A technique for visual confirmation of intrathoracic placement of tube thoracostomy using a fiberoptic laryngoscope in a cadaver.

PURPOSE: Safe intrathoracic placement of chest tubes is a continual challenge. Current techniques for determining the intrathoracic location of the thoracostomy site include blunt dissection and digital exploration, with subsequent tube placement. Using current techniques, complication rates for this procedure approach 30%. We present a novel technique using available endotracheal intubation technology for determining intrathoracic placement of tube thoracostomy. METHODS: One cadaver was used for placement of tube thoracostomy. Both sides of the thorax were prepared in the standard fashion for tube thoracostomy placement, and tube thoracostomy was performed on each hemithorax at interspaces 3 through 7. The right side of the thorax was used for standard thoracostomy placement, and the left side was used for fiberoptic visualization of thoracostomy placement using a video laryngoscope. Thoracic wall thickness was measured at all thoracostomy sites. Proper placement and any injuries were documented for each site. RESULTS: Chest wall thickness ranged from 2.4 to 3.8 cm on the right and 2.8 to 4.0 cm on the left. With use of fiberoptic thoracostomy, no injuries were generated. During the standard thoracostomy placement in the sixth intercostal space, a pulmonary laceration was caused using blunt dissection. CONCLUSIONS: Use of a fiberoptic laryngoscope offers a novel technique for direct visualization the thoracic space during tube thoracostomy. Further studies are needed to determine the safety of this technique in patients.

Surgical stabilization of flail chest: the impact on postoperative pulmonary function.

OBJECTIVES: Flail chest results in significant morbidity. Controversies continue regarding the optimal management of flail chest. No clear guidelines exist for surgical stabilization. Our aim was to examine the association of bedside spirometry values with operative stabilization of flail chest. METHODS: IRB approval was obtained to identify patients with flail chest who underwent surgical stabilization between August 2009 and May 2011. At our institution, all rib fracture patients underwent routine measurement of their forced vital capacity (FVC) using bedside spirometry. Formal pulmonary function tests were also obtained postoperatively and at three months in patients undergoing stabilization. Both the Synthes and Acute Innovations plating systems were utilized. Data is presented as median (range) or (percentage). RESULTS: Twenty patients (13 male: 65 %) with median age of 60 years (30-83) had a median of four ribs (2-9) in the flail segment. The median Injury Severity Score was 17 (9-41) and the median Trauma and Injury Severity Score was 0.96 (0.04-0.99). Preoperative pneumonia was identified in four patients (20 %) and intubation was required in seven (35 %). Median time from injury to stabilization was four days (1-33). The median number of plates inserted was five (3-11). Postoperative median FVC (1.8 L, range 1.3-4 L) improved significantly as compared to preoperative median value (1 L, range 0.5-2.1 L) (p = 0.003). This improvement continued during the follow-up period at three months (0.9 L, range 0.1-3.0) (p = 0.006). There were three deaths (15 %), none of which were related to the procedure. Subsequent tracheostomy was required in three patients (15 %). The mean hospital stay and ventilator days after stabilization were nine days and three days, respectively. Mean follow-up was 5.6 ± 4.6 months. CONCLUSION: Operative stabilization of flail chest improved pulmonary function compared with preoperative results. This improvement was sustained at three months follow-up.

Chemical components separation with botulinum toxin A: a novel technique to improve primary fascial closure rates of the open abdomen.

