8:30 SEXUALLY TRANSMITTED DISEASES: STATEWIDE TRENDS AND INTERVENTION MODELS
Tyronda Reynolds*, Remonica Hightower*, Linda Williams*, and Carol Jones, Delta State University, Cleveland, MS 38733
Data from the Mississippi State Department of Health and the University of Mississippi Medical Center demonstrated that some sexually transmitted diseases (STD's) have recently shown disproportionate increases in rural Mississippi. Statistical trends for four STD's (AIDS, Chlamydia, Gonorrhea, and Syphilis) were presented. Means, correlations, and significance tests (p < .01) were used to make regional comparisons among the nine public health districts in the state. Recommended interventions for health professionals and educators were presented, based on a "consensus" statement (April 17, 1997) published by the National Institutes of Health.
8:45 A COMPARISON OF CA50 WITH CA195, CA125, CAl9-9, CA72-4, CYFRA 21-1, AND CEA FOR THB SERODIAGNOSIS OF GASTROINTESTINAL CANCERS
Jiarong Ying1*, Paul A. Sykes1*, Margaret Jackson2, James T. Johnson1, Harold Schultze3, and Margot Hall1, 1University of Southern Mississippi, Hattiesburg, MS 39406, 2Wright Patterson AFB, OH 45433, and 3Puckett Laboratory, Hattiesburg, MS 39402
Gastrointestinal cancers (GI CA) represent a serious health hazard in the United States. Many diagnostic methods exist but often the diagnosis is made in late stage disease when the prognosis is poor. A noninvasive early detection method would be most beneficial. In this study, sera from 554 patients (16 pancreatic CA, 101 colorectal CA, 27 other GI CA, 216 other CA, and 194 non CA) were assayed in a random blind manner for the presence of tumor antigens and the results correlated with diagnoses established pathologically. We used immunoassay test kits from CIS Bio International (CA50), Hybritech, Inc (CA195, CEA), and Centocor, Inc (CA125, CA19-9, CA72 -4, CYFRA 21-1) to test for the concentration of these antigens. The diagnostic efficiencies for combined GI cancers were: CA50 (72.6%), CA195 (73.1%), CA19-9 (75.6%), CA125 (72.1%), CEA (69.6%), CA72-4 (74.2%), and CYFRA 21-1 (74.6%). The diagnostic sensitivity of CA50 for pancreatic CA was comparable to CA19-9 (66.7%) and superior to the other markers with the exception of CA195 (100%). We conclude that CA50 has potential as a marker of pancreatic and other GI cancers.
9:00 LUMPED PARAMETER MODELING OF THE MOULDER ENABLING CARDIOVASCULAR DEVICE IN THE CANINE CIRCULATORY SYSTEM
Asim Haque1*, R. Wayne Barbee2, Robert Zone1, David Sailor1, and P. Michael Lynch1, 1Tulane University, New Orleans, LA 70118, and 2Carolinas Medical Center, Charlotte, NC 28203
Heart failure is a leading cause of hospitalization in America. Unfortunately, many patients' conditions deteriorate, requiring an organ transplant or some other form of cardiac assistance. The Moulder Enabling Cardiovascular Device (MECAD) was developed to serve as a viable option for transplant patients. Unlike other ventricular assist devices (VADs), the MECAD is placed downstream of the aortic arch, just distal to the left subclavian artery, to reduce the chance of embolism to the head, heart, or arms. A lumped parameter model of the canine circulatory system was adapted and tested to contain both the shunt and the MECAD pump paths. The MECAD pump was approximated using a DC current source with a characteristic internal resistance. With no measurements of the ascending aortic pressure or flow available, aortic pressure was derived from the distal brachiocephalic artery pressure using various circuit analysis techniques, which inherently produced some error. Control runs showed good similarities with preliminary experimental data from anesthetized canines. Comparison of model results to the shunt and MECAD pump runs were favorable using lowered peripheral resistance values. Due to a pressure decrease downstream with the addition of the MECAD, the peripheral resistance in the lower body decreases accordingly as a result of autoregulation in the periphery to maintain constant flow.
9:15 SYSTEMIC ARTERIAL PRESSURE (AP) RESPONSES TO CHRONIC VERTEBRAL ARTERY (VA) ANGIOTENSIN II (ANGII) INFUSION DURING ADRENERGIC BLOCKADE (AB)
David Kirk* and Drew Hildebrandt, University of Mississippi Medical Center, Jackson, MS 39216
One of ANGII's sites of action is thought to be the central nervous system, in that ANGII moving from the circulation into the brain helps control AP. We have shown that chronic VA ANGII increases AP for the duration of the infusion. The mechanism of this increased AP is unknown, but could be the result of increased sympathetic (SNS) activity. This study was designed to examine the effect of VA ANGII infusion with and without AB in conscious, chronically-instrumented dogs (n=7). When ANGII was infused into the VA of normal dogs at 0.5 ng/kg/min, AP increased 13+3 mmHg on day (D) 1, averaging 15+3 mmHg above control for the 7D infusion period. When ANGII was infused into dogs in which alpha- and beta-AB was achieved by prazocin and propranolol administration, AP increased 5+2 mmHg on D1, and 11 mmHg by D7, not different from the ultimate rise in pressure in normal dogs. The pattern of changes in AB dogs is similar to when ANGII is infused i.v., suggesting the chronic AP changes resulted from spillover from the cerebral into the peripheral circulation, not increased SNS activity. Thus, ANGII's central actions may play a role in the acute response to pathophysiological increases in ANGII, but are of little importance in the chronic physiological response to ANGII.
9:30 CHRONIC HOMOCYSTEINE ELEVATION AS A CAUSE LEADING TO CARDIAC FIBROSIS AND CONTRACTILE DYSFUNCTION IN HYPERTENSION
Lane M. Smiley*, Vibhas S. Mujumdar, and Suresh C. Tyagi, University of Mississippi Medical Center, Jackson, MS 39216
Hypertensive heart disease is one of the leading causes of cardiovascular related morbidity and mortality. Elevated plasma levels of homocysteine, hyperhomocysteinemia, H(e), has emerged as an independent risk factor for hypertensive heart disease. The cardiovascular hypertrophy and fibrosis lead to heart disease. The mechanisms by which H(e) induces cardiovascular functional abnormalities are largely unknown. We hypothesized that chronic homocysteine elevation leads to cardiac fibrosis and contractile dysfunction in hypertension. To test this hypothesis we prepared cardiac rings from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar Kyoto (WKY) rats. Thirty-two weeks old male SHR and WKY rats (Charles Rever Laboratories) were employed in this study. At this age SHR developed significant LV hypertrophy. However, WKY are normal. The levels of H(e) were found to be elevated in SHR (9.8 ± 0.8 µM) compared to WKY (4.6 ± 0.01 µM). The ventricular extracellular matrix (ECM) collagen was estimated by biochemical methods. The contractility of left and right ventricular rings in response to homocysteine from SHR and WKY rats was measured by isometric tension myobath. The results suggested that in LV endocardial rings the homocysteine (100 µM) generated 2 grams tension per gram of tissue (g/g) in SHR as compared to 3 g/g in WKY. In RV endocardial rings the homocysteine (100 µM ) generated 0.6 g/g in SHR as compared to 3.4 g/g in WKY. The cardiac collagen in SHR was 0.075 µg/µl and in WKY was 0.035 µg/µl. These results suggested: (1) there was increased collagen expression in SHR as compared to WKY; (2) homocysteine is a cardioactive agent and induces contraction in normal heart; (3) in chronic hypertension, homocysteine, may be partly responsible for deterioration of cardiac contractile function; and (4) cardiac fibrosis (i.e., collagen) may in part be responsible for reduced cardiac contractile function observed in SHR as compared to their normotensive WKY.
