Identification of Glandular (Acinar)/Tubule Formation in Invasive Carcinoma of the Breast: A Study to Determine Concordance Using the World Health Organization Definition.

CONTEXT.­: The Nottingham Grading System (NGS) developed by Elston and Ellis is used to grade invasive breast cancer (IBC). Glandular (acinar)/tubule formation is a component of NGS. OBJECTIVE.­: To investigate the ability of pathologists to identify individual structures that should be classified as glandular (acinar)/tubule formation. DESIGN.­: A total of 58 hematoxylin-eosin photographic images of IBC with 1 structure circled were classified as tubules (41 cases) or nontubules (17 cases) by Professor Ellis. Images were sent as a PowerPoint (Microsoft) file to breast pathologists, who were provided with the World Health Organization definition of a tubule and asked to determine if a circled structure represented a tubule. RESULTS.­: Among 35 pathologists, the κ statistic for assessing agreement in evaluating the 58 images was 0.324 (95% CI, 0.314-0.335). The median concordance rate between a participating pathologist and Professor Ellis was 94.1% for evaluating 17 nontubule cases and 53.7% for 41 tubule cases. A total of 41% of the tubule cases were classified correctly by less than 50% of pathologists. Structures classified as tubules by Professor Ellis but often not recognized as tubules by pathologists included glands with complex architecture, mucinous carcinoma, and the "inverted tubule" pattern of micropapillary carcinoma. A total of 80% of participants reported that they did not have clarity on what represented a tubule. CONCLUSIONS.­: We identified structures that should be included as tubules but that were not readily identified by pathologists. Greater concordance for identification of tubules might be obtained by providing more detailed images and descriptions of the types of structures included as tubules.

Validation of prediction tools for GI bleeding in patients on dual anti-platelet therapy after percutaneous coronary intervention.

BACKGROUND AND AIMS: The management of dual anti-platelet therapy after percutaneous coronary intervention (PCI) and GI bleeding (GIB) remains a clinical dilemma. We sought to identify predictors of GIB and recurrent bleeding and to determine whether recurrent bleeding increases the risk of major adverse cardiovascular events (MACEs). METHODS: In this single-center retrospective study, patients undergoing PCI were identified. The primary and secondary endpoints were GIB at 180 days and recurrent bleeding or MACE at 365 days. Logistic regression was used to identify predictors of GIB and recurrent bleeding. Cox proportional hazards modeling was used to determine whether recurrent bleeding can predict a MACE. RESULTS: Five hundred thirty-six patients were included. On multivariable analysis, PCI for acute coronary syndrome was associated with a 95% increased odds of GIB (P < .001). The P2Y12 inhibitor was continued in >90% of patients, which trended toward significance for recurrent bleeding (P < .10). The HAS-BLED score (Hypertension, Abnormal renal and liver function, Stroke, Bleeding tendency or predisposition, Labile INRs, Elderly, Drugs), including a labile international normalized ratio and prior major bleeding, was strongly associated with recurrent bleeding (P ≤ .009). Recurrent bleeding was associated with a 115% increased risk of MACEs (P = .02). We derived a novel risk score, named the SIGE score ([S]TEMI at PCI, having a labile [I]NR at PCI, index [G]IB within 180 days of PCI, and previous precatheterization [E]ndoscopy within 6 months), to predict recurrent bleeding at 365 days with a high predictive accuracy (area under the curve, .773; 95% confidence interval, .702-.845). CONCLUSIONS: The SIGE score may help to predict recurrent bleeding, which was shown to be associated with an increased risk of MACEs. Further external validation is needed.

Pathological response in mucinous carcinoma of breast after neoadjuvant therapy - a multi-institutional study.

