Immunological effects of ractopamine in rabbits receiving the viral inactivated rabbit hemorrhagic disease vaccine.

Background: There is an obvious lack of information about the effects of ractopamine, a ß-adrenergic agonist, on the growth performance and immune responses of rabbits, particularly in those receiving the viral rabbit hemorrhagic disease (RHD) vaccine. Aim: The current study was undertaken to study the effects of ractopamine on growth performances and immunological parameters in rabbits inoculated with the viral RHD vaccine. Methods: Experimental rabbits were grouped into four groups, the first acted as a control and received distilled water, the second received ractopamine, the third received inactivated RHD vaccine, and the fourth received both ractopamine, and inactivated RHD vaccine. Then, blood analysis, histopathological, histomorphometric, and immunohistochemistry (IHC) examinations were followed. Results: The obtained results demonstrated that ractopamine induced significant increases in body weight gain, neutrophils, monocytes, nitric oxide, lysosome, and improved feed conversion rate. A significant decrease in lymphocytes with insignificant decreases in eosinophils, phagocytic % and index, serum total protein, α, ß, and γ globulin were observed. Vaccinated rabbits only showed a marked rise in WBCs, neutrophils, monocytes, eosinophils, basophils, phagocytic index and activity, nitric oxide, lysosome activity, total protein, albumin, γ globulin, and a decrease in lymphocytes. Rabbits that received ractopamine and then vaccinated had insignificant increases in body weight, weight gain, WBCs, neutrophils, monocyte, eosinophils, basophils, phagocytic activity, and index, globulins besides a significant decrease in lymphocytes. Pathologically, rabbits that received ractopamine alone, with a vaccine or vaccinated only showed an increase in villus length, villus width, and absorption surface area. IHC of rabbits' liver and kidneys of the control and vaccinated group showed negative expression for caspase-3, but rabbits received ractopamine only or rabbits vaccinated and received ractopamine showed diffuse positive moderate expression for caspase-3. Conclusion: Ractopamine induced several adverse effects on the immune responses of the rabbits inoculated with the viral HRD vaccine.

Development and preclinical evaluation of equine-derived hyperimmune serum against SARS-CoV-2 infection in K-18 hACE2 transgenic (Tg) mice.

This study aimed to develop an equine-derived hyperimmune serum against SARS-CoV-2 and evaluate its efficacy as a potential immunotherapy tool for the treatment of known and potential variants of COVID-19 in preclinical trials. The novelty of this study is the whole virus and ALUM gel adjuvant formula. The horses were immunized using a whole inactivated SARS-CoV-2 antigen, and the final purified hyperimmune serum showed high plaque reduction neutralization (PRNT 50) neutralizing titers. The efficacy of the hyperimmune serum was evaluated histopathologically and biochemically in the lungs, hearts, and serum of K18 hACE2 transgenic mice (n=45), which is an accepted model organism for SARS-CoV-2 studies and was challenged with live SARS-CoV-2. Serum treatment improved the general condition, resulting in lower levels of proinflammatory cytokines in the blood plasma, as well as reduced viral RNA titers in the lungs and hearts. Additionally, it reduced oxidative stress significantly and lessened the severity of interstitial pneumonia in the lungs when compared to infected positive controls. The study concluded that equine-derived anti-SARS-CoV-2 antibodies could be used for COVID-19 prevention and treatment, especially in the early stages of the disease and in combination with antiviral drugs and vaccines. This treatment will benefit special patient populations such as immunocompromised individuals, as specific antibodies against SARS-CoV-2 can neutralize the virus before it enters host cells. The rapid and cost-effective production of the serum allows for its availability during the acute phase of the disease, making it a critical intervention in preventing the spread of the disease and saving lives in new variants where a vaccine is not yet developed.

Single MVA-SARS-2-ST/N Vaccination Rapidly Protects K18-hACE2 Mice against a Lethal SARS-CoV-2 Challenge Infection.

