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The increased prevalence of opioid use disorder (OUD) makes it imperative to disentangle the biological mechanisms contributing to individual differences in OUD vulnerability. OUD shows strong heritability, however genetic variants contributing toward vulnerability remain poorly defined. We performed a genome-wide association study using over 850 male and female heterogeneous stock (HS) rats to identify genes underlying behaviors associated with OUD such as nociception, as well as heroin-taking, extinction and seeking behaviors. By using an animal model of OUD, we were able to identify genetic variants associated with distinct OUD behaviors while maintaining a uniform environment, an experimental design not easily achieved in humans. Furthermore, we used a novel non-linear network-based clustering approach to characterize rats based on OUD vulnerability to assess genetic variants associated with OUD susceptibility. Our findings confirm the heritability of several OUD-like behaviors, including OUD susceptibility. Additionally, several genetic variants associated with nociceptive threshold prior to heroin experience, heroin consumption, escalation of intake, and motivation to obtain heroin were identified. Tom1, a microglial component, was implicated for nociception. Several genes involved in dopaminergic signaling, neuroplasticity and substance use disorders, including Brwd1, Pcp4, Phb1l2 and Mmp15 were implicated for the heroin traits. Additionally, an OUD vulnerable phenotype was associated with genetic variants for consumption and break point, suggesting a specific genetic contribution for OUD-like traits contributing to vulnerability. Together, these findings identify novel genetic markers related to the susceptibility to OUD-relevant behaviors in HS rats.

Background: Little is known about the specific roles of cortical and accumbal oxytocin receptors in drug use disorders. To better understand the importance of the endogenous oxytocin system in cocaine relapse behavior, we developed an adeno-associated viral vector-expressing short hairpin (sh) RNAs to selectively degrade the rat oxytocin receptor (OxyR) mRNA in vivo., Methods: Male (Sprague-Dawley) rats received bilateral infusions of the shRNA for the oxytocin receptor (shOxyR) or an shRNA control virus into the prefrontal cortex (PFC) or the nucleus accumbens core (NAc). Rats self-administered cocaine on an escalating FR ratio for 14 days, lever responding was extinguished, and rats were tested for cued and cocaine-primed reinstatement of drug seeking., Results: OxyR knockdown in the PFC delayed the acquisition of lever pressing on an fixed ratio 1 schedule of reinforcement. All rats eventually acquired the same level of lever pressing and discrimination, and there were no differences in extinction. OxyR knockdown in the NAc had no effect during acquisition. In both the PFC and NAc, the shOxyR decreased cued reinstatement relative to shRNA control virus but was without effect during drug-primed reinstatement. OxyR knockdown in the PFC increased chamber activity during a social interaction task., Conclusions: This study provides critical new information about how endogenous OxyRs function to affect drug seeking in response to different precipitators of relapse. The tool developed to knockdown OxyRs in rat could provide important new insights that aid development of oxytocin-based therapeutics to reduce return-to-use episodes in people with substance use disorder and other neuropsychiatric disorders., (© The Author(s) 2023. Published by Oxford University Press on behalf of CINP.)

