Prenatal inflammation exposure-programmed hypertension exhibits multi-generational inheritance via disrupting DNA methylome

The multi-generation heredity trait of hypertension in human has been reported, but the molecular mechanisms underlying multi-generational inheritance of hypertension remain obscure. Recent evidence shows that prenatal inflammatory exposure (PIE) results in increased incidence of cardiovascular diseases, including hypertension. In this study we investigated whether and how PIE contributed to multi-generational inheritance of hypertension in rats. PIE was induced in pregnant rats by intraperitoneal injection of LPS or Poly (I:C) either once on gestational day 10.5 (transient stimulation, T) or three times on gestational day 8.5, 10.5, and 12.5 (persistent stimulation, P). Male offspring was chosen to study the paternal inheritance. We showed that PIE, irrespectively induced by LPS or Poly (I:C) stimulation during pregnancy, resulted in multi-generational inheritance of significantly increased blood pressure in rat descendants, and that prenatal LPS exposure led to vascular remodeling and vasoconstrictor dysfunction in both thoracic aorta and superior mesenteric artery of adult F2 offspring. Furthermore, we revealed that PIE resulted in global alteration of DNA methylome in thoracic aorta of F2 offspring. Specifically, PIE led to the DNA hypomethylation of G beta gamma (Gβγ) signaling genes in both the F1 sperm and the F2 thoracic aorta, and activation of PI3K/Akt signaling was implicated in the pathologic changes and dysregulated vascular tone of aortic tissue in F2 LPS-P offspring. Our data demonstrate that PIE reprogrammed DNA methylome of cells from the germline/mature gametes contributes to the development of hypertension in F2 PIE offspring. This study broadens the current knowledge regarding the multi-generation effect of the cumulative early life environmental factors on the development of hypertension.     

The alterations of degree centrality in the frontal lobe of patients with panic disorder

Objective: The brain network in panic disorder (PD) is still an intriguing issue for research. In this study, we hoped to investigate the role of DC (degree centrality) for the pathophysiology of PD, especially for the fear network.Methods: We enrolled 60 patients with PD and 60 controls in the current study. The gender and age were matched for two groups. All participants received the resting-state functional magnetic resonance imaging to survey the baseline brain activity. Then the DC values of all participants were using REST toolbox. We also compared the DC values between PD and controls. The statistical threshold was set as FDR (false discovery rate) < 0.05.Results: The DC values were significantly lower in the right superior frontal gyrus of PD patients compared to controls (FDR < 0.05). In addition, a negative correlation between the DC values and panic severity was observed in the right superior frontal gyrus and left inferior frontal gyrus. However, there was no significant association between the DC values and illness duration.Conclusion: The DC seemed significantly altered in the frontal lobe of PD patients. The role of the frontal lobe might be more emphasized in the pathophysiology research for PD.     

三七茎基与须根含量测定方法的研究

目的:建立三七茎基与须根中皂苷的含量测定方法,测定不同药龄和不同月份生育期的广西三七茎基与须根中皂苷的含量.方法:采用RP-HPLC法,Shim pack CLC-ODS柱分离,流动相:乙腈与水梯度洗脱,检测波长203 nm,柱温:室温,流速1.0 ml/min.结果:广西三七茎基与须根中人参皂苷Rg1、Re、Rb1和三七皂苷R1的含量随药龄及月份的变化而变化;所含的Rg1和Rb1的总量均符合<中国药典>2000年版中三七的质量要求.结论:所建立的高效液相色谱法灵敏度高,专属性强,重现性好,能准确、快速的进行含量测定;广西三七茎基和须根中Rg1、Re、Rb1和R1的含量普遍较高,完全可与三七主根一起入药.     

利多卡因治疗脑卒中后顽固性呃逆疗效观察

目的观察利多卡因静脉滴注治疗脑卒中所致顽固性呃逆的疗效。方法选取脑卒中合并顽固性呃逆患者53例,男29例,女24例。随机分为常规治疗组26例和利多卡因治疗组27例。利多卡因治疗组在治疗原发病的同时,采用利多卡因静脉滴注,治疗中观察用药前后呃逆症状轻重变化,发生频率及持续时间。结果利多卡因治疗组27例中,治愈15例,好转10例,总有效率为92.6%;而常规治疗组总有效率为63.5%,治疗组疗效明显高于对照组(P<0.01)。结论利多卡因静滴治疗顽固性呃逆,具有操作简单,用药量小,起效快,疗效高,无明显不良反应及价格低廉等优点,是治疗呃逆的有效方法之一。     

LS‐106, a novel EGFR inhibitor targeting C797S,exhibits antitumor activities both in vitro and in vivo

