C. Bruce Alexander

Nitric Oxide-dependent Generation of Reactive Species in Sickle Cell Disease ACTIN TYROSINE NITRATION INDUCES DEFECTIVE CYTOSKELETAL POLYMERIZATION

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO2Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO2Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO2Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO2Tyr content and increased levels of hepatic and renal protein NO2Tyr derivatives in SCD (21.4 ± 2.6 and 37.5 ± 7.8 ng/mg) versus wild type mice (8.2 ± 2.2 and 10 ± 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO2Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr91, Tyr198, and Tyr240) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.

Loss of Fibroblast Thy-1 Expression Correlates with Lung Fibrogenesis

Fibroblasts consist of heterogeneous subpopulations that have distinct roles in fibrotic responses. Previously we reported enhanced proliferation in response to fibrogenic growth factors and selective activation of latent transforming growth factor (TGF)-beta in fibroblasts lacking cell surface expression of Thy-1 glycoprotein, suggesting that Thy-1 modulates the fibrogenic potential of fibroblasts. Here we report that compared to controls Thy-1-/- C57BL/6 mice displayed more severe histopathological lung fibrosis, greater accumulation of lung collagen, and increased TGF-beta activation in the lungs 14 days after intratracheal bleomycin. The majority of cells demonstrating TGF-beta activation and myofibroblast differentiation in bleomycin-induced lesions were Thy-1-negative. Histological sections from patients with idiopathic pulmonary fibrosis demonstrated absent Thy-1 staining within fibroblastic foci. Normal lung fibroblasts, in both mice and humans, were predominantly Thy-1-positive. The fibrogenic cytokines interleukin-1 and tumor necrosis factor-alpha induced loss of fibroblast Thy-1 surface expression in vitro, which was associated with Thy-1 shedding, Smad phosphorylation, and myofibroblast differentiation. These results suggest that fibrogenic injury promotes loss of lung fibroblast Thy-1 expression, resulting in enhanced fibrogenesis.

Spatial mapping of pulmonary and vascular nitrotyrosine reveals the pivotal role of myeloperoxidase as a catalyst for tyrosine nitration in inflammatory diseases

Nitrotyrosine (NO(2)Tyr) formation is a hallmark of acute and chronic inflammation and has been detected in a wide variety of human pathologies. However, the mechanisms responsible for this posttranslational protein modification remain elusive. While NO(2)Tyr has been considered a marker of peroxynitrite (ONOO(-)) formation previously, there is growing evidence that heme-protein peroxidase activity, in particular neutrophil-derived myeloperoxidase (MPO), significantly contributes to NO(2)Tyr formation in vivo via the oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (.NO(2)). Coronary arteries from a patient with coronary artery disease, liver and lung tissues from a sickle cell disease patient, and an open lung biopsy from a lung transplant patient undergoing rejection were analyzed immunohistochemically to map relative tissue distributions of MPO and NO(2)Tyr. MPO immunodistribution was concentrated along the subendothelium in coronary tissue and hepatic veins as well as in the alveolar epithelial compartment of lung tissue from patients with sickle cell disease or acute rejection. MPO immunoreactivity strongly colocalized with NO(2)Tyr formation, which was similarly distributed in the subendothelial and epithelial regions of these tissues. The extracellular matrix protein fibronectin (FN), previously identified as a primary site of MPO association in vascular inflammatory reactions, proved to be a major target protein for tyrosine nitration, with a strong colocalization of MPO, NO(2)Tyr, and tissue FN occurring. Finally, lung tissue from MPO(-/-) mice, having tissue inflammatory responses stimulated by intraperitoneal zymosan administration, revealed less subendothelial NO(2)Tyr immunoreactivity than tissue from wild-type mice, confirming the significant role that MPO plays in catalyzing tissue nitration reactions. These observations reveal that (i) sequestration of neutrophil-derived MPO in vascular endothelial and alveolar epithelial compartments is an important aspect of MPO distribution and action in vivo, (ii) MPO-catalyzed NO(2)Tyr formation occurs in diverse vascular and pulmonary inflammatory pathologies, and (iii) extracellular matrix FN is an important target of tyrosine nitration in these inflammatory processes.