Although mucinous carcinoma (MC) is considered a favorable histologic subtype of invasive breast cancer (BC), a subset of MC is managed with neoadjuvant therapy (NAT). The clinical and pathologic features of MC following NAT are not well characterized. The aim of this study is to characterize pathologic response in patients with MC treated with NAT, including neoadjuvant endocrine therapy (NET), neoadjuvant chemotherapy (NCT), and Herceptin-targeted NCT (H-NCT). We conducted a retrospective cohort study of 28 patients with MC who received preoperative adjuvant therapy followed by resection from three institutions between 2010 and 2020. Demographic and clinical information were retrieved from the medical records. Pathologic review of the post NAT resection specimens was performed including tumor grading, tumor size, staging, residual tumor cellularity, estrogen receptor (ER) and HER2 status. Nine (32 %) patients with ER+/HER2- MC received NET, 8 (29 %) ER+/HER2- MC were treated with NCT only and 11 (39 %) HER2+ MC received HER2-targeted NCT (H-NCT). The HER2+ MC patients were younger (45 vs. 64 years; p = 0.006). The HER2+ MC were of higher grade (p = 0.03) and more likely to be multifocal (p = 0.008). Only 2 of 28 (7 %) MC (both HER2+) showed complete pathologic response with residual acellular mucin pools. Persistent mass-forming mucin pools were present in 26 (93 %) cases. The residual tumor cellularity was markedly reduced (≤5 %) in H-NCT treated MC (11/11, 100 %), followed by NET group (6/9, 67 %) and NCT only group (4/8, 50 %) (p = 0.011). Similarly, a higher rate of pathologic response (pCR/RCB-I) was observed in H-NCT (7/11, 64 %), followed by NET group (5/9, 56 %), and NCT only group (1/7, 13 %) (p = 0.053). Post-therapy, all HER2+ MC were smaller than 2 cm and ypT size was significantly smaller in H-NCT group (11/11, 100 %) versus combined NET (5/9, 55 %) and NCT only groups (4/8, 50 %) (p = 0.029). We conclude that ER-/HER2+ and ER+/HER2-mucinous carcinomas of the breast show robust pathological response to neoadjuvant HER2 targeted and endocrine therapy, respectively. Our findings suggest that MC may show good response to endocrine therapy.

Metabolic Unhealthiness is Associated With Increased Risk of Critical COVID-19 Pneumonia and Inpatient Mortality in Hospitalized Patients with Obesity or Overweight.

Background and aims Being metabolically unhealthy (MU) is defined as having either hypertension, hyperlipidemia, type 2 diabetes mellitus/pre-diabetes, or fatty liver disease. We aimed to determine if MU was associated with severe COVID-19 pneumonia (severe disease). Methods We performed a single-center retrospective study between March 2020 and August 2021 for patients with overweight or obesity hospitalized with COVID-19 pneumonia. Logistic regression analysis was utilized to derive a risk score for severe disease. The accuracy of the model was assessed using the area under the receiver operating characteristic curve (AUROCC) and bootstrap resampling. Results A total of 334 of 450 patients hospitalized with COVID-19 pneumonia (74.2%) were MU. Patients who were MU had higher in-hospital mortality (10.5% vs. 2.6%) and longer length of hospitalization (median 6 vs. 4 days). MU was not associated with severe disease, p=0.311. On multivariable analysis, older age, male sex, and Asian race were associated with severe disease. Not being vaccinated was associated with doubled odds of severe disease. The AUROCC of the final model was 0.66 (95% CI: 0.60 to 0.71). The risk score at the lowest quintile had a 33.1% to 65.5% predicted risk and a 58.7% observed risk of severe disease, whereas, at the highest quintile, there was an 85.7% to 97.7% predicted risk and an 89.7% observed risk of severe disease. Conclusion Being MU was not a predictor of severe disease, even though mortality was higher despite having higher rates of vaccination. This risk score may help to predict severe disease in hospitalized patients with obesity or overweight. External validation is recommended.

