Diffusible cytotoxic metabolites contribute to the in vitro bystander effect associated with the cyclophosphamide/cytochrome P450 2B1 cancer gene therapy paradigm.

Tumor cells become sensitive to the inert prodrug cyclophosphamide (CPA) after transfer of the gene encoding cytochrome P450 2B1. This enzyme activates CPA into 4-hydroxycyclophosphamide, which ultimately degrades into acrolein and phosphoramide mustard, the anticancer and DNA-alkylating metabolite. It is imperative that any prodrug-activating gene therapy strategy against cancer possess the capacity to affect the proliferation of tumor cells even when they do not express the transgene (bystander effect), because current methodologies cannot achieve gene transduction in all tumor cells. Prodrug-activating gene therapy schemes described to date exhibit a bystander effect that is not mediated by conditioned medium in culture and may depend on cell contact. In contrast, we find that CPA-sensitized, P450-expressing C6 glioma cells (C6-P450) transfer cytotoxicity to nonexpressing cells by releasing diffusible metabolites through the medium. A 3-h exposure to the prodrug is necessary and sufficient to achieve killing of the transfected cells, and medium conditioned by these cells can kill untransfected cells with similar potency. This bystander effect occurs in the presence of CPA even when only 10% of cells in culture express the P450 2B1 gene, and it is not reproduced by cells that have been irradiated. In an animal model of intracerebral brain tumors, expression of the P450 2B1 gene within the neoplastic cells enhanced significantly the antitumor effect of CPA, even when it was administered systemically. This study shows that CPA/P450 2B1 gene therapy represents a novel tumor-killing strategy that displays an expanded range of cytotoxic action both spatially and temporally within tumor cells and significantly potentiates the anticancer action of CPA when administered i.v.

Differential expression of type I adrenal steroid receptors in immune tissues is associated with tissue-specific regulation of type II receptors by aldosterone.

We examined the influence of the type I adrenal steroid receptor agonist, aldosterone, on type II adrenal steroid receptor binding in the rat spleen and thymus after adrenalectomy. In the spleen, adrenalectomy was associated with a significant up-regulation of type II receptors, which was blocked by the concurrent administration of aldosterone (1 microgram/h) via sc osmotic minipumps. Neither adrenalectomy nor aldosterone treatment altered type II receptor binding in the thymus. Despite high doses of aldosterone (10 micrograms/h), which resulted in supra-physiological blood concentrations of this hormone, there was no evidence of type II receptor decreases in spleen or thymus below receptor levels found in sham-adrenalectomized rats. The effect of aldosterone on type II receptor binding appeared to be mediated by the type I receptor, since there was no aldosterone effect on the thymus, which did not exhibit detectable levels of type I receptor binding. Moreover, there was no evidence that aldosterone competed for the type II receptor in vivo or in vitro as determined by measurements of the type II receptor dissociation constant in the spleen of adrenalectomized, aldosterone-treated animals. Since selective activation of the type I receptor occurs in the spleen under physiological conditions, these results indicate that type I receptors may play a tonic inhibitory role in type II receptor expression in immune cells which express both receptor subtypes and reside in this tissue. Furthermore, the findings suggest that there may be different mechanisms involved in the up-regulation vs. the down-regulation of type II adrenal steroid receptors, and effects mediated solely via the type I adrenal steroid receptor appear only to influence the former process.

Effects of selective type I and II adrenal steroid agonists on immune cell distribution.