Return to methamphetamine (meth) use is part of an overarching addictive disorder hallmarked by cognitive sequela and cortical dysfunction in individuals who use meth chronically. In rats, long access meth self-administration produces object recognition memory deficits due to drug-induced plasticity within the perirhinal cortex (PRH). PRH projections are numerous and include the medial prefrontal cortex (mPFC). To evaluate the role of the PRH-mPFC reciprocal circuit in novel object recognition memory, a rgAAV encoding GFP-tagged Cre recombinase was infused into the PRH or the mPFC and rats were tested for recognition memory. On test day, one group explored both familiar and novel objects. A second group explored only familiar objects. GFP and Fos expression were visualized in the mPFC or PRH. During exploration, PRH neurons receiving input from the mPFC were equally activated by exploration of novel and familiar objects. In contrast, PRH neurons that provide input to the mPFC were disproportionately activated by novel objects. Further, the percent of Fos + cells in the PRH positively correlated with recognition memory. As such, the flow of communication appears to be from the PRH to the mPFC. In agreement with this proposed directionality, chemogenetic inhibition of the PRH-mPFC circuit impaired object recognition memory, whereas chemogenetic activation in animals with a history of long access meth self-administration reversed the meth-induced recognition memory deficit. This finding informs future work aimed at understanding the role of the PRH, mPFC, and their connectivity in meth associated memory deficits. These data suggest a more complex circuitry governing recognition memory than previously indicated with anatomical or lesion studies., Competing Interests: Declaration of competing interest The authors have no declarations of interest to report., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: There are numerous pharmacologic treatments for opioid use disorder (OUD), but none that directly target the underlying addictive effects of opioids. Oxytocin, a peptide hormone produced in the paraventricular nucleus (PVN) of the hypothalamus, has been investigated as a potential therapeutic for OUD. Promising preclinical and clinical results have been reported, but the brain region(s) and mechanism(s) by which oxytocin impacts reward processes remain undetermined., Methods: Here, we assess peripherally administered oxytocin's impacts on cued reinstatement of heroin seeking following forced abstinence and its effects on neuronal activation in the PVN and key projection regions. We also examine how designer receptors exclusively activated by designer drug (DREADD)-mediated activation or inhibition of oxytocinergic PVN neurons alters cued heroin seeking and social interaction., Results: As predicted, peripheral oxytocin administration successfully decreased cued heroin seeking on days 1 and 30 of abstinence. Oxytocin administration also led to increased neuronal activity within the PVN and the central amygdala (CeA). Activation of oxytocinergic PVN neurons with an excitatory (Gq) DREADD did not impact cued reinstatement or social interaction. In contrast, suppression with an inhibitory (Gi) DREADD reduced heroin seeking on abstinence day 30 and decreased time spent interacting with a novel conspecific., Discussion: These findings reinforce oxytocin's therapeutic potential for OUD, the basis for which may be driven in part by increased PVN-CeA circuit activity. Our results also suggest that oxytocin has distinct signaling and/or other mechanisms of action to produce these effects, as inhibition, but not activation, of oxytocinergic PVN neurons did not recapitulate the suppression in heroin seeking., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Purpose: Expected toxicity from chemoradiation (CRT) is an important factor in treatment decisions but is poorly understood in older adults with lower gastrointestinal (GI) malignancies. Our objective was to compare acute adverse events (AAEs) of older and younger adults with lower GI malignancies treated on NRG studies., Methods: Data from 6 NRG trials, testing combined modality therapy in patients with anal or rectal cancer, were used to test the hypothesis that older age was associated with increased AAEs. AAEs and compliance with protocol-directed therapy were compared between patients aged ≥70 and < 70. Categorical variables were compared across age groups using the chi-square test. The association of age on AAEs was evaluated using a covariate-adjusted logistic regression model, with odds ratio (OR) reported. To adjust for multiple comparisons, a p-value <0.01 was considered statistically significant., Results: There were 2525 patients, including 380 patients ≥70 years old (15%) evaluable. Older patients were more likely to have worse baseline performance status (PS 1 or 2) (23% vs. 16%, p = 0.001), but otherwise baseline characteristics were similar. Older patients were less likely to complete their chemotherapy (78% vs. 87%, p < 0.001), but had similar RT duration. On univariate analysis, older patients were more likely to experience grade ≥ 3 GI AAEs (36% vs. 23%, p < 0.001), and less likely to experience grade ≥ 3 skin AAEs (8% vs. 14%, p = 0.002). On multivariable analysis, older age was associated with grade ≥ 3 GI AAE (OR 1.93, 95% CI: 1.52, 2.47, p < 0.001) after adjusting for sex, race, PS, and disease site., Conclusions: Older patients with lower GI cancers who underwent CRT were less likely to complete chemotherapy and had higher rates of grade 3+ GI AAEs. These results can be used to counsel older adults prior to treatment and manage expected toxicities throughout pelvic CRT., Competing Interests: Declaration of Competing Interest Ms. Moughan, Drs. Ajani, Anne, Arora, Berger, Crane, Ellsworth, Green, Hopkins, Kachnic, Lee, and Mohiuddin, have nothing to disclose. Dr. Ballo discloses employment, and a leadership role with the West Cancer Clinic, and Honoraria, a Consulting or Advisory Role, and Travel, Accommodations, or Expenses from Novocure, LLC. Dr. Jagsi discloses stock or other ownership and a Consulting or Advisory Role with Equity Quotient. Honoraria with Amgen & Vizient. Dr. Lichtman discloses Honoraria with Remedy One and Magellan. Dr. Meropol discloses employment with Flatiron Health, Stock or Other Ownership with Flatiron Health, and Roche, a US Patent for Methods of Therapy for Cancers Characterized by Overexpression of HER2 Receptor Protein, along with Research Funding for this patent. Dr. Meyer discloses Honoraria from Varian Medical Systems, and Sirtex Medical, Ltd., and Research Funding from Varian Medical Systems. Dr. Ari VanderWalde discloses Honoraria from AstraZeneca, a Consulting or Advisory Role with Bristol-Myers Squibb, Caris Life Sciences, Compugen, ImmunoCore, and Concerto HealthAI, Research Funding from Amgen, AstraZeneca, Bristol-Myers Squibb, Genentech, Caris Life Sciences, Merck, Eli Lilly, EMD Serono, Replimune, Polynoma, and Millenium. Dr. Noam VanderWalde discloses a Consulting or Advisory Role with Concerto HealthAI., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

