Michelle Tucci

A preliminary study of CA15-3, c-erbB-2, epidermal growth factor receptor, cathepsin-D, and p53 in saliva among women with breast carcinoma.

Abstract A panel of markers used to aid in the diagnosis and treatment of breast cancer was examined in the saliva of a cohort of healthy women, women with benign lesions of the breast, and women with diagnosed breast cancer. We found recognized tumor markers c-erbB-2 (erb), cancer antigen 15-3 (CA15-3), and tumor suppressor oncogene protein 53 (p53) in the saliva of all three groups of women. The levels of erb and CA15-3 in the cancer patients evaluated, however, were significantly higher than the salivary levels of healthy control subjects and benign tumor patients. Conversely, pantropic p53 levels were higher in control subjects compared with those women with breast cancer and those with benign tumors. Although cathepsin-D and epidermal growth factor receptor were detected, they did not demonstrate any clear correlation with disease status. The results of the pilot suggest that these markers have potential use in initial detection and/or follow-up screening for the detection of breast cancer in women.

A preliminary report on the effects of sustained administration of corticosteroid on traumatized disc using the adult male rat model.

Study Design A novel degenerative disc disease model and sustained delivery method for corticosteroid in male Sprague-Dawley albino rats. Objectives To develop a model of degenerative disc disease and to determine the effect of continuous sustained release of corticosteroid on the process of degeneration within the traumatized disc. Summary of Background Data The current modalities of treating symptomatic degenerative disc disease are either conservative or surgical. However, there is no cure for the degenerative process and prevention, therefore, is the ideal treatment. An understanding of the mechanisms involved in disc degeneration is crucial to develop new methods for prevention and treatment, including appropriate delivery systems and dosages of repair factors. Methods The L5-L6 intervertebral disc was pierced with a 23-gauge needle in 18 rats. The animals received either sham or corticosterone-charged tricalcium phosphate ceramic capsules. The rats were euthanized at 4 weeks. Chondrocytes in the transition zone areas were counted and compared statistically. Results The surgical technique induced degeneration of the nucleus without evidence of inflammation at adjacent levels when compared with nontraumatized controls. The number of chondrocytes per area was significantly less in the sham group than in the control group. Corticosteroid treatment showed chondrocyte numbers similar to control in 4 of 5 different views of the disc. The anterior region of the disc had 50% less chondrocytes per area than the control; however, the chondrocyte numbers were 50% greater than in the same site from discs of sham animals. Conlcusions The results show the development of a degenerative disc animal model that can be used to test the effects of growth enhancing factors in disc repair. Administration of continuous sustained release of corticosterone can slow the process of degeneration within the traumatized disc in the rat model.

Comparison of NPY Y1 Receptors in the Spines of Ovariectomized and Ovary Intact Rats

Introduction Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that acts as a neurotransmitter in the brain and in the autonomic nervous system of humans. In the autonomic system it is produced mainly by neurons of the sympathetic nervous system and serves as a strong vasoconstrictor. Recently, NPY has been shown to increase during stress and in patients with chronic back pain. NPY has the potential to innervate 5 subtypes of receptors Y1-Y5. The Y1 receptor has been shown to play a role in cell survival and contribute to pain hypersensitivity. In addition, Y1 receptors are also thought to contribute to bone loss leading to disc degeneration in osteoporosis. The goal of our study was identify the location of the Y1 receptor in the disc of osteoporotic animals and to compare those to ovary intact control. Methods Eight OVX animals and eight ovary intact control animals were followed weekly for 8 weeks. Four animals in each group were sacrificed at 4 and 8 weeks and blood and spines were harvested. Spines were decalcified and processed for histological analysis. Routine hematoxylin and eosin staining along with immunhistochemical analysis of the spine Y1 receptors. Results The results showed the intervertebral discs in the control group appeared normal. The nucleus pulposus contained abundant notochordal cells, surrounded by large zones of extracellular matrix, and the cartilage endplates were hyaline cartilage composed of chondrocytes. In the OVX animals, the discs showed degenerative changes, where the nucleus pulposus appeared reduced in size and comprised relatively few, chondrocyte-like cells. Mucoid degeneration could also be seen. An increased number of small chondrocytes appeared in the inner layer of the annulus. Bony tissues that contained bone marrow, hematopoietic lineage cells and mineralized bone, became more obvious in the deep zone of middle cartilage endplate. Immunohistochemical analysis for Y1 receptors showed positive staining in the outer annulus in the control with defined staining of chondrocyte cells. Y1 receptor staining was diminished in the OVX animals. Higher levels of circulating NPY was found in the plasma of OVX animals and may contribute to internalization of the Y1 receptor as a compensatory mechanism. Conclusions Y1 receptors are located in the outer portion of the annulus of rats. The portion of cellular staining differed between the OVX and intact ovary control animals. More experiments are needed to clarify the role of NPY in the normal and osteoporotic spine.

