Neuregulin1 and ErbB expression in the uninjured and regenerating olfactory mucosa.

Neuregulin1, a protein involved in signaling through the ErbB receptors, is required for the proper development of multiple organ systems. A complete understanding of the expression profile of Neuregulin1 is complicated by the presence of multiple isoform variants that result from extensive alternative splicing. Remarkably, these numerous protein products display a wide range of divergent functional roles, making the characterization of tissue-specific isoforms critical to understanding signaling. Recent evidence suggests an important role for Neuregulin1 signaling during olfactory epithelium development and regeneration. In order to understand the physiological consequences of this signaling, we sought to identify the isoform-specific and cell type-specific expression pattern of Neuregulin1 in the adult olfactory mucosa using a combination of RT-qPCR, FACS, and immunohistochemistry. To complement this information, we also analyzed the cell-type specific expression patterns of the ErbB receptors using immunohistochemistry. We found that multiple Neuregulin1 isoforms, containing predominantly the Type I and Type III N-termini, are expressed in the uninjured olfactory mucosa. Specifically, we found that Type III Neuregulin1 is highly expressed in mature olfactory sensory neurons and Type I Neuregulin1 is highly expressed in duct gland cells. Surprisingly, the divergent localization of these Neuregulin isoforms and their corresponding ErbB receptors does not support a role for active signaling during normal turnover and maintenance of the olfactory mucosa. Conversely, we found that injury to the olfactory epithelium specifically upregulates the Neuregulin1 Type I isoform bringing the expression pattern adjacent to cells expressing both ErbB2 and ErbB3 which is compatible with active signaling, supporting a functional role for Neuregulin1 specifically during regeneration.

An atypical proprotein convertase in Giardia lamblia differentiation.

Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Nonviral gene transfer into primary cultures of human and porcine mesothelial cells.

Due to their abundance and accessibility, mesothelial cells may be suitable tools for recombinant reagent expression by gene transfer. Genetically modified porcine mesothelial cells (PMCs) may have the potential for the treatment of vascular diseases in humans. We studied the effect of various transfection reagents on the primary culture of PMCs and human mesothelial cells (HMCs). The cells were transfected with a plasmid encoding a reporter gene (luciferase or green fluorescent protein [GFP]) under the control of the cytomegalovirus promoter. Transfection was achieved using cationic lipids (DOSPER and DOTAP) or calcium phosphate/deoxyribonucleic acid coprecipitation or Fugene 6. Results showed that Fugene 6 was the most efficient and reproducible transfection reagent with both PMCs and HMCs. With Fugene 6, luciferase activity in PMCs (1.5 x 10(8) relative light units [RLU]/10(6) cells) was at least 2.5-fold higher than with the other transfection reagents, and it was 100-fold higher than in HMCs. However, the proportion of transfected cells expressing GFP was only 1%. These preliminary findings open up new avenues for developing experimental studies on the use of genetically modified PMCs.

Human mesothelial cells express tissue factor when switched to proliferating state. Pharmacological modulation in vitro.

Covering the inner surface of small-diameter arterial prostheses with endothelial cells (ECs) has been proposed as a means of improving biocompatibility and thrombosis resistance. Because the availability of autologous ECs is limited, autologous human mesothelial cells (HMCs) have been suggested as a substitute for ECs. However, HMCs express tissue factor (TF) in vitro, a deleterious characteristic in vivo. We investigated the distribution of TF antigen and of its inhibitor, tissue factor pathway inhibitor, on HMCs and the effect of pharmacological agents on TF expression. TF antigen was measured by enzyme-linked immunosorbent assay and localized by confocal microscopy. Three distinct pools of TF antigen were demonstrated: within the cells, at the cell surface, and in the extracellular matrix. The effects of ilomedin (10 microg/ml) and heparin (500 U/ml), known to affect procoagulant activity, were evaluated by incubating HMCs for 24 h with or without these agents. Ilomedin, but not heparin, decreased TF antigen expression by 30% (P < 0.05). Despite the theoretical potential of HMCs as a vascular prosthesis lining, TF expression by HMCs remains a major drawback. A technique capable of blocking TF expression until the HMCs return to their resting state is needed. Genetic manipulation of HMCs may hold promise for such a technique.

Persistent infection of Myxobolus cerebralis, the causative agent of salmonid whirling disease, in Tubifex tubifex.

