Multiple tissues express alpha 1-antitrypsin in transgenic mice and man.

Hepatocytes are considered to be the predominant source of alpha 1-antitrypsin (AAT), the major antiprotease in human plasma. The development of emphysema in the hereditary PiZ AAT deficiency state suggests that inhibition of leukocyte elastase in the lung is a major function of this protein. In addition, patients with AAT deficiency are at increased risk for developing cholestasis in infancy and chronic liver disease as adults. The mechanism for hepatic cell injury, however, is not understood. Transgenic mice that express the normal human AAT gene demonstrate abundant AAT in hepatocytes and specific cell types of numerous nonhepatic tissues. Immunoperoxidase techniques have previously disclosed AAT in many of the cell types seen in transgenic mice; however, the issue of local synthesis vs. endocytosis in these cell types has remained unresolved. In this study, AAT mRNA was seen in a variety of tissues in the transgenic mouse. Immunoelectron microscopy of renal tubular and small intestinal epithelial cells in the transgenic mice demonstrated AAT within the cisternae of the rough endoplasmic reticulum, as in hepatocytes. These findings support the possibility of local synthesis in the various cell types. The results suggest that in addition to maintaining tissue integrity in the lung, the protease/antiprotease balance may have physiological functions in other organs as well.

Regulation of intercellular adhesion molecule-1 (ICAM-1) in ischemic and reperfused canine myocardium.

Previous studies in vitro have shown an important role for intercellular adhesion molecule-1 (ICAM-1) in adherence interactions of canine neutrophils with canine jugular vein endothelial cells and in cytotoxicity of canine neutrophils for adult cardiac myocytes. To evaluate the regulation of ICAM-1 in myocardial inflammation and its role in the pathogenesis of myocardial ischemia and reperfusion, a series of in vivo and ex vivo studies were performed in canine animals. Systemic administration of LPS elicited ICAM-1 mRNA in several tissues, including myocardium, which demonstrated increasing ICAM-1 staining on intercalated discs of cardiac myocytes. In ischemia and reperfusion protocols: (a) ICAM-1 mRNA was found in ischemic segments within 1 h of reperfusion and in both ischemic and normally perfused segments by 24 h of reperfusion; (b) expression of ICAM-1 was detected in cardiac myocytes in the ischemic region by 6 h of reperfusion; increased expression was seen thereafter as a function of time; (c) post-ischemic (but not preischemic) cardiac lymph collected at intervals from 1 to 24 h after reperfusion elicited ICAM-1 mRNA, ICAM-1 expression, and ICAM-1-dependent neutrophil adhesion in canine jugular vein endothelial cells and in cardiac myocytes with peak cytokine activity seen by 1 h; (d) extravascular localization of neutrophils was detected in ischemic areas only, and was associated with endothelium bearing high levels of ICAM-1 within 1 h of reperfusion; infiltration increased thereafter in association with increasing levels of ICAM-1 mRNA in myocardial segments and increasing levels of ICAM-1 expression on cardiac myocytes. These findings provide the first direct evidence for inflammatory regulation of ICAM-1 in ischemic and reperfused canine myocardium. They support the hypothesis that ICAM-1 participates in neutrophil-mediated myocardial damage.

Adherence of neutrophils to canine cardiac myocytes in vitro is dependent on intercellular adhesion molecule-1.

The adhesiveness of isolated canine cardiac myocytes for neutrophils is greatly increased by stimulation with cytokines such as tumor necrosis factor alpha (TNF alpha). Since this adhesion is significantly inhibited by an anti-CD18 MAb, experiments were performed to test the hypothesis that the newly expressed adhesion molecule on the cardiac myocytes was intercellular adhesion molecule-1 (ICAM-1). A newly developed MAb, CL18/6, was found to exhibit the functional and binding characteristics with canine neutrophils and canine jugular vein endothelial cells expected of an antibody recognizing ICAM-1. MAb CL18/6 also bound to isolated cardiac myocytes after stimulation of the myocytes with cytokines, and it blocked by greater than 90% the adhesion of neutrophils to stimulated myocytes. A partial cDNA clone for canine ICAM-1 was isolated, and ICAM-1 mRNA was found to be increased in both endothelial cells and cardiac myocytes after cytokine stimulation. Cytokines that both increased the CL18/6-inhibitable adhesion of neutrophils to myocytes and induced expression of ICAM-1 were IL-1 beta, TNF alpha, and LPS. These results are consistent with the conclusion that canine endothelial cells and cardiac myocytes express ICAM-1 in response to cytokine stimulation, and that ICAM-1 functions as an adhesive molecule for neutrophils on both cell types.