DNA repair and DNA damage signaling pathways are critical for the maintenance of genomic stability. Defects of DNA repair and damage signaling contribute to tumorigenesis, but also render cancer cells vulnerable to DNA damage and reliant on remaining repair and signaling activities. Here, we review the major classes of DNA repair and damage signaling defects in cancer, the genomic instability that they give rise to, and therapeutic strategies to exploit the resulting vulnerabilities. Furthermore, we discuss the impacts of DNA repair defects on both targeted therapy and immunotherapy, and highlight emerging principles for targeting DNA repair defects in cancer therapy., (© 2022 Hopkins et al.; Published by Cold Spring Harbor Laboratory Press.)

Methamphetamine (meth) causes enduring changes within the medial prefrontal cortex (mPFC) and the nucleus accumbens (NA). Projections from the mPFC to the NA have a distinct dorsal-ventral distribution, with the prelimbic (PL) mPFC projecting to the NAcore, and the infralimbic (IL) mPFC projecting to the NAshell. Inhibition of these circuits has opposing effects on cocaine relapse. Inhibition of PL-NAcore reduces cued reinstatement of cocaine seeking and IL-NAshell inhibition reinstates cocaine seeking. Meth, however, exhibits a different profile, as pharmacological inhibition of either the PL or IL decrease cued reinstatement of meth-seeking. The potentially opposing roles of the PL-NAcore and IL-NAshell projections remain to be explored in the context of cued meth seeking. Here we used an intersectional viral vector approach that employs a retrograde delivery of Cre from the NA and Cre-dependent expression of DREADD in the mPFC, in both male and female rats to inhibit or activate these parallel pathways. Inhibition of the PL-NAcore circuit reduced cued reinstatement of meth seeking under short and long-access meth self-administration and after withdrawal with and without extinction. Inhibition of the IL-NAshell also decreased meth cued reinstatement. Activation of the parallel circuits was without an effect. These studies show that inhibition of the PL-NAcore or the IL-NAshell circuits can inhibit reinstated meth seeking. Thus, the neural circuitry mediating cued reinstatement of meth seeking is similar to cocaine in the dorsal, but not ventral, mPFC-NA circuit., (© 2021 Society for the Study of Addiction.)