A Rare Case of Breast Metastatic Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is one of the most common spindle cell neoplasms of the alimentary system, and can arise anywhere along the gastrointestinal tract (GI). Its incidence rate is up to 22 cases per million, with a minor geographic variation. GIST is thought to originate from interstitial cell of Cajal, and its pathogenesis is related to molecular defects, such as KIT receptor tyrosine kinase or platelet-derived growth receptor alpha gene activation. While the majority of GISTs are known to show a benign disease course, metastases of high-grade forms to different organ systems have been seldom reported. We present a case with an unprecedented metastasis of GIST to the breast. The patient is a 62-year-old female with a history of the primary resection of GIST from the small intestine. Her disease course was initially complicated by multiple metastases, solely localized to the liver for which she had a living-donor liver transplant. The tumor harbored both KIT exon 11 and exon 17 mutation. Fourteen months post-transplant, the patient was found to have metastatic GIST on her breast biopsy. GIST metastasis to the breast is extremely rare. A consideration of this spindle cell neoplasm as a differential is recommended when clinical suspicion arises. The pathophysiology, current diagnostic tool, grading system, and treatment of this tumor are discussed.

Triple-Positive Breast Carcinoma: Histopathologic Features and Response to Neoadjuvant Chemotherapy.

CONTEXT.­: It is unclear whether HER2+ tumors expressing both estrogen receptor (ER) and progesterone receptor (PR), that is, triple-positive breast carcinomas (TPBCs), show unique morphologic and clinical features and response to neoadjuvant chemotherapy (NAC). OBJECTIVE.­: To study the morphologic and immunohistochemical features of TPBCs from patients who underwent NAC. DESIGN.­: We retrospectively reviewed core biopsy and post-NAC slides of 85 TPBCs. H-scores were calculated for ER and PR. HER2 slides and fluorescence in situ hybridization (FISH) reports were reviewed. Residual cancer burden was calculated for post-NAC specimens. RESULTS.­: Eighty-one of the 85 tumors (95.3%) showed ductal histology, 3 (3.5%) were invasive lobular carcinomas, and 1 (1.2%) showed mixed ductal and lobular features. A subset showed mucinous (n = 7, 8.2%), apocrine (n = 5, 5.9%), and/or micropapillary (n = 4, 4.7%) differentiation. Fifty-four TPBCs (63.5%) showed high ER expression (H-score >200), including 27 (31.8%) with high expression of ER and PR. Fifty-two tumors (61.1%) showed HER2 3+ staining. Mean HER2/CEP17 ratio by FISH was 3.6 (range, 2-12.2) and mean HER2 signals per cell was 8 (range, 3.7-30.4). Pathologic complete response (pCR) rate was 35.3% (30 of 85). HER2 3+ staining was the only significant predictor of pCR on multivariate analysis (odds ratio = 9.215; 95% CI, 2.401-35.371; P < .001). The ER/PR expression did not correlate with response to therapy. CONCLUSIONS.­: TPBCs are heterogeneous with some showing mucinous, lobular, or micropapillary differentiation. The pCR rate of TPBCs is similar to that reported for ER+/PR-/HER2+ tumors. HER2 overexpression by IHC was associated with significantly better response to therapy and may help select patients for treatment in the neoadjuvant setting.

Screening for Bacterial Vaginosis in Pregnant Adolescents and Women to Prevent Preterm Delivery: Updated Evidence Report and Systematic Review for the US Preventive Services Task Force.