The sudden emergence of SARS-CoV-2 demonstrates the need for new vaccines that rapidly protect in the case of an emergency. In this study, we developed a recombinant MVA vaccine co-expressing SARS-CoV-2 prefusion-stabilized spike protein (ST) and SARS-CoV-2 nucleoprotein (N, MVA-SARS-2-ST/N) as an approach to further improve vaccine-induced immunogenicity and efficacy. Single MVA-SARS-2-ST/N vaccination in K18-hACE2 mice induced robust protection against lethal respiratory SARS-CoV-2 challenge infection 28 days later. The protective outcome of MVA-SARS-2-ST/N vaccination correlated with the activation of SARS-CoV-2-neutralizing antibodies (nABs) and substantial amounts of SARS-CoV-2-specific T cells especially in the lung of MVA-SARS-2-ST/N-vaccinated mice. Emergency vaccination with MVA-SARS-2-ST/N just 2 days before lethal SARS-CoV-2 challenge infection resulted in a delayed onset of clinical disease outcome in these mice and increased titers of nAB or SARS-CoV-2-specific T cells in the spleen and lung. These data highlight the potential of a multivalent COVID-19 vaccine co-expressing S- and N-protein, which further contributes to the development of rapidly protective vaccination strategies against emerging pathogens.

Kinetic Ensemble of Tau Protein through the Markov State Model and Deep Learning Analysis.

The ordered assembly of Tau protein into filaments characterizes Alzheimer's and other neurodegenerative diseases, and thus, stabilization of Tau protein is a promising avenue for tauopathies therapy. To dissect the underlying aggregation mechanisms on Tau, we employ a set of molecular simulations and the Markov state model to determine the kinetics of ensemble of K18. K18 is the microtubule-binding domain of Tau protein and plays a vital role in the microtubule assembly, recycling processes, and amyloid fibril formation. Here, we efficiently explore the conformation of K18 with about 150 µs lifetimes in silico. Our results observe that all four repeat regions (R1-R4) are very dynamic, featuring frequent conformational conversion and lacking stable conformations, and the R2 region is more flexible than the R1, R3, and R4 regions. Additionally, it is worth noting that residues 300-310 in R2-R3 and residues 319-336 in R3 tend to form sheet structures, indicating that K18 has a broader functional role than individual repeat monomers. Finally, the simulations combined with Markov state models and deep learning reveal 5 key conformational states along the transition pathway and provide the information on the microsecond time scale interstate transition rates. Overall, this study offers significant insights into the molecular mechanism of Tau pathological aggregation and develops novel strategies for both securing tauopathies and advancing drug discovery.

Immunization with V987H-stabilized Spike glycoprotein protects K18-hACE2 mice and golden Syrian hamsters upon SARS-CoV-2 infection.

Safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are crucial to fight against the coronavirus disease 2019 pandemic. Most vaccines are based on a mutated version of the Spike glycoprotein [K986P/V987P (S-2P)] with improved stability, yield and immunogenicity. However, S-2P is still produced at low levels. Here, we describe the V987H mutation that increases by two-fold the production of the recombinant Spike and the exposure of the receptor binding domain (RBD). S-V987H immunogenicity is similar to S-2P in mice and golden Syrian hamsters (GSH), and superior to a monomeric RBD. S-V987H immunization confer full protection against severe disease in K18-hACE2 mice and GSH upon SARS-CoV-2 challenge (D614G or B.1.351 variants). Furthermore, S-V987H immunized K18-hACE2 mice show a faster tissue viral clearance than RBD- or S-2P-vaccinated animals challenged with D614G, B.1.351 or Omicron BQ1.1 variants. Thus, S-V987H protein might be considered for future SARS-CoV-2 vaccines development.

Serum protein electrophoresis in European mink (Mustela lutreola): reference intervals and comparison of agarose gel electrophoresis and capillary zone electrophoresis.

