The site of an immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type 1 does not constitute the neutralization epitope.

We previously reported the in vitro generation of a neutralization-resistant variant of the molecularly cloned isolate of human immunodeficiency virus type 1 (HIV-1), HXB2D. The molecular basis for the resistance was shown to be a point mutation in the env gene, causing the substitution of threonine for alanine at position 582 of gp41. Here, we show the variant to be resistant to syncytium inhibition as well as to neutralization by the immune-selecting serum. Moreover, 30% of HIV-positive human sera able to neutralize the parental virus have significantly decreased ability to neutralize the variant. As the A-to-T substitution thus has general relevance to the interaction of HIV-1 with the host immune system, we investigated further the biologic and immunologic bases for the altered properties. Synthetic peptides corresponding to the 582 region failed to compete in infectivity, neutralization, or syncytium inhibition assays and did not elicit neutralizing antibodies. Furthermore, human antibodies, affinity purified on synthetic peptide resins, bound to gp41 and peptides from the 582 region but did not possess neutralizing antibody activity. Some viral constructs in which the AVERY sequence in the 582 region was altered by site-directed mutagenesis were not infectious, indicating that the primary structure in this region is crucial for viral infectivity. Constructs predicted to possess a local secondary structure similar to that of the variant nevertheless behaved like the parental virus and remained neutralization sensitive. These results suggest that the requirements for neutralization resistance in this region are very precise. Our results with synthetic peptides show that the 582 region does not by itself constitute a neutralization epitope. Moreover, the degree of flexibility in amino acid substitution which allows maintenance of neutralization sensitivity suggests that position 582 does not form part of a noncontiguous neutralization epitope. The basis for neutralization resistance of the immune-selected variant is more likely a conformational change altering a neutralization epitope at a distant site.     

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1 ^a . Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1^b- and Qa-1^c-encoded determinants. Qa-1 ^d target cells are unique in that they express determinants recognized by anti-Qa-1^a CTL and by anti-Qa-1^b CTL. Killing of Qa-1 ^d targets by anti-Qa-1^a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1 ^d cells by one anti-Qa-1^b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1^a CTL and Qa-1.1-specific antibodies did not have identical specificities.Abbreviations used in this paper B6 C57BL/6J - CAB concanavalin A stimulated lymphoblasts - CML cell-mediated lympholysis - CTL cytotoxic T lymphocyte - NMS normal mouse serum - MHC major histocompatibility complex - MLC mixed leukocyte culture - MR maximum release - SMDM supplemented Mishell-Dutton medium - SR spontaneous release     

Gating kinetics of four classes of voltage-dependent K+ channels in pheochromocytoma cells

Clonal pheochromocytoma (PC-12) cells have four different types of voltage-dependent K+ channels whose activation does not require high concentrations of Ca++ on the cytoplasmic side of the membrane (Hoshi, T., and R. W. Aldrich, 1988, Journal of General Physiology, 91:73-106). The durations of open and closed events of these four different types of voltage-dependent K+ channels were measured using the excised configuration of the patch-clamp method. The open durations of a class of K+ channels termed the Kz channel, which activates rapidly and inactivates slowly in response to depolarizing pulses, had two exponential components. The closed durations of the Kz channel had at least four exponential components. The time constants of the fastest of the two exponential components in the closed durations were very similar to those of the two exponential components present in the first-latency distribution. The first latencies of the Kz channel decreased steeply with depolarization, contributing to the increased probability of the channel being open with depolarization. The Kz channel also had a very slow gating process that resulted in a clustering of blank sweeps. A gating scheme containing two open states and five closed states is consistent with the observations. The Ky channel had one exponential component in the open durations and three exponential components in the closed durations. The first latencies varied greatly depending on the prepulse voltage and duration. The results were consistent with a sequential model with a large number of closed states and one open state. The Kx channel, which required large hyperpolarizing prepulses to remove steady state inactivation and did not show inactivation with maintained depolarization, had two exponential components in the open durations and three exponential components in the closed durations. The burst behavior of the Kx channel involved many more than two states. The transient Kw channel had one exponential component in the open durations and the mean open time increased with depolarization. The first latencies of the Kw channel were steeply dependent on the voltage, decreasing with depolarization.     

