Effect of host age on tumor-associated angiogenesis in mice.

Previous reports on the slower growth of tumors in senescent mice have suggested a decrease in tumor angiogenesis in these animals, but such an observation has not yet been documented quantitatively. In this study, we report the relative amount of tumor angiogenesis and tumor volume for two different types of tumor in 11 young (8-9-wk old) versus nine older (19-mo old) male C57BL/10 mice. B16 melanoma or SP1 methylcholanthrene-induced fibrosarcoma cells were injected into the ventral skin of mice. After 3 days, the mice were killed and the injection sites were examined for angiogenesis surrounding the tumor (centrally directed tumor angiogenesis), nerve-associated angiogenesis, and tumor volume. In the older mice, there was significantly less centrally directed tumor angiogenesis for both tumors tested, and nerve-associated angiogenesis was decreased for B16 melanoma. The mean tumor volume for the B16 implants was smaller for the older animals, but the mean SP1 tumor volumes were identical for both age groups. These findings support the hypothesis that tumor growth in older animals is associated with less formation of new blood vessels, and this may explain the slower tumor growth observed in aged animals with certain experimental tumors.

Hydrogen peroxide activates NFkappaB and the interleukin-6 promoter through NFkappaB-inducing kinase.

Aging is associated not only with oxidant stress, but also with increased interleukin-6 (IL-6) levels. To determine if oxidative stress could contribute to the age-associated increase IL-6 expression, we exposed LNCaP prostate carcinoma cells and HeLa cervical carcinoma cells to H2O2 as an oxidant challenge. We found that H2O2 induced IL-6 expression through activation of the IL-6 promoter. Furthermore, H2O2-induced activation of the promoter was mediated through nuclear factor-kappaB (NFkappaB) secondary to H2O2-induced phosphorylation and degradation of IkappaBalpha. NFkappaB-inducing kinase (NIK) is upstream of the IkappaB kinase complex that induces IkappaBalpha degradation. Accordingly, we explored if H2O2 induces IL-6 expression through NIK. In addition to H2O2 inducing NIK autophosphorylation, transfection of LNCaP cells with a dominant negative NIK diminished H2O2-mediated NFkappaB and IL-6 promoter activity. Taken together, these results demonstrate that H2O2 induces the IL-6 promoter by activating NFkappaB through NIK. These data provide a candidate mechanism through which oxidant challenge induces IL-6 gene expression with age.

Ovariectomy does not induce osteopenia through interleukin-6 in rhesus monkeys (Macaca mulatta).

To characterize the role of interleukin-6 (IL-6) in estrogen (E2)-depletion bone loss, we utilized a nonhuman primate model of human skeletal physiology. Adult female rhesus monkeys were sham-operated (S; n = 5), ovariectomized (ovx; n = 10), or ovx followed by E2 replacement (ovx + E2; n = 10) and evaluated for the indicated parameters at 0, 3, 6, and 9 months post-ovx. Lumbar spine bone mineral density (BMD) decreased by 3 months and continued to decline through 9 months in the ovx, but not in the ovx + E2 or S groups. Middle and distal radius BMD was decreased at 9 months in the ovx, but not in the ovx + E2 or S groups. The S group had marked fluctuations in bone remodeling parameters, and cytokine levels in S animals were consistent with menstrual cycling, and therefore only those values in the ovx and ovx + E2 groups are reported. Serum osteocalcin and skeletal-specific alkaline phosphatase were elevated in the ovx group compared with the ovx + E2 group. There was no difference in serum or bone marrow plasma IL-6 levels between the ovx and ovx + E2 groups. Similarly, there was no difference in basal or phorbol ester-stimulated IL-6 levels of peripheral blood mononuclear cell or bone marrow cell culture supernatants between groups. There was no difference in serum or bone marrow soluble IL-6 receptor between groups. However, the bone marrow plasma soluble IL-6 receptor levels were transiently increased from baseline at 3 months in the ovx but not in the ovx + E2 group. In summary, there was no bone loss in the ovx + E2 group, although the serum and bone marrow IL-6 levels were similar to those of the ovx group. These data suggest that modulation of IL-6 is not the key mechanism through which estrogen deprivation mediates bone loss in rhesus monkeys.

Quantitative computed tomography of bone.