Exposure to acute stress can increase vulnerability to develop or express many psychiatric disorders, including post-traumatic stress disorder. We hypothesized that stress-induced psychiatric vulnerability is associated with enduring neuroplasticity in the nucleus accumbens core because stress exposure can alter drug addiction-related behaviors that are associated with accumbens synaptic plasticity. We used a single 2-h stress session and 3 weeks later exposed male and female rats to stress-conditioned odors in a modified defensive burying task, and quantified both active and avoidant coping strategies. We measured corticosterone, dendritic spine and astrocyte morphology in accumbens, and examined reward sensitivity using a sucrose two-bottle choice and operant sucrose self-administration. Exposure to stress odor increased burying (active coping) and immobility (avoidant coping) in the defensive burying task in female and male rats. Systemic corticosterone was transiently increased by both ongoing acute restraint stress and stress-conditioned odors. Three weeks after administering acute restraint stress, we observed increased dendritic spine density and head diameter, and decreased synaptic association with astroglia and the astroglial glutamate transporter, GLT-1. Exposure to conditioned stress further increased head diameter without affecting spine density or astroglial morphology, and this increase by conditioned stress was correlated with burying behavior. Finally, we found that stress-exposed females have a preference for sweet solutions and higher motivation to seek sucrose than stressed male rats. We conclude that acute stress produced enduring plasticity in accumbens postsynapses and associated astroglia. Moreover, conditioned stress odors induced active behavioral coping strategies that were correlated with dendritic spine morphology., (© 2021. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Hypoxic pulmonary vasoconstriction (HPV) is a well-characterized vascular response to low oxygen pressures and is involved in life-threatening conditions such as high-altitude pulmonary edema (HAPE) and pulmonary arterial hypertension (PAH). While the efficacy of oral therapies can be affected by drug metabolism, or dose-limiting systemic toxicity, inhaled treatment via pressured metered dose inhalers (pMDI) may be an effective, nontoxic, practical alternative. We hypothesized that a stable water-in-perfluorooctyl bromide (PFOB) emulsion that provides solubility in common pMDI propellants, engineered for intrapulmonary delivery of pulmonary vasodilators, reverses HPV during acute hypoxia (HX). Male Sprague Dawley rats received two 10-min bouts of HX (13% O 2 ) with 20 min of room air and drug application between exposures. Treatment groups: intrapulmonary delivery (PUL) of (1) saline; (2) ambrisentan in saline (0.1 mg/kg); (3) empty emulsion; (4) emulsion encapsulating ambrisentan or sodium nitrite (NaNO 2 ) (0.1 and 0.5 mg/kg each); and intravenous (5) ambrisentan (0.1 mg/kg) or (6) NaNO 2 (0.5 mg/kg). Neither PUL of saline or empty emulsion, nor infusions of drugs prevented pulmonary artery pressure (PAP) elevation (32.6 ± 3.2, 31.5 ± 1.2, 29.3 ± 1.8, and 30.2 ± 2.5 mmHg, respectively). In contrast, PUL of aqueous ambrisentan and both drug emulsions reduced PAP by 20-30% during HX, compared to controls. IL6 expression in bronchoalveolar lavage fluid and whole lung 24 h post-PUL did not differ among cohorts. We demonstrate proof-of-concept for delivering pulmonary vasodilators via aerosolized water-in-PFOB emulsion. This concept opens a potentially feasible and effective route of treating pulmonary vascular pathologies via pMDI.

In this issue of Cancer Cell, Quereda and colleagues report that a newly developed specific inhibitor of CDK12/13, SR-4835, sensitizes triple-negative breast cancer cells to PARP inhibitors and DNA-damaging chemotherapeutics by reducing expression of the genes in the DNA damage response pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Background: The prevalence of metabolic syndrome (MetS) generally rises with increasing adiposity, but tends to plateau at the highest levels of body mass index (BMI) with some individuals, even with severe obesity, expressing few or no components of MetS. We examined factors associated with the expression of MetS in severely obese women participating in a large observational study., Methods: Anthropometrics, including Heath equation-adjusted bioimpedance-determined fat-free mass (FFM) and fat mass (FM), lipids and related laboratory measurements, resting energy expenditure (REE), and respiratory quotient (RQ), were studied in 949 women with severe obesity., Results: Even though the mean BMI was 45.7 kg/m 2 and all participants met MetS criteria for increased waist circumference, 30% of subjects did not have MetS. Unadjusted FM (P = 0.0011), FFM (P < 0.0001), and REE (P < 0.0001) were greater in the women with MetS. Surprisingly, in multivariate logistic regression FFM was positively associated with MetS (P = 0.0002), while FM was not (P = 0.89). Moreover, FFM, not FM, was significantly associated with all five components of MetS except for triglyceride levels. REE and RQ were higher in those with MetS, and REE was strongly associated with multiple components of MetS., Conclusions: In women with severe obesity, higher FFM and REE were paradoxically associated with increased rather than decreased risk of MetS, while FFM-adjusted FM was unrelated to MetS.

