Reversible temperature-sensitive alterations in lung fluid balance.

Hypothermia is intentionally imposed during the harvesting of lungs for transplantation. The aim of this study was to investigate the fluid balance alterations in rat lung preparations exposed to hypothermic perfusion. Lowering perfusate temperature from 37 degrees C to values between 27 and 7 degrees C caused an immediate, marked pulmonary hypertension and vasoconstriction accompanied by rapid development of pulmonary edema (+1.15 g, or approximately 90%, gain in lung weight within 5 min). However, on rewarming, vasoconstriction was immediately reversed. Edema was resolved, but along a two-component time course: an immediate reduction of lung weight on rewarming (t 1/2 of 0.5 min) that mirrored the recovery of pulmonary artery pressure and vasoconstriction, and also a slower pressure-independent component of recovery (t 1/2 of 3.5 min). Ouabain (300 microM) markedly inhibited the lung's ability to recover from edema, indicating that fluid clearance from lung tissue was the result of activation of ouabain-sensitive (Na+,K+)-ATPase pump. Results could not be explained by vascular or airspace injury as lung sections from hypothermic lungs appeared normal. The findings indicate that hypothermia induces pulmonary edema formation, which can be rapidly cleared upon rewarming by activation of ouabain-sensitive (Na+,K+)-ATPase pump. Thus, impaired fluid clearance from lung extravascular spaces may be a critical factor limiting gas exchange in transplanted lungs exposed to hypothermia.

VIP receptors as molecular targets of breast cancer: implications for targeted imaging and drug delivery.

Receptors for vasoactive intestinal peptide (VIP-R) are overexpressed in human breast cancer. This phenomenon may have important diagnostic and therapeutic implications because carrier systems loaded with imaging or therapeutic agents, and with surface ligands to VIP-R could potentially be actively targeted to breast cancer. Previously, we have prepared sterically stabilized liposomes (SSL) with VIP non-covalently associated on their surface. However, these liposomes were not able to actively target to breast cancer in rats in situ, most probably due to dissociation of non-covalently associated VIP from SSL. Hence, there is a need to conjugate VIP covalently to SSL. This study aims to begin to address this issue and to test the targeting ability of VIP-SSL to n-methyl nitrosourea (MNU)-induced rat breast cancer in vitro. First, VIP was conjugated to DSPE-PEG(3400)-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethylene glycol)]-N-hydroxy succinamide, PEG M(w) 3400] under mild conditions to obtain a predominantly 1:1 conjugate of VIP and DSPE-PEG(3400) (DSPE-PEG(3400)-VIP), as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, DSPE-PEG(3400)-VIP was inserted into preformed fluorescent cholesterol (BODIPY-Chol) labeled SSL by incubation at 37 degrees C. To test breast cancer targeting ability in vitro, these VIP-SSL were subsequently incubated with MNU-induced rat breast cancer tissue sections. The results showed that when compared to fluorescent SSL without VIP or non-covalently attached VIP, significantly more VIP-SSL were attached to rat breast cancer tissues indicating that SSL with covalently attached VIP can be actively targeted to rat breast cancer tissues. This targeted carrier system is currently being explored for functional imaging and targeted chemotherapy of breast cancer.

Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice.

The in vivo contributions of CD18 integrin-dependent and -independent mechanisms in mediating the increases in lung neutrophil (polymorphonuclear leukocyte; PMN) sequestration and microvascular permeability are not well understood. We determined the time course of these responses to Gram-negative sepsis in the mouse lung and addressed the specific contributions of CD18 integrins and ICAM-1. PMN sequestration in the lung was assessed by morphometric analysis, and transalveolar PMN migration was assessed by bronchoalveolar lavage. Lung tissue PMN number increased by 6-fold within 1 h after i.p. Escherichia coli challenge; this value peaked at 3 h (7-fold above control) and decreased at 12 h (3.5-fold above control). PMN migration into the airspace was delayed; the value peaked at 6 h and remained elevated up to 12 h. Saturating concentrations of anti-CD18 and anti-ICAM-1 mAbs reduced lung tissue PMN sequestration and migration; however, peak responses at 3 and 6 h were inhibited by 40%, indicating that only a small component of PMN sequestration and migration was CD18 dependent at these times. In contrast to the time-dependent decreased role of CD18 integrins in mediating PMN sequestration and migration, CD18 and ICAM-1 blockade prevented the increase in lung microvascular permeability and edema formation at all times after E. coli challenge. Thus, Gram-negative sepsis engages CD18/ICAM-1-independent mechanisms capable of the time-dependent amplification of lung PMN sequestration and migration. The increased pulmonary microvascular permeability induced by E. coli is solely the result of engagement of CD18 integrins even when PMN accumulation and migration responses are significantly CD18 independent.