INTRODUCTION: Failure to definitively close the open abdomen (OA) after damage control laparotomy leads to considerable morbidity and mortality. We have developed a novel technique, the "chemical components separation," which incorporates injection of botulinum toxin A (BTX), a long-term flaccid paralytic, into the lateral abdominal wall musculature. METHODS: This is a retrospective review of all OA patients (age ≥18) from December 2009-June 2010 who underwent BTX injection. Under ultrasound guidance, a total of 300 units of BTX were injected into the external oblique, internal oblique and transversus abdominus. RESULTS: A total of 18 patients were injected with a median age of 66 years (56 % male). Indications for OA treatment included questionable bowel viability (39 %), shock (33 %), loss of abdominal domain (6 %) and feculent contamination (17 %). Median ASA score was 3 with an APACHE 3 score of 85. Patients underwent a median of 4 serial abdominal explorations. The primary fascial closure rate was 83 % with a partial fascial closure rate of 6 % and planned ventral hernia rate of 11 %. Of the 9 patients injected within 24 h of their initial OA procedure, 89 % achieved primary fascial closure. Mortality was 11 %; death was unrelated to BTX injection. The overall complication rate was 67 %; specific complications rates included fascial dehiscence (11 %), enterocutaneous fistula development (0 %), intra-abdominal abscess (44 %) and deep surgical site infection (33 %). CONCLUSION: The "chemical components separation" technique described is safe and avoids the extensive dissection necessary for mechanical components separation in critically ill patients with infected/contaminated abdominal domains. While further evaluation is required, the described technique provides potential to improve delayed primary fascial closure rates in the OA setting.

[Sarcomatoid renal cell carcinoma. A rare and aggressive variation of primary renal cell carcinoma]. / Das sarkomatoide Nierenzellkarzinom. Eine seltene und aggressive Variante des primären Nierenzellkarzinoms.

Every year, renal cell carcinoma (RCC) is responsible for the highest proportion of cancer-associated deaths in relation to all other malignant urological diseases. Initially called carcinosarcoma, the sarcomatoid differentiation confers higher aggressiveness on any of the different subtypes of RCC, with a frequency of ca. 1%. The presence of a sarcomatoid component makes the disease locally aggressive, which typically presents an advanced grade that is associated with fast progression and fatal outcome in a vast proportion of cases, with median survival lower than 1 year. This is important for predicting the outcome for patients undergoing nephrectomy due to RCC, since chemotherapy in a certain group of patients with progressive disease can be a reasonable alternative to the failure of immunotherapy in sarcomatoid renal carcinoma. We report our experience with sarcomatoid RCC in four patients with extensive tumor progression in comparison to the literature.

Highly sensitive biosafety model for stem-cell-derived grafts.

BACKGROUND: The recent success in the derivation of differentiated cell types from stem cells has raised prospects for the application of regenerative cell therapy. In particular, embryonic stem cells are attractive sources for cell transplantation, due to their immortality and rapid growth. These cells, however, also possess tumorigenic properties, which raises serious safety concerns and makes biosafety testing mandatory. Our goal was to establish a highly sensitive animal model for testing the proliferative potential of stem-cell grafts. METHODS: BALB/c nude mice received cell grafts of non-neoplastic MRC-5 cells containing defined numbers of mouse embryonic stem cells. We either injected 1 million viable cells into the kidney capsule, or mixed 2 million cells with Matrigel for s.c. transplantation. To analyze the possible impact of an intact immune response on tumor development, we also transplanted the cells into immunocompetent mice. Animals were sacrificed when the tumors became >1 cm and were analyzed in detail. RESULTS: The nude mouse model reproducibly allowed detection of 20 tumorigenic cells, and even as few as 2 ES cells were found to form teratoma. Interestingly, the administration of cell grafts at two different application sites resulted in different growth kinetics and tumor phenotypes. The highest level of sensitivity (100% detection of 20 tumorigenic ES cells) was achieved by s.c. injection of cells mixed with Matrigel. The influence of the immune system on tumor-cell development was demonstrated by a higher tumor rate of transplants in immunodeficient nude mice compared with immunocompetent mice. DISCUSSION: We have established a reliable animal model for routine assessment of the biosafety profile of stem-cell-derived cell transplants. This model will facilitate the generation of homogenous non-tumorigenic cell populations, and will help to integrate standardized safety systems into the application of stem-cell-derived grafts for clinical purposes.

Scalable production of embryonic stem cell-derived cardiomyocytes.

