Re-evaluation of mouse tissue factor pathway inhibitor and comparison of mouse and human tissue factor pathway inhibitor physiology.

Essentials Mouse models are often used to define roles of tissue factor pathway inhibitor (TFPI) in man. TFPI isoform-specific KOs reveal unexpected differences between mouse and human TFPI physiology. Mouse plasma contains 20 times more TFPI than man, derived from TFPIγ, a form not found in man. TFPIγ null mice, expressing only TFPI isoforms α and ß, may better reflect the human situation. SUMMARY: Background Mouse models can provide insight into the pathophysiology of human thrombosis and hemostasis. Tissue factor pathway inhibitor (TFPI) regulates coagulation through protein S (PS)-enhanced factor (F) Xa inhibition and FXa-dependent inhibition of FVIIa/tissue factor (TF) activity. TFPI is expressed as isoforms α and ß in man, and α, ß and γ in the mouse. Objective Assess the reliability of extending TFPI-related studies in mice to humans. Method Compare mouse and human TFPI physiology using a variety of methods. Results Mouse TFPI and human TFPI are similar in regard to: (i) the mechanisms for FVIIa/TF and FXa inhibition; (ii) TFPIα is a soluble form and TFPIß is glycosyl phosphatidyl inositol (GPI) membrane anchored; (iii) the predominant circulating form of TFPI in plasma is lipoprotein-associated; (iv) low levels of TFPIα circulate in plasma and increase following heparin treatment; and (v) TFPIα is the isoform in platelets. They differ in that: (i) mouse TFPI circulates at a ~20-fold higher concentration; (ii) mouse lines with isolated isoform deletions show this circulating mouse TFPI is derived from TFPIγ; (iii) sequences homologous to the mouse TFPIγ exon are present in many species, including man, but in primates are unfavorable for splicing; and (iv) tandem mass spectrometry (MS/MS) detects sequences for TFPI isoforms α and ß in human plasma and α and γ in mouse plasma. Conclusion To dissect the pathophysiological roles of human TFPIα and TFPIß, studies in TFPIγ null mice, expressing only α and ß, only α or only ß should better reflect the human situation.

Airway proteins involved in bacterial clearance susceptible to cathepsin G proteolysis.

Serine proteases released from neutrophils are central to the pathogenesis of cystic fibrosis lung disease and are considered to be obvious therapeutic targets. Neutrophil elastase digests key opsonins present in the lung and disrupts phagocytosis, allowing bacteria to persist despite established pulmonary inflammation. We have found that cathepsin G, an abundant serine protease found in human and murine neutrophils, has other roles in the development of suppurative lung diseases. Murine models of endobronchial inflammation indicate that cathepsin G inhibits airway defences and interferes with the host's ability to clear Pseudomonas aeruginosa from the lung with effects distinct from neutrophil elastase. We hypothesise that differences in bacterial killing are due to defects in innate defences created by proteolysis. Protein profiles of bronchoalveolar lavage of infected wild-type and cathepsin G-deficient mice were compared using two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry. Four proteins in bronchoalveolar lavage were cleaved by cathepsin G. Serum amyloid P component leaked into the lung during acute infection and was digested by cathepsin G. Its cleavage products had greater binding to lipopolysaccharide and interfered with phagocytosis. These results indicate that cleaved serum amyloid P component acts as an anti-opsonin and interferes with bacterial clearance from the lung.

Mutational status of overexpressed p16 in head and neck cancer: evidence for germline mutation of p16/p14ARF.

Inactivation of the p16 tumor suppressor gene is a common phenomenon in squamous cell carcinoma of the head and neck (SCCHN). Less commonly described is the observation of p16 overexpression in SCCHN. Since overexpression of p16 is a potent predictor of outcome in other cancers, we were interested in determining the level of expression of p16 in our SCCHN specimens as a prerequisite to later prognostic studies. We were also interested in determining the mutational status of p16 in these tumors, in order to determine whether the combination of overexpression and gene alteration may predict a different clinical outcome from overexpression alone. A total of 84 specimens of SCCHN were selected for study. These specimens were obtained from all major sites within the oral cavity, oropharynx, pharynx and larynx. The level of expression of p16 in SCCHN specimens was measured by semi-quantitative RT-PCR. In 35 cases, RNA was also isolated from matched normal tissue obtained from a negative tumor margin. In the other 49 cases, the expression level was compared with the level of expression measured in pooled normal RNA obtained from 10 specimens of normal epithelial tissue. Overexpression of p16 was documented when the level of expression in the tumor specimen was 2-fold or greater above the level of expression found in normal tissue. A total of 46 specimens demonstrated overexpression of p16 (55%). All specimens demonstrating overexpression were then subject to sequence analysis. Thirty specimens (65%) showed p16-specific gene alterations, ranging from intragenic deletions to single point mutations, and 15 of these cases concomitantly affect p14ARF. A single specimen demonstrated a silent point mutation within the p16 reading frame. This mutation produces a stop codon at residue 85 in the context of the p14ARF reading frame, predicting premature termination of p14ARF within a previously determined nucleolar localization signal. This observation suggests that in some cases at least, p14ARF may be a selective target for alteration, independently of p16. Analysis of a normal tissue specimen obtained from a negative tumor margin, and a blood sample obtained approximately five years after surgery indicate that this p14ARF-specific alteration may represent a germline mutation.