The objective of this study was to quantify and determine the periodicity in the release of the triactinomyxon (TAM) stage of Myxobolus cerebralis, the causative agent of salmonid whirling disease, by its aquatic oligochaete host Tubifex tubifex. For this, 24 individual T. tubifex (infected as a group at 15 C) were examined daily for the release of M. cerebralis TAMs, and the number of waterborne TAMs released by each worm was quantified. The duration of the infection in these worms was also monitored using a polymerase chain reaction (PCR) diagnostic test. TAMs were first released 74 days postexposure (PE) and continued to be released until 132 days PE. During this period, each worm released on average, 1.5 x 10(3) waterborne TAMs 12 times; however, no pattern or periodicity was noted. The results of the PCR diagnostic tests conducted at 5, 7, 9, and 15 mo PE were positive, and the persistent infection was confirmed at 606 days PE (approximately 20 mo) when the remaining worms began releasing TAMs again. Similar results were observed in naturally infected T. tubifex, indicating that these worms remain infected for the duration of their natural lifespan and are capable of shedding viable TAMs, in temporally separate periods. These findings open the possibility of a seasonal periodicity in TAM release by T. tubifex.

Phenotypic and functional characteristics of porcine peritoneal mesothelial cells.

The objective of this study was to establish a technique to isolate porcine mesothelial cells (PMCs) from omental tissue and to compare them to human mesothelial cells (HMCs). The PMCs were dispersed by collagenase digestion and isolated on a Ficoll layer. Their morphologic and ultrastructural features were assessed at confluence by light and electronic microscopy, and they were characterized by immunohistochemistry using specific HMC markers. PMC proliferation was studied in the presence of growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or transforming growth factors beta1, beta2, or beta3 (TGF). Fibrinolytic PMC activity was detected by zymography for tissue plasminogen activator (tPA) and by reverse zymography for plasminogen activator inhibitor-1 (PAI-1). The recalcification time of cell lysates was used to define PMC procoagulant activity, and gelatinase zymography was used to detect metalloproteinase production. At confluence, PMCs formed typical cobblestone monolayers and exhibited structural features characteristic of HMCs. Weibel Palade bodies were never seen. Specific HMC markers (HBME1, ME1, WT1) cross-reacted with PMCs. As HMCs and PMCs coexpressed cytokeratin and vimentin, and also expressed vinculin and alpha-actin. Addition of PDGF or EGF to the culture medium stimulated PMC proliferation. PMCs constitutively expressed fibrinolytic and procoagulant activity and secreted MMP9 and MMP2. The technique described in this study allows isolation of mesothelial cells from porcine omental tissue. These porcine cells exhibit a mesothelial phenotype and functional properties similar to those of HMCs. Our data warrant an evaluation of mesothelial cells as targets in several therapeutic strategies with porcine models.

Macro and microheterogeneity in normal endothelial cells: differential composition of luminal glycocalyx and functional implications.

Endothelial cells (EC) are involved in various physiological and pathological processes through the expression of their surface glycoproteins. They are covered by the glycocalyx, composed of glucidic residues from cell surface membrane glycoproteins, glycoplipids and proteoglycans. Glucidic sequences can be specifically characterized by their binding to lectins. Eight lectins were used to investigate the distribution and regulation of EC surface glucidic residues in various blood vessels of adult and newborn pigs. EC lectin binding was compared to von Willebrand factor (vWF) expression as EC reference marker. Six out of eight lectins (BSI-B4, DBA, EEA, HP, MAL I and PNA) were helpful for this determination. Considering only the intensity of labelings, vWF and DBA gave the best stainings of adult pig ECs. In newborn pigs, the best labelings were obtained with EEA and MAL I. Furthermore, the distribution of lectin binding to ECs and EC vWF expression was heterogeneous depending on the EC location along vascular tree and age. Beside this macroheterogeneity this study highlights a microheterogeneity of EC lectin binding and vWF expression in situ, defined as a staining of equal intensity by individual ECs, scattered among negative ones, in a given vascular segment. EC surface sugar residues were differently modulated in newborn and adult pig ECs and differently according to EC vWF expression. The functional involvement of EC glycocalyx was reflected by EC lectin binding in the spleen and liver. This study emphasizes the high level of EC heterogeneity for various markers. The EC macro- and microheterogeneity reflect the "plasticity" or "unstability" of EC phenotypes and suggests that ECs are subject to several levels of regulation and are probably grouped in functional clusters to best adjust their functions to microenvironmental requirements. This concept must be considered in further investigations notably in in vitro studies where EC phenotype can be altered.

Gene therapy in lung transplantation: feasibility of ex vivo adenovirus-mediated gene transfer to the graft.

