 оС and thawing at room temperature (20-22 оС) had no appreciable effect on the qualitative character of cytopathogenicity but had a negative effect on the preservation of virus titer. Data on the effects of chemical factors when working with pathogens are necessary to create conditions of biological safety for personnel and the environment, as well as the design of technologies for the manufacture of biological products for diagnostic and prophylactic purposes. In this study, we studied the effect of formaldehyde to the virus. The results obtained show that inactivation of Sars-CoV-2 virus with formalin depends on formaldehyde concentration, reaction temperature, incubation time.

Aktivitas Ramuan Herbal sebagai Antagonis Reseptor H1, Relaksan Otot Polos dan Penghambat Degranulasi Sel Mast sebagai Antiasmatik

Asthma is still become a major health problem worldwide, affected all ages from pediatrics to 
 adults. The severity of the disease vary from mild to severe and leads to the risk of death. The 
 uses of medicinal plants as antiasthmatic have often been conducted by the local community. 
 The present study was conducted in vitro and in vivo to determine the activities of herbs 
 containing of sembung leaves (Blumea balsamifera L.), kemukus seeds (Piper cubeba L.), teki 
 rhizome (Cyperus rotundus L.), and patikan kebo herbs (Euphorbia hirta L.) with certain ratio 
 as an antiasthmatic. The in vitro test was carried out on isolated smooth muscle cells of guinea 
 pig. Histamine was used to stimulate the contraction of smooth muscle cells of guinea pig 
 trachea. The in vivo test was carried out using guinea pigs which sensitized with ovalbumin 
 and administered with the herbs formula. The trachea of guinea pigs were prepared for 
 histopathological examination and stained on toluidine blue. The result revealed that the 
 antiasthmatic herbs of 1, 2, and 3 showed activities as H1 receptor antagonists. The 
 antiasthmatic herbs of 1, 2, and 3 were able to relax the smooth muscle of guinea pig’s trachea 
 that was experiencing contraction, but in lower level compared to theophylline. The
 antiasthmatic herbs formula in this study did not have the activity to inhibit mast cell 
 degranulation.

Comparative evaluation of fracture healing potential of differentiated and undifferentiated guinea pig and canine bone marrow-derived mesenchymal stem cells in a guinea pig model

Background and aimThis work was conducted to compare the therapeutic potential of undifferentiated and osteogenic differentiated canine (xenogeneic) and guinea pig (allogeneic) BMSCs in fracture healing using guinea pig as a model. Materials and methodsA well-characterized homogenous population of third passage mesenchymal stem cells of bone marrow origin was used in all the experiments. MSCs from both the species, i.e., canine and guinea pigs, were differentiated and characterized. Expression of MHC I and II along with co-stimulatory molecules was assessed based on relative mRNA expression. The osteogenic differentiated and undifferentiated MSCs from both species were used for evaluating fracture healing in the guinea pig model. The healing potential was assessed based on radiographic, histopathology, and clinical observations. ResultsBMSCs from both species expressed MSC surface antigens and successfully differentiated to osteogenic, chondrogenic, and adipogenic lineages. The mRNA expression of class I and II MHC molecules in all the three lineages showed no significant (p > 0.05) differences after differentiating to adipogenic, chondrogenic, and osteogenic lineages. Radiographic and clinical examination revealed that MSCs therapy significantly improved bone fracture healing with a non-significant (p > 0.05) difference between differentiated and undifferentiated BMSCs. In addition, allogeneic MSCs therapy performed better than xenogeneic therapy. ConclusionMSCs remained hypo immunogenic after differentiation and have comparable fracture healing potential though allogeneic MSCs have better therapeutic potential than xenogenic MSCs.