Detection of VIP receptors in MNU-induced breast cancer in rats: implications for breast cancer targeting.

Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide with a wide range of biological activities. Receptors for VIP (VIP-R) are overexpressed in breast cancer, where they may have diagnostic and therapeutic implications. Although N-methyl nitrosourea (MNU)-induced breast cancer in rats is used extensively as a model to study mammary carcinogenesis, there is no information about the expression of VIP-R in this model. Therefore, the purpose of this study was to investigate the presence of VIP-R in MNU-induced breast cancer in rats so that this model can be used to perform studies involving VIP-R. Breast cancer was induced in 36-day-old virgin female Sprague-Dawley rats, by a single intravenous injection of MNU (50 mg/kg body weight). The breast tumors were detected 100-150 days after injection. The normal and cancerous rat breast tissue were excised and 20 micro sections were incubated with 40 nM fluorescein-labeled VIP (Fluo-VIP(TM)), in the presence and absence of 1000-fold excess unlabeled VIP, pituitary adenylate cyclase activating polypeptide (PACAP) or secretin. The sections were visualized under a fluorescence microscope and photographed. Fluo-VIP(TM) stained rat breast cancer tissue homogeneously and to a much greater extent than normal rat breast tissue (p < 0.05). This staining was specific as indicated by displacement of Fluo-VIP(TM) by excess unlabeled VIP and PACAP. Displacement of Fluo-VIP(TM) by secretin indicated the probable presence of VIP receptors of type VPAC1 (VIP receptor subtype 1) in the rat breast. These data suggest that, as in human breast cancer, VIP-R, predominantly of type VPAC1, are overexpressed in MNU-induced rat breast cancer tissue as compared to the normal rat breast tissue. Thus, MNU-induced rat breast cancer model can be used as a tool to study the functional role of VIP-R in human mammary carcinogenesis and VIP-R mediated active breast cancer targeting. This could have implications in the diagnosis, prognosis and therapy of human breast cancer.

Disseminated Pseudallescheria boydii infection in a nonimmunocompromised host.

We present a highly unusual case of pulmonary Pseudallescheria boydii infection in a nonimmunocompromised host with a cavitating mass lesion. The diagnosis was confirmed by open lung biopsy. The patient was treated at another institution with course of amphotericin B, considered an ineffective therapy for this infection, and presented to us with direct extension and invasion of the left atrial appendage and the pulmonary artery, followed by massive pulmonary embolization and hematogenous dissemination to the liver, spleen, kidney, pancreas, and brain.

Wegener's granulomatosis and alpha1-antitrypsin-deficiency emphysema: proteinase-related diseases.

Wegener's granulomatosis (WG) and alpha1-antitrypsin (alpha1-AT)-deficiency emphysema are both uncommon disorders. A relationship may exist between these diseases involving the proteinase and antiproteinase balance in the lung. A case is presented of WG and alpha1-AT-deficiency emphysema occurring in the same patient. Previous studies concerning the correlation between abnormal alpha1-AT alleles and WG are discussed. Potential mechanisms for the relationship and recommendations for screening are given.

Thoracic blastomycosis and empyema.

Blastomycosis is endemic in river valley areas of the southeastern and Midwestern United States. Pulmonary manifestations include chronic cough and pleuritic pain. Radiographic appearance of the infection can mimic bronchogenic lung carcinoma. Pleural effusion is rarely associated with this pulmonary infection, and empyema has not been previously reported. We report a case of pulmonary and pleural Blastomyces dermatitidis infection presenting as empyema thoracis. Diagnosis and treatment were attained with video-assisted thoracoscopic (VATS) pleural and lung biopsy and debridement.

