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BackgroundIn the phase III IMpassion130 study, atezolizumab plus nab-paclitaxel (A+nP) showed clinical benefit in advanced or metastatic triple-negative breast cancer patients who were programmed death-ligand 1 (PD-L1)+ (tumor-infiltrating immune cells [IC] ≥1%) using the SP142 immunohistochemistry assay. Here we evaluate 2 other PD-L1 assays for analytical concordance with SP142 and patient-associated clinical outcomes.MethodsSamples from 614 patients (68.1% of intention-to-treat population) were centrally evaluated by immunohistochemistry for PD-L1 status on IC (VENTANA SP142, SP263, Dako 22C3) or as a combined positive score (CPS; 22C3).ResultsUsing SP142, SP263, and 22C3 assays, PD-L1 IC ≥1% prevalence was 46.4% (95% confidence interval [CI] = 42.5% to 50.4%), 74.9% (95% CI = 71.5% to 78.3%), and 73.1% (95% CI = 69.6% to 76.6%), respectively; 80.9% were 22C3 CPS ≥1. At IC ≥1% (+), the analytical concordance between SP142 and SP263 and 22C3 was 69.2% and 68.7%, respectively. Almost all SP142+ cases were captured by other assays (double positive), but several SP263+ (29.6%) or 22C3+ (29.0%) cases were SP142- (single positive). A+nP clinical activity vs placebo+nP in SP263+ and 22C3+ patients (progression-free survival [PFS] hazard ratios [HRs] = 0.64 to 0.68; overall survival [OS] HRs = 0.75 to 0.79) was driven by double-positive cases (PFS HRs = 0.60 to 0.61; OS HRs = 0.71 to 0.75) rather than single-positive cases (PFS HRs = 0.68 to 0.81; OS HRs = 0.87 to 0.95). Concordance for harmonized cutoffs for SP263 (IC ≥4%) and 22C3 (CPS ≥10) to SP142 (IC ≥1%) was subpar (approximately 75%).Conclusions22C3 and SP263 assays identified more patients as PD-L1+ (IC ≥1%) than SP142. No inter-assay analytical equivalency was observed. Consistent improved A+nP efficacy was captured by the SP142 PD-L1 IC ≥1% subgroup nested within 22C3 and SP263 PD-L1+ (IC ≥1%) populations.

We measured interferon γ-induced protein 10 (IP-10) levels in 428 patients at baseline, week 1, and week 2 of all-oral treatment for hepatitis C virus (HCV) infection. An increased baseline IP-10 level was associated with a T allele in the IL28B gene, an increased alanine aminotransferase level in treatment-naive but not experienced patients, and an increased body mass index. At week 1, the mean decline in plasma IP-10 levels was the same in treatment-naive and treatment-experienced patients (-49%), whereas during week 2 the mean decline in IP-10 levels in treatment-naive patients (-14%) was significantly larger than in treatment-experienced patients (-2%; P = .0176). IP-10 thus may be a surrogate marker of the rate of intracellular viral replication complex decay.

We measured interferon γ-induced protein 10 (IP-10) levels in 428 patients at baseline, week 1, and week 2 of all-oral treatment for hepatitis C virus (HCV) infection. An increased baseline IP-10 level was associated with a T allele in the IL28B gene, an increased alanine aminotransferase level in treatment-naive but not experienced patients, and an increased body mass index. At week 1, the mean decline in plasma IP-10 levels was the same in treatment-naive and treatment-experienced patients (-49%), whereas during week 2 the mean decline in IP-10 levels in treatment-naive patients (-14%) was significantly larger than in treatment-experienced patients (-2%; P = .0176). IP-10 thus may be a surrogate marker of the rate of intracellular viral replication complex decay.

Cysteine scanning mutagenesis of the transmembrane domains 5 and 6 reveal that phenylalanine204 and tyrosine266 are important for receptor signaling. Data are consistent with the role of these aromatic residues near the ends of the transmembrane segments in the alpha-receptor promoting ligand activation.

The residues that contribute to alpha-factor receptor (Ste2p) activation are investigated by isolating second-site suppressor mutations that restored function to defective receptors carrying either an F204S or Y266C substitution. Thirty-five different suppressor mutations were identified, which on their own, caused a range of phenotypes, including hypersensitivity, constitutive activity, altered ligand binding, and loss of function.

The α-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae. Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation. In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phc[sup 204] and Tyr[sup 266], that were important for receptor signaling. These residues play a specific role in responding to α-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin). To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues. A drop in reactivity coincided with residues likely to be buried in the membrane. Interestingly, both Phe[sup 204] and Tyr[sup 266] are located very near the interface region. However, these assays predict that Phe[sup 204] is accessible at the surface of the receptor, consistent with the strong defect in binding α-factor caused by mutating this residue. In contrast, Tyr[sup 266] was not accessible. This correlates with the ability of Y266C mutant receptors to bind α-factor and suggests that this residue is involved in the subsequent triggering of receptor activation. These results highlight the role of aromatic residues near the ends of the transmembrane segments in the α-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors. [ABSTRACT FROM AUTHOR]

Human brucellosis is a common zoonosis worldwide. Here we present a case of focal vertebral brucellosis in a 71-year-old Mexican-American woman who contracted infection from unpasteurized goat milk. Standard agglutination serology was negative; the diagnosis was established by the isolation of Brucella melitensis from abscess fluid. A B. melitensis protein microarray comprised of nearly all proteins encoded by the bacterial genome was used to determine the kinetics of this patient's antibody responses to the complete collection of open reading frames existing in the genome (ORFeome). Three patterns of antibody responses against B. melitensis antigens were seen for serum samples obtained on days 0 (pretreatment), 14, 49, 100, and 180: (i) stable titers over time, (ii) a steady fall in titers, and (iii) an initial rise in titers followed by declining titers. Sera from this patient with chronic brucellosis recognized some of the same B. melitensis proteins as those recognized by sera from acute/subacute, blood culture-positive brucellosis patients but also recognized a distinct set of proteins. This study is the first to determine the kinetics of the human antibody responses to the complete repertoire of proteins encoded by a bacterial genome and demonstrates fundamentally different immunopathogenetic mechanisms between acute human brucellosis and chronic human brucellosis. While an extension of these findings to a larger patient population is necessary, these findings have important clinical and diagnostic implications and lead toward new insights into the fundamental immunopathogenesis of brucellosis.

The alpha-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

The frog Xenopus laevis has provided significant insights into developmental and cellular processes. However, X. laevis has an allotetraploid genome precluding its use in forward genetic analysis. Genetic analysis may be applicable to Xenopus (Silurana) tropicalis, which has a diploid genome and a shorter generation time. Here, we show that many tools for the study of X. laevis development can be applied to X. tropicalis. By using the developmental staging system of Nieuwkoop and Faber, we find that X. tropicalis embryos develop at similar rates to X. laevis, although they tolerate a narrower range of temperatures. We also show that many of the analytical reagents available for X. laevis can be effectively transferred to X. tropicalis. The X. laevis protocol for whole-mount in situ hybridization to mRNA transcripts can be successfully applied to X. tropicalis without alteration. Additionally, X. laevis probes often work in X. tropicalis--alleviating the immediate need to clone the X. tropicalis orthologs before initiating developmental studies. Antibodies that react against X. laevis proteins can effectively detect the X. tropicalis protein by using established immunohistochemistry procedures. Antisense morpholino oligonucleotides (MOs) offer a new alternative to study loss of gene activity during development. We show that MOs function in X. tropicalis. Finally, X. tropicalis offers the possibility for forward genetics and genomic analysis., (Copyright 2002 Wiley-Liss, Inc.)