BACKGROUND: Knowledge of reference intervals for blood analytes, including serum protein fractions, is of great importance for the identification of infectious and inflammatory diseases and is often lacking in wild animal species. MATERIAL AND METHODS: Serum samples were obtained from European minks enrolled in the breeding program (n = 55). Agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) were used to separate and identify protein fractions. Albumin, α1, α2, ß, and γ-globulins fractions were identified in all mink sera by both electrophoresis methods. Reference intervals (90% CI) were determined following the 2008 guidelines of the Clinical Laboratory Standard Institute. The methods were compared using Passing-Bablok regression, Bland-Altman analysis, and Lin's concordance correlation. RESULTS: A significant bias was found between methods for α1, α2, and γ-globulin. Lin's concordance correlation was considered unacceptable for α1, α2, and ß-globulins. Differences for gender between methods were found for albumin and α2-globuins, which were higher for males than females. γ-globulins were higher for adults than young minks using both methods; however, α1 and α2-globulins were lower. CONCLUSION: Both methods are adequate for identifying serum protein disorders, but the AGE and CZE methods are not equivalent. Therefore, reference intervals for each technique are required.

A cell-adapted SARS-CoV-2 mutant, showing a deletion in the spike protein spanning the furin cleavage site, has reduced virulence at the lung level in K18-hACE2 mice.

Here we investigated the virulence properties of a unique cell-adapted SARS-CoV-2 mutant showing a ten-amino acid deletion encompassing the furin cleavage site of the spike protein (Δ680SPRAARSVAS689; Δ680-689-B.1) in comparison to its parental strain (wt-B.1) and two Delta variants (AY.122 and AY.21) of concern. After intranasal inoculation, transgenic K18-hACE2 mice were monitored for 14 days for weight change, lethality, and clinical score; oral swabs were daily collected and tested for the presence of N protein subgenomic RNA. At 3 and 7 dpi mice were also sacrificed and organs collected for molecular, histopathological, and immune response profile investigations. The Δ680-689-B.1-infected mice exhibited reduced shedding, lower virulence at the lung level, and milder pulmonary lesions. In the lung, infection with Δ680-689-B.1 was associated with a significant lower expression of some cytokines at 3 dpi (IL-4, IL-27, and IL-28) and 7 dpi (IL-4, IL-27, IL-28, IFN-γ and IL-1α).

Soluble Angiotensin-Converting Enzyme 2 Protein Improves Survival and Lowers Viral Titers in Lethal Mouse Model of Severe Acute Respiratory Syndrome Coronavirus Type 2 Infection with the Delta Variant.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) utilizes angiotensin-converting enzyme 2 (ACE2) as its main receptor for cell entry. We bioengineered a soluble ACE2 protein termed ACE2 618-DDC-ABD that has increased binding to SARS-CoV-2 and prolonged duration of action. Here, we investigated the protective effect of this protein when administered intranasally to k18-hACE2 mice infected with the aggressive SARS-CoV-2 Delta variant. k18-hACE2 mice were infected with the SARS-CoV-2 Delta variant by inoculation of a lethal dose (2 × 104 PFU). ACE2 618-DDC-ABD (10 mg/kg) or PBS was administered intranasally six hours prior and 24 and 48 h post-viral inoculation. All animals in the PBS control group succumbed to the disease on day seven post-infection (0% survival), whereas, in contrast, there was only one casualty in the group that received ACE2 618-DDC-ABD (90% survival). Mice in the ACE2 618-DDC-ABD group had minimal disease as assessed using a clinical score and stable weight, and both brain and lung viral titers were markedly reduced. These findings demonstrate the efficacy of a bioengineered soluble ACE2 decoy with an extended duration of action in protecting against the aggressive Delta SARS-CoV-2 variant. Together with previous work, these findings underline the universal protective potential against current and future emerging SARS-CoV-2 variants.

Age associated susceptibility to SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still an ongoing global health crisis. Clinical data indicate that the case fatality rate (CFR) is age dependent, with a higher CFR percentage in the elderly population. We compared the pathogenesis of SARS-CoV-2 in young and aged K18-hACE2 transgenic mice. We evaluated morbidity, mortality, viral titers, immune responses, and histopathology in SARS-CoV-2-infected young and old K18-hACE2 transgenic mice. Within the limitation of having a low number of mice per group, our results indicate that SARS-CoV-2 infection resulted in slightly higher morbidity, mortality, and viral replication in the lungs of old mice, which was associated with an impaired IgM response and altered cytokine and chemokine profiles. Results of this study increase our understanding of SARS-CoV-2 infectivity and immuno-pathogenesis in the elderly population.

