Optimising magnetic resonance imaging-based evaluation of the ossification of the medial clavicular epiphysis: a multi-centre study.

Evaluation of the ossification of the medial clavicular epiphysis plays a key role in forensic age estimation, particularly in determining whether the age of 18 has been attained. A key research objective in the forensic age estimation field at present is to establish non-X-ray methods for investigating the clavicle. This paper looks at the use of magnetic resonance imaging for evaluating the developmental state of the medial clavicular epiphysis. Clavicle specimens obtained from autopsies of 125 female and 270 male subjects aged from 10 to 30 were examined using a 3-T magnetic resonance scanner. One FFE-3D-T1 gradient echo sequence and one 2D-T2 turbo spin echo sequence were acquired. In each case, two investigators undertook a consensual determination of the ossification stage of the medial clavicular epiphysis using recognised classification systems. To determine intra-observer and inter-observer agreement, 80 clavicle specimens were subjected to repeat evaluation. We present statistics relating to the ossification stages. The inclusion of established sub-stages of clavicular ossification offers an additional option for determining whether a subject has attained the age of 18 which is applicable in both sexes. For both sexes, the minimum ages for ossification stages 4 and 5 allow conclusions to be drawn about a subject's age at a point in time lying several years in the past. Magnetic resonance imaging is a valid investigatory procedure for determining the ossification stage of the medial clavicular epiphysis. This paper makes a contribution to expanding the range of methods available for forensic age estimation.

CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix.

Microvascular endothelial cell invasion into the fibrin provisional matrix is an integral component of angiogenesis during wound repair. Cell surface receptors which interact with extracellular matrix proteins participate in cell migration and invasion. Malignant cells use CD44-related chondroitin sulfate proteoglycan (CSPG) as a matrix receptor to mediate migration and invasion. In this study, we examine whether cell surface CSPG can mediate similar events in nonmalignant wound microvascular endothelial cells or whether use of CSPG for migration and invasion is a property largely restricted to malignant cells. After inhibiting CSPG synthesis with p-nitrophenyl beta-d xylopyranoside (beta-d xyloside), wound microvascular endothelial cells were capable of attaching and spreading on the surface of a fibrin gel; however, their ability to invade the fibrin matrix was virtually eliminated. To begin to examine the mechanism by which endothelial cells use CSPG to invade fibrin matrices, cell adhesion and migration on fibrinogen was examined. Endothelial cell adhesion and migration on fibrinogen were inhibited by both beta-d xyloside and after cleavage of chondroitin sulfate from the core protein by chondroitinase ABC. We have determined that wound microvascular endothelial cells express the majority of their proteoglycan as CSPG and that the CSPG core protein is immunologically related to CD44. PCR studies show that these cells express both the "standard" (CD44H) isoform and an isoform containing the variably spliced exon V3. In addition, anti-CD44 antibody blocks endothelial cell migration on fibrinogen. Affinity chromatography studies reveal that partially purified microvascular endothelial cell CSPG binds fibrinogen. These findings suggest that CD44-related CSPG, a molecule implicated in the invasive behavior of tumor cells, is capable of binding fibrinogen/fibrin, thereby mediating endothelial cell migration and invasion into the fibrin provisional matrix during wound repair.

Acute lung injury. Pathogenesis of intraalveolar fibrosis.

In patients dying with acute lung injury, interstitial mesenchymal cells migrate into the airspace where they replicate and deposit connective tissue. We therefore hypothesized that peptides capable of promoting mesenchymal cell migration and replication would be present in the alveolar airspace. To examine this hypothesis, patients with severe acute diffuse lung injury (n = 26) underwent bronchoalveolar lavage. Acutely ill patients without lung injury served as controls (n = 12). Recovered effluent was examined for mesenchymal cell growth-promoting and migration-promoting activity. Lavage cell supernates from both patients and controls were devoid of bioactivity. However, substantial growth-promoting and migration-promoting activity was present in lavage fluid from nearly every patient, whereas little or none was present in fluid from controls. Characterization of the bioactivity indicated a significant proportion consisted of three peptides related to PDGF: (a) a 14-kD peptide that shared with PDGF several biophysical, biochemical, receptor-binding, and antigenic properties; (b) a 29-kD peptide that appeared identical to PDGF of platelet origin; and (c) a 38-kD peptide that was biophysically and antigenically similar to PDGF. These data indicate that peptide moieties are present in the airspace of patients after acute lung injury that can signal mesenchymal cell migration and replication.