Importance: Preterm delivery results in adverse outcomes; identifying and treating bacterial vaginosis may reduce its occurrence. Objective: To update the evidence on screening and treatment of asymptomatic bacterial vaginosis in pregnancy for the US Preventive Services Task Force. Data Sources: MEDLINE, Cochrane Library, and trial registries through May 29, 2019; bibliographies from retrieved articles, experts, and surveillance of the literature through December 31, 2019. Study Selection: Fair- or good-quality English-language studies evaluating diagnostic accuracy of tests feasible within primary care; randomized clinical trials (RCTs); nonrandomized controlled intervention studies (for harms only); or meta-analyses of metronidazole or clindamycin. Data Extraction and Synthesis: Two reviewers independently assessed titles/abstracts and full-text articles, extracted data, and assessed study quality; when at least 3 similar studies were available, meta-analyses were conducted. Main Outcomes and Measures: Sensitivity, specificity, preterm delivery, maternal adverse effects, congenital birth defects, childhood cancer. Results: Forty-four studies (48 publications) were included. No studies evaluated the benefits or harms of screening. Twenty-five studies (n = 15 785) evaluated the accuracy of screening tests; across individual studies and tests, sensitivity ranged from 0.36 to 1.0 and specificity ranged from 0.49 to 1.0. Among trials reporting findings from general obstetric populations (n = 7953), no significant association was observed between treatment and spontaneous delivery before 37 weeks (pooled absolute risk difference [ARD], -1.44% [95% CI, -3.31% to 0.43%]; 8 RCTs, n = 7571) or any delivery before 37 weeks (pooled ARD, 0.20% [95% CI, -1.13% to 1.53%]; 6 RCTs, n = 6307). Among 5 trials reporting findings among women with a prior preterm delivery, findings were inconsistent; 3 showed a significant beneficial effect, while 2 did not. Maternal adverse events from treatment were infrequent and minor (eg, candidiasis) but were slightly more common with active treatment compared with placebo across 8 RCTs. Two meta-analyses of observational studies reported no significant association between metronidazole exposure and congenital malformations (odds ratio, 0.96 [95% CI, 0.75 to 1.22]; odds ratio, 1.08 [95% CI, 0.90 to 1.29]). One cohort study reported no significantly increased incidence of childhood cancer among metronidazole-exposed children (adjusted relative risk, 0.81 [95% CI, 0.41 to 1.59]). However, studies of in utero exposure had important limitations. Conclusions and Relevance: Accuracy of screening tests for bacterial vaginosis varies. The evidence suggests no difference in the incidence of preterm delivery and related outcomes from treatment for asymptomatic bacterial vaginosis in a general obstetric population but was inconclusive for women with a prior preterm delivery. Maternal adverse events from treatment appear to be infrequent and minor, but the evidence about harms from in utero exposure was inconclusive.

Hormone receptor and HER2 assessment in breast carcinoma metastatic to bone: A comparison between FNA cell blocks and decalcified core needle biopsies.

BACKGROUND: Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) guide the clinical management of breast cancer metastases. Decalcification of bone core needle biopsies (CNBs) can affect IHC. In the current study, the authors sought to define whether fine-needle aspiration (FNA) would be a better alternative to CNB for reliable IHC. METHODS: Patients with breast cancer metastases to bone that were sampled by both CNB and FNA were selected. ER, PR, and HER2 were performed in FNA cell blocks (FNA-CBs) and concurrent decalcified CNBs. Discrepancies were classified as minor when there was a difference of up to 30% nuclear staining in IHC for ER and PR between paired samples and as major when a clinically relevant change was observed (ie, positive vs negative). Quantitative reverse transcriptase-polymerase chain reaction of ESR1 messenger RNA levels was performed on FNA/CNB pairs with discrepancies for ER IHC. IHC status of the primary breast carcinoma was recorded. RESULTS: Concordance rates for ER, PR, and HER2 were 89%, 67%, and 93%, respectively, between FNA-CB and CNB pairs from 27 patients. Major discrepancies were noted in approximately 11% of FNA/CNB pairs for ER IHC and in 33% of FNA/CNB pairs for PR. ESR1 messenger RNA levels of FNA/CNB matched samples were similar and did not explain the differences in ER IHC expression in the majority of cases. Two of 27 FNA/CNB pairs had different results for HER2 IHC that changed from negative on CNB to equivocal (2+) on FNA-CB. Both cases had prior HER2 amplification by fluorescence in situ hybridization. CONCLUSIONS: FNA-CB and CNB appear to constitute acceptable methods for the assessment of ER, PR, and HER2 for clinical decision making.