Interleukin-8 gene induction in the myocardium after ischemia and reperfusion in vivo.

Neutrophil adhesion and direct cytotoxicity for cardiac myocytes require chemotactic stimulation and are dependent upon CD18-ICAM-1 binding. To characterize the potential role of IL-8 in this interaction, canine IL-8 cDNA was cloned and the mature recombinant protein expressed in Escherichia coli BL21 cells. Recombinant canine IL-8 markedly increased adhesion of neutrophils to isolated canine cardiac myocytes. This adhesion resulted in direct cytotoxicity for cardiac myocytes. Both processes were specifically blocked by antibodies directed against CD18 and IL-8. In vivo, after 1 h of coronary occlusion, IL-8 mRNA was markedly and consistently induced in reperfused segments of myocardium. IL-8 mRNA was not induced in control (normally perfused) myocardial segments. Minimal amounts of IL-8 mRNA were detected after 3 or 4 h of ischemia without reperfusion. Highest levels of induction were evident in the most ischemic myocardial segments. IL-8 mRNA peaked in the first 3 h of reperfusion and persisted at high levels beyond 24 h. IL-8 staining was present in the inflammatory infiltrate near the border between necrotic and viable myocardium, as well as in small veins in the same area. These findings provide the first direct evidence for regulation of IL-8 in ischemic and reperfused canine myocardium and support the hypothesis that IL-8 participates in neutrophil-mediated myocardial injury.

Myocardial contrast echocardiography: relation of collateral perfusion to extent of injury and severity of contractile dysfunction in a canine model of coronary thrombosis and reperfusion.

OBJECTIVES: This study sought to determine whether myocardial contrast echocardiography could be used to detect and quantitate collateral blood flow capable of limiting the effects of ischemia in an experimental model of coronary thrombosis and reperfusion. BACKGROUND: Myocardial contrast echocardiography has been used to assess collateral blood flow in humans, but this technique has not been extensively validated in the experimental laboratory. METHODS: Myocardial ischemia occurred after electrically induced left circumflex coronary artery thrombosis in a canine model. Ischemia was intensified by administration of vasodilators. Reperfusion was induced with recombinant tissue-type plasminogen activator. Myocardial perfusion was assessed with contrast echocardiography and radiolabeled microspheres. Infarct size was determined by histochemical staining methods. Myocardial samples were evaluated histologically. RESULTS: The dogs were classified into two groups on the basis of contrast echocardiographic detection of perfusion in the ischemic region: those with (n = 13) and without collateral flow (n = 10). Collateral perfusion detected by contrast echocardiography paralleled changes detected by radiolabeled microspheres during thrombosis and vasodilator administration. A 91% agreement was observed between the two techniques in detecting collateral flow > 0.3 ml/min per g (p < 0.0001). Collateral perfusion correlated directly with radial shortening fractions of the ischemic myocardium (p < 0.01). Recovery of function after reperfusion was faster, infarct size was smaller (mean [+/- SD] 4 +/- 1% vs. 11 +/- 3%, p = 0.05), and histopathologic injury was less in dogs with than without collateral flow, respectively (p < 0.05). CONCLUSIONS: Myocardial contrast echocardiography can identify physiologically significant collateral vessels capable of limiting the degree of ischemic damage during coronary thrombosis. The magnitude of collateral flow and the change in flow induced by vasodilators can be assessed and compares favorably with the microsphere standard.

Prenatal diagnosis of dominant and recessive dystrophic epidermolysis bullosa: application and limitations in the use of KF-1 and LH 7:2 monoclonal antibodies and immunofluorescence mapping technique.