Lung transplantation is associated with complications such as reperfusion injury and graft rejection. Gene therapy targeted to the graft offers a promising approach to the prevention of these complications. Because adenovirus vectors can transfer genes in vivo to the lung vasculature, we evaluated the feasibility of adenovirus-mediated gene transfer to the lung graft in a porcine model of left lung allotransplantation. Following removal of the donor lung, an adenovirus vector encoding the beta-galactosidase (beta-Gal) gene was injected ex vivo into the lumen of the upper lobe pulmonary artery of the graft. After 2 hr of incubation at 10 degrees C, the lung graft was implanted into the recipient animal. Three days later, the animals were sacrificed and the lung graft was evaluated for beta-Gal activity. No beta-Gal activity was detected in the left lower lobe used as a control. In contrast, beta-Gal activity was detected in endothelial cells of the left upper lobe pulmonary circulation, and was also observed in airway and alveoli epithelial cells. However, less than 1% of cells of the graft expressed beta-Gal. In vitro experiments showed that this may be explained in part by the low temperature and the short duration of adenovirus incubation within the graft, and by the low susceptibility of porcine cells to human adenovirus. Furthermore, expression of the exogenous gene occurred in several organs of recipient animals. Thus, adenovirus-mediated gene transfer to the lung graft is feasible ex vivo, but several parameters limit gene transfer efficiency and need to be improved before clinical application is attempted.

Fluid compartments in hemorrhaged rats after hyperosmotic crystalloid and hyperoncotic colloid resuscitation.

Postresuscitation organ failure may be associated with detrimental changes in body fluid compartments. We measured how shock and resuscitation acutely alters the interstitial, cellular, and plasma compartments in different organs. Nephrectomized, anesthetized rats were bled to 50 mmHg mean arterial pressure for 1 h, followed by 60 min of resuscitation to restore blood pressure using 0.9% normal saline (NS,n = 10), 7.5% hypertonic saline (HS,n = 8), 10% hyperoncotic albumin (HA, n = 8), or 7.5% hypertonic saline and 10% hyperoncotic albumin (HSA, n = 7). A 2-h 51Cr-EDTA distribution space estimated extracellular fluid volume (ECFV), and a 5-min 125I-labeled albumin distribution space measured plasma volume (PV). Total tissue water (TW) was measured from wet and dry weights; interstitial fluid volume (ISFV) and cell water were calculated. NS resuscitation required 7 times more fluid (50.9 +/- 7.7 vs. 8.6 +/- 0.7 for HA, 5.9 +/- 0.4 for HS, and 3.9 +/- 0.5 ml/kg for HSA), but there were no differences between solutions in whole animal PV, ECFV, or ISFV. Fluid shifts within tissues depended on resuscitation solution and type of tissue. TW was significantly reduced by hypertonic saline groups in heart, muscle, and liver (P < 0.05). ISFV was significantly reduced by HA groups in the skin. In all tissues, mean cell water in groups receiving HS was smaller; this was significant for heart, lung, muscle, and skin. In conclusion, 1) HS solutions mobilize fluid from cells while expanding both PV and ISFV, and 2) TW and cellular water increase with both isotonic crystalloids and hyperoncotic colloids in many tissues.

Effects of isotonic crystalloid resuscitation on fluid compartments in hemorrhaged rats.

Redistribution of fluid after isotonic crystalloid resuscitation from hemorrhage may result not only in interstitial edema but also in cellular edema. We measured the extent to which shock and resuscitation altered fluid compartments in different organs. Nephrectomized, anesthetized rats were randomly divided into a Control group (n = 10) and a Hemorrhage plus Resuscitation group (H/R, n = 10). Rats were subjected to 60 min hemorrhagic hypotension (50 mmHg) followed by a 60 min resuscitation period with .9% NaCl infused as needed to maintain mean arterial pressure at 80% of baseline. A 2 h 51Cr-EDTA distribution space was used to estimate extracellular fluid volume (ECFV) and a 5 min 125I-albumin distribution space was used to measure plasma volume (PV). After euthanasia, total tissue water was measured by wet/dry weight analysis and interstitial fluid volume (ISFV) and cell water were calculated for selected organs. Resuscitation volume was two times the shed blood volume, but resulted in a PV equal to that of the Controls. There were no significant differences in whole animal ECFV or ISFV, although the mean values in the H/R group were greater than that of the Control group. The mean values for total tissue water for each tissue in the H/R group were larger than the respective means of the Control tissues but was significantly greater for only the heart (3639 +/- 56 microL/g vs. 3493 +/- 24 microL/g, mean +/- S.E., p < .05). In all H/R tissues, mean values for ISFV were also larger; this difference was significant for only the liver and small intestines (744 +/- 62 vs. 518 +/- 29 microL/g and 1117 +/- 155 vs. 706 +/- 58 microL/g, respectively). Heart cell water was significantly larger in H/R than Controls (2900 +/- 60 microL/g vs. 2738 +/- 27 microL/g). These data suggest that resuscitation of hemorrhage using isotonic crystalloid normalizes overall PV and ECFV but also causes interstitial expansion in selected gut tissues and cellular edema in the heart.

