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Aim: Cell division cycle 25B (CDC25B) belongs to the CDC25 family of phosphatases that regulate cell cycle progression. CDC25B also contributes to tumor initiation and progression, but no connection between CDC25B levels and drug sensitivity in pancreatic cancer has been reported. Based on our finding that bromodomain and extraterminal domain (BET) inhibitors decrease levels of CDC25B, we aim to compare the sensitivity of models expressing contrasting levels of CDC25B to the BET inhibitor JQ1, in pancreatic cancer cell lines in vitro and in patient-derived xenograft (PDX) models of pancreatic ductal adenocarcinoma (PDAC) in vivo . Methods: We compared the efficacy of the standard of care agent gemcitabine with the BET inhibitor JQ1, using alamarBlue assays to determine IC 50 s of three pancreatic cancer cell lines in vitro . We used immunohistochemistry (IHC) and immunoblot (IB) to detect CDC25B. We also compared the effect of each agent on the progression of PDX models of PDAC in vivo with contrasting levels of CDC25B. Results: Immunohistochemical data demonstrated that levels of CDC25B differed by ~2- to 5-fold in cell lines and PDX models used. In vitro data showed that the level of CDC25B paralleled sensitivity to JQ1. Similarly, in vivo data showed that tumors with high-level CDC25B were more sensitive to JQ1 than tumors with lower CDC25B. The combination of JQ1 + a pan CDC25 inhibitor was synergistic in gemcitabine-resistant Panc1.gemR cells that had relatively high levels of CDC25B expression compared to parent cells. Conclusion: The data suggest that CDC25B may be an independent indicator of sensitivity to BET inhibitors and that CDC25B may contribute to gemcitabine insensitivity in this tumor type., Competing Interests: All authors declared that there are no conflicts of interest., (© The Author(s) 2024.)

Conjugation of steroids and sterol compounds with a sulfonate group is a major pathway in the regulation of their activity, synthesis and excretion. Three human cytosolic sulfotransferases are highly involved in the sulfonation of sterol compounds. SULT1E1 has a low nM affinity for estrogen sulfonation and also conjugates non-aromatic steroids with a significantly lower affinity. SULT2A1 is responsible for the high levels of fetal and adult dehydroepiandrosterone (DHEA) sulfate synthesis in the adrenal gland as well as many 3α and 3ß-hydroxysteroids and bile acids. SULT2B1b is responsible for the majority of cholesterol sulfation in tissues as well as conjugating 3ß-hydroxysteroids. Although there are multiple methods for assaying cytosolic SULT activity, two relatively simple, rapid and versatile assays for steroid sulfonation are described. The first method utilizes radiolabeled substrates and organic solvent extraction to isolate the radiolabeled product from the aqueous phase. The second assay utilizes 35 S-3'-phosphoadenosine 5'-phosphosulfate (PAPS) to generate 35 S-conjugated products that are resolved by thin layer chromatography. Both assays useful in situations requiring measurement of SULT activity in a timely manner., (Copyright © 2023. Published by Elsevier Inc.)

We reported previously that the BET inhibitor (BETi) JQ1 decreases levels of the DNA repair protein RAD51 and that this decrease is concomitant with increased levels of DNA damage. Based on these findings, we hypothesized that a BETi would augment DNA damage produced by radiation and function as a radiosensitizer. We used clonogenic assays to evaluate the effect of JQ1 ± ionizing radiation (IR) on three pancreatic cancer cell lines in vitro . We performed immunofluorescence assays to assess the impact of JQ1 ± IR on DNA damage as reflected by levels of the DNA damage marker γH2AX, and immunoblots to assess levels of the DNA repair protein RAD51. We also compared the effect of these agents on the clonogenic potential of transfectants that expressed contrasting levels of the principle molecular targets of JQ1 (BRD2, BRD4) to determine whether levels of these BET proteins affected sensitivity to JQ1 ± IR. The data show that JQ1 + IR decreased the clonogenic potential of pancreatic cancer cells more than either modality alone. This anticlonogenic effect was associated with increased DNA damage and decreased levels of RAD51. Further, lower levels of BRD2 or BRD4 increased sensitivity to JQ1 and JQ1 + IR, suggesting that pre-treatment levels of BRD2 or BRD4 may predict sensitivity to a BETi or to a BETi + IR. We suggest that a BETi + IR merits evaluation as therapy prior to surgery for pancreatic cancer patients with borderline resectable disease., Competing Interests: EY serves as an advisory consultant for AstraZeneca, Bayer, Clovis, and Strata Oncology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Garcia, Miller, Zeng, van Waardenburg, Yang and Yoon.)

