Epithelial stem cells of the prostate and their role in cancer progression.

Prostate cancer is a leading cause of cancer-related death in adult men. It can regress dramatically upon antihormonal therapy, but it often recurs in a more aggressive, androgen-independent form. Defining the prostate tissue stem cells (PrSCs) and their involvement in cancer initiation and maintenance may lead to better therapeutics. Using a tissue-regeneration model in which dissociated prostate epithelial cells mixed with inductive mesenchyme give rise to prostatic tubules, we have identified a small population of prostate cells that contains multiple stem cell characteristics. In this system, prostate cancer can be initiated by autocrine or paracrine growth factor signaling and intracellular overexpression of genes often found mutated in human prostate cancer. Using an in vitro prostate sphere assay, we further defined the PrSC population and demonstrated their self-renewal and multilineage differentiation capabilities. Microarray analyses of the stem- and non-stem-cell populations have assisted us in finding and evaluating additional markers that can better define the PrSC population and further delineate the different cell types of the prostate, including those that serve as the target cell for tumor initiation.

Effect of seeding osteoprogenitor cells as dense clusters on cell growth and differentiation.

One approach for forming tissue equivalents involves seeding of cells into porous scaffolds followed by culture in vitro. Within this paradigm, the strategy by which cells are initially seeded may dictate the ultimate properties of the tissue equivalent. In particular, low cell densities may suffer from poor intercellular communication, whereas high densities may result in an unfavorable microenvironment due to transport limitations. A third alternative is to seed cells as dense clusters, which might benefit from intercellular contact without the high nutrient demand. To test this approach, planar substrates were seeded with 10(4) osteoprogenitor marrow stromal cells either as a diffuse subconfluent dispersion (2.6 x 10(3) cells/cm(2)) or as a single dense cluster (8 x 10(4) cells/cm(2)). In this study, the densely clustered cells demonstrated significantly diminished cell growth and collagen synthesis. However, a significantly higher level of alkaline phosphatase activity--a measure of bone-forming potential--and moderately more mineralization were observed with these dense cultures. These findings show that clustering can enhance the differentiation phase while diminishing the proliferating phase of these diploid cells without requiring large cell numbers. Thus, this seeding strategy may improve the quality of engineered tissues.

Testosterone delivery using glutamide-based complex high axial ratio microstructures.

Complex high axial ratio microstructures (CHARMs) were evaluated for delivery of testosterone in vivo. Methods to incorporate testosterone included noncovalent mixing and covalent attachment of testosterone to the lipid to form a prodrug monomer. When prepared by covalent attachment, testosterone-loaded CHARMs were resistant to in vitro spontaneous hydrolysis; when injected into rats, testosterone was released with biphasic kinetics consisting of a burst followed by a much slower phase. Some CHARM material associated with testosterone persisted at the site of injection for at least 9 days.

Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds.

Culture of seeded osteoblastic cells in three-dimensional osteoconductive scaffolds in vitro is a promising approach to produce an osteoinductive material for repair of bone defects. However, culture of cells in scaffolds sufficiently large to bridge critical-sized defects is a challenge for tissue engineers. Diffusion may not be sufficient to supply nutrients into large scaffolds and consequently cells may grow preferentially at the periphery under static culture conditions. Three alternative culturing schemes that convect media were considered: a spinner flask, a rotary vessel, and a perfusion flow system. Poly(DL-lactic-co-glycolic acid) (PLGA) foam discs (12.7 mm diameter, 6.0 mm thick, 78.8% porous) were seeded with osteoblastic marrow stromal cells and cultured in the presence of dexamethasone and L-ascorbic acid for 7 and 14 days. Cell numbers per foam were found to be similar with all culturing schemes indicating that cell growth could not be enhanced by convection, but histological analysis indicated that the rotary vessel and flow system produced a more uniform distribution of cells throughout the foams. Alkaline phosphatase (ALP) activity per cell was higher with culture in the flow system and spinner flask after 7 days, while no differences in osteocalcin (OC) activity per cell were observed among culturing methods after 14 days in culture. Based on the higher ALP activity and better cell uniformity throughout the cultured foams, the flow system appears to be the superior culturing method, although equally important is the fact that in none of the tests did any of the alternative culturing techniques underperform the static controls. Thus, this study demonstrates that culturing techniques that utilize fluid flow, and in particular the flow perfusion system, improve the properties of the seeded cells over those maintained in static culture.

