Mostafa Fraig

Dose-Dependent Activity of Pyrazinamide in Animal Models of Intracellular and Extracellular Tuberculosis Infections

ABSTRACT Recent in vitro pharmacokinetic data suggest that the currently recommended dose of pyrazinamide may be suboptimal for killing intracellular bacilli in humans. We evaluated a range of pyrazinamide doses against intracellular and extracellular Mycobacterium tuberculosis in chronically infected mice and guinea pigs, respectively. Antibiotics were given five times weekly for 4 weeks beginning 28 days after infection. Human-equivalent doses of isoniazid reduced lung bacterial counts 10-fold in each species. Pyrazinamide given at 1/4 and 1/2 the human-equivalent dose was minimally active, while human-equivalent doses reduced lung bacterial counts by ∼1.0 log10 in each species. Doubling the human-equivalent dose of pyrazinamide reduced the lung bacillary burden by 1.7 and 3.0 log10 in mice and guinea pigs, respectively. As in humans and mice, pyrazinamide showed significant synergy with rifampin in guinea pigs. Clinical studies are warranted to investigate the sterilizing activity and tolerability of higher doses of pyrazinamide in combination tuberculosis regimens.

Effectiveness of tuberculosis chemotherapy correlates with resistance to Mycobacterium tuberculosis infection in animal models

OBJECTIVES It is widely believed that persistent Mycobacterium tuberculosis inhabits necrotic lung granulomas in humans and that the microenvironmental conditions encountered therein render the bacilli phenotypically tolerant to antibiotics, accounting for the long duration required for successful treatment of tuberculosis (TB). To validate this belief, we directly compared the activity of rifampicin/isoniazid/pyrazinamide (RHZ) against chronic TB infection in guinea pigs, which exhibit caseous granulomas histologically resembling human caseous foci, and in mice, which lack necrotic granulomas. METHODS Guinea pigs and mice were aerosol-infected with M. tuberculosis CDC1551 and twice weekly treatment with RHZ was started 4 weeks later. Culture-positive relapse was assessed in subgroups of guinea pigs after 3 months and 4 months of treatment. RESULTS All guinea pig lungs exhibited histological evidence of granulomas with central caseation, while mouse lungs exhibited cellular lesions at the initiation of antibiotic treatment. Guinea pig lungs became culture-negative after 2 months of RHZ given twice weekly at human-equivalent doses. Relapse rates in guinea pigs were 0% (0/10) both after 3 months and 4 months of treatment. In contrast, all mouse lungs remained culture-positive after 4 months of equivalent RHZ exposures. CONCLUSIONS Caseous necrosis does not reduce the sterilizing activity of the standard antituberculosis regimen of RHZ. Our findings have important implications for the use of alternative animal models in testing novel TB drug regimens and for modelling M. tuberculosis persistence.

Severe pulmonary hypertension in idiopathic nonspecific interstitial pneumonia

Pulmonary hypertension (PH) is a common complication of interstitial lung disease (ILD), particularly in idiopathic pulmonary fibrosis (IPF) and ILD associated with connective tissue disease, where the underlying pathology is often a nonspecific interstitial pneumonia (NSIP) pattern. The degree of PH in ILD is typically mild to moderate and radiographic changes of ILD are usually prominent. We describe four patients with idiopathic NSIP and severe PH (mPAP > 40 mmHg). The average mean pulmonary artery pressure was 51±7 mmHg and pulmonary vascular resistance was 13±4 Woods units. Pulmonary function was characterized by mild restriction (total lung capacity 63–94% predicted) and profound reductions in DLCO (19–53% predicted). Computed tomographic imaging revealed minimal to moderate interstitial thickening without honeycombing. In two of the cases, an initial clinical diagnosis of idiopathic pulmonary arterial hypertension was made. Both were treated with intravenous epoprostenol, which was associated with worsening of hypoxemia. All four patients died or underwent lung transplant within 4 years of PH diagnonsis. Lung pathology in all four demonstrated fibrotic NSIP with medial thickening of the small and medium pulmonary arteries, and proliferative intimal lesions that stained negative for endothelial markers (CD31 and CD34) and positive for smooth muscle actin. There were no plexiform lesions. Severe pulmonary hypertension can therefore occur in idiopathic NSIP, even in the absence of advanced radiographic changes. Clinicians should suspect underlying ILD as the basis for PH when DLCO is severely reduced or gas exchange deteriorates with pulmonary vasodilator therapy.