Introduction: [ 18 F]PSMA-1007 has potential advantages over [ 68  Ga]Ga-PSMA-11, although limited prospective data evaluating diagnostic performance exist. The aims of this study are to describe the concordance of [ 18 FPSMA-1007 and [ 68  Ga]Ga-PSMA-11 for TNM with the American Joint Committee on Cancer (AJCC) prognostic stage and assess differences in tracer uptake., Methods: Fifty men (mean age 71.8) were imaged with [ 68  Ga]Ga-PSMA-11 and [ 18 F]PSMA-1007 < 4 weeks apart. Images were independently reported according to TNM by two experienced nuclear medicine specialists blinded to the other scan and prior imaging. Discordant results were resolved by a third independent nuclear medicine specialist. Quantitative analysis of lesion uptake and physiologic tissue for each tracer was performed by one experienced reader., Results: Scan indications were initial staging (n = 12), biochemical recurrence (n = 27) and metastatic disease evaluation (n = 11). Most patients had ISUP grade group 3 or higher. Median PSA value was 2.7 ng/ml (IQR 0.7-12.0), and a minority of patients (28%) were currently treated with androgen deprivation therapy. [ 18 F]PSMA-1007 uptake was significantly higher than [ 68 Ga]Ga-PSMA-11 in local recurrence, nodal and distant metastases and most physiologic sites (including bone) except for urinary bladder which was significantly lower. [ 18 F]PSMA-1007 upstaged local prostate staging in 5/17 patients, local recurrence in 3/33 patients, regional nodal disease in 3/50 patients and 1 distant metastasis (bladder). [ 68 Ga]Ga-PSMA-11 upstaged regional nodal disease in 1/50 patients and distant metastasis in one patient (right adrenal). Overall AJCC prognostic stage was concordant in 46/50 (92%) patients, with two patients upstaged for both [ 18 F]PSMA-1007 and [ 68 Ga]Ga-PSMA-11. [ 18 F]PSMA-1007 had more equivocal results (one regional node; six equivocal bone lesions, one of which was subsequently confirmed metastatic) than [ 68 Ga]Ga-PSMA-11 (one equivocal local recurrence)., Conclusion: Overall AJCC prognostic stage was similar (92%) between [ 18 F]PSMA-1007 and [ 68 Ga]Ga-PSMA-11. [ 18 F]PSMA-1007 demonstrates higher uptake within involved nodes and distant metastases and most physiologic sites except urinary bladder which aided [ 18 F]PSMA-1007 local staging of the prostate primary/local recurrence and regional nodal disease adjacent ureters. However, [ 18 F]PSMA-1007 liver uptake obscured a solitary right adrenal metastasis, and more equivocal bone lesions were identified. Trial registration The study was registered with Australia New Zealand Clinical Trials Registry (ACTRN12618000665235) on 24 April 2018., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Data regarding COVID-19 vaccine efficacy and adverse events (AE) in patients with autoimmune and inflammatory rheumatic diseases (AIIRD) have been published recently although these mostly include the mRNA vaccines (Pfizer-BioNTech and Moderna) and the ChAdOx1 nCoV-19/AZD1222 (Oxford-AstraZeneca). This research aimed to study the prevalence of AE presented with six different SARS-CoV-2 vaccines {ChadOX1 nCoV-19 (AZD1222), Ad5-nCoV2, Ad26.COV2.S, mRNA-1273, BNT162b2, and CoronaVac} in Mexican patients with AIIRD. We performed a cross-sectional study about vaccine history. Two hundred and twenty five consecutive patients were recruited, mean age was 50.7 years and the majority (n = 213; 94.6%) were females. One hundred and seven (47.5%) received BNT162b2 mRNA, 34 (15.1%) Ad5-nCoV, 29 (12.8%) mRNA-1273, 28 (12.4%) ChAdOX1 nCoV-19 (AZD1222), 22 (9.7%) CoronaVac and 5 (2.2%) Ad26.COV2.S. The vaccines that had the most AE proportionally to the number of patients vaccinated were Janssen (5; 100%) followed by Pfizer-BioNTEch (86; 80%) and CanSinoBIO (27; 79.4%). Localized pain was the most frequent (158; 70.2%) AE. Fatigue (78; 34.7%), headache (69; 30.6%) and muscle ache (66; 29.3%) were the most common systemic symptoms. No serious AE that required medical attention or hospitalization were reported. The current results support the safety of different COVID-19 vaccines in patients with AIIRD. This information can help fight vaccine hesitancy in this population., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: [ 18 F]PSMA-1007 offers advantages of low urinary tracer excretion and theoretical improved spatial resolution for imaging prostate cancer. However, non-specific bone lesions (NSBLs), defined as mild to moderate focal bone uptake without a typical morphological correlate on CT, are a common finding on [ 18 F]PSMA-1007 PET/CT. The purpose of this study was to investigate the clinical outcomes of patients with [ 18 F]PSMA-1007 avid NSBLs, to determine whether patients with NSBLs represent a higher risk clinical cohort, and to determine whether SUVmax can be used as a classifier of bone metastasis., Methods: A retrospective audit of 214 men with prostate cancer was performed to investigate the clinical outcomes of [ 18 F]PSMA-1007 avid NSBLs according to defined criteria. We also compared the serum PSA, Gleason score, and uptake time of patients with [ 18 F]PSMA-1007 avid NSBLs to patients without [ 18 F]PSMA-1007 avid bone lesions. Finally, we analysed an SUVmax threshold to identify bone metastases using ROC curve analysis., Results: Ninety-four of 214 patients (43.9%) demonstrated at least one NSBL. No [ 18 F]PSMA-1007 avid NSBLs met criteria for a likely malignant or definitely malignant lesion after a median 15.8-month follow-up interval (11.9% definitely benign, 50.3% likely benign, and 37.7% equivocal). There were no statistically significant differences in serum PSA, Gleason score, and uptake time between patients with [ 18 F]PSMA-1007 avid NSBLs and those without [ 18 F]PSMA-1007 avid bone lesions. All NSBLs with adequate follow-up had SUVmax ≤ 11.1. The value of the highest SUVmax distinguished between NSBLs and definite prostate cancer bone metastases, whereby an SUVmax threshold of ≥ 7.2 maximized the Youden's index., Conclusion: [ 18 F]PSMA-1007 avid NSBLs rarely represent prostate cancer bone metastases. When identified in the absence of definite metastatic disease elsewhere, it is appropriate to classify those with SUVmax < 7.2 as likely benign. NSBLs with SUVmax 7.2-11.1 may be classified as equivocal or metastatic, with patient clinical risk factors, scan appearance, and potential management implications used to guide interpretation., (© 2021. Crown.)