Prenatal diagnosis is now possible for junctional and recessive dystrophic forms of epidermolysis bullosa (EB); however, there is no similar published experience for dominant dystrophic EB, although data with KF-1 monoclonal antibody suggests that both forms of dystrophic EB can be identified at least postnatally with this unique probe. We now report our experience with light microscopy, electron microscopy, immunofluorescence mapping, and KF-1 and LH 7:2 monoclonal antibodies, in both a mother with dominant dystrophic EB and her fetus at risk, and in a fetus previously shown to be affected with recessive dystrophic EB. KF-1 and LH 7:2 antigens were absent in recessive dystrophic EB fetal skin, identical to findings observed postnatally. LH 7:2 was normally expressed in a mother with dominant dystrophic EB and in her fetus at risk for this disease. In contrast, while KF-1 antigen was abnormally expressed in the affected mother, it was normally expressed in only 1/7 fetal biopsies despite the fact that this fetus was shown by light and electron microscopy and immunofluorescence mapping to be unaffected with dominant dystrophic EB. We conclude that 1) transmission electron microscopy can be used to prenatally exclude the diagnosis of dominant dystrophic EB (Cockayne-Touraine variety), 2) immunofluorescence mapping is an accurate technique for prenatal as well as postnatal diagnosis of EB, and 3) KF-1 cannot by itself be used as an accurate probe for the prenatal diagnosis of dominant dystrophic EB, due to the apparent variability in the time for the normal expression of KF-1 in fetal skin during the second trimester.

Rhabdomyosarcoma in children. Correlation of form and prognosis in one institution's experience.

All cases of rhabdomyosarcoma and undifferentiated sarcoma accessioned at Texas Children's Hospital since 1954 were reviewed. Electron microscopy and immunohistochemistry were used as aids to diagnosis. Rhabdomyosarcoma was diagnosed by study of biopsy specimens obtained prior to therapy in 63 of these 105 cases. Clinical information extending for at least 1 year was available in 47 cases; these cases were used for correlative study. Cellular anaplasia--defined as nuclear enlargement, hyperchromasia, and abnormal mitoses--was noted in 14 cases; 12 of these 14 died an average of 1.53 years after diagnosis. Only 10 of the 33 remaining patients died, including only one of eight with well-differentiated tumors. The mean duration of follow-up for survivors was 4.8 years. By chi-square analysis, the difference in survival between the groups with and without anaplasia was highly significant (p = 0.0009). The relative risk of death within 5 years was 2.48 for the group with anaplasia. Patients with well-differentiated tumors were significantly more likely to survive (p = 0.03). In this study, cellular anaplasia correlated strongly with unfavorable prognosis.

Calcification of soft contact lenses in patient with dry eye and elevated calcium concentration in tears.

Following radiotherapy for an extensive intraepithelial carcinoma of the conjunctiva and eyelid, a patient developed opacification of soft contact lenses used on the same side. Calcilm was demonstrated within the contact lenses by cytochemical methods and by energy-dispersive x-ray microanalysis. The phenomenon was associated with unilateral tear insufficiency and an elevated tear calcium level as determined by atomic absorption spectrophotometry.

Nasopharyngeal carcinoma in children--a retrospective review and demonstration of Epstein-Barr viral genomes in tumor cell cytoplasm: a report of the Pediatric Oncology Group.

Twenty-seven nasopharyngeal carcinomas were entered in the Pediatric Oncology Group Rare Tumor Registry from 1973 to 1988 (15 males, 12 females; 10 white, 15 black, two unknown; aged 8 to 17 years). Eight tumors were non-keratinizing carcinomas (World Health Organization 2) and 19 were undifferentiated (World Health Organization 3). The overall 3-year survival rate was 70% (SE 11%). Nine children developed distant metastases and two were salvaged. We found that localized tumor (P = .02) and black race (P = .05) were associated with a better outcome. In situ hybridization using a biotinylated probe demonstrated Epstein-Barr virus DNA in the cytoplasm of the neoplastic epithelial cells in nine of 11 tumors examined, firmly establishing the presence of Epstein-Barr virus within the malignant cells of nasopharyngeal carcinomas of both World Health Organization 2 and World Health Organization 3 histology, rather than in the surrounding lymphocytes.

The broadly based pericardial flap. A tissue for atrial wall replacement that grows.