Quantitative applications: forecasting and costing nursing service at an Army community hospital.

The utilization of a quantitative method of forecasting, coupled with an existing patient classification system (the Army's Workload Management System for Nursing), provides a creative costing tool for managing nursing resources at military medical facilities. Although the nursing management options discussed in this article are focused on a downsizing situation at an Army Community Hospital, they are applicable throughout military medical facilities.

Mechanism of entry of a xenotropic MMuLV-derived recombinant retrovirus into porcine cells using the expression of the reporter nlslacZ gene.

A xenotropic Moloney murine leukemia virus-derived recombinant retrovirus (MMuLVSVnlslacZ) has been utilized to study the mechanism of virus entry into endothelial and epithelial porcine cells. In the genome of this recombinant retrovirus, the nlslacZ reporter gene is under the transcriptional control of both LTR and SV40 early promoter. The entry of the retrovirus has been determined from the expression of this transduced reporter gene after its integration into the infected cells. This allows the detection of a very low level of viral infection and hence entry of the virus. Exposure of the virus-cell mixture to acidic pH (less than 6) during the early phase of interaction reduces the level of internalization. Cellular infection in presence of weak bases, ammonium chloride and amantadine and an ionophore monensin at concentrations sufficient to neutralize the endosomal pH does not modify the extent of viral entry into the cells. The results indicate that the entry of the recombinant retrovirus into porcine cells takes place by a pH-independent viral membrane-cell plasma membrane fusion mechanism.

Orthopaedic trauma in men: the relative risk among drinkers and the prevalence of problem drinking in male orthopaedic admissions.

Admissions to an acute male orthopaedic ward (n = 369) were asked about their accident, their alcohol consumption, and alcohol-related problems in the past 2 years. Comparing their consumption with that of males from a community survey revealed an increased risk of orthopaedic admission in drinkers consuming 21 units of alcohol/week or over, relative to drinkers consuming less than 21 units/week, in the age group 31-50 years. In all, 34% of the sample met a criterion for problem drinking based on self-reported alcohol consumption and/or medical and social problems associated with alcohol. In 13%, alcohol was viewed by the patient as having contributed to the accident, and in 19% according to the interviewer's perception of whom 76% were classifiable as problem drinkers. Twenty-six men said the accident had made them think about changing their drinking habits. Detection of problem drinking in orthopaedic male admissions is possible and could be usefully linked to a counselling service.

A review of three incidents involving the release of 125I from seeds interstitially implanted within the prostate gland.

Three cases have occurred at the Medical College of Virginia in which 125I seeds were accidentally ruptured while being interstitially implanted within cancerous prostate-gland tumors. During the first incident the physician performing the surgery was contaminated. In the second and third cases, the iodine seeds were post-operatively discovered to be leaking. Both patients demonstrated significant thyroid uptakes and were administered potassium iodide as a blocking agent. Routine urine analyses and thyroid bioassays were performed during a period of several months to study the removal of 125I from the thyroid and to determine the effect potassium iodide had on the iodine-removal rate. Data obtained during the follow-up studies emphasize the necessity of post-operative monitoring and point out the value of administering a blocking agent in cases involving the release of 125I from a ruptured seed. The findings also indicate the risks during this type of therapeutic procedure may warrant the prophylactic use of exogenous iodine prior to implanting the iodine seeds.

Performance evaluations for diagnostic x-ray equipment.

Technical factors influencing the performance of x-ray equipment are: focal spot size, focal spot growth, leakage radiation, accuracy of the exposure timer, kilovoltage, exposure reproducibility, linearity of exposure, x-ray beam quality, and collimation. An evaluation of these factors is necessary to determine x-ray equipment performance characteristics.

Radiation exposure during panoramic radiography in children.

In an effort to determine the amount of radiation exposure a child receives during a panoramic radiograph and to evaluate the effect of a protective thyroid collar, dosimeter readings were made on twenty-nine child patients at fourteen anatomic sites. The levels of radiation recorded were similar to those previously recorded for adults and phantoms, except for greater doses in the thyroid area. Placement of a lead-lined thyroid collar resulted in significant reduction of the thyroid doses.

Personality profiles and leadership potential of medical-surgical and psychiatric nursing graduate students.

When personality profiles and leadership potential of psychiatric and medical-surgical nursing graduate students were compared, using the California Psychological inventory (CPI) and the Managerial Key for the CPI, medical-surgical majors scored significantly higher on the CPI scale (p greater than .05). Overall, both groups scored higher than the norm and showed a more optimal personality development than has been observed in earlier studies of this kind. In the Managerial Key for the CPI, the test of leadership potential on both groups received a high rating, comparable to that of the top-management group in the original research.