Fast on-rates allow short dwell time ligands to activate T cells

Two contrasting theories have emerged that attempt to describe T-cell ligand potency, one based on the t_1/2 of the interaction and the other based on the equilibrium affinity (K_D). Here, we have identified and studied an extensive set of T-cell receptor (TCR)-peptide-MHC (pMHC) interactions for CD4⁺ cells that have differential K_Ds and kinetics of binding. Our data indicate that ligands with a short t_1/2 can be highly stimulatory if they have fast on-rates. Simple models suggest these fast kinetic ligands are stimulatory because the pMHCs bind and rebind the same TCR several times. Rebinding occurs when the TCR-pMHC on-rate outcompetes TCR-pMHC diffusion within the cell membrane, creating an aggregate t_1/2 (t_a) that can be significantly longer than a single TCR-pMHC encounter. Accounting for t_a, ligand potency is K_D-based when ligands have fast on-rates (k_on) and t_1/2-dependent when they have slow k_on. Thus, TCR-pMHC k_on allow high-affinity short t_1/2 ligands to follow a kinetic proofreading model.     

Antigen-specific T lymphocyte proliferation decreases over time in advanced chronic hepatitis C

To evaluate T cell immunity in advanced liver disease, antigen-specific lymphoproliferative (LP) responses were prospectively studied in the context of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial. Peripheral blood responses to hepatitis C virus (HCV), tetanus and Candida protein antigens were measured at baseline, month 12 (M12), M24, M36 and M48 in 186 patients randomized to either low-dose peginterferon-alfa-2a (PEG-IFN) only or observation. Liver histology was evaluated at baseline, M24 and M48. Patients with cirrhosis (Ishak 5-6) were less likely to have positive LP responses to HCV at baseline than patients with fibrosis (15%vs 29%, P = 0.03) and had lower levels of HCV c100 responses at baseline, M24 and M48 (P = 0.11, P = 0.05, P = 0.02, respectively). For 97 patients with complete longitudinal data, the frequency of positive LP responses to HCV, tetanus and Candida antigens declined over time (P < 0.003), and the slope of this decline was greater in the PEG-IFN treatment group than the observation group (P < 0.02). Lower levels of tetanus LP responses were associated with fibrosis progression and clinical outcomes (P = 0.009). Poorer CD4+ T cell proliferative function was associated with more advanced liver disease in chronic hepatitis C and may be further affected by long-term PEG-IFN treatment.     

Quantitative assessment of MRI T2 relaxation time of thigh muscles in juvenile dermatomyositis

Objective. The aim of the study was to examine the validityand reliability of a quantifiable measure of inflammation usingmagnetic resonance imaging (MRI) in children with juvenile dermatomyositis(JDM). Methods. Children with active JDM, inactive JDM and healthychildren received detailed assessments of recognized measuresof muscle inflammation including muscle strength (manual muscletesting and myometry) and function (Childhood Myositis AssessmentScale, Childhood Health Assessment Questionnaire), the muscleenzymes lactate dehydrogenase (LDH) and creatine kinase (CK)and T₂-weighted MRI scans of the thigh muscles, and these valueswere correlated with each other. Results. Ten children with active JDM, 10 with inactive JDMand 20 healthy children completed the study. There was no significantdifference in ages between the three groups. The MRI T₂ relaxationtimes were significantly increased in active JDM compared withinactive JDM and healthy children (P = 0.05), indicating a detectableincrease in inflammation within the muscles. There were alsogood correlations between the MRI scores and the measures ofmuscle strength and function; however, there was no correlationbetween the MRI and muscle enzymes. Conclusions. The MRI T₂ relaxation time can be used as a quantitativemeasure of muscle inflammation and it has good correlationswith other measures of disease activity. KEY WORDS: Magnetic resonance imaging, Juvenile dermatomyositis, Childhood Myositis Assessment Scale, Muscle inflammation.     

急性心肌梗死后患者不同时间的微伏级T波电交替

目的观察急性心肌梗死(简称心梗)后不同时间的微伏级T波电交替(MTWA)。方法用带有TWA移动平均修正技术(TWA MMA)的动态心电图检测系统,以时域法检测45例急性心梗后患者小于1个月、6个月时MTWA。判断标准是:在V3导联Val≥t47μV为阳性,Val≤t47μV为阴性。2次检测均为阴性的归为A组;2次检测,其中1次为阳性的归为B组。结果小于1个月时MTWA阴性占80%,阳性占20%;6个月时MTWA阴性占60%,阳性占40%;6个月时有11例(24.4%)MTWA由阴性转变为阳性,有3例(6.7%)由阳性转变为阴性。这两个时期检测MTWA,结果一致的病人有29例,符合率为64.4%。阴性转阳性患者左室射血分数降低多于持续阴性患者。结论急性心梗后6个月内MTWA处于动态变化之中,对急性心梗后6个月内,MTWA阴性的患者应予更多的关注。     