Mass spectrometry compatibility of two-dimensional gel protein stains.

As proteomic technology evolves, protein staining sensitivity is constantly being improved, enabling researchers to better visualize the proteome of their system. The current challenge is to balance the limits of detection of protein visualization with those of the mass spectrometric methods. In this report, mass spectra generated from human serum or rat liver proteins stained with either colloidal Coomassie blue, Daiichi silver, SYPRO Orange, SYPRO Red, SYPRO Ruby, or SYPRO Tangerine are compared. It has been concluded that the newest generation of fluorescent protein stains, compared with traditional staining methods, are more compatible to matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The number of database matches obtained using each mass spectrometry method and the percent sequence coverage obtained from trypsin digested proteins stained using these six methods is provided.

Practical aspects of fluorescent staining for proteomic applications.

SYPRO Orange and SYPRO Ruby staining methods, modified for use with large-format two dimensional (2-D) gels, are compared to the manufacturer's recommended protocols to determine sensitivity and reproducibility of the new methods. This study examines the critical aspects of fixation, washing, and staining to develop an optimized fluorescent staining method. It was determined that careful control of sodium dodecyl sulfate (SDS) levels and pH in the gel was critical for successful staining with SYPRO Orange. Overnight fixation in 40% ethanol/2% acetic acid/0.0005% SDS preserved protein content, eliminated ampholyte-generated staining artifacts, and had no detrimental effects on staining. Three one-hour washes in 2% acetic acid/0.0005% SDS, followed by staining with SYPRO Orange diluted 1:5,000 with washing solution for 3 or more hours, produced high sensitivity, low background images using a STORM 860 laser scanner. Gels viewed two years after staining showed no significant changes with respect to the initial protein patterns, and allowed successful mass spectrometric postgel characterization of protein spots. Protocol changes applied to SYPRO Ruby staining improved the contrast of STORM 860-generated images, but had little impact on staining sensitivity. A comparison of the cost benefits of staining with SYPRO Orange vs. SYPRO Ruby is also discussed.

Safety and efficacy of transcervical resection of parapharyngeal space neoplasms.

There are several surgical approaches for resection of parapharyngeal space (PPS) neoplasms. The purpose of this study was to evaluate local disease control, facial nerve injury, and need for mandibulotomy associated with resection of PPS neoplasms via the transcervical approach with submandibular gland excision. A retrospective chart review of 33 patients who underwent resection of a PPS neoplasm between October 1991 and July 2000 was performed. Of the 33 patients, 3 patients developed local recurrence after a median follow-up of 24 months. None of the patients experienced facial nerve paresis or paralysis. Three patients (9.1%) required a mandibulotomy for further exposure. This study demonstrated that the transcervical approach with submandibular gland excision for resection of PPS neoplasms provides excellent local disease control with minimal risk of facial nerve injury or need for mandibulotomy and/or tracheotomy.

Basal cell carcinoma metastatic to the parotid: report of a new case and review of the literature.

Basal cell carcinoma is the most common of the cutaneous malignancies, accounting for 65 to 75% of all skin cancers. The natural history of this disease is one of chronic local invasion. Metastatic basal cell carcinoma is a rare clinical entity, with a reported incidence of only 0.0028 to 0.5%. Approximately 85% of all metastatic basal cell carcinomas arise in the head and neck region. We present a case of basal cell carcinoma that spread to the parotid gland in a man who had multiple lesions on his scalp and face. We also review the literature on metastatic basal cell carcinoma of the head and neck, and we discuss its epidemiology, etiology, histopathology, and treatment.

Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1.

