Inhibition of CXCR7 extends survival following irradiation of brain tumours in mice and rats.

BACKGROUND: In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour's response to IR has not been addressed. METHODS: We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro. RESULTS: CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs. CONCLUSIONS: These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.

Membrane association and destabilization by Aggregatibacter actinomycetemcomitans leukotoxin requires changes in secondary structures.

Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis, and localized aggressive periodontitis. Aggregatibacter actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and destabilization. In this study, we tested this hypothesis by analysing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans.

Bone morphogenetic protein function is required for terminal differentiation of the heart but not for early expression of cardiac marker genes.

To examine potential roles for bone morphogenetic proteins (BMPs) in cardiogenesis, we used intracellular BMP inhibitors to disrupt this signaling cascade in Xenopus embryos. BMP-deficient embryos showed endodermal defects, a reduction in cardiac muscle-specific gene expression, a decrease in the number of cardiomyocytes and cardia bifida. Early expression of markers of endodermal and precardiac fate, however, was not perturbed. Heart defects were observed even when BMP signal transduction was blocked only in cells that contribute primarily to endodermal, and not cardiac fates, suggesting a non-cell autonomous function. Our results suggest that BMPs are not required for expression of early transcriptional regulators of cardiac fate but are essential for migration and/or fusion of the heart primordia and cardiomyocyte differentiation.

CaM kinase IV regulates lineage commitment and survival of erythroid progenitors in a non-cell-autonomous manner.

Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

In vitro labeling of gonadal steroid hormone receptors in brain tissue sections.

Autoradiographic methods have been developed for measurement of gonadal steroid receptors in situ in brain tissue sections. Based on principles established previously for estrogen receptors in the rat brain using a 125I-labeled ligand, procedures have been developed for in vitro labeling of estrogen, androgen, and progestin receptors with commercially available tritiated ligands. Addition of protamine sulfate to the incubation buffer precipitates the receptors in situ in the tissue sections, allowing them to be detected autoradiographically after incubation with labeled steroid and subsequent washing to remove unbound and nonspecifically bound ligand. Occupied and unoccupied estrogen receptors can be measured selectively using appropriately modified incubation conditions. In the case of androgen and progestin receptors, unoccupied receptors are readily detected by in vitro labeling of tissue sections, but occupied receptors do not appear to label efficiently. Preliminary data suggest that these methods should be equally applicable to a variety of laboratory animals, including the rat, mouse, guinea pig, and monkey.

Aromatase in the cerebral cortex, hippocampus, and mid-brain: ontogeny and developmental implications.

Aromatase activity was measured in explant cultures from the newborn mouse and rat brain and in homogenates of regions of the rat brain sampled between birth and 51 days of age. Conversion of 19-[3H]hydroxy-androstenedione to estradiol and estrone was detected in explant cultures from the mouse preoptic/septal region, anterior cingulate cortex, and midbrain, as well as from the rat preoptic area, septum, hippocampus, anterior cingulate cortex, and midbrain central grey. No detectable estrogen biosynthesis was observed in explants from the cerebellum and spinal cord of either species. Measurements of aromatase in tissue homogenates using 1 beta[3H]androstenedione as substrate revealed detectable enzyme activity in the hypothalamus + preoptic area, amygdala, hippocampus, anterior cingulate cortex, and midbrain, from birth onward. Aromatase activity per milligram of tissue protein was highest in the hypothalamus-preoptic area and amygdala, followed by the hippocampus, midbrain, and cingulate cortex. In all brain regions, aromatase activity was markedly higher at Postnatal Days 1 and 7 than later in life. In both the cingulate cortex and the hippocampus, aromatase was barely detectable above the assay blank in adult (51 day) animals. These results demonstrate that regions of the developing rodent neo- and archicortex have the capacity to convert androgen to estrogen, consistent with a role for local estrogen biosynthesis in the sexual differentiation of higher brain functions.

In vitro autoradiographic visualization of occupied estrogen receptors in the rat brain with an iodinated estrogen ligand.

