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Background: Inflammatory cytokines dysregulate microvascular function, yet how cytokines affect lymphatic endothelial cells (LEC) are unclear., Methods and Results: We examined effects of TNF-α, IL-1 beta, and IFN-gamma on LEC proliferation, endothelial cell adhesion molecule (ECAM) expression, capillary formation, and barrier changes in murine (SV-LEC) and human LECs (HMEC-1a)., Results: All cytokines induced ICAM-1, VCAM-1, MAdCAM-1, and E-selectin in SV-LECs; TNF-α, IL-1 beta; and IFN-gamma induced ECAMs (but not MAdCAM-1) in HMEC-1a. IL-1 beta increased, while IFN-gamma and TNF-α reduced SV-LEC proliferation. While TNF-α induced, IFN-gamma decreased, and IL-1 beta did not show any effect on HMEC-1a proliferation. TNF-α, IL-1 beta, and IFN-gamma each reduced capillary formation in SV-LEC and in HMEC-1a. TNF-α and IL-1 beta reduced barrier in SV-LEC and HMEC-1a; IFN-gamma did not affect SV-LEC barrier, but enhanced HMEC-1a barrier. Inflammatory cytokines alter LEC growth, activation and barrier function in vitro and may disturb lymphatic clearance increasing tissue edema in vivo., Conclusion: Therapies that maintain or restore lymphatic function (including cytokines blockade), may represent important strategies for limiting inflammation.

Cytochrome P450 (CYP) and hydroxysteroid dehydrogenase enzymes are involved in the conversion of cholesterol to steroid hormones. These enzymes are primarily expressed in the placenta, adrenal and gonads. Interestingly, some of these enzyme activities have been demonstrated in non-endocrine tissues, where they may be involved in important paracrine and autocrine actions. This is particularly the case in the human fetus where steroid precursors circulate at high levels and could be metabolized within tissues to produce active steroid hormones. Herein, we tested the hypothesis that transcripts for steroidogenic enzymes are expressed in fetal tissues other than the classical steroidogenic organs. To test this hypothesis, real-time reverse transcription polymerase chain reaction (RT-RTPCR) assays were developed that quantify mRNA levels for steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A), 3beta-hydroxysteroid dehydrogenase types 1 and 2 (HSD3B1 and HSD3B2), 17alpha-hydroxylase (CYP17), 21-hydroxylase (CYP21), 11beta-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2) and aromatase (CYP19). The use of RT-RTPCR allows the specific detection of these transcripts at levels that would not be detectable using northern analysis. In addition, this method can detect levels of transcript that would not lead to sufficient protein for detection of enzymatic activity of protein by western analysis. Thus, this methodology can detect low levels of expression that could play a role in regulating intra-tissue concentrations of steroid hormone. Total RNAs used for RT-RTPCR analysis were isolated from several human fetal tissues, including adrenal, testis, ovary, placenta, aorta, brain, liver, kidney, heart, lung, pancreas, prostate, stomach, and thymus. Our findings suggest that RT-RTPCR is a powerful tool for the examination of steroidogenic enzyme mRNA expressions. Using this approach, we have identified and quantified transcript levels of StAR and steroidogenic enzymes in several endocrine and non-endocrine fetal tissues. Even though some of the mRNA levels measured in these peripheral tissues are extremely lower in respect to the steroidogenic tissues, they could be sufficient to produce local (i.e. autocrine and paracrine) effects because produced steroids are not diluted into the entire circulation. These findings open new perspectives on the role of steroid hormones synthesized locally as probable regulatory factors of the development of several organ systems.

Percutaneous vertebroplasty (PV) is a safe and effective treatment for relieving pain in patients complaining of severe back pain induced by osteoporotic or neoplastic compression fractures. The success rate exceeds 90% and the complication rate is lower than 1%. Most of the complications are transient. The classic indication for PV is severe, persistent, and incapacitating focal back pain not responding to standard medical therapy and related to one or more collapsed vertebral bodies of 4 to 12 weeks duration.

