Single-cell landscape of immunological responses in patients with COVID-19.

In coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the relationship between disease severity and the host immune response is not fully understood. Here we performed single-cell RNA sequencing in peripheral blood samples of 5 healthy donors and 13 patients with COVID-19, including moderate, severe and convalescent cases. Through determining the transcriptional profiles of immune cells, coupled with assembled T cell receptor and B cell receptor sequences, we analyzed the functional properties of immune cells. Most cell types in patients with COVID-19 showed a strong interferon-α response and an overall acute inflammatory response. Moreover, intensive expansion of highly cytotoxic effector T cell subsets, such as CD4+ effector-GNLY (granulysin), CD8+ effector-GNLY and NKT CD160, was associated with convalescence in moderate patients. In severe patients, the immune landscape featured a deranged interferon response, profound immune exhaustion with skewed T cell receptor repertoire and broad T cell expansion. These findings illustrate the dynamic nature of immune responses during disease progression.

Expected Eight-Week Prenatal Versus Twelve-Week Perinatal Tenofovir Alafenamide Prophylaxis to Prevent Mother-To-Child Transmission of Hepatitis B Virus: A Multicenter, Prospective, Open-label, Randomized Controlled Trial.

INTRODUCTION: The course of maternal antiviral prophylaxis to prevent mother-to-child transmission of hepatitis B virus (HBV-MTCT) varies greatly, and it has not been demonstrated in a randomized controlled study. METHODS: In this multicenter, open-label, randomized controlled trial, eligible pregnant women with HBV DNA of 5.3-9.0 log10 IU/mL who received tenofovir alafenamide fumarate (TAF) from the first day of 33 gestational weeks to delivery (expected eight-week) or to four-week postpartum (expected twelve-week) were randomly enrolled at a 1:1 ratio and followed until six-month postpartum. All infants received standard immunoprophylaxis (hepatitis B immunoglobulin and vaccine). The primary endpoint was the safety of mothers and infants. The secondary endpoint was infants' HBV-MTCT rate at seven months of age. RESULTS: Among 119 and 120 intention-to-treat pregnant women, 115 and 116 women were followed until delivery, and 110 and 112 per-protocol mother-infant dyads in two groups completed the study. Overall, TAF was well tolerated, no one discontinued therapy due to adverse events (0/239, 0%, 95% confidence interval [CI] 0%-1.6%), and no infant had congenital defects or malformations at delivery (0/231, 0%, 95% CI 0%-1.6%). The infants' physical development at birth (n=231) and at seven months (n=222) were normal. Furthermore, 97.0% (224/231, 95% CI 93.9%-98.5%) of women achieved HBV DNA <5.3 log10 IU/mL at delivery. The intention-to-treat and per-protocol infants' HBV-MTCT rates were 7.1% (17/239, 95% CI 4.5%-11.1%) and 0% (0/222, 95% CI 0%-1.7%) at seven months of age. Comparatively, 15.1% (18/119, 95% CI 9.8%-22.7%) versus 18.3% (22/120, 95% CI 12.4%-26.2%) of women in the two groups had mildly elevated alanine aminotransferase levels at three-month and six-month postpartum, respectively (P=0.507); notably, no one experienced alanine aminotransferase flare (0% [0/119, 95% CI 0%-3.1%] versus 0% [0/120, 0%-3.1%]). DISCUSSION: Maternal TAF prophylaxis to prevent HBV-MTCT is generally safe and effective, and expected eight-week prenatal duration is feasible. ClinicalTrials.gov, NCT04850950.

Feasibility and safety of endoscopic resection for the jejunoileal lesions.