Peripancreatic paraganglioma mimics pancreatic/gastrointestinal neuroendocrine tumor on fine needle aspiration: Report of two cases and review of the literature.

Cytologic diagnosis of extra-adrenal paraganglioma presenting as a peripancreatic mass is challenging with a high error rate due to its rarity. We report two cases of peripancreatic masses identified by radiology. Endoscopic ultrasound-guided fine needle aspiration (FNA) of the masses showed a moderately cellular tumor composed of small to medium sized neoplastic cells with round to oval nuclei, arranged singly and in loose clusters. Focal rosette-like structures were present. The cells were positive for neuroendocrine markers (synaptophysin and chromogranin). A diagnosis of a neoplasm with neuroendocrine differentiation and neuroendocrine tumor was made respectively on FNA for each case. The subsequent surgical resection of the tumors revealed peripancreatic paraganglioma. Although paraganglioma has been reported in the literature, the detailed comparison of perpancreatic paraganglioma versus pancreatic/gastrointestinal neuroendocrine tumor is still lacking. Therefore using these two cases with literature review, we wish to illustrate the differential diagnosis between these two entities based on cytomorphology and immunohistochemical study.

Patients' confidence in methods of blood pressure assessment and their reported adherence to antihypertensive medications.

OBJECTIVE: Adherence to antihypertensive medications is often less than optimal. Research suggests that patients have limited confidence regarding whether office blood pressure (BP) assessments represent their 'true' BP, which may further promote poor adherence to BP-lowering medication. We assessed peoples' confidence in the methods of BP assessment and examined the associations between patients' confidence levels and medication adherence comparing office and home BP-monitoring techniques. METHODS: We surveyed US adults aged 30 years or older (N=1010), all of whom had undergone an office BP measurement within the past 6 months. Respondents who indicated being prescribed antihypertensive medication (N=429) were asked to indicate their level of confidence on a 1-9 scale that BP measurements represented their true BP, and their adherence to antihypertensive medication using the eight-item Morisky Medical Adherence Scale (MMAS-8). RESULTS: Respondents had equal confidence that both office BP measurements and home monitoring measurements reflected their true BP (median=7). Respondents indicated that they would have slightly more confidence in ambulatory BP monitoring (median=8). As respondents' confidence in the assessments of BP from office measurements and home monitoring increased from 1 to 9, the mean MMAS-8 score, adjusted for age, race, and education, increased from 5.38 to 6.25 (P=0.053) and from 5.50 to 6.14 (P=0.25), respectively. CONCLUSION: As patients' confidence in a BP assessment method increases, so too does their reported adherence to prescribed antihypertensive medications. This finding further supports the incorporation of methods in which patients can feel confident that the measurements are representative of their 'true' BP.

Distribution and potential significance of intravillous and intrafibrinous particulate microcalcification.

Radiologic studies indicate that placental calcifications seen at 28-32 weeks' gestation are associated with adverse fetal outcome. One type of placental calcification is typically located at the basement membrane of chorionic villi. It has a fine particulate appearance and can only be seen microscopically. We have designated these calcifications as Intravillous and Intrafibrinous Particulate MicroCalcification (IPMC). In this study we examined the distribution and potential significance of IPMC. Placentas from 14 groups of fetal and maternal outcomes are examined histologically for IPMC. These groups were preterm birth, post term birth, intrauterine fetal demise, fetuses with non-reassuring heart rates, intrauterine growth restriction, fetal anomalies, mothers with gestational hypertension, gestational diabetes, placental abruption, pre-eclampsia and placentas of normal spontaneous vaginal births and placentas with chorioamnionitis, chronic villitis and infarcts. We observed fine dust-like particulates deposited in continuous and discrete patches. The particulates were predominantly located in the basement membranes of fibrotic chorionic villi and in perivillous fibrin. Compared to placentas without adverse outcomes, a higher incidence of IPMC was seen in intrauterine fetal demise cases and in cases with infarcts which suggests that hypoxia played a role in the etiology of IPMC.