Rare isolation of human-tropic recombinant porcine endogenous retroviruses PERV-A/C from Göttingen minipigs

BackgroundPorcine endogenous retroviruses (PERVs) can infect human cells and pose a risk for xenotransplantation when pig cells, tissues or organs are transplanted to human recipients. Xenotransplantation holds great promise to overcome the shortage of human donor organs after solving the problems of rejection, functionality and virus safety. We recently described the transmission of a human-tropic recombinant PERV-A/C, designated PERV-F, from peripheral blood mononuclear cells (PBMCs) of a Göttingen Minipig (GöMP) to human 293 cells (Krüger et al., in Viruses 12(1):38, 2019). The goal of this study was to characterize PERV-F in more detail and to analyze the probability of virus isolation from other animals.MethodsThe recombination site in the envelope (env) gene, the long terminal repeats (LTR), the proteins and the morphology of the recombinant PERV-F were characterized by polymerase chain reaction (PCR), sequencing, Western blot analysis, immunofluorescence, and transmissible electron microscopy. Mitogen-stimulated PBMCs from 47 additional pigs, including 17 new GöMP, were co-cultured with highly susceptible human 293 T cells, and the PERV-A/C prevalence and PERV transmission was analyzed by PCR.ResultsPERV-F, isolated from a GöMP, is an infectious human-tropic PERV-A/C virus with a novel type of recombination in the env gene. The length of the LTR of PERV-F increased after passaging on human cells. In a few minipigs, but not in German landrace pigs, PERV-A/C were found. There was no transmission of human-tropic PERV-A/C from additional 47 pigs, including 17 GöMP, to human cells.ConclusionThese data show that human-tropic recombinant PERV-A/C proviruses can only be found in a very small number of minipigs, but not in other pigs, and that their isolation as infectious virus able to replicate on human cells is an extremely rare event, even when using highly susceptible 293 cells.

Integrative Analysis of the lncRNA-Associated ceRNA Regulatory Network Response to Hypoxia in Alveolar Type II Epithelial Cells of Tibetan Pigs.

The function of alveolar type II epithelial (ATII) cells is severely hampered by oxygen deficiency, and understanding the regulatory mechanisms controlling responses to hypoxia may assist in relieving injury induced by hypoxia. In this study, we cultured ATII cells from Tibetan pigs and Landrace pigs under hypoxic and normoxic environments to screen for differentially expressed (DE) lncRNAs, DEmiRNAs, and construct their associated ceRNA regulatory networks in response to hypoxia. Enrichment analysis revealed that target genes of DElncRNAs of Tibetan pigs and Landrace pig between the normoxic (TN, LN) and hypoxic (TL, LL) groups significantly enriched in the proteoglycans in cancer, renal cell carcinoma, and erbB signaling pathways, while the target genes of DEmiRNAs were significantly enriched in the axon guidance, focal adhesion, and mitogen-activated protein kinase (MAPK) signaling pathways. Hypoxia induction was shown to potentially promote apoptosis by activating the focal adhesion/PI3K-Akt/glycolysis pathway. The ssc-miR-20b/MSTRG.57127.1/ssc-miR-7-5p axis potentially played a vital role in alleviating hypoxic injury by regulating ATII cell autophagy under normoxic and hypoxic conditions. MSTRG.14861.4-miR-11971-z-CCDC12, the most affected axis, regulated numerous RNAs and may thus regulate ATII cell growth in Tibetan pigs under hypoxic conditions. The ACTA1/ssc-miR-30c-3p/MSTRG.23871.1 axis is key for limiting ATII cell injury and improving dysfunction and fibrosis mediated by oxidative stress in Landrace pigs. Our findings provide a deeper understanding of the lncRNA/miRNA/mRNA regulatory mechanisms of Tibetan pigs under hypoxic conditions.

The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells Ex Vivo Without Stimulation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

Transcriptome and Metabolome Integration Provides New Insights Into the Regulatory Networks of Tibetan Pig Alveolar Type II Epithelial Cells in Response to Hypoxia

