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Purpose: Recruit and sequence breast cancer subjects in Guatemalan and US Hispanic populations. Identify optimum strategies to recruit Latin American and Hispanic women into genetic studies of breast cancer., Methods: We used targeted gene sequencing to identify pathogenic variants in 19 familial breast cancer susceptibility genes in DNA from unselected Hispanic breast cancer cases in the US and Guatemala. Recruitment across the US was achieved through community-based strategies. In addition, we obtained patients receiving cancer treatment at major hospitals in Texas and Guatemala., Results: We recruited 287 Hispanic US women, 38 (13%) from community-based and 249 (87%) from hospital-based strategies. In addition, we ascertained 801 Guatemalan women using hospital-based recruitment. In our experience, a hospital-based approach was more efficient than community-based recruitment. In this study, we sequenced 103 US and 137 Guatemalan women and found 11 and 10 pathogenic variants, respectively. The most frequently mutated genes were BRCA1, BRCA2, CHEK2, and ATM. In addition, an analysis of 287 US Hispanic patients with pathology reports showed a significantly higher percentage of triple-negative disease in patients with pathogenic variants (41% vs. 15%). Finally, an analysis of mammography usage in 801 Guatemalan patients found reduced screening in women with a lower socioeconomic status (p < 0.001)., Conclusion: Guatemalan and US Hispanic women have rates of hereditary breast cancer pathogenic variants similar to other populations and are more likely to have early age at diagnosis, a family history, and a more aggressive disease. Patient recruitment was higher using hospital-based versus community enrollment. This data supports genetic testing in breast cancer patients to reduce breast cancer mortality in Hispanic women., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Lung cancer is the leading cause of cancer death in men and women in the United States. Recent studies have implicated the tumor microenvironment as a new chemotherapeutic target by demonstrating the importance of tumor cell-stromal interactions in cancer progression. However, the exact mechanisms by which tumor cell-stromal interactions drive lung cancer progression remain undefined, particularly in the lung. We suspect host fibroblasts represent an important component of the tumor microenvironment that drives tumor progression. We found that human non-small cell lung carcinoma cell lines show alterations in cell morphology, proliferation, migration, and colony formation on soft agar when exposed to fibroblast-conditioned media (FCM). Interestingly, FCM also promoted tumor cell resistance to cisplatin-induced apoptosis. These effects varied depending on the cancer cell line used. Similar observations were made when exposing murine Lewis Lung Carcinoma cells to conditioned media harvested from primary murine lung fibroblasts. Certain effects of FCM, but not all, could be prevented by using a cMET inhibitor. In vivo, we observed enhanced growth of the primary tumors when treated with FCM, but no changes in metastatic behavior. Although the identity of the stimulating agent(s) in the fibroblast-conditioned media was not unveiled, further studies revealed that the activity is more than one factor with a high-molecular weight (over 100 kDa). These studies implicate lung fibroblast-derived factors in lung cancer progression. These data suggest that targeting the lung tumor stroma alone, or in combination with other interventions, is a promising concept that warrants further study in the setting of lung cancer., (Copyright © 2022. Published by Elsevier Inc.)

Background: Chronic heavy alcohol consumption is a major risk factor for the development of liver steatosis, fibrosis, and cirrhosis, but the mechanisms by which alcohol causes liver damage remain incompletely elucidated. This group has reported that α4 nicotinic acetylcholine receptors (α4 nAChRs) act as sensors for alcohol in lung cells. This study tested the hypothesis that α4 nAChRs mediate the effects of alcohol in the liver., Methods: Expression of acetylcholine receptor subunits in mouse liver was determined by RNA sequencing (RNA-seq). α4 nAChR knockout (α4 KO) mice were generated in C57BL/6J mice by introducing a mutation encoding an early stop codon in exon 4 of Chrna4, the gene encoding the α4 subunit of the nAChR. The presence of the inactivating mutation was established by polymerase chain reaction and genomic sequencing, and the lack of α4 nAChR function was confirmed in primary fibroblasts isolated from the α4 KO mice. Wild-type (WT) and α4 KO mice were fed the Lieber-DeCarli diet (with 36% of calories from alcohol) or pair fed an isocaloric maltose-dextrin control diet for a 6-week period that included a ramping up phase of increasing dietary alcohol., Results: Chrna4 was the most abundantly expressed nAChR subunit gene in mouse livers. After 6 weeks of alcohol exposure, WT mice had elevated serum transaminases and their livers showed increased fat accumulation, decreased Sirt1 protein levels, and accumulation of markers of oxidative stress and inflammation including Cyp2E1, Nos2, Sod1, Slc7a11, TNFα, and PAI1. All these responses to alcohol were either absent or significantly attenuated in α4 KO animals., Conclusion: Together, these observations support the conclusion that activation of α4 nAChRs by alcohol or one of its metabolites is one of the initial events promoting the accumulation of excess fat and expression of inflammatory mediators. Thus, α4 nAChRs may represent viable targets for intervention in chronic alcohol-related liver disease., (© 2022 Research Society on Alcoholism.)