Adenovirus-mediated gene therapy is a promising new approach for treatment of ovarian cancer. In animal models, complete elimination of cancer cells is often achieved, although the therapeutic gene has not been delivered to all these cells. This is referred to as a bystander effect, because tumor cells near those that receive the therapeutic gene are also eliminated. Several mechanisms have been proposed for the bystander effect, including intercellular communication within the tumor via gap junctions, apoptosis, antiangiogenesis, cytokines or other soluble mediators, and immunological mechanisms. There are two well-documented antitumor effector cell populations in athymic nude mice: macrophages and natural killer (NK) cells. We hypothesize that peritoneal populations of NK cells in nude mice treated with adenoviruses are involved in the observed bystander effect in this in vivo model. We investigated the role of NK cells as immunological mediators for the bystander effect using the p53 tumor suppressor as the therapeutic anticancer gene. Most ovarian cancer cell lines tested were sensitive to lysis by NK cells, although different ovarian cancer cell lines exhibited different sensitivities to NK cell-mediated lysis. To determine the importance of NK cells in the overall efficacy and in the bystander effect of gene therapy, NK cells were depleted in mice by administration of anti-NK1.1 monoclonal antibodies. To study the efficacy of NK depletion, C57BL/6 (nu/nu) mice were given injections i.v. by a single tail vein injection or i.p. with increasing doses of anti-NK1.1 IgG. All doses of anti-NK1.1 antibody, from 100-500 micrograms, essentially eliminated cytotoxic NK activity. To assess the duration of depletion after a single dose of anti-NK1.1 IgG, a time-course experiment was performed. NK 1.1 antibody was effective in completely depleting cytotoxic NK cell activity in the mice for up to 7 days, whether given as 500 micrograms (i.p.) or 200 micrograms (i.v.). Flow cytometric analysis performed on peritoneal cell populations confirmed depletion of NK cells by approximately 80%. Finally, a survival study was performed, in which animals were depleted of NK cells. In this experiment, NK cell-depleted mice were injected with anti-NK1.1 IgG, and control mice were mice were treated with normal saline. Two days later, all mice were inoculated with a lethal i.p. dose of NIH:OVCAR-3 ovarian cancer cells. After 3 days, the mice were divided into two treatment groups; one treatment group received three consecutive daily i.p. injections of Ad-CMV-p53 (SCH58500), and the second treatment group received three consecutive daily i.p. injections of control adenovirus construct, rAd-null. All of the NK cell-depleted animals, whether treated with rAd-null or with Ad-CMV-p53 (SCH58500) were dead of disease by 116 and 138 days, respectively, after initiation of adenovirus treatment, and no statistically significant difference in survival was observed (P = 0.349). A significant survival advantage was seen in control (NK-competent) mice treated with rAd-null (P = 0.04), although all were dead of disease by day 184. Importantly, control NK-competent mice treated with Ad-CMV-p53 (SCH58500) showed no tumor growth or ascites production, and all animals survived. These results indicate that immunological mechanisms involving natural killer cells play an important role in the bystander effect involving adenovirus-p53 gene therapy for ovarian cancer.

Study Design: Ex vivo biomechanical study using osteoporotic cadaveric vertebral bodies., Objective: To determine the association between the volume of cement injected during percutaneous vertebroplasty and the restoration of strength and stiffness in osteoporotic vertebral bodies, two investigational cements were studied: Orthocomp (Orthovita, Malvern, PA) and Simplex 20 (Simplex P with 20% by weight barium sulfate content; Stryker-Howmedica-Osteonics, Rutherford, NJ)., Summary of Background Data: Previous biomechanical studies have shown that injections of 8-10 mL of cement during vertebroplasty restore or increase vertebral body strength and stiffness; however, the dose-response association between cement volume and restoration of strength and stiffness is unknown., Methods: Compression fractures were experimentally created in 144 vertebral bodies (T6-L5) obtained from 12 osteoporotic spines harvested from female cadavers. After initial strength and stiffness were determined, the vertebral bodies were stabilized using bipedicular injections of cement totaling 2, 4, 6, or 8 mL and recompressed, after which post-treatment strength and stiffness were measured. Strength and stiffness were considered restored when post-treatment values were not significantly different from initial values., Results: Strength was restored for all regions when 2 mL of either cement was injected. To restore stiffness with Orthocomp, the thoracic and thoracolumbar regions required 4 mL, but the lumbar region required 6 mL. To restore stiffness with Simplex 20, the thoracic and lumbar regions required 4 mL, but the thoracolumbar region required 8 mL., Conclusion: These data provide guidance on the cement volumes needed to restore biomechanical integrity to compressed osteoporotic vertebral bodies.