Methods have been developed for the selective measurement of occupied estrogen receptors (ER) in brain tissue sections. Cryostat sections of unfixed tissue were incubated with radiolabeled estrogen at physiological temperatures, displacing endogenous receptor-bound estrogen by radioligand and thereby allowing the receptor complexes to be visualized autoradiographically after washing to remove nonspecifically bound steroid. The resultant autoradiographs were analyzed by computer-assisted densitometry. Synthetic 11 beta-methoxy-substituted radiolabeled estrogens gave the best autoradiographic images, as a result of reduced nonspecific labeling, although [3H]-estradiol was also used successfully. With the synthetic ER ligand 11 beta-methoxy 16 alpha-[125I]-iodo-estradiol, exposure times of less than 24 hr generated acceptable autoradiographs; with 3H-labeled estrogens, exposures of 3 months or more may be required. The method is sufficiently sensitive to detect physiological changes in ER occupation and to allow determination of receptor affinities and saturation binding capacities in discrete cell groups identified in sections from individual animals.

The importance of stabilizing the specimen taken at needle localized biopsy of the breast for microcalcifications.

The specimen from a needle localized biopsy of the breast must be compared with its roentgenogram to locate the area of microcalcification within the specimen. Any errors in aligning the specimen to its roentgenogram could result in failure to sample and microscopically examine the area of the microcalcifications. The results of the current study show the importance of maintaining the orientation and position of the specimen from the time the roentgenogram is taken until it is examined by the pathologist. Each of 45 consecutive specimens taken at biopsy were secured to a square of cardboard immediately after excision from the breast to maintain the orientation of the specimen during the process of roentgenography. The histologic findings of this group (group 2) were compared with the preceding 87 biopsies (group 1) in which no effort had been made to immobilize the specimen for a roentgenogram. The presence of microcalcifications was confirmed roentgenographically in all the specimens of each group. Histologic confirmation of the presence of microcalcifications was obtained in 42 of 45 specimens in group 2 and only 71 of 87 in group 1 (p = 0.035). Thirty-one per cent of specimens in group 2 contained a carcinoma compared with 10 per cent in group 1. We conclude that fixing the position of the specimen after excision improves the ability of the pathologist to locate the suspicious area of microcalcifications within the specimen. This may lead to an increase in the yield of these biopsies and the identification of occult carcinomas that might otherwise be missed.

Properitoneal synthetic mesh repair of recurrent inguinal hernias.

Safe reconstruction of the inguinal floor is the goal of any operation for repair of groin herniation. Operating in the properitoneal space avoids dissection of the scarred cord, and the incidence of testicular complications is markedly lowered. This study reports our experience with placing synthetic mesh between the peritoneum and the deficient inguinal floor for the repair of recurrent hernias of the groin area. During a five year period, 84 men underwent repair of 100 recurrent inguinal hernias using the properitoneal approach. Fifty-four patients had repair of a unilateral recurrent hernia, 16 had repair of a bilateral recurrent hernia and 14 had repair of both a recurrent hernia and a contralateral primary hernia. Postoperative complications occurred in six patients. No testicular complications were observed. Postoperative follow-up study ranged from six months to five years. There were only three recurrent hernias after this repair. All occurred within the first six months postoperatively. The properitoneal approach for repair of recurrent groin hernias using prosthetic mesh safely creates a new "fascia transversalis" with a low rate of recurrence and effectively eliminates testicular complications.

Analysis of illegally distributed anabolic steroid products by liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry.

Anabolic steroid products found in the illegal market are primarily oil-based injectables or tablets and often do not contain the ingredients declared on the label. An analytical scheme based on a reverse-phase liquid chromatographic (LC) system for screening, tentative identification, and quantitation is presented. Methanolic sample extracts are chromatographed on an octadecyl column using 2 mobile phases (methanol and (75 + 25) methanol-water) and tracked at 3 wavelengths (240, 210, and 280 nm) or with a photodiode array UV detector. Retention time ratios (RR) relative to testosterone and UV data are used for tentative identification. The same LC system serves as a cleanup and isolation step for identity confirmation by direct insertion probe mass spectrometry (MS) or Fourier transform infrared spectrophotometry (FTIR). Recoveries range from 96.2 to 100.2% for 11 different steroids extracted from sesame oil. LC RR values for over 40 steroids, analytical results for typical products, and MS and FTIR spectra for selected compounds are presented.