The repair of many congenital heart anomalies would be facilitated by a tissue replacement for the atrial wall or pulmonary artery which would grow with the child. Such a tissue has not previously been identified. In 12 puppies a broadly based flap of pericardium was sutured over the right atrial free wall. The atrial wall was excised from beneath the flap. In four animals the flap was then cut away from its pericardial attachments superiorly, inferiorly, and along the phrenic nerve, leaving an autogenous pericardial patch. In the other eight animals the flap was left intact, allowing retention of neural and vascular supply. The animals were put to death 263 +/- 23 days later. In four animals having a pericardial patch, the area of the patch did not increase (94% of original size, NS) despite an increase in body surface area (BSA) to 169% (p less than 0.05) of original BSA. In eight animals with a pericardial flap, the area of the flap increased to 214% (p less than 0.01) of the original size with an increase in BSA to 199% (p less than 0.01) of original BSA. The flap size index (size/BSA) increased to 109% of the original index while the patch size index decreased to 54% of the original, a significant difference (p less than 0.01). The broadly based pericardial flap grew in a manner parallel to BSA increase in these puppies.

Gene expression profiles of giant hairy naevi.

BACKGROUND: Congenital neomelanocytic naevi appear in nearly 1% of newborns. Giant hairy naevi (GHN) are uncommon lesions covering large areas of the body. They are of concern because they have the potential to transform into malignant melanomas. AIMS: To describe gene expression profiles of GHN and nearby normal skin from patients with GHN and normal control skin (from patients with cleft lip/palate). METHODS: Tissues from three patients with GHN and two normal controls were studied for differences in gene expression profiles. Total RNA was isolated from normal skin near the hairy naevus, GHN, and skin from normal controls. The RNA samples were subjected to probe labelling, hybridisation to chips, and image acquisition according to the standard Affymetrix protocol. RESULTS: There were 227 genes affected across all samples, as determined by DNA microarray analysis. There was increased expression of 22 genes in GHN compared with nearby normal skin. Decreased expression was noted in 73 genes. In addition, there was increased expression of 36 genes in normal skin near GHN compared with normal control skin, and decreased expression of five genes. Categories of genes affected were those encoding structural proteins, proteins related to developmental processes, cell death associated proteins, transcription factors, growth factors, stress response modulators, and collagen associated proteins. Changes in mRNA expression were checked by reverse transcription polymerase chain reaction. CONCLUSIONS: Genetic profiles of GHN may provide insight into their pathogenesis, including their potential for malignant transformation. Such information may be useful in improving the understanding and management of these lesions.

Role of L-selectin in physiological manifestations after burn and smoke inhalation injury in sheep.

The effects of a monoclonal antibody against L-selectin [leukocyte adhesion molecule (LAM)1-3] on microvascular fluid flux were determined in conscious sheep subjected to a combined injury of 40% third-degree burn and smoke inhalation. This combined injury induced a rapid increase in systemic prefemoral lymph flow (sQlymph) from the burned area and a delayed-onset increase in lung lymph flow. The initial increase in sQlymph was associated with an elevation of the lymph-to-plasma oncotic pressure ratio; consequently, it leads to a predominant increase in the systemic soft tissue permeability index (sPI). In an untreated control group, the increased sPI was sustained beyond 24 h after injury. Pretreatment with LAM1-3 resulted in earlier recovery from the increased sPI, although the initial responses in sQlymph and sPI were identical to those in the nontreatment group. The delayed-onset lung permeability changes were significantly attenuated by pretreatment with LAM1-3. These findings indicate that both leukocyte-dependent and -independent mechanisms are involved in the pathogenesis that occurs after combined injury with burn and smoke inhalation.

Effect of multiple gene transfers of insulinlike growth factor I complementary DNA gene constructs in rats after thermal injury.