9:45 DETECTION OF THE ESTRUS CYCLE BY MEANS OF PAP STAIN IN ADULT FEMALE RATS
Zelma Cason*, Kenneth Butler, Michelle Tucci, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
It is well documented that the consistency and reliability in staining are the cornerstones of cytological interpretation. Subtle cell appearances are constantly being compared and assessed during screening, and cytologists rely heavily on the quality and appearance of the stain. In the research setting, estrus cycle (4 days) in rodents is normally detected by means of using several stains such as hematoxylin and eosin (H&E) and Giemsa. The specific objective of this study is to evaluate the morphological changes associated with the estrus cycle by means of Papanicolaou stain (PAP) method and compare that with existing methods used in investigational purposes (H&E). Adult Sprague Dawley female rats were randomly divided into three groups (n=3). Group I animals were left as our intact controls, group II were implanted with TCPL loaded with estrogen (8 pg/ml), and group III animals were implanted with TCPL loaded with progestrone and estrogen. The data obtained from this study demonstrate the following: (1) the use of PAP stain retain the transparent quality of the cytoplasm, (2) nuclear chromatin was easily distinguished, (3) unlike the use of conventional staining procedures, the use of PAP stain have shown to be decisive and can give very consistent results.
10:00 Break
10:15 TUMOR NECROSIS FACTOR STIMULATES THE PRODUCTION OF INTERLEUKIN 8 AND MONOCYTE CHEMOATTRACTANT PROTEIN-1 IN CULTURED RABBIT SYNOVIAL AND MACROPHAGE CELLS
Michelle Tucci*, James Hughes, and Rodney Baker, University of Mississippi Medical Center, Jackson, MS 39216
Rheumatoid arthritis is an autoimmune disease that causes inflammation
mainly in synovial tissues. The inflammatory sites are characterized by
infiltration of activated lymphocytes and macrophages into the synovial
membrane, and the proliferation of synovial cells. The local production
of a number of cytokines by proliferative synovial cells as well as by
infiltrating cells appears to account for many of the pathological and
clinical manifestations in rheumatoid arthritis. Among the cytokines secreted
by infiltrating macrophages, tumor necrosis factor seems to be important
in the development of local inflammation and tissue damage. Tumor necrosis
factor added to either rabbit synovial cells or peripheral macrophages
increased the levels of Monocyte chemotatic protein -1 (MCP-1) and IL-8
in a dose dependent fashion. IL-8 induces the infiltration of leukocytes
and MCP-1 promotes monocyte/macrophage infiltration and continued activation
to sustain the inflammatory response. The release of IL-8 and MCP-1 from
cells occurred after 10 hours and was measurable for 35 to 40 hours after
stimulation for IL-8 and MCP-1, respectively. The data suggest that tumor
necrosis factor induces the expression of pro-inflammatory cytokines in
both rabbit synovial and peripheral macrophage cells indicating that both
cell types may be responsible for the pathological changes observed in
rheumatoid arthritis.
10:30 HISTOPATHOLOGICAL EVALUATIONS OF PROSTATIC TISSUES EXPOSED TO LONG TERM DELIVERY OF ESTROGEN AND ANDROGEN USING ADULT RATS
Willie Cavett*, Zelma Cason, Michelle Tucci, Aaron Puckett, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
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 morphological changes of prostatic tissue. Adult male rats were randomly divided into four equal groups: group I were implanted subcutaneously with TCPL loaded with estrogen (10 pq/ml/day), group II were implanted with TCPL loaded with DHT (2 ng/ml/day), group III 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. At the end of 8 weeks post implantation, all animals were sacrificed and the prostate tissues were collected, weighed and embedded for histopathological evaluations. 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. Light microscopic evaluation of the animals implanted with estrogen loaded TCPL revealed an atrophic pattern in the epithelium with loss of the cellular structure. In addition, other observations such as low cuboidal and thin glands, pleomorphism, severe angiogenesis, presence of abundant connective tissue stroma and granulomatous conditions were also detected. In contrast, ventral prostate collected from animals implanted with TCPL filled with DHT (Group II) 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.
10:45 EFFECT OF TURMERIN ON ENDOTHELIAL DENUDATION BY AIR DRYING
Hari H.P. Cohly, C. Hammett, Suman Das, Michael F. Angel, Vijaya Kanji, Annelle Taylor*, Hamad Benghuzzi, and Angel K. Markov, University of Mississippi Medical Center, Jackson, MS, 39216-4505
The objective of this study is to determine if arterial endothelial injury can be attenuated by local application of 80 ng/ml turmerin or by oral administration of the same dose. Anesthetized Lewis rats (n =12) weighing 200 gms randomly were assigned to two groups. After 5 min of air drying a segment of right carotid artery 6 rats were treated locally 80 µg/ml with turmerin and the rest were treated with 0.9% NaCl. Thereafter turmerin was administered by gavage (80 µg) every 24 hrs for 14 days. Animals were sacrificed on day 14 and carotid artery removed from the injured and the non-injured site was removed for histological analysis and serum collected for lipid peroxidation analysis by measuring malondialdehyde (MDA) and conjugated dienes. We showed in one rat no proliferation in the intma while there was significant variation in controls. MDA for control was .5925 ± .02 nanomoles/ml while turmerin was .187± .04; conjugated diene for control was .402 ± .03 nanomoles/ml while turmerin was .212 ± .04 nanomole/ml (p<0.05). Although there was significant reduction in serum peroxidation activity the histological findings indicate that attenuation of carotid artery injury may involve other factors than decreased lipid peroxidation.
11:00 EFFECT OF TURMERIN AND AZT ON MITOGEN STIMULATED HIV-INFECTED HUMAN MONONUCLEAR CELLS
Hari H.P. Cohly*, Sabah Asad, Suman Das, Michael F. Angel, Rajeswara Rao, and Stan Reed, University of Mississippi Medical Center, Jackson, MS, 39216-4505, and University of Toronto, Toronto, M5G 1X8 Canada
It has been suggested that plant antioxidants may offer protection from viral replication and cell death associated with oxidative stress in patients with HIV/AIDS (Immunology Today 15:209-213, 1994). Pilot studies showed that turmerin, a water soluble portion of turmeric, an antioxidant, inhibits human CEM-T cell-line infected with HIV. To further pursue these observations HIV-infected human mononuclear cells were tested for responsiveness to phytohemagglutinin, concanavalin A and pokeweed mitogen in conjunction with AZT or turmerin. Human mononuclear cells from AIDS patients were isolated by ficoll-hypaque and incubated with 0.5 µg/ml and 0.05 µg/ml of turmerin or 5 µM AZT in conjunction with Concanavalin A at 10 µg/ml, pokeweed mitogen at 10 µg/ml and phytohemagglutinin at 10 ng/ml for 72 hrs at 37°C and pulsed with 3H thymidine for 18 hrs and then harvested using a scintillation counter. Human mononuclear cells from 6 patients showed that turmerin inhibits the responsiveness of all the mitogens tested in a dose response fashion and that turmerin at 0.5 µg/ml concentration inhibits mitogen responsiveness better than AZT at 5 µM. Thus, turmerin inhibits HIV-infected mononuclear cells from proliferation.
