Durability of SARS-CoV-2-Specific T-Cell Responses at 12 Months Postinfection.

BACKGROUND: Characterizing the longevity and quality of cellular immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enhances understanding of coronavirus disease 2019 (COVID-19) immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Analysis of the function, durability, and diversity of cellular response long after natural infection, over a range of ages and disease phenotypes, is needed to identify preventative and therapeutic interventions. METHODS: We identified participants in our multisite longitudinal, prospective cohort study 12 months after SARS-CoV-2 infection representing a range of disease severity. We investigated function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry, and compared magnitude of SARS-CoV-2-specific antibodies. RESULTS: SARS-CoV-2-specific antibodies and T cells were detected 12 months postinfection. Severe acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12 months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. CONCLUSIONS: SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12 months postinfection, with higher frequency noted in those who experienced severe disease.

Transcriptomic analyses suggest a dominant role of insulin in the coordinated control of energy metabolism and ureagenesis in goat liver.

BACKGROUND: The ureagenesis plays a central role in the homeostatic control of nitrogen metabolism. This process occurs in the liver, the key metabolic organ in the maintenance of energy homeostasis in the body. To date, the understanding of the influencing factors and regulators of ureagenesis in ruminants is still poor. The aim of this study was to investigate the relationship between energy metabolism and ureagenesis and detect the direct regulators of ureagenesis in the liver by using RNA-seq technology. RESULTS: Eighteen four-month-old male goats were divided into two groups randomly and received a diet containing 10% (LNFC group, n = 9) or 30% non-fiber carbohydrate (MNFC group, n = 9), respectively, for four weeks. The global gene expression analysis of liver samples showed that, compared with a LNFC diet, the MNFC diet promoted the expression of genes required for synthesis of fatty acid and glycerol, whereas it suppressed those related to fatty acid oxidation, gluconeogenesis from amino acids and ureagenesis. Additionally, gene expression for rate-limiting enzymes of ureagenesis were highly correlated to the gene expression of key enzymes of both fatty acid synthesis and glycerol synthesis (Spearman correlation coefficient > 0.8 and p < 0.05). In the differentially expressed signaling pathways related to the endocrine system, the MNFC diet activated the insulin and PPAR signaling pathway, whereas it suppressed the leptin-JAK/STAT signaling pathway, compared with the LNFC diet. Reverse transcription quantitative PCR analyses of 40 differentially expressed genes confirmed the RNA-seq results (R2 = 0.78). CONCLUSION: Our study indicated that a dietary NFC-induced increase of energy supply promoted lipid anabolism and decreased ureagenesis in the caprine liver. By combining our results with previously published reports, insulin signaling can be suggested to play the dominant role in the coordinated control of hepatic energy metabolism and ureagenesis.

Overexpression of BmFoxO inhibited larval growth and promoted glucose synthesis and lipolysis in silkworm.

Forkhead box O (FoxO) is a downstream transcription factor of the insulin-signaling pathway, which plays vital roles in the growth and metabolism of organisms. In this study, BmFoxO was overexpressed in BmE cells, in which proliferation was inhibited and apoptosis was increased. The transgenic vector overexpressing BmFoxO was constructed, and the transgenic silkworm line A4FoxO was generated via embryonic microinjection. The body size of A4FoxO silkworm was smaller than that of non-transgenic silkworm (WT). The quantitative polymerase chain reaction results revealed that the insulin pathway was enhanced and the growth-related TOR pathway was suppressed. Furthermore, the translation of proteins in the fat body of A4FoxO silkworm was inhibited. The expression level of genes involved in the glucose synthesis and lipolysis pathways was increased, whereas that of genes involved in fat synthesis was decreased. Oil red O staining revealed that the amount of lipid droplets was reduced in A4FoxO silkworms compared with WT. Further analysis showed that the content of triglyceride and glycogen was significantly decreased in fat body, but the content of glucose and trehalose was increased in the hemolymph of A4FoxO silkworms. These results suggest that the enhanced expression of BmFoxO disturbs glycolipid metabolism and affects silkworm growth.

Effects of glucagon-like peptides 1 and 2 and epidermal growth factor on the epithelial barrier of the rumen of adult sheep.

