Fluorescence immunohistochemistry and confocal scanning laser microscopy

We have developed a reliable technique for labeling and examining neural structures in soft tissues associated with articular joints and have tested it in human wrist joints under various specimen-related conditions. The labeling protocol employs an immunohistochemical process with a panneuronal marker (PGP 9.5) as the primary antibody and Alexa Fluor 488 as the fluorescing secondary antibody. Imaging was done using a confocal laser scanning microscope, which produced exceptionally detailed three-dimensional images of nerve endings and transiting nerve fibers from thick sections of wrist joint ligaments obtained from human cadavers. The protocol provided a practical postmortem window for specimen acquisition and processing without significant apparent worsening of image quality. The images produced are resistant to fading with repeated exposure to a fluorescent light source, which gives many opportunities for observation. Background staining is minimal, producing high contrast labeling of target tissues, which, in turn, enhances image analysis.     

Hepatocyte paraffin 1 antibody does not distinguish primary ovarian tumors with hepatoid differentiation from metastatic hepatocellular carcinoma.

Anti-hepatocyte antibody, hepatocyte paraffin 1, is a monoclonal antibody that is highly specific for normal and neoplastic hepatocytes and that can differentiate hepatocytic from nonhepatocytic tumors. This marker has been rarely studied in extra-hepatic tumors and to our knowledge has not been investigated in ovarian tumors with hepatoid differentiation. We studied hepatocyte paraffin 1 immunoreactivity in a series of ovarian hepatoid carcinomas, ovarian hepatoid yolk sac tumors (YSTs), and hepatocellular carcinomas metastatic to the ovary to assess the potential utility of hepatocyte paraffin 1 in differential diagnosis. Hepatocyte paraffin 1 positivity was seen in three of seven ovarian hepatoid carcinomas, five of eight hepatoid yolk sac tumors, and six of eight metastatic hepatocellular carcinomas. The extent of positivity ranged from <25% to >50% of the tumor cells. There was strong coarsely granular cytoplasmic staining in all three tumor types without a distinctive staining pattern in any group. The degree of hepatic differentiation correlated with hepatocyte paraffin 1 positivity in the three groups: 83% of the well differentiated tumors, 50% of the moderately differentiated tumors, and none of the poorly differentiated tumors were positive. All ovarian hepatoid carcinomas were either immunoreactive for alpha-fetoprotein or had an elevated serum alpha-fetoprotein level; more than half of these tumors were hepatocyte paraffin 1 negative. All but one hepatocyte paraffin 1 negative hepatoid yolk sac tumor and ovarian hepatocellular carcinoma were also negative for alpha-fetoprotein. In conclusion, hepatocyte paraffin 1 is positive in primary ovarian tumors with hepatoid differentiation, with the degree of hepatocyte paraffin 1 positivity correlating with the degree of hepatoid differentiation. Hepatocyte paraffin 1, however, is not useful in distinguishing metastatic hepatocellular carcinoma from primary ovarian hepatoid carcinoma or hepatoid yolk sac tumor.     

Proteolytic Processing Mechanisms in the Biosynthesis of Neuroendocrine Peptides: The Subtilisin-like Proprotein Convertases

The recent discovery of a novel family of precursor processing endoproteases has greatly accelerated progress in understanding the complex mechanisms underlying the maturation of prohormones, neuropeptides, and many other precursor-derived proteins. At least six members of this family have been found thus far in mammalian species, several having alternatively spliced isoforms, and related enzymes have been identified in many invertebrates, including molluscs, insects, nematodes, and coelenterates. The proprotein convertases are all dependent on calcium for activity and all possess highly conserved subtilisin-like domains with the characteristic catalytic triad of this serine protease (ordered Asp, His, and Ser along the polypeptide chain). Two members of this family, PC2(SPC2) and PC1/PC3(SPC3), appear to play a preeminent role in neuroendocrine precursor processing. Both convertases are expressed only in the brain and in the extended neuroendocrine system, while another important family member—furin/PACE (SPC1)—is expressed more ubiquitously, in almost all tissues, and at high levels in liver. SPC2 and SPC3 exhibit acidic pH optima and other properties which enhance their activity in the acidic, calcium-enriched environment of the dense-core secretory granules of the regulated pathway in neuroendocrine cells, while furin has a neutral pH optimum and is localized predominantly to the trans Golgi network where it is retained by a C-terminal transmembrane domain. Furin processes a wide variety of precursors in the constitutive pathway, such as those of growth factors, receptors, coagulation factors, and viral glycoproteins. Recent findings on the processing of proopiomelanocortin, proinsulin, proglucagon, and several other neuroendocrine precursors by SPC2 and SPC3 are discussed, along with information on the structure, properties, evolution, developmental expression, and regulation or the convertases. An inherited defect in the fat/fat mouse which affects the processing of proinsulin, and probably also many other prohormones, due to a point mutation in carboxypeptidase E has recently been identified and has begun to provide new insights into the functional integration of the individual processing steps.     