Background and Purpose: Previous ex vivo biomechanical studies have shown that kyphoplasty with polymethylmethacrylate cement increases vertebral body (VB) strength and restores VB stiffness and height after compression fracture. The purpose of the current study was to determine if a hydroxyapatite cement used as a void filler during kyphoplasty provides mechanical stabilization similar to that of a polymethylmethacrylate cement., Methods: Simulated compression fractures were experimentally created in 33 osteoporotic VBs harvested from female cadaver spines. VBs were assigned to one of three groups: 1) kyphoplasty with a custom mixture of Simplex P; 2) kyphoplasty with BoneSource; and 3) no treatment. The kyphoplasty treatment consisted of inserting a balloon-like device into the VB via both pedicles, inflating the tamp, and filling the created void with Simplex P bone cement or BoneSource. VBs in the no-treatment group received no interventions. Pre- and posttreatment heights were measured, and the repaired VBs were recompressed to determine posttreatment strength and stiffness values., Results: Kyphoplasty with altered Simplex P restored strength, whereas kyphoplasty with BoneSource and the no-treatment protocol both resulted in significantly weaker VBs relative to initial strength. All treatments resulted in significantly less stiff VBs relative to their initial condition. All VBs lost significant height after initial compression, but a significant amount of lost height was restored by kyphoplasty with either cement., Conclusion: Kyphoplasty with either cement significantly restored VB height. Kyphoplasty with altered Simplex P resulted in stronger repairs than did no treatment or kyphoplasty with BoneSource.

Objective: The aim of this study was to assess the toxicity of concomitant topotecan and radiation therapy in a Phase I study. Primary treatment for cervical carcinoma usually consists of surgery or radiation, with chemotherapy used in a neoadjuvant or concomitant fashion. There are in vitro data to suggest that topotecan is a radiosensitizing agent., Methods: Six patients with cervical cancer were recruited to this study. All patients had completed whole pelvic radiation therapy and were scheduled for low-dose brachytherapy. The patients were administered topotecan IV during their low-dose brachytherapy. The initial dose of topotecan was 0.5 mg/m2/day for 5 days concomitant with low-dose brachytherapy for two brachytherapy applications., Results: Three patients were accrued to the initial dose level. No major toxicity was noted at this dose level. Three patients were treated at the 1.0 mg/m2/day dose level; however, significant toxicity was noted at this level. (Two patients experienced grade 4 and one a grade 3 hematologic toxicity)., Conclusion: Significant marrow toxicity was noted with concomitant topotecan and intracavitary radiation at 1.0 mg/m2/day. The maximum tolerated dose in this trial was 0.5 mg/m2/day for 5 days of topotecan concomitant with low-dose brachytherapy.