HYPOTHESIS: Multiple subcutaneous injections of cholesterol-containing cationic liposomes encapsulating the complementary DNA (cDNA) gene for insulinlike growth factor I (IGF-I) increase the rate of transfected skin cells and result in increased IGF-I protein levels in the skin with subsequent improvement in wound healing when compared with a single injection. SETTING: Laboratory. INTERVENTION: Twenty-four adult male Sprague-Dawley rats (350-375 g) received a full-thickness scald burn on 60% of their body surface. These rats were randomly divided to receive either 1 injection of liposomes containing 2.2 microg-cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct, or 2 injections of liposomes containing 2.2 microg cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct. MAIN OUTCOME MEASURES: Transfection rates and IGF-I protein levels in the skin and physiological responses to the IGF-I gene therapy, evaluated from changes in body weight, protein content in serum and liver, and the rate of burn wound healing. RESULTS: There was a significant decrease in transfection rate and IGF-I protein expression distal from the injection site in animals receiving 1 injection, as compared with a consistent increase in rats receiving multiple injections. Multiple injections improved the response to thermal trauma by increasing the extent of the healed burn wound 33 days after thermal injury (single injection, 31% +/- 1% vs multiple injections, 38% +/- 2%), total serum protein (single injection, 52 +/- 0.5 g/L vs multiple injections, 55 +/- 0.6 g/L), and total liver protein (single injection, 82.0 +/- 0.3 mg/mL vs multiple injections, 91.0 +/- 3.8 mg/mL), P<.05. CONCLUSIONS: Gene transfer rates can be increased by multiple injections of liposomes encapsulating IGF-I cDNA constructs. Increased transfer results in greater IGF-I protein skin concentrations, accelerated wound healing, and increased serum and liver protein concentrations. The clinical relevance of these findings is that liposomal gene constructs should be applied in well-defined distances to improve gene transfer in the skin, and thus clinical outcome after thermal injury.

Wilms tumor arising in horseshoe kidney.

Tumors originating in horseshoe kidneys are rare; only 21 cases of Wilms tumor reported in horseshoe kidneys. A seven-month-old girl with Wilms tumor arising from the isthmus of a horseshoe kidney is reported. The literature is reviewed, and surgical considerations are discussed.

Optimal technique to diagnose primary ciliary dyskinesia.

OBJECTIVE: To develop a cost-effective protocol for diagnosing primary ciliary dyskinesia (PCD). STUDY DESIGN: Retrospective chart review in a tertiary care academic medical center. METHODS: A review of the electron microscopy logbook identified all patients who had a suspected diagnosis of PCD. Biopsy of respiratory tract mucosa was performed using a cytology brush or a cup forceps in the outpatient clinic or operating room (OR). Outcome measures were to determine the diagnostic adequacy of cytological evaluation compared with tissue biopsy and to determine whether an adequate nasal mucosa sample can be collected in the outpatient clinic setting as compared with the OR and the use of general anesthesia RESULTS: Twenty-seven patients underwent 31 biopsies. Fifteen specimens were collected with a cup forceps, and 16 with a cytology brush. The sampling sites were nasal mucosa in 28 cases and trachea in the rest. Twelve specimens (39%) were collected in the clinic; the rest were obtained in the OR in conjunction with another procedure. Neither method of specimen collection nor mode of anesthesia made a significant difference in the probability of obtaining an adequate specimen. Ten samples were nondiagnostic: cytological evaluation, 31% (n = 16); tissue biopsy, 27% (n = 15); clinic, 42% (n = 12); and OR, 31% (n = 16). The cost of evaluating ciliary motion at our institution was $150, with an additional charge of $1,297 for electron microscopic evaluation. The nonprofessional fee for an outpatient nasal biopsy was $98; in the OR the cost of anesthesia supplies, surgical suite, recovery room, and day-surgery bed was at least $1,860. CONCLUSION: Our results suggest that the optimal method for diagnosis of PCD is in the outpatient clinic with specimen collection by means of either a cup forceps or a cytology brush.

Tracheal agenesis. A case report and literature review.

Tracheal agenesis (TA) is a rare congenital anomaly that is incompatible with prolonged life. It occurs in isolation or in association with other anomalies. The affected neonate presents with respiratory distress and is unable to produce an audible cry despite obvious physical effort. A difficult intubation ensues, and often only during a postmortem examination is the diagnosis of TA made. It is hoped that in addition to adding the 47th case of TA to the literature, this article will provide some insight into the pathogenesis, presentation, and management of this anomaly. It must be emphasized that as TA carries a fatal prognosis, great care must be taken in establishing the diagnosis. If the diagnosis is confirmed, complex reconstructive surgery is not recommended as it has not been shown to change the prognosis or clinical course of affected neonates.

Lymphocyte autofluorescence: a screening procedure for neurodegenerative diseases.