Role of vasopressin on excitatory amino acids mediated pressor responses in the periaqueductal gray area

In order to evaluate the role played by vasopressin on pressor responses elicited by stimulation of the periaqueductal gray (PAG) area by excitatory amino acids we carried out in vivo studies in genetically vasopressin deficient rats (Brattleboro). Microinjections of l-glutamic acid (glutamate, 0.6 to 60 nmol/rat) or N-methyl-d-aspartic acid (NMDA, 0.07 to 7 nmol/rat) into the PAG area of freely moving Brattleboro rats induced increases of arterial blood pressure values significantly lower than those obtained in Long Evans rats (control) (glutamate in Brattleboro rats: from +2±1 mmHg to 16±3 mmHg; glutamate in Long Evans rats: from +16±2 mmHg to +36±4 mmHg; NMDA in Brattleboro rats: from +5±2 mmHg to +34 ±8 mmHg; NMDA in Long Evans rats: from +18±7 mmHg to 80±9 mmHg; n=5). Similarly, in anaesthetized Brattleboro rats (urethane 1.2 g/kg i.p.) pressor responses to NMDA microinjections (0.7 nmol/rat) into the PAG area were significantly lower than in Long Evans rats (controls) (+15±3 mmHg vs +24±4 mmHg). In Long Evans rats NMDA injection also reversed blood pressure decrease induced by ganglionic blocker, hexamethonium and/or losartan (3 mg/kg i.v.), an AT1 receptor antagonist. In Brattleboro rats, NMDA injection did not reverse blood pressure decreases induced by hexamethonium (5 mg/kg i.v.). Moreover, hexamethonium induced blood pressure decrease was not reversed by acetylcholine injection (137 nmol/rat) into the PAG area of anaesthetized Long Evans rats, but if injected before hexamethonium, acetylcholine was able to increase blood pressure (+25±3 mmHg). Our results document: i) the importance of the PAG area in the control of cardiovascular system; ii) the involvement of excitatory amino acids in the neural control of vasopressin release; iii) the close relationship between glutamate and vasopressin in the central blood pressure regulation. Received: 1 April 1997 / Accepted: 2 February 1998     

Managing COPD: no more nihilism!

This special issue of Patient Education and Counseling is long overdue. During most of the last two decades asthma, and notably asthma self-management has been in the spotlight, while COPD has had to endure a nihilistic approach. The first sign that interest was shifting to the treatment of COPD came from a few large randomized trials on the use of inhaled corticosteroids (ICS) in COPD. Although these studies demonstrated a moderate effect of ICS in COPD, it has become clear that true improvements in the management of this chronic disease will have to come from behavioral interventions. This special issue of Patient Education and Counseling is dedicated solely to the non-pharmaceutical management of COPD. It addresses many issues related to behavioral therapy, such as smoking cessation, exercise training, nutritional aspects, and self-management programs, including action plans to self-treat exacerbations. With the availability of all the treatment and management options, described in this special issue, a nihilistic attitude toward the patient with COPD is no longer justified.     

Depletion of long-lived lymphocytes in old New Zealand black mice

Labelling studies with tritiated thymidine in NZB mice revealed that old mice of this strain with established Coombs positive haemolytic anaemia were depleted predominantly of long-lived small lymphocytes. Lymphopoiesis in the bonemarrow was relatively less affected suggesting that the peripheral destruction or deviation of re-circulating lymphocytes and not a precursor cell deficiency may account for the depletion of these cells. Old NZB mice in which the development of Coombs positive haemolytic anaemia, but not other disease features, has been prevented by thymectomy and treatment with anti-lymphocyte globulin several months earlier were equally deficient in this population of lymphocytes. C57BL mice, subjected to the same regime largely regenerated this population during the subsequent 12 months and rejected skin homografts in the same time as young controls of the same strain. Loss of lymphocytes responsible for antigen recognition would account for the impaired cellular immunity of old NZB mice observed in this study and described earlier by other authors. This process appears to accompany the progression of the spontaneous disease in this strain.     