2型糖尿病患者心率变异性时域指标与肾病的关系

研究了正常白蛋白尿（n=113，NAU）、微量白蛋白尿（n=107，MAU）和临床白蛋白尿（n=88，CAU）2型糖尿病患者，发现：CAU组心率变异时域指标（SDNN，SDANN，SDNNI）高于NAU和MAU组。     

Congenital goitrous hypothyroidism: discordant systolic time intervals, pituitary and peripheral responses to high daily doses of T4 or T3 therapy

Left ventricular performance was studied by a noninvasive technique through the measurement of the systolic time intervals (total eletromechanical systole, left ventricular ejection (LVET) time, preejection period (PEP) and PEP/LVET ratio (Systolic Quotient) in 8 young adults with congenital goitrous hypothyroidism. All subjects showed lengthening of PEP, shortening of LVET and an increased PEP/LVET ratio associated with low serum T3 and T4, an exaggerated TSH response to TRH, high levels of serum cholesterol, triglycerides and carotene. They were treated with increasing L-T4 at monthly intervals (100, 200 and 400 micrograms daily), followed by L-T3 (50 and 200 micrograms daily) after stopping medication for another month. Systolic time intervals and the systolic quotient promptly reversed to the normal range with physiologic L-T4 (100 micrograms) or L-T3 (50 micrograms) replacement, but the TSH peak response to TRH was still present and exaggerated. Further reductions of the systolic quotient occurred with 200 micrograms L-T4, but not with supraphysiological doses (400 micrograms L-T4 or 200 micrograms L-T3) of thyroid hormones. The highest dose of L-T3 (200 micrograms/day) induced a significantly lower mean systolic quotient than 400 micrograms L-T4 daily, while 5 patients still had a significant TSH response to TRH. This was interpreted as discordant pituitary and cardiac response to L-T3 and L-T4 therapy. Serum cholesterol and triglycerides were considered as very sensitive index of thyroid hormone peripheral action. These had a significant positive correlation with changes in the left ventricular performance. Serum carotene, although decreasing significantly with L-T4 or L-T3 treatment, had no significant correlation with the systolic quotient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.As part of a virus life cycle, genetic information needs to be incorporated into the newly produced virus particles. Tailed bacteriophages, which probably form the largest biomass of the planet (1), and many eukaryotic viruses such as herpes viruses use powerful ATPase motors to achieve this (2). These motors generate forces as high as 80–100 pN and translocate DNA into a preformed prohead until a DNA condensate of near crystalline density fills the interior (3).The viral packaging motors share a common architecture with the ASCE (additional strand, conserved E) superfamily of multimeric ring ATPases that perform diverse functions such as chromosome segregation (helicases), protein remodeling (chaperones and proteasomes), and cargo transport (dyneins) (4). Although much has been learned about the mechanochemistry of these motors, little is known about how a functional motor is assembled and its activity is initiated. The packaging motors have the difficult task of precisely inserting the end of a viral genome into the capsid at the time of initiation.In a general virus assembly pathway shared by dsDNA viruses, assembly starts at a unique fivefold vertex of the prohead called the portal vertex, which is formed from 12 molecules of the portal protein (5). A protein shell assembles around a protein scaffold and later becomes an empty prohead after the scaffold leaves, or is degraded (6). In most dsDNA bacteriophages as well as herpes viruses a complex of two proteins, known as small and large “terminase” proteins, recognize a specific sequence of DNA in the concatemeric viral genome (e.g., cos site in phage λ and pac site in phage P22) and make a cut to create a dsDNA end (7, 8). The small terminase is responsible for binding to the cos or pac site, whereas the large terminase makes the cut. However, phage phi29 and adenoviruses do not require DNA cutting because the genome is a unit-length molecule with a covalently attached “terminal protein” at the ends (9). The large terminase, which is also an ATPase, then attaches to the protruding end of the portal and assembles into an oligomeric motor that translocates the DNA genome into the empty prohead through the ∼3.5-nm-diameter portal channel using energy from ATP hydrolysis (7, 8). After packaging one unit-length viral genome (headful packaging), the motor dissociates from the full head and the neck and tail proteins assemble on the portal to make an infectious virus.Bacteriophage T4 has been an important model for tailed bacteriophages as well as herpes viruses (10, 11). The T4 packaging motor, a pentamer of gp17 (70 kDa) (large terminase protein) assembled on the gp20 portal dodecamer (12) is the fastest (packaging rate up to ∼2,000 bp/s) of the viral packaging motors studied (13). Gp17 possesses all of the basic enzymatic activities necessary for generating a DNA-full head: ATPase, nuclease, and translocase (14, 15). An oligomeric small terminase protein, gp16, that forms 11-mer and 12-mer rings recognizes the viral genome in vivo, although it lacks strict sequence specificity and is dispensable for packaging in vitro (16). Cryo-EM reconstruction of the packaging motor in complex with the capsid portal, which we will call the “packaging machine,” shows a ring of five gp17 molecules assembled on the prohead portal into a pentameric configuration with the translocation groove facing the channel (12). An electrostatic force-driven translocation mechanism was proposed in which gp17 subunits alternate between the “tensed” (compact) and “relaxed” (extended) conformational states that is coupled to translocation of DNA in a piston-like fashion (12).Genetic and biochemical studies of several packaging systems have delineated the mechanisms of genome recognition and DNA cutting (17, 18). Structural studies (12, 16, 19) and single-molecule optical tweezers (3, 13) and fluorescence spectroscopy (20, 21) approaches have been used to dissect the mechanochemical steps of DNA translocation. However, the transient nature of DNA and protein interactions at the initiation stage, which involve insertion of the dsDNA end into the prohead and triggering of translocation, has been a major challenge (22). The dynamics of motor assembly, timescales of motor–DNA–portal interactions, and mechanism of initiation are poorly understood in any system.Here, we report a single-molecule fluorescence assay that allowed us to dissect packaging initiation starting from a dsDNA end, in real time, by the phage T4 DNA packaging machine. We reconstituted a fully functional minimal T4 packaging complex and imaged individual packaging machines in real time by total internal reflection fluorescence microscopy. Each machine carried out successive DNA translocations and the times for motor assembly and packaging initiation were quantified. Using this assay we found that packaging initiations occurred in bursts with long pauses in between. We discovered that packaging initiation shows unusual plasticity. It can occur through multiple pathways: motor assembly on the portal followed by interaction with DNA and, unexpectedly, direct interaction of DNA with the portal followed by recruitment of the motor subunits. Finally, subtle changes in the ATP binding Walker A P-loop residues that lower the rate of ATP hydrolysis lead to severe defects in packaging initiation. These results provided insights into the dynamics of interactions that lead to single-molecule encapsidation of DNA by a viral packaging machine.     