Lung transplant pathology. A comparative study of pulmonary acute rejection and cytomegaloviral infection.

This article describes a comparative study performed to determine the histologic features of pulmonary rejection and cytomegaloviral (CMV) infection following lung transplantation. Rejection was defined clinically by findings of new pulmonary symptoms or radiographic changes or decreased oxygenation in the absence of documented infection in patients who were treated for rejection and improved. These patients also had negative CMV cultures. CMV infection was studied in a group of non-lung and non-bone marrow transplant patients and was defined by the presence of characteristic nuclear inclusions in lung biopsies. Ten rejection biopsies and nine CMV biopsies were examined. No histologic feature was unique to rejection, however; perivascular lymphocytic infiltrates occurred more frequently and more intensely in rejection than in CMV infection (p = 0.0029). Endothelialitis also occurred more frequently in rejection (p = 0.0331), but it was always seen in association with perivascular lymphocytic inflammation. In rejection, the inflammatory infiltrate was primarily perivascular, with extension into the interstitium in several cases. In contrast, CMV infection was characterized predominantly by interstitial inflammation with involvement of associated vessels. We conclude that although overlapping features are present in both processes, pulmonary rejection can be distinguished from CMV infection on the basis of histology.

Single lung transplantation for severe emphysema.

UNLABELLED: Lung transplantation is effective therapy for patients with severe obstructive lung disease. We reviewed seven patients with severe emphysema (age, 48 +/- 5 years; forced expiratory volume in 1 second [FEV1] 0.76 +/- 0.26 liters) who received single-lung transplants (SLT) at our institution between August 1989 and September 1990. Studies to assess the adequacy of cardiac function before transplantation showed moderately reduced right ventricular function (by multiple gated acquisition, 34 +/- 6%), moderately elevated pulmonary artery pressure (25 +/- 3 mm Hg), and normal left ventricular function (by multiple gated acquisition 65% +/- 12%) and coronary arteriograms. Time on the waiting list before transplantation was reduced compared with heart-lung transplant (HLT) recipients (waiting time, 2.9 +/- 1.5 months for SLT, 9.6 +/- 10.2 months for HLT). Six of the SLT recipients are currently alive (after transplantation interval, 17 +/- 5 months); the remaining recipient died of pulmonary embolism 21 days after SLT. Number of ventilator days, intensive care unit days, and days to hospital discharge after transplantation did not differ significantly from HLT recipients. Cardiopulmonary bypass was necessary in four SLT recipients. Pulmonary function was markedly improved after SLT (FEV1, 1.78 +/- 0.73 L/min after SLT versus 0.75 +/- 0.3 L/min before SLT; p less than 0.01), and functional status is correspondingly improved. CONCLUSIONS: SLT constitutes effective therapy for patients with severe emphysema, including those with moderate reduction of right ventricular function; and SLT offers distinct advantages over HLT, including decreased waiting time before transplantation, improved donor organ utilization, and less frequent need for cardiopulmonary bypass.

Mechanisms of alveolar fibrosis after acute lung injury.

In patients who die after severe acute lung injury, a dramatic fibroproliferative response occurs within the alveolar air space, interstitium, and microvessels. Profound shunt physiology, dead space ventilation, and pulmonary hypertension are the physiologic consequences of this fibroproliferative response. The anatomic pattern of the response is unique within each alveolar compartment. For example, the air space is obliterated by granulation tissue, with replicating mesenchymal cells, their connective tissue products, and an expanding network of intra-alveolar capillaries. In contrast, the vascular fibroproliferative response is dominated by mesenchymal cell replication and connective tissue deposition within the walls of microvessels. Despite the unique anatomic features of these fibroproliferative processes, the regulatory signals involved are likely to be similar. Although our current understanding of the signals regulating the fibroproliferative response to acute lung injury is limited, inferences can be made from in vitro studies of mesenchymal cell behavior and several better understood fibroproliferative processes, including wound healing and chronic fibrotic lung diseases. As clinicians, our future ability to enhance effective lung repair will likely utilize therapeutic strategies specifically targeted to the signals that regulate the fibroproliferative process within the alveolar microenvironment.

Intrapleurally administered streptokinase in the treatment of acute loculated nonpurulent parapneumonic effusions.