Background: Convalescent plasma has been widely used to treat COVID-19 and is under investigation in numerous randomized clinical trials, but results are publicly available only for a small number of trials. The objective of this study was to assess the benefits of convalescent plasma treatment compared to placebo or no treatment and all-cause mortality in patients with COVID-19, using data from all available randomized clinical trials, including unpublished and ongoing trials (Open Science Framework, https://doi.org/10.17605/OSF.IO/GEHFX )., Methods: In this collaborative systematic review and meta-analysis, clinical trial registries (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform), the Cochrane COVID-19 register, the LOVE database, and PubMed were searched until April 8, 2021. Investigators of trials registered by March 1, 2021, without published results were contacted via email. Eligible were ongoing, discontinued and completed randomized clinical trials that compared convalescent plasma with placebo or no treatment in COVID-19 patients, regardless of setting or treatment schedule. Aggregated mortality data were extracted from publications or provided by investigators of unpublished trials and combined using the Hartung-Knapp-Sidik-Jonkman random effects model. We investigated the contribution of unpublished trials to the overall evidence., Results: A total of 16,477 patients were included in 33 trials (20 unpublished with 3190 patients, 13 published with 13,287 patients). 32 trials enrolled only hospitalized patients (including 3 with only intensive care unit patients). Risk of bias was low for 29/33 trials. Of 8495 patients who received convalescent plasma, 1997 died (23%), and of 7982 control patients, 1952 died (24%). The combined risk ratio for all-cause mortality was 0.97 (95% confidence interval: 0.92; 1.02) with between-study heterogeneity not beyond chance (I 2  = 0%). The RECOVERY trial had 69.8% and the unpublished evidence 25.3% of the weight in the meta-analysis., Conclusions: Convalescent plasma treatment of patients with COVID-19 did not reduce all-cause mortality. These results provide strong evidence that convalescent plasma treatment for patients with COVID-19 should not be used outside of randomized trials. Evidence synthesis from collaborations among trial investigators can inform both evidence generation and evidence application in patient care., (© 2021. The Author(s).)

Gemcitabine is used to treat pancreatic cancer (PC), but is not curative. We sought to determine whether gemcitabine + a BET bromodomain inhibitor was superior to gemcitabine, and identify proteins that may contribute to the efficacy of this combination. This study was based on observations that cell cycle dysregulation and DNA damage augment the efficacy of gemcitabine. BET inhibitors arrest cells in G1 and allow increases in DNA damage, likely due to inhibition of expression of DNA repair proteins Ku80 and RAD51. BET inhibitors (JQ1 or I-BET762) + gemcitabine were synergistic in vitro, in Panc1, MiaPaCa2 and Su86 PC cell lines. JQ1 + gemcitabine was more effective in vivo than either drug alone in patient-derived xenograft models ( P < 0.01). Increases in the apoptosis marker cleaved caspase 3 and DNA damage marker γH2AX paralleled antitumor efficacy. Notably, RNA-seq data showed that JQ1 + gemcitabine selectively inhibited HMGCS2 and APOC1 ~6-fold, compared to controls. These proteins contribute to cholesterol biosynthesis and lipid metabolism, and their overexpression supports tumor cell proliferation. IPA data indicated that JQ1 + gemcitabine selectively inhibited the LXR/RXR activation pathway, suggesting the hypothesis that this inhibition may contribute to the observed in vivo efficacy of JQ1 + gemcitabine.

Stroke is a leading cause of death and disability. The pathophysiological mechanisms associated with stroke are very complex and not fully understood. Lysosomal function has a vital physiological function in the maintenance of cellular homeostasis. In neurons, CTSD (cathepsin D) is an essential protease involved in the regulation of proteolytic activity of the lysosomes. Loss of CTSD leads to lysosomal dysfunction and accumulation of different cellular proteins implicated in neurodegenerative diseases. In cerebral ischemia, the role of CTSD and lysosomal function is not clearly defined. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons and the middle cerebral artery occlusion (MCAO) model of stroke to assess the role of CTSD in stroke pathophysiology. Our results show a time-dependent decrease in CTSD protein levels and activity in the mouse brain after stroke and neurons following OGD, with concurrent defects in lysosomal function. We found that shRNA-mediated knockdown of CTSD in neurons is sufficient to cause lysosomal dysfunction. CTSD knockdown further aggravates lysosomal dysfunction and cell death in OGD-exposed neurons. Restoration of CTSD protein levels via lentiviral transduction increases CTSD activity in neurons and, thus, renders resistance to OGD-mediated defects in lysosomal function and cell death. This study indicates that CTSD-dependent lysosomal function is critical for maintaining neuronal survival in cerebral ischemia; thus, strategies focused on maintaining CTSD function in neurons are potentially novel therapeutic approaches to prevent neuronal death in stroke. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; FTD: frontotemporal dementia, HD: Huntington disease; LAMP1: lysosomal associated membrane protein 1; LSD: lysosomal storage disease; MCAO: middle cerebral artery occlusion; OGD: oxygen glucose deprivation; OGR: oxygen glucose resupply; PD: Parkinson disease; SQSMT1: sequestosome 1; TCA: trichloroacetic acid; TTC: triphenyl tetrazolium chloride.

Pancreatic cancer (PC) is anticipated to be second only to lung cancer as the leading cause of cancer-related deaths in the United States by 2030. Surgery remains the only potentially curative treatment for patients with pancreatic ductal adenocarcinoma (PDAC), the most common form of PC. Multiple recent preclinical studies focus on identifying effective treatments for PDAC, but the models available for these studies often fail to reproduce the heterogeneity of this tumor type. Data generated with such models are of unknown clinical relevance. Patient-derived xenograft (PDX) models offer several advantages over human cell line-based in vitro and in vivo models and models of non-human origin. PDX models retain genetic characteristics of the human tumor specimens from which they were derived, have intact stromal components, and are more predictive of patient response than traditional models. This review briefly describes the advantages and disadvantages of 2D cultures, organoids and genetically engineered mouse (GEM) models of PDAC, and focuses on the applications, characteristics, advantages, limitations, and the future potential of PDX models for improving the management of PDAC.