Use of single nucleotide polymorphisms identifies backcrossing and species misidentifications among three San Francisco estuary osmerids

Two threatened osmerid species native to the San Francisco Estuary (SFE)—Delta Smelt (Hypomesus transpacificus) and Longfin Smelt (Spirinchus thaleichthys)—are subject to broad human influence, including significant habitat alteration and the presence of the introduced osmerid, Wakasagi (Hypomesus nipponensis). The identification of these closely related species and their hybrids is difficult in field collected specimens which are subject to damage through handling and may be difficult to identify morphologically, especially when young. In addition, it is known that these three species hybridize, but the extent and effect of hybridization is difficult to quantify and monitor. We developed assays for 24 species-specific single nucleotide polymorphisms (SNPs) that identify whether a sample is a pure species (Delta Smelt, Longfin Smelt, or Wakasagi), a first generation (F₁) hybrid, or a backcross. We used this SNP panel to genetically identify wild osmerids collected in Yolo Bypass from 2010 to 2016 and detected nine Delta Smelt × Wakasagi F₁ hybrids and two Wakasagi × (Delta Smelt × Wakasagi) backcross hybrids; all assayed hybrids had Wakasagi as the maternal parent. The backcrossing into Wakasagi suggests that hybridization may only occur in one direction and thus preclude introgression to Delta Smelt. We also found substantial morphological field misidentifications (32.7%) in the Yolo Bypass samples resulting in more Wakasagi and fewer Delta Smelt than previously recorded when based on morphology. The SNP panel described in this study constitutes a valuable resource for monitoring hybridization in the SFE and assigning species identifications with accuracy and efficiency.     

Sprouted and Freeze‐Dried Wheat and Oat Seeds – Phytochemical Profile and in Vitro Biological Activities

This research was carried out to study phytochemical profile, in vitro antioxidant capacity, reducing power, anti‐hyperglycemic, anti‐inflammatory activities and simulated gastrointestinal digestion of 7‐day old cereal sprouts: spelt wheat ‘Nirvana’ (WSSpe), wheat ‘Simonida’ (WSSim), oat ‘Golozrni’ (OSG) and oat ‘Jadar’ (OSJ). OSG expressed significantly higher (P ≤ 0.05) total phenols (TPC) and flavonoids content (TFC), antioxidant capacities (DPPH and ABTS assays) and reducing power (EC₅₀^DPPH = 2.12 mg/ml; EC₅₀^ABTS = 0.87 mg/ml; EC_0.5^RP = 12.24 mg/ml) as well as anti‐hyperglycemic activity (EC₅₀^AHgA = 0.96 mg/ml). WSSpe had the highest content of chlorophyll (131.23 mg/100 g) and carotenoids (22.84 mg/100 g). WSSim possessed the most potent anti‐inflammatory activity (2.71 mg/ml), though not significantly different from OSG (2.77 mg/ml). The in vitro simulation of gastro‐intestinal digestion showed higher release of phenolic compounds in intestinal than in gastric fluid.     

Improved patient-reported outcomes in patients with psoriatic arthritis treated with abatacept: results from a phase 3 trial

Background

To explore the effect of abatacept treatment on patient-reported outcomes (PROs) in psoriatic arthritis (PsA).

Methods

Patients with PsA were randomised (1:1) to subcutaneous abatacept 125?mg weekly/placebo for 24?weeks with early escape (EE) to open-label abatacept (week 16). Adjusted mean changes from baseline to weeks 16 (all patients) and 24 (non-EE responders) in Health Assessment Questionnaire-Disability Index (HAQ-DI), Short Form-36 (SF-36; physical and mental component summary and domains), Dermatology Life Quality Index and Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) were evaluated. Subpopulations were analysed by baseline C-reactive protein (CRP) level (> vs?≤?upper limit of normal [ULN]) and prior tumour necrosis factor inhibitor (TNFi) exposure. Proportions of patients reporting improvements ≥ minimal clinically important differences (MCIDs) and?≥?normative values (NVs) in HAQ-DI, SF-36 and FACIT-F (week 16 before EE) were analysed.

Results

In total population, numerically higher improvements in most PROs were reported with abatacept (n?=?213) versus placebo (n?=?211) at both time points (P?>?0.05). Higher proportions of abatacept versus placebo patients reported PRO improvements ≥ MCID and?≥?NV at week 16. At week 16, all PRO improvements were numerically greater (P?>?0.05) in patients with baseline CRP?>?ULN versus CRP?≤?ULN (all significant [95% confidence interval] for abatacept vs placebo); improvements in SF-36 component summaries and FACIT-F were greater in TNFi-naïve versus TNFi-exposed patients (abatacept > placebo). Week 24 subgroup data were difficult to interpret due to low patient numbers.

Conclusions

Abatacept treatment improved PROs in patients with PsA versus placebo, with better results in elevated baseline CRP and TNFi-naïve subpopulations.

Trial registration

ClinicalTrials.gov number, NCT01860976 (funded by Bristol-Myers Squibb); date of registration: 23 May 2013.