Epidermal growth factor (EGF) and glucagon-like peptides (GLP) modulate the tight junctions (TJ) of the intestinal epithelial barrier (EB) of monogastric animals. This work tried to elucidate whether GLP-1, GLP-2 and EGF can affect the EB of the rumen. Ovine ruminal epithelia were incubated in Ussing chambers for 7 hr with 25 or 250 nM of either GLP-1 or GLP-2 on the serosal side, with 2.5 nM of EGF on the serosal side or with 0.25 or 2.5 nM EGF on the mucosal side. No treatment affected tissue conductance. Short-circuit current (Isc ) was affected by time and treatment and their interactions. Only 250 nM of either GLP-1 or GLP-2 decreased Isc in certain periods compared with 25 nM GLP-1 or 0.25 nM mucosally applied EGF; however, not when compared to control epithelia. Fluorescein flux rates (Jfluor ) of ruminal epithelia were affected by treatment, time and time × treatment interaction. The time × treatment interaction was based on an increase in Jfluor between the first and last hour in epithelia incubated with 25 nM GLP-1 or GLP-2 and in epithelia incubated with EGF. After 7 hr incubation, claudin-7 mRNA expression was downregulated in all treatments. Claudin-1 mRNA was upregulated after incubation with 2.5 nM EGF on the serosal side, claudin-4 mRNA was downregulated by 2.5 nM EGF on the mucosal side, and occludin mRNA was increased after incubation with 250 nM GLP-2. The protein abundance of all tested TJ proteins was not influenced by treatment. We conclude that GLP-1, GLP-2, and EGF have no obvious acute effects on the EB of ruminal epithelia under simulated physiological conditions ex vivo. However, by decreasing the mRNA expression of claudin-7 and partly affecting other TJ proteins, they may modulate EB in the longer term or under certain conditions.

High-Concentrate Diet-Induced Change of Cellular Metabolism Leads to Decreases of Immunity and Imbalance of Cellular Activities in Rumen Epithelium.

BACKGROUND/AIMS: In animals, the immune and cellular processes of tissue largely depend on the status of local metabolism. However, in the rumen epithelium, how the cellular metabolism affects epithelial immunity, and cellular processes, when the diet is switched from energy-rich to energy-excess status, with regard to animal production and health, have not as yet been reported. METHODS: RNA-seq was applied to compare the biological processes altered by an increase of dietary concentration from 10% to 35% with those altered by an increase of dietary concentration from 35% to 65% (dietary concentrate: the non-grass component in diet, including corn, soya bean meal and additive. High concentrate diet composed of 35% grass, 55% corn, 8% soya bean meal and 2% additive). In addition to the functional analysis of enriched genes in terms of metabolism, the immune system, and cellular process, the highly correlated genes to the enriched metabolism genes were identified, and the function and signaling pathways related to the differentially expressed neighbors were compared among the groups. RESULTS: The variation trends of molar proportions of ruminal SCFAs and those of enriched pathways belonging to metabolism, immune system, and cellular process were altered with the change of diets. With regard to metabolism, lipid metabolism and amino acid metabolism were most affected. According to the correlation analysis, both innate and adaptive immune responses were promoted by the metabolism genes enriched under the 65% concentrate diet. However, the majority of immune responses were suppressed under the 35% concentrate diet. Moreover, the exclusive upregulation of cell growth and dysfunction of cellular transport and catabolism were induced by the metabolism genes enriched under the 65% concentrate diet. On the contrary, a balanced regulation of cellular processes was detected under the 35% concentrate diet. CONCLUSIONS: These results indicated that the alterations of cellular metabolism promote the alterations in cellular immunity, repair, and homeostasis in the rumen epithelium, thereby leading to the switch of concentrate effects from positive to negative with regard to animal production and health.

Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain.

UNLABELLED: The porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replication in vitro, it enhanced virus replication in vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses. IMPORTANCE: The tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (residues 291 and 295) reduced virus replication in vitro but enhanced virus replication and induced higher-level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

Cross protective immune responses in nursing piglets infected with a US spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original US PEDV strain.

We investigated cross-protective immunity of a US spike-insertion deletion porcine epidemic diarrhea virus (PEDV) Iowa106 (S-INDEL) strain against the original US PEDV (PC21A) strain in nursing piglets. Piglets were inoculated orally with S-INDEL, PC21A or mock. At 20-29 days post-inoculation (dpi), all pigs were challenged with the PC21A strain. The S-INDEL-inoculated pigs had lower ileal IgA antibody secreting cells, serum IgA and neutralizing antibody titers compared with PC21A-inoculated pigs. No pigs in the PC21A-group developed diarrhea, whereas 81 and 100% of pigs in the S-INDEL and mock-groups had diarrhea post challenge, respectively. S-INDEL induced partial protective immunity against the original US PEDV strain.

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus.

UNLABELLED: Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. IMPORTANCE: Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination.

Acidic pH and short-chain fatty acids activate Na+ transport but differentially modulate expression of Na+/H+ exchanger isoforms 1, 2, and 3 in omasal epithelium.