EGFR T790M testing through repeated liquid biopsy over time: a real-world multicentric retrospective experience

BackgroundAbout 15% of non-small cell lung cancers (NSCLCs) harbor epidermal growth factor receptor (EGFR) mutations. Upfront treatment with first and second generation EGFR tyrosine kinase inhibitors (1-2gen TKIs) is superior to chemotherapy. The most frequent resistance mechanism to 1-2gen TKIs is EGFR T790M mutation, which is targeted by osimertinib. T790M mutation can be revealed by liquid biopsy (LB) or by tissue rebiopsy (TB). LB is easily feasible but less sensitive than TB. We focused on repeated LBs and analyzed clinical features associated with EGFR T790M detection.MethodsThis is a retrospective multicenter observational study including EGFR-mutant NSCLC consecutive patients with disease progression (PD) after 1-2gen TKIs and with a first EGFR LB negative for T790M mutation, referred between 2016 and 2019. Aims of the study were to determine the prevalence of T790M mutation using LB in a real-life setting and the prevalence of T790M mutation by repeated LBs. We explored the association of T790M with clinical-pathological features and, through a survey, we evaluated the decision-making process behind LB request. Data on TBs were also collected.ResultsOne hundred and ten patients were included in the study, for a total of 326 LBs. Median number of LB per patient was 3.0. The T790M prevalence through LB was 34.5%. Over time, significantly more LBs were requested “at clinical and radiological PD” and “at radiological PD” compared to “arbitrarily”. The probability of finding the T790M mutation for a patient across each subsequent LB did not significantly change. Liver and lymph node PD were significantly correlated to T790M positivity. Notably, “at PD” compared to “arbitrarily” LB request and liver, bone or lymph node PD were correlated to the detection of any EGFR mutation in cfDNA. TB was performed in 59.7% of patients with a T790M negative LB and 18.8% of them were T790M positive. In most cases, TB was not feasible due to anatomical reasons. In our study population, the overall T790M prevalence—detected with both LB and TB—was 42.7%.ConclusionsRepeated LB testing can be useful in a real-life scenario to detect EGFR T790M mutation.     