Secondary hypogammaglobulinemia in adults—A large retrospective cohort study / Hipogammaglobulinemia secundaria en adultos: estudio de cohorte retrospectiva

Background and objective: IgG replacement therapy (IgG-RT) has radically changed the clinical evolution of primary immunodeficiencies, yet the information regarding secondary hypogammaglobulinemia (SHG) is insufficient or conflicting. We aim to describe clinical features, evolution and treatment of SHG patients in our center. Methods: Dynamic retrospective cohort between January 2001 and July 2021 of adults with gamma globulin fraction <0.6g/dL in a serum protein electrophoresis and a coincident decrease of IgG levels – with a disease-related SHG or treatment that reduces serum immunoglobulins. Results: We included 1012 patients with SHG with a median follow-up of 5 years (IQR 2–8). Hematological diseases were identified in 95% of the patients and 61% received drugs related to SHG. Sixty five percent had more than one etiological factor associated with SHG. Infectious diseases were present in 69% of the patients, 48% had respiratory infections and 17% had severe infections. There was statistical association between respiratory and severe infections with multiple myeloma (MM), lymphoma and rituximab. MGUS had less infections and death compared with other etiologies. IgG-RT was indicated in 18.7% of the patients and 4.6% received it for more than 6 months with variable intervals. Among the latter group, there was a significant reduction of all-type infections and respiratory infections with IgG-RT (p<0.001), and it was consistent with similar findings in lymphoma, MM and all IgG levels subgroups. Conclusion: SHG was associated with more than one etiological factor and a high frequency of infections. IgG-RT indication was irregular yet still effective. It is relevant to consider IgG levels screening, monitoring and accurate indication of IgG-RT.(AU)

Antecedentes y objetivos: La IgG sustitutiva ha cambiado radicalmente la evolución de las inmunodeficiencias primarias, mientras que la información sobre hipogammaglobulinemia secundaria (HGS) es insuficiente y discordante. El objetivo del estudio es describir las características clínicas, evolución y tratamiento de pacientes con HGS. Métodos: Cohorte retrospectiva dinámica entre enero de 2001 y julio de 2021 de adultos con proteinograma y fracción de gammaglobulina <0,6g/dL y dosaje disminuido de IgG, con enfermedad o tratamiento que produzcan HGS. Resultados: Se incluyó a 1.012 pacientes con HGS con una mediana de seguimiento de 5 años (IIC 2-8). El 95% tenía enfermedad hematológica y el 61% recibió fármacos asociados a HGS. El 65% tenía más de un factor etiológico asociado con HGS. El 69% presentó infecciones de cualquier tipo, el 48% infecciones respiratorias y el 17%, infecciones graves. Hubo asociación significativa entre infecciones respiratorias y graves entre los subgrupos de mieloma múltiple, linfoma y rituximab. Los pacientes con MGUS tuvieron menor frecuencia de infecciones y muerte comparados con otros factores etiológicos. El 18,7% de los pacientes recibió IgG sustitutiva y el 4,6% de forma crónica, con intervalos variables. Los últimos tuvieron disminución significativa de infecciones de cualquier tipo e infecciones respiratorias con IgG sustitutiva (p<0,001), que se mantuvo en los subgrupos con mieloma múltiple, linfoma y todos los niveles de IgG. Conclusión: La HGS asoció más de un factor etiológico y alta frecuencia de infecciones. La indicación de IgG sustitutiva fue irregular, pero, aún así, efectiva. Se plantea considerar el dosaje de inmunoglobulinas, monitoreo y la adecuada indicación de IgG sustitutiva.(AU)

Cutting Edge: Characterization of Low Copy Number Human Angiotensin-Converting Enzyme 2-Transgenic Mice as an Improved Model of SARS-CoV-2 Infection.