Most mitochondrial protein complexes include both nuclear and mitochondrial gene products, which coevolved to work together. This coevolution can be disrupted due to disparity in genetic ancestry between the nuclear and mitochondrial genomes in recently admixed populations. Such mitonuclear DNA discordance might result in phenotypic effects. Several nuclear-encoded proteins regulate expression of mitochondrial DNA (mtDNA) genes. We hypothesized that mitonuclear DNA discordance affects expression of genes encoded by mtDNA. To test this, we utilized the data from the GTEx project, which contains expression levels for ∼100 African Americans and >600 European Americans. The varying proportion of African and European ancestry in recently admixed African Americans provides a range of mitonuclear discordance values, which can be correlated with mtDNA gene expression levels (adjusted for age and ischemic time). In contrast, European Americans did not undergo recent admixture. We demonstrated that, for most mtDNA protein-coding genes, expression levels in energetically-demanding tissues were lower in African Americans than in European Americans. Furthermore, gene expression levels were lower in individuals with higher mitonuclear discordance, independent of population. Moreover, we found a negative correlation between mtDNA gene expression and mitonuclear discordance. In African Americans, the average value of African ancestry was higher for nuclear-encoded mitochondrial than non-mitochondrial genes, facilitating a match in ancestry with the mtDNA and more optimal interactions. These results represent an example of a phenotypic effect of mitonuclear discordance on human admixed populations, and have potential biomedical applications., Competing Interests: The 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 Torres-Gonzalez and Makova.)

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and β-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

Purpose: Fibroproliferation and excess deposition of extracellular matrix (ECM) are the pathologic hallmarks of idiopathic pulmonary fibrosis (IPF), a chronic progressive disorder with high mortality and suboptimal treatment options. Although the etiologic mechanisms responsible for the development and progression of IPF remain unclear, cell-ECM interactions and growth factors are considered important. Cilengitide is a cyclic RGD pentapeptide with anti-angiogenic activity that targets αvβ3, αvβ5 and α5β1, integrins known to mediate cell-ECM interactions and activate the pro-fibrotic growth factor Transforming Growth Factor beta (TGF-β)., Methods: Cilengitide was studied in vitro with the use of NIH/3T3 cells and primary lung fibroblasts, and in vivo in the well-characterized bleomycin-induced lung injury model. The extent of ECM deposition was determined by RT-PCR, Western blot, histologic analysis and hydroxyproline assay of lung tissue. Bronchoalveolar lavage analysis was used to determine cell counts., Results: Cilengitide treatment of cultured fibroblasts showed decreased adhesion to vitronectin and fibronectin, both integrin-dependent events. Cilengitide also inhibited TGF-β-induced fibronectin gene expression and reduced the accumulation of mRNAs and protein for fibronectin and collagen type I. Both preventive and treatment effects of daily injections of cilengitide (20 mg/kg) failed to inhibit the development of pulmonary fibrosis as determined by histological analysis (Ashcroft scoring), bronchoalveolar lavage (BAL) fluid cell counts, and hydroxyproline content., Conclusions: Overall, our data suggest that, despite its in vitro activity in fibroblasts, daily injections of cilengitide (20 mg/kg) did not inhibit the development of or ameliorate bleomycin-induced pulmonary fibrosis in mice.

Lung cancer remains the leading cause of cancer death in the United States. Since most lung cancers occur in aged individuals with chronic lung disorders characterized by inflammation and/or fibrosis, we hypothesized that aging and tissue inflammation/remodeling act in concert to promote lung cancer progression. To test this, we engaged in studies using young and aged C57BL/6 mice in conjunction with bleomycin treatment in a syngeneic model of lung cancer. Wildtype young (3 months) and aged (9 months) C57BL/6 mice were injected with Lewis Lung Carcinoma (LLC) cells at day 14 after injection with phosphate-buffered saline or bleomycin. Untreated aged mice were found to develop more lung metastases than young mice. Bleomycin induced weight loss and lung inflammation/remodeling in both young and aged mice, and it increased the number of lung metastases in aged lungs, but not in young lungs. Since aged lungs show alterations in the expression of fibronectin EDA, we repeated studies in aged WT and aged FN EDA KO mice. In the absence of tissue remodeling/inflammation, WT and FN EDA KO mice developed the same number of metastases when injected with LLC cells. However, the increase in lung metastasis due to bleomycin treatment was abolished in FN EDA KO mice, but only in aged and injured lungs. Together, these studies show increased lung cancer metastasis in aging animals and point to the influence of FN EDA and injury in this process.

Age, sex and diet are well-established risk factors for several diseases. In humans, each of these variables has been linked to differences in plasma redox potentials (E h ) of the glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox couples. Mice have been very useful for modeling human disease processes, but it is unknown if age, sex and diet affect redox couples in mice as they do in humans. The purpose of the present study was to examine the effects of these factors on plasma redox potentials in C57BL/6J mice. We found that age had no effect on either redox couple in either sex. Plasma E h Cys/CySS and E h GSH/GSSG were both more oxidized (more positive) in females than in males. A 24-hour fast negated the sex differences in both redox potentials by oxidizing both redox couples in male mice, while having no effect on E h Cys/CySS and a smaller effect on E h GSH/GSSG in female mice. A diet with excess sulfur amino acids reduced the plasma E h Cys/CySS in females to a level comparable to that seen in male mice. Thus, sex-specific differences in plasma E h Cys/CySS could be normalized by two different dietary interventions. Some of these findings are consistent with reported human studies, while others are not. Most strikingly, mice do not exhibit age-dependent oxidation of plasma redox potentials. Care must be taken when designing and interpreting mouse studies to investigate redox regulation in humans., (Copyright © 2020. Published by Elsevier Inc.)

Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 ( IRF4 ) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. SIGNIFICANCE: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers., (©2020 American Association for Cancer Research.)

Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI.

Background: A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process., Methodology/principal Findings: The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process., Conclusions/significance: CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 μL instillate volume can be introduced into mouse lung.

Objective: Age-related changes in the larynx lead to significant voice impairment and reduced quality of life. There is a need for aged animal models that have practical generation times to study the fundamental changes and new therapeutics for the aging voice. The senescence accelerated prone mouse strain (SAMP) animals experience rapid aging without any experimental manipulation. The main objective of this study was to demonstrate the use of senescence accelerated mice to study aging in the larynx., Study Design: Murine model., Setting: Department of Animal Resources, Emory University., Subjects and Methods: Larynges from five senescence accelerated prone mice, five normal aging senescence resistant mice, and five C57BL/6 mice were harvested and processed for paraffin sections. Histomorphometry was performed for assessment of collagen and hyaluronic acid distribution. In addition, frozen laryngeal tissue was harvested for transcriptional and translational assessment of collagen-1, using real-time polymerase chain reaction with specific primers and Western blots. Myofibroblast assessment was performed by immunostaining for the presence of alpha-smooth muscle actin., Results: The deposition of collagen increased at six months of age in the SAMP vocal fold, and the level of collagen-1 mRNA increased with age. The myofibroblast protein alpha-smooth muscle actin was also found at a higher concentration in the SAMP vocal tissue. In contrast, the levels of hyaluronic acid in the vocal folds of SAMP mice decreased with age when compared to age-matched C57BL/6 mice., Conclusion: SAMP mice show accelerated, age-related changes in the vocal fold that were evident at as early as six months of age. The use of senescence accelerated mice offers promise as a model to study age-related laryngeal changes., (Copyright 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.)

Objectives/hypothesis: There is a need for a slow-release system for local delivery of therapeutics to the larynx. Most therapeutic substances, such as steroids or chemotherapeutic agents that are injected into the larynx are cleared rapidly. Repeated laryngeal injection of these substances at short intervals is impractical. Injectable encapsulated poly(lactide-co-glycolide) (PLGA) nanoparticles offer a potential slow-release delivery system for biologically active substances in the larynx., Study Design: Controlled animal study., Methods: PLGA nanoparticles were fabricated using a double emulsion method and were loaded with Texas Red-dextran (NPTR), hepatocyte growth factor (NPHGF), and bovine serum albumin (NPBSA). In vitro release of NPTR, NPBSA, and NPHGF was determined over approximately 2 weeks to assess potential duration of PLGA nanoparticle delivery. In vivo release of NPTR was assessed in a murine vocal fold injection model. The transcriptional effect of NPHGF on procollagen was measured in vitro to assess whether released growth factor retained functionality., Results: In vitro release kinetics demonstrated slow release of NPTR, NPBSA, and NPHGF over 12 to 14 days. In vitro NPTR release correlated with in vivo results. In vivo presence of NPTR occurred up to 7 days compared to 1 day for Texas Red control. In addition, NPHGF ameliorated transforming growth factor-beta induced procollagen in vitro in 3T3 fibroblast cells., Conclusions: The results demonstrate the potential utility of nanoparticle encapsulation as an effective method for long-term delivery of specific drugs and biologically active substances to the larynx.

Bone marrow-derived mesenchymal stem cells (BMDMSC) are emerging as a therapeutic modality in various inflammatory disease states, including acute lung injury (ALI). A hallmark of inflammation, and a consistent observation in patients with ALI, is a perturbation in the systemic redox environment. However, little is known about the effects of BMDMSC on the systemic redox status. The objective of the present study was to determine whether exogenously infused BMDMSC protect against endotoxin-induced oxidation of plasma cysteine (Cys) and glutathione (GSH) redox states. To determine the effect on the redox state if BMDMSC, mice received endotoxin intraperitoneally (1 mg/kg), followed by intravenous infusion of either 5 × 10(5) BMDMSC or an equal volume of saline solution. Control mice received intraperitoneal endotoxin followed by 5 × 10(5) lung fibroblasts given intravenously. Cys, cystine (CySS), GSH, and glutathione disulfide (GSSG) concentrations were determined by HPLC. Results showed sequential preservation of plasma Cys and GSH levels in response to BMDMSC infusion. The data show that BMDMSC infusion leads to a more reducing Cys and GSH redox state. The findings are the first to demonstrate that BMDMSC have antioxidant effects in vivo, and add to our understanding of the systemic effects of BMDMSC in lung injury.

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.