BACKGROUND: Endoscopic resection (ER) for jejunoileal lesions (JILs) has been technically challenging. We aimed to characterize the clinicopathologic characteristics, feasibility, and safety of ER for JILs. METHOD: We retrospectively investigated 52 patients with JILs who underwent ER from January 2012 to February 2022. We collected and analyzed clinicopathological characteristics, procedure-related parameters, outcomes, and follow-up data. RESULTS: The mean age was 49.4 years. Of the 52 JILs, 33 ileal tumors within 20 cm from the ileocecal valve were resected with colonoscopy, while 19 tumors in the jejunum or the ileum over 20 cm from the ileocecal valve received enteroscopy resection. The mean procedure duration was 49.0 min. The en bloc resection and en bloc with R0 resection rates were 86.5% and 84.6%, respectively. Adverse events (AEs) included one (1.9%) major AE (delayed bleeding) and five (9.6%) minor AEs. During a median follow-up of 36.5 months, two patients had local recurrence (3.8%), while none had metastases. The 5-year recurrence-free survival (RFS) and disease-specific survival (DSS) were 92.9% and 94.1%, respectively. Compared with the enteroscopy group, overall AEs were significantly lower in the colonoscopy group (P < 0.05), but no statistical differences were observed in RFS (P = 0.412) and DSS (P = 0.579). There were no significant differences in AEs, RFS, and DSS between the endoscopic submucosal dissection (ESD) and the endoscopic mucosal resection (EMR) group. CONCLUSIONS: ER of JILs has favorable short-term and long-term outcomes. Both ESD and EMR can safely and effectively resect JILs in appropriately selected cases.

Single-cell RNA sequencing reveals intrahepatic and peripheral immune characteristics related to disease phases in HBV-infected patients.

OBJECTIVE: A comprehensive immune landscape for HBV infection is pivotal to achieve HBV cure. DESIGN: We performed single-cell RNA sequencing of 2 43 000 cells from 46 paired liver and blood samples of 23 individuals, including six immune tolerant, 5 immune active (IA), 3 acute recovery (AR), 3 chronic resolved and 6 HBV-free healthy controls (HCs). Flow cytometry and histological assays were applied in a second HBV cohort for validation. RESULTS: Both IA and AR were characterised by high levels of intrahepatic exhausted CD8+ T (Tex) cells. In IA, Tex cells were mainly derived from liver-resident GZMK+ effector memory T cells and self-expansion. By contrast, peripheral CX3CR1+ effector T cells and GZMK+ effector memory T cells were the main source of Tex cells in AR. In IA but not AR, significant cell-cell interactions were observed between Tex cells and regulatory CD4+ T cells, as well as between Tex and FCGR3A+ macrophages. Such interactions were potentially mediated through human leukocyte antigen class I molecules together with their receptors CANX and LILRBs, respectively, contributing to the dysfunction of antiviral immune responses. By contrast, CX3CR1+GNLY+ central memory CD8+ T cells were concurrently expanded in both liver and blood of AR, providing a potential surrogate marker for viral resolution. In clinic, intrahepatic Tex cells were positively correlated with serum alanine aminotransferase levels and histological grading scores. CONCLUSION: Our study dissects the coordinated immune responses for different HBV infection phases and provides a rich resource for fully understanding immunopathogenesis and developing effective therapeutic strategies.

Characteristics of blood immune cell profile and their correlation with disease progression in patients infected with HIV-1.

BACKGROUND: Antiretroviral therapy (ART) can reduce viral load in individuals infected with human immunodeficiency virus (HIV); however, some HIV-infected individuals still cannot achieve optimal immune recovery even after ART. Hence, we described the profile of peripheral immune cells and explored the association with disease progression in patients infected with HIV-1. METHODS: Mass cytometry analysis was used to characterize the circulating immune cells of 20 treatment-naïve (TNs), 20 immunological non-responders (INRs), 20 immunological responders (IRs), and 10 healthy controls (HCs). Correlation analysis was conducted between cell subpopulation percentages and indicators including HIV-1 cell-associated (CA)-RNA, DNA, CD4+ T cell count, and CD4/CD8 ratio. RESULTS: Global activation, immunosenescence, and exhaustion phenotypes were observed in myeloid cells and T cells from individuals with HIV-1 infection. We also found that specific subsets or clusters of myeloid, CD4+ T, and CD8+ T cells were significantly lost or increased in TN individuals, which could be partially restored after receiving ART. The percentages of several subpopulations correlated with HIV-1 CA-RNA, DNA, CD4+ T cell count, and CD4/CD8 ratio, suggesting that changes in immune cell composition were associated with therapeutic efficacy. CONCLUSION: These data provide a complete profile of immune cell subpopulations or clusters that are associated with disease progression during chronic HIV-1 infection, which will improve understanding regarding the mechanism of incomplete immune recovery in INRs.