The purpose of this project was to determine whether a nurse residency program was effective in improving satisfaction with new graduates' performance competence in interprofessional collaboration. This was a cross-sectional survey design, comparing the satisfaction ratings of nurse leaders and staff nurses at a mid-western academic medical center to national benchmark data obtained from the 2007 Nursing Practice Readiness Tool. The sample consisted of 149 nurses who worked in inpatient units where new graduates practice. Thirty-five had 1 year or less of experience in nursing and 114 had at least 2 years of experience. Managers, experienced nurses, and new graduate nurses varied in their satisfaction ratings regarding interprofessional collaboration. Satisfaction of new graduates' competencies by nurse managers and staff nurses were rated higher in each category, compared with the national study, with 63% of nurse leaders satisfied with new graduates' ability to communicate with the interprofessional team, compared with the national average of 38%. Participants reported 56% satisfaction in the ability to work as a team, compared with 37% reported in the national study., (Copyright 2016, SLACK Incorporated.)

Although humpback whale (Megaptera novaeangliae) calves are reported to vocalize, this has not been measurably verified. During March 2006, an underwater video camera and two-element hydrophone array were used to record nonsong vocalizations from a mother-calf escort off Hawaii. Acoustic data were analyzed; measured time delays between hydrophones provided bearings to 21 distinct vocalizations produced by the male calf. Signals were pulsed (71%), frequency modulated (19%), or amplitude modulated (10%). They were of simple structure, low frequency (mean=220 Hz), brief duration (mean=170 ms), and relatively narrow bandwidth (mean=2 kHz). The calf produced three series of "grunts" when approaching the diver. During winters of the years 2001-2005 in Hawaii, nonsong vocalizations were recorded in 109 (65%) of 169 groups with a calf using an underwater video and single (omnidirectional) hydrophone. Nonsong vocalizations were most common (34 of 39) in lone mother-calf pairs. A subsample from this dataset of 60 signals assessed to be vocalizations provided strong evidence that 10 male and 18 female calves vocalized based on statistical similarity to the 21 verified calf signals, proximity to an isolated calf (27 of 28 calves), strong signal-to-noise ratio, and/or bubble emissions coincident to sound.

The glucocorticoid receptor (GR) is involved in the transcriptional regulation of genes associated with inflammation, glucose homeostasis, and bone turnover through the association with ligands, such as corticosteroids. GR-mediated gene transcription is regulated or fine-tuned via the recruitment of co-factors including coactivators and corepressors. Current therapeutic approaches to targeting GR aim to retain the beneficial anti-inflammatory activity of the corticosteroids while eliminating negative side effects. Towards achieving this goal the experiments discussed here reveal a mechanism of co-factor binding in the presence of either bound agonist or antagonist. The GR ligand binding domain (GR-LBD(F602S)), in the presence of agonist or antagonist, utilizes different modes of binding for coactivator versus corepressor. Coactivator binding to the co-effector binding pocket of GR-LBD(F602S) is driven both by favorable enthalpic and entropic interactions whereas corepressor binding to the same pocket is entropically driven. These data support the hypothesis that ligand-induced conformational changes dictate co-factor binding and subsequent trans-activation or trans-repression.

Purpose: In preclinical studies, sequential exposure to irinotecan (CPT-11) then fluorouracil (5-FU) is superior to concurrent exposure or the reverse sequence; a 24-hour infusion of CPT-11 may be better tolerated than shorter infusions., Experimental Design: CPT-11 was first given at four levels (70-140 mg/m(2)/24 hours), followed by leucovorin 500 mg/m(2)/0.5 hours and 5-FU 2,000 mg/m(2)/48 hours on days 1 and 15 of a 4-week cycle. 5-FU was then increased in three cohorts up to 3,900 mg/m(2)/48 hours., Results: Two patients had dose-limiting toxicity during cycle 1 at 140/3,900 of CPT-11/5-FU (2-week delay for neutrophil recovery; grade 3 nausea despite antiemetics); one of six patients at 140/3,120 had dose-limiting toxicity (grade 3 diarrhea, grade 4 neutropenia). Four of 22 patients with colorectal cancer had partial responses, two of which had prior bolus CPT-11/5-FU. The mean 5-FU plasma concentration was 5.1 micromol/L at 3,900 mg/m(2)/48 hours. The end of infusion CPT-11 plasma concentration averaged 519 nmol/L at 140 mg/m(2)/24 hours. Patients with UDP-glucuronosyltransferase (UGT1A1; TA)6/6 promoter genotype had a lower ratio of free to glucuronide form of SN-38 than in patients with >/=1 (TA)7 allele. Thymidylate synthase genotypes for the 28-base promoter repeat were 2/2 (13%), 2/3 (74%), 3/3 (13%); all four responders had a 2/3 genotype., Conclusions: Doses (mg/m(2)) of CPT-11 140/24 hours, leucovorin 500/0.5 hours and 5-FU 3,120/48 hours were well tolerated.