Adrenal steroids exert their effects through two distinct adrenal steroid receptor subtypes; the high affinity type I, or mineralocorticoid, receptor and the lower affinity type II, or glucocorticoid, receptor. Adrenal steroids have well known effects on immune cell distribution, and although both type I and II receptors are expressed in immune cells and tissues, few data exist on the relative effects mediated through these two receptor subtypes. Accordingly, we administered selective type I and II adrenal steroid receptor agonists to young adult male Sprague-Dawley rats for 7 days and then measured immune cell distribution in the peripheral blood and spleen. Results were compared with those of similar studies using the naturally occurring glucocorticoid of the rat, corticosterone, which binds both type I and II receptors. The majority of the well characterized effects of adrenal steroids on peripheral blood immune cells (increased neutrophils and decreased lymphocytes and monocytes) were reproduced by the type II receptor agonist, RU28362. RU28362 decreased the numbers of all lymphocyte subsets [T-cells, B-cells, and natural killer (NK) cells] to very low absolute levels. The largest relative decrease (i.e. in percentage) was seen in B-cells, whereas NK cells exhibited the least relative decrease and actually showed a 2-fold increase in relative percentage during RU28362 treatment. Similar to RU28362, the type I receptor agonist, aldosterone, significantly reduced the number of lymphocytes and monocytes. In contrast to RU28362, however, aldosterone significantly decreased the number of neutrophils. Moreover, aldosterone decreased the number of T-helper cells and NK cells, while having no effect on the number of B-cells or T-suppressor/cytotoxic cells. Corticosterone at physiologically relevant concentrations had potent effects on immune cell distribution, which were indistinguishable from those of the type II receptor agonist, RU28362. Taken together, these results indicate that effects of adrenal steroids on immune cell distribution are dependent on the receptor subtype involved as well as the specific cell type targeted. These factors allow for varied and complex effects of adrenal steroids on the immune system under physiological conditions.

Neurologic disturbances in Paget disease of bone: response to calcitonin.

The neurologic manifestations of Paget disease and the therapeutic effect of calcitonin were studied in 49 patients. Twenty-four patients (49%) had neurologic disorders involving cranial nerves other than the auditory system, brainstem, spinal cord, or spinal roots and nerves. Eighteen of the 24 patients (75%) showed significant subjective or objective improvement after calcitonin treatment. The effect of calcitonin treatment on spinal cord compression was dramatic in three of six patients. The observations made of these patients support previous data suggesting that the neurologic signs and symptoms of Paget disease have their pathogenesis in both mechanical impingement and vascular distortion. The importance of early detection of neurologic signs and symptoms is emphasized, since prompt treatment with calcitonin may prevent severe complications.

Transcutaneous interruption of ultrasound contrast agents for blood flow evaluation.

RATIONALE AND OBJECTIVES: The ability to create short boluses in targeted arteries with rapid rise times is limited by the transport of bubbles from the venous to arterial portion of the circulation. Acoustic interruption of contrast agent in arteries may create the short boluses necessary for simple wash-in/wash-out measures of blood flow. METHODS: An ultrasound contrast agent was used with spectral Doppler ultrasound to observe contrast interruption in femoral arteries and VX2 carcinoma in a rabbit model. At an upstream location in the femoral artery, single, sinusoidal ultrasound tone bursts at 1.8 MHz with durations of 0.25 to 1 seconds were applied to interrupt the flow of contrast agent injected intravenously. RESULTS: In VX2 carcinoma, bursts as short as 40 cycles produced contrast interruption lasting only one cardiac cycle within the tumor periphery and I(SPPA) <3 W/cm2 produced measurable interruptions. CONCLUSIONS: Acoustic fields applied transcutaneously interrupted flow of contrast agents to form temporally short negative boluses.

Management of splenic artery aneurysms: the significance of portal and essential hypertension.

