Histological and Biomechanical Evaluation of the Preserved Degenerative Dermis in Rat Autologous Skin Transplant Models after a Deep Second Degree Burn

To describe the histological and biomechanical changes of the preserved degenerative dermis in rat  autologous skin transplant models after a deep second-degree burn. 50 SD rats were divided into 5 groups  randomly of 10 rats of each: 7-days group, 9-days group, 14-days group, 21-days group, and 60-days group.  Deep second-degree burn wounds were prepared on the back of rats sized 3.5cm×3.5cm. Super tangential  excision was performed on the burn wound to preserve the degenerative dermis. Then, autologous epidermis  was grafted on the wound. After that, the histological changes of the preserved degenerative dermis tissues  and the graft areas were observed by macroscopic, light microscope and electron microscope in the 7, 9, 14,  21, 60 days after the operation. Moreover, the tensile properties of healing deeply burned rat skin were also  tested for each group at the same time points mentioned above. Results: (1) According to the macroscopic  observation, 7 days after the operation, the grafted skin was fused with the area of burn wound; A few hairs  were growing out on the skin at the 14th day; the injured skin recovered to normality by the 60th day. (2)  Hyaline change occurred in the preserved degenerative dermis tissues based on the observation by light  microscope. At the 7th day after operation, the dermis papillae and reticular layer could be discerned; by  the 21st day, the thickness, structures and morphology of grafted skin were similar to the normal tissues. (3)  7 days after operation, ballooning changes were observed by the electron microscope in the mitochondria  and endoplasmic reticulum of damaged cells and the number of the ribosomes was obviously reduced. The  subcellular wound improved continuously and approached normality by the 21st day. (4) 9 days after the  operation, the tensible strength and maximal strain of the grafting rat skin approached 70% and 90% of  natural skin, respectively. (5) 60 days after the operation, the tensile performance of the healing rat skin  recovered to the natural level. Conclusion: The histological and biomechanical changes of the denatured dermis of a deep second  degree burn wound may gradually recover to normality after being covered by autologous skin.

IFN-γ– and IL-10–expressing virus epitope-specific Foxp3+ T reg cells in the central nervous system during encephalomyelitis

Pathogen-specific Foxp3+ T reg cells can be identified on the basis of cytokine production, are detected in naive T cell populations, and exhibit suppressive ability toward effector T cells with the same antigen specificity

Lung directed antibody gene transfer confers protection against SARS-CoV-2 infection

The novel coronavirus disease (COVID-19) pandemic continues to be a worldwide threat and effective antiviral drugs and vaccines are being developed in a joint global effort. However, some elderly and immune-compromised populations are unable to raise an effective immune response against traditional vaccines. We hypothesised that passive immunity engineered by the in vivo expression of anti SARS-CoV-2 monoclonal antibodies (mAbs), an approach termed vectored immunoprophylaxis (VIP), could offer sustained protection against COVID-19 in all populations irrespective of their immune status or age. We developed three key reagents to evaluate VIP for SARS-CoV-2: (i) we engineered standard laboratory mice to express human ACE2 via rAAV9 in vivo gene transfer, to allow in vivo assessment of SARS-CoV-2 infection, (ii) to simplify in vivo challenge studies, we generated SARS-CoV-2 Spike protein pseudotyped lentiviral vectors as a simple mimic of authentic SARS-CoV-2 that could be used under standard laboratory containment conditions; and (iii) we developed in vivo gene transfer vectors to express anti-SARS-CoV-2 mAbs. A single intranasal dose of rAAV9 or rSIV.F/HN vectors expressing anti-SARS-CoV 2 mAbs significantly reduced SARS-CoV-2 mimic infection in the lower respiratory tract of hACE2-expressing mice. If translated, the VIP approach could potentially offer a highly effective, long-term protection against COVID-19 for highly vulnerable populations; especially immune-deficient/senescent individuals, who fail to respond to conventional SARS-CoV 2 vaccines. The in vivo expression of multiple anti-SARS71 CoV-2 mAbs could enhance protection and prevent rapid mutational escape

Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Regulates Cell Stress Response and Apoptosis

Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE

Original Article - Erythropoietin preconditioning on hippocampus neuronal apoptosis following status epilepticus induced by Li-pilocarpine in rats through anti-caspase-3 expression