Management of delayed bleeding of upper gastrointestinal endoscopic submucosal tunneling procedures: a retrospective single-center analysis and brief meta-analysis.

OBJECTIVES: Delayed bleeding is a rare but important major adverse event (mAE) after endoscopic submucosal tunneling procedures (ESTP), which is scarcely reported. We aimed to characterize the clinical characteristics of delayed bleeding and provide better management of this mAE. METHOD: From August 2010 to October 2022, we reviewed 3852 patients with achalasia receiving peroral endoscopic myotomy (POEM) and 1937 patients with upper gastrointestinal tumors receiving submucosal tunneling endoscopic resection (STER). Among these, records of 22 patients (15 POEM, 7 STER) with delayed bleeding were collected. Clinical characteristics, treatment, and outcomes of delayed bleeding were analyzed. RESULTS: The mean age was 43.6 years. Ten patients (45.5%) were intratunnel bleeding, seven (31.8%) were intratunnel bleeding accompanied by mucosal bleeding, and five (22.7%) were mucosal bleeding. The most common accompanied symptoms were hematemesis, fever, and melena. The most common accompanied mAEs were fistula, pulmonary inflammation, and pleural effusion with atelectasis. The mean duration from ESTP to endoscopic intervention was 5.3 ± 4.9 days. Active bleeding was identified in 21 patients (95.5%). The bleeding was successfully controlled by electrocoagulation (19 cases), endoscopic clipping (six cases), and Sengstaken-Blakemore tube insertion (three cases), and no patient required surgical intervention. The mean hemostatic procedure duration was 61.8 ± 45.8 min. The mean post-bleeding hospital stay was 10.0 ± 6.2 days. A brief meta-analysis of previous studies showed the pooled estimate delayed bleeding rate after POEM, STER, and G-POEM was 0.4%. CONCLUSIONS: Delayed bleeding is uncommon and could be effectively managed by timely emergency endoscopic procedures without requiring subsequent surgical interventions.

Tenofovir Alafenamide for Pregnant Chinese Women With Active Chronic Hepatitis B: A Multicenter Prospective Study.

BACKGROUND & AIMS: Data on long-term tenofovir alafenamide (TAF) therapy for pregnant women with active chronic hepatitis B (CHB) (immune clearance and reactivation phases, currently and previously diagnosed) and their infants are lacking. METHODS: Pregnant women with active CHB treated with TAF and tenofovir disoproxil fumarate (TDF) were enrolled in this multicenter prospective study, and infants received immunoprophylaxis. The primary outcomes were rates of adverse (safety) events in pregnant women and defects in infants and fetuses. The secondary outcomes were virologic responses in pregnant women, infants' safety, hepatitis B surface antigen (HBsAg) status, and growth conditions. RESULTS: One hundred three and 104 pregnant women were enrolled and 102 and 104 infants were born in the TAF and TDF groups, respectively. In the TAF group, the mean age, gestational age, alanine aminotransferase level, and viral loads at treatment initiation were 29.3 years, 1.3 weeks, 122.2 U/L, and 5.1 log10 IU/mL, respectively. TAF was well-tolerated, and the most common adverse event was nausea (29.1%) during a mean of 2 years of treatment. Notably, 1 (1.0%) TAF-treated pregnant woman underwent induced abortion due to noncausal fetal cleft lip and palate. No infants in either group had birth defects. In the TAF group, the hepatitis B e antigen seroconversion rate was 20.7% at postpartum month 6, infants had normal growth parameters, and no infants were positive for HBsAg at 7 months. The TDF group had comparable safety and effectiveness profiles. CONCLUSIONS: TAF administered throughout or beginning in early pregnancy is generally safe and effective for pregnant women with active CHB and their infants.