Low sodium content in feed and large amounts of salivary sodium secretion are essential requirements to efficient sodium reabsorption in the dairy cow. It is already known that Na(+)/H(+) exchange (NHE) of the ruminal epithelium plays a key role in Na(+) absorption, and its function is influenced by the presence of short-chain fatty acids (SCFA) and mucosal pH. By contrast, the functional role and regulation of NHE in omasal epithelium have not been completely understood. In the present study, we used model studies in small ruminants (sheep and goats) to investigate NHE-mediated Na(+) transport and the effects of pH and SCFA on NHE activity in omasal epithelium and on the expression of NHE isoform in omasal epithelial cells. Conventional Ussing chamber technique, primary cell culture, quantitative PCR, and Western blot were used. In native omasal epithelium of sheep, the Na(+) transport was electroneutral, and it was inhibited by the specific NHE3 inhibitor 3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride, which decreased mucosal-to-serosal, serosal-to-mucosal, and net flux rates of Na(+) by 80% each. The application of low mucosal pH (6.4 or 5.8) in the presence of SCFA activated the Na(+) transport across omasal epithelium of sheep compared with that at pH 7.4. In cultured omasal epithelial cells of goats, mRNA and protein of NHE1, NHE2, and NHE3 were detected. The application of SCFA increased NHE1 mRNA and protein expression, which was most prominent when the culture medium pH decreased from 7.4 to 6.8. At variance, the mRNA and protein expression of NHE2 and NHE3 were decreased with low pH and SCFA, which was contrary to the published data from ruminal epithelial studies. In conclusion, this paper shows that (1) NHE1, NHE2, and NHE3 are expressed in omasal epithelium; (2) NHE3 mediates the major portion of transepithelial Na(+) transport in omasal epithelium; and (3) SCFA and acidic pH acutely activate Na(+) transport but suppress the expression of NHE2 and NHE3 in the longer term. By contrast, the expression of NHE1 is increased by SCFA and acidic pH, indicating a prominent role for NHE1 in the regulation of intracellular pH of omasal epithelium. Our results suggest a regulatable Na(+) absorption in ruminal and omasal epithelium. It is of benefit for intracellular pH homeostasis and highly relevant to dairy cows fed on high-concentrate diets.

Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs.

To verify whether porcine deltacoronavirus infection induces disease, we inoculated gnotobiotic pigs with 2 virus strains (OH-FD22 and OH-FD100) identified by 2 specific reverse transcription PCRs. At 21-120 h postinoculation, pigs exhibited severe diarrhea, vomiting, fecal shedding of virus, and severe atrophic enteritis. These findings confirm that these 2 strains are enteropathogenic in pigs.

Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in nursing piglets. Following its first detection in the United States in February 2014, additional PDCoV strains have been identified in the United States and Canada. Currently, no treatments or vaccines for PDCoV are available. In this study, U.S. PDCoV strain OH-FD22 from intestinal contents of a diarrheic pig from Ohio was isolated in swine testicular (ST) and LLC porcine kidney (LLC-PK) cell cultures by using various medium additives. We also isolated PDCoV [OH-FD22(DC44) strain] in LLC-PK cells from intestinal contents of PDCoV OH-FD22 strain-inoculated gnotobiotic (Gn) pigs. Cell culture isolation and propagation were optimized, and the isolates were serially propagated in cell culture for >20 passages. The full-length S and N genes were sequenced to study PDCoV genetic changes after passage in Gn pigs and cell culture (passage 11 [P11] and P20). Genetically, the S and N genes of the PDCoV isolates were relatively stable during the first 20 passages in cell culture, with only 5 nucleotide changes, each corresponding to an amino acid change. The S and N genes of our sequenced strains were genetically closely related to each other and to other U.S. PDCoV strains, with the highest sequence similarity to South Korean strain KNU14-04. This is the first report describing cell culture isolation, serial propagation, and biological and genetic characterization of cell-adapted PDCoV strains. The information presented in this study is important for the development of diagnostic reagents, assays, and potential vaccines against emergent PDCoV strains.

Short-chain fatty acids and acidic pH upregulate UT-B, GPR41, and GPR4 in rumen epithelial cells of goats.