Cytomegalovirus-specific T-cell reactivity in biliary atresia at the time of diagnosis is associated with deficits in regulatory T cells

Biliary atresia (BA) is a progressive, inflammatory cholangiopathy that culminates in fibrosis of extrahepatic and intrahepatic bile ducts. A leading theory on the pathogenesis of BA is that the bile duct damage is initiated by a virus infection, followed by a bile duct-targeted autoimmune response. One mechanism of autoimmunity entails a diminished number or function of regulatory T cells (Tregs). The aim of this study was to identify potential virus-specific liver T cells from infants with BA at the time of diagnosis, implicating the virus involved in early bile duct damage. A subaim was to determine if the presence of virus infection was associated with quantitative changes in Tregs. Liver T cells from BA and control patients were cultured with antigen-presenting cells in the presence of a variety of viral or control proteins. 56% of BA patients had significant increases in interferon-gamma-producing liver T cells in response to cytomegalovirus (CMV), compared with minimal BA responses to other viruses or the control group CMV response. In addition, a positive correlation between BA plasma CMV immunoglobulin M (IgM) and liver T-cell CMV reactivity was identified. Investigation of peripheral blood Tregs revealed significant deficits in Treg frequencies in BA compared with controls, with marked deficits in those BA patients who were positive for CMV. Conclusion: Liver T-cell responses to CMV were identified in the majority of BA patients at diagnosis, suggesting perinatal CMV infection as a plausible initiator of bile duct damage. Deficiency of Tregs in BA implies decreased inhibition of inflammation and autoreactivity, potentially allowing for exaggerated bile duct injury.     

CD45RA on human CD8 T cells is sensitive to the time elapsed since the last antigenic stimulation

The expression of CD45RA on CCR7- human CD8+ memory T cells is widely considered to be a marker of terminal differentiation. We studied the time course of CD45RA and CCR7 expression on human antitumoral cytotoxic T lymphocyte (CTL) clones and blood CD8+ T cells after antigenic stimulation. Our results indicate that CD45RA+ CCR7- CD8+ T cells are resting memory cells that, upon antigenic stimulation and during the next 10 days, proliferate, lose CD45RA, and transiently acquire CCR7. In the absence of further antigenic stimulation, they progressively re-express CD45RA and become CD45RA+ CCR7-. We conclude that the expression of CD45RA on these cells is indicative of the time elapsed since the last antigenic stimulation rather than the incapacity to proliferate or particularly high lytic potential. This concept leads to a reinterpretation of the significance of the presence of CD45RA+ CD8+ memory cells in patients affected by viral infections or by cancer.     

Regulatory T cells control the CD8 adaptive immune response at the time of ductal obstruction in experimental biliary atresia

CD8 T-lymphocytes are effector cells of cholangiocyte injury in human and in rhesus rotavirus (RRV)-induced experimental biliary atresia (BA). Here we hypothesize that neonatal deficiency in CD25(+) CD4(+) regulatory T cells (Tregs) leads to aberrant activation of hepatic T-lymphocytes in BA. We found that adoptive transfer of total CD4 cells, but not of CD25-depleted CD4 cells, prior to RRV inoculation reduced expansion of CD8 cells, plasma bilirubin levels, ductal inflammation, and bile duct epithelial injury at 7 days postinfection (dpi) compared with age-matched infected controls without adoptive transfer. Searching for mechanisms, we found that in vitro production of interferon-gamma (IFN-γ) by na?ve CD8 cells upon polyclonal stimulation was enhanced in coculture with hepatic dendritic cells (DCs) from RRV-infected, but not with DCs from noninfected mice, which was correlated with an increased proportion of CD11b(+) myeloid (m)DCs and up-regulation of the costimulatory molecule CD86 on RRV-primed DCs. Furthermore, DC-dependent T-lymphocyte activation was blocked by anti-CD86 antibody in dose-dependent fashion. Importantly, expression of CD86 on mDCs was down-regulated by Tregs in vitro, and adoptive transfer of Treg-containing CD4 cells decreased expression of CD86 on hepatic mDCs at 7 dpi. On the contrary, in mice resistant to experimental BA, CD25+ cell depletion aggravated bile duct injury at 12 dpi after RRV inoculation, as plasma bilirubin levels were elevated by >20-fold compared with nondepleted infected controls. Increased susceptibility to hepatobiliary injury in Treg-depleted mice was linked to hepatic CD8 expansion and enhanced stimulatory capacity of hepatic DCs. CONCLUSION: Activation of hepatic T-lymphocytes driving biliary obstruction in BA is regulated by mDCs by way of CD86-dependent costimulation and is susceptible to inhibition by Tregs.