Compromised long-lived memory CD8+ T cells are associated with reduced IL-7 responsiveness in HIV-infected immunological nonresponders.

Immune deficiency is one of the hallmarks of HIV infection and a major cause of adverse outcomes in people living with HIV (PLWH). Long-lived memory CD8+ T cells (LLMCs) are essential executors of long-term protective immunity; however, the generation and maintenance of LLMCs during chronic HIV infection are not well understood. In the present study, we analyzed circulating LLMCs in healthy controls (HCs) and PLWH with different disease statuses, including treatment naïve patients (TNs), complete responders (CRs), and immunological nonresponders (INRs). We found that both TNs and INRs showed severely compromised LLMCs compared with HCs and CRs, respectively. The decrease of LLMCs in TNs correlated positively with the reduction of their precursors, namely memory precursor effector T cells (MPECs), which might be associated with elevated pro-inflammatory cytokines. Strikingly, INRs showed an accumulation of MPECs, which exhibited diminished responsiveness to interleukin 7 (IL-7), thereby indicating abrogated differentiation into LLMCs. Moreover, in vitro studies showed that treatment with dexamethasone could improve the IL7-phosphorylated (p)-signal transducer and activator of transcription (STAT5) response by upregulating the expression of the interleukin 7 receptor (IL-7Rα) on MPECs in INRs. These findings provide insights that will encourage the development of novel therapeutics to improve immune function in PLWH.

Immunometabolism at the service of traditional Chinese medicine.

To enhance therapeutic efficacy and reduce adverse effects, ancient practitioners of traditional Chinese medicine (TCM) prescribe combinations of plant species/animal species and minerals designated "TCM formulae" developed based on TCM theory and clinical experience. TCM formulae have been shown to exert curative effects on complex diseases via immune regulation but the underlying mechanisms remain unknown at present. Considerable progress in the field of immunometabolism, referring to alterations in the intracellular metabolism of immune cells that regulate their function, has been made over the past decade. The core context of immunometabolism is regulation of the allocation of metabolic resources supporting host defense and survival, which provides a critical additional dimension and emerging insights into how the immune system and metabolism influence each other during disease progression. This review summarizes research findings on the significant association between the immune function and metabolic remodeling in health and disease as well as the therapeutic modulatory effects of TCM formulae on immunometabolism. Progressive elucidation of the immunometabolic mechanisms involved during the course of TCM treatment continues to aid in the identification of novel potential targets against pathogenicity. In this report, we have provided a comprehensive overview of the benefits of TCM based on regulation of immunometabolism that are potentially applicable for the treatment of modern diseases.

Randomised controlled trial: effect of metformin add-on therapy on functional cure in entecavir-treated patients with chronic hepatitis B.

INTRODUCTION AND OBJECTIVES: Hepatitis B surface antigen (HBsAg) clearance, indicating functional cure or resolved chronic hepatitis B (CHB), remains difficult to achieve via nucleos(t)ide analogue monotherapy. We investigated whether metformin add-on therapy could help achieve this goal in entecavir-treated patients with hepatitis B e antigen (HBeAg)-negative CHB. PATIENTS AND METHODS: Patients with HBeAg-negative CHB who met eligibility criteria (entecavir treatment for > 12 months, HBsAg < 1000 IU/mL) were randomly assigned (1:1) to receive 24 weeks of either metformin (1000 mg, oral, once a day) or placebo (oral, once a day) add-on therapy. The group allocation was blinded for both patients and investigators. Efficacy and safety analyses were based on the intention-to-treat set. The primary outcome, serum HBsAg level (IU/mL) at weeks 24 and 36, was analysed using mixed models. RESULTS: Sixty eligible patients were randomly assigned to the metformin (n = 29) and placebo (n = 31) groups. There was no substantial between-group difference in the HBsAg level at week 24 (adjusted mean difference 0.05, 95% confidence interval -0.04 to 0.13, p = 0.278) or week 36 (0.06, -0.03 to 0.15, p = 0.187), and no significant effect of group-by-time interaction on the HBsAg level throughout the trial (p = 0.814). The occurrence of total adverse events between the two groups was comparable (9 [31.0%] of 29 vs. 5 [16.1%] of 31, p = 0.227) and no patient experienced serious adverse events during the study. CONCLUSION: Although it was safe, metformin add-on therapy did not accelerate HBsAg clearance in entecavir-treated patients with HBeAg-negative CHB.