Inhibition of Breast Cancer Metastasis by Presurgical Treatment with an Oral Matrix Metalloproteinase Inhibitor: A Preclinical Proof-of-Principle Study.

Breast cancer has the second highest death toll in women worldwide, despite significant progress in early diagnosis and treatments. The main cause of death is metastatic disease. Matrix metalloproteinases (MMP) are required for the initial steps of metastasis, and have therefore been considered as ideal pharmacologic targets for antimetastatic therapy. However, clinical trials of MMP inhibitors were unsuccessful. These trials were conducted in patients with advanced disease, beyond the stage when these compounds could have been effective. We hypothesized that early treatment with a selective MMP inhibitor between the time of diagnosis and definitive surgery, the so-called "window-of-opportunity," can inhibit metastasis and thereby improve survival. To investigate our hypothesis, we used the 4T1 mouse model of aggressive mammary carcinoma. We treated the animals with SD-7300, an oral inhibitor of MMP-2, -9, and -13, starting after the initial detection of the primary tumor. Seven days later, the primary tumors were excised and analyzed for MMP activity, and the SD-7300 treatment was discontinued. After 4 weeks, the animals were sacrificed and their lungs analyzed histologically for number of metastases and metastatic burden (metastases' area/lung section area). SD-7300 treatment inhibited 70% to 80% of tumor-associated MMP activity (P = 0.0003), reduced metastasis number and metastatic burden by 50% to 60% (P = 0.002 and P = 0.0082, respectively), and increased survival (92% vs. 66.7%; P = 0.0409), relative to control vehicle. These results show that treatment of early invasive breast cancer with selective MMP inhibitors can lower the risk of recurrence and increase long-term disease-free survival. Mol Cancer Ther; 15(10); 2370-7. ©2016 AACR.

Sclerosing hemangioma: A diagnostic dilemma in fine needle aspiration cytology.

Sclerosing hemangioma of the lung is a benign neoplasm with a widely debated histogenesis. It has a polymorphic histomorphology characterized by a biphasic cell population of "surface cells" and "round cells" arranged in four general patterns: Papillary, solid, angiomatous, and sclerotic. This variability in histomorphology makes it difficult to diagnose sclerosing hemangioma by fine needle aspiration (FNA). We present a case of sclerosing hemangioma diagnosed on FNA with immunohistochemistry performed on an accompanied cell block. The clinical presentation, cytomorphology, immunohistochemistry, and differential diagnoses are discussed.

Dollars and Discomfort: What Will People Be Willing to Give for Better Blood Pressure Assessment?

Recently, the US Preventive Services Task Force issued a draft recommendation to utilize 24-hour ambulatory blood pressure (BP) monitoring (ABPM) to confirm the diagnosis of hypertension after screening. However, ABPM can be inconvenient and has some adverse effects such as pain and bruising from the repeated cuff inflations. In this national survey, we asked adults 30 years and older how much physical discomfort they would be willing to undergo to have the most accurate test available for evaluating possible high BP. We also asked how much they would be willing to pay to have the test. Among 1010 respondents, 95% of participants indicated willingness to undergo at least mild physical discomfort. The median amount people would be willing to pay was $25. These findings suggest that people are willing to undergo a bit of discomfort, and even pay a small amount, for the benefit of accurate BP assessment.

A Note of Caution: Variable Cytokeratin Staining in Sentinel Node Metastases.

Sentinel lymph node biopsy is the current standard procedure used to stage patients with breast cancer. The best histological method in evaluating sentinel nodes is highly debated among institutions and is thus not standardized. The optimal histological analysis is a balance between comprehensive evaluation of the sentinel nodes and cost effectiveness. One commonly used approach is serial sectioning and alternately staining with hemotoxylin and eosin and AE1/AE3 cytokeratin immunohistochemistry analysis. We report 2 cases of metastatic carcinoma demonstrating negative staining for AE1/AE3. This observation highlights a rare but potential pitfall to this commonly used strategy in assessing sentinel lymph node biopsies in breast cancer.