An indandione-containing class of inhibitors abrogates DNA replication of human papillomavirus (HPV) types 6 and 11 by binding reversibly to the transactivation domain (TAD) of the viral E2 protein and inhibiting its interaction with the viral E1 helicase. To locate the binding site of this class of protein-protein interaction inhibitors, a benzophenone derivative was used to generate an irreversibly labeled E2-TAD polypeptide. The single site of covalent modification of the E2-TAD was identified by proteolytic digestions using trypsin, LysC, and V8 proteases and characterization of the resulting peptides by LC-MS procedures. Through this methodology, the benzophenone attachment point was located at the terminal methyl of residue Met101. Evidence further pinpointed the site of photoaffinity attachment to the terminal carbon atom, which is significant in providing a definitive example of the ability to locate photoinduced cross-linking to a polypeptide with atomic resolution using solely mass spectrometric detection. The location of the inhibitor binding site vis-à-vis the Glu39 and Glu100 residues sensitive to mutation for HPV 11 E2-TAD is discussed in relation to the crystal structure of the E2-TAD from the related HPV type 16.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.

The allosteric inhibition of the lymphocyte function associated antigen-1/intercellullar adhesion molecule (LFA-1/ICAM-1) interaction, by a class of small molecules, is characterized by a battery of mass spectrometric techniques. Binding of hydantoins to the I domain of LFA-1 is observed by size exclusion chromatography/mass spectrometry (SEC/MS) and by direct electrospray ionization mass spectrometry (ESI/MS). A photoactive hydantoin analog specifically labels an amino acid residue of LFA-1 I domain. Competition with this photoaffinity labeling by a panel of inhibitors is correlated with their Kd's for inhibition of the LFA-1/ICAM interaction. Alterations to the tertiary structure of LFA-1 I domain, upon compound binding, are inferred from perturbation in the ESI mass spectrum of the polypeptide's charge state distribution and by an altered level of nonspecific multimer formation. The results demonstrate specific, stoichiometric, reversible binding of the hydantoins to LFA-1. They further show correlation of this binding with activity and indicate alterations in the polypeptide's tertiary structure, on hydantoin binding, consistent with the proposed mechanism for inhibition of the protein-protein interaction.

The specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.

Autoimmune paraneoplastic syndromes are commonly encountered in patients with myelodysplastic syndromes (MDS). A review of case reports and small series suggest as many as 10% of MDS patients may experience various autoimmune syndromes. Clinical manifestations of such phenomena may include an acute systemic vasculitic syndrome, skin vasculitis, fever, arthritis, pulmonary infiltrates, peripheral polyneuropathy, inflammatory bowel disease, glomerulonephritis, and even classical connective tissue disorders, such as relapsing polychondritis. On the other hand, asymptomatic immunologic abnormalities have also been reported in these patients. These autoimmune manifestations frequently respond to immunosuppressive agents including steroids and occasional hematologic responses to steroid therapy have also been reported. We report five patients with history of MDS who manifested different spectrums of autoimmune phenomena including: pyoderma gangrenosum (PG), vasculitis, Coombs negative hemolytic anemia, idiopathic thrombocytopenia, and chronic inflammatory demyelinating polyneuropathy (CIDP). We also review the incidence, nature, course and response to therapy of these manifestations and discuss potential pathogenic mechanisms.

The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.

The proteinase encoded by human immunodeficiency virus type 1 (HIV-1) cleaves peptide substrates of sequences derived from processing sites in HIV-1 gag-pol polypeptide. Based on this cleavage, assays that utilize HPLC to measure activity of HIV-1 proteinase are reported herein. In the assay first described, a baseline separation of unlabeled substrate and products is achieved with a run time of 10 min and UV detection. Enzyme concentrations as low as 1 nM, which is the lowest reported for an assay employing underivatized peptide substrate, are attained. Even more powerful, versatile and sensitive, a second method that takes advantage of a peptide substrate labeled at its N-terminus with the fluorescein derivative is described as well. Because of the fluorescein label, this method offers several superior features, including very fast analysis of substrate and product in less than 3 min and fluorescence detection which provides essentially total freedom from interference. Synthesis of fluorescein-labeled peptide substrate is accomplished by solid-phase peptide synthesis.