BACKGROUND: Splenic artery aneurysm(s) (SAA) are rare. But the incidence and significance of SAA among patients with portal hypertension (PHTN), especially among those who undergo orthotopic liver transplantation (OLT), have not been clearly delineated. STUDY DESIGN: An 11-year (February 1987 to June 1998) retrospective review of our experience with treated SAA was performed. Patient characteristics, risk factors, clinical presentation, surgical management, aneurysm characteristics, and patient outcomes were assessed. Patients were separated according to a history of PHTN for analysis. Patients were also subdivided into ruptured versus elective presentations. RESULTS: Thirty-four patients (22 in the PHTN group) were treated for SAA during the study period. Sixty-two percent (21 of 34) were women; the average age was 50.6 years. In patients without a history of PHTN (n = 12), essential hypertension was a significant risk factor (p < 0.001) for development of SAA. All patients underwent surgical treatment for SAA: resection with splenectomy (n = 23), ligation with splenectomy (n = 5), ligation of SAA only (n = 4), and vascular reconstruction (n = 2). The average size of all treated SAA was 4.8 +/- 2.6 cm, ranging from 1.5 to 12cm. Operative mortality after SAA rupture (n = 15) was 40%, compared with zero mortality for elective SAA repair (n = 19, p < 0.005). Rupture of SAA was associated with a higher mortality in patients with PHTN compared with patients without such history (56% versus 17%, respectively). After a mean followup period of 46 months, survival after rupture was 60% in contrast to 84% after elective repair. The majority of our patients with a history of PHTN (20 of 22) has undergone OLT, representing 0.46% of all OLT recipients (n = 4,374) during the study period. In four patients, SAA were repaired concurrently during transplantation. Of the 7 patients presented with rupture of SAA after OLT, 6 patients presented within 3 to 16 days postoperatively, with a median of 6 days and an overall mortality of 57%. CONCLUSIONS: Essential hypertension and PHTN appear to be significant risk factors for development of SAA. Rupture of SAA is associated with a significant mortality, highest among patients with PHTN. Elective repair remains a safe and effective method of treatment. The significance of SAA is recognized among patients undergoing liver transplantation. A decision should be made to screen and electively treat SAA found in liver transplant patients, especially if the aneurysm is larger than 1.5 cm. Awareness of the increased rupture risk is crucial in management during the immediate posttransplant period.

The experience of an academic medical center with endovascular treatment of abdominal aortic aneurysms.

BACKGROUND: Endovascular repair of abdominal aortic aneurysms (AAA) is gaining momentum although it is not yet approved in the United States by regulatory agencies. The Endovascular Grafting System (EGS), the first device to enter clinical trials in 1993, is now in phase III testing. METHODS: We reviewed the first 50 patients to undergo an EGS repair of AAA over 24 months at our institution. Results were compared with 69 patients who underwent open repair during the same time period by the same surgeon. RESULTS: Devices were successfully implanted in 47 of 50 (94%) patients. Three were converted to standard repair. Although length of stay was shorter, costs were similar. Follow-up was 3 to 24 months. Perigraft flow was noted in 33% at discharge; 73% of those stopped either spontaneously or with coiling. Three graft limbs occluded, requiring thrombolytic therapy. CONCLUSIONS: The EGS repair of AAA is feasible and effective. Cooperation between surgery and radiology is important for the success of a new endovascular program.

Iliocaval complications of retroperitoneal fibrosis.

BACKGROUND: Retroperitoneal fibrosis can compress ureters, nerves, and blood vessels in the abdomen. However, clinically significant large-vein obstruction secondary to this process is rare. METHODS: Three hundred forty patients with retroperitoneal fibrosis were treated at our institution between 1976 and 1993. The clinical data from seven of these patients, who were treated for iliocaval complications of retroperitoneal fibrosis, were reviewed. RESULTS: Six patients had signs and symptoms of chronic obstruction, and one patient presented with acute iliocaval thrombosis and underwent attempt at venous thrombectomy in the referring hospital. All patients exhibited extremity edema and three had venous claudication. Iliocaval occlusion was confirmed in all patients by venography, computed tomography, or magnetic resonance imaging. The obstructive process involved the iliocaval tree (four patients), the inferior vena cava alone (two patients), and the iliac vein alone (one patient). Five patients were managed conservatively with leg elevation, compression stockings, and anticoagulation. Two patients received prednisone. One patient underwent an iliocaval bypass from the external iliac vein to the juxtarenal cava using a ringed polytetrafluoroethylene graft with a femoral arteriovenous fistula. A second patient with an isolated left common iliac vein obstruction underwent a left-to-right femorofemoral saphenous vein bypass. Four patients treated conservatively continued to have extremity edema. The two patients managed surgically remain asymptomatic from venous insufficiency, with patent grafts at 25 and 12 months after surgery, respectively. CONCLUSION: Iliocaval obstruction is an unusual complication of retroperitoneal fibrosis. Although most cases can be managed conservatively, reconstruction is an option for patients who have failed medical treatment and are symptomatic secondary to chronic venous obstruction. Lifelong anticoagulation should be considered for all patients with progressive iliocaval obstruction secondary to retroperitoneal fibrosis.

