Dani S. Zander

Inhibition of Arterial Thrombus Formation by ApoA1 Milano

The mutant form of human apoA1, known as apoA1 Milano, is formed as a result of arginine 173 to cysteine substitution and inhibits experimental atherosclerosis in cholesterol-fed animals. This study was designed to determine if apoA1 Milano would modify arterial thrombogenesis. Sprague Dawley rats were intravenously administered the carrier alone (n=8) or apoA1 Milano (20 mg. kg-1. d-1 for 4 to 10 days, n=17). The abdominal cavity was opened, and the abdominal aorta was isolated. Whatman paper impregnated with 35% FeCl3 was wrapped around the surface of the aorta, and aortic flow was recorded continuously. In carrier-treated rats, an occlusive platelet-fibrin-rich thrombus was formed in 21.2+/-4.1 (mean+/-SD) minutes. Treatment of rats with apoA1 Milano markedly delayed time to thrombus formation (38.8+/-11.9 versus 21.2+/-4.1 minutes, P<0. 01), inhibited platelet aggregation (25+/-7% versus 50+/-11%, P<0. 01), and reduced weight of the thrombus (18.5+/-1.8 versus 23.7+/-2. 3 mg/cm, P<0.01). Total cholesterol and HDL levels remained similar in both groups of rats, but plasma apoA1 Milano levels were elevated in apoA1 Milano-treated rats. In in vitro studies, incubation of platelets with apoA1 Milano reduced ADP-induced platelet aggregation by about 50%, but apoA1 Milano had no direct effect on vasoreactivity. This study provides further evidence for critical role of platelets in thrombosis. Use of apoA1 Milano offers a novel approach to inhibit arterial thrombosis.

Diagnostic importance of 9p21 homozygous deletion in malignant mesotheliomas

Definitive diagnosis of malignant mesothelioma in small specimens can be extremely difficult based on morphology alone. Homozygous deletion of 9p21, the locus harboring the p16 gene, has been reported as the most common genetic alteration in malignant mesotheliomas. Recent studies demonstrated that this alteration may be useful for differentiating benign from malignant mesothelial proliferations in cytology specimens. The aim of this study was to evaluate the diagnostic utility of homozygous deletion of 9p21 assessed by fluorescence in situ hybridization (FISH) in mesothelial proliferations involving serosal surfaces in paraffin-embedded tissue. p16 protein immunoexpression was also explored as a potential diagnostic aid. FISH analysis demonstrated homozygous deletion of the 9p21 locus in 35 of 52 cases (67%) of pleural mesothelioma and in 5 of 20 cases of peritoneal mesothelioma (25%) (P<0.005). None of 40 cases of reactive pleural mesothelial proliferations showed p16 deletion (P<0.005). Loss of immunoexpression of p16 was observed in 71% of the peritoneal mesotheliomas, 40% of the pleural malignant mesotheliomas and 15% of the reactive mesothelial cells. Homozygous deletion did not correlate with p16 protein expression in any of the studied groups. Our study suggests that 9p21 homozygous deletion assessed by FISH on paraffin-embedded tissue may be helpful for differentiating between malignant mesotheliomas and reactive mesothelial proliferations. A discrepancy between p16 protein expression and homozygous deletion suggests that other molecular mechanisms may play a role in p16 protein expression in mesothelial proliferations.

TTF-1 and p63 for distinguishing pulmonary small-cell carcinoma from poorly differentiated squamous cell carcinoma in previously pap-stained cytologic material

In histology and cell block sections, antibodies to thyroid transcription factor-1 (TTF-1) and p63 have been demonstrated to be useful markers for distinguishing between small-cell lung carcinoma and poorly differentiated pulmonary squamous cell carcinoma. In this study, we assessed the utility of TTF-1 and p63, as an antibody panel, for differentiating between these two neoplasms in previously Papanicolaou (Pap)-stained cytologic smears and cytospin slides. Twenty-six lung carcinomas (13 small-cell lung carcinomas, 13 poorly differentiated pulmonary squamous cell carcinomas) were evaluated. One or two previously 95% ethanol-fixed, Pap-stained smears or cytospin slides were selected from each case. The cytologic material from these slides was transferred to positively charged slides. Unstained recuts were obtained from the corresponding histologic specimens or cell blocks. Immunohistochemical staining for TTF-1 and p63 was performed on the paired samples from each tumor. All (13/13) small-cell lung carcinomas were negative for p63 and 92% (12/13) were positive for TTF-1. Conversely, all (13/13) poorly differentiated pulmonary squamous cell carcinomas expressed p63 and did not express TTF-1. Immunoreactivity for p63 was also noted in bronchial reserve cells and metaplastic squamous cells. The immunostaining results obtained from the cytology slides were concordant with those of the histology or cell block sections in all cases. The results of this study show that TTF-1 and p63 immunostaining can be successfully applied to previously Pap-stained cytologic material, as an antibody panel, to facilitate pathologic differentiation between small-cell lung carcinomas and poorly differentiated pulmonary squamous cell carcinomas. p63 immunostaining, however, must be interpreted in conjunction with cytomorphology to distinguish between poorly differentiated pulmonary squamous cell carcinomas and benign cellular constituents of the lung.