Pancreatic cancer is a fatal disease. The five-year survival for patients with all stages of this tumor type is less than 10%, with a majority of patients dying from drug resistant, metastatic disease. Gemcitabine has been a standard of care for the treatment of pancreatic cancer for over 20 years, but as a single agent gemcitabine is not curative. Since the only therapeutic option for the over 80 percent of pancreatic cancer patients ineligible for surgical resection is chemotherapy with or without radiation, the last few decades have seen a significant effort to develop effective therapy for this disease. This review addresses preclinical and clinical efforts to identify agents that target molecular characteristics common to pancreatic tumors and to develop mechanism-based combination approaches to therapy. Some of the most promising combinations include agents that inhibit transcription dependent on BET proteins (BET bromodomain inhibitors) or that inhibit DNA repair mediated by PARP (PARP inhibitors)., Competing Interests: Declaration of Competing Interest The authors declare that there is no competing interest related to this review manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Our previous finding that the BET inhibitor (BETi) JQ1 increases levels of the DNA damage marker γH2AX suggested that JQ1 might enhance the sensitivity of tumor cells to PARP inhibitors (PARPi), which are selectively toxic to cells that harbor relatively high levels of DNA damage. To address this hypothesis, we evaluated the effect of a BETi (JQ1 or I-BET762) combined with a PARPi (olaparib or veliparib) in KKU-055 and KKU-100 cholangiocarcinoma (CCA) cell lines and of JQ1 with olaparib in a xenograft model of CCA. Each combination was more effective than any of the four drugs as single agents. Combination indices ranged from 0.1 to 0.8 at the ED50 for all combinations, indicating synergy and demonstrating that synergy was not limited to a specific combination. Mechanistically, downregulation of BETi molecular targets BRD2 or BRD4 by shRNA sensitized CCA cells to BETi as single agents as well as to the combination of a BETi + a PARPi. Our data indicate that combinations of a BETi with a PARPi merit further evaluation as a promising strategy for CCA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aim: Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma (PDAC), but neither gemcitabine alone nor in combination produces durable remissions of this tumor type. We developed three PDAC patient-derived xenograft (PDX) models with gemcitabine resistance (gemR) acquired in vivo , with which to identify mechanisms of resistance relevant to drug exposure in vivo and to evaluate novel therapies., Methods: Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly. Tumors initially responded, but regrew on treatment and were designated gemR. We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance [ribonucleotide reductase subunit M1 (RRM1), RRM2, human concentrative nucleoside transporter 1 (hCNT1), human equilibrative nucleoside transporter 1 (hENT1), cytidine deaminase (CDA), and deoxycytidine kinase (dCK)] in gemR and respective gemcitabine-naive parental tumors., Results: Parental and gemR tumors did not differ in tumor cell morphology, amount of tumor-associated stroma, or expression of stem cell markers. No consistent pattern of expression of the six gemR marker proteins was observed among the models. Increases in RRM1 and CDA were consistent with in vitro -derived gemR models. However, rather than the expected decreases of hCNT1, hENT1, and dCK, gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors., Conclusion: These models are the first PDAC PDX models with gemcitabine resistance acquired in vivo . The data indicate that mechanisms identified in models with resistance acquired in vitro are unlikely to be the predominant mechanisms when resistance is acquired in vivo . Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models., Competing Interests: Conflicts of interest All authors declared that there are no conflicts of interest.

Sulfotransferase 4A1 (SULT4A1), a member of cytosolic sulfotransferases (SULT), is exclusively expressed in neurons with no known function. Severe phenotype and early postnatal death in SULT4A1 knockout mice revealed that SULT4A1 is an essential neuronal protein. Localization of SULT4A1 in different cytosolic compartments, including mitochondria, suggests multiple roles for this protein. We observed that knockdown of SULT4A1 results in the accumulation of reactive oxygen species in primary cortical neurons, suggesting a potential role of SULT4A1 in regulating redox homeostasis. Expression of SULT4A1 in the human neuroblastoma SH-SY5Y cells revealed a defused but nonuniform staining pattern in the cytoplasm, with increased density around mitochondria. Subcellular fractionation of SULT4A1 expressing SH-SY5Y cells confirms the presence of SULT4A1 in mitochondrial fractions. SULT4A1 expressing cells display significant protection against H 2 O 2 -mediated defects in mitochondrial function and loss of mitochondrial membrane potential. Expression of SULT4A1 in SH-SY5Y cells also protects against H 2 O 2 -induced cell death. These data indicate that SULT4A1 protects mitochondria against oxidative damage and may serve as a potential pharmacological target in neural diseases involving mitochondrial dysfunction and oxidative stress. SIGNIFICANCE STATEMENT: Studies on SULT4A1 knockout mice suggest that SULT4A1 plays a vital role in neuronal function and survival via yet undefined mechanisms. Our data demonstrate that depletion of SULT4A1 induces oxidative stress in neurons and expression of SULT4A1 in SH-SY5Y cells protects against oxidative-stress-induced mitochondrial dysfunction and cell death. These results suggest that SULT4A1 may have a crucial protective function against mitochondrial dysfunction and oxidative stress, and may serve a potential therapeutic target in different neurological diseases involving mitochondrial dysfunction and oxidative stress., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: DNA repair deficiency accumulates DNA damage and sensitizes tumor cells to PARP inhibitors (PARPi). Based on our observation that the BET inhibitor JQ1 increases levels of DNA damage, we evaluated the efficacy of JQ1 + the PARPi olaparib in preclinical models of pancreatic ductal adenocarcinoma (PDAC). We also addressed the mechanism by which JQ1 increased DNA damage., Methods: The effect of JQ1 + olaparib on in vivo tumor growth was assessed with patient-derived xenograft (PDX) models of PDAC. Changes in protein expression were detected by immunohistochemistry and immunoblot. In vitro growth inhibition and mechanistic studies were done using alamarBlue, qRT-PCR, immunoblot, immunofluorescence, ChIP, and shRNA knockdown assays., Findings: Tumors exposed in vivo to JQ1 had higher levels of the DNA damage marker γH2AX than tumors exposed to vehicle only. Increases in γH2AX was concomitant with decreased expression of DNA repair proteins Ku80 and RAD51. JQ1 + olaparib inhibited the growth of PDX tumors greater than either drug alone. Mechanistically, ChIP assays demonstrated that JQ1 decreased the association of BRD4 and BRD2 with promoter loci of Ku80 and RAD51, and shRNA data showed that expression of Ku80 and RAD51 was BRD4- and BRD2-dependent in PDAC cell lines., Interpretation: The data are consistent with the hypothesis that JQ1 confers a repair deficient phenotype and the consequent accumulation of DNA damage sensitizes PDAC cells to PARPi. Combinations of BET inhibitors with PARPi may provide a novel strategy for treating PDAC. FUND: NIH grants R01CA208272 and R21CA205501; UAB CMB T32 predoctoral training grant., (Copyright © 2019. Published by Elsevier B.V.)