Tibetan pigs show a widespread distribution in plateau environments and exhibit striking physiological and phenotypic differences from others pigs for adaptation to hypoxic conditions. However, the regulation of mRNAs and metabolites as well as their functions in the alveolar type II epithelial (ATII) cells of Tibetan pigs remain undefined. Herein, we carried out integrated metabolomic and transcriptomic profiling of ATII cells between Tibetan pigs and Landrace pigs across environments with different oxygen levels to delineate their signature pathways. We observed that the differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) profiles displayed marked synergy of hypoxia-related signature pathways in either Tibetan pigs or Landrace pigs. A total of 1,470 DEGs shared between normoxic (TN, ATII cells of Tibetan pigs were cultured under 21% O2; LN, ATII cells of Landrace pigs were cultured under 21% O2) and hypoxic (TL, ATII cells of Tibetan pigs were cultured under 2% O2; LL, ATII cells of Landrace pigs were cultured under 2% O2) groups and 240 DAMs were identified. Functional enrichment assessment indicated that the hypoxia-related genes and metabolites were primarily involved in glycolysis and aldosterone synthesis and secretion. We subsequently constructed an interaction network of mRNAs and metabolites related to hypoxia, such as guanosine-3′, 5′-cyclic monophosphate, Gly-Tyr, and phenylacetylglycine. These results indicated that mitogen-activated protein kinase (MAPK) signaling, aldosterone synthesis and secretion, and differences in the regulation of MCM and adenosine may play vital roles in the better adaptation of Tibetan pigs to hypoxic environments relative to Landrace pigs. This work provides a new perspective and enhances our understanding of mRNAs and metabolites that are activated in response to hypoxia in the ATII cells of Tibetan pigs.

Study of the CRISPR/Cas3 System for Xenotransplantation

The CRISPR/Cas3 system, classified in class I system, was recently focused as a new technology. For application of this system to porcine cells, the plasmids of bpNLS-Cascade, BPNLS-hCas3, and pBS-U6icrRNA were prepared. Initially, 2 crRNAs were established in the exon 9 of pig Gal-T (GGTA1) as #45 and #86. Next, hCas3+#45+#86 (group 1, control), Cascade+ hCas3+#45 (group 2), Cascade+hCas3+#86 (group 3), and Cascade+hCas3+#45+#86 (group 4) were set and transfected into pig fibroblasts. Transfected cells were analyzed for bulk expression of α1,3Gal epitope by fluorescence-activated cell sorting (FACS), using a GSI-B4 lectin 2 days after the transfection. As the results, changes of expression are observed in order of G4>G2>G3, indicating the effect of the Cas3 system. Therefore, the nested polymerase chain reaction (PCR) for target region of GGTA1 was performed. Next, the PCR products from each group were checked in blotting, and the products were placed into the cloning sit of TOPO vector and transformed into Escherichia coli. Sixteen colonies of each group were checked by PCR, and clones containing PCR product with slightly varying length were evaluated. The direct sequence of these PCR changes were demonstrated as 294 to 754 bp deletions. In conclusion, we confirmed the effect of the CRISPR/Cas3 system on pig cell, especially in xenotransplantation.

The response of pig erythrocytes to the technological stress, depending on gender

The article considers the issues of the peculiarities of how the technological stress influences the indicators of red blood cells in pigs. Clinically healthy animals were selected at the stage of mobilization of the development of stress response according to the principle of pairs of analogues: group I - breeding boars, group II - breeding sows. The oxygen budget of the body, determined by the indicators of red blood cells, during the development of a stress reaction in breeding pigs of “mature age” is also determined by their gender. Boars had average values of erythrocytes and hemoglobin in reference values, but the degree of saturation of erythrocytes with hemoglobin was lower than normal and lower than that of sows. This may indicate that boars experience a greater shortage of oxygen supply to the tissues. Sows, in general, are more stress-resistent, as evidenced by the values of the ratio of lymphocytes and neutrophils, as well as their erythrocyte indicators. They differed from boars by a significantly lower level of erythrocytes and the reference level of hemoglobin in all examined individuals. In sows, the indicator of erythrocyte saturation with hemoglobin was significantly higher than in boars.

Selective human cells in vitro transduction with porcine endogenous retrovirus (PERV).

Porcine xenograft transplantation raises concerns in humans about the risk of infection with porcine endogenous retroviruses (PERV) as they are an integral part of the pig genome and are therefore very difficult to exclude. In this study, for the first time, arelationship between the provirus genes sequences and released virions from pig cell line and the embedded sequence of this retrovirus in infected human cells was analyzed. PERV infection of human cells HEK-293 and HeLa and detection of PERV in pig PK-15 cells and supernatant were assessed by QPCR or RT-QPCR using primers specific for envA, envB, gag, pol genes and LTR region. Sequence analysis was performed at the DNA level and changes in the amino acid sequence were deduced in silico. Fifty nucleotide substitutions (45 in pol, 3 in gag and one each in envA and envB) were detected and most of these were heterozygous (42), which were present mainly in PK-15 cells. Our results show that sequence of the pol gene and the Pol protein is less conserved compared to the other PERV genes and PERV with some polymorphisms were not released from pig cells or/and do not infect human cells. PERV virions with ahomozygous allele system were released from PK-15 cells, although their sequence replicated on the basis of the heterozygous PERV provirus sequence in PK-15. The newly discovered selective transduction of human cells with PERV will be helpful in studying the characteristics and genetic variability of the retrovirus genes to ensure safe xenotransplantation. Keywords: PERV; porcine endogenous retroviruses; infection; genetic polymorphism; xenotransplantation.