Distinction of pulmonary small cell carcinoma from poorly differentiated squamous cell carcinoma: An immunohistochemical approach

Accurate morphologic distinction between small cell carcinoma and poorly differentiated squamous cell carcinoma has critical therapeutic significance, but can be limited by crush artifact, tumor necrosis, limited tumor representation, and overlapping morphologic features. We evaluated a panel of antibodies for their efficacy in distinguishing between these neoplasms. Formalin-fixed paraffin-embedded tissue sections of small cell carcinomas and poorly differentiated squamous cell carcinomas underwent immunohistochemical staining with antibodies to thyroid transcription factor-1, p63, high molecular weight keratin, and p16(INK4A). Of 28 small cell carcinomas, 26 (93%) small cell carcinomas showed diffuse moderate or strong staining for thyroid transcription factor-1 with no staining for high molecular weight keratin and p63. In contrast, 27/28 (96%) poorly differentiated squamous cell carcinomas manifested opposite immunoreactivities, with diffuse moderate or strong staining for high molecular weight keratin and p63, and no or minimal staining for thyroid transcription factor-1. In two additional cases originally interpreted as small cell carcinoma, high molecular weight keratin highlighted small numbers of neoplastic large cells, leading to reclassification as combined small cell and non-small cell carcinomas. p16(INK4A) expression varied widely in poorly differentiated squamous cell carcinomas, but was consistently moderate or strong and diffuse in small cell carcinomas, and proved helpful in the two thyroid transcription factor-1-negative small cell carcinomas. This study demonstrates that a panel consisting of antibodies to thyroid transcription factor-1, p63, high molecular weight keratin, and p16(INK4A) is highly effective for distinguishing between small cell carcinoma and poorly differentiated squamous cell carcinoma. This panel also facilitates diagnosis of combined small cell and non-small cell carcinomas.

Results of a randomized, prospective, multicenter trial of mycophenolate mofetil versus azathioprine in the prevention of acute lung allograft rejection

Background. Although the use of mycophenolate mofetil (MMF) has reduced the incidence of acute rejection in heart and kidney allograft recipients, its role in lung transplantation remains controversial. Therefore, we conducted a randomized, prospective, open-label, multicenter study in lung transplant recipients to determine whether MMF decreases episodes of acute allograft rejection when compared with azathioprine (AZA). Methods. Between March of 1997 and January of 1999, 81 consecutive lung transplant recipients from two centers were prospectively randomized to receive cyclosporine, corticosteroids, and either 2 mg/kg per day of AZA or 1 g twice daily of MMF. The primary study endpoint was biopsy-proven acute allograft rejection over the first 6 months posttransplant. Secondary endpoints included clinical rejection, cytomegalovirus (CMV) infection, adverse events, and survival. Surveillance bronchoscopies were performed at 1, 3, and 6 months, or if clinically indicated. Pathologists interpreting the biopsy results were blinded to the randomization. Results were analyzed according to intention-to-treat. Between group comparisons of means and proportions were made by using two sample t tests and Fisher’s exact tests, respectively. Six-month survival was calculated by the Kaplan-Meier method and compared by the log rank test. Results. Thirty-eight patients were prospectively randomized to receive AZA, and 43 MMF. The incidence of biopsy proven grade II or greater acute allograft rejection at 6 months was 58% in the AZA group and 63% in the MMF group (P =0.82). The 6-month survival rates in the MMF and AZA groups were 86% and 82%, respectively (P =0.57). Rates of CMV infection and adverse events were not significantly different between the two groups. Conclusions. Acute rejection rates and overall survival at 6 months are similar in lung transplant recipients treated with either MMF- or AZA-based immunosuppression.

Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron

In many models, a protective role for heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, has been demonstrated. Also, HO-1 null mice (KO) are more susceptible to inflammation and hypoxia and transplant rejection. Nonetheless, their response to hyperoxia (> 95% O(2)) has not yet been evaluated. Surprisingly, after acute hyperoxic exposure, KO had significantly decreased markers of lung oxidative injury and survived chronic hyperoxia as well as wild-type (WT) controls. Disrupted HO-1 expression was associated with decreased lung reactive iron and iron-associated proteins, decreased NADPH cytochrome cp450 reductase activity, and decreased lung peroxidase activity compared to WT. Injection of tin protoporphyrin, an inhibitor of HO, in the WT decreased acute hyperoxic lung injury, whereas transduction of human HO-1 in the KO reversed the relative protection of the KO to acute injury and worsened hyperoxic survival. This suggests that disruption of HO-1 protects against hyperoxia by diminishing the generation of toxic reactive intermediates in the lung via iron and H(2)O(2).