Computed tomography (CT) is well accepted as an imaging procedure, but comparatively little effort has been made to utilize the potential capability of CT to quantify tissue densities and composition. There are two reasons for this. First, precision and accuracy of quantification are limited by nonlinear effects. These effects are nonlocal and are object and scanner dependent. Second, intraindividual and interindividual variations of tissue compositions are considerable. Single energy measurements require restrictive assumptions on tissue compositions. The diagnosis and treatment monitoring of osteopenic bone diseases with low-dose CT is given as an example of a successful application of quantitative CT. With a special-purpose CT system and an analytic procedure for the quantification of bone at peripheral measuring sites, longitudinal examinations were performed. Low-dose quantitative CT permitted quantification, on an individual basis, of the bone loss of immobilization osteoporosis on a week-by-week basis. Changes due to postmenopausal osteoporosis are less drastic, and so measurement at intervals of months is adequate. In women after menopause, 3-month intervals were used in evaluating the natural course of osteoporosis and in quantifying the effects of sodium fluoride treatment on trabecular bone. Low-dose quantitative CT has proved to be a sensitive and highly reproducible procedure for the noninvasive evaluation of bone loss or bone accretion. During a disease or therapy, each patient can be evaluated individually.

A prototype high-purity germanium detector system with fast photon-counting circuitry for medical imaging.

A data-acquisition system designed for x-ray medical imaging utilizes a segmented high-purity germanium (HPGe) detector array with 2-mm wide and 6-mm thick elements. The detectors are contained within a liquid-nitrogen cryostat designed to minimize heat losses. The 50-ns pulse-shaping time of the preamplifier electronics is selected as the shortest time constant compatible with the 50-ns charge collection time of the detector. This provides the detection system with the fastest count-rate capabilities and immunity from microphonics, with moderate energy resolution performance. A theoretical analysis of the preamplifier electronics shows that its noise performance is limited primarily by its input capacitance, and is independent of detector leakage current up to approximately 100 nA. The system experimentally demonstrates count rates exceeding 1 million counts per second per element with an energy resolution of 7 keV for the 60-keV gamma ray photon from 241Am. The results demonstrate the performance of a data acquisition system utilizing HPGe detector systems which would be suitable for dual-energy imaging as well as systems offering simultaneous x-ray transmission and radionuclide emission imaging.

Interleukin-2 and syngeneic bone marrow transplantation in a murine fibrosarcoma model.

Mice received interleukin-2 (IL-2) either before and after, or just after intravenous inoculation of syngeneic fibrosarcoma cells. Fewer pulmonary tumor colonies were observed in those animals treated with IL-2, and the best results were observed when IL-2 was administered prior to tumor inoculation. When mice were lethally irradiated and reconstituted with tumor-contaminated bone marrow, IL-2 treatment was also associated with fewer tumor lung colonies. IL-2 may prove to be a useful adjuvant therapy, particularly in the setting of autologous bone marrow transplantation when the infused marrow is contaminated with tumor cells.

Initial description of a tumor enhancing activity produced by murine splenocytes.

We have shown that certain murine tumors grow more slowly and spread less readily in immune deficient animals. We have also demonstrated that immunologic factors explain certain aspects of this difference. In the work presented we demonstrate that a subpopulation of splenocytes produce a factor(s) that enhances tumor cell proliferation in vitro. We also describe an in vitro model to determine the level of tumor stimulatory activity. We found that the tumor cell growth-enhancing activity (TEA) is heat stable but sensitive to trypsin digestion, low pH and beta-mercaptoethanol. TEA production is found to be insensitive to mitogen stimulation such as concanavalin A, lipopolysaccharide, and phytohemagglutinin. Among the known growth factors and interleukins we have tested (interleukin 1-7, basic FGF, EGF, TGF-beta PDGF, GM-CSF, and MCSF), none appear to account for TEA activity.

Effect of diet on hyperthermia-induced cell lethality and prostaglandin release.

Hyperthermia-induced cell lethality is thought to be mediated through injury to the cell membrane. Membrane perturbation results in the release of prostaglandins (PG) and leukotrienes (LT). These compounds are potent biological mediators and may modify the tumor microenvironment and therapeutic efficacy. Membrane composition and PG/LT release are influenced by the dietary fatty acids. The relationship between these variables and response to hyperthermia was examined in vitro using murine P388 leukemia cells grown as an ascites in mice provided either saturated fatty acid diet (SFA; 16% beef tallow) or unsaturated fatty acid diet (UFA; 16% safflower oil). Cells were harvested and exposed in vitro to either 37 degrees C or 43.5 degrees C for periods up to 2 hours. Hyperthermic exposure for 2 hours resulted in 40% cell lethality in SFA cells and 55% in UFA cells. The phospholipid and total cholesterol content was higher (33% and 50% respectively) in the UFA versus the SFA cells. Hyperthermia produced a six-fold increase in prostaglandin E2 PGE2 release by SFA cells and a 4.5-fold increase by UFA cells. No LTC4 was detected. Alteration of dietary fat affects cell lethality and PG release following hyperthermic treatment. The increase in phospholipid and cholesterol content of UFA cells may be a response to reduced membrane fluidity.

Orchiectomy increases bone marrow interleukin-6 levels in mice.