Adequate pleural drainage is believed to be an essential component of the management of low pH-low glucose parapneumonic effusion. Parapneumonic effusions may become loculated rapidly, preventing adequate drainage with a single chest tube. Administration of intrapleural streptokinase may be effective in promoting drainage for loculated, nonpurulent low pH-low glucose parapneumonic effusions when fibrin adhesions may not yet be organized. Intrapleural streptokinase was used in 12 patients with relatively large, symptomatic, loculated, nonpurulent parapneumonic effusions in whom the initial thoracentesis demonstrated a pH less than or equal to 7.0 and/or glucose less than or equal to 40 mg/dl, and when inadequate drainage was demonstrated roentgenographically despite tube thoracostomy. Mean pleural fluid WBC was 9,750/mm3 (range, 1 to 27 K), and pleural fluid glucose and pH were 33 +/- 21 mg/dl and 6.95 +/- 0.19, respectively. A solution of streptokinase, 250,000 units in normal saline, was given intrapleurally via the chest tube. Effectiveness of intrapleural streptokinase was assessed radiographically and by monitoring the volume of fluid drained from the chest tube after streptokinase instillation. A greater than 50% improvement in the CXR was seen in nine of 12 patients after intrapleural administration of streptokinase. The volume of fluid out in the first 48 h post-streptokinase was 849 +/- 836 ml (range, 100 to 3,000). In addition, clinical improvement (decreased chest discomfort, less dyspnea, or reduced fever) was noted in eight of 12 patients after streptokinase treatment. We conclude that intrapleural administration of streptokinase is an effective adjunct to the management of nonpurulent, loculated parapneumonic effusions that may reduce the need for multiple chest tubes or surgical drainage.

Induction of lung fibroblast apoptosis by soluble fibronectin peptides.

Despite the importance of fibroproliferative lung disorders, no safe and effective therapies exist for reducing the size of the fibroblast population in existing fibrotic lesions. Recent work suggests that therapies that promote fibroblast apoptosis during the repair phase following lung injury may facilitate lung repair by eliminating excess fibrotic tissue. We report here our finding that three soluble fibronectin peptides (RGD, CS-1, and FN-C/H-V) induce apoptosis in lung fibroblasts. Fibroblast susceptibility to these peptides was dose and time dependent, with a maximal effect observed at 96 h (87 +/- 16% [mean +/- SEM] apoptosis). The peptides were able to induce fibroblast apoptosis in fibrin gels, an in vitro model of early fibroproliferative lesions. Fibroblasts were difficult to kill. All three peptides were required for maximal apoptosis of anchored cells. Apoptosis occurred by disruption of adhesion (anoikis). Treatment of fibroblasts with peptides caused proteolysis of pp125FAK, a tyrosine kinase involved in integrin-mediated signaling related to cell survival. These data show that soluble fibronectin peptides trigger nontransformed fibroblast apoptosis in routine culture and in fibrin gels by a mechanism that includes disruption of an integrin-mediated survival signaling pathway. The use of small fibronectin peptides to promote fibroblast apoptosis warrants further study as possible antifibrotic therapy.

Integrin mediation of type II cell adherence to provisional matrix proteins.

Lung injury causes alveolar type I epithelial cell death, basement membrane denudation, and alveolar flooding with serum fibronectin and fibrinogen. For successful restoration of normal architecture, the epithelium must be regenerated from progenitor type II alveolar cells. Using adhesion assays, we examined whether type II alveolar cells adhere to the provisional matrix proteins fibronectin, fibrinogen, and fibrin, and whether integrins mediate this adherence. Rat type II cells adhered to fibronectin, vitronectin, fibrinogen, and fibrin. Synthetic RGD (arginine-glycine-aspartic acid) peptide blocked this adhesion. Flow cytometry and Western analysis indicated that type II cells expressed beta 1- and alpha v beta 3-integrins. Anti-beta 1-and anti-alpha v beta 3-integrin antibodies blocked type II cell adhesion to fibronectin and to fibronectin and fibrinogen, respectively. In summary, type II cells adhered to fibronectin, fibrinogen, and fibrin, and adhesion was partially mediated by integrins. This study provides the first evidence of type II cell adhesion to fibrin gels and vitronectin, beta 1- and alpha v beta 3-integrin mediation of type II cell adhesion, and the presence of the alpha v beta 3-integrin on type II epithelial cells.