Lower Respiratory Tract Infection of the Ferret by 2009 H1N1 Pandemic Influenza A Virus Triggers Biphasic, Systemic, and Local Recruitment of Neutrophils

ABSTRACT Infection of the lower respiratory tract by influenza A viruses results in increases in inflammation and immune cell infiltration in the lung. The dynamic relationships among the lung microenvironments, the lung, and systemic host responses during infection remain poorly understood. Here we used extensive systematic histological analysis coupled with live imaging to gain access to these relationships in ferrets infected with the 2009 H1N1 pandemic influenza A virus (H1N1pdm virus). Neutrophil levels rose in the lungs of H1N1pdm virus-infected ferrets 6 h postinfection and became concentrated at areas of the H1N1pdm virus-infected bronchiolar epithelium by 1 day postinfection (dpi). In addition, neutrophil levels were increased throughout the alveolar spaces during the first 3 dpi and returned to baseline by 6 dpi. Histochemical staining revealed that neutrophil infiltration in the lungs occurred in two waves, at 1 and 3 dpi, and gene expression within microenvironments suggested two types of neutrophils. Specifically, CCL3 levels, but not CXCL8/interleukin 8 (IL-8) levels, were higher within discrete lung microenvironments and coincided with increased infiltration of neutrophils into the lung. We used live imaging of ferrets to monitor host responses within the lung over time with [18F]fluorodeoxyglucose (FDG). Sites in the H1N1pdm virus-infected ferret lung with high FDG uptake had high levels of proliferative epithelium. In summary, neutrophils invaded the H1N1pdm virus-infected ferret lung globally and focally at sites of infection. Increased neutrophil levels in microenvironments did not correlate with increased FDG uptake; hence, FDG uptake may reflect prior infection and inflammation of lungs that have experienced damage, as evidenced by bronchial regeneration of tissues in the lungs at sites with high FDG levels. IMPORTANCE Severe influenza disease is characterized by an acute infection of the lower airways that may progress rapidly to organ failure and death. Well-developed animal models that mimic human disease are essential to understanding the complex relationships of the microenvironment, organ, and system in controlling virus replication, inflammation, and disease progression. Employing the ferret model of H1N1pdm virus infection, we used live imaging and comprehensive histological analyses to address specific hypotheses regarding spatial and temporal relationships that occur during the progression of infection and inflammation. We show the general invasion of neutrophils at the organ level (lung) but also a distinct pattern of localized accumulation within the microenvironment at the site of infection. Moreover, we show that these responses were biphasic within the lung. Finally, live imaging revealed an early and sustained host metabolic response at sites of infection that may reflect damage and repair of tissues in the lungs.

Do liquid-based preparations of pulmonary bronchial brushing specimens perform differently from classically prepared cases for the diagnosis of malignancies?: Observations from the college of American pathologists interlaboratory comparison program in nongynecologic cytology

CONTEXT Pulmonary bronchial brushing specimens can be processed by liquid-based or conventional methods. The ability to accurately diagnose a pulmonary malignancy with a liquid-based preparation (LBP) versus a conventional preparation may differ. OBJECTIVE To compare the performance of LBPs of malignant pulmonary bronchial brushing specimens with the performance of conventional preparations. DESIGN Participant responses from 553 malignant pulmonary bronchial brushing samples were evaluated for concordance with the general diagnosis. The performance of LBPs was compared with that of classic preparations. A nonlinear mixed model was used to analyze the performance by reference diagnosis, preparation type, program years, participant type, and the interaction terms between these 4 factors. RESULTS Concordance with the general category of malignant disease was observed in 95.2% of conventional Papanicolaou-stained, 90.9% of modified Giemsa-stained, and 96.9% of LBP (P < .001) samples. The results were significantly different between individual reference diagnoses (P < .001). The performance of LBPs was consistently higher for most diagnoses and was significantly better for squamous cell carcinoma (P = .005), small cell carcinoma (P < .001), and metastatic carcinoma not otherwise specified (P < .001). All participant types performed significantly better with LBPs of small cell carcinoma. Pathologists and cytotechnologists performed significantly better with LBPs of squamous cell carcinoma. A significantly higher concordance was observed between the general diagnosis and program years 2007-2011 versus 2001-2006 (P = .006). CONCLUSIONS Liquid-based preparations performed better than conventional methods, with significantly higher performance in squamous cell, small cell, and metastatic carcinomas. Improved performance over time may reflect more frequent use of LBP methods and increased familiarity with interpreting the morphologic findings.

Baclofen, a GABABR Agonist, Ameliorates Immune-Complex Mediated Acute Lung Injury by Modulating Pro-Inflammatory Mediators

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.

Primary Pulmonary Non–Small Cell Carcinomas: The College of American Pathologists Interlaboratory Comparison Program Confirms a Significant Trend Toward Subcategorization Based Upon Fine-Needle Aspiration Cytomorphology Alone

