重组人IL-2对感染弓形虫孕鼠T细胞亚群及NK细胞的影响

目的　研究妊娠期感染弓形虫的小鼠脾脏T细胞亚群和NK细胞水平的变化及IL - 2对二者的影响。方法　BALB/c孕鼠被随机分为 4组 :空白对照、感染对照、两个IL - 2处理组。于妊娠第 8d ,空白对照腹腔注射 0 2ml生理盐水 ,其余组腹腔接种 4 0 0个弓形虫速殖子。在感染的 - 1、0、1d ,IL - 2处理组分别腹腔注射rhuIL - 2 5 0 0u(低剂量组 )和 10 0 0u(高剂量组 )。妊娠第 10、12、14、16d取孕鼠脾 ,流式细胞术检测T细胞亚群和NK细胞水平。结果与空白组相比 ,在妊娠各期感染组的脾CD4 + T细胞水平显著降低 ,而CD8+ T细胞水平显著升高 (第 10、12、14d) ,二者比值明显倒置 ;NK细胞水平明显下降。高剂量IL - 2处理组的CD4 + T细胞水平在妊娠第 10、12d高于感染组 ,CD8+ T细胞水平在妊娠第 10、14d低于感染组 ,CD4 + /CD8+ T细胞比值在妊娠第 10、12、14d高于感染组。低剂量IL - 2处理组CD4 + T细胞与感染组无显著性差异 ,CD8+T细胞水平仅在妊娠第 14d显著性低于感染组。结论　感染弓形虫孕鼠的外周免疫受到一定抑制 ,妊娠各期脾CD4 + /CD8+T细胞比例失衡 ;感染孕鼠NK细胞数量减少 ;注射适量rhuIL - 2可以使倒置的CD4 + /CD8+ T细胞比值逆转 ,促进NK细胞增殖 ,提高母体免疫水平。     

The global succinylation of SARS-CoV-2–infected host cells reveals drug targets

SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.

Since its emergence in 2019, SARS-CoV-2 has caused an ongoing pandemic of COVID-19 worldwide (1). SARS-CoV-2, together with the bat-origin coronavirus SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), usually induce respiratory diseases (2). By comparison, SARS-CoV-2 has high transmissibility, and has continued to evolve, leading to the emergence of vaccine-breakthrough variants (3–5). Effective and safe antiviral drugs are limited in the treatment of COVID-19. Thus, it is necessary to develop effective and broad-spectrum antiviral drugs for SARS-CoV-2 and emerging variants (6–8).Protein posttranslational modifications (PTMs) are the critical regulation mechanisms of many cellular processes, which makes them attractive viral targets. As viruses cannot replicate themselves, they have developed abilities to alter cellular pathways and proteins required for virus replication, including protein PTMs. Lysine residues in proteins are vulnerable to a variety of PTMs—such as methylation, acetylation, ubiquitination, and succinylation—which play important roles in cellular physiology and pathology. Succinylation, a newly identified PTM, endows lysine group with two negative charges, which leads to more changes in protein structure and function (9). Quantitative proteomics has been widely used to clarify viral pathogenesis by explaining the changed cellular processes and identifying the pivotal pathways or proteins that are potential antiviral targets (10). Here we present the global succinylation and protein abundance in SARS-CoV-2–infected cells and map these succinylation changes to the disturbed pathways. We also determine the possible mechanism of host succinylation regulated by SARS-CoV-2 and evaluate potential drugs to treat COVID-19.     

Ex vivo expansion of CD34<Superscript>+</Superscript> and T and NK cells from umbilical cord blood for leukemic BALB/C nude mouse transplantation

CBSCT with low incidence of GVHD is associated with higher rates of delayed engraftment and relapse compared with other stem cells transplants. The immune-mediated effect of NK and cytotoxic T cells against residual tumor cells was shown to prevent relapse and reinduce remission after bone marrow transplantation. We expanded CD34⁺ and T and NK cells ex vivo in cord blood with different cytokines combination and transplanted into leukemic BALB/C nude mouse. The results showed significant expansion of MNCs and CD34⁺ cells. The CD3⁺ T cells increased in the groups containing cytokines cocktail, especially in the group with IL-7 or IL-2. CD56⁺ NK cells number increased significantly only in a medium containing IL-2. Of the 20 engrafted BALB/C nude mice, 14 survived after 6 weeks transplantation, and the numbers in each group were from 3 to 4. Human CD3⁺ cells in the bone marrow of the survived mice were analyzed by flow cytometry and showed existing evidences. RT-PCR was used to detect leukemic fusion bcr/abl gene; all mice that experienced expanded cord blood transplantation could not be found to have expression of fusion bcr/abl gene. These suggest that T, NK cells as well as CD34+ cells could be expanded from CB MNCs in the same medium with the combination of cytokines. The expanded CB MNCs could reconstitute hematopoiesis and eliminate minimal residue leukemia disease in transplanted mice.     

Disentangling the lifespans of hepatitis C virus‐infected cells and intracellular vRNA replication‐complexes during direct‐acting anti‐viral therapy

The decay rate of hepatitis C virus (HCV)‐infected cells during therapy has been used to determine the duration of treatment needed to attain a sustained virologic response, but with direct‐acting anti‐virals (DAA), this rate has been difficult to estimate. Here, we show that it is possible to estimate it, by simultaneously analysing the viral load and alanine aminotransferase (ALT) kinetics during combination DAA therapy. We modelled the HCV RNA and ALT serum kinetics in 26 patients with chronic HCV genotype 1b infection, under four different sofosbuvir‐based combination treatments. In all patients, ALT decayed exponentially to a set point in the normal range by 1‐3 weeks after initiation of therapy. The model indicates that the ALT decay rate during the first few weeks after initiation of therapy reflects the death rate of infected cells, with an estimated median half‐life of 2.5 days in this patient population. This information allows independent estimation of the rate of loss of intracellular replication complexes during therapy. Our model also predicts that the final ALT set point is not related to the release of ALT by dying HCV‐infected cells. Using ALT data, one can separately obtain information about the rate of ‘cure’ of HCV‐infected cells versus their rate of death, something not possible when analysing only HCV RNA data. This information can be used to compare the effects of different DAA combinations and to rationally evaluate their anti‐viral effects.     

Viral RNA but no evidence of replication can be detected in the peripheral blood mononuclear cells of hepatitis E virus–infected patients

Summary. Hepatitis E virus (HEV) infection is an important cause of acute viral hepatitis in several developing countries but has recently been shown to cause chronic hepatitis in immunosuppressed persons. Other hepatotropic viruses that cause chronic infection have been shown to infect peripheral blood mononuclear cells (PBMCs) and to persist in those cells. We therefore decided to look for evidence of replication of HEV in PBMCs obtained from patients with acute hepatitis E, using strand‐specific assays for positive and negative HEV RNA. Of the 44 patients with acute hepatitis E during an outbreak in India, including 27 with detectable IgM anti‐HEV and 19 with detectable serum HEV RNA, 11 had detectable HEV RNA in their PBMCs. However, of the six PBMC specimens with strong HEV RNA signal, none had detectable negative‐strand HEV RNA, a marker of viral replication. These findings indicate the presence of HEV RNA but the absence of its replication in PBMCs from patients with acute hepatitis E.