11:15 EFFECT OF ANTIOXIDANTS (TURMERIC, TURMERIN AND CURCUMIN) ON HUMAN IMMUNODEFICIENCY VIRUS
Hari H.P. Cohly*, Sabah Asad, Suman Das, Michael F. Angel, Rajeswara Rao, and Stan Reed, University of Mississippi Medical Center, Jackson, MS, 39216-4505, and University of Toronto, Toronto, M5G 1X8 Canada
Turmeric and its components were tested for anti-HIV activity. The responsiveness of T cells infected with HIV IIIB strain was tested with turmeric (T), turmerin (Tm) and curcumin (Cu) in the absence and presence of AZT for p-24 antigen release. CEM human T cell line was infected with HIV IIIB strain at 37°C for 2 h and non adsorbed virus was then removed by washing. Infected cells were then aliquoted out at 1 x105 cells per well. Various concentrations of turmeric (0.1 mg/ml-0.01 mg/ml), turmerin (0.5 µg/ml-0.05 µg/ml), curcumin (0.1 mg/ml-0.01 mg/ml) were incubated in the presence and absence of AZT (5 µM) for 7 days at 37°C with 5% CO2. p-24 release and cell viability was measured at day 3 and day 7. Our results showed turmeric and curcumin does not reduce p-24 antigen release from the infected cell while adding 5 µM of AZT showed a dramatic change in the amount of p-24 antigen. Turmerin in combination with AZT at 0.5 µg/ml and 0.05 µg/ml concentration cause a noticeable reduction in p-24 antigen release but most importantly cause a significant increase in cell viability. Thus, turmerin in combination with AZT provides better protection by decreasing the toxicity of AZT.
11:30 PROTECTIVE EFFECT OF FRUCTOSE-1,6-DIPHOSPHATE IN HYPOGLYCEMIC COMA IN RABBITS
Lorenzo A. Farias2, James W. Stephens1*, Russell Young1, P.H. Mantellini2, and Angel K. Markov1, 1University of Mississippi Medical Center, Jackson, MS 39216, and 2Institute for Experimental Surgery, Central University of Venezuela, Caracas, VZ
Brain injury due to hypoglycemia is of paramount importance in the treatment of diabetes. Since fructose-1,6-diphosphate (FDP) has been shown to be neuroprotective in hypoglycemic coma, we investigated if it will afford brain protection when given 10 min after isoelectric EEG (ISO-EEG). In anesthetized rabbits (n=12), hypoglycemic coma was induced and maintained with insulin. Ten min after ISO-EEG, randomly 6 rabbits received slow IV bolus of 500 mg/Kg 10% FDP followed by an infusion of 10 mg/Kg/min for 30 min, while the remaining rabbits received 0.9% NaCl in the same manner. After 30 min of ISO-EEG, hypoglycemia was terminated with glucose 75 mg/Kg (50%) IV. All rabbits survived the 6 h post-hypoglycemic observation period. Treated rabbits recovered partial EEG activity during FDP infusion and the EEG became normal after glucose. No EEG activity was noted during 0.9% NaCl infusion,and after glucose those were sporadic. Arterial pressure and blood gases, body temperature and blood glucose were not different between the groups, however arterial pH was lower in the FDP group during hypoglycemia (p<0.05). Neurologic recovery (neurologic deficit score, totally abnormal = 100, normal = 0) was better in the FDP group. The results indicate FDP is a neuroprotective agent.
| GLUC(mg/dl) | PaO2(mmHg) | Neuro. Def. Score | ||||
| S | F | S | F | S | F | |
| ISO-EEG | 20.7±1.3 | 21±1.3 | 186±13 | 176±8 | ||
| 30 min
Iso-EEG |
20±1.1 | 20±1.4 | 183±6 | 183±10 | ||
| 2 h
post-gluc |
156±15 | 245±16 | 115±3 | 121±4 | 34.5±8.24 | 13±3.58* |
| 4 h post | 165±39 | 203±12 | 110±2 | 117±7 | 30.4±6.58 | 6±2.17** |
| 6 h post | 165±29 | 124±11 | 105±3 | 102±8 | 34±7.22 | 6.4±2.25+ |
| *p<0.04,**p<0.007, +p<0.005 | ||||||
2:00 Divisional Business Meeting and
Douglas-Walker Award Presentation
2:15 Divisional Poster Session
FATAL PULMONARY BLASTOMYCOSIS AND ADULT RESPIRATORY DISTRESS SYNDROME
James A. Grantham*, Leigh Jones, and Luciano Lemos, University of Mississippi Medical Center, Jackson, MS 39216
A 51-year-old immunocompetent black male was admitted to UMMC with the initial diagnosis of upper lobe pneumonia and started on erythromycin for possible community-acquired pneumonia. Direct examination of the sputum revealed Blastomyces dermatidis and was later confirmed by culture. Amphotericin B was initiated as treatment. Five days after admission, the patient died with a terminal clinical picture of Adult Respiratory Distress Syndrome (ARDS). Blastomycosis is a poorly understood fungal disease that is more prominent in Mississippi than any other state. There are many obscure areas of the disease such as epidemiology, immunology of the etiologic agent, and ecology of the fungus. Blastomycosis classically carries a chronic progressive clinical course. Anti-fungal agents have been successful treating the disease, however ARDS secondary to blastomycosis is a serious life threatening condition. Among 106 patients with blastomycosis reviewed within the past seventeen years, this is the only case of ARDS occurring at UMMC.
EVALUATION OF THREE SERUM IRON BINDING CAPACITY METHODS IN IRON OVERDOSE
Phillip T. Smith*, Will John Martin, and William L. Roberts University of Mississippi Medical Center, Jackson, MS 39216
Serum iron binding capacity measurements are used to diagnose iron deficiency and overload conditions. One method that uses magnesium carbonate to measure serum total iron binding capacity (TIBC) overestimates TIBC in acute iron overdose. It is important to accurately determine serum iron and TIBC since patients whose iron exceeds TIBC are candidates for deferoxamine therapy. Three methods for measuring IBC were evaluated using a serum pool spiked with increasing concentrations of ferric chloride to simulate iron overload. Iron and TIBC measurements were made using Vitros 250 and aca Star analyzers and unbound IBC (UIBC) measurements were made using Boehringer Mannheim reagents and instrumentation. The two TIBC methods first saturate all iron binding sites with excess Fe+3, remove unbound iron with an alumina column, and finally measure protein bound iron. The UIBC method adds excess Fe+3 to saturate iron binding sites, reduces unbound Fe+3 to Fe+2, and measures Fe+2. The UIBC method gave constant UIBC values as expected until the added iron exceeded 1000 µg/dL at which point the UIBC began to increase. Both TIBC methods showed linear increases in TIBC with added iron (TIBC = 0.94 x Fe + 163). We conclude that the alumina column methods are inaccurate for TIBC measurement in overdose situations. The UIBC method is satisfactory until the serum iron concentration exceeds 1000 µg/dL.