Here we describe the derivation of novel cell lines from spontaneous mammary tumors that arose in mouse mammary tumor virus-polyomavirus (MMTV-PyV) Middle T (MidT) transgenic mice. Clonal cell lines from four mixed cell populations were tested for adenovirus transducibility and sensitivity to p53 tumor suppressor gene therapy mediated by SCH58500, a replication-deficient adenovirus that expresses human p53. The MidT2-1 cell line was selected for further characterization in vitro and in vivo. This cell line carried the PyV MidT antigen, had wild-type p53 DNA, and was sensitive to suppression of proliferation by MMAC/PTEN tumor suppressor gene therapy. MidT2-1 cells gave rise to highly aggressive tumors in syngeneic FVB mice in both the mammary fat pad and the peritoneal cavity. The histopathology of MidT2-1 tumors closely resembled the histopathology of the primary transgenic tumors. Tumor growth in vivo was inhibited by p53 gene therapy or by MMAC gene therapy. In addition, combination therapy with a number of anticancer agents had synergistic or additive efficacy in vitro. In particular, MMAC gene therapy synergized with SCH58500 or paclitaxel. In the i.p. MidT2-1 tumor model p53 gene therapy enhanced the survival benefits of paclitaxel/cisplatin chemotherapy. Combination therapy has become a mainstay in cancer treatment. In this report, we use a novel transgenic mouse tumor cell line to suggest new combinations that might be explored in clinical cancer care. These include gene therapy using the tumor suppressors MMAC and p53, chemotherapy using farnesyl transferase inhibitors, the microtubule stabilizing taxanes, and the DNA synthesis disruptors gemcitabine and cisplatin. The precise biological mechanisms by which these therapies induce their antitumor effects are not fully elucidated. However, the work presented here suggests that many of these therapeutic approaches have synergistic antitumor activity when used in combination.

Objective: Most precancerous lesions of the cervix are treated with surgery or ablative therapy. Chemoprevention, using natural and synthetic compounds, may intervene in the early precancerous stages of carcinogenesis and prevent the development of invasive disease. Our trial used indole-3-carbinol (I-3-C) administered orally to treat women with CIN as a therapeutic for cervical CIN., Methods: Thirty patients with biopsy proven CIN II-III were randomized to receive placebo or 200, or 400 mg/day I-3-C administered orally for 12 weeks. If persistent CIN was diagnosed by cervical biopsy at the end of the trial, loop electrocautery excision procedure of the transformation zone was performed. HPV status was assessed in all patients., Results: None (0 of 10) of the patients in the placebo group had complete regression of CIN. In contrast 4 of 8 patients in the 200 mg/day arm and 4 of 9 patients in the 400 mg/day arm had complete regression based on their 12-week biopsy. This protective effect of I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI, 0. 25 to 0.99) P = 0.023) for the 200 mg/day group and a RR of 0.55 ((95% CI, 0.31 to 0.99) P = 0.032) for the 400 mg/day group. HPV was detected in 7 of 10 placebo patients, in 7 of 8 in the 200 mg/day group, and in 8 of 9 in the 400 mg/day group., Conclusions: There was a statistically significant regression of CIN in patients treated with I-3-C orally compared with placebo. The 2/16 alpha-hydroxyestrone ratio changed in a dose-dependent fashion., (Copyright 2000 Academic Press.)

Study Design: Comparative ex vivobiomechanical study., Objectives: To determine the strength and stiffness of osteoporotic vertebral bodies subjected to compression fractures and subsequently stabilized via bipedicular injection of one of two bone cements: one is a commercially available polymethylmethacrylate (Simplex P) and one is a proprietary glass-ceramic-reinforced BisGMA/BisEMA/TEGDMA matrix composite that is being developed for use in vertebroplasty (Orthocomp)., Summary of Background Data: Osteoporotic compression fractures present diagnostic and therapeutic challenges for the clinician. Vertebroplasty, a new technique for treating such fractures, stabilizes vertebral bodies by injection of cement. Little is known, however, about the biomechanics of this treatment., Methods: Five vertebral bodies (L1-L5) from each of four fresh spines were harvested from female cadavers (age, 80 +/- 5 years), screened for bone density using DEXA (t = -3.4 to -6.4), disarticulated, and compressed in a materials testing machine to determine initial strength and stiffness. The fractures then were repaired using a transpedicular injection of either Orthocomp or Simplex P and recrushed., Results: For both cement treatments, vertebral body strength after injection of cement was significantly greater than initial strength values. Vertebral bodies augmented with Orthocomp recovered their initial stiffness; however, vertebral bodies augmented with Simplex P were significantly less stiff than they were in their initial condition., Conclusions: Augmentation with Orthocomp results in similar or greater mechanical properties compared with Simplex P, but these biomechanical results have yet to be substantiated in clinical studies.