Continuous and highly variable rate controlled release of model drugs from sphingolipid-based complex high axial ratio microstructures.

Sphingolipids have been synthesized that contain as polar headgroups, model drugs ester-linked to the primary hydroxyl group of the ceramide core. These lipids, when allowed to self assemble below their chain-melting temperatures, either as single molecular species or in combination with other sphingolipid-derived amphiphiles, are shown to form supramolecular assemblies of varying morphologies including complex high axial ratio microstructures (CHARMs). Within these microstructures, the lipid esters are highly resistant to hydrolysis as compared to the esters dispersed as solitary monomers in aqueous solution or in a matrix of fluid phosphatidylcholine vesicles. The rate of headgroup hydrolysis within CHARMs may be manipulated over a broad range (days to years) by varying the length of the amide-linked fatty acyl chain in the ceramide core or the distance between the ester and the C-1 ceramide of the core. These microstructures, which have exceptionally high surface area display of attached headgroups, may be useful for controlled release of pharmacological agents.

The relationship between the structure of the headgroup of sphingolipids and their ability to form complex high axial ratio microstructures.

Ceramides with chemically modified polar headgroups were prepared and examined for their ability to form complex high axial ratio microstructures (CHARMS), potential drug delivery vehicles. In general, if the modified ceramide had either a hydrogen bond donor or acceptor at C-1 and C-3, including hydrophobic or hydrophilic groups attached to C-1 microstructures formed. Tolerated groups include amides, esters, sulfonates, and ethers. If modification at C-3 added significant bulk (greater than four carbons regardless of hydrophilicity), then amorphous aggregates formed. Ceramides with C-1 and C-3 bridged through a cyclic structure also made microstructures. By using a sphingolipid with an amine headgroup, CHARMs may be modified covalently after formation.

Effect of osteoblastic culture conditions on the structure of poly(DL-lactic-co-glycolic acid) foam scaffolds.

Poly(DL-lactic-co-glycolic acid) (PLGA) foams are an osteoconductive support that holds promise for the development of bone tissue in vitro and implantation into orthopedic defects. Because it is desirable that foams maintain their shape and size, we examined a variety of foams cultured in vitro with osteoblastic cells. Foams were prepared with different porosities and pore sizes by the method of solvent casting/porogen leaching using 80, 85, and 90 wt% NaCl sieved with particle sizes of 150-300 and 300-500 microm and characterized by mercury intrusion porosimetry. Foams seeded with cells were found to have volumes after 7 days in static culture that decreased with increasing porosity: the least porous exhibited no change in volume while the most porous foams decreased by 39 +/- 10%. In addition, a correlation was observed between decreasing foam volume after 7 days in culture and decreasing internal surface area of the foams prior to seeding. Furthermore, foams prepared with the 300-500 microm porogen had lower porosities, greater mean wall thicknesses between adjacent pores, and larger volumes after 7 days in culture than those prepared with the smaller porogen. Two culture conditions for maintaining cells, static and agitated (in a rotary vessel), were found to have similar influences on foam size, cell density, and osteoblastic function for 7 and 14 days in culture. Finally, we examined unseeded foams in aqueous solutions of pH 3.0, 5.0, and 7.4 and found no significant decrease in foam size with degradation. This study demonstrates that adherent osteoblastic cells may collapse very porous PLGA foams prepared by solvent casting/particulate leaching: a potentially undesirable property for repair of orthopedic defects.

Application of fluid mechanic and kinetic models to characterize mammalian cell detachment in a radial-flow chamber.

The strength of adhesion and dynamics of detachment of murine 3T3 fibroblasts from self-assembled monolayers were measured in a radial-flow chamber (RFC) by applying models for fluid mechanics, adhesion strength probability distributions, and detachment kinetics. Four models for predicting fluid mechanics in a RFC were compared to evaluate the accuracy of each model and the significance of inlet effects. Analysis of these models indicated an outer region at large radial positions consistent with creeping flow, an intermediate region influenced by inertial dampening, and an inner region dominated by entrance effects from the axially-oriented inlet. In accompanying experiments patterns of the fraction of cells resisting detachment were constructed for individual surfaces as a function of the applied shear stress and evaluated by comparison with integrals of both a normal and a log-normal distribution function. The two functions were equally appropriate, yielding similar estimates of the mean strength of adhesion. Further, varying the Reynolds number in the inlet, Re(d), between 630 and 1480 (corresponding to volumetric flow rates between 0.9 and 2.1 mL/s) did not affect the mean strength of adhesion. For these same experiments, analysis of the dynamics of detachment revealed three temporal phases: 1) rapid detachment of cells at the onset of flow, consistent with a first-order homogeneous kinetic model; 2) time-dependent rate of detachment during the first 30 sec. of exposure to hydrodynamic shear, consistent with the first-order heterogeneous kinetic model proposed by Dickinson and Cooper (1995); and 3) negligible detachment, indicative of pseudo-steady state after 60 sec. of flow. Our results provide rigorous guidelines for the measurement of adhesive interactions between mammalian cells and prospective biomaterial surfaces using a RFC. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 616-629, 1997.