THE EFFECT OF ATTENUATED TRYPANASOMA MUSCULI ON IMMUNOGLOBULIN G ANTIBODY LEVELS OF INBRED MICE
Jeremy Allen*, Vicki Jenson, Barrett Jones, Matthew Jones, Jeniffer McMurry, Melissa Morris, Trey Polk, and Jason Spellings, Belhaven College, Jackson, MS 39202
Trypanosoma musculi is a blood parasite infective only to mice. Parasites appear in the blood approximately four days after initial infection and reach a plateau on about day eight. This plateau phase lasts about two weeks. It is thought to be maintained by a dynamic equilibrium between the host's immune killing of the parasite and the trypanosome's ability to evade the immune system and reproduce. It has been postulated that a trypanocidal IgG antibody, ablastin, prevents the dividing forms from being viable in the blood. At day twelve after inoculation an immune response is first detected. Prior to day twelve, little immunity to the parasite can be detected except for the presence of antibodies. At the end of three weeks, the host immune system is able to destroy all trypanosomes. In this study we introduced an attenuated strain of T. musculi into mice and monitored the IgG levels to see if there was an increase of IgG to the infective parasites. We also monitored the levels of T. musculi in the blood to detect if there was a connection between antibody levels and parasite infection rate. The results of the study indicated that as the infection of T. musculi increased IgG antibody levels in the mouse blood also increased.
EWING'S SARCOMA OF THE BONE DETECTED BY FINE NEEDLE ASPIRATION
Shauna Crouse*, Zelma Cason, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
Ewing's sarcoma is an aggressive, malignant neoplasm that occurs in the vertebrae, ribs and long bones of the lower extremities. According to literature, this tumor has a male predilection within the second and third decades of life. In his study, we report a unique case showing the prevalence of this rare pathological disturbance among older individuals. In this study, we report a fine needle aspiration from a 42 year-old white male with existing Paget's disease and a primary Ewing's sarcoma of the right hip metastatic to the rib and spine. The histologic, cytologic, special and immunohistochemical stains, electron microscopic features and therapeutic protocol were discussed. A review of the one such case previously reported in the literature is presented. The fine needle aspiration displayed an abundance of double-celled population with noncohesive morphology. In addition, large light cells with round to oval nuclei and small cells with hyperchromatic nuclei with scant, vacuolated cytoplasm were observed. Intracytoplasmic glycogen contents were detected by the use of periodic acid-Shiff stain. This confirms the characteristic of Ewing's Sarcoma.
THE EFFECT OF SUSTAINED DELIVERY OF REPRODUCTIVE HORMONES ON THE ADRENAL GLANDS USING ADULT MALE RATS AS A MODEL
Kenneth Butler*, Hamed A. Benghuzzi, Michelle Tucci, Aaron Puckett, and Zelma Cason, University of Mississippi Medical Center, Jackson, MS 39216
The effectiveness of sustained delivery of reproductive hormones is often determined by the morphological and chemical changes observed in target organs. In this investigation, porous implants of tricalcium phosphate-lysine (TCPL), loaded with estrogen and testosterone, were implanted into the peritoneal cavity of adult male rats. The specific objective of this experiment is to determine the morphological and immunohistochemical changes that are suspected to occur when the aforementioned loaded TCPL ceramics are introduced into the peritoneal cavity. The adrenal glands were harvested, processed in histology, and submitted for microscopic evaluation. Evaluation of routine and specially stained sections (5 µm, H&E, IL-1, IL-6, TNF) of the adrenal gland revealed several significant findings. These include the following: (1) there was a significant change in the architecture and function of the adrenal glands of animals treated with estrogen and testosterone, (2) immunohistochemical staining was negative for both the experimental and control groups, (3) there is a remarkable reduction in the overall adrenal mass, and (4) the regression of the three zones in cortical tissue was found to be consistent in all animal treated with testosterone or estrogen. In conclusion, this study suggests that the application of exogenous testosterone and estrogen through a sustained delivery device cause significant changes in the adrenal gland. The atrophy in adrenal tissue could be a major factor in suppressing the endogenous levels of glucocorticoids and mineral-corticoids. The mechanism behind this phenomenon is unknown and is the focus of further investigation.
GROWTH HORMONE ENHANCES THE PRODUCTION OF IL-1 AND IL-6 FROM HL-60 CELLS
Quanikka Allen*, Yanika Daniels, Michelle Tucci, Leon Anderson, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
Growth Hormone (GH) is well known to increase the proliferation activity of several cell types, such as, cells in the gastrointestinal tract, cartilage progenitor cells, and hepatoma cells to name a few. In this study growth hormone was administered to (1 x 105 Cells/Well) HL-60 cells at concentrations of 0, 0.14, 1.4, and 2.10 µg/ml. Hl-60 are monocytic cells that are capable of secreting factors such as cytokines and growth factors. Morphological evaluation was conducted throughout the experiment. A total of 100 µl aliquot of supernatant were removed from the cells at 24 and 48 hours. Biochemical analysis was performed by measuring markers for cell damage (Lactate dehydrogenase (LDH)), and cytokine production. The results show that after 48 hours of GH administration cell proliferation increased in a dose dependent fashion. Cell number in the presence of GH at concentrations of 0.14, 1.4 and 2.10 ng/ml were increased by 3.2, 5.6, and 6.2 fold, respectively. Microscopic evaluation revealed that there was no significant damage in treated cells. This was confirmed by LDH activity levels (p<0.05). Regardless of GH concentration, application of GH induced the production of both IL-1 and IL-6. The data suggest that GH can: (1) stimulate proliferation rate of HL-60 cells in culture, and (2) GH induces the cells to secrete inflammatory cytokines such as IL-1 and IL-6 without causing cellular disturbances.