Histopathologic Evaluation of the Substained Delivery of Ovarian Hormones and Neuropeptide Y Antagonist on the Body Weights and Vital Organs of Ovarectomized Rats

The route of administration of ovarian hormones and other biologicals represent a key factor in the treatment, prevention or eradication of different diseases in humans. Many studies have documented that the use of sustained delivery of biologicals is more effective compared to conventional route of administration. To date, the literature is lacking studies that conclusively demonstrate the change on body weights and vital organ morphology associated with sustained delivery of Neuropeptide Y antagonist and estrogen compared to intact and ovariectomized adults as a model. Therefore, the purpose of this study represents a histopathologic evaluation of the sustained delivery of ovarian hormones and neuropeptide y antagonist on the body weight and vital organs of ovariectomized rats. A total of 25 Sprague Dawley rats were obtained and divided into five groups, intact control with ovaries, sham, OVX + estrogen, and NPY antagonist. Animals in three of the five groups were surgically implanted with a TCP drug delivery device loaded with 1.6 mg of estrogen and 1.6 mg of NPY antagonist, and the sham animals were implanted with empty capsules. The capsules were surgically inserted into the muscle adjacent to the six lumbar vertebra. The animals were euthanized at two weeks, post-implantation phases. An analysis of the body weights were compared, and the OVX + estrogen and the OVX + NPY antagonist were similar to the intact control animals. Histopathologic evaluation of the major body organs revealed no histopathological change in the adrenals, spleen, liver, lungs or heart muscles of the intact, OVX, sham and NPY antagonist low and high dose treated animals. The only significant difference was observed in the kidney, where the glomeruli appeared much larger in the estrogen treated animals. Overall conclusion obtained from this study demonstrated that TCPL delivery can be used effectively to administer NPY at sustained level with remarkable reduction in side effects that associated with conventional means.

Subcutaneous Fibroblast Migration is Altered by Amino Acid Coated UHMW-PE Implants

The purpose of this investigation was to determine fibroblast behavior after implantation of ultra-high molecular weight polyethylene (UHMW-PE) rinsed with saline (control) or coated with poly-L-lysine (PLL), arginine-glycine-aspartic acid (RGD), or arginine-glycine-glutamic acid (RGE) into 16 adult male rats subcutaneously. At 90 days post-implantation, fibroblast counts were highest in the saline rinsed group (34±2 cells/HPF) and significantly reduced in RGD (19±10), RGE (2±3), and PLL (0) treated groups. These findings indicate fibroblast migration in surrounding fibrous tissue can be strongly influenced using various amino acid combination coatings in subcutaneous applications.

The Effect of Glucocorticoids and LPS on the Functional Activity of RAW Cell Line

It is well documented that glucocorticoids are potent anti-inflammatory and immunosuppressive agents that are known to affect cell mediated inflammation by the inhibition of cellular proliferation and cytokine production. The literature is lacking knowledge in elucidating the mode of action of such agents on the transformed like cells in culture. Therefore, RAW (macrophage like) cells was selected as a model to determine the effects of cortisol administration or cortisol in the presence of LPS on the cells metabolic functions. Cells were treated with physiological concentrations of cortisol or cortisol + LPS for periods of 24, 48 and 72 hours. After each phase, cell numbers, cellular damage and cellular morphology were determined. The results indicated cortisol and cortisol + LPS treated cells inhibited cellular proliferation as well as increased cellular MDA levels as early as 24 hours. Overall, the results indicate that cortisol has a remarkable effect on RAW cellular proliferation similar to the reduction seen in our previous findings using HEP-2 cells. In addition to reduction in cellular number the cells ability to adjust to a bacterial challenge may be directly altered. These results provided important information for patients who are immunosuppressed or chronically exposed to stressful conditions.