Formation of high-axial-ratio-microstructures from natural and synthetic sphingolipids.

Amphiphiles that form high-axial-ratio-microstructures (HARMs) are being considered as novel materials for controlled release of drugs and other biologically functional molecules. HARMs consisting of tubules, ribbons, solid rods and helices are formed from sphingolipids by addition of water to a solution of amphiphile in DMF. Single molecular species of galactocerebroside (GalCer) containing long unsaturated fatty acid chains or natural GalCer containing mixed-length, non-hydroxy fatty acids (NFA-GalCer) or alpha-hydroxy fatty acids (HFA-GalCer) form cylindrical structures. In contrast, single molecular species of GalCer containing long saturated fatty acids form ribbons and helices. GalCer HARMs are typically under 100 nm in diameter and have lengths of several microns. The importance of the amide of GalCer for HARM formation was evaluated using psychosine, which forms solid fibers, whereas sphingosine and an analog of GalCer in which the amide is reduced to a secondary amine form amorphous aggregates. Single molecular species of ceramide containing long unsaturated fatty acid chains form cylindrical structures, whereas those with long saturated fatty acids form ribbons and helices. Short chain saturated ceramide also forms cylindrical structures. GalCer analogs with N-acetyl-glycine in place of the galactose form fibers whereas those with N-acetyl-proline yield amorphous material. The N-acetyl-proline-containing amphiphile can de doped into pure GalCer or NFA-GalCer without perturbing tubule formation.

Evaluation of a system using oral mucosal transudate for HIV-1 antibody screening and confirmatory testing. OraSure HIV Clinical Trials Group.

OBJECTIVE: To determine accuracy of a human immunodeficiency virus type 1 (HIV-1) antibody testing system using a device to collect and stabilize oral mucosal transudate (OMT), a fluid with increased levels of IgG; an enzyme immunoassay (EIA) screening test optimized for OMT; and a Western blot confirmatory test designed for use with OMT. DESIGN: The OMT specimens were tested by EIA and, if indicated, confirmatory Western blot according to a standard testing algorithm. The OMT results were compared with true HIV status as determined by serum testing and/or clinical diagnosis. PATIENTS: Specimens from 3570 subjects (2382 at low risk, 698 at high risk, 242 with acquired immunodeficiency syndrome [AIDS], and 248 "nonspecificity" [persons with diseases associated with an increased frequency of false-positive results in HIV testing]) were collected at 11 geographically diverse sites (including blood banks, public health clinics, general medical clinics, HIV clinics, sexually transmitted disease clinics, and a hemophilia center) in the United States. MAIN OUTCOME MEASURES: Overall accuracy of testing OMT for HIV-1 antibodies compared with true HIV-1 antibody status; sensitivity and specificity of OMT EIA and Western blot. RESULTS: Sensitivity of OMT EIA testing in 673 true-positive subjects was 99.9% (672/673). The OMT Western blot results in the 673 true-positive subjects were positive in 665 and indeterminate in 8. The EIA followed by Western blot (if EIA was repeatedly reactive) yielded a negative result in 99.9% (2893/2897) of OMT samples from true negatives and an indeterminate result in 4. The OMT testing system provided the correct result or would trigger appropriate follow-up testing in 3569 (>99.9%) of 3570 cases. CONCLUSION: HIV-1 antibody testing of OMT samples is a highly accurate alternative to serum testing.

Synthesis and bioevaluation of delta 7-5-desaturase inhibitors, an enzyme late in the biosynthesis of the fungal sterol ergosterol.