Nursing constraint models for electronic health records: a vision for domain knowledge governance

Various forms of electronic health records (EHRs) are currently being introduced in several countries. Nurses are primary stakeholders and need to ensure that their information and knowledge needs are being met by such systems information sharing between health care providers to enable them to improve the quality and efficiency of health care service delivery for all subjects of care. The latest international EHR standards have adopted the openEHR approach of two-level modelling. The first level is a stable information model determining structure, while the second level consists of constraint models or 'archetypes' that reflect the specifications or clinician rules for how clinical information needs to be represented to enable unambiguous data sharing. The current state of play in terms of international health informatics standards development activities is providing the nursing profession with a unique opportunity and challenge. Much work has been undertaken internationally in the area of nursing terminologies and evidence-based practice. This paper argues that to make the most of these emerging technologies and EHRs we must now concentrate on developing a process to identify, document, implement, manage and govern our nursing domain knowledge as well as contribute to the development of relevant international standards. It is argued that one comprehensive nursing terminology, such as the ICNP or SNOMED CT is simply too complex and too difficult to maintain. As the openEHR archetype approach does not rely heavily on big standardised terminologies, it offers more flexibility during standardisation of clinical concepts and it ensures open, future-proof electronic health records. We conclude that it is highly desirable for the nursing profession to adopt this openEHR approach as a means of documenting and governing the nursing profession's domain knowledge. It is essential for the nursing profession to develop its domain knowledge constraint models (archetypes) collaboratively in an international context.     

HLA antigens in ulcerative colitis: a study in the Sicilian population

HLA antigens were investigated in 41 Sicilian patients with ulcerative colitis and in 151 healthy controls. Frequencies of HLA-B5 and DR2 were increased in the group of patients with ulcerative colitis whereas the DR3 antigen frequency was decreased. However the corrected p values were not significant. Thus, present results indicate that in ulcerative colitis HLA linked genetic factors play a marginal role, if any.     

Skill needs for nurses in their role as health informatics professionals: a survey in the context of global health informatics education

In the process of developing global health informatics education, a common understanding of educational outcomes is required. Therefore, an educational framework for health informatics professionals is desirable to support student mobility, trans-national and borderless education. Nurses form a significant part of the health workforce and need to be properly educated for their roles in health informatics. To ascertain their perceptions of needs and priorities, we developed a web-based questionnaire and surveyed Australian nurses on the preferred knowledge/skills set for health informatics professionals. Among others, the questionnaire is based on the International Medical Informatics Association's (IMIA) set of recommendations on education and IMIA's scientific map. Benner's five levels of competencies were applied to measure the degree of competency required for each skill/knowledge. Altogether, 82 Australian nurses completed the questionnaire. The nurses' perceived degree of competency required for a total of 74 specific skills and knowledge in five skill categories is presented in this paper as well as the overall results for each of the five categories. Further, significant differences between the nurses' primary roles and primary interest in health informatics are discussed. The development of a comprehensive health informatics education framework needs to take into account nurses as well as other health professionals. Repeating the survey in other countries and for various professions is essential to develop an international educational framework.     

Immunoglobulin G1 enzyme-linked immunosorbent assay for diagnosis of Johne's Disease in red deer (Cervus elaphus)

This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne's disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured protoplasmic antigen (PpAg). ELISA development was based on the antigen reactivity of the immunoglobulin G1 (IgG1) isotype, which is a highly specific marker for mycobacterial disease seroreactivity in deer. Sensitivity estimates and test parameters were established using 102 Mycobacterium paratuberculosis-infected animals from more than 10 deer herds, and specificity estimates were determined using 508 uninfected animals from 5 known disease-free herds. A receiver-operated characteristic analysis determined that at a cut point of 50 ELISA units, there was a specificity of 99.5% and sensitivities of 84.0% with PPDj antigen, 88.0% with PpAg, and 91.0% when the antigens were used serially in a composite test. Estimated sensitivity was further improved using recombinant protein antigens unique for M. paratuberculosis, which identified infected animals that were unreactive to PPDj or PpAg. While 80% of animals that were seropositive in the IgG1 ELISA had detectable histopathology, the assay could also detect animals with subclinical disease. The test was significantly less sensitive (75%) for animals that were culture positive for M. paratuberculosis but with no detectable pathology than for those with pathological evidence of JD (>90%). When the IgG1 ELISA was used annually over a 4-year period in a deer herd with high levels of clinical JD, it eliminated clinical disease, increased production levels, and reduced JD-related mortality.