Prostatic Tissue Manipulation By Continous Delivery Of Estrogen/Dihydrotestoterone Uisng Ceramic Drug Delivery System

Tricalcium phosphate lysine drug delivery system (TCPL) has been used successfully to deliver various organic compounds at a sustained manner for long duration. Several studies conducted in our laboratories have used TCPL to treat various pathological disturbances. The objective of this study was to evaluate the effect of estrogen (E) and Dihydrotestosterone (DHT) delivered in a sustained manner by means of tricalcium phosphate-lysine (TCPL) delivery system on the morphological changes of ventral prostatic tissue. Adult male rats (BW 300350 gm, n = 24) were randomly divided into four equal groups: group I were implanted subcutaneously with TCPL loaded with estrogen (10 pq/ml), group I1 were implanted with TCPL loaded with DHT (2 ng/ml), group I11 were implanted with sham TCPL capsules and rats in group IV served as intact unimplanted controls. Surgical aseptic techniques were performed according to standard laboratory procedures. The animals were maintained at the University of Mississippi Medical Center Animal Facilities following the rules and regulations established by NIH on the Care and Use of Laboratory Animals. At the end of 8 weeks post implantation, all animals were sacrificed and the prostate tissues were collected, weighed and embedded for histopathological evaluations. Statistical analysis was conducted by using standard computer programs (STATVIEW). Data collected from this study have shown that exogenous intake of estrogen and DHT delivered in a sustained manner for eight weeks induced several pathophysiological conditions in ventral prostatic tissue in comparison to prostatic tissue collected from sham and control animals. Light microscopic evaluation of cross sections obtained from animals implanted with estrogen loaded TCPL revealed an atrophic pattem in the epithelium with loss of the cellular structure. In addition, 1 other observations such as low cuboidal and thin glands, pleomorphism, severe angiogenesis, presence of abundant connective tissue stroma and granulomatous conditions were detected. In contrast, cross section of ventral prostate tissue collected from animals implanted with TCPL filled with DHT (Group 11) revealed the following: (i) pleomorphism, (ii) occasional presence of connective tissue stroma, (iii) prostatic hypertrophy alone, or in conjunction with hyperplasia of the ,epithelial cell and (iv) involvement of inflammatory cells. In conclusion, the results of this study suggest that the use of TCPL capsules loaded with estrogen and androgens can be used effectively to regulate the growth of prostatic tissue.

Detection of nonspecific immunohistochemical response caused by tissue necrosis and the presence of metallic wear debris

Tissues retrieved during revision arthroplasty (n=25) were processed for routine immunohistochemical techniques to localize the presence of bone resorbing cytokines (IL-1 and IL-6), and to identify areas of significant necrosis and metallic deposition. Tissues were processed and embedded according to standard laboratory techniques. Several slides were prepared for each tissue sample taken. Several sequential slides were stained for Hematoxylin and Eosin to demonstrate cell type. Other slides of the same tissues were processed for localization of cytokines, IL-1 and IL-6. To identify specific cytokine reactions, the tissues were first blocked with proteins to ensure that all. The results revealed that there were several locations on the tissues that appeared highly reactive for cytokines IL-1 and IL-6. However, some of these areas turned out to be nonspecific when compared with negative controls and the Hematoxylin and Eosin stained slides. Areas of extreme necrosis or where metal debris was presence demonstrated sites of nonspecific reactivity. Areas of necrosis have a high nonspecific affinity for antibodies and these areas of necrosis can be demonstrated on the Hematoxylin and Eosin sections, and should be avoided in determining the amount of specific cellular reactivity. In addition, new immunohistochemical techniques use cobalt ions as enhancers of peroxidase response, in areas on slides labeled as negative controls where metallic debris was trapped, there was an increase in nonspecific positive reactivity. These factors must be carefully controlled in determining true cellular responses that causes resorption of bone and implant failure.

The effect of P-Trp, P-Ala and P-Val on the proliferation rate of HeLa cells in culture

The objective of this study was to investigate the effect of various biomedical polymers on the adhesion rate of HeLa cells as a model. The HeLa cells used in this study seeded by following our standard laboratory procedure. A total of 1/spl times/10/sup 5/ cells were plated in each of the pretreated wells with various concentrations of (0.01, 0.1, and 1% wt/vol) polyvaline (P-Val), polyalanine (p-Ala), polytryptophan (P-Trp) and buffered control. At the end of 1,4, and 24 hours the assay was developed by following standard lab procedure. The final determination was achieved by means of ELIZA. The data obtained from this study suggest that (i) the rate of HeLa spreading was strongly influenced by both incubation time and the polymer concentration, (ii) it is apparent that polymer treated wells within the first hour facilitated attachment of HeLa cells to their surfaces for all concentrations tested with the exception of 1% alanine. This observation confirms our previous results using different cell lines and provides a valuable information for experiments that need to be performed in a short period of time with the maximum amount of cells attached, (iii) the surface attachment of HeLa to P-Ala, P-Val, and P-Trp were demonstrated to vary depending on their chemical structure and level of microporosity. Thus, overall observation led us to conclude that the surface reactivity of P-Ala, P-Val, and P-Trp be always taken into account in discussing their biocompatibility using transformed cells such as HeLa cells as a model.