Effect of mass screening mammography on staging of carcinoma of the breast in women.

In 1980, our institution began a screening mammography program. A retrospective review was done to document whether or not implementation of this program significantly changed the method of discovery of carcinoma of the breast and the stage of carcinoma of the breast at presentation in the patients we studied. Women diagnosed with carcinoma of the breast during a three year period, prior to the institution of the screening program (group 1) were compared with a similar set of patients diagnosed after establishment of the program (group 2). There were 165 patients in group 1 and 181 in group 2. The vast majority of cancers for those in group 1, 84.2 per cent, were discovered at self-examination, as compared with 46.3 per cent for those in group 2. Forty-eight per cent of the cancers for those in group 2 were first discovered by mammography as compared with 6.1 per cent in group 1. There was no statistical difference in the number of patients presenting with stage O, III and IV disease. The percentage of patients presenting with stage I disease rose significantly after the establishment of the mammography program (16.4 per cent in group 1 to 41.5 per cent in group 2). Findings from this study confirm that a mammographic screening program can have a marked effect on lowering the stage of carcinoma of the breast at presentation. The current recommendations for screening mammography should be implemented. Doing so will result in earlier detection and improved survival in this group of patients.

Hormonal specificity and activation of social behavior in male red-winged blackbirds.

Five groups of male red-winged blackbirds were observed 12 times over 14 days in an aviary setting. An estradiol-treated female was present during the last two observations of each group. Each group of males consisted of a castrated (CA) and an intact (IN) control and six castrated males given one of the following hormone treatments: the aromatizable androgens, testosterone (T) and androstenedione (AE); the nonaromatizable androgens, 5 alpha-dihydrotestosterone (DHT), and androsterone (AN); the estrogen, estradiol (E); or a combination of E + DHT. Castration significantly reduced the frequency of singing and three common vocalizations, chucks, checks, and ips, below the levels shown by IN males. Only hormone treatments which provided both androgenic and estrogenic metabolites (A + E = T, AE, E + DHT) restored normal levels of these behaviors in castrated males. Endocrine status also modulated epaulet display. IN and A + E males were more likely than other males to keep their epaulets constantly exposed. The frequency of sidling and supplanting also varied significantly across hormone-treatment groups, with A + E males showing higher frequencies of these behaviors than other males. T was clearly the most effective hormone treatment in activating hormone-sensitive behaviors in this species. Hormone treatment was more important than size or plumage pattern in determining the outcome of aggressive interactions. In some groups, the dominant male clearly inhibited the performance of hormone-sensitive behaviors by other males. Among A + E males, the frequency of higher intensity song spread displays was highly correlated with the frequency of high-intensity aggressive behaviors and negatively correlated with the tendency to withdraw from other males. Patterns of correlations among behaviors suggest that some calls are more hormone dependent than others, and thus may serve different signal functions.

Estrogen receptor levels in hypothalamic and vocal control nuclei in the male zebra finch.

Estrogens play an important role in the activation and differentiation of vocal behavior in male zebra finches. In the present experiment, we conducted a series of in vitro binding assays to quantify estrogen receptor concentrations in individual hypothalamic and vocal control nuclei. Receptor concentrations were measured in cytosol fractions obtained from castrated males and, since adrenalectomy is not a viable possibility in this species, in castrated males treated with 1,4,6-androstatriene-3,17-dione (ATD), an inhibitor of estrogen synthesis. Specific, high-affinity estrogen binding was detected in both untreated castrates and castrates treated with ATD. Although ATD treatment had no effect on estrogen receptors in hypothalamic-preoptic tissue, ATD-treated males had significantly higher levels of [3H]estrogen binding in 3 vocal control nuclei: the dorsomedial portion of the intercollicular nucleus (DM), the magnocellular nucleus of the anterior neostriatum (MAN), and Area X. Low levels of estrogen binding were also detected in cytosol from the caudal portions of the ventral hyperstriatum (HVc) and the robust nucleus of the archistriatum (RA) of both untreated and ATD-treated castrates. In most brain regions examined, estrogen receptor levels were lower than androgen receptor levels measured in previous experiments. The presence of both androgen- and estrogen-concentrating neurons in these areas provides compelling evidence for the interaction of androgens and estrogens in the neural control of male vocal behavior in this species.