Sulfotransferase 4A1 (SULT4A1) belongs to the cytosolic sulfotransferase (SULT) superfamily of enzymes that catalyze sulfonation reactions with a variety of endogenous and exogenous substrates. Of the SULTs, SULT4A1 was shown to have the highest sequence homology between vertebrate species, yet no known function or enzymatic activity has been identified for this orphan SULT. To better understand SULT4A1 function in mammalian brain, two mutant SULT4A1 mouse strains were generated utilizing clustered regulatory interspaced short palindromic repeats (CRISPR)-content-addressable storage (Cas) 9 technology. The first strain possessed a 28-base pair (bp) deletion (Δ28) in exon 1 that resulted in a frameshift mutation with premature termination. The second strain possessed a 12-bp in-frame deletion (Δ12) immediately preceding an active site histidine 111 common to the SULT family. Homozygous pups of both strains present with severe and progressive neurologic symptoms, including tremor, absence seizures, abnormal gait, ataxia, decreased weight gain compared with littermates, and death around postnatal days 21-25. SULT4A1 immunostaining was decreased in brains of heterozygous pups and not detectable in homozygous pups of both SULT4A1 mutants. SULT4A1 localization in subcellular fractions of mouse brain showed SULT4A1 associated with mitochondrial, cytosolic, and microsomal fractions, a novel localization pattern for SULTs. Finally, primary cortical neurons derived from embryonic (E15) CD-1 mice expressed high levels of SULT4A1 throughout the cell except in nuclei. Taken together, SULT4A1 appears to be an essential neuronal protein required for normal brain function, at least in mammals. Mouse models will be valuable in future studies to investigate the regulation and functions of SULT4A1 in the mammalian brain., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Cholangiocarcinoma (CCA) is a fatal disease with a 5-year survival of <30%. For a majority of patients, chemotherapy is the only therapeutic option, and virtually all patients relapse. Gemcitabine is the first-line agent for treatment of CCA. Patients treated with gemcitabine monotherapy survive ∼8 months. Combining this agent with cisplatin increases survival by ∼3 months, but neither regimen produces durable remissions. The molecular etiology of this disease is poorly understood. To facilitate molecular characterization and development of effective therapies for CCA, we established a panel of patient-derived xenograft (PDX) models of CCA. We used two of these models to investigate the antitumor efficacy and mechanism of action of the bromodomain inhibitor JQ1, an agent that has not been evaluated for the treatment of CCA. The data show that JQ1 suppressed the growth of the CCA PDX model CCA2 and demonstrate that growth suppression was concomitant with inhibition of c-Myc protein expression. A second model (CCA1) was JQ1-insensitive, with tumor progression and c-Myc expression unaffected by exposure to this agent. Also selective to CCA2 tumors, JQ1 induced DNA damage and apoptosis and downregulated multiple c-Myc transcriptional targets that regulate cell-cycle progression and DNA repair. These findings suggest that c-Myc inhibition and several of its transcriptional targets may contribute to the mechanism of action of JQ1 in this tumor type. We conclude that BET inhibitors such as JQ1 warrant further investigation for the treatment of CCA. Mol Cancer Ther; 17(1); 107-18. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Neuroblastoma is a pediatric tumor characterized by histologic heterogeneity, and accounts for ~15% of childhood deaths from cancer. The five-year survival for patients with high-risk stage 4 disease has not improved in two decades. We used whole exome sequencing (WES) to identify mutations present in three independent high-risk stage 4 neuroblastoma tumors (COA/UAB-3, COA/UAB -6 and COA/UAB -8) and a stage 3 tumor (COA/UAB-14). Among the four tumors WES analysis identified forty-three mutations that had not been reported previously, one of which was present in two of the four tumors. WES analysis also corroborated twenty-two mutations that were reported previously. No single mutation occurred in all four tumors or in all stage 4 tumors. Three of the four tumors harbored genes with CADD scores ≥20, indicative of mutations associated with human pathologies. The average depth of coverage ranged from 39.68 to 90.27, with >99% sequences mapping to the genome. In summary, WES identified sixty-five coding mutations including forty-three mutations not reported previously in primary neuroblastoma tumors. The three stage 4 tumors contained mutations in genes encoding protein products that regulate immune function or cell adhesion and tumor cell metastasis.

The primary aim of this study was to evaluate the antitumor efficacy of the bromodomain inhibitor JQ1 in pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (tumorgraft) models. A secondary aim of the study was to evaluate whether JQ1 decreases expression of the oncogene c-Myc in PDAC tumors, as has been reported for other tumor types. We used five PDAC tumorgraft models that retain specific characteristics of tumors of origin to evaluate the antitumor efficacy of JQ1. Tumor-bearing mice were treated with JQ1 (50 mg/kg daily for 21 or 28 days). Expression analyses were performed with tumors harvested from host mice after treatment with JQ1 or vehicle control. An nCounter PanCancer Pathways Panel (NanoString Technologies) of 230 cancer-related genes was used to identify gene products affected by JQ1. Quantitative RT-PCR, immunohistochemistry and immunoblots were carried out to confirm that changes in RNA expression reflected changes in protein expression. JQ1 inhibited the growth of all five tumorgraft models (P<0.05), each of which harbors a KRAS mutation; but induced no consistent change in expression of c-Myc protein. Expression profiling identified CDC25B, a regulator of cell cycle progression, as one of the three RNA species (TIMP3, LMO2 and CDC25B) downregulated by JQ1 (P<0.05). Inhibition of tumor progression was more closely related to decreased expression of nuclear CDC25B than to changes in c-Myc expression. JQ1 and other agents that inhibit the function of proteins with bromodomains merit further investigation for treating PDAC tumors. Work is ongoing in our laboratory to identify effective drug combinations that include JQ1.