Effects of Paramyxoviral Infection on Airway Epithelial Cell Foxj1 Expression, Ciliogenesis, and Mucociliary Function

To elucidate molecular mechanisms underlying the association between respiratory viral infection and predisposition to subsequent bacterial infection, we used in vivo and in vitro models and human samples to characterize respiratory virus-induced changes in airway epithelial cell morphology, gene expression, and mucociliary function. Mouse paramyxoviral bronchitis resulted in airway epithelial cell infection and a distinct pattern of epithelial cell morphology changes and altered expression of the differentiation markers beta-tubulin-IV, Clara cell secretory protein, and Foxj1. Furthermore, changes in gene expression were recapitulated using an in vitro epithelial cell culture system and progressed independent of the host inflammatory response. Restoration of mature airway epithelium occurred in a pattern similar to epithelial cell differentiation and ciliogenesis in embryonic lung development characterized by sequential proliferation of undifferentiated cells, basal body production, Foxj1 expression, and beta-tubulin-IV expression. The effects of virus-induced alterations in morphology and gene expression on epithelial cell function were illustrated by decreased airway mucociliary velocity and impaired bacterial clearance. Similar changes in epithelial cell Foxj1 expression were also observed in human paramyxoviral respiratory infection. Taken together, these model systems of paramyxoviral respiratory infection mimic human pathology and identify epithelial cell Foxj1 expression as an early marker of epithelial cell differentiation, recovery, and function.

MiR-365 regulates lung cancer and developmental gene thyroid transcription factor 1

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is an essential fetal lung developmental factor, which can be recurrently activated by gene amplification in adult lung cancer. We have discovered the first microRNA (i.e., miR-365) that directly regulates TTF-1 by interacting with its 3’-untranslated region. By gene expression profiling, we identified other putative targets of miR-365 and miR-365*. In line with the microRNA/target relationship, the expression patterns of miR-365 and TTF-1 were in an inverse relationship in human lung cancer. Exploration of human lung cancer genomics data uncovered that TTF-1 gene amplification was significantly associated with DNA copy number loss at one of the two genomic loci encoding the precursor RNA of mature miR-365 (i.e., mir-365-1). This implies the existence of genetic selection pressure to lose the repressive miR-365 that would otherwise suppress amplified TTF-1. We detected a signaling loop between transforming growth factor beta (TGFb) and miR-365 and this loop reinforced suppression of TTF-1 via miR-365. Mir-365 also targeted an epithelial mesenchymal transition (EMT)-promoting gene HMGA2. In summary, these data connect the lung transcriptional program to the microRNA network.

Mesalamine-related lung disease: clinical, radiographic, and pathologic manifestations.

Lung injury related to mesalamine (5-aminosalicylic acid) has rarely been reported in patients with inflammatory bowel diseases. Patients present with progressive respiratory symptoms and radiographic abnormalities whose genesis may occur from days to years after initiation of therapy. Although pathologic features overlap with other pulmonary disorders, findings of chronic interstitial pneumonia and poorly formed nonnecrotizing granulomas should prompt consideration of mesalamine-related lung disease in a patient receiving this medication. The authors describe the clinical, radiographic, and pathologic manifestations of mesalamine-related lung disease in three patients and review the literature related to this topic.

Causes of pulmonary granulomas: a retrospective study of 500 cases from seven countries

Background The frequencies of various causes of pulmonary granulomas in pathological material are unknown, as is the influence of geographical location on aetiology. The aim of this study was to identify the causes of pulmonary granulomas in pathological specimens, to define their frequencies, and to determine whether these causes vary by geographical location. Methods 500 lung biopsies and resections containing granulomas were reviewed retrospectively by expert pulmonary pathologists from 10 institutions in seven countries. Fifty consecutive cases from each location were assigned a diagnosis based on histological features and available clinical/microbiological data. Results A specific cause was identified in 58% of cases (290/500), most commonly sarcoidosis (136, 27%) and mycobacterial or fungal infections (125, 25%). Mycobacteria were identified in 19% of cases outside the USA versus 8% within the USA. In contrast, fungi accounted for 19% cases in the USA versus 4% in other locations. Fungi were mostly detected by histology, whereas most mycobacteria were identified in cultures. In 42% of cases (210/500) an aetiology could not be determined. Conclusions Across several geographical settings, sarcoidosis and infections are the most common causes of pulmonary granulomas diagnosed in pathological specimens. Fungi are more commonly identified than mycobacteria in the USA, whereas the reverse is true in other countries. A definite aetiology cannot be demonstrated in more than a third of all cases of pulmonary granulomas, even after histological examination. These findings highlight the need to submit material for histology as well as cultures in all cases in which granulomatous disease enters the differential diagnosis.