PHYSIOLOGICAL RESPONSE ASSOCIATED WITH TARGETED DELIVERY OF ANDROGENIC HORMONE USING TRAUMATIZED RAT FEMURS AS A MODEL
Stephanie Burks*, Michelle Tucci, Audrey K. Tsao, James Hughes, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
Previous results from our laboratories have suggested that the porous architecture of the TCPL ceramic materials with interconnecting apertures allows and directs osseous ingrowth. The objective of this experiment was to characterize TCPL as a ceramic carrier that can release anabolic steroid at the site of trauma to augment bone healing, and to determine the cellular response associated with this delivery system. Thirty-two male Sprague-Dawley rats (250-300 g) were randomly divided into four equal groups. Group I rats served as intact control, group II and III animals had surgical defects in the right femur and were treated with TCPL ceramics alone or TCPL ceramic carrier system containing testosterone (5 mg) and Gentamicin, respectively. Group IV rats had unmanaged surgical defects. Bone densities of all animals were determined at the end of 1, 2, 3, and 6 weeks. Bone densities increased at faster rates (p < 0.05 ) in rats treated with TCPL ceramics containing androgen. Immunochemical analysis of bone tissue revealed that animals treated with testosterone had higher numbers of cells expressing interleukin-1 (>53%), in comparison with animals treated with TCPL-ceramic-carrier (<15%) or unmanaged defects (<2%). The data obtained suggests that presence of the androgen receptors on the bone surface, allows for ample amounts of testosterone binding which may induce the synthesis of IL-1 and can contribute to the early healing. This information is considered critical for understanding the mechanisms that are associated with bone healing.
THE ROLE OF REPRODUCTIVE HORMONES ON THE PROLIFERATION RATE OF HL60 CELLS AND THE SECRETION OF CYTOKINES
Yanika Daniels*, Quannika Allen, Michelle Tucci, Leon Anderson, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
The specific objective of this study was to investigate the effect of Testosterone (T) and Estradiol (E) on the viability, proliferation rate, and expression of cytokines in HL60 cells. The cells utilized in this experiment were obtained from the American Type Culture Collection, and were placed in culture at a density of 0.5 X 106 cells per ml. The cells were treated with various concentrations of the steroid ranging from 2 pg/ml to 20 ng/ml media. The cells were maintained in a sterile environment and cell viability, proliferation rates, and production of cytokines IL-1 and IL-6 were determined at 24 and 48 hours using standard laboratory protocols. The results of this study showed that the cells treated with E had the highest cell proliferation rate ranged from 24 to 65% increase compared to the control untreated wells. This trend was found to be a dose dependent (726% increase). Estradiol and Testosterone at all concentrations stimulated the production of cytokines IL-1 and IL-6. LDH measurement revealed that there was no evidence of cellular damage. The mechanism of action of T and E on HL60 cells has to be further investigated at the molecular and biochemical levels. Data obtained from this investigation suggest that HL60 cells are selectively responsive to steroid hormones and such a response was found to be dose dependent.
LOCALIZATION OF CYTOKINES ON EPIDIDYMAL TISSUE EXPOSED TO SUSTAINED DELIVERY OF ANDROGENS IN ADULT RATS
Alaina Coleman*, Michelle Tucci, Zelma Cason, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
Recent studies reported from our laboratory have established that the sustained delivery of danazol in combination with androgens resulted in the remarkable reduction of epididymal mass. The specific objective of this investigation is to evaluate the cytological as well as immunohistochemical characteristics of epididymal tissues exposed to sustained delivery of Testosterone (T) and Estrogen (E) by means of TCPL delivery system. Adult Sprague-Dawley intact male rats were used for this study (n=40). Surgical aseptic techniques were performed using our standard protocol. Histopathological evaluations were conducted on collected epididymal tissues at the end of one, three and six months post-implantation of TCPL implants. The data obtained in this investigation demonstrated the following: (1) remarkable reduction in sperm counts and motility obtained from epididymal tubules in all experimental groups, (2) the lumen of the epididymal tubules were devoid of sperm in animals treated with T at the end of six month phase, (3) a decrease in the diameter of tubules with occasional hypertrophic epithelium in all experimental animals, (4) immunohistopathological evaluation has shown a stimulation of interleukin-6 producing cells in animals treated with T during the entire investigation. In contrast, a negative response for interleukin-1 and tumor necrosis factor (TNF) in all experimental and treated epididymal tissues was observed. The IL-6 positive response could be attributed to the proliferative behavior of this cytokine in comparison to the inflammatory response of IL-1 and TNF.
COMPARISON OF CELL TYPES AND CYTOKINES PRODUCED IN THE SYNOVIAL LINING OF RHEUMATOID ARTHRITIS PATIENTS UNDERGOING JOINT ARTHROPLASTY
Shannon Morgan1*, Dianne Cooper1, Monica Lemos1, Adel Mohamed2, Michelle Tucci1, Hamed A. Benghuzzi1, and Audrey K. Tsao1, 1University of Mississippi Medical Center, Jackson, MS 39216, and 2University of Saskatoon, Saskatchewan, Canada
Rheumatoid arthritis (RA) is an immune-mediated disease manifesting as chronic polyarthritis with intermittent acute inflammatory episodes. The target tissue in RA is synovial lining of articular joints, with synovial proliferation and damage to articular cartilage and bone. These permanent changes cause joint deformity and functional losses. The proliferative synovium contains fibroblast-like cells, macrophages, dendritic morphology cells, mast cells, B, and T lymphocytes. Synovial cells are capable of secreting cytokines, leading to acute inflammation (IL-1, IL-6, IL-8, and TNF), and chronic inflammation (IL-2, IL-4, and IL-10). This study looks at tissue from twelve RA patients undergoing primary joint arthroplasty. These tissues were assayed, both quantitatively by ELISA and qualitatively by immunohistochemical analysis, for the factors of acute and chronic inflammation to show which are most prevalent. Results showed that macrophages deriving acute inflammation cytokines (IL-1, IL-6 and IL-8) were easily detected at levels of 60 pg/mg protein + 8 pg/mg protein; 115 pg/mg protein + 21 pg/mg protein, and 87 pg/mg protein + 17 pg/mg protein; respectively. T-cells deriving chronic inflammation cytokines (IL-2, IL-4 and IL-10) were rarely detected. This suggests that induction of cytokines in a cascade like manner can account for amplification of acute inflammation, but the initial stimulus for cytokine release in RA, is unknown.
BIOCHEMICAL ANALYSIS ASSOCIATED WITH HL-60 CELLS INCUBATED WITH VARIOUS CERAMIC MATERIALS
Leigh Jones*, Michelle Tucci, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
The purpose of this experiment was to determine the effect of TCP, ALCAP, and HA ceramic particles on the viability and proliferation rate of HL-60 cells in culture. Previous experiments have suggested that growing cell line such as macrophages, fibroblasts, and endothelial cells on discs made of TCP, ALCAP, and HA induced immunochemical and morphological changes. The effect of ceramic particles (<38 µm) on HL-60 cells in culture have never been investigated. A total of 1 x 105 (n=5) cells were placed in culture with 2 mg of ALCAP, TCP, or HA particles. At the end of 24, 48, and 96 hours, the cells were evaluated morphologically and then counted. The supernatants were collected and biochemical analysis for LDH and ALP was performed. The data of this investigation revealed the following: (1) At the end of the 24 hours, the cell number in wells containing HA and ALCAP particles were statistically lower (p<.05) than the control cells, whereas cells in the presence of TCP were similar to the control. (2) Microscopic evaluation of cells treated with HA showed hydropic change at 24, 48, and 96 hours. (3) At 96 hours, there were no significant changes in cell number between the groups. This phenomenon could be attributed to the depletion of nutrients within the wells or possibly the cells reaching confluence. (4) Biochemical analysis of markers for cellular disturbances revealed that there were no significant differences between the different group at any time point. The mechanism of action of HL-60 cells toward the ceramic material at a specific particle size is unknown, and further investigation is recommended.