Currently, the mechanism(s) responsible for the regulation of urea transporter B (UT-B) expression levels in the epithelium of the rumen remain unclear. We hypothesized that rumen fermentation products affect ruminal UT-B expression. Therefore, the effects of short-chain fatty acids (SCFA), pH, ammonia, and urea on mRNA and protein levels of UT-B were assayed in primary rumen epithelial cell cultures and in rumen epithelium obtained from intact goats. In vitro, SCFA and acidic pH were found to synergetically stimulate both mRNA and protein expression of UT-B, whereas NH4Cl decreased mRNA and protein levels of UT-B at pH 6.8. Treatment with urea increased both levels at pH 7.4. When goats received a diet rich in nitrogen (N) and nonfiber carbohydrates (NFC), their rumen epithelium had higher levels of UT-B, and the rumen contained higher concentrations of SCFA and NH3-N with a lower pH. An increase in plasma urea-N concentration was also observed compared with the plasma of the goats that received a diet low in N and NFC. In a second feeding trial, goats that received a NFC-rich, but isonitrogenous, diet had higher mRNA and protein levels of UT-B, and higher levels of G protein-coupled receptor (GPR) 41 and GPR4, in their rumen epithelium. The ruminal concentrations of SCFA and NH3-N also increased, while a lower pH was detected. In contrast, the serum urea-N concentrations remained unchanged. These data indicate that ruminal SCFA and pH are key factors, via GPR4 and GPR41, in the dietary regulation of UT-B expression, and they have priority over changes in plasma urea.

Experimental infection of a US spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original US PEDV infection.

Although the original US porcine epidemic diarrhea virus (PEDV) was confirmed as highly virulent by multiple studies, the virulence of spike-insertion deletion (S-INDEL) PEDV strains is undefined. In this study, 3-4 day-old conventional suckling piglets were inoculated with S-INDEL PEDV Iowa106 (4 pig litters) to study its virulence. Two litters of age-matched piglets were inoculated with either the original US PEDV PC21A or mock as positive and negative controls, respectively. Subsequently, all pigs were challenged with the original US PEDV PC21A on 21-29 days post-inoculation (dpi) to assess cross-protection. All S-INDEL Iowa106- and the original US PC21A-inoculated piglets developed diarrhea. However, the severity of clinical signs, mortality (0-75%) and fecal PEDV RNA shedding titers varied among the four S-INDEL Iowa106-inoculated litters. Compared with the original PC21A, piglets euthanized/died acutely from S-INDEL Iowa106 infection had relatively milder villous atrophy, lower antigen scores and more limited intestinal infection. Two of four S-INDEL Iowa106-infected sows and the original PC21A-infected sow showed anorexia and watery diarrhea for 1-4 days. After the original PC21A challenge, a subset (13/16) of S-INDEL Iowa106-inoculated piglets developed diarrhea, whereas all (5/5) and no (0/4) pigs in the mock and original PC21A-inoculated pigs had diarrhea, respectively. Our results suggest that the virulence of S-INDEL PEDV Iowa106 was less than the original US PEDV PC21A in suckling pigs, with 100% morbidity and 18% (6/33) overall (0-75%) mortality in suckling pigs depending on factors such as the sow's health and lactation and the piglets' birth weight. Prior infection by S-INDEL Iowa106 provided partial cross-protection to piglets against the original PC21A challenge at 21-29 dpi.

Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain.

The infectious dose of a virus pool of original US PEDV strain PC22A was determined in 4-day-old, cesarean-derived, colostrum-deprived (CDCD) piglets. The median pig diarrhea dose (PDD50) of the virus pool was determined as 7.35 log10 PDD50/mL, similar to the cell culture infectious titer, 7.75 log10 plaque-forming units (PFU)/mL. 100 PDD50 caused watery diarrhea in all conventional suckling piglets (n = 12) derived from a PEDV-naive sow, whereas 1000 and 10 000 PDD50 did not cause diarrhea in piglets derived from two PEDV-field exposed-recovered sows. This information is important for future PEDV challenge studies and validation of PEDV vaccines.

Pathology of US porcine epidemic diarrhea virus strain PC21A in gnotobiotic pigs.

To understand the progression of porcine epidemic diarrhea virus infection, we inoculated gnotobiotic pigs with a newly emerged US strain, PC21A, of the virus. At 24­48 hours postinoculation, the pigs exhibited severe diarrhea and vomiting, fecal shedding, viremia, and severe atrophic enteritis. These findings confirm that strain PC21A is highly enteropathogenic.

Modulation of sheep ruminal urea transport by ammonia and pH.

Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (< 7), uptake of NH4 (+) into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4 (+). Implications for epithelial and ruminal homeostasis are discussed.

Transcription factor STAT enhanced antimicrobial activities in Bombyx mori.