Integrin mediation of alveolar epithelial cell migration on fibronectin and type I collagen.

Acute lung injury leads to type I alveolar epithelial cell (AEC) death, denudation of the alveolar basement membrane, and formation of an alveolar provisional matrix from fibronectin, fibrinogen, and type I collagen. The provisional matrix provides a scaffold for alveolar repair. To restore normal lung architecture, surviving type II AECs must reepithelialize denuded alveoli. We examined whether AECs migrate on provisional matrix proteins and whether integrins mediate this migration using a Boyden chemotaxis chamber. Cultured AECs migrated on fibronectin-coated filters by haptotaxis (defined as movement on a solid-phase substrate) more than one type I collagen-coated filters, and they did not migrate on fibrinogen-coated filters. Soluble fibronectin augmented migration on type I collagen-coated filters, but not on fibronectin-coated filters. Anti-alpha v beta 3-integrin monoclonal antibody (MAb) inhibited migration on substrate-bound fibronectin by 62-77%, whereas anti-beta 1-integrin MAb inhibited migration by 48%. Anti-alpha 2-integrin MAb almost completely inhibited migration on substrate-bound type I collagen, but not on fibronectin. The novel findings in this study are as follows: 1) AECs migrate by haptotaxis more effectively on substrate-bound fibronectin than on type I collagen; 2) alpha v beta 3- and beta 1-integrins partially mediate AEC haptotaxis on fibronectin; and 3) the alpha 2 beta 1-integrin mediates AEC migration on type I collagen. These results support the importance of type II cell migration on provisional matrix proteins during repair of lung injury.

A comparison of the ability of frog and rat S-9 to activate promutagens in the Ames test.

A mutagenesis assay employing the frog, Rana pipiens, is currently under development [McKinnell et al, 1979]. A question that must be answered is whether the frog is metabolically capable of activating a large number of promutagens. The Ames assay offers a simple means of comparing the metabolism of mutagens by different animal species. The Ames response obtained with frog-liver S-9 was compared to the response with rat-liver S-9, using the following compounds: Benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, 2-amino-fluorene, azobenzene, Sudan II, dibutylnitrosamine, hydrazine sulfate, hydroxyethylhydrazine, cyclophosphamide, 1,2-dichloroethane, tris(2,3 dibromopropyl)phosphate, diallate, quinoline, quercetin, aflatoxin B 1, emodin, and safrole. Of these compounds, activation by rat S-9 was observed for all except hydrazine sulfate and safrole. All except Sudan II, 1,2-dichloroethane, quinoline, and safrole gave positive Ames responses with frog S-9. In general, the responses with frog S-9 were quantitatively lower than those obtained with Aroclor-induced rat S-9; however, the optimum procedure for frog-liver induction has not been determined. The response to dichloroethane is very sensitive to the amount of activating enzyme present; it might be positive with optimally induced frog S-9. Thus, only two of the 15 compounds positive with rat S-9 were definitely missed when tested with frog S-9. We feel that the frog assay appears to be promising from the standpoint of false-negatives.

Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor.

Fibroproliferative disorders are characterized by accumulations of mesenchymal cells and connective tissue in critical locations, leading to organ dysfunction. We examined the role of platelet-derived growth factor (PDGF) in the pathogenesis of obliterative bronchiolitis, a fibroproliferative process that occurs after lung transplantation and results in small airway occlusion. Bronchoalveolar lavage fluid from obliterative bronchiolitis patients significantly stimulated fibroblast migration, whereas fluid from patient controls did not. Quantitation by radioligand binding assay demonstrated increased concentrations of PDGF in lavage fluid from obliterative bronchiolitis patients (patients, 104 +/- 26.9 pM; controls, 8.4 +/- 6.9 pM; P < 0.01). Heparin affinity, gel filtration, and Western blot analysis confirmed the presence of PDGF in lavage fluid. Immunohistochemical and in situ hybridization studies of histologic sections and bronchoalveolar lavage cells suggest that alveolar macrophages are one cellular source. Prospective evaluation of sequential bronchoalveolar lavage samples from a patient who developed obliterative bronchiolitis demonstrated markedly increased PDGF concentrations before the onset of irreversible airflow obstruction. These findings are consistent with a role for PDGF in the fibroproliferative changes observed in obliterative bronchiolitis.