Reprogramming host myocytes by human embryonic stem cells derived cardiac progenitors improves remodeling and contractility of failed overloaded right ventricle

Cell based myocardium regeneration should restore overloaded right ventricular (RV) function in grown-up congenital heart diseases. The graft of human cardiac progenitors engineered to express MLC2vGFP and differentiated from embryonic stem cells, decrease fibrosis and improve RV remodeling by a still unknown mechanism. After 5 months of an overloaded RV of a pig model of repaired Tetralogy of Fallot, a collagen patch containing 107 NKX2.5+ cardiac progenitors was applied on the epicardium of the whole RV free wall (4 treated pigs). A control group (n = 4) received a cell-free or no patch. Myocardial contractility was assessed by echocardiography using strain parameters before and 2 months after cell graft. Myocardial fibrosis was quantified by red Sirius staining and fibrosis genes QPCR analysis. The fate of progenitors was tracked by GFP and human-and cardiac specific markers. RV contractility improved in the treated group by contrast to control. Expression of collagen genes, and in turn RV fibrosis were significantly lower in treated animals. While differentiating human cardiac progenitors remained in the patch, other migrated through the fibrosis and differentiated in cardiomyocytes within the myocardium. These cells featured MLC2v+ sarcomeres and were surrounded by small pigproliferating Ki-67+ TnT+ cells as well as myocytes with fetal sarcomeres. Nfkb+ Oct4+ pig cells were also observed in the neighborhood. In vitro experiments revealed that expression of Nfkb+ in neonatal myocytes or their activation by IL6 reprogrammed them into Oct4+ cells that redifferentiated into cardiomyocytes. We propose that migration of progenitors through the fibrosis triggers a local inflammation. Immune cells secrete cytokines acting on a cardiomyocytic inflammasome at the origin of cell reprogramming. Reprogrammed cells redifferentiate into cardiomyocytes while proliferating, thus regenerating the myocardium.

Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Fibroblast growth factor 11 inhibits foot-and-mouth disease virus gene expression and replication in vitro.

Foot-and-mouth disease virus (FMDV) causes highly contagious disease of cloven-hoofed animals such as cattle, swine, and sheep. Although FMD vaccine is the traditional way to protect against the disease, the use of FMD vaccines to protect early infection is limited. The alternative strategy of applying antiviral agents is required to control the spread of FMDV in outbreak situations. Fibroblast growth factor 11 (FGF11) is a member of the intracellular FGF. Here, we identified the inhibitory effect of FGF11 on FMDV gene expression through the transcriptional and translational regulation. For the quantitative analysis of FMDV transcription/translation level, we firstly constructed a plasmid reporter system (FMDV five prime untranslated region (5′ UTR) -luci) conjugating luciferase encoding gene with FMDV 5′ UTR region, which is a non-coding region to control FMDV transcription/translation and includes cis-acting replication element (CRE) and internal ribosome entry site (IRES). FGF11 decreased the gene expression of FMDV 5′ UTR-luci reporter in a dose-dependent manner. We further confirmed the inhibitory function of FGF11 on FMDV gene expression a replication in the FMDV-infected pig cells. FGF11 expression inhibited RNA production of FMDV RNA polymerase 3D gene in the FMDV-infected cells. In addition, while FMDV cell infection induced cytopathic effect (CPE) within 24 hr, FGF11 expression dramatically repressed CPE at the basal level. These results indicate that FGF11 inhibits FMDV gene expression and replication in vitro, implicating to provide intervention strategy for FMDV pathogenesis and transmission.

Observation in inner ear of tree shrew using scanning electron microscope and the Atoh1 distribution in cochlea.