Pharmacologic testing is a method that may be employed to predict the permanent outcome of arterial embolization. Various drugs used for these tests and their differential indications are discussed. Pharmacologic testing provides an additional margin of safety and is useful as an adjunctive tool when performing embolizations of vessels supplying the head, brain, or spinal cord.

Study Design: Cadaveric study on the biomechanics of osteoporotic vertebral bodies augmented and not augmented with polymethylmethacrylate cement., Objectives: To determine the strength and stiffness of osteoporotic vertebral bodies subjected to compression fractures and 1) not augmented, 2) augmented with unipedicular injection of cement, or 3) augmented with bipedicular injection of cement., Summary of Background Data: Percutaneous vertebroplasty is a relatively new method of managing osteoporotic compression fractures, but it lacks biomechanical confirmation., Methods: Fresh vertebral bodies (L2-L5) were harvested from 10 osteoporotic spines (T scores range, -3.7 to -8.8) and compressed in a materials testing machine to determine intact strength and stiffness. They were then repaired using a transpedicular injection of cement (unipedicular or bipedicular), or they were unaugmented and recrushed., Results: Results suggest that unipedicular and bipedicular cement injection restored vertebral body stiffness to intact values, whereas unaugmented vertebral bodies were significantly more compliant than either injected or intact vertebral bodies. Vertebral bodies injected with cement (both bipedicular and unipedicular) were significantly stronger than the intact vertebral bodies, whereas unaugmented vertebral bodies were significantly weaker. There was no significant difference in loss in vertebral body height between any of the augmentation groups., Conclusions: This study suggests that unipedicular and bipedicular injection of cement, as used during percutaneous vertebroplasty, increases acute strength and restores stiffness of vertebral bodies with compression fractures.

The purpose of this study was to determine the strength and stiffness of osteoporotic vertebral bodies (VBs) subjected to compression fractures and subsequently treated with bipedicular injections of various polymethylmethacrylate cements. Ten spines were harvested from nonembalmed female cadavers (age 68.6 +/- 13.7 years) and evaluated for bone mineral density using the dual energy X-ray absorptiometry method (t-score = -2.3 +/- 2.4). The 50 VBs (L1-L5) were disarticulated, compressed in a materials testing machine to determine initial strength and stiffness, and then assigned to one of six groups. Two of these groups (n = 8, n = 9) concerned experimental cements, the results of which are not reported here. The 33 vertebral bodies in the remaining four groups were left untreated or were repaired using a transpedicular injection of one of three commercially available polymethylmethacrylate cements. These four groups were: a) no treatment (no cement, n = 8); b) Simplex P (n = 9); c) Cranioplastic (n = 8); and d) Osteobond (n = 8). The VBs were then compressed again according to the initial protocol, and posttreatment strength and stiffness were measured. Results suggested that bipedicular injection of Simplex P and Osteobond restored VB stiffness to initial values, whereas VBs injected with Cranioplastic were significantly less stiff than in their initial state. VBs injected with cement (regardless of type) were significantly stronger than they were initially.

Percutaneous vertebroplasty consists of injecting polymethylmethacrylate cement into the cancellous bone of vertebral bodies for the treatment of various lesions of the spine, including osteoporotic compression fractures. Clinicians practicing vertebroplasty commonly alter the mixture of monomer-to-powder recommended by the manufacturer in an effort to decrease viscosity and increase the working time. The purpose of the current study was to measure the effect that varying the monomer-to-powder ratio has on the compressive material properties of a cement (Cranioplastic) commonly used in vertebroplasty. Cylindrical specimens were prepared varying a monomer-to-polymer ratio of 0.40 to 1.07 ml/g and tested per the American Society for Testing and Materials standard F451. Specimens prepared at 0.53 mL/g, which is near the manufacturer's recommended monomer-to-polymer mixture of 0.57 mL/g, exhibited the greatest mean values for ultimate compressive stress, yield stress, and elastic modulus. Specimens prepared at higher or lower ratios exhibited diminished strength, in some cases by as much as 24%. Although altering the monomer-to-powder ratio affects the cement's material properties, it is as yet unknown if the decrease is clinically significant.