Rejection pattern of simultaneously transplanted cardiac and skeletal muscle grafts in a rat model.

BACKGROUND: We studied the correlation of cardiac and skeletal muscle allograft rejection in a rat model to assess the feasibility of using biopsies from simultaneously transplanted skeletal muscle for surveillance of cardiac graft rejection. METHODS: Thirty Lewis rats (RT1l)) underwent simultaneous heterotopic heart and cutaneous maximus flap (HHCM) allotransplant. Seven recipient rats (control) received syngeneic HHCM grafts from Lewis donors while the remaining 23 (study group) received HHCM grafts from Brown Norway (RT1n) donors. Control rats were sacrificed after 7 days while rats in the study group were serially sacrificed at days 1-7 after transplantation. No immunosuppression was given. The tissue sections from the HHCM grafts were assessed for acute rejection based on the grading system adopted by the International Society for Heart and Lung Transplantation. RESULTS: As expected, all the control rats had no evidence of rejection. One study animal developed an infection in the skeletal muscle allograft and was excluded. Two study animals had no evidence of rejection when sacrificed 1 day after transplant. The remaining 20 rats developed acute cellular rejection in their graft(s). Upon comparison of acute cellular rejection between the heterotopic heart and the cutaneous maximus flap grafts, rejection correlated grade for grade in 75% (15 of 20 rats). All five rats that did not have identical grades of rejection had mild rejection (grades IA, IB and II). Presence or absence of rejection, therefore, correlated in 20/22 rats: 15/20 rats with cardiac rejection and 2/2 rats without cardiac rejection. CONCLUSION: Cardiac and skeletal muscle allografts have similar pattern of rejection with little grade to grade variability. The clinical implications for surveillance of cardiac rejection warrants further investigation.

Staining tissue with methacrylate casts for light microscopy.

Scanning electron microscopy (SEM) of methyl methacrylate casts and light microscopy (LM) of tissue are well-established methods for studying the microcirculation. The two are complimentary, but methacrylate is transparent and thus its presence is often not appreciated by LM. Applying histologic stains to sections of tissue embedded in methyl methacrylate would allow the relationships of light microscopic and scanning electron microscopic views of cast vasculature to be better appreciated. We sought to test different stains on cast tissue to find one that would accent the cast. Surgically removed and autopsied human lungs were cast with methacrylate and processed by routine light microscopic methods. They were stained with the hematoxylin and eosin, Masson trichome, elastic--van Gieson, Grocott methenamine silver, Brown-Brennan, and Ziehl-Neelsen methods. The Ziehl-Neelsen procedure stained the methacrylate best, giving it a red color. This procedure also worked well without heating. We conclude that (1) cast methacrylate lung can be processed for routine LM with excellent results; (2) methacrylate stains well with the Ziehl-Neelsen technique; (3) the acid--fast stained cast lung shows capillaries and cells in both normal and diseased lung better than the routine hematoxylin and eosin stain; (4) this technique can be used to assess filling and correlate findings on the same tissue with the two different microscopic methods.

A new rat model to study the correlation of cardiac and skeletal muscle allograft rejection.

As a first step to study the correlation of cardiac and skeletal muscle allograft rejection, we describe a new experimental rat model of simultaneous heterotopic heart and cutaneous maximus muscle flap allotransplant. Brown Norway rats were used as donors and Lewis rats as recipients. No immunosuppression was given. The grafts were revascularized with sequential end-to-side anastomosis of each vascular pedicle to the infrarenal aorta and vena cava. Syngeneic heart and cutaneous maximus muscle grafts remained functional and showed no sign of rejection at 7 days after the transplant. In contrast, both allografts developed severe rejection and functional compromise at 7 days after the transplant. Our experimental model is technically feasible and reproducible and may provide important information about the pattern of rejection of cardiac and skeletal muscle allografts.

Giant cell arteritis manifesting as chronic cough and fever of unknown origin.