This study aimed to observe the ultrastructure on the surface of the inner ear of a normal tree shrew using scanning electron microscope (SEM). The specimens of cochlea, macula utriculi, macula sacculi, and crista ampullaris of the normal adult tree shrew were collected and observed by SEM. We used immunofluorescence for cochlear protein Atoh1 staining. We observed that cochlea of the tree shrew is centered on the cochlear axis, circling about 3.5 times from bottom to top of the cochlea. The organ of Corti is located between medial and lateral grooves, including inner and outer hair cells as well as supporting cells. Maculae staticae include macula of saccule and macula of utricle, and the surface of macula is covered with a large number of otoliths. We found a gelatinous layer below the otoliths, followed by the layer of the honeycomb structure. The hair cell cilia of macula and crista ampullaris include one kinocilium and more stereocilia. There is no obvious cross structure but numerous hair cell cilia on semicircular canal crista ampullaris. Immunofluorescence staining showed that protein Atoh1 is mainly distributed in the nucleus of the cochlea's inner and outer hair cells. The observation of the inner ear structure under SEM elucidate the fine surface morphological structure of the entire cochlea, the vestibular maculae staticae, and crista ampullaris, providing new insight into the structure and function of the inner ear of tree shrew. HIGHLIGHTS: This article is the first to describe the inner ear ultrastructure of a small primate tree shrew by scanning electron microscopy (SEM). Under an SEM, the phalangeal processes of Deiter cells in tree shrews were observed to be connected to the tip of a neighboring hair cell, which was different from that of Deiters' cells in guinea pigs, and this crossed one hair cell, and connected to the tip of the third hair cell. It was observed that the crista ampullaris of tree shrews were horseshoe-shaped, and similar to that of humans and monkeys, this had no obvious "cross-shaped hump" structure. Tree shrew's ABR threshold value curve conforms to the mammalian U-shaped curve, wave III is the main wave of ARB, its sensory frequency may be higher 8 kHz, and the characteristics of the stereocilia of tree shrew we have observed may be related to the perception of higher frequency hearing.

The variable lipoprotein family participates in the interaction of Mycoplasma hyorhinis with host extracellular matrix and plasminogen

Mycoplasma hyorhinis (Mhr) infects pigs, typically causing polyserositis and polyarthritis. It has also been reported in various human tumors. The variable lipoprotein (Vlp) family is a vital surface component mediating the immune evasion of Mhr. We have previously reported its functions in the adherence of Mhr to pig cells. Herein, we further evaluated its role in interacting with host extracellular matrix (ECM) components (fibronectin, collagen type Ⅳ and laminin) and plasminogen. Consequently, the recombinant Vlp proteins of all the seven members (VlpA-VlpG) were able to bind most of the tested host molecules. Further experiment showed that region Ⅱ of all Vlp members has a strong binding ability, while the binding ability of region Ⅲ of each member varied between different host molecules. Comparing the Vlps containing short (rVlpX3) or long (rVlpX12) region Ⅲ, we found that the ability of most Vlps binding NCI-H292 cell membrane proteins became weaker as the molecule grows, except VlpG. However, the binding of VlpA, VlpB, VlpC and VlpG to tested ECM components and plasminogen tended to increase as Vlps became longer, and those of VlpE and VlpF decreased, and that of VlpD did not change. Furthermore, the activation of Vlp-bound plasminogen was proved. In summary, the Vlp family participates in the interaction of Mhr with host ECM and plasminogen in addition to cytoadhesion. The size variation of Vlps is likely to further regulate these interactions. The results may help to elucidate the roles of Vlps in the persistent infection of Mhr.

Virological and Parasitological Characterization of Mini-LEWE Minipigs Using Improved Screening Methods and an Overview of Data on Various Minipig Breeds.