With the wide clinical use of the third‐generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR‐mutated non–small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth‐generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS‐106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell‐free assay, LS‐106 potently inhibited the kinase activities of EGFR^{19del/T790M/C797S} and EGFR^{L858R/T790M/C797S} with IC₅₀ values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS‐106 exhibited comparable kinase inhibitory effect to osimertinib on EGFR^L858R/T790M and wild‐type EGFR. Results from cellular experiments demonstrated that LS‐106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR^{19del/T790M/C797S} or EGFR^{L858R/T790M/C797S}, and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR^{19del/T790M/C797S} (named PC‐9‐OR cells) using the CRISPR/Cas9 system and found that LS‐106 markedly suppressed the activation of EGFR^{19del/T790M/C797S} and the proliferation of PC‐9‐OR cells. Moreover, cells harboring EGFR^{19del/T790M/C797S} underwent remarkable apoptosis upon LS‐106 treatment. In vivo experiments further demonstrated that oral administration of LS‐106 caused significant tumor regression in a PC‐9‐OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS‐106 as a novel fourth‐generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S–triple‐mutant tumor models.     

The spatial distribution of immune cell subpopulations in hepatocellular carcinoma

Infiltrating immune cells in the tumor microenvironment (TME) influence tumor progression and patient prognosis, making them attractive therapeutic targets for immunotherapy research. A deeper understanding of immune cell distributions in the TME in hepatocellular carcinoma (HCC) is needed to identify interactions among different immune cell types that might impact the effectiveness of potential immunotherapies. We performed multiplex immunohistochemistry using a tissue microarray of samples from 302 patients with HCC to elucidate the spatial distributions of immune cell subpopulations (CD3⁺, CD4⁺, CD8⁺, CD66b⁺, and CD68⁺) in HCC and normal liver tissues. We analyzed the associations between different immune subpopulations using Pearson''s correlation. G(r) functions, K(r) functions and Euclidean distance were applied to characterize the bivariate distribution patterns among the immune cell types. Cox regression and Kaplan‐Meier analysis were used to evaluate the associations between tumor infiltration by different immune cells and patient outcomes after curative surgery. We also analyzed the relationship between the spatial distribution of different immune cell subpopulations with HCC patient prognosis. We found that the immune cell spatial distribution in the HCC TME is heterogeneous. Our study provides a theoretical basis for HCC immunotherapy.     

Complex decay dynamics of HIV virions,intact and defective proviruses,and 2LTR circles following initiation of antiretroviral therapy

In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t_1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4⁺ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t_1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After ∼3 mo, the decay slope changes, and CD4⁺ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5′ or 3′ end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.