Interleukin-6 (IL-6) appears to be an important factor in disease states associated with bone resorption. There is both in vitro and in vivo evidence supporting the fact that androgens down-regulate interleukin-6 production. These observations, in combination with the fact that osteoblasts and bone marrow stromal cells produce IL-6, led us to hypothesize that orchiectomy-induced androgen loss will result in increased IL-6 expression in the bone microenvironment. To prove our hypothesis we assessed the effect of orchiectomy on IL-6 protein and mRNA expression in bone marrow and spleen. We found that orchiectomy was associated with increased serum IL-6 levels at 3 and 28 days postsurgery. Phorbol ester-stimulated IL-6 levels were also higher in supernatants from bone marrow and spleen cell cultures from orchiectomized mice compared with unoperated or sham-operated mice. Additionally, we found that steady state IL-6 mRNA levels were increased in bone marrow but not spleen cells. Finally, we found that orchiectomized mice had splenomegaly and increased bone marrow cellularity. Histopathology of the spleen revealed lymphoid hyperplasia accompanied by a marked mononuclear cell infiltration of the red pulp. We conclude that orchiectomy induces IL-6 expression in the bone marrow. These findings suggest that endocrine and cytokine interactions contribute to bone pathophysiology.

Estrogen inhibits phorbol ester-induced I kappa B alpha transcription and protein degradation.

Estrogen (E2) is known to prevent bone loss and the mechanism is, at least in part, mediated by inhibition of expression of cytokines such as interleukin-6 (IL-6). Expression of IL-6 is tightly regulated and the transcription factor NF kappa B can upregulate IL-6 gene expression by binding to its promoter region. NF kappa B is kept in an inactive state by associating with its cytoplasmic inhibitor I kappa B alpha. Upon mitogenic stimulation, I kappa B alpha becomes phosphorylated, followed by a rapid protein degradation. As a result, NF kappa B is released and translocate to the nucleus where DNA binding occurs. It has been shown that E2 treatment downregulates mitogen-induced IL-6 expression by inhibiting NF kappa B activity. Here, we sought to determine whether E2 regulates IL-6 gene expression by modulating the levels of I kappa B alpha. Our results show that E2 treatment almost completely inhibits phorbol ester-induced I kappa B alpha protein degradation. In addition, E2 inhibits phorbol ester-stimulated I kappa B alpha gene expression. Taken together, our results suggest that E2 maintains steady state levels of I kappa B alpha upon mitogen stimulation, resulting in inhibition of NF kappa B activation and IL-6 gene expression. This may explain the protective effect of E2 on bone loss.

Smectic-A*-smectic-C* transition in a ferroelectric liquid crystal without smectic layer shrinkage.

The smectic layer spacing of a nonfluorinated ferroelectric liquid crystal (FLC) compound with almost no shrinkage and only minor tendency to form zigzag defects was characterized by small angle x-ray diffraction. The material lacks a nematic phase. The smectic-A*-smectic-C* phase transition was studied by measuring the thermal and electric field response of the optical tilt and the electric polarization. These properties are described very well by a Landau expansion even without introduction of a higher-order Theta(6) term. This result suggests a pure second-order phase transition far from tricriticality and differs considerably from the typical behavior of the A*-C* transition in most FLC materials.

Effects of heat and other agents on amino acid uptake in Escherichia coli.

In Escherichia coli K1060 grown at 37 degrees C we observed that the uptake of both L-[3H]leucine and L-[35S]methionine was inhibited by exposure of the cells to 48 degrees C. The calcium channel blockers diltiazem and verapamil, and the anti-arrhythmic agent quinidine, inhibited the uptake of L-[3H]leucine at both 37 degrees C and 48 degrees C. Verapamil also inhibited the uptake of L-[35S]methionine at 37 degrees C, but at 48 degrees C protected against some of the heat-induced decrease in the uptake of this amino acid. The local anaesthetic procaine markedly inhibited the uptake of both labelled amino acids at temperatures between 37 degrees C and 48 degrees C. Amino acid uptake and cell killing were not correlated.

Intermolecular and intramolecular reorientations in nonchiral smectic liquid-crystalline phases studied by broadband dielectric spectroscopy

Molecular dynamics has been studied by broadband dielectric relaxation spectroscopy in the Sm-A, Sm-B, and Sm-E phases (Sm denotes smectic) of a homologous series of nonchiral stilbenes. An assignment of modes is presented based on their dependence on temperature and molecular length, and, as far as they obey the Arrhenius law, their activation energy has been determined. In general, reorientations of entire molecules around their short axis are active, whereas reorientations of entire molecules around their long axis are locked out in the Sm-E phase of shorter homologs, yet intramolecular reorientations of polar sites have been established. Strong evidence is presented for an interdependence of reorientations of entire molecules around the short and long axes within the biaxial Sm-E phase of longer homologs.