A popular mouse model of COVID-19, the K18-hACE2 mouse, expresses the SARS-coronavirus entry receptor, human angiotensin-converting enzyme 2 (hACE2) driven by the keratin-18 promoter. SARS-CoV-2-infected K18-hACE2 mice exhibit neuropathology not representative of human infection. They contain eight transgene (Tg) copies, leading to excess hACE2 expression and rampant viral replication. We generated two new lines of K18-hACE2 mice encoding one and two copies of hACE2 (1-hACE2-Tg and 2-hACE2-Tg, respectively). Relative to the original strain (called 8-hACE2-Tg in this study), 2-hACE2-Tg mice exhibited lower mortality, with less viral replication in the lung and brain. Furthermore, 1-hACE2-Tg mice exhibited no mortality and had no detectable virus in the brain; yet, they exhibited clear viral replication in the lung. All three strains showed SARS-CoV-2-related weight loss commensurate with the mortality rates. 1-hACE2-Tg mice mounted detectable primary and memory T effector cell and Ab responses. We conclude that these strains provide improved models to study hACE2-mediated viral infections.

Design, synthesis and biological evaluation of 1,2,3-triazole benzothiazole derivatives as tubulin polymerization inhibitors with potent anti-esophageal cancer activities.

In this work, we utilized the molecular hybridization strategy to design and synthesize novel 1,2,3-triazole benzothiazole derivatives K1-26. The antiproliferative activities against MGC-803, Kyse30 and HCT-116 cells were explored, and their structure-activity relationship were preliminarily conducted and summarized. Among them, compound K18, exhibited the strongest proliferation inhibitory activity, with esophageal cancer cells Kyse30 and EC-109 being the most sensitive to its effects (IC50 values were 0.042 and 0.038 µM, respectively). Compound K18 effectively inhibited tubulin polymerization (IC50 = 0.446 µM), thereby hindering tubulin polymerize into filamentous microtubules in Kyse30 and EC-109 cells. Additionally, compound K18 induced the degradation of oncogenic protein YAP via the UPS pathway. Based on these dual molecular-level effects, compound K18 could induce G2/M phase arrest and cell apoptosis in Kyse30 and EC-109 cells, as well as regulate the expression levels of cell cycle and apoptosis-related proteins. In summary, our findings highlight a novel 1,2,3-triazole benzothiazole derivative K18, which possesses significant potential for treating esophageal cancers.

Antiviral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models.

SARS-CoV-2 variants of concern (VOCs) continue to evolve and reemerge with chronic inflammatory long COVID sequelae, necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the receptor for advanced glycation end products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an antiviral and anti-inflammatory therapeutic effect in the COVID-19 system. The protective therapeutic effect of RAGE-Ig was determined in vivo in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six VOCs of SARS-CoV-2. The underlying antiviral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). Following treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated (1) significant dose-dependent protection (i.e., greater survival, less weight loss, lower virus replication in the lungs); (2) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (3) a RAGE-Ig dose-dependent increase in the expression of type I IFNs (IFN-α and IFN-ß) and type III IFN (IFNλ2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (4) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. Our preclinical findings revealed type I and III IFN-mediated antiviral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.

Vaccine-associated respiratory pathology correlates with viral clearance and protective immunity after immunization with self-amplifying RNA expressing the spike (S) protein of SARS-CoV-2 in mouse models.

In this study, we evaluated the immunogenicity and protective immunity of in vitro transcribed Venezuelan equine encephalitis virus (VEEV TC-83 strain) self-amplifying RNA (saRNA) encoding the SARS-CoV-2 spike (S) protein in wild type (S-WT) and stabilized pre-fusion conformations (S-PP). Immunization with S-WT and S-PP saRNA induced specific neutralizing antibody responses in both K18-Tg hACE2 (K18) and BALB/c mice, as assessed using SARS-CoV-2 pseudotyped viruses. Protective immunity was assessed in challenge experiments. Two immunizations with S-WT and S-PP induced protective immunity, evidenced by lower mortality, lower weight loss and more than one log10 lower subgenomic virus RNA titers in the upper and lower respiratory tracts in both K18 and BALB/c mice. Histopathologic examination of lungs post-challenge showed that immunization with S-WT and S-PP resulted in a higher degree of immune cell infiltration and inflammatory changes, compared with control mice, characterized by high levels of T- and B-cell infiltration. No substantial differences were found in the presence and localization of eosinophils, macrophages, neutrophils, and natural killer cells. CD4 and CD8 T-cell depletion post immunization resulted in reduced lung inflammation post challenge but also prolonged virus clearance. These data indicate that immunization with saRNA encoding the SARS-CoV-2 S protein induces immune responses that are protective following challenge, that virus clearance is associated with pulmonary changes caused by T-cell and B-cell infiltration in the lungs, but that this T and B-cell infiltration plays an important role in viral clearance.