Dynamics of HIV reservoir decay and naïve CD4 T-cell recovery between immune non-responders and complete responders on long-term antiretroviral treatment.

BACKGROUND: The dynamics of viral reservoir decay and naïve CD4 T-cell recovery between immunological non-responders (INR) and complete responders (CR) during long-term antiretroviral treatment (ART) are not fully known. METHODS: Twenty-eight chronic HIV-infected individuals on 5-year ART were divided into two groups: INR (CD4 counts ≤350 cells/µL, n = 13) and CR (CD4 counts ≥500 cells/µL, n = 15). The levels of HIV DNA and cell-associated HIV RNA (CA-RNA), CD4 counts, naïve CD4 counts and their correlations were analyzed at baseline, years 1, 3 and 5 of ART between the two groups. Expression of PD-1 on CD4 T-cells was quantified by flow cytometry. Linear mixed effect models were used to estimate the change procession in repeated measurements over 5 years. Slopes of the above-mentioned indicators were estimated using participant-specific linear regressions, respectively. RESULTS: INR maintained higher levels of HIV DNA and CA-RNA with higher percentages of PD-1+CD4 T-cells compared with CR during 5-year ART, concurrent with lower naïve CD4 T-cells. However, the rates of HIV DNA and CA-RNA decay in INR were not different from that in CR over time, and INR had higher rates of naïve CD4 T-cell percentage recovery. The baseline levels of HIV DNA were positively associated with the 5-year levels of HIV DNA, but negatively associated with the 5-year naïve CD4 counts. CONCLUSIONS: INR maintained significantly higher viral reservoir and lower naïve CD4 T-cells compared with CR during 5-year ART, however, the rates of reservoir decay and naïve CD4 T-cell percentage growth within INR were not lower than that in CR over time.

Transforming growth factor-ß promotes the function of HIV-specific CXCR5+ CD8 T cells.

HIV replication can be inhibited by CXCR5+ CD8 T cells (follicular cytotoxic T cell [TFC]) which transfer into B-cell follicles where latent HIV infection persists. However, how cytokines affect TFC remain unclear. Understanding which cytokines show the ability to affect TFC could be a key strategy toward curing HIV. Similar mechanisms could be used for the growth and transfer of TFCs and follicular helper T (TFH) cells; as a result, we hypothesized that cytokines IL-6, IL-21, and transforming growth factor-ß (TGF-ß), which are necessary for the differentiation of TFH cells, could also dictate the development of TFCs. In this work, lymph node mononuclear cells and peripheral blood mononuclear cells from HIV-infected individuals were cocultured with IL-6, IL-21, and TGF-ß. We then carried out T-cell receptor (TCR) repertoire analysis to compare the differences between CXCR5- and CXCR5+ CD8 T cells. Our results showed that the percentage and function of TFC can be enhanced by stimulation with TGF-ß. Besides, TGF-ß stimulation enhanced the diversity of TCR and complementarity-determining region 3 sequences. HIV DNA showed a negative correlation with TFC. The use of TGF-ß to promote the expression of CXCR5+ CD8 T cells could become a new treatment approach for curing HIV.

Granulocytic myeloid-derived suppressor cell population increases with the severity of alcoholic liver disease.