A 57-year-old white man sought medical attention because of chronic cough and fever of unknown origin. An extensive work-up over 4 weeks, including repeated blood cultures, chest roentgenograms, a gallium scan, and computed tomographic scans of the sinuses, chest, and abdomen, was nondiagnostic. The patient was referred to our institution for bronchoscopy. Further analysis of his history revealed that he had a headache in conjunction with the cough and an episode of a flashing color design in his left eye 1 week before assessment. The erythrocyte sedimentation rate was 115 mm in 1 hour. A biopsy of the temporal artery showed granulomatous inflammation of the vessel wall with multinucleated giant cells, histiocytes, lymphocytes, plasma cells, and few eosinophils. The multinucleated giant cells were closely related to the fragmented elastic lamina. Corticosteroid therapy resulted in prompt resolution of the chronic cough and fever. Giant cell arteritis should be considered in the differential diagnosis of chronic cough.

Detection of neutral endopeptidase 24.11 (neprilysin) in human hepatocellular carcinomas by immunocytochemistry.

BACKGROUND: We have reported previously that neutral endopeptidase 24.11 (neprilysin; NEP; CALLA, CD10) activity was very high in rat hepatomas and a cultured human hepatocarcinoma cell line (SK-HEP1). MATERIALS AND METHODS: While continuing these studies, we detected the presence of NEP in SK-HEP 1 cells by immunocytochemistry and in paraffin-embedded human hepatocellular carcinomas as well. IgG purified from polyclonal antisera to human NEP was employed as a source of antibody. RESULTS: SK-HEP 1 cells gave a strong positive reaction to the IgG fraction of the antisera. In control studies, where IgG was preabsorbed with recombinant NEP, the results were negative. Of the 18 hepatocellular carcinomas tested, NEP was expressed in 14 (78%) malignant tumors, while adjacent liver tissue did not show the presence of NEP. CONCLUSIONS: It is suggested that, because none of the known hepatocellular carcinoma markers are highly specific, the detection of NEP in these malignant cells can be an additional useful diagnostic tool.

Decrease in immunoreactive neutral endopeptidase in uvula epithelium of patients with obstructive sleep apnea.

The purpose of this study was to determine whether neutral endopeptidase (NEP; EC3.4.24.11) is decreased in the uvula epithelium of patients with obstructive sleep apnea (OSA). Tissues were obtained by uvulopharyngopalatoplasty in seven patients with moderate OSA and by autopsy in five individuals not known to have OSA. Using antisera to human NEP and immunoperoxidase staining, we found that NEP was localized in uvula epithelial cells of both patients with OSA and controls. However, there was a significant decrease in the number of epithelial cells staining for NEP in patients with OSA relative to controls (67 +/- 10 cells versus 261 +/- 33 cells, in 5 randomly selected high-power microscopic fields, respectively; mean +/- SEM; p < .05). The intensity of staining for NEP was similar in both groups. We conclude that immunoreactive NEP is significantly decreased in the uvula epithelium of patients with OSA.

Inflammation in the uvula mucosa of patients with obstructive sleep apnea.

This study was conducted to determine whether inflammation is present in the uvula mucosa of patients with obstructive sleep apnea (OSA). Uvulas were obtained by uvulopalatopharyngoplasty in 21 patients with moderate OSA (mean apnea/hypopnea index and standard error of the mean: 32 +/- 4) and by autopsy in 5 individuals not known to have OSA. Using point counting in five randomly selected high-power microscopic fields (X100), the authors found that the number of leukocytes in the lamina propria of the uvula mucosa was significantly higher in patients with OSA than in the controls (179 +/- 12 cells vs. 71 +/- 4 cells, respectively; P < .05). This was due to a significant increase in the number of plasma cells in patients with OSA as compared with controls (89 +/- 15 cells vs. 21 +/- 5 cells, respectively; P < .05). The thickness of the lamina propria (an index of interstitial edema) was also significantly increased in patients with OSA compared with controls (0.99 +/- 0.12 mm vs. 0.27 +/- 0.02 mm, respectively; P < 0.05). The authors conclude that inflammation, characterized by plasma cell infiltration and interstitial edema, is present in the uvula mucosa of patients with moderate OSA. They also suggest that soft palate inflammation contributes to upper airway occlusion observed during sleep in these patients.