Oxygen-dependent chronic obstructive pulmonary disease does not prohibit aortic aneurysm repair.

BACKGROUND: Severe oxygen-dependent chronic obstructive pulmonary disease (COPD) is considered by many to be a contraindication to open abdominal aortic aneurysm (AAA) repair. We reviewed our own experience with this patient population. METHODS: From July 1995 to March 1999, 14 consecutive patients limited by home oxygen-dependent COPD underwent elective open infrarenal AAA repair. Their medical records were reviewed. RESULTS: The mean aortic aneurysm size was 6.3 cm. The mean PaO2 = 70 mm Hg, PaCO2 = 45 mm Hg, forced expiratory volume in 1 second (FEV1) = 34% of predicted, and forced vital capacity (FVC) = 67% of predicted. All 14 patients were extubated within 24 hours, mean length of hospital stay was 5.9 days, and there were no perioperative deaths. CONCLUSIONS: Severe home oxygen-dependent COPD is not a contraindication to safe elective open AAA repair.

Mesenteric venous thrombosis.

MVT is an uncommon form of visceral ischemia. Symptoms and signs of MVT are usually nonspecific and should not be relied on for accurate diagnosis. A simple, logical diagnostic algorithm can be used to manage most of these patients (Fig. 6). CT or MRI appears to be the most sensitive diagnostic test and should be obtained early for any patient suspected of harboring MVT. Patients with peritonitis require prompt abdominal exploratory laparotomy to rule out ischemic bowel. Once the diagnosis of acute MVT is confirmed, the patient should be anticoagulated with heparin. During operation, all nonviable bowel should be resected with intent for a second-look laparotomy after 24 hours if there is any question of ongoing ischemia. We recommend using fluorescein-assisted evaluation of marginally viable bowel intraoperatively. After the operation, anticoagulation is continued with heparin and then oral warfarin sodium when the patient's bowel function returns. For those patients without peritonitis, we recommend prompt anticoagulation followed by at least a 48- to 72-hour period of close observation. All patients who have had an episode of acute MVT and do not have a contraindication to anticoagulation should be anticoagulated on a life-long basis with warfarin sodium. Despite our increased awareness of acute MVT, the 30-day mortality rate remains high. Acute MVT typically has a more insidious and unpredictable course than do other forms of visceral ischemic syndromes, with a mortality rate as high as that of its arterial counterpart. Although there has been a slight improvement in survival during the last 20 years, the recurrence rate remains high and the long-term prognosis is poor in this group of patients. Survival of patients with chronic MVT is better than that of those with acute MVT and appears to be determined by the underlying disease.

Cavitation damage of pyrolytic carbon in mechanical heart valves.

A summary of the observations of cavitation-related damage is presented for over a hundred mechanical heart valves (MHV) containing pyrolytic carbon components. Valves were obtained from several types of simulators, animal studies and clinical explantations, and were primarily of the bileaflet type. Damage on these valves was documented as to location, type, and severity. This report focuses on the damage location where cavitation bubbles have been observed in vitro. Pitting and microcracking are the forms of damage observed that can be associated with cavitation. The pitting is primarily of a focal nature and is thought to be due to cavitation bubble collapse or, possibly, initiation. Certain features of the deposited material appear to be important relative to cavitation damage resistance and the so-called cavitation threshold of a MHV. A highly polished surface with few micropores provides few nucleation sites for cavitation bubbles and will better withstand cavitation forces. Attributing certain observations of in vivo damage to cavitation is done by inference from; 1. the similarity of the damage features observed on explants to those produced by cavitation in vitro, and 2. the identity of the location of this damage with the location of cavitation as observed by high speed video (HSV). In addition, confirmation was obtained in a number of instances by in vitro observation of cavitation coinciding with a specific damage location on the same explanted MHV. In most of the fractures, a focal pit was usually present on the fracture line at or near the fracture origin, indicating pitting as a primary damage mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin-induced thrombocytopenia: pathophysiology.