We examined the buffy coats from 12 consecutive patients with neurodegenerative diseases to determine the value of autofluorescence study by fluorescence microscopy in parallel with electron microscopy, as a rapid and inexpensive screening procedure for lymphocyte inclusions characteristic of neuronal ceroidlipofuscinoses. Four patients (3 with neuronal ceroidlipofuscinoses and 1 with Hallervorden-Spatz syndrome) had discrete, yellow-orange, 1-2 microns autofluorescent granules in the cytoplasm of some lymphocytes. These granules corresponded to characteristic inclusions of neuronal ceroidlipofuscinoses demonstrated by electron microscopy. Five patients (4 undiagnosed or nonspecific disease, and 1 with Leber congenital amaurosis) had hazy green cytoplasmic autofluorescence which correlated with parallel tubular arrays in lymphocytes. The 3 patients with no autofluorescence had no ultrastructural lymphocytic inclusions. We conclude that buffy coat autofluorescence is a rapid and inexpensive method of identifying specimens that require electron microscopic confirmation, and the absence of autofluorescence of lymphocytes in a buffy coat specimen eliminates the necessity for electron microscopic examination. However, characteristic ultrastructural inclusions associated with neurodegenerative diseases may occur in other tissues, such as skin and conjunctiva.

Life-threatening congestive heart failure as the presentation of centronuclear myopathy.

Myocardiopathy is associated infrequently with centronuclear myopathy. We present biopsy studies of a 15 1/2-year-old black male who presented with profound acute congestive heart failure and diffuse muscular atrophy. Cardiac symptoms had been present for 6 months; limb weakness had been unassociated with either infantile hypotonia or developmental delay. Cardiac catheterization demonstrated a dilated myocardiopathy and poor left ventricular contractility. Biopsies of both ventricles revealed striking hydropic degeneration and fibrosis. Right triceps biopsy disclosed centronuclear myopathy. Because the spectrum of disease expression in centronuclear myopathy is extensive, an association with cardiac disease always should be considered in these patients. In addition, we recommend that patients who present with idiopathic myocardiopathy should be evaluated for this and other skeletal muscle diseases.

Effects of 5-aminolaevulinic acid on human ovarian cancer cells and human vascular endothelial cells in vitro.

Results are reported on the cellular effects and the sensitivity of cultured tumor epithelial cells (TEC) derived from human ovarian cystadenocarcinoma and human umbilical vein-derived endothelial cells (HUVEC) to exogenous 5-aminolaevulinic acid (ALA) and ALA-induced photodynamic therapy (PDT). Cellular alterations and PDT efficiency were evaluated using colorimetric thiazolyl blue (MTT) assay, trypan blue exclusion assay, electron microscopy, and gel electrophoresis. ALA-induced protoporphyrin IX (PpIX) accumulation in TEC was associated with a concentration and time-dependent significant decrease in mitochondrial activity, increase in cell membrane permeability, and dark toxicity. Maximum PpIX loaded TEC demonstrated a high sensitivity to PDT. Neither cellular alterations nor PDT effects were observed in HUVEC under identical experimental conditions. These results indicate a potential clinical value for the use of ALA-mediated PDT to treat minimal residual disease in mucinous ovarian carcinoma. In addition, the ALA-induced PpIX cytotoxicity may be exported to a new chemotherapeutic regimen via a conventionally viewed photochemotherapeutic agent.

Fulminant Rocky Mountain spotted fever. Its pathologic characteristics associated with glucose-6-phosphate dehydrogenase deficiency.

Three patients with documented fulminant Rocky Mountain spotted fever (RMSF) (death on or before day 5 of illness) had severe multisystemic injury as shown by clinical signs and laboratory data, but on microscopic examination showed minimal evidence of the typical mononuclear leukocytic response to rickettsial vascular infection and injury. Thrombosis was more extensive than in classic RMSF, with fibrin thrombi located in foci of rickettsial infection. These patients had a rash either preterminally or not at all, particularly severe Rickettsia-associated pulmonary lesions, and other shock-related lesions, eg, centrilobular hepatic necrosis. All three patients were male blacks with glucose-6-phosphate dehydrogenase deficiency, a condition recently associated with severity of RMSF. Diagnosis of fulminant RMSF requires awareness of its pathologic and epidemiologic aspects, and use of rickettsial isolation or specific immunofluorescence.