INTERACTION OF CELLS WITH UHMWPE IMPREGNATED WITH THE BIOACTIVE PEPTIDES RGD, RGE OR POLY-L-LYSINE
Donnis Harrison*, Richard Johnson, Michelle Tucci, Aaron Puckett, and Hamed A. Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
Several reports have suggested that encapsulation of orthopaedic and dental implants with fibrous tissue can lead to implant failure. The binding of cells to the surface of the implants is to specific amino-acid sequences, typically RGD. The specific objective of this study was to investigate the interaction of cultured human peripheral macrophages with specific amino-acid sequences to determine if adherence is due to the specificity of the sequence. Macrophages were seeded at a density of 1x105 cells on ultra high molecular weight proteins (UHMWPE) coated with either amino-acid heteropolymers 1x10-6 M RGE, 1x10-9 M RGD, or amino-acid homopolymer 2.5% Poly-L-Lysine. Cells were observed daily and morphology was recorded. The results showed that cells growing in the presence of RGD had significantly (p<0.05) higher numbers of cells adhering and remaining viable, in comparison to cells growing on Poly-L-Lysine or RGE. Cells growing on UHMWPE coated with RGE appeared irregularly (elongated and spindle) shaped and unevenly spaced. The cells growing in the presence of Poly-L-Lysine showed cellular disruption and lysis, whereas cells growing on the RGD appeared intact, regularly spaced and began fusing into giant cells. The results show macrophages can interact with coating on the material surface, and these surfaces can affect adherence. Use of RGE which inhibits binding of the cells may be a factor that can be used to coat implants to increase their longevity.
8:45 SYMPOSIUM: CARDIOVASCULAR DISEASES IN MISSISSIPPI
Introduction
8:50 OBESITY AND HYPERTENSION
Dr. John E. Hall, Chairman and Guyton Professor of Physiology and Biophysics, University of Mississippi Medical Center
9:20 Questions
9:25 CARDIOVASCULAR DISEASE AND HEART FAILURE
Dr. Charles K. Moore, Assistant Professor of Medicine and Medical Director, Heart Failure/Cardiac Transplant Program, University of Mississippi Medical Center
9:55 Questions
10:00 Break
10:10 CARDIOVASCULAR DISEASE AND DIABETES
Dr. Donald McClain, Professor of Medicine and Director, Division of Endocrinology, University of Mississippi Medical Center
10:40 Questions
10:45 CARDIOVASCULAR DISEASE AND STROKE
Dr. David L. Gordon, Associate Professor of Neurology and Director, Acute Stroke Unit, University of Mississippi Medical Center
11:15 Questions
11:20 THE JACKSON HEART STUDY
Dr. Daniel W. Jones, Associate Professor of Medicine and Director, Division of Hypertension, University of Mississippi Medical Center
11:50 Questions
1:30 NEONATAL OUTCOME OF INFANTS BORN TO MOTHERS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AT UMC (19861996)
Nina T. Washington* and Linda I. Ray*, University of Mississippi Medical Center, Jackson, MS 39216
Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disorder which is associated with increased morbidity and mortality. SLE usually affects women of childbearing years. Lupus pregnancies have been associated with prematurity, intrauterine growth retardation (IUGR), and neonatal lupus syndrome. The objective of this retrospective chart study was to determine the outcome of lupus pregnancies at UMC from January 1986 through December 1996. Medical records were identified by pairing diagnostic codes for SLE and intrauterine pregnancy. Forty-six medical records were identified. The pregnancies under review reflected medical management at UMC. Nine pregnancies (20%) resulted in abortion. Two abortions were elective (Cydophosphamide exposure and congenital anomaly). Seven were spontaneous abortions. Two mothers had positive anticardiolipins (ACL) which is associated with spontaneous abortions. Three had negative ACLs; ACL status was unknown in three. Nineteen pregnancies (41%) were associated with prematurity ( 37 weeks gestation). Micropreemies were unusual; the average weight was 1857 gms. (range 855 gms. - 2690 gms.). APGARS were usually good; nursery course was usually uneventful except for respiratory distress syndrome. Two neonates had arrhythmias, one also had thrombocytopenia, and one had pericardial effusion in utero; these findings could be associated with neonatal lupus. Three infants were noted to have IUGR. Fifteen pregnanices (32%) resulted in term pregnancies; no complications were noted. Three mothers were lost to follow-up. Pregnancy loss and preterm birth were the primary adverse outcomes. Given that 60% of the pregnancies resulted in prematurity or pregnancy loss, these mothers require prenatal care throughout pregnancy. Even though the premature infants usually had a relatively uneventful nursery course, longterm consequences of prematurity may not be apparent in the neonatal period. We next plan to contact the children and perform developmental testing.
1:45 SYNERGISTIC ENHANCEMENT OF COLLAGENOUS PROTEIN SYNTHESIS BY NIFEDIPINE AND INTERLEUKIN-1-
Roger B. Johnson1*, Edward J. Zebrowski2, and Xiaoli Dai1, 1University of Mississippi Medical Center, Jackson, MS 39216, and 2University of Manitoba, Winnipeg, MB R3E OW2 Canada
Gingival overgrowth commonly occurs coincident to therapy with calcium channel blockers. The biologic mechanism for this condition is unknown; anecdotal evidence suggests that gingivitis may contribute to development of the overgrowth. This study tests the hypothesis that collagenous protein synthesis is synergistically enhanced by nifedipine (N) and the pro-inflammatory cytokine, interleukin-1- (IL-1-). Fibroblasts were isolated from biopsies of normal gingiva and cells separated into two groups. Group 1 were exposed to media containing 0, 5, 50, or 500 pg/mL IL-1-, or 10-7M N for 7 days; Group 2 were exposed to those concentrations of IL-1-+10-7M N. 3H-proline was added to the medium for the final 24 hours. Cells and matrix were harvested and radioactivity determined by liquid scintillation analysis. Means (dpm/104 cells) were compared by factorial ANOVA and Sheffè comparisons. Collagenous protein synthesis was significantly reduced by 5 pg/mL+10-7M N and enhanced by 500 pg/mL IL-1-+10-7M N as compared to N or IL-1- alone. Thus, patients may be more susceptible to overgrowth during nifedipine therapy, due to the synergism between IL-1- and nifedipine on collagenous protein synthesis by gingival fibroblasts.