Progressive metastatic disease is a major cause of mortality for patients diagnosed with multiple types of solid tumors. One of the long-term goals of our laboratory is to identify  molecular interactions that regulate metastasis, as a basis for developing agents that inhibit this process. Toward this goal, we recently demonstrated that intercellular adhesion molecule-2 (ICAM-2) converted neuroblastoma (NB) cells from a metastatic to a non-metastatic phenotype, a previously unknown function for ICAM-2. Interestingly, ICAM-2 suppressed metastatic but not tumorigenic potential in preclinical models, supporting a novel mechanism of regulating metastasis. We hypothesized that the effects of ICAM-2 on NB cell phenotype depend on the interaction of ICAM-2 with the cytoskeletal linker protein α-actinin. The goal of the study presented here was to evaluate the impact of α-actinin binding to ICAM-2 on the phenotype of NB tumor cells. We used in silico approaches to examine the likelihood that the cytoplasmic domain of ICAM-2 binds directly to α-actinin. We then expressed variants of ICAM-2 with mutated α-actinin-binding domains, and compared the impact of ICAM-2 and each variant on NB cell adhesion, migration, anchorage-independent growth, co-precipitation with α-actinin and production of localized and disseminated tumors in vivo. The in vitro and in vivo characteristics of cells expressing ICAM-2 variants with modified α-actinin-binding domains differed from cells expressing ICAM-2 wild type (WT) and also from cells that expressed no detectable ICAM-2. Like the WT protein, ICAM-2 variants inhibited cell adhesion, migration and colony growth in vitro. However, unlike the WT protein, ICAM-2 variants did not completely suppress development of disseminated NB tumors in vivo. The data suggest the presence of α-actinin-dependent and α-actinin-independent mechanisms, and indicate that the interaction of ICAM-2 with α-actinin is critical to conferring an ICAM-2-mediated non-metastatic phenotype in NB cells.

Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer.  Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types.  Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing.  Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models.  We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation.  Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained <10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies.

Background: Cell adhesion molecules (CAMs) are expressed ubiquitously. Each of the four families of CAMs is comprised of glycosylated, membrane-bound proteins that participate in multiple cellular processes including cell-cell communication, cell motility, inside-out and outside-in signaling, tumorigenesis, angiogenesis and metastasis. Intercellular adhesion molecule-2 (ICAM-2), a member of the immunoglobulin superfamily of CAMs, has six N-linked glycosylation sites at amino acids (asparagines) 47, 82, 105, 153, 178 and 187. Recently, we demonstrated a previously unknown function for ICAM-2 in tumor cells. We showed that ICAM-2 suppressed neuroblastoma cell motility and growth in soft agar, and induced a juxtamembrane distribution of F-actin in vitro. We also showed that ICAM-2 completely suppressed development of disseminated tumors in vivo in a murine model of metastatic NB. These effects of ICAM-2 on NB cell phenotype in vitro and in vivo depended on the interaction of ICAM-2 with the cytoskeletal linker protein α-actinin. Interestingly, ICAM-2 did not suppress subcutaneous growth of tumors in mice, suggesting that ICAM-2 affects the metastatic but not the tumorigenic potential of NB cells. The goal of the study presented here was to determine if the glycosylation status of ICAM-2 influenced its function in neuroblastoma cells., Methods: Because it is well documented that glycosylation facilitates essential steps in tumor progression and metastasis, we investigated whether the glycosylation status of ICAM-2 affected the phenotype of NB cells. We used site-directed mutagenesis to express hypo- or non-glycosylated variants of ICAM-2, by substituting alanine for asparagine at glycosylation sites, and compared the impact of each variant on NB cell motility, anchorage-independent growth, interaction with intracellular proteins, effect on F-actin distribution and metastatic potential in vivo., Results: The in vitro and in vivo phenotypes of cells expressing glycosylation site variants differed from cells expressing fully-glycosylated ICAM-2 or no ICAM-2. Most striking was the finding that mice injected intravenously with NB cells expressing glycosylation site variants survived longer (P ≤ 0.002) than mice receiving SK-N-AS cells with undetectable ICAM-2. However, unlike fully-glycosylated ICAM-2, glycosylation site variants did not completely suppress disseminated tumor development., Conclusions: Reduced glycosylation of ICAM-2 significantly attenuated, but did not abolish, its ability to suppress metastatic properties of NB cells.

A simple and sensitive analytical method for simultaneous determination of anastrozole, bicalutamide, and tamoxifen as well as their synthetic impurities, anastrozole pentamethyl, bicalutamide 3-fluoro-isomer, and tamoxifen e-isomer, was developed and validated by using high performance liquid chromatography (HPLC). The separation was achieved on a Symmetry(®) C-8 column (100×4.6 mm i.d., 3.5 μm) at room temperature (±24 °C), with a mobile phase consisting of acetonitrile/water containing 0.18% N,N dimethyloctylamine and pH adjusted to 3.0 with orthophosphoric acid (46.5/53.5, v/v) at a flow rate of 1.0 mL min(-1) within 20 min. The detection was made at a wavelength of 270 nm by using ultraviolet (UV) detector. No interference peaks from excipients and relative retention time indicated the specificity of the method. The calibration curve showed correlation coefficients (r) >0.99 calculated by linear regression and analysis of variance (ANOVA). The limit of detection (LOD) and limit of quantitation (LOQ), respectively, were 2.2 and 6.7 μg mL(-1) for anastrozole, 2.61 and 8.72 μg mL(-1) for bicalutamide, 2.0 and 6.7 μg mL(-1) for tamoxifen, 0.06 and 0.22 μg mL(-1) for anastrozole pentamethyl, 0.02 and 0.07 μg mL(-1) for bicalutamide 3-fluoro-isomer, and 0.002 and 0.007 μg mL(-1) for tamoxifen e-isomer. Intraday and interday relative standard deviations (RSDs) were <2.0% (drugs) and <10% (degradation products) as well as the comparison between two different analysts, which were calculated by f test., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a RecQ-type DNA helicase and a 3'-5' exonuclease, but its exact role in DNA metabolism is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutSalpha), MSH2/MSH3 (MutSbeta) and MLH1/PMS2 (MutLalpha) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutSalpha and MutSbeta can strongly stimulate the helicase activity of WRN specifically on forked DNA structures with a 3'-single-stranded arm. The stimulatory effect of MutSalpha on WRN-mediated unwinding is enhanced by a G/T mismatch in the DNA duplex ahead of the fork. The MutLalpha protein known to bind to the MutS alpha-heteroduplex complexes has no effect on WRN-mediated DNA unwinding stimulated by MutSalpha, nor does it affect DNA unwinding by WRN alone. Our data are consistent with results of genetic experiments in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequences.