Selective neuronal loss following status epilepticus(SE) was first described just under 100 years ago. The acute pathology following SE was shown to be 'ischemic cell change' and was assumed to arise through hypoxia/ischemia. Recently, erythropoietin (Epo) has been shown to have potent anti-apoptosis activity in central nervous system neurons in animal models of ischaemic injury. Aims: In this report, in order to determine Epo preconditioning on hippocampus neuronal apoptosis, we examined caspase-3 expression following SE caused by Li-pilocarpine in rats. Settings and Design: Animals were classified into three groups: EP group (pilocarpine group), rhEpo-pilocarpine group and control group. Four hours after preconditioning with Epo intraperitoneally, pilocarpine hydrochloride was administered intraperitoneally and observed for behavioral manifestations of SE. The animals were sacrificed at one hour after SE onset. Materials and Methods: At the above-mentioned time point, animals were deeply anesthetized and were perfused through the left ventricle. Detection of hippocampus neuronal apoptosis was performed with caspase-3 immunohistochemical technique on three groups. To further confirm which cell population upregulates caspase-3, brain sections were stained for NeuN (green) and caspase-3 (red).Statistical analysis ANOVA and Fisher's post hoc test was used. Results Quatification of hippocampus neurons revealed that the number of caspase-3-positive cells in the CA1/CA3 area and dentate gyrus(DG)of three groups had a significant difference. In comparison with control group, there was an increase by 74% and 534%, 42% and 272% in the CA1/CA3 area and DG of EP group and rhEpo-treated group respectively. There was a decrease by 18% and 26% in the CA1/CA3 area and DG of rhEpo-treated group compared with those in EP group. In addition, colocalization of caspase-3 with NeuN was shown. Conclusions Systemic rhEpo therapy reduced caspase-3 expression in SE induced by Li-pilocarpine

Identification and Characterization of Dominant Helper T-Cell Epitopes in the Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus▿

By using a series of overlapping synthetic peptides covering 98% of the amino acid sequence of the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus (SARS-CoV), four helper T-cell (Th) epitopes (NP11, residues 11 to 25; NP51, residues 51 to 65; NP61, residues 61 to 75; and NP111, residues 111 to 125) in C57BL mice (H-2b), four (NP21, residues 21 to 35; NP91, residues 91 to 105; NP331, residues 331 to 345; and NP351, residues 351 to 365) in C3H mice (H-2k), and two (NP81, residues 81 to 95; and NP351, residues 351 to 365) in BALB/c mice (H-2d) have been identified. All of these peptides were able to stimulate the proliferation of NP-specific T-cell lines or freshly isolated lymph node cells from mice immunized with recombinant NP. Immunization of mice with synthetic peptides containing appropriate Th epitopes elicited strong cellular immunity in vivo, as evidenced by delayed-type hypersensitivity. Priming with the helper peptides (e.g., NP111 and NP351) significantly accelerated the immune response induced by recombinant NP, as determined by the production of NP-specific antibodies. When fused with a conserved neutralizing epitope (SP1143-1157) from the spike protein of SARS-CoV, NP111 and NP351 assisted in the production of high-titer neutralizing antibodies in vivo. These data provide useful insights regarding immunity against SARS-CoV and have the potential to help guide the design of peptide-based vaccines

Comparison of Immunoglobulin G Responses to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome (SARS) Coronavirus in Patients with SARS▿

Both the nucleocapsid (N) and the spike (S) proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) are able to induce strong humoral responses in humans following an infection. To compare the immunoglobulin G (IgG) responses to the S and N proteins of SARS-CoV in SARS patients during the manifestation/convalescent period with those during the postinfection period, serum samples were collected from hospitalized SARS patients within 6 weeks after the onset of illness (set 1; 57 sequential samples from 19 patients) or 2 to 3 months after their recovery (set 2; 33 postinfection samples from 33 subjects). Serum samples from 100 healthy blood donors (set 3), collected in 2002, were also included. The specific IgG response to whole virus, the fragment from positions 450 to 650 of the S protein (S450-650), and the full-length N protein of SARS-CoV were measured by enzyme-linked immunosorbent assays (ELISAs). Western blot assays were carried out to confirm the ELISA results. Fifty-one of the serum samples in set 1 (89%) bound to the N protein, a proportion similar to that which recognized whole virus (79%) and the S-protein fragment (77%). All 33 serum samples from set 2 were strongly positive for N-protein-specific IgG, while 27 (82%) were positive for anti-S450-650 IgG. Two of the serum samples from set 3 were strongly positive for anti-N-protein IgG but not anti-S450-650 IgG. Similar levels of IgG responses to the S and N proteins were observed in SARS patients during the manifestation and convalescent stages. In the postinfection period, however, a number of patients had much lower serum IgG levels against S450-650 than against the N protein

Clinical nomogram prediction model to assess the risk of prolonged ICU length of stay in patients with diabetic ketoacidosis: a retrospective analysis based on the MIMIC-IV database