Alcoholic liver disease (ALD) is a progressive liver disease that can cause a series of complications, including cirrhosis, liver failure and hepatocellular carcinoma. Granulocytic myeloid-derived suppressor cell (gMDSC) populations have been observed to expand in various liver diseases and to inhibit innate and adaptive immunity in patients with liver disease. However, the characteristics of gMDSCs in patients with ALD have not been studied. We studied 24 healthy controls (HCs) and 107 patients with ALD and found an accumulation of gMDSCs in the peripheral blood of patients with alcoholic liver cirrhosis (ALC). Furthermore, ALC patients with a poor prognosis displayed a significant increase in peripheral gMDSCs and showed an increased capacity for arginase I production compared to HCs. In contrast, plasma arginase I levels in ALC patients were negatively correlated with total bilirubin and international normalized ratio, two key parameters of liver damage. Importantly, gMDSCs accumulated in the livers of ALC patients, and the frequency of liver gMDSCs significantly correlated with that of peripheral gMDSCs. In addition, gMDSC enrichment in vitro significantly inhibited the function of natural killer (NK) cells, perhaps preventing the NK-induced apoptosis of hepatic stellate cells. In summary, increased peripheral and intrahepatic gMDSC populations are present in patients with ALC and may contribute to enhancing the severity of liver cirrhosis.

Plasma levels of soluble ST2, but not IL-33, correlate with the severity of alcoholic liver disease.

Alcoholic liver disease (ALD) is a complication that is a burden on global health and economy. Interleukin-33 (IL-33) is a newly identified member of the IL-1 cytokine family and is released as an "alarmin" during inflammation. Soluble suppression of tumourigenicity 2 (sST2), an IL-33 decoy receptor, has been reported as a new biomarker for the severity of systemic and highly inflammatory diseases. Here, we found the levels of plasma sST2, increased with the disease severity from mild to severe ALD. Importantly, the plasma sST2 levels in ALD patients not only correlated with scores for prognostic models (Maddrey's discriminant function, model for end-stage liver disease and Child-Pugh scores) and indexes for liver function (total bilirubin, international normalized ratio, albumin, and cholinesterase) but also correlated with neutrophil-associated factors as well as some proinflammatory cytokines. In vitro, lipopolysaccharide-activated monocytes down-regulated transmembrane ST2 receptor but up-regulated sST2 mRNA and protein expression and produced higher levels of tumour necrosis factor-α (TNF-α). By contrast, monocytes pretreated with recombinant sST2 showed decreased TNF-α production. In addition, although plasma IL-33 levels were comparable between healthy controls and ALD patients, we found the IL-33 expression in liver tissues from ALD patients was down-regulated at both RNA and protein levels. Immunohistochemical staining further showed that the decreased of IL-33-positive cells were mainly located in liver lobule area. These results suggested that sST2, but not IL-33, is closely related to the severity of ALD. Consequently, sST2 could be used as a potential biomarker for predicting the prognosis of ALD.

Activated γδ T cells exhibit cytotoxicity and the capacity for viral clearance in patients with acute hepatitis B.

γδ T cells are a unique population of lymphocytes that have regulatory roles in patients with chronic hepatitis B (CHB); however, their role in acute hepatitis B (AHB) infection remains unclear. Phenotype and function of γδ T cells were analyzed in 29 AHB patients, 28 CHB patients, and 30 healthy controls (HCs) using immunofunctional assays. Compared with HCs and CHB patients, decreased peripheral and increased hepatic γδ T cells were found in AHB patients. Increased hepatic γδ T cells in AHB patients were attributed to elevated hepatic chemokine levels. Peripheral γδ T cells exhibited highly activated and terminally differentiated memory phenotype in AHB patients. Consistently, peripheral γδ T cells in AHB patients showed increased cytotoxic capacity and enhanced antiviral activity which was further proved in longitudinal study. Activated γδ T cells in AHB patients exhibited increased cytotoxicity and capacity for viral clearance associated with liver injury and the control of infection.

[Quantitative determination of febrifugine by proton nuclear magnetic resonance spectroscopy with internal standard method].