Human alveolar capillaries undergo angiogenesis in pulmonary veno-occlusive disease.

The bronchial circulation undergoes angiogenesis in several pathological conditions, such as lung neoplasm and bronchiectasis, but whether the pulmonary circulation can do this has been questioned. A woman treated with mitomycin C and 5-fluorouracil developed progressive, fatal pulmonary hypertension over 5 months. In addition to light and transmission electron microscopic examination of her lung, her pulmonary vasculature was cast and the casts were studied with scanning electron microscopy. Light microscopy showed that she had pulmonary veno-occlusive disease and angiomatoid capillary growth in the alveolar walls. Transmission electron microscopy confirmed the presence of pulmonary hypertension and showed thickened endothelial basement membrane. Scanning electron microscopy of the cast blood vessels showed distortion and destruction of alveolar capillaries prohibiting the passage of erythrocytes. Large new capillaries developed on top of, and were connected to, the shrivelled capillaries that made up the alveolar wall. The new capillaries were larger and fewer, which reduced the alveolar-capillary interface. Arteries and veins were irregularly narrowed and the veins had broad muscularity. Oedema was present, and the pulmonary lymphatics were extensively cast, especially in the lobular septa, but the lymphatics had a normal appearance. It appears that this patient suffered extensive capillary damage and venous occlusion and that the response was extensive new capillary formation, sometimes in angiomatoid configurations, and hypertrophy of pulmonary veins and arteries. Casting the microvasculature and viewing it with scanning electron microscopy identified new alveolar capillaries in this patient with acquired pulmonary hypertension.

Carboxypeptidase M activity is increased in bronchoalveolar lavage in human lung disease.

Carboxypeptidase M (CPM) cleaves the C-terminal arginine and lysine of peptides; it is expressed in the lung, especially on the plasma membrane of alveolar type I cells. Here, we report on CPM in human bronchoalveolar lavage (BAL) collected from 69 patients and analyzed for activity, cell number and type, and protein level. Seventy-six percent of CPM activity, measured at pH 7.5 with 5-dimethylamino-naphthalene-1-sulfonyl-alanyl-arginine (Dansyl-Ala-Arg) substrate, was immunoprecipitated with polyclonal antibody to purified human enzyme. In patients without active lung disease, CPM activity in BAL was 7.69 (+/- 2.12) nmol/h/mg protein, but in patients with acute pneumonia, it was 29.25 (+/- 4.06) (p < 0.01). In patients with Pneumocystis carinii pneumonia, CPM activity was elevated to 26.00 (+/- 4.85) (p < 0.01) and in patients with lung cancer, to 30.95 (+/- 4.12) (p < 0.01). The activity was not associated with the cellular elements of BAL. The highest specific activity was in the large aggregate fraction of surfactant, which also contained the highest concentration of phosphorus. Transmission electron microscopy of this fraction revealed the presence of typical lamellar bodies and tubular myelin structures. The high CPM activity may stem from its induction and release in acute lung disease. In addition, CPM may be a marker of infection with certain pathogens and an indicator of type I cell injury in parenchymal lung diseases.

Intrasplenic venous thrombosis: CT findings.

OBJECTIVE: To describe the CT findings of intrasplenic venous thrombosis in three cases. MATERIALS AND METHODS: Three adult patients had abdominal CT indicative of short-segment thrombosis of an intrasplenic blood vessel. Two patients underwent splenectomy; thrombosis of a segmental splenic vein was confirmed histologically in both cases. RESULTS: Segmental splenic vein thrombosis appeared on CT as a short, 1-2 cm, linear lucency surrounded by normal splenic parenchyma. The probable etiology of intrasplenic venous thrombosis was main splenic vein occlusion related to pancreatitis in two cases and splenic compression and inflammation by perisplenic abscess in the third case. CONCLUSION: Thrombosis of segmental intrasplenic veins is detectable with CT and can be a secondary CT finding of main splenic vein thrombosis.