Minipigs play an important role in biomedical research and have also been used as donor animals in xenotransplantation. To serve as a donor in xenotransplantation, the animals must be free of potential zoonotic viruses, bacteria and parasites. Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated as most of the other pig viruses can. PERV-A and PERV-B infect human cells in cell culture and are integrated in all pigs, whereas PERV-C infects only pig cells and it is found in many, but not all pigs. Minipigs are known for a high prevalence of recombinant PERV-A/C viruses able to infect human cells (Denner and Schuurman, Viruses, 2021;13:1869). Here, Mini-LEWE minipigs are screened for the first time for pig viruses including PERV. Peripheral blood mononuclear cells (PBMCs) from 10 animals were screened using PCR-based methods (PCR, RT-PCR, and real-time PCR). In comparison with our previous screening assays, numerous improvements were introduced, e.g., the usage of gene blocks as a PCR standard and foreign RNA to control reverse transcription in RT-PCR. Using these improved detection methods, Mini-LEWE pigs were found to be negative for porcine cytomegalovirus (PCMV), porcine lymphotropic herpesviruses (PLHV-1, -2 and -3), porcine circoviruses (PCV1, 2, 3 and 4), porcine parvovirus (PPV) and hepatitis E virus (HEV). All animals carried PERV-A, PERV-B and PERV-C in their genome. PERV-A/C was not found. In contrast to all other minipig breeds (Göttingen minipigs, Aachen minipigs, Yucatan micropig, Massachusetts General Hospital miniature pigs), Mini-LEWE minipigs have less viruses and no PERV-A/C. Parasitological screening showed that none of the Mini-LEWE minipigs harbored ecto- and gastrointestinal parasites, but at least one animal tested positive for anti-Toxoplasma gondii antibodies.

The cathelicidin CATH-2 efficiently neutralizes LPS- and E. coli-induced activation of porcine bone marrow derived macrophages

Infectious diseases in pigs cause monetary loss to farmers and pose a zoonotic risk. Therefore, it is important to obtain more porcine specific immunological knowledge as a measure to protect against infectious diseases, for example by exploring immunomodulators that are usable as vaccine adjuvants. Cathelicidins are a class of host defence peptides (HDPs) able to directly kill microbes as well as exert a diverse range of effects on the immune system. The peptides have shown promise as immunomodulatory peptides in many applications, including vaccines. However, it is currently unknown what the precise effect of these peptides is on porcine immune cells and whether peptides of other species might also have a strong immunomodulatory effect on porcine macrophages. Mononuclear bone marrow cells of pigs, aged 5–6 months, were cultured into M1 or M2 macrophages and stimulated with LPS or whole bacteria in the presence of host defence peptides (HDPs).CATH-2 and LL-37 strongly inhibited LPS-induced activation of M1 macrophages, the inhibition of LPS-induced activation of M2 macrophages by HDPs was milder, showing that the peptides have selective effects on different cell types. Upon stimulation with whole bacteria, only CATH-2 could effectively inhibit macrophage activation, showing the potent anti-inflammatory potential of this peptide. These results show that porcine peptides are not necessarily the most active in a porcine system, and that CATH-2 is effective in a porcine system as an anti-inflammatory immune modulator, which can be used, for example, in inactivated pathogen vaccines.

Vaccination with a Brucella ghost developed through a double inactivation strategy provides protection in Guinea pigs and cattle

Vaccination can prevent and control animal brucellosis. Currently, live attenuated vaccines are extensively used to prevent Brucella infection. However, traditional vaccines such as live attenuated vaccines are associated with biological safety risks for both humans and animals. The bacterial ghost (BG) is a new form of vaccine with great prospects. However, bacterial cells cannot be completely inactivated by biological lysis, conferring a safety risk associated with the vaccine. In this study, we developed a Brucella abortus A19 bacterial ghost (A19BG) through a double inactivation strategy with sequential biological lysis and hydrogen peroxide treatment. This strategy resulted in 100% inactivation of Brucella, such that viable bacterial cells were not detected even at an ultrahigh concentration of 1010 colony-forming units/mL. Furthermore, A19BG had a typical BG morphology and good genetic stability. Moreover, it did not induce adverse reactions in guinea pigs. The levels of antibodies, interferon-γ, interleukin-4, and CD4+ T cells in guinea pigs inoculated with the A19BG vaccine were similar to those inoculated with the existing A19 vaccine. Immunization with A19BG conferred a similar level of protection with that of A19 against Brucella melitensis M28 in both guinea pigs and cattle. In conclusion, the combination of biological lysis and H2O2-mediated inactivation is a safe and effective strategy that can serve as a reference for the preparation of BG vaccines.