Ergosterol, the predominant sterol of fungi, is postulated to have many cellular functions which include a bulk membrane role and a regulatory role. Studies with sterol auxotrophs show that, even in the presence of sterols which can fulfill the bulk membrane requirements, a small concentration of ergosterol is absolutely necessary for growth. The delta 5-double bond appears to be required for the regulatory role of ergosterol; therefore, development of inhibitors of the enzyme that introduce this double bond, delta 7-sterol 5-desaturase (5-desaturase), may lead to effective antifungal agents. Within is the first reported synthesis of inhibitors of fungal 5-desaturase and the development of an in vitro tritium efficacy radioassay. The inhibitors were of the general structure 7,22(E)-ergostadien-3 beta-ol with alpha-face heteroatom substituents in the vicinity of C-5. They exhibited IC50 values of 47-149 microM.

Characterization of the epitope recognized by a monoclonal antibody highly specific for blood group M antigen.

The mouse monoclonal antibody M2A1 of IgG1 class, which is highly specific for blood group M antigen, was obtained and characterized by means of hemagglutination, enzyme-linked immunosorbent assay, immunoblotting, and inhibition assays. The use of modified M glycoprotein preparations for inhibition tests and of variant McN and Henshaw red cell membranes for immunoblotting showed that M2A1 recognized an epitope including the NH2-terminal serine and sialic acid residues of glycophorin A, whereas the fifth glycine residue was not involved. The reactivity of the antibody with M antigen was distinctly dependent on ionic strength and pH; the optimum was at pH 8 to 9. The alpha-amino group of terminal serine residue was not necessary for the reaction with M2A1 antibody, and the results obtained suggested that the positive charge of this group contributed to decreasing antigen-antibody reactions at pH below 8. The reaction of the antibody with blood group N antigen was not detectable in any of the assays used.

Health care quality: the new marketing challenge.

More and more buyers of health care are evaluating the quality of care as well as the cost. To stay competitive, providers must now learn how to sell quality.

Injuries missed at operation: nemesis of the trauma surgeon.

Injuries missed at initial operation have the potential to cause the most disastrous complications in trauma patients. Over the past 5 years, 12 patients have required re-operation for 14 injuries missed at initial laparotomy and/or thoracotomy. Six missed injuries were vascular, two each in the thorax, pelvis, and retroperitoneum. The other eight were visceral: three small bowel (one patient), two pancreatic, and one each of the heart, ureter, and diaphragm. Five patients (42%) died, three with missed vascular and two with missed visceral injuries. Three died due to complications directly related to their missed injuries, while the unrecognized injury did not play a significant role in the other two. Indications for re-operation in patients with vascular injuries were hypotension in two patients, persistent output from drains in three, and refractory acidosis in one. Re-exploration in visceral injuries was for clinical sepsis in three patients, DIC in one, cardiac tamponade in one, and persistent chest tube drainage in one. Eleven of the 12 patients presented to the E.D. in shock. All patients had multiple injuries with a mean of 3.25 organ systems injured. Hypotension, coagulopathy, and/or hypothermia (T less than 92 degrees) were felt to have contributed to missing the injury in five of the patients with vascular, and three of the patients with visceral injuries. In the four other patients, injuries were missed due to inadequate exploration or a low index of suspicion in the presence of multiple injuries.(ABSTRACT TRUNCATED AT 250 WORDS)

Central venous blood oxygen saturation: an early, accurate measurement of volume during hemorrhage.

Accurate and relatively simple monitoring is essential in managing patients with multiple injuries, and becomes particularly important when there is substantial occult blood loss. Tachycardia, said to occur following a 15% blood loss, is generally regarded as the first reliable sign of hemorrhage. However, heart rate is a nonspecific parameter which is affected by factors other than changing intravascular volume. The purpose of this study was to evaluate available means of monitoring volume status and to identify the parameter which is the earliest and most reliable indication of blood loss. Sixteen mongrel dogs were anesthetized and bled by increments of 3% of their total blood volume until the onset of sustained hypotension or a 25% blood loss. All dogs were monitored with a Swan-Ganz catheter and an arterial line. Vital signs, full hemodynamic parameters, and arterial and mixed venous blood gases were measured after each 3% blood loss. Statistical analysis of the data demonstrated that only Cardiac Index and Mixed Venous Oxygen Saturation showed linearity as function of measure blood loss. Linear regression analysis generated r values that ranged from 0.85-0.99 with a mean of 0.95 for Mixed Venous Oxygen Saturation; r values for Cardiac Index ranged from 0.39-0.98 with a mean of 0.85. Furthermore, all dogs had increased tissue oxygen extraction after 3-6% blood loss. Because Central Venous Blood Oxygen Saturation mirrors Mixed Venous Oxygen Saturation and is easily and rapidly measured, we extended our study by repeating all of the previously measured parameters, with the addition of CVP blood gases in an unanesthetized animal model.(ABSTRACT TRUNCATED AT 250 WORDS)

Lumbar arterial injury: radiologic diagnosis and management.