A quantitative nuclear magnetic resonance method(qNMR) was established for determination of the absolute content of febrifugine. Proton nuclear magnetic resonance spectroscopy of febrifugine was obtained in DMSO-d6 with hydroquinone as the internal standard substance on a Bruker Ascend 600 MHz superconducting nuclear resonance spectrometer at 298 K. The specific parameters were as follows: the observing frequency was 600 MHz,spectra width was 7 211 Hz, pulse width was 9.70 µs, pulse sequence was zg30,scan times was 32 and relaxation time was 2 s. The proton signal peaked at δ 7.71 for febrifugine and δ 6.55 for hydroquinone were selected as the quantification peaks. Linear regression of quantitative peak area ratio of febrifugine-hydroquinone versus their mass ratio yielded a correlation coefficient of 0.999 6 and a regression equation of Y=0.083 3X+0.008 6．The linear range of febrifugine was 2.17-17.07 g·L⁻¹,the precision RSD was 0.78%(n=6),the repeatability RSD was 1.2%(n=6),and the contents of three batches of febrifugine sample were 94.91%,95.09% and 95.52%,respectively. The content of febrifugine was 96.44% determined by high performance liquid chromatography(HPLC). The relative error of the content of febrigugine determinted by qNMR and HPLC methods was 1.27%. The results showed that the internal standard method of proton nuclear magnetic resonance spectroscopy could be used to determine the absolute content of febrifugine.

Generic ledipasvir-sofosbuvir for patients with chronic hepatitis C: A real-life observational study.

BACKGROUND & AIMS: Few patients from developing countries can afford brand name direct-acting antiviral agents for treating hepatitis C virus (HCV) infection, and controversy regarding the bioequivalence of generics exists. This study aimed to observe the safety and efficacy of 8 or 12weeks of generic ledipasvir-sofosbuvir with or without ribavirin for Chinese genotype 1b HCV-infected patients. METHODS: In this open-labelled observational study, 63 cirrhotic (group 1) and 65 non-cirrhotic (group 2) patients were administered generic ledipasvir-sofosbuvir plus 1000-1200mg of ribavirin daily for 12 and 8weeks, respectively; and 64 non-cirrhotic patients (group 3) received ledipasvir-sofosbuvir for 8weeks. The primary efficacy endpoint was undetectable HCV RNA at week 12 (SVR12) after cessation of therapy. Safety and pharmacokinetic data were collected. RESULTS: One hundred and eighty-seven patients completed treatment, and the latest undetectable HCV RNA was observed in three patients with cirrhosis at week 5 during treatment. Intention-to-treat analysis revealed 96.8% (61/63), 96.9% (63/65), and 96.9% (62/64) of SVR12 rates in groups 1, 2, and 3, respectively. One patient in group 3 relapsed at post-treatment week 4. The regimens were generally well-tolerated. The most common adverse events were fatigue (17.8%), diarrhea (10.9%), and headache (9.9%). Four patients discontinued therapy due to diarrhea and vomiting. One patient from group 2 discontinued treatment on day 29 because of drug-unaffordability; fortunately, she achieved SVR12. CONCLUSION: This study demonstrated that 8 or 12weeks of generic ledipasvir-sofosbuvir with or without ribavirin are safe and effective for patients with genotype 1b HCV infection. LAY SUMMARY: The price of Harvoni® has led to restrictions and access limitations in many developing and even developed countries with limited healthcare budgets. Gilead approved generic ledipasvir-sofosbuvir costs far less than Harvoni® and presents a similar cure rate for patients with chronic hepatitis C.

[Investigation on stability and degradation kinetics of febrifugine].