A longitudinal molecular and cellular lung atlas of lethal SARS-CoV-2 infection in K18-hACE2 transgenic mice.

BACKGROUND: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to approximately 500 million cases and 6 million deaths worldwide. Previous investigations into the pathophysiology of SARS-CoV-2 primarily focused on peripheral blood mononuclear cells from patients, lacking detailed mechanistic insights into the virus's impact on inflamed tissue. Existing animal models, such as hamster and ferret, do not faithfully replicate the severe SARS-CoV-2 infection seen in patients, underscoring the need for more relevant animal system-based research. METHODS: In this study, we employed single-cell RNA sequencing (scRNA-seq) with lung tissues from K18-hACE2 transgenic (TG) mice during SARS-CoV-2 infection. This approach allowed for a comprehensive examination of the molecular and cellular responses to the virus in lung tissue. FINDINGS: Upon SARS-CoV-2 infection, K18-hACE2 TG mice exhibited severe lung pathologies, including acute pneumonia, alveolar collapse, and immune cell infiltration. Through scRNA-seq, we identified 36 different types of cells dynamically orchestrating SARS-CoV-2-induced pathologies. Notably, SPP1+ macrophages in the myeloid compartment emerged as key drivers of severe lung inflammation and fibrosis in K18-hACE2 TG mice. Dynamic receptor-ligand interactions, involving various cell types such as immunological and bronchial cells, defined an enhanced TGFß signaling pathway linked to delayed tissue regeneration, severe lung injury, and fibrotic processes. INTERPRETATION: Our study provides a comprehensive understanding of SARS-CoV-2 pathogenesis in lung tissue, surpassing previous limitations in investigating inflamed tissues. The identified SPP1+ macrophages and the dysregulated TGFß signaling pathway offer potential targets for therapeutic intervention. Insights from this research may contribute to the development of innovative diagnostics and therapies for COVID-19. FUNDING: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020M3A9I2109027, 2021R1A2C2004501).

N-dihydrogalactochitosan serves as an effective mucosal adjuvant for intranasal vaccine in combination with recombinant viral proteins against respiratory infection.

Mucosal vaccinations for respiratory pathogens provide effective protection as they stimulate localized cellular and humoral immunities at the site of infection. Currently, the major limitation of intranasal vaccination is using effective adjuvants capable of withstanding the harsh environment imposed by the mucosa. Herein, we describe the efficacy of using a unique biopolymer, N-dihydrogalactochitosan (GC), as a nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV), an MF-59 equivalent. In contrast to AV, intranasal application of GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. Moreover, GC+S+NC-vaccinated animals were largely resistant to the lethal SARS-CoV-2 challenge and experienced drastically reduced morbidity and mortality, with animal weights and behavior returning to normal 22 days post-infection. In contrast, animals intranasally vaccinated with AV+S+NC experienced severe weight loss, mortality, and respiratory distress, with none surviving beyond 6 days post-infection. Our findings demonstrate that GC can serve as a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses. STATEMENT OF SIGNIFICANCE: We demonstrated that a unique biopolymer, N-dihydrogalactochitosan (GC), was an effective nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV). In contrast to AV, GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. About 90 % of the GC+S+NC-vaccinated animals survived the lethal SARS-CoV-2 challenge and remained healthy 22 days post-infection, while the AV+S+NC-vaccinated animals experienced severe weight loss and respiratory distress, and all died within 6 days post-infection. Our findings demonstrate that GC is a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses.

[Preparation of a block copolymer-based temperature-responsive affinity chromatography stationary phase for antibody separation and purification].