Abstract Background The duration of hospitalization, especially in the intensive care unit (ICU), for patients with diabetic ketoacidosis (DKA) is influenced by patient prognosis and treatment costs. Reducing ICU length of stay (LOS) in patients with DKA is crucial for optimising healthcare resources utilization. This study aimed to establish a nomogram prediction model to identify the risk factors influencing prolonged LOS in ICU-managed patients with DKA, which will serve as a basis for clinical treatment, healthcare safety, and quality management research. Methods In this single-centre retrospective cohort study, we performed a retrospective analysis using relevant data extracted from the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. Clinical data from 669 patients with DKA requiring ICU treatment were included. Variables were selected using the Least Absolute Shrinkage and Selection Operator (LASSO) binary logistic regression model. Subsequently, the selected variables were subjected to a multifactorial logistic regression analysis to determine independent risk factors for prolonged ICU LOS in patients with DKA. A nomogram prediction model was constructed based on the identified predictors. The multivariate variables included in this nomogram prediction model were the Oxford acute severity of illness score (OASIS), Glasgow coma scale (GCS), acute kidney injury (AKI) stage, vasoactive agents, and myocardial infarction. Results The prediction model had a high predictive efficacy, with an area under the curve value of 0.870 (95% confidence interval [CI], 0.831–0.908) in the training cohort and 0.858 (95% CI, 0.799–0.916) in the validation cohort. A highly accurate predictive model was depicted in both cohorts using the Hosmer–Lemeshow (H-L) test and calibration plots. Conclusion The nomogram prediction model proposed in this study has a high clinical application value for predicting prolonged ICU LOS in patients with DKA. This model can help clinicians identify patients with DKA at risk of prolonged ICU LOS, thereby enhancing prompt intervention and improving prognosis

A potent and broad‐spectrum neutralizing nanobody for SARS‐CoV‐2 viruses, including all major Omicron strains

Abstract SARS‐CoV‐2 viruses are highly transmissible and immune evasive. It is critical to develop broad‐spectrum prophylactic and therapeutic antibodies for potential future pandemics. Here, we used the phage display method to discover nanobodies (Nbs) for neutralizing SARS‐CoV‐2 viruses especially Omicron strains. The leading nanobody (Nb), namely, Nb4, with excellent physicochemical properties, can neutralize Delta and Omicron subtypes, including BA.1, BA.1.1 (BA.1 + R346K), BA.2, BA.5, BQ.1, and XBB.1. The crystal structure of Nb4 in complex with the receptor‐binding domain (RBD) of BA.1 Spike protein reveals that Nb4 interacts with an epitope on the RBD overlapping with the receptor‐binding motif, and thus competes with angiotensin‐converting enzyme 2 (ACE2) binding. Nb4 is expected to be effective for neutralizing most recent Omicron variants, since the epitopes are evolutionarily conserved among them. Indeed, trivalent Nb4 interacts with the XBB1.5 Spike protein with low nM affinity and competes for ACE2 binding. Prophylactic and therapeutic experiments in mice indicated that Nb4 could reduce the Omicron virus loads in the lung. In particular, in prophylactic experiments, intranasal administration of multivalent Nb4 completely protected mice from Omicron infection. Taken together, these results demonstrated that Nb4 could serve as a potent and broad‐spectrum prophylactic and therapeutic Nb for COVID‐19

Analysis of differences in tobacco leaf microbial communities after redrying in Chinese provinces and from abroad

Abstract Microorganisms play an important role in the tobacco aging process. Before the aging process, raw tobacco leaves must be threshed and redried. In order to explore the differences of microbial community structure of threshed and redried tobacco leaves from different origins at home and abroad, 14 groups of tobacco leaves from 8 different countries were tested by high-throughput DNA sequencing and microbiology analysis. Then, through amplicon sequence variants (ASV) cluster analysis, Venn diagram and species labeling and other microbial diversity analysis, the dominant bacteria and fungi on the surface of threshed and redried tobacco leaves were obtained. The results showed that there were significant differences in the composition of tobacco bacteria and fungi after threshing and redrying from different geographical areas. The relative abundance of Microbacterium and Sphingomonas in domestic tobacco leaves was significantly higher than that of foreign tobacco leaves. The relative abundance of Pseudomonas in foreign tobacco bacterial colonies was significantly higher than that of domestic tobacco leaves. In terms of fungi, the relative abundance of Aspergillus and Alternaria in domestic tobacco leaves was significantly higher than that of foreign tobacco leaves. Septoria, Sampaiozyma, Cladosporium and Phoma account for significantly higher proportions of foreign tobacco leaves. These microorganisms may be indispensable in aging process to form different flavors of tobacco leaves. It provides an important theoretical basis for the further use of microorganisms to promote tobacco leaf aging