The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance.

Mitochondria are known to be functional organelles, but their role as a signaling unit is increasingly being appreciated. The identification of a short open reading frame (sORF) in the mitochondrial DNA (mtDNA) that encodes a signaling peptide, humanin, suggests the possible existence of additional sORFs in the mtDNA. Here we report a sORF within the mitochondrial 12S rRNA encoding a 16-amino-acid peptide named MOTS-c (mitochondrial open reading frame of the 12S rRNA-c) that regulates insulin sensitivity and metabolic homeostasis. Its primary target organ appears to be the skeletal muscle, and its cellular actions inhibit the folate cycle and its tethered de novo purine biosynthesis, leading to AMPK activation. MOTS-c treatment in mice prevented age-dependent and high-fat-diet-induced insulin resistance, as well as diet-induced obesity. These results suggest that mitochondria may actively regulate metabolic homeostasis at the cellular and organismal level via peptides encoded within their genome.

Requirement for NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition.

Treatment of PARP-1-expressing cells with the combination of a DNA methylating agent (MMS) and the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) leads to an ATR/Chk1-dependent S phase checkpoint and cell death by apoptosis. Activation of ATM/Chk2 is involved in sustaining the S phase checkpoint, and double strand break (DSB) accumulation was demonstrated. NBS1, part of the MRN complex that responds to DSBs, is known to modulate ATR- and ATM-dependent checkpoint responses to UV and IR, but a role in the response to PARP inhibition has not been addressed. Here we show that the S phase checkpoint observed 4-8h after MMS+4-AN treatment was absent in cells deficient in NBS1, but was present in NBS1-complemented (i.e., functionally wild-type) cells, indicating a critical role for NBS1 in this checkpoint response. NBS1 was phosphorylated in response to MMS+4-AN treatment, and this was partially ATR- and ATM-dependent, suggesting involvement of both upstream kinases. NBS1 expression had little effect on ATR-mediated phosphorylation of Chk1 and ATM-mediated phosphorylation of Chk2 in response to MMS+4-AN. Phosphorylation of SMC1 was also observed in response to MMS+4-AN treatment. In the absence of ATM and NBS1, phosphorylation of SMC1 was weak, especially at early times after MMS+4-AN treatment. In the absence of ATR activation, reduced SMC1 phosphorylation was seen over a 24h time course. These results suggested that both ATR and ATM phosphorylate SMC1 in response to MMS+4-AN and that this phosphorylation is enhanced by phospho-NBS1. The loss of the MMS+4-AN-induced S phase checkpoint in NBS1-deficient cells may be due to a reduced cellular level of the critical downstream effector, phospho-SMC1.

Cell cycle arrest by transforming growth factor beta1 enhances replication of respiratory syncytial virus in lung epithelial cells.

Respiratory syncytial virus (RSV) is a common respiratory viral infection in children which is associated with immune dysregulation and subsequent induction and exacerbations of asthma. We recently reported that treatment of primary human epithelial cells (PHBE cells) with transforming growth factor beta (TGF-beta) enhanced RSV replication. Here, we report that the enhancement of RSV replication is mediated by induction of cell cycle arrest. These data were confirmed by using pharmacologic inhibitors of cell cycle progression, which significantly enhanced RSV replication. Our data also showed that RSV infection alone resulted in cell cycle arrest in A549 and PHBE cells. Interestingly, our data showed that RSV infection induced the expression of TGF-beta in epithelial cells. Blocking of TGF-beta with anti-TGF-beta antibody or use of a specific TGF-beta receptor signaling inhibitor resulted in rescue of the RSV-induced cell cycle arrest, suggesting an autocrine mechanism. Collectively, our data demonstrate that RSV regulates the cell cycle through TGF-beta in order to enhance its replication. These findings identify a novel pathway for upregulation of virus replication and suggest a plausible mechanism for association of RSV with immune dysregulation and asthma.