Context.-Subtyping of non-small cell lung carcinomas (NSCLCs) is necessary for optimal patient management with specific diagnoses triggering specific molecular tests and affecting therapy. Objective.-To assess the accuracy of the participants of the College of American Pathologists Interlaboratory Comparison Program in diagnosing and subtyping NSCLC fine-needle aspiration (FNA) slides, based on morphology alone, considering preparation and participant type and trends over time. Design.-The performance of program participants was reviewed for the 5-year period spanning 2007-2011. Lung FNA challenges with reference diagnoses of adenocarcinoma and squamous cell carcinoma (SCC) were evaluated for diagnostic concordance by using a nonlinear mixed model analysis. Results.-There were 10 493 pathologist and 6378 cytotechnologist responses with concordance rates of 97.4% and 97.9% for malignancy, respectively. Overall concordance rates for subcategorization were 54.6% for adenocarcinoma and 74.9% for SCC. For the exact reference diagnoses, pathologists performed better for adenocarcinoma and cytotechnologists performed better for SCC. Accurate subcategorization of adenocarcinomas significantly increased over time with 31.5% of adenocarcinomas classified as NSCLC in 2007 and 25.5% of adenocarcinomas classified as NSCLC in 2011 (P < .001). In comparing preparation types, modified Giemsa-stained smears showed the lowest overall concordance (46.8%). Modified Giemsa-stained smears with SCCs were the least likely to be accurately subcategorized (36.4%). Conclusions.-Participants are proficient at interpreting NSCLCs as malignant by FNA but are less successful at subcategorization with cytomorphology alone. During the study period, a statistically significant trend was confirmed toward greater accuracy of subcategorization of adenocarcinomas, suggesting that participants are cognizant of the impact that more specific cytomorphologic interpretations have in directing molecular triage and therapy.

Circulating Adipose Fatty Acid Binding Protein Is a New Link Underlying Obesity-Associated Breast/Mammary Tumor Development

It is clear that obesity increases the risk of many types of cancer, including breast cancer. However, the underlying molecular mechanisms by which obesity is linked to cancer risk remain to be defined. Herein, we report that circulating adipose fatty acid binding protein (A-FABP) promotes obesity-associated breast cancer development. Using clinical samples, we demonstrated that circulating A-FABP levels were significantly increased in obese patients with breast cancer in comparison with those without breast cancer. Circulating A-FABP released by adipose tissue directly targeted mammary tumor cells, enhancing tumor stemness and aggressiveness through activation of the IL-6/STAT3/ALDH1 pathway. Importantly, genetic deletion of A-FABP successfully reduced tumor ALHD1 activation and obesity-associated mammary tumor growth and development in different mouse models. Collectively, these data suggest circulating A-FABP as a new link between obesity and breast cancer risk, thereby revealing A-FABP as a potential new therapeutic target for treatment of obesity-associated cancers.

Strong NFκB Expression is Associated With High-grade Dysplasia in Barrett's Esophagus.

Nuclear factor kappa B (NF&kgr;B) is a transcription factor that regulates the activation of genes involved in proinflammatory response and growth. In this study, we utilized immunohistochemical stains for 2 of the NF&kgr;B molecules (RELA and NF&kgr;B-1) to evaluate the expression of NF&kgr;B in Barrett’s esophagus (BE). Forty-three cases of BE [17 cases with no dysplasia, 16 cases with low-grade dysplasia (LGD), and 10 cases with high-grade dysplasia (HGD)], 10 normal esophageal biopsies, and 9 cases of esophageal adenocarcinoma were evaluated. Expression of NF&kgr;B-1 and RELA did not occur in normal esophageal squamous mucosa. BE without dysplasia showed weak expression of RELA and NF&kgr;B-1 in 35% and 65% of cases, respectively. BE with LGD showed weak expression of RELA and NF&kgr;B-1 in 50% and 75% of cases, respectively. Strong expression of RELA and NF&kgr;B-1 did not occur in BE without dysplasia or with LGD. BE with HGD showed strong expression of RELA and NF&kgr;B-1 in 80% and 90% of cases, respectively. All cases of adenocarcinoma showed strong expression of both RELA and NF&kgr;B-1. There was a progressive increase in staining intensity of RELA and NF&kgr;B-1 along the metaplasia-dysplasia-adenocarcinoma pathway. Strong expression of NF&kgr;B is associated with HGD and adenocarcinoma (P<0.0001). We showed that strong expression of NF&kgr;B-1 and RELA correlates highly with BE with HGD and adenocarcinoma.

Cytomorphologic findings and differential diagnosis of pulmonary papillary adenoma: A case report and literature review

Pulmonary papillary adenoma is a rare tumor of the lung. Some authors refer to it as papillary adenoma of type II pneumocytes. It demonstrates benign behavior, although some references suggest that this tumor may rarely exhibit invasive characteristics. We report a case of pulmonary papilloma adenoma of the lung diagnosed by fine‐needle aspiration biopsy and transbronchial biopsy. The patient is a 78‐year‐old woman, who presented to an outside facility with complaint of confusion after a missed episode of dialysis. On further workup, she was found to have a 3.8 cm irregular mass in the upper lobe of her right lung as visualized on chest CT. Fine‐needle aspiration and a concurrent forceps‐assisted transbronchial biopsy of the mass were performed. On microscopical examination, tumor cells formed small cohesive papillary fronds. On cytological evaluation, tumor cells were uniform medium‐sized epithelial cells with moderate cytoplasm, fine chromatin, and inconspicuous nucleoli. The biopsies showed papillary arrangement of epithelial cells in a background of mild fibrosis and chronic inflammation. There was no piling up of cells and the nuclei were uniform with bland appearance. No mitoses were appreciated, and Ki‐67 activity was low. The clinical decision was for observation. The patient suffered no complications after the procedures during 26 months of follow‐up. We hereby present this case with a review of the literature. Diagn. Cytopathol. 2016;44:543–547.