The effects of an aromatization inhibitor on the reproductive behavior of male zebra finches.

Recent evidence indicates that aromatizable androgens are more effective than nonaromatizable androgens in restoring normal levels of sexual behavior in castrated male zebra finches (Poephila guttata). To determine whether the efficacy of treatment with aromatizable androgens, is in part due to their conversion to estrogens, castrated male finches were treated with androstenedione (AE), an aromatizable androgen, and their sexual and aggressive behavior was compared with that of castrates treated with AE plus 1,4,6-androstatriene-3,17-dione (ATD), an aromatization inhibitor. Males treated with AE + ATD showed less courtship activity and less copulatory behavior than AE-treated males, and were unlikely to have nests. Estradiol (E), when given concurrently with AE + ATD, reversed the inhibitory effects of ATD and restored levels of courtship and copulation to those observed in AE-treated males. Only AE- and AE + ATD + E-treated males displayed aggressive behaviors, but the frequency of such behaviors was so low that there were no significant differences across groups. These data affirm the importance of estrogen in the control of reproductive activities in male zebra finches and indicate that aromatization may be an obligatory step for maintaining normal levels of sexual and aggressive behavior.

Chemometric classification of reversed-phase high-performance liquid chromatography columns.

Cluster analysis and principal components analysis have been used to classify nine octadecyl (C18) high-performance liquid chromatography (HPLC) columns into three general groups displaying similar chromatographic behaviour. Principal components analysis was also able to identify the key test compounds on which the classification was most highly dependent. These identifications agreed with the classification and test compound selection by an HPLC specialist. In addition, the chemometric techniques can easily be extended to many more columns and test measurements than could be conveniently examined by a human expert.

Classification of octadecyl-bonded liquid chromatography columns.

The differences in performance characteristics exhibited by columns available under the same octadecyl (C-18) label cause difficulties for the authors and users of official methods. This paper presents a simple scheme for classifying C-18 columns on the basis of the 2 predominant reverse phase retention mechanisms-hydrophobic and silanophilic interactions, which are dependent on the hydrocarbon coverage and the unreacted silanol sites, respectively. Hydrophobic interactions are represented by the ratio of anthracene k:benzene k in an acetonitrile-water (65 + 35) mobile phase. The unreacted silanols are represented by the N,N-diethyltoluamide k:anthracene k ratio with acetonitrile as the mobile phase. Column efficiency, which is largely dependent on particle size and packing technique, is included as a third classification criterion. Twelve brands of C-18 packings were subjected to this test scheme and qualitatively classified into 3 major groups on the basis of 3 criteria:hydrophobicity index, free silanol index, and efficiency. This scheme is intended as an aid in the selection of similar or equivalent columns and in defining the type of column suitable for an analytical method.

Corporate quality assurance programs expanding.

The Sisters of Charity Health Care System (SCHCS) of Cincinnati, before expanding its quality assurance (QA) activities, conducted a survey of 29 Catholic multi-institutional systems to learn more about their QA functions. Of those surveyed, nine systems already had a corporate QA program and seven more were considering developing one. The existing programs have four general areas of responsibility: assisting member facilities in complying with Joint Commission on Accreditation of Hospitals standards, coordinating QA activities among facilities, facilitating development of a monitoring system, and providing consultation and education on quality issues. Seven of the nine systems with established QA programs had a single person in charge, generally someone with at least a master's degree and clinical, management, and actual QA experience. They usually were called "directors of quality assurance" and reported to vice-presidents. Their salary ranged from $49,000 to $65,000. Program budgets were an average of $88,809, and average staff size was 2.6 persons. The seven systems that had not yet established a QA program but were considering it had plans similar to existing programs. Based on these trends, SCHCS launched a corporate QA program in July 1986 by hiring a director of quality assurance.

Preperitoneal synthetic mesh placement for recurrent hernias of the groin.

In order to improve the results and avoid the testicular complications attending the standard repair of recurrent hernias of the groin, we have used the preperitoneal route to reduce the hernia and place a veil of Teflon to act as a new "fascia transversalis." This technique has been used in 50 such operations and the results have been presented herein.