Injury of the lumbar arteries is a cause of potentially life-threatening retroperitoneal hemorrhage. Twelve patients who sustained massive hemorrhage of the lumbar arteries associated with lumbar spinal fractures and/or pelvic fractures are described. Computed tomography (CT) was helpful by revealing a distinct separation of the lumbar hemorrhage from the hematomas associated with pelvic fracture. On arteriograms, stasis within lumbar extravasation was manifested as globular or streaky accumulations of contrast medium, pseudoaneurysms or diffuse "staining," or opacification of a fracture site. Multiple lumbar bleeding sites were seen frequently. Embolization with pledgets of absorbable gelatin sterile sponge controlled bleeding in ten patients. Abdominal aortography should be an essential part of the arteriographic evaluation of retroperitoneal hematomas associated with pelvic fractures, especially when there are lumbar fractures. Selective lumbar arteriography should be performed for confirmation when there is suspicion of lumbar artery injury on the basis of aortographic findings. Embolization with pledgets of surgical gelatin is effective in controlling hemorrhage from these injuries.

Intussusception following abdominal trauma.

We reviewed the charts of 21 patients on the Trauma Service who were operated on for intestinal obstruction for the years 1983 through 1985. Six (28.6%) of the 21 patients had intussusception as the cause of their obstruction post-laparotomy for trauma. All were males ages 17 to 25 years. The mechanisms of injury were gunshot wounds in three, stab wounds in two, and blunt trauma in one. Five patients were hypotensive on admission with systolic BP less than 70, and two patients received uncrossmatched blood preoperatively. Injuries at exploration included liver laceration (six patients), gastric perforation (two patients), and diaphragmatic lacerations, splenic laceration, renal injury, and ventricular injury, one each. No patient suffered small intestinal injuries and we cannot explain the occurrence of intussusception. Intussusception occurred in the first 8 postoperative days in four patients and at 21 days, and 10 months, in the remaining two. The diagnosis was made twice by CT scan preoperatively. Jejunojejunal intussusception was common (five patients), jejunoileal in one and ileocolic in one (who also had a jejunojejunal intussusception). All patients were treated with manual reduction alone and none recurred. There were no postoperative complications and all patients were discharged by the eighth postoperative day. Our study suggests that early postoperative obstruction is caused by intussusception with unexpected frequency in trauma patients, and can be diagnosed by CT scan in some cases. Treatment with operative reduction has an excellent prognosis.

Arterial trauma: diagnostic and therapeutic angiography.

Between 1978 and 1982, 1,200 patients underwent angiography following acute traumatic injury, and arterial injuries were detected in 182 cases (15.2%). Two-thirds of the cases were associated with penetrating trauma; half the injuries occurred in the pelvis or lower extremities. The most common and reliable sign of arterial injury was the presence of extraluminal contrast media. Other signs included occlusion, laceration, transection, arteriovenous fistula, intimal flap, and intraluminal thrombus. Luminal narrowing was difficult to interpret and resulted from a variety of causes, some of which do not require surgical intervention. Embolotherapy to control arterial bleeding was attempted in 79 patients (43%), and hemostasis was achieved in 69 of them (87%). Transcatheter closure was attempted in 19 of 34 arteriovenous fistulas, resulting in complete success in 15 cases and partial success in two.

Use of sutureless intraluminal aortic prostheses in traumatic rupture of the aorta.

Despite wide acceptance of use of sutureless intraluminal prosthetic grafts in surgical management of acute and chronic aortic dissection, their use in traumatic aortic rupture appears not to have been specifically addressed. This report describes the use of intraluminal prostheses in two cases of aortic disruption due to blunt trauma. Aortic cross-clamp times were 21 and 28 minutes, respectively. Both patients have recovered without sequelae from their aortic injuries. Although not all traumatic aortic disruptions are suitable for repair with intraluminal prostheses, the ease and safety with which this device may be employed recommends consideration of their use in appropriate cases of traumatic rupture of the aorta.