Small cell carcinoma of the esophagus: Evidence for a unified histogenesis

Four cases of small cell carcinoma of the esophagus, histologically indistinguishable from oat cell carcinoma of the lung, were studied by electron microscopy. Three were composed largely of small cells with neurosecretory granules characteristic of APUD (amine precursor uptake and decarboxylation) cells. Two of these three cases also contained foci of squamous or glandular differentiation, or both. The fourth case was classified as reserve cell carcinoma with squamous differentiation, since the tumor cells were devoid of neurosecretory granules but contained desmosomes and bundles of intracytoplasmic tonofilaments. The evidence provided by these four cases and also by the 48 cases previously reported in the literature supports our hypothesis that a totipotent primitive cell serves as the common precursor for squamous cell, adeno-, and small cell carcinoma of the esophagus. The latter can be subdivided into oat cell and reserve cell carcinomas, both having the propensity for further squamous and glandular differentiation. The hypothesis can also be applied to lung cancer.

Pelvic trauma in rapidly fatal motor vehicle accidents.

Objective To study the incidence and nature of pelvic fractures in rapidly fatal automobile accidents. Design Retrospective. Setting County Medical Examiners Office. Patients The files of 255 consecutive motor vehicle accident fatalities examined at the Jefferson County Coroner/Medical Examiners office (study period 1996–1998) were reviewed. We orrelated this information with our previous findings, derived from a review of 392 such cases (study period 1994–1996). Results Approximately 25% of decedents involved in rapidly fatal automobile accidents sustained pelvic fractures. In 93% of the cases, postmortem radiographs were available and suitable for scoring according to the Orthopaedic Trauma Association nomenclature. The distribution of pelvic fractures by type was type A, 16%; type B, 32%; and type C, 52%, with the most common pelvic fracture being type C1 (26%). Additionally, pedestrians and motorcyclists were twice as likely to sustain a pelvic fracture, and the severity of pelvic fracture type seemed to correlate with increasing speed of the automobile. No correlation between drug use or direction of impact and incidence or type of pelvic fracture was observed. Compared with published studies on survivors of automobile accidents, our data suggest that pelvic injuries may tend to be more severe in victims who do not survive to hospitalization. Conclusions Our data indicate that current estimates about the mortality of pelvic fractures may be faulty due to exclusion of victims who fail to survive to hospitalization. This series suggests that an appreciation of the full spectrum of pelvic ring disruptions requires collaboration between orthopaedic surgeons and forensic pathologists.

Black thyroid: clinical manifestations, ultrastructural findings, and possible mechanisms.

Ultrastructural examination of three black thyroid glands showed lysosomal accumulations of lipofuscin-like pigment and granular electron-dense material in association with 1) a minocycline-associated black thyroid with normal thyroid function; 2) a minocycline-associated black thyroid with a significant inflammatory component, fibrosis, and primary hypothyroidism; and 3) a black thyroid gland with no exposure to minocycline. The deposition of the pigments in the three cases resulted in macroscopically recognizable black thyroid glands. It is speculated that an imbalance in lysosomal function accounts for this abnormality. The glandular hypofunction documented in case 2 is unique and confirms the need to monitor function carefully in patients who are receiving minocycline. In one case electrondense deposits were identified in the thyroid gland interstitium.