Cardiomyocyte transplantation could offer a new approach to replace scarred, nonfunctional myocardium in a diseased heart. Clinical application of this approach would require the ability to generate large numbers of donor cells. The purpose of this study was to develop a scalable, robust, and reproducible process to derive purified cardiomyocytes from genetically engineered embryonic stem (ES) cells. ES cells transfected with a fusion gene consisting of the alpha-cardiac myosin heavy chain (MHC) promoter driving the aminoglycoside phosphotransferase (neomycin resistance) gene were used for cardiomyocyte enrichment. The transfected cells were aggregated into embyroid bodies (EBs), inoculated into stirred suspension cultures, and differentiated for 9 days before selection of cardiomyocytes by the addition of G418 with or without retinoic acid (RA). Throughout the culture period, EB and viable cell numbers were measured. In addition, flow cytometric analysis was performed to monitor sarcomeric myosin (a marker for cardiomyocytes) and Oct-4 (a marker for undifferentiated ES cells) expression. Enrichment of cardiomyocytes was achieved in cultures treated with either G418 and retinoic acid (RA) or with G418 alone. Eighteen days after differentiation, G418-selected flasks treated with RA contained approximately twice as many cells as the nontreated flasks, as well as undetectable levels of Oct-4 expression, suggesting that RA may promote cardiac differentiation and/or survival. Immunohistological and electron microscopic analysis showed that the harvested cardiomyocytes displayed many features characteristic of native cardiomyocytes. Our results demonstrate the feasibility of large-scale production of viable, ES cell-derived cardiomyocytes for tissue engineering and/or implantation, an approach that should be transferable to other ES cell derived lineages, as well as to adult stem cells with in vitro cardiomyogenic activity.

Thrombin as a survival factor for cancer cells: thrombin activation in malignant effusions in vivo and inhibition of idarubicin-induced cell death in vitro.

OBJECTIVES: The aim of the experiments shown here, is to demonstrate exemplarily that thrombin can be a survival factor for malignant cells. METHODS: Activation of the coagulation system has been examined in patients with acute myeloid leukemia (AML) and non-Hodgkin lymphoma (NHL) before and after chemotherapy as well as in malignant effusions of heavily pretreated patients with solid tumors. Thrombin receptor expression (PAR-I) has been examined on HL-60 cells; the effect ofthrombin on the proliferation of the cells and inhibition of apoptosis induction by idarubicin has been shown. RESULTS: Using fibrinopeptide A as an indirect parameter for thrombin activation, we found elevated levels in patients with AML and NHL before and a significant 2-fold increase after chemotherapy (p < 0.02 for the AML group; p < 0.0006 for the NHL group). Apparently, this does not only affect patients with hematological diseases, but also with solid tumors. In order to find out if the tumor cells directly activate thrombin, we examined malignant effusions of patients with different solid tumors. Comparing prothrombin fragment 1 + 2 in ascites and pleural effusions with the patients' serum levels, we found it significantly increased in all cases (mean of 1.96 +/- 0.5 nmol/l in the serum vs. 12.1 +/- 3.6 nmol/l in effusions; p < 0.001). The majority of patients presented elevated serum levels. Additionally, we incubated HL-60 cells (human promyelocytic leukemia) with thrombin prior to treatment with idarubicin. Expression of thrombin receptor (PAR-1) could be verified by FACS-analysis using a monoclonal antibody. HL-60 cells responded with increased proliferation to thrombin exposure with concentrations between 0.3 and 3 U/ml. This effect could be abolished by the addition of hirudin, demonstrating thrombin specificity. In these concentrations, thrombin was able to abrogate the induction of apoptosis by idarubicin completely (p < 0.005). CONCLUSIONS: Here we give evidence for the role of thrombin as a resistance factor for tumor cells towards chemotherapy. In the light of the fact that thrombin is regularly activated in cancer patients, these findings indicate that thrombin is a clinically relevant cellular resistance factor. A number of pre-clinical and clinical studies imply that inhibition of the coagulation system, e.g. by low-molecular weight heparins or warfarin, increases the effect of chemotherapy.