HIT is a procoagulant disorder that is associated with significant morbidity and mortality if undetected and untreated. It occurs in approximately 5% of all patients receiving heparin therapy. HIT can be separated into two different types based on the clinical presentation and the pathophysiological mechanism. Type I HIT is an early, mild form of thrombocytopenia that is thought to be non-immune-mediated. No therapy is necessary for this type of HIT. Type II HIT has a delayed onset and is immunologically mediated. It is the more severe form and is associated with the development of HITT. Once suspected or diagnosed, all heparin therapy must be withdrawn. The thrombocytopenia will generally resolve within several days to a week. Minimizing the risk to the patient for developing HIT is the best form of prevention currently available.

Doppler US gating of cardiac MR imaging.

RATIONALE AND OBJECTIVES: Electrocardiographic (ECG) gating of cardiac magnetic resonance (MR) imaging has been problematic for many reasons. The purpose of this study was to demonstrate the feasibility of using Doppler ultrasound (US) gating, either directly off the moving cardiac wall or the systolic upstroke of the arterial signal from the great vessels in neck, in alternative gating modes. MATERIALS AND METHODS: A 2.5-MHz, range-gated Doppler US device was used with A-mode guidance for gating directly off left ventricular wall motion. A 4- or 8.1-MHz, continuous-wave (CW) Doppler US device was used for gating off the systolic upstroke from the great vessels in the neck. The subject undergoing imaging held the transducer against his chest for range-gated Doppler US and against his neck for 8.1-MHz CW Doppler US. The 4-MHz transducer was strapped to the subject's neck. Modified Doppler signals were fed back into the gating circuitry of the MR imager to achieve cardiac synchrony. RESULTS: Cardiac gating was achieved by using both the range-gated technique directly off the cardiac wall and the CW method off blood flow from the great vessels. Problems occurred with radiofrequency shielding during the range-gated method; however, these problems were almost completely removed by use of the CW Doppler probes. CONCLUSION: Doppler US gating of MR images is possible and potentially could overcome many shortcomings of ECG gating. Subsequent embodiments of the technique will require improved radiofrequency shielding in the range-gated technique.

The effects of nimodipine on ischemic injury of the spinal cord during thoracic aortic cross-clamping.

BACKGROUND: The purpose of this study was to evaluate the protective effects of a neuroselective calcium antagonist, nimodipine on spinal cord ischemia during and after thoracic aortic cross-clamping. METHODS: Thirty adult dogs underwent 60 minutes of thoracic aortic cross-clamping via a left thoracotomy. The dogs were randomized into 3 groups (n = 10) and received either intravenous control (normal saline), sham (polyethylene glycol) or nimodipine solution during a period of 150 minutes. Spinal cord perfusion (SCP) was directly monitored using a laser doppler flowmeter. After 48 hours, neurologic status was assessed using Tarlov scores and the spinal cords evaluated histologically for evidence of ischemia (grades 1-4: severe to mild ischemia). RESULTS: Twenty-six dogs survived the operation. Proximal (carotid) blood pressure increased 30-40% and cerebrospinal fluid (CSF) pressure increased 50% during cross-clamping for all 3 groups. The SCP decreased predictably during cross-clamping in all dogs but after unclamping, the nimodipine group had significantly less hyperperfusion than the saline and sham control groups (30 min after unclamping, control: 74.1 +/- 12.6 ml/min, sham: 51.8 +/- 4.15 ml/min, nimodipine: 33.1 +/- 3.9 ml/min, p = 0.04). This hyperperfusion phenomenon correlated with adverse neurologic (Tarlov score) outcome (p = 0.01). Paraplegia rates were 78% (control), 70% (sham) and 71% (nimodipine) (p = NS). The histologic grades of the spinal cords from those dogs which received nimodipine tended to correspond to better tissue preservation (control: 1.72 +/- 0.49, sham: 1.75 +/- 0.46, nimodipine: 2.14 +/- 0.56, p = NS). CONCLUSIONS: Nimodipine used as single agent therapy failed to show a statistically significant clinical neurologic benefit. However, nimodipine significantly decreased postischemic reperfusion hyperemia in the spinal cord as measured by laser doppler flowmetry. This reduced hyperperfusion, which significantly correlated with functional outcome, may be responsible for dampening neural cell damage. Thus, nimodipine should be considered as an adjunct to a multimodality approach in the prevention of spinal cord ischemia during thoracic and thoracoabdominal aortic reconstructions.