Dentine matrix protein 1 (DMP1) is an important component of the non-collagenous extracellular matrix of developing teeth and bones. Functions of DMP1 other than a putative role in the initiation of mineralization are largely unknown. A first report on the DNA and deduced amino acid sequence showed that DMP1 has a single Arg-Gly-Asp (RGD) sequence. Here, whether the RGD sequence functions as a cell-attachment domain was tested. Using site-directed mutagenesis, two mutant recombinant DMP1 proteins with specific alterations at the RGD site were created. In the first mutant protein the RGD sequence was altered to a RGE (RGE) sequence; in the second the RGD domain was deleted (DEL). Mutated proteins were confirmed to be DMP1 by partial protein sequencing and dot-blot analysis with an anti-DMP1 antibody. Attachment of RPC-C2A (dental pulp cells), MC3T3-E1 (calvarial cells) or CHO (Chinese hamster ovary cells) to non-tissue-culture plastic coated with either DMP1, RGE or DEL proteins was compared. Bovine serum albumin and fibronectin served as negative and positive controls, respectively. The RGD-containing native DMP1 protein effectively allowed cell attachment and spreading. The RGE and DEL proteins with the altered and deleted RGD sites were significantly less effective in promoting cell attachment than the recombinant DMP1. Both RPC-C2A pulp cells and MC3T3-E1 cells showed similar reductions in attachment to mutated proteins. Treatment of RPC-C2A cells with a RGD-containing peptide prior to plating on DMP1-coated chambers abolished DMP1-mediated cell attachment. In contrast to RPC-C2A and MC3T3-E1cells, CHO cells, which normally do not express DMP1, failed to attach to DMP1. These data demonstrate that DMP1 promotes cell attachment through the RGD domain and that the attachment is cell- and tissue-specific. A basis for these observations is proposed using computer-generated models of the polypeptides within the DMP1 protein containing the RGD, RGE or DEL sequences.

Application of near-infrared spectroscopy in the pharmaceutical solid dosage form.

Near-infrared (NIR) spectroscopy, one of the most rapidly growing methodologies in pharmaceutical analysis, has been used to analyze the pharmaceutical solid dosage form. The objective of this study was to examine the information that can be gathered from NIR spectroscopy and demonstrate the potential utility of the technique as an alternative to current methods of tablet performance testing. The tablet formulation included active drug (acetaminophen or theophylline), binder (hydroxyethylcellulose), filler (lactose, calcium sulfate, dibasic calcium phosphate dihydrate, or microcrystalline cellulose), and lubricant (magnesium stearate). The compression forces were varied from 5 to 25 kN. A Foss/NIRSystems scanning near-infrared spectrometer was used to measure the diffuse reflectance from the tablet surface. Each tablet was scanned on opposite sides to reduce the effects of positioning. First derivative and multiplicative scatter correction data treatments were explored. A calibration for compression force, independent of the filler, was developed. In addition, the spectra were able to distinguish among the fillers used. A comparison of these spectra with data collected earlier suggests that the technique could differentiate among drugs as well. Near-infrared diffuse reflection spectroscopy, when properly calibrated, can determine the compression force used to prepare a tablet. This measurement may be independent of the different active drugs or fillers used in the tablet formulations.

Arachidonic acid metabolites in saliva of patients with squamous cell carcinoma of the head and neck.

Alterations in the production of arachidonic acid metabolites by squamous epithelium of the upper aerodigestive tract may play a role in the pathogenesis of squamous cell carcinoma of the head and neck. The levels of cyclooxygenase and lipoxygenase metabolites of arachidonic acid metabolism were measured by radioimmunoassay in the saliva of 51 patients with squamous cell carcinoma of the head and neck and compared with a control group of 27 patients with no history of cancer. Levels of leukotriene B4 were significantly increased in cancer patients (p = 0.02), whereas there were no significant differences in levels of prostaglandin E2 or 15-hydroxy-eicosatetranoic acid. Levels of metabolites did not correlate with a history of tobacco use and did not change in a consistent manner after surgery. The significance of arachidonic acid metabolites in the saliva of patients with squamous cell carcinoma of the head and neck is unknown.

Non-fatal deliberate self-poisoning in Dublin's north inner city--an overview.