The role of the human RECQ5beta helicase in the maintenance of genomic stability remains elusive. Here we show that RECQ5beta promotes strand exchange between arms of synthetic forked DNA structures resembling a stalled replication fork in a reaction dependent on ATP hydrolysis. BLM and WRN can also promote strand exchange on these structures. However, in the presence of human replication protein A (hRPA), the action of these RecQ-type helicases is strongly biased towards unwinding of the parental duplex, an effect not seen with RECQ5beta. A domain within the non-conserved portion of RECQ5beta is identified as being important for its ability to unwind the lagging-strand arm and to promote strand exchange on hRPA-coated forked structures. We also show that RECQ5beta associates with DNA replication factories in S phase nuclei and persists at the sites of stalled replication forks after exposure of cells to UV irradiation. Moreover, RECQ5beta is found to physically interact with the polymerase processivity factor proliferating cell nuclear antigen in vitro and in vivo. Collectively, these findings suggest that RECQ5beta may promote regression of stalled replication forks to facilitate the bypass of replication-blocking lesions by template-switching. Loss of such activity could explain the elevated level of mitotic crossovers observed in RECQ5beta-deficient cells.

Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIalpha (TOPO IIIalpha) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine-1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution.

The product of the gene mutated in Bloom's syndrome, BLM, is a 3'-5' DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg(2+), is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks.

Proteins belonging to the highly conserved RecQ helicase family are essential for the maintenance of genomic stability. Here, we describe the biochemical properties of the human RECQ5beta protein. Like BLM and WRN, RECQ5beta is an ATP-dependent 3'-5' DNA helicase that can promote migration of Holliday junctions. However, RECQ5beta required the single-stranded DNA-binding protein RPA in order to mediate the efficient unwinding of oligonucleotide-based substrates. Surprisingly, we found that RECQ5beta possesses an intrinsic DNA strand-annealing activity that is inhibited by RPA. Analysis of deletion variants of RECQ5beta revealed that the DNA helicase activity resides in the conserved N-terminal portion of the protein, whereas strand annealing is mediated by the unique C-terminal domain. Moreover, the strand-annealing activity of RECQ5beta was strongly inhibited by ATPgammaS, a poorly hydrolyzable analog of ATP. This effect was alleviated by mutations in the ATP-binding motif of RECQ5beta, indicating that the ATP-bound form of the protein cannot promote strand annealing. This is the first demonstration of a DNA helicase with an intrinsic DNA strand-annealing function residing in a separate domain.

Bloom (BLM) and Werner (WRN) syndrome proteins are members of the RecQ family of SF2 DNA helicases. In this paper, we show that restricting the rotational DNA backbone flexibility, by introducing vinylphosphonate internucleotide linkages in the translocating DNA strand, inhibits efficient duplex unwinding by these enzymes. The human single-stranded DNA binding protein replication protein A (RPA) fully restores the unwinding activity of BLM and WRN on vinylphosphonate-containing substrates while the heterologous single-stranded DNA binding protein from Escherichia coli (SSB) restores the activity only partially. Both RPA and SSB fail to restore the unwinding activity of the SF1 PcrA helicase on modified substrates, implying specific interactions of RPA with the BLM and WRN helicases. Our data highlight subtle differences between SF1 and SF2 helicases and suggest that although RecQ helicases belong to the SF2 family, they are mechanistically more similar to the SF1 PcrA helicase than to other SF2 helicases that are not affected by vinylphosphonate modifications.

Bloom syndrome protein forms an oligomeric ring structure and belongs to a group of DNA helicases showing extensive homology to the Escherichia coli DNA helicase RecQ, a suppressor of illegitimate recombination. After over-production in E.coli, we have purified the RecQ core of BLM consisting of the DEAH, RecQ-Ct and HRDC domains (amino acid residues 642-1290). The BLM(642-1290) fragment could function as a DNA-stimulated ATPase and as a DNA helicase, displaying the same substrate specificity as the full-size protein. Gel-filtration experiments revealed that BLM(642-1290) exists as a monomer both in solution and in its single-stranded DNA-bound form, even in the presence of Mg(2+) and ATPgammaS. Rates of ATP hydrolysis and DNA unwinding by BLM(642-1290) showed a hyperbolic dependence on ATP concentration, excluding a co-operative interaction between ATP-binding sites. Using a lambda Spi(-) assay, we have found that the BLM(642-1290) fragment is able to partially substitute for the RecQ helicase in suppressing illegitimate recombination in E.coli. A deletion of 182 C-terminal amino acid residues of BLM(642-1290), including the HRDC domain, resulted in helicase and single-stranded DNA-binding defects, whereas kinetic parameters for ATP hydrolysis of this mutant were close to the BLM(642-1290) values. This confirms the prediction that the HRDC domain serves as an auxiliary DNA-binding domain. Mutations at several conserved residues within the RecQ-Ct domain of BLM reduced ATPase and helicase activities severely as well as single-stranded DNA-binding of the enzyme. Together, these data define a minimal helicase domain of BLM and demonstrate its ability to act as a suppressor of illegitimate recombination.