For persons living with HIV-1 (PLWH), lifelong adherence to antiretroviral therapy (ART) is critical for maintaining suppression of viral replication and forestalling the development of fatal immunodeficiency. Following initiation of ART, plasma virus levels decay rapidly to below the limit of detection of clinical assays (1–6). Because antiretroviral drugs block new infection of susceptible cells, but not virus production by cells that have an integrated viral genome, this decay must reflect the loss of productively infected cells, cells that were infected prior to the initiation of ART. Productively infected cells could die from viral cytopathic effects, cytolytic host effector mechanisms, or virus-independent T cell turnover. In principle, the decay of plasma virus could also be explained by transition to a nonproductive or latent state of infection. Importantly, the decay is biphasic, indicating the presence of two populations of productively infected cells with different half-lives. Most of the plasma virus is produced by cells that decay very rapidly, with a half-life of less than 1 d. Perelson et al. (4) showed that after most of these cells have decayed, the slope changes, reflecting the slower elimination of a second population of productively infected cells. This population decays with a variable half-life (mean ∼ 2 wk). Although this biphasic decay is a consistent feature of the response to ART, there is still uncertainty about the nature, anatomic location, and fate of the cells responsible for virus production during the first and second phases of decay (referred to here as first- and second-phase cells, respectively). The differences between these two populations have never been elucidated.The first and second phases of decay bring viremia down to below the limit of detection of clinical assays (typically 20 to 50 copies of HIV-1 RNA per mL of plasma) within months of ART initiation, initially raising hope for eradication. However, a latent form of the virus persists in resting memory CD4⁺ T cells (7–14). Initial studies used a quantitative viral outgrowth assay (QVOA) to demonstrate that latently infected resting CD4⁺ T cells purified from PLWH on long-term suppressive ART could be induced to produce replication-competent virus by global T cell activation (8, 9). Longitudinal studies using the QVOA demonstrated that the half-life of the latent reservoir in resting CD4⁺ T cells is 44 mo in PLWH who are adherent to ART. This half-life is long enough to guarantee lifetime persistence of HIV-1 despite ART (12–14). Strategies targeting the latent reservoir in resting CD4⁺ T cells are a major focus of HIV cure research (15–17). In addition to resting CD4⁺ T cells, other cell types may contribute to HIV-1 persistence (18–20).Prior to and immediately following initiation of ART, the frequency of latently infected cells detected by QVOA is substantially higher than frequencies observed in PLWH on long-term ART (21). In principle, several different types of decay processes occurring over the first 6 to 12 mo of treatment could reduce the frequency of latently infected cells to the more stable frequencies observed in PLWH on long-term ART. Early studies by Jerome Zack and Mario Stevenson demonstrated that infected resting CD4⁺ T cells could harbor linear, unintegrated HIV-1 DNA in a state of preintegration latency (22, 23). Following cellular activation, linear unintegrated HIV-1 DNA can be integrated and transcribed, allowing production of virus (22, 23). The half-life of linear, unintegrated forms of the viral genome is not clear, with some studies suggesting that these forms are labile (22, 24–26). Some reverse-transcribed viral genomes can undergo homology-dependent or end-to-end ligation, generating one-long-terminal repeat or two-long-terminal repeat (2LTR) circles, respectively (reviewed in ref. 27). The stability of these forms is also controversial, but they are clearly replication-defective (27–31). Following integration of linear viral cDNA, decay dynamics depend on dynamics of the infected host cells, which can be eliminated by viral cytopathic effects, immune cytolytic effector mechanisms, and normal contraction-phase death of previously activated CD4⁺ T cells (32, 33).While the QVOA provides a definitive minimal estimate of the frequency of latently infected cells, it underestimates reservoir size because not all proviruses in resting CD4⁺ T cells are induced upon one round of maximum T cell activation (34–36). Many replication-competent proviruses require multiple rounds of stimulation for induction. As an alternative to the QVOA, many studies use PCR-based assays to measure proviral DNA. However, the vast majority of HIV-1 proviruses are defective due to apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC)-mediated hypermutation or large internal deletions (34, 37–39). PCR-based assays do not distinguish between defective and intact proviruses (40, 41). Although infected cell dynamics have been explored using PCR-based assays (42), the results likely reflect the dynamics of defective proviruses (41). The recently developed intact proviral DNA assay (IPDA) uses two carefully chosen amplicons to probe informative regions of individual proviruses to provide better discrimination between intact and defective proviruses (41, 43). This assay has proven useful in evaluating the long-term dynamics of cells with intact and defective proviruses, demonstrating differences in decay rates that may reflect some vulnerability of cells with intact proviruses to immune effector mechanisms (41, 44, 45).In this study, we use the IPDA to explore the decay of intact and defective proviruses at early time points following initiation of ART. We identify decay processes occurring over intermediate time scales, but with pronounced differences between intact and defective proviruses. Of particular importance is the second-phase decay because infected cells that survive second-phase decay may down-regulate HIV-1 gene expression and enter the stable latent reservoir. Our findings also provide insight into mechanisms for the elimination of the cells with intact viral genomes and into the proper use of assays for the latent reservoir.     

Common occurrence of an antiidiotypic antibody that recognizes T14+ anti-DNA antibodies in patients with systemic lupus erythematosus

Objective. To investigate whether antibodies to a T14 anti-DNA antibody can be found in patients with systemic lupus erythematosus (SLE). Methods. Seventy-six serum samples (37 from patients with SLE) were randomly selected from among sera submitted for routine antinuclear antibody testing. Short, overlapping peptides based on the partial V_H (variable region of the heavy chain) sequence of the T14 antibody were synthesized on multipins and screened for reactivity with SLE sera. In addition, selected peptides from T14 and related proteins were synthesized in bulk and screened for reactivity with both SLE and control sera. A monoclonal antibody was generated to determine the prevalence of the T14 idiotype (T14+ Id) in the different study populations. Results. Antibodies were detected by a peptide based on the third complementarity-determining region (CDR3) of the T14 protein in 15 (41%) of 37 patients with SLE or 15 (54%) of 28 who had anti-DNA antibodies, in 3 (9%) of 34 patients without anti-DNA antibodies (9 of whom had SLE), and in 6 (10%) of 57 healthy controls. In SLE sera, the antiidiotypic (anti-Id) responses (IgM and IgG) correlated well with the anti-DNA responses (IgG), and both responses correlated well with the T14+ Id activity in SLE sera. Control peptides based on the 18/2 (16/6+ Id) and S107 proteins detected low antibody activities in SLE sera, attributable to cross-reactivity with the T14 peptide. A peptide based on an unrelated human antibody was not reactive with these sera. Conclusion. Anti-Id antibodies directed to T14 V_HCDR3 were found commonly in the sera of patients with SLE, and they appeared to be induced by the anti-DNA antibodies present in the sera. Based on these findings, these secondary antibodies may be pathogenic in SLE.