2:00 DOUBLE HETEROZYGOSITY FOR THE CODON 39 CT NONSENSE MUTATION AND A TRIPLICATED -GLOBIN GENE LOCUS CAN CAUSE "DOMINANTLY" INHERITED THALASSEMIA INTERMEDIA
S.L. Rhodes1*, M. Plonczynski1, A. Harrell1, J. Li1, S. Safaya1, J.C. Files2, and M.H. Steinberg1,2, 1VA Medical Center, Jackson, MS, and 2University of Mississippi, Jackson, MS 39216
Thalassemia intermedia is a phenotype produced by different genotypes including dominantly inherited -thalassemic hemoglobinopathies, homozygosity for "mild" -thalassemia alleles, and double heterozygosity for thalassemia and -globin gene triplications. A Caucasian woman, presented while pregnant with a PCV of 21.3, MCV 73 fl, reticulocytes 4-7%, and nucleated red cells in the blood. Her father had similar hematological findings. Hb A2 levels (4.7-5.7%) and Hb F (4.9-5.5%) were elevated in the proband and her father; no abnormal hemoglobins were present. The probands' mother had normal hematological and electrophoretical findings. Sequence of the -globin genes of all family members showed that both the proband and her father were heterozygous for the codon 39 nonsense mutation, CAGTAG. This mutation introduces a Mae I site into the gene and its presence was confirmed by restriction analysis in the proband and father. Homozygotes for the codon 39 nonsense mutation have thalassemia major. Heterozygotes may have minimal anemia and microcytosis. Analysis of the -globin genes by blot hybridization showed a triplicated -globin gene arrangement (anti -3.7) in both the proband and her father and normal -globin genes in the mother. An additional -globin gene compounds the unbalanced chain synthesis responsible for the features of thalassemia. The proband received both the abnormal -globin gene and -globin locus from her father. While the transmission of the phenotype in this family appears to be dominantly inherited, since the - and -globin genes segregate independently, it is, more precisely, an example of polygenic inheritance.
2:15 ALTERED EXPRESSION OF PARVALBUMIN AND CALBINDIN IN PURKINJE CELLS IN SPINOCEREBELLAR ATAXIA-1 (SCA-1)
P.J.S. Vig1*, S.H. Subramony1, J.D. Fratkin, E.N. Burright2 , D.O. McDaniel1, D. Desaiah1, and Z. Qin1, 1University of Mississippi Medical Center, Jackson, MS 39216, and 2University of Minnesota, Minneapolis, MN 55455
Neuronal calcium binding proteins (CBPs) are involved in Ca2+ - buffering and transport. Since neuronal degeneration may be accompanied by impaired Ca2+- homeostasis, a protective role for CBPs has been postulated. Therefore, to understand the role of CBPs in the pathogenesis of SCA-1, the expression of calbindin D-28k (CaB) and parvalbumin (PV) was investigated by immunohistochemistry in the cerebellum and in other brain areas of control and SCA-1 patients. We also determined PV and CaB expression (by immunohistochemistry and immunoblot analysis) in Purkinje cells of transgenic mice (TM) (transgenic line B05 and A02 expressing the human SCA-1 gene with expanded CAG and normal CAG tract respectively) and age-matched nontransgenic mice (nTM). Animals at different postnatal ages were used. All surviving Purkinje cells in SCA-1 patients were strongly immunoreactive to CaB similar to those in controls. The number of PV-immunoreactive Purkinje cells was markedly reduced in SCA-1. In addition , there was a significant decrease in the intensity of PV immunostaining within the individual Purkinje cells compared with controls. However, in the hippocampus, temporal cortex, and lateral geniculate scattered PV-positive neurons were seen in SCA-1 patients, similar to those in controls. In 9 and 12 wks old TM-B05 there was a marked decrease in PV immunoreactivity in Purkinje cells as compared to the nTM controls and TM-A02. PV immunostaining in interneurons was well preserved in all the groups. There was also a decrease in CaB immunoreactivity in Purkinje cells of 9 and 12 wks old TM-B05. Purkinje cells of 4 and 6 wks of TM-B05 which exhibit no ataxia and even lack demonstrable Purkinje cell loss also revealed reduction in PV immunoreactivity. CaB immunohistochemistry did not detect any marked changes in CaB immunoreactivity within Purkinje cells at 4 wks. However, at 6 wks, immunostaining and immunoblot analysis revealed a significant decrease in CaB in TM-B05 compared to controls. These data clearly demonstrate that the decrease in CBPs immunoreactivity in the surviving Purkinje cells in SCA-1 reflect biochemical alterations preceding Purkinje cell degeneration. Whether a decrease in the levels of PV and CaB is due to the alterations in their mRNA expression is currently being pursued.
2:30 CONTROL OF EYELID MOVEMENTS BY VERTICAL GAZE CENTERS
Bing-Zhong Chen*, Yan Pan, and Paul J. May, University of Mississippi Medical Center, Jackson, MS 39216
The rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) and the interstitial nucleus of Cajal (InC) are vertical gaze centers that project to the oculomotor nucleus. Coordination of vertical eye and eyelid movements during gaze shifts suggests that the caudal central subdivision (CCS), which contains levator motoneurons that direct upward eyelid movements, might receive inputs from these two centers. To examine this issue, biotinylated dextran amine (BDA) was injected into either the riMLF or InC of cats that also had their levator motoneurons retrogradely labeled with HRP. BDA labelled axons from the riMLF ran through the MLF and terminated in the oculomotor nucleus, as expected. Terminal arbors were also present bilaterally in the CCS. They displayed primarily en passant boutons, which were often closely associated with HRP labeled levator motoneurons. Electron microscopic examination of riMLF terminals revealed direct synaptic contacts with motoneurons. The terminal density in the CCS increased relative to the oculomotor nucleus with mediocaudally located riMLF injections. InC injections also labeled terminal arbors bilaterally in the CCS that were closely associated with levator motoneurons, suggesting synaptic contact. This projection was denser than the riMLF projection, perhaps due to labeling of fibers of passage. In conclusion, our results strongly suggest that both the riMLF, especially its caudomedial portion, and the InC contain premotor neurons that directly control eyelid movements during vertical gaze changes.
2:45 Break
3:00 A LIGHT AND ELECTRON MICROSCOPIC EXAMINATION OF COMMISSURAL PREMOTOR NEURONS CONTROLLING TRIGEMINAL MOTOR FUNCTION
Yan Pan* and Paul J. May, University of Mississippi Medical Center, Jackson, MS 39216
The act of mastication involves considerable coordination of the movement between the two sides of the jaw. We investigated the premotor substrate for this coordination by making a neuronal tracer (BDA) injection into the trigeminal motor nucleus and overlying supratrigeminal area. The contralateral trigeminal motor nucleus contained dense terminal label, and lighter terminal label was observed in the supratrigeminal nucleus. In addition, retrogradely labelled neurons were found dorsolateral to the contralateral trigeminal motor nucleus in the supratrigeminal nucleus, and medially in the pontine reticular formation. This group of neurons extended caudally into pars oralis of the spinal trigeminal nucleus. The medial neurons were uniformly small, but an additional population of very large neurons was present in the supratrigeminal nucleus. These large isodendritic neurons had up to eight, poorly branched dendrites that could be followed up to 300 µm. Ultrastructural examination of the supratrigeminal nucleus revealed retrogradely labelled somata and dendrites. A variety of terminal types synapsed on these labelled neurons, including profiles with spherical, pleomorphic and frankly flattened vesicles. The density of these synaptic contacts increased dramatically on distal dendrites. Anterogradely labelled terminals containing round vesicles were also present, and in some cases were observed to synapse on retrogradely labelled neurons. These results indicate that the perinuclear areas, particularly the supratrigeminal nucleus, are not only directing masticatory motoneurons bilaterally, they are interconnected as well.