Antibodies play an essential role in cancer diagnosis and treatment because of the specificity for target biomolecules and reduction of side effects. However, antibodies separation and purification still face some challenges. Antibody elution from columns using a low-pH aqueous solution leads to aggregation or loss of activity of the antibody drugs. In this paper, a block copolymer-based temperature-responsive affinity chromatography (TRAC) stationary phase, SiO2-P[NIPAM-b-4VP]-MEP using the block temperature-responsive copolymer poly(N-isopropylacrylamide-b-4-vinylpyridine) (P[NIPAM-b-4VP]) as the space arms and 4-mercaptoethyl pyridine (MEP) as the ligand was prepared for antibody separation. The TRAC column was tested using bovine serum albumin (BSA) and γ-globulin as model proteins, and the effects of salt concentration in the mobile phase and temperature on their separation were studied in detail. At 40 ℃, the TRAC stationary phase only selectively retained γ-globulin due to the specific affinity interaction between antibodies and the ligand MEP. At 5 ℃, γ-globulin can be eluted from the column with a mass recovery of 92.7% using a Tris-HCl buffer (pH 8.0) solution containing 0.6 mol/L NaCl. The adsorption capacity of γ-globulin on this stationary phase was (71.5 ±2.1) mg/g (n=3), which was twice that of a traditional temperature-sensitive affinity chromatography stationary phase SiO2-PNIPAM-MEP. The stationary phase was also used to separate and purify immunoglobulin (IgG) in human serum in one step by altering the temperature and ion strength of the mobile phase, resulting in a purity of 97.4%±0.7%. Thus, this new technology has specific selectivity for antibodies, as well as mild and green elution conditions, ultimately resolving the problem of traditional affinity chromatography using acid elution, which can lead to the antibodies aggregation/inactivation. This technology has great application potential for the industrial production of antibody drugs.

[Analysis of adverse neonatal outcomes in pregnant women with positive anti-Ro/SSA and anti-La/SSB antibodies].

Objective: To investigate the relationship between positive anti-Ro/Sjögren syndrome antigen type A (SSA) antibody and anti-La/Sjögren syndrome antigen type B (SSB) antibody in pregnant women and neonatal adverse outcomes. Methods: This study was a retrospective study, and 145 deliveries of 136 anti-Ro/SSA and anti-La/SSB antibody positive pregnant women were selected who had prenatal examination and delivered in Peking University First Hospital from January 2017 to June 2022. According to whether adverse neonatal outcomes occurred, 145 deliveries were divided into adverse outcome group (26 cases) and no adverse outcome group (119 cases). According to the time when anti-Ro/SSA and anti-La/SSB antibodies were found positive, 145 deliveries were divided into the antibody positive during pregnancy group (69 cases) and the pre-pregnancy antibody positive group (76 cases). The pregnancy outcomes, treatment and maternal and infant antibody levels of pregnant women between the adverse outcome group and no adverse outcome group, between antibody positive during pregnancy group and the pre-pregnancy antibody positive group were compared. Results: (1) Most of the pregnant women with positive anti-Ro/SSA and anti-La/SSB antibodies were diagnosed as undifferentiated connective tissue disease, accounting for 40.4% (55/136), followed by Sjogren's syndrome (25.0%, 34/136), systemic lupus erythematosus (23.5%, 32/136), antiphospholipid antibody syndrome (6.6%, 9/136), idiopathic thrombocytopenic purpura (1.5%, 2/136), and 4 cases were not diagnosed. (2) The titers of anti-Ro/SSA and anti-La/SSB antibodies in the first trimester and the second trimester were compared, and there were no statistical significances (all P>0.05). (3) The proportion of high level anti-Ro/SSA antibody (>100 kU/L), positive level of anti-La/SSB antibody and positive rate of anti-La/SSB antibody in the adverse outcome group were higher than those in the no adverse outcome group, and the birth weight of newborns and live birth rate in the adverse outcome group were lower than that in the no adverse outcome group, all with statistical significances (all P<0.05). The anti-Ro/SSA antibody level, the proportion of drug treatment (hydroxychloroquine, glucocorticoid, gamma globulin), the incidence of fetal growth restriction (FGR), the rate of preterm birth, and the positive level of anti-Ro/SSA and anti-La/SSB antibodies in newborns were compared between the two groups, and there were no statistically significant differences (all P>0.05). (4) The anti-Ro/SSA antibody level of pregnant women in the pre-pregnancy antibody positive group, the proportion of hydroxychloroquine and glucocorticoid treatment, and the anti-Ro/SSA antibody positive rate of newborns were higher, while the incidence of FGR and gamma globulin treatment rate of newborns in the antibody positive during pregnancy group were higher, respectively, and the differences were statistically significant (all P<0.05). The levels of anti-La/SSB antibodies in pregnant women, anti-Ro/SSA and anti-La/SSB antibodies in newborns, the positive rate of anti-La/SSB antibodies in newborns and the incidence of adverse outcomes were compared between the antibody positive during pregnancy group and the pre-pregnancy antibody positive group, and there were no statistical significances (all P>0.05). Conclusions: High concentrations of anti-Ro/SSA antibodies and co-positive anti-La/SSB antibodies during pregnancy may increase the incidence of adverse neonatal outcomes. There is no significant difference in the incidence of adverse neonatal outcomes between antibody positive pregnant women and antibody positive pregnant women who were first found during pregnancy after comprehensive treatment in the rheumatology and immunology department.