STAT, a transcription factor in the JAK/STAT signaling pathway, regulates immune response to pathogens. In the silkworm (Bombyx mori), STAT exists as two split-forms, STAT-S and STAT-L. However, the role of STAT in silkworm immunity remains unclear. Our purpose was to investigate the effect of STAT on the expression of antimicrobial peptide (AMP) genes and resistance against pathogens. The expression levels of STAT-S and STAT-L were significantly up-regulated after induction by pathogenic microorganisms. In BmE cells, lipopolysaccharide (LPS), peptidoglycan (PGN) and ß-glucan stimulated STAT-S and STAT-L to transfer from the cytoplasm to the nucleus. We found that overexpression of STAT-S and STAT-L in cells could promote the expression of AMPs. We generated transgenic silkworm lines overexpressing STAT-L or STAT-S (OE-STAT-S; OE-STAT-L) or interfering with STAT (A4-dsSTAT). Overexpression of STAT-S and STAT-L upregulated the expression of AMP genes in the OE-STAT-S and OE-STAT-L, increased the survival rates of the OE-STAT-S silkworms and lowered the mortality of OE-STAT-L silkworms infected with S. aureus or Beauveria bassiana. By contrast, the death rate of A4-dsSTAT silkworms was higher after infection with these pathogenic microorganisms. These findings may provide insights into the role of STAT in the antimicrobial immune response of silkworms.

Dissociation protocols influence the phenotypes of lymphocyte and myeloid cell populations isolated from the neonatal lymph node.

Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings.

Acid tolerance of lactate-utilizing bacteria of the order Bacteroidales contributes to prevention of ruminal acidosis in goats adapted to a high-concentrate diet.

The rapid accumulation of organic acids, particularly lactate, has been suggested as the main cause of ruminal acidosis (RA) for ruminants fed high-concentrate diets. Previous research has shown that a gradual shift from low-to high-concentrate diets within 4 to 5 weeks effectively reduces the risk for RA. However, the mechanisms remain unknown. In this study, 20 goats were randomly allocated into four groups (n = 5) and fed with a diet containing a weekly increasing concentrate portion of 20%, 40%, 60%, and 80% over 28 d. At d 7, 14, 21, and 28, one group (named C20, C40, C60, and C80 according to the last concentrate level that they received) was killed and the ruminal microbiome was collected. Ruminal acidosis was not detected in any of the goats during the experiment. Nonetheless, ruminal pH dropped sharply from 6.2 to 5.7 (P < 0.05) when dietary concentrate increased from 40% to 60%. A combined metagenome and metatranscriptome sequencing approach identified that this was linked to a sharp decrease in the abundance and expression of genes encoding nicotinamide adenine dinucleotide (NAD)-dependent lactate dehydrogenase (nLDH), catalyzing the enzymatic conversion of pyruvate to lactate (P < 0.01), whereas the expression of two genes encoding NAD-independent lactate dehydrogenase (iLDH), catalyzing lactate oxidation to pyruvate, showed no significant concomitant change. Abundance and expression alterations for nLDH- and iLDH-encoding genes were attributable to bacteria from Clostridiales and Bacteroidales, respectively. By analyzing the gene profiles of 9 metagenome bins (MAG) with nLDH-encoding genes and 5 MAG with iLDH-encoding genes, we identified primary and secondary active transporters as being the major types of sugar transporter for lactate-producing bacteria (LPB) and lactate-utilizing bacteria (LUB), respectively. Furthermore, more adenosine triphosphate was required for the phosphorylation of sugars to initiate their catabolic pathways in LPB compared to LUB. Thus, the low dependence of sugar transport systems and catabolic pathways on primary energy sources supports the acid tolerance of LUB from Bacteroidales. It favors ruminal lactate utilization during the adaptation of goats to a high-concentrate diet. This finding has valuable implications for the development of measures to prevent RA.

Increased innate immune activation induces protective RSV-specific lung-resident memory T cells in neonatal mice.

Young infants frequently experience respiratory tract infections, yet vaccines designed to provide mucosal protection are lacking. Localizing pathogen-specific cellular and humoral immune responses to the lung could provide improved immune protection. We used a well-characterized murine model of respiratory syncytial virus (RSV) to study the development of lung-resident memory T cells (TRM) in neonatal compared to adult mice. We demonstrated that priming with RSV during the neonatal period failed to retain RSV-specific clusters of differentiation (CD8) TRM 6 weeks post infection, in contrast to priming during adulthood. The reduced development of RSV-specific TRM was associated with poor acquisition of two key markers of tissue residence: CD69 and CD103. However, by augmenting both innate immune activation and antigen exposure, neonatal RSV-specific CD8 T cells increased expression of tissue-residence markers and were maintained in the lung at memory time points. Establishment of TRM correlated with more rapid control of the virus in the lungs upon reinfection. This is the first strategy to effectively establish RSV-specific TRM in neonates providing new insight into neonatal memory T cell development and vaccine strategies.