Metalloproteinase inhibition prevents acute respiratory distress syndrome.

BACKGROUND: The acute respiratory distress syndrome (ARDS) occurs in patients with clearly identifiable risk factors, and its treatment remains merely supportive. We postulated that patients at risk for ARDS can be protected against lung injury by a prophylactic treatment strategy that targets neutrophil-derived proteases. We hypothesized that a chemically modified tetracycline 3 (COL-3), a potent inhibitor of neutrophil matrix metalloproteinases (MMPs) and neutrophil elastase (NE) with minimal toxicity, would prevent ARDS in our porcine endotoxin-induced ARDS model. METHODS: Yorkshire pigs were anesthetized, intubated, surgically instrumented for hemodynamic monitoring, and randomized into three groups: (1) control (n = 4), surgical instrumentation only; (2) lipopolysaccharide (LPS) (n = 4), infusion of Escherichia coli lipopolysaccharide at 100 microg/kg; and (3) COL-3 + LPS (n = 5), ingestion of COL-3 (100 mg/kg) 12 h before LPS infusion. All animals were monitored for 6 h following LPS or sham LPS infusion. Serial bronchoalveolar lavage (BAL) samples were analyzed for MMP concentration by gelatin zymography. Lung tissue was fixed for morphometric assessment at necropsy. RESULTS: LPS infusion was marked by significant (P < 0.05) physiological deterioration as compared with the control group, including increased plateau airway pressure (P(plat)) (control = 15.7 +/- 0.4 mm Hg, LPS = 23.0 +/- 1.5 mm Hg) and a decrement in arterial oxygen partial pressure (P(a)O(2)) (LPS = 66 +/- 15 mm Hg, Control = 263 +/- 25 mm Hg) 6 h following LPS or sham LPS infusion, respectively. Pretreatment with COL-3 reduced the above pathophysiological changes 6 h following LPS infusion (P(plat) = 18.5 +/- 1.7 mm Hg, P(a)O(2) = 199 +/- 35 mm Hg; P = NS vs control). MMP-9 and MMP-2 concentration in BAL fluid was significantly increased between 2 and 4 h post-LPS infusion; COL-3 reduced the increase in MMP-9 and MMP-2 concentration at all time periods. Morphometrically LPS caused a significant sequestration of neutrophils and monocytes into pulmonary tissue. Pretreatment with COL-3 ameliorated this response. The wet/dry lung weight ratio was significantly greater (P < 0.05) in the LPS group (10.1 +/- 1.0 ratio) than in either the control (6.4 +/- 0.5 ratio) or LPS+COL-3 (7.4 +/- 0.6 ratio) group. CONCLUSIONS: A single prophylactic treatment with COL-3 prevented lung injury in our model of endotoxin-induced ARDS. The proposed mechanism of COL-3 is a synergistic inhibition of the terminal neutrophil effectors MMPs and NE. Similar to the universal practice of prophylaxis against gastric stress ulceration and deep venous thromboses in trauma patients, chemically modified tetracyclines may likewise be administered to prevent acute lung injury in critically injured patients at risk of developing ARDS.

Preparation protocols for high-activity photosystem II membrane particles of green algae and higher plants, pH dependence of oxygen evolution and comparison of the S2-state multiline signal by X-band EPR spectroscopy.

Photosystem II (PS II) membrane particles are particularly well suited for various types of spectroscopic investigations on the PS II manganese complex. Here we present: (1) a preparation protocol for PS II membrane particles of higher plants, which yields exceptionally high oxygen-evolution activity due to the use of glycinebetaine as a PS II-stabilizing agent; (2) preparation protocols for highly active PS II membrane particles for the green algae Scenedesmus obliquus and Chlamydomonas reinhardtii; (3) a determination of pH dependence of oxygen evolution for spinach and Scenedesmus; (4) a comparison of the EPR multiline signal observed in the S2-state of green algae and higher plants of PS II membrane particles. A clearly broader type of multiline EPR signal is observed in green algae.