Effects of egg yolk and salt on Micrococcaceae heat resistance.

The heat resistance and growth possibilities of various members of the Micrococcaceae in egg yolk and egg yolk with added salt were determined. Egg yolk alone protected members of the Micrococcaceae considerably against heat. Whereas in water Staphylococcus aureus S6 had a decimal reduction time (D) value of 66 s at 55 degrees C, its D value in egg yolk at the same temperature was 246 s. In salted egg yolk (water activity, 0.95), S. aureus S6 had a D value of 180 s at 66 degrees C and was largely inactivated during the pasteurization processes currently applied. Micrococcus saprophyticus and S. epidermidis (D value of each under the same conditions, 390 s) could survive such treatments to a certain extent and can thus spoil commercial egg yolk.

[Ultrasound determination of sacral position in the frontal plane]. / Die sonographische Bestimmung der Kreuzbeinstellung in der Frontalebene.

AIM OF THE STUDY: The position of the sacrum in the frontal plane is important for the statics of the spine. The most common method of investigating the sacrum is its visualisation on an antero-posterior X-ray film. The aim of this study was to find a method of determining the position of the sacrum in the frontal plane by sonography. METHOD: Criteria for the correct image of the upper rim of the sacrum are presented and the procedure for measuring sacral tilt is introduced. The results of the sonographic measurements of 107 patients are compared with the results of X-ray measurements. The intraindividual differences are described and statistically evaluated. RESULTS: Almost 90% of the patients revealed a difference between the sonography and the radiology measurement of less than three millimeters. The overall sensibility was 80%. The specificity was 82%. The results were calculated for different age groups. They turned out to be better in children and adolescents than in adult patients. Obese adults had significantly worse results than patients with normal body weight. CONCLUSIONS: This study suggests that the method presented can help to provide reliable information on the position of the sacrum, especially in children and adolescents. By using sonography we can partly reduce the necessary X-ray exposure, especially in the diagnosis and treatment of leg length discrepancies.

Heat inactivation of staphylococcus epidermidis at various water activities.

Members of the family Micrococcaceae play an important role in food spoilage and even in food poisoning. In contrast to members of the family Enterobacteriaceae, these bacteria can grow in media with low water activities. Therefore, the heat resistance of Staphylococcus epidermidis, a rather resistant member of the family Micrococcaceae, was studied at water activities between 0.87 and 1.00. The heat inactivation curves were clearly biphasic at all temperatures and water activities tested. Especially at low water activities, the D-values of the tail phase were extremely high (at 0.87 water activity, a D-value at 70 degrees C of 500 s was recorded).

A biotinylated MutS fusion protein and its use in a rapid mutation screening technique.

The Escherichia coli DNA mismatch repair protein, MutS, binds single base pair mismatches and short deletions in vivo and in vitro. To adapt this protein for mutation detection, a fusion protein of E. coli MutS with a biotinylated peptide domain has been constructed (MutSb). The biotinylation tag facilitates MutS detection and binding by avidin without significantly altering the DNA mismatch binding properties of MutS in vitro. We describe a novel and rapid mutation detection method with MutSb using streptavidin-coated magnetic beads and demonstrate that MutSb can also be used to remove mismatch containing DNA fragments from a mixture of DNA fragments in solution.