3:15 CHARACTERIZATION OF PROCESSED HAIR PROTEINS BY POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE), ISOELECTROFOCUSING (IEF), AND WESTERN BLOTTING
George E. Abraham III*, Hari H.P. Cohly, Jim Thompson, Robert Allen Smith, Cheryl Blanchard, Barbara Rogers, and Michael F. Angel, University of Mississippi Medical Center, Jackson, MS 39216, and South West Research Institute, San Antonio, TX 78217-5675
The objective of this study was to define the keratinous protein composition of processed hair using PAGE, IEF, and western blotting. Processed hair (2%) was prepared by suspending hair powder in Dulbecco's Modified Eagles Medium and homogenizing the sample in a liquid nitrogen bath. The homogenate was then centrifuged at 1500 rpm. The supernatant was filtered through a 0.22 µm filter and microfuged at 12,000 g and further sterilized by 0.22 µm filtration. PAGE was run on the sample using a SDS gel. The gel was stained, destained, fixed, and dried. The protein bands were counted and their molecular weights were compared to that of a standard marker which was run on both ends of the gel. Twelve bands were identified in the samples. The band molecular weights corresponded to the following: 300k, 250k, 210k,160k, 150k, 90k, 80k, 75k, 70k, 65k, 60k, and 40k. The six most significant bands corresponded to the following: 210k, 90k, 80k, 70k, 60k, and 40k. The PAGE successfully located twelve proteins in the sample, six of which were distinct. Further characterization of these bands will be possible through IEF and western blotting using the monoclonal antibodies Ks8.60, Ks8.12, antihuman filaggrin, AE13, and BT115.
3:30 EFFECT OF PROCESSED HAIR (PH) ON PARTIAL THICKNESS WOUNDS IN RATS
Hari H.P. Cohly*, Robert Allen Smith, Mike Adams, Cheryl Blanchard, Barbara Rogers, and Michael F. Angel, University of Mississippi Medical Center, Jackson, MS 39216, and South West Research Institute, San Antonio, TX 78217-5675
This study determines if treatment with PH enhances wound healing in partial thickness wounds. CD/Hairless rats (N=28) were randomly divided into two groups. Each group of 14 rats was anesthetized and two .12 inch (in-depth) partial thickness wounds were created on either side of the dorsal midline with a hand dermatome (surface dimensions 2.0 x 4.0 cm). Group A wounds were untreated while Group B wounds were treated with PH powder every other day. The wounds were covered with a semipermeable, transparent dressing (Tegaderm R; 3M Corp., St. Paul, MN). Photographs, acetate tracings and tissue biopsies were taken immediately after wounding and every other day through day 6. Two animals from each group were selected for wound biopsy (4mm punch). Tissue specimens were evaluated by H&E stain, tissue lipid peroxidation and histochemical analysis. The wound areas were determined by planimetry. At day 2, 4, and day 6 the PH treated wounds continued to show significantly advanced maturation as compared to the control group. Clinically observed epithelialization in the planimetry studies at day 4 indicate 98.5% coverage in the treated and 76.7% in the non-treated rats (p<0.05). This study shows that PH is beneficial in treating partial thickness wounds.
3:45 ROLE OF PROCESSED HAIR ON HYDROGEN PEROXIDE-INDUCED HUMAN DERMAL FIBROBLAST (F) CELL INJURY
Hari H.P. Cohly, Andrew Smith*, Crystal Paige, Robert Allen Smith, Cheryl Blanchard, Barbara Rogers, and Michael F. Angel, University of Mississippi Medical Center, Jackson, MS 39216, and South West Research Institute, San Antonio, TX 78217-5675
This study determines if processed hair (PH) offers protection from
oxidative injury in F. Semi-confluent F in 24-well plate were labeled with
3H-arachidonic acid (0.1 µCi/ml) and selected concentration
of PH (2,000 µg/ml-0.5 µg/ml). The cells were then washed and
additionally labeled with 51Cr (0.1 µCi/ml) and incubated
for 3 hrs at 37°C. The adherent cells were further washed and incubated
for 3 hrs with 5.0 mM H202 at 37°C. 3H-arachidonic
acid release, 51Cr release and lipid peroxidation by the thiobarbituric
acid reaction was determined. Specific 51Cr release for controls
was 45.8 ± 1.93, K10 µg/ml 42.3 ± 2.9%, K5 µg/ml
47.8 ± 1.4, K2 µg/ml 57.2 ± 2.8 and K1 µg/ml
54.1 ± 8.4 compared to 5 mM H202 which was
70.9 ± 2.1%. 3H-arachidonic acid release for controls
was 112 ± 23%, K10µg/ml 265 ± 27%, K5 µg/ml 176
± 6%, K2 µg/ml 188 ± 40 and K1 µg/ml 174 ±
8% compared to 5 mM H202 which was 396 + 31%. There
was a statistically significant difference (p<0.05) in 3H-arachidonic
acid and 51Cr release assay with PH 10 µg/ml-1.0 µg/ml.
This study shows that 5 mM H202 causes oxidative
stress to F. PH at concentrations 10 µg/ml -1.0 µg/ml provide
protection against oxidative stress by H202.
4:00 EFFECT OF PROCESSED HAIR SCAFFOLDING ON WOUND HEALING PARAMETERS
Hari H.P. Cohly*, Robert Allen Smith, Cheryl Blanchard, Philip Hawner, Barbara Rogers, Michael F. Angel, University of Mississippi Medical Center, Jackson, MS 39216, and South West Research Institute, San Antonio, TX 78217-5675
Keratinous Protein (KP) from human hair was studied to determine cell proliferation of keratinocytes (K), fibroblasts (F), endothelial cells (E) and T cells. K, F, and E were separately grown to 30% confluency, starved for 12 hrs and then incubated for 2 days in the presence and absence of KP and the appropriate growth medium. T cells were incubated in the presence and absence of a T cell mitogen, Con A for 3 days with KP. Cell proliferation was assessed by tritiated thymidine uptake. K peaks with KP at 50 µg/ml; Stimulation index (SI)=3.25 and with growth medium SI = 3.10. F peaks with KP at 1.5 µg/ml; SI=15 and with growth medium SI= 24.6. E had peak response to KP at 100 µg/ml; SI =2.15 that is continuous through 1.5 µg/ml;SI= 1.6 and with growth medium SI= 1.6. T cells peaked at 2500 µg/ml; SI=10 and decreasing to basal level at 79 µI/ml. KP in the presence of Con A has inhibitory concentrations at 2500 µg/ml-156 µg/ml (SI=6) but reach values similar to Con A at 20 µg/ml (SI=16) (p<0.01). KP stimulates K, E, and F activity at similar concentrations, e.g., 1.5 µg/ml. Cellular hyperesponsiveness coupled with low antigenicity demonstrates enhanced wound healing.