Videomicroscopy provides accurate in vivo assessment of pulmonary microvascular reactivity in rabbits.

When defining the mechanism of hypoxic pulmonary vasoconstriction (HPV), investigators have employed ex vivo preparations because of the belief that accurate, quantitative assessment of pulmonary microvessels could not be obtained in vivo. We hypothesize that accurate, quantitative assessment of pulmonary microvascular reactivity can be performed using a simple, in vivo preparation. Our aim was to provide this quantitative assessment in a defined animal model, and to confirm that the chosen preparation could discriminate changes in microvascular reactivity as influenced by endogenous mediators. New Zealand rabbits were instrumented for in vivo microscopy and direct measurement of subpleural arterioles. Rabbits were first randomized to either control (n = 7) or endotoxin (n = 5), infusion of Escherichia coli lipopolysaccharide (200 Fg/kg). All rabbits were then exposed to a repeated protocol of normoxia (21% O2) for 20 min and then hypoxia (15% O2) for 10 min over 2 h. The changes in arteriole diameter were measured at the end of each interval. Normal pulmonary arterioles repeatedly constrict 15+/-3.5% during hypoxia. Altering endogenous vasoactive mediators, as with infusion of endotoxin, caused a loss of hypoxia-induced vasoconstriction. The results of our study validate this experimental preparation for the reliable quantification of pulmonary microvascular reactivity and investigation of hypoxic pulmonary vasoconstriction under both normal and pathologic conditions.

Soluble tumor necrosis factor receptor prevents post-pump syndrome.

Post-pump syndrome is an acute lung injury following cardiopulmonary bypass (CPB) which is indistinguishable from the adult respiratory distress syndrome (ARDS). Tumor necrosis factor (TNF) is central to the inflammatory process and is capable of triggering the entire pathophysiologic response leading to ARDS. We hypothesized that treatment with a soluble TNF receptor-binding protein (TNFbp) would reduce the increase in serum TNF and prevent acute lung injury in our sequential insult model of ARDS following CPB. Anesthetized pigs were randomized to one of three groups: Control (n = 3), surgical preparation only; CPB + LPS (n = 6), femoral-femoral hypothermic bypass for 1 h followed by infusion of low dose Escherichia coli lipopolysaccharide (LPS; 1 microg/kg); and TNFbp + CPB + LPS (n = 4), pretreatment with intravenous TNFbp (2 mg/kg) followed immediately by both insults. CPB + LPS caused severe lung injury demonstrated by a significant fall in PaO2 and an increase in both intrapulmonary shunt and peak airway pressure as compared to all groups (P < 0.05). These changes were associated with a significant increase in plasma TNF level and pulmonary neutrophil sequestration. TNFbp significantly reduced plasma levels of TNF and prevented the lung injury typically observed with this ARDS model, but did not reduce pulmonary neutrophil sequestration. Thus, elevated serum TNF is not responsible for neutrophil sequestration but does play a role in neutrophil activation which causes lung injury. Prophylactic use of TNFbp in CPB patients may prevent neutrophil activation and reduce the incidence of post-pump ARDS.

Hyperparathyroidism and pregnancy.