It is almost three decades since a survey of attempted suicide in Dublin City Hospitals was published. In 1962, 159 cases in the City of Dublin and Borough of Dun Laoghaire were studied and reported. Over a six month period (November 1989-April 1990) 147 cases of deliberate self-poisoning were admitted to the Mater Hospital, Dublin, of which 100 case notes were appropriate for examination. In 1973, a six month survey of attempted suicide was carried out at the Mater Hospital, Dublin where 38 patients were admitted following non-fatal deliberate self-poisoning. In our study, the female to male ratio was 2:1, 50% of cases took more than one drug type,, benzodiazepines being the most commonly ingested compound. Alarmingly over a quarter (28%) of females under 25 had overdosed on paracetamol either alone or in conjunction with other compounds. Alcohol was consumed in 46% of all cases. 40% of cases involved a past history of deliberate self-poisoning and 60% had a past history of psychiatric intervention. Our survey outlines current trends in deliberate self-poisoning in Dublin's North Inner City. We have compared our 1990 survey to the similar survey conducted by our unit seventeen years ago. Several characteristics remain unaltered. The number of overdoses has almost trebled. We have noted a sharp rise in unemployment, alcohol abuse and psychiatric history among female overdoses. We have noted the disturbing introduction of paracetamol overdose particularly among young females (25%). Finally we have documented the introduction of overdosing amongst the homeless population which did not exist in the 1973 survey, but which accounts for 12% of our survey.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct mechanical ventricular actuation for cardiac arrest in humans. A clinical feasibility trial.

Direct mechanical ventricular actuation (DMVA) is a non-blood-contacting method of biventricular cardiac massage which may be applied expediently for total circulatory support. The purpose of this study was to assess the feasibility of DMVA application for patients suffering refractory cardiac arrest. Following informed consent, DMVA was applied in 22 patients. Vascular access for hemodynamic monitoring was possible in only 12 patients, whose outcomes serve as the basis for this report. The mean age of the patients was 48.2 +/- 4.2 years (seven men; five women). The average time from witnessed cardiac arrest to DMVA application was 81 +/- 9 minutes. Application took less than two minutes from the time of skin incision and resulted in immediate hemodynamic improvement. Systolic and diastolic blood pressures averaged 78 +/- 4 and 41 +/- 4 mm Hg, respectively, with a mean cardiac output of 3.14 +/- 0.18 L/min during a mean of 228 +/- 84 minutes of circulatory support (range, 25 minutes to 18 hours). In selected cases the device was temporarily removed for 2 to 3 minutes and open-chest cardiac massage (OCCM) performed at similar compression rates. DMVA increased arterial pressures 65 percent and cardiac output 190 percent compared to OCCM. Initial arterial pH (7.12 +/- 0.04) improved by the time the device was removed (7.24 +/- 0.05). Serum lactate levels decreased from 18.0 +/- 2.3 mumol/L to 14.9 +/- 2.9 mumol/L. Four patients were successfully defibrillated: two had inadequate cardiac function and died within 1 h, and two were successfully resuscitated, but later died from cardiac failure and respiratory insufficiency. Another patient regained normal neurologic function during DMVA and was successfully bridged to cardiopulmonary bypass for emergent coronary artery bypass grafting, but died later from myocardial infarction. There were only two complications: (1) a cardiac laceration during pericardiotomy (1/22 patients); and (2) a ventricular rupture during OCCM (1/22). No complication resulted from the device. We found DMVA to be a feasible method for acute cardiovascular stabilization in victims suffering refractory cardiac arrest. Human clinical trials employing earlier DMVA application are required to determine its resuscitative potential.

Psychogenic skin disease: a review of 35 cases.

In three years of clinical practice, the authors saw 35 cases of dermatological disorders of strictly psychological origin--8 patients with dermatitis artefacta, 8 with delusional parasitosis, and 19 who presented with skin complaints but showed no dermatological pathology ('dermatological non-disease'). All but two initially presented for dermatological opinion rather than psychiatric assessment, and 12 refused psychiatric referral. Demographic and clinical details of all 35 cases are given, including possible related factors, course, treatment, and outcome, and the cases are discussed in the context of the existing literature. Liaison between dermatologists and psychiatrists is strongly advocated.

Effect of (+)-gossypol on fertility in male hamsters.

(+)-Gossypol was isolated from the bark of Thespesia populnea and tested for its ability to inhibit the fertility of male hamsters. Male hamsters of proven fertility were treated orally for 54 days with 40 mg/kg of (+)-gossypol, 40 mg/kg of racemic gossypol, or 5% gum acacia (vehicle control) and were mated with estrous female hamsters during the fourth and seventh weeks of treatment. Both the control and the (+)-gossypol-treated animals exhibited normal fertility throughout the experiment. The racemic gossypol-treated animals were infertile when evaluated during both the fourth and seventh weeks of treatment. Morphologic examination of the testicular tissue could not explain the loss of fertility. These data demonstrate the inability of (+)gossypol to decrease male fertility and suggest that the activity of racemic gossypol may be due primarily to the presence of the (-) optical isomer.