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.

A woman reported painful thrombosis of the superficial femoral artery 16 months after a transfemoral microcatheter was glued into a cerebral arteriovenous malformation and transected at the groin. When the catheter was removed, a portion was found to be incorporated into the wall of the carotid artery. This case demonstrates that portions of a retained microcatheter may be incorporated into the arterial wall while other portions may remain mobile and cause late peripheral arterial symptoms.

Objective: Glutamine is proposed to protect bowel from radiation. However, glutamine may decrease cancer's radiosensitivity. We evaluate glutamine's effect on the growth rate and radiosensitivity of two cervical carcinoma cell lines in vitro., Methods: HeLa and CaSki cells were seeded at 3000 cells/well in glutamine-free medium. An increasing amount of glutamine (0.4, 10, and 20 mM) was added to the respective plates, incubated, and irradiated with a single fraction of 0.5, 1, 3, and 6 Gy. Using a growth inhibition assay and photometric analysis, the viable cells were counted on day 8. Cell counts represent a mean +/- standard deviation from six experiments and are expressed in 10(3) cells. Analysis of variance was performed., Results: In nonirradiated HeLa plates, absence of glutamine results in 5.7 +/- 1.2 cells/well. Addition of glutamine at 0.4, 10, and 20 mM to nonirradiated cells significantly (P < 0.0001) increased growth to 79.1 +/- 10.0, 122.5 +/- 9.0, and 114.3 +/- 13.9 cells/well, respectively. In culture plates irradiated with 6 Gy, HeLa cells supplemented with 0.4, 10, and 20 mM of glutamine showed lower cell counts (P < 0.008). A similar significant growth suppression at 6 Gy in comparison to 0.5, 1, and 3 Gy was observed (P < 0.01). CaSki cells showed similar patterns., Conclusions: Growth of HeLa and CaSki cells in vitro requires a minimum of 0.4 mM of glutamine in the medium. Supraphysiologic glutamine concentration does not increase tumor growth or radioresistance. Glutamine should be evaluated further as a potential bowel radioprotector., (Copyright 1998 Academic Press.)

Background and Purpose: Published reports of controlled experiments designed to evaluate the performance of over-the-wire microcatheter systems are rare and have often been based on subjective impressions from small clinical series. This investigation was designed to compare the load forces required to propel state-of-the-art, hydrophilically coated microcatheters from each of four manufacturers through a standardized tortuous pathway constructed of polytetrafluoroethylene tubing., Methods: Currently available hydrophilically coated microcatheters were provided by four manufacturers. A 20-cm long, three-dimensional pathway simulating the intracranial carotid circulation was constructed of 0.065-in. (inner diameter) polytetrafluoroethylene tubing and immersed in a water bath at 37 degrees C. Testing was performed using an Instron tabletop load frame fitted with a 2-lb load cell. Durability and load force tests were conducted using a 0.014-in. stainless steel noncoated guidewire, with the wire tip protruding 1 cm beyond the catheter tip. At least four samples of microcatheters from each manufacturer were tested., Results: Extensive trackability testing of the guidewire alone established reproducible performance with maximum load forces of less than 8 g. Maximum gram forces for the four reinforced microcatheters were not greatly different, measuring between 9 and 14 g. Excessive buckling of the only nonreinforced catheter was initially overcome early in the pathway in a staccato, stepwise fashion. After reaching a critical load, however, the catheter and guidewire prolapsed., Conclusion: All reinforced microcatheters tested established good and reproducible performance in our model. Reinforced microcatheters provided superior trackability over the one nonreinforced device tested.