BACKGROUND: Hyperparathyroidism during pregnancy can carry significant morbidity to both mother and fetus. Surgery is the definitive treatment of choice although medical management is occasionally warranted. This report looks at 6 cases of hyperparathyroidism during pregnancy and reviews the morbidity and mortality of this entity. METHODS: We retrospectively reviewed all parathyroidectomies performed over a 21-year period (May 1975 to May 1996). A total of 750 cases were performed. In 6 cases, hyperparathyroidism occurred during pregnancy. Serum calcium was measured in all patients before and after parathyroidectomy. RESULTS: A total of 6 of 750 patients (0.8%) were found to have hyperparathyroidism during pregnancy. In this review, fetal mortality rate was 17% (1 of 6). Two infants experienced neonatal tetany. Maternal morbidity included 1 woman who developed hypercalcemic crisis and acute pancreatitis. Two patients presented with hyperemesis gravidarum, 2 were asymptomatic, and 1 had recurrent urinary tract infection. CONCLUSION: Hyperparathyroidism during pregnancy, while rare, remains a preventable cause of fetal morbidity and mortality. Based on our case series and review of the literature, surgery is the definitive treatment of choice and is considered safe and effective if performed during the second trimester of pregnancy. Those involved with the care of the pregnant patient need to be diligent to diagnose and treat this entity promptly.

Immunoaffinity localization of the enzyme xanthine oxidase on the outside surface of the endothelial cell plasma membrane.

BACKGROUND: Reactive oxygen metabolites generated from endothelial xanthine oxidase (XO) trigger reperfusion injury in many organs. We evaluated the possibility that endothelial XO was localized on the endothelial cell surface, as well as within the cytoplasm. METHODS: Primary cultures of bovine (BAECs) and porcine (PAECs) aortic endothelial cells were grown in media documented to be free of XO. Polyclonal and monoclonal antibodies were developed against XO. These antibodies were used to evaluate BAEC and PAEC for cell surface XO through immunofluorescence staining, hybridoma cell surface labeling, and endothelial cell surface binding. RESULTS: These antibodies bound specifically to the surface of these cells when the membrane was shown to be intact and impermeable (and the cytoplasm inaccessible) to immunoglobulins Moreover, hybridoma cells expressing monoclonal antibody to XO bound specifically to the endothelial cell surface. Finally, intact endothelial cells bound specifically to the anti-XO polyclonal antibodies immobilized to the surface of a Petri dish. The integrity of these endothelial cell plasma membranes was demonstrated by the subsequent growth and replication of these cells in culture. CONCLUSIONS: These findings indicate that XO is present on the outside surface of the endothelial cell plasma membrane. This would not only explain the known in vivo efficacy of intravascularly administered large molecular weight antioxidants (such as superoxide dismutase) but could have important implications for inflammatory signaling.

Pharmacologic approach to tissue injury mediated by free radicals and other reactive oxygen metabolites.

Highly toxic metabolites of oxygen are generated normally by aerobic metabolism in most cells, and this generation is often greatly increased in pathologic conditions. When this oxidant flux exceeds the capability of the multiple endogenous antioxidant mechanisms, tissue injury ensues. The pharmacologic modification of this injury process, with agents that scavenge these reactive oxygen metabolites, block their generation, or enhance the endogenous antioxidant capability, has shown great promise in animal models of common clinical conditions, and has already been successfully applied in controlled clinical trials. This approach represents an interruption of tissue injury at its most basic level.

Metabolism of steroids by human brain tumors.

Hormonal steroids or their precursors can be metabolized in the CNS to products with altered hormonal activity. The importance of the intracerebral transformation of steroids has been demonstrated, particularly with regard to neuroendocrine regulation and sexual behavior. These studies were carried out on normal brain tissues, but the ability of neoplastic tissues of CNS origin to metabolize steroids is unknown. We investigated the in vitro metabolism of tritiated pregnenolone, testosterone, and estradiol-17 beta by homogenates of four brain tumors defined as astrocytomas. In three tumors of cortical origin, removed from adult patients, the only enzymic activity found was the conversion of estradiol to estrone. In one tumor of cerebellar origin removed from an 11-year-old boy, the following conversions were found: pregnenolone to progesterone, testosterone to either androstenedione or estradiol, and estradiol to estrone. These results demonstrate that human astrocytomas can transform steroids to compounds with modified hormonal activity. These compounds formed by the tumorous tissue can affect brain function, which may be of clinical significance. Furthermore, these results may add important parameters for biochemical characterization of neoplastic brain tissues.