To investigate the stability and degradation kinetics of febrifugine. The results showed that within 24 hours, febrifugine content was decreased by only 1% in mobile phase solvent, but its content was decreased to be 90% of the initial content in the water, methanol, 50% methanol and 10% acetonitrile solution. When the pH value of the solution was between 3 and 7, the retention rate of febrifugine in 24 hours was over 98%, but its content was decreased by about 12% in alkaline solution (pH 9.0). The higher the temperature, the worse the stability of febrifugine. At 40-80 ℃, the content of febrifugine was decreased to be 60% of its initial content in 10 hours, but the content was decreased by only 5% in 10 h at 20 ℃.However, no matter 40 ℃or 60 ℃, febrifugine was mainly transformed into isofebrifugine, and the total content of febrifugine and isofebrifugine was equal to their initial total content in 10 hours, while incase of 80 ℃, the total content was decreased to be 83.33% in 10 h, which suggested that the structure of febrifugine was absolutely changed, not just isomerized to be isofebrigugine at high temperature. Light had a significant impact on the stability of febrifugine. Under bright light, the content of febrifugine was reduced by about 23% in 108 h, but it only decreased by about 10% in the natural light or darkness. In artificial gastric fluid (pH 1.4) and artificial intestinal fluid (pH 6.8), the content of febrifugine was decreased by less than 5% in 10 h. After storage at high temperature(60 ℃), high humidity [(75±1)%] and strong light (3 000 lx) conditions for 10 d, the content of solid febrifugine was decreased by 0.27%, 7.6% and 5.39%, respectively. The degradation of febrifugine basically complied with the first-order reaction kinetic process in the following conditions: in water, methanol, 50%methanol and 10% acetonitrile solvents, alkaline solution (pH>7), different light intensity and different temperatures (20,40 ℃). Therefore, no matter the isolation and purification of febrifugine or the production of the related preparations, it should be done fast in the acidic solution, low temperature and dark conditions, while the febrifugine solid should be kept in dry and dark conditions.

[Simultaneous determination of febrifugine and isofebrifugine in Dichroa febrifuga root by HPLC method].

To develop the HPLC method for simultaneous determination of febrifugine and isofebrifugine in Dichroa febrifuga root, and on the basis of this, the feasibility of quantitative analysis of multi-component by a single-marker (QAMS) model for the determination of the two alkaloids was investigated. The chromatographic separation was performed on an octadecyl bonded silica gel column with mixed solvent consisting of acetonitrile-water-glacial acetic acid-triethylamine (9∶91∶0.36∶0.745) as mobile phase at a flow rate of 1.0 mL•min⁻¹. The detection wavelength was set at 225 nm, and the column temperature was set at 30 ℃. The linear range of febrifugine and isofebrifugine were 10.7-426 ng and 10.6-424 ng, respectively. Their average recovery were 98.33% (RSD 2.7%) and 100.4% (RSD 1.8%), respectively. On the basis of this established method, febrifugine was used as the internal reference substance to calculate the relative correction factors (RCF) and the relative retention values (RRV) of isofebrifugine to febrifugine. Through a series of methodology evaluations, the two alkaloids were simultaneously assayed only by quantitative determination of febrifugine. This result played the part of demonstration role for the application of QAMS model in the determination of isomers.

CXCR5+ CD4+ T follicular helper cells participate in the pathogenesis of primary biliary cirrhosis.

UNLABELLED: There is increasing interest in the role of T follicular helper (Tfh) cells in autoimmunity from the perspective of both their role in breach of tolerance and their effects on the natural history of disease progression. Indeed, the critical role of Tfh cells in autoimmunity is further highlighted based on their location in the germinal center (GC), a pathogenic hot spot for development of autoreactivity. To address the role of Tfh cells in primary biliary cirrhosis (PBC), we comprehensively evaluated the immunobiology of CXCR5(+) CD4(+) Tfh cells in 69 patients with PBC, including a nested subgroup of 16 autoimmune hepatitis (AIH) and 20 healthy controls (HC), followed for 1 year. We report herein several key observations. First, there was an increased frequency of circulating Tfh cells in patients with PBC compared to AIH (P < 0.05) and HC (P < 0.01). Second, the function of circulating Tfh cells from PBC patients, including interleukin (IL)-21 production (P < 0.05), the ability to promote B-cell maturation, and autoantibody production, were greater than HC. Third, the frequency of these cells was significantly decreased in ursodeoxycholic acid (UDCA) responders compared to UDCA-treated nonresponders, in both cross-sectional (P = 0.023) and longitudinal studies (P = 0.036), respectively. Indeed, similar increases of Tfh cells were noted in liver and spleen. CONCLUSION: These results significantly extend our understanding of lymphoid subpopulations in PBC and their relative role in disease expression. Our data also provide a novel biomarker for evaluation of the effectiveness of new therapeutic approaches.