Intrapulmonary teratoma : a case report and review of the literature

Intrapulmonary teratomas are rare; only 30 cases have been reported in the world literature. These tumors are thought to originate from the third pharyngeal pouch. They occur equally in men and women and usually are diagnosed in the second to fourth decade of life. They are more often benign than malignant, although malignant lesions may have a favorable postoperative prognosis and benign lesions may exhibit high morbidity and mortality because of their size and location. These tumors present radiographically as lobulated masses that may contain calcification or peripheral collections of air. They most often occur in the upper lobes. The computed tomographic findings of intrapulmonary teratoma are less well known but have been described in two cases. An additional case is presented with a review of the literature and a tabular summary of the characteristics of this unusual entity.

The utility of open lung biopsy following lung transplantation.

BACKGROUND Most pulmonary complications associated with lung transplantation have non-specific clinical characteristics. Furthermore, common diagnostic modalities, including bronchoscopy with transbronchial biopsy (TBB), often do not render a definitive diagnosis. In this study, we reviewed our experience with open lung biopsy (OLB) following lung transplantation, specifically regarding its ability to safely provide clinically relevant information that affects therapeutic decisions. METHODS From October 1989 to March 2000, 202 patients underwent lung transplantation at our institution. We reviewed the clinical course of the 42 patients who received 48 OLBs. Of these patients, we determined the pre-operative clinical condition, preceding TBB histologic information, OLB histology, treatment changes, and procedural complications as a result of the OLB. RESULTS A new, clinically unsuspected diagnosis was made in 14 biopsies (29% of all OLB), and all of these resulted in therapy changes. Thirty-two biopsies (67% of all OLB) confirmed our clinical suspicions, and new therapy was initiated in 30 of these patients. Two patients (4% of all OLB) had non-diagnostic OLB. Four biopsies (8% of all OLB), including the 2 non-diagnostic OLBs, did not result in any therapy changes or initiation of new therapy. Complications occurred in 3 patients, all of whom had an air leak for >7 days. CONCLUSION Open lung biopsy in lung transplant patients renders a new, unsuspected diagnosis in nearly one third of patients and leads to specific, directed therapy in the vast majority of patients. Open-lung biopsy can be performed safely and should be considered when diagnosis is uncertain in clinically deteriorating patients.

Analysis of the incidence of pelvic trauma in fatal automobile accidents

Pelvic fractures are frequent complications of motor vehicle accidents, and motor vehicle accidents are the most common cause of pelvic fracture. Although pelvic fractures are associated with considerable morbidity and mortality, there has traditionally been no attempt to grade or classify pelvic fractures during postmortem examination. The authors performed a retrospective study of cases examined at the Jefferson County Coroner/Medical Examiner Office from 1994 to 1996, reviewing investigative reports and autopsy findings. Radiographs were examined for the presence and Tile type of pelvic fracture. Pelvic fractures were identified in 88 of 392 cases (23%). In most (89%), the pelvic fracture was readily classified according to Tile type on the basis of radiographs and the inferred mechanism of injury. This study indicates that current estimates of the mortality of pelvic fractures are low because of the exclusion of individuals who do not survive to hospitalization. Furthermore, pelvic fractures in rapidly fatal motor vehicle accidents tend to be more severe than fractures in individuals who have a significant interval of survival. The presence and classification of pelvic fractures may be readily determined by radiographs in most cases. The Tile classification scheme is easy to apply and has important implications in the comparison of study groups.

2008 ACLPS Panel Discussion on Resident Education in Clinical Pathology

A curriculum in clinical pathology (CP) was developed under the auspices of the Academy of Clinical Laboratory Physicians and Scientists (ACLPS) in 2006. At the 2008 ACLPS meeting in Philadelphia, PA, a panel was convened to address the current challenges in resident education and how to overcome them. Current challenges include the heterogeneity of the discipline (which requires analytical, medical, and managerial knowledge), the diverse repertoire of clinical laboratory testing, and the need to better integrate the resident into the work flow of the laboratory, especially with respect to clinical consultation. Recommendations of the panel include the incorporation of active learning, clinical consultation, and competency assessment into CP resident training. A summary of the panel discussion is presented herein.