The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.

Ten patients harboring a small, intra-vitreal, copper foreign body in one eye for 4 months to 29 years have been studied clinically and electrophysiologically. Nine of the ten affected eyes had a visual acuity of 20/60 or better. Some asymmetry existed in the electrophysiologic responses of seven affected eyes as compared to the controls, but all of these responses were within the normal range. The electroretinogram was reduced in one eye but was consistent with vitreous opacification. Vitreous opacification and copper deposition in the macula were potentially serious sequelae observed in four eyes. Small, intraocular copper foreign bodies can be tolerated for long periods of time without retinal toxicity, but vitreous changes of maculopathy may necessitate intervention.

Videotaped programs that could be used for screening of visual acuity and of visual fields by means of broadcast television have been developed and tested. The tests are designed to be effective regardless of the size of the viewer's television screen. Inevitably, false-positive and false-negative results would occur, which would have to be carefully explained at the time the tests were broadcast.

In an attempt to prevent the intense inflammatory response to intravitreal copper foreign bodies in the rabbit eye, varying dosages of dexamethasone sodium phosphate were administered daily via the subconjunctival route. The steroid appeared to behave systemically and had similar effects in both the injected and the noninjected eyes. Its effect was similar also regardless of the concentration of copper within the foreign body. Nine days of dexamethasone therapy prevented a substantial number of copper foreign bodies from becoming totally encapsulated during that time. Furthermore, a delay in the onset of the inflammatory response to the foreign body was noted. In a few instances, inflammation was completely suppressed during the entire course of therapy.

The detection and recognition masking-level differences for the 36 individual CID W-1 spondaic words (Technisonic Studios) were established on 36 listeners with normal hearing. The masking for two conditions, SoNo and S pi No, was accomplished with 70-dB-SPL speech-spectrum noise. The word lists were administered in 2-dB decrements with the 36 words presented at each signal-to-noise ratio, which ranged from 9 dB to -19 dB for SoNo and from 3 dB to -25 dB for S pi No. The detection and recognition thresholds determined for each spondaic word under both masking conditions are presented. Additionally, the slopes of the psychometric functions for each word under both conditions are given. The mean masking-level difference (SoNo-S pi No) for the detection-response task was 9.4 dB (SD = .7 dB). The mean masking-level difference for the recognition task was 7.2 dB (SD = 1.4 dB). The recognition masking-level differences for the individual spondaic words ranged from 4.4 dB (stairway) to 10.0 dB (oatmeal). A list of 10 words that had the largest masking-level differences for recognition is suggested to enhance the clinical utility of the masking-level difference for speech.

Chloroquine was administered intramuscularly 5 days a week to rhesus monkeys for as long as 4 1/2 years. No clinical, fluorescein angiographic, or electrophysiological evidence of retinal damage was observed. Yet chloroquine/chloroquine byproduct analysis of the ocular tissues revealed an enormous binding capacity of the pigmented tissues of the eye (choroid plus RPE, ciliary body, and iris) with eventual accumulation observed in the retina. Despite the normal ophthalmic appearance and function, extensive pathological changes occurred in the retinas and choroids of these experimental monkeys. The chloroquine caused an initial dramatic effect on the ganglion cells, with the photoreceptors affected shortly thereafter. Patching degeneration of the ganglion cells and photoreceptors then progressed over several years, with the choroid and pigment epithelium ultimately deteriorating as well.

A platelet-fibrinogen-thrombin mixture utilizing autologous platelets was studied for its potential to seal perforating corneal wounds. In rabbits, the mixture demonstrated sufficient adhesive properties to permit 75% of penetrating keratoplasties to remain in place without the use of sutures. All 12 grafts held initially; after 48 h three grafts extruded. In control eyes only two out of 12 (16%) of the corneal grafts remained in place. The material is simple to prepare and apply and is not toxic to the cornea. It does not cause inflammation or lid irritation. Intraocular complications observed with its use were transient anterior synechiae and retrocorneal membrane formation.