Measurement of plasma protein and whole body protein metabolism using [15N]glycine in a young adult man - a pilot study.

A novel simplified method is presented for the estimation of the metabolism of plasma proteins (albumin, fibrinogen, α, ß and γ-globulin, glycoprotein) with regard to the whole body protein metabolism in a young male volunteer (22 years, 81 kg body mass). This method is based on multiple oral administration of [15N]glycine followed by measurement of 15N in plasma proteins, total free amino acids, urea and excreted urinary N. The fractional synthesis rate of albumin was estimated to 6.8 % d-1 based on amino acids and 3.3 % d-1 based on urea, respectively. The fractional synthesis rate of the other plasma proteins ranged from 4.3 % d-1 (γ-globulin) to 26.4 % d-1 (α-globulin, fibrinogen). We conclude that the simplified approach using [15N]glycine provides results which are similar to results based on the simultaneously applied 131I-human serum albumin technique as 'gold standard' and to those reported in literature. The compartmental analysis considering comprehensive tracer kinetic data ensures reliable data treatment and enables statistical evaluation. The analytical effort is minimal because the 15N enrichment of plasma protein after chemical digestion may be directly used. Therefore, the novel stable isotope 15N method is suitable for studies in clinical and nutritional research and practice.

Unintended findings: Therapeutic effects of hormones or gamma globulins on Lentiform Fork sign in 3 diabetic uremic patients: Case report/case series.

INTRODUCTION: The lentiform fork sign (LFS) is a unique magnetic resonance imaging (MRI) finding characterized by a bright hyperintense rim delineating the lentiform nucleus as a fork associated with metabolic acidosis in end-stage renal disease. PATIENT CONCERNS: We report 3 cases of LFS in diabetic uremic patients. In one case of uremia, intensive hemodialysis treatment was not effective. Given our poor understanding of LFS, it was regarded as bilateral basal ganglia pathology, and pulse hormone and gamma globulins therapy was initiated. The patient neurological symptoms improved, and the pathological signs on imaging subsided. Based on our experience with the first LFS case, 2 diabetic uremic cases presenting with LFS were successfully treated with hormone or gamma globulin pulse therapy in addition to intensive hemodialysis. DIAGNOSIS: Based on the clinical manifestations, past medical history and MRI imaging changes of the 3 cases reported here, the diagnosis of LFS was established. INTERVENTIONS: Our experience from these 3 cases suggests that hormone supplementation and gamma globulin therapy may be indicated for treating LFS. LESSONS: Our findings highlight that in diabetic uremic dialysis patients with neurological symptoms, LFS should be suspected. The clinical manifestations, past medical history and MRI imaging findings are essential for diagnosing LFS. Hormone supplementation and gamma globulin therapy may be the effective treatment for LFS.