Background: The mechanisms underlying progressive renal fibrosis are unknown, but the common association of fibrosis and microvascular loss suggests that hypoxia per se may be a fibrogenic stimulus., Methods: To determine whether human renal fibroblasts (HRFs), the primary matrix-producing cells in the tubulointerstitium, possess oxygen-sensitive responses relevant to fibrogenesis, cells were exposed to 1% O2 in vitro., Results: Hypoxia simultaneously stimulated extracellular matrix synthesis and suppressed turnover with increased production of collagen alpha1(I) (Coll-I), decreased expression of collagenase, and increased tissue inhibitor of metalloproteinase (TIMP)-1. These effects are time dependent, require new RNA and protein synthesis, and are specific to hypoxia. The changes in Coll-I and TIMP-1 gene expression involve a heme-protein O2 sensor and protein kinase- and tyrosine kinase-mediated signaling. Although hypoxia induced transforming growth factor-beta1 (TGF-beta1), neutralizing anti-TGF-beta1-antibody did not block hypoxia-induced Coll-I and TIMP-1 mRNA expression. Furthermore, hypoxic-cell conditioned-medium had no effect on the expression of these mRNAs in naive fibroblasts, suggesting direct effects on gene transcription. Transient transfections identified a hypoxia response element (HRE) in the TIMP-1 promoter and demonstrated HIF-1-dependent promoter activation by decreased ambient pO2., Conclusions: These data suggest that hypoxia co-ordinately up-regulates matrix production and decreases turnover in renal fibroblasts. The results support a role for hypoxia in the pathogenesis of fibrosis and provide evidence for novel, direct hypoxic effects on the expression of genes involved in fibrogenesis.

Background: Previous research in our laboratory revealed that during the first year of life, infants who had more exposure to alcohol, as inferred from questionnaires about parental alcoholism and alcohol intake, mouthed an ethanol-scented toy more compared with less exposed infants. The present study focused on older children (3.8-6.0 years) to determine whether their hedonic response to the odor of alcohol was related to the drinking habits of their parents., Methods: Age-appropriate, game-like tasks that were fun for children and minimized the impact of language development were used to examine their preferences and identification of a variety of odors, one of which was beer., Results: The children's preference for the odor of beer varied as a function of the escape drinking of their mothers alone or both parents. That is, children who lived in a household in which one or both parents drank alcohol to escape were significantly more likely to dislike the odor bottle that contained alcohol when compared with children whose parents did not drink to escape. This difference between the groups was odor specific. Additional analyses also revealed that the fathers of children who rejected the beer odor reported drinking significantly more than the fathers of those who liked the odor., Conclusions: These findings suggest that some early learning about alcohol is based on sensory experiences and anchor it to children's experiences at home and the emotional context in which their parents experience alcohol.

Alveolitis is thought to precede permanent lung derangement in a variety of interstitial lung diseases (ILD). Bronchoalveolar lavage (BAL) cell differentials can be used to evaluate the intensity of alveolitis, whereas clinical, roentgenographic, and functional studies are insensitive monitors of lung inflammation. As ILD is generally diffuse, unilobar lavage is widely used and presumed to gauge overall lung inflammation. Consistency of lobe to lobe cell differential findings has not been systematically addressed. We analyzed 53 bilateral lobar lavages (right middle lobe and lingula) in 34 patients with sarcoidosis, idiopathic pulmonary fibrosis-collagen vascular disease (IPF-CV) and a group of mixed interstitial diseases. Cellular differentials from each lobe were independently assessed and compared. The sarcoid group, with predominantly T-lymphocytes in BAL, showed excellent interlobar correlation (r = 0.92, p less than 0.001), with only 17% showing a discrepancy of greater than 10% in percentage of T-lymphocytes. In contrast, the IPF-CV group, with predominantly neutrophils in BAL, showed good interlobar correlation (r = 0.79; p less than 0.01), but 35% had a greater than 10% discrepancy in percentage of neutrophils. Finally, the mixed group, with lymphocytes as the predominant cell in BAL, showed poor interlobar correlation (r = 0.58; p greater than 0.10), with 42% showing a greater than 10% discrepancy in percentage of lymphocytes. These results were not explicable on the basis of differences in volume of lavage fluid or total cells recovered, these parameters being remarkably consistent between lobes in the 3 patient groups and in a control group of 8 subjects without ILD. The chest roentgenogram was markedly insensitive to lavage interlobar variation.(ABSTRACT TRUNCATED AT 250 WORDS)

Rheumatoid arthritis (RA), a systemic disorder of unknown cause, is associated with a variety of well-recognized pulmonary abnormalities including interstitial lung disease. To investigate possible pathogenic events in this disorder, we performed bronchoalveolar lavage (BAL) in 24 patients with classic or definite RA. Using radiographic and physiologic parameters as well as BAL cell differentials, 3 distinct groups emerged. Group I consisted of 9 patients with evidence of clinical interstitial lung involvement. Group II consisted of 5 patients without evidence of clinical interstitial lung disease (normal chest roentgenogram and functional testing) but who had abnormal BAL cellular differentials. The 10 remaining patients (Group III) had no evidence of clinical interstitial lung disease and had normal BAL cell differentials. Bronchoalveolar lavage in Group I had a significantly increased percentage of neutrophils (12.4 +/- 4.2; p less than 0.05) compared with Group II (2.4 +/- 0.8) and Group III (2.9 +/- 0.6). All patients in Group II had elevated BAL lymphocytes (24.4 +/- 6.4) compared with Group I (10.1 +/- 3.8; p greater than 0.05) and Group III (5.4 +/- 0.7; p less than 0.01). Both Groups I and II had detectable IgM in BAL (0.02 +/- 0.01 and 0.04 +/- 0.02 mg/mg of albumin, respectively), whereas Group III patients and normal control subjects did not (p less than 0.01). There was a marked reduction in BAL T-lymphocyte Leu 3/Leu 2A (helper/suppressor) cell ratios in Group I (0.92 +/- 0.02; p less than 0.05) compared with Group II (2.6 +/- 0.6) and Group III (1.6 +/- 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)