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Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder characterised by the degeneration of motor neurons and progressive muscle weakness. It is caused by homozygous deletions in the survival motor neuron gene on chromosome 5. SMA shows a wide range of clinical severity, with SMA type I patients often dying before 2 years of age, whereas type III patients experience less severe clinical manifestations and can have a normal life span. Here, we describe the design, setup and utilisation of the TREAT-NMD national SMA patient registries characterised by a small, but fully standardised set of registry items and by genetic confirmation in all patients. We analyse a selection of clinical items from the SMA registries in order to provide a snapshot of the clinical data stratified by SMA subtype, and compare these results with published recommendations on standards of care. Our study included 5,068 SMA patients in 25 countries. A total of 615 patients were ventilated, either invasively (178) or non-invasively (437), 439 received tube feeding and 455 had had scoliosis surgery. Some of these interventions were not available to patients in all countries, but differences were also noted among high-income countries with comparable wealth and health care systems. This study provides the basis for further research, such as quality of life in ventilated SMA patients, and will inform clinical trial planning.

Background: The World Health Organization (WHO) has recommended measurement of neonatal thyroid-stimulating hormone (TSH) as a marker of population iodine status. A population is considered iodine sufficient when <3% of neonatal blood samples collected 3-4 days after birth have TSH concentrations >5 mIU/L. However, changes in technology and clinical practices have opened the WHO criteria to various interpretations., Aim: This study aimed to investigate the effects of time of sampling, weight, and sex on neonatal TSH concentrations by analyzing neonatal TSH data, based on the WHO criteria for population iodine sufficiency., Methods: We analyzed the Western Australian (WA) Newborn Screening Program records for 198,826 babies born in WA between 2005 and 2011, to determine the relationship between neonatal TSH concentrations and time of sampling, weight, and sex., Results: The proportion of TSH results above the WHO cut-off was higher for samples collected 48-72 h after birth rather than later, for males, for birth weights below 2500 g, and when a cut-off of 5.0 mIU/L was used., Conclusion: Following changes in newborn screening protocols and earlier collection of blood samples, the WHO criteria appear inappropriate. We recommend that WHO revise current guidelines regarding use of neonatal TSH for monitoring population iodine status.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease, caused by the absence of the dystrophin protein. Although many novel therapies are under development for DMD, there is currently no cure and affected individuals are often confined to a wheelchair by their teens and die in their twenties/thirties. DMD is a rare disease (prevalence <5/10,000). Even the largest countries do not have enough affected patients to rigorously assess novel therapies, unravel genetic complexities, and determine patient outcomes. TREAT-NMD is a worldwide network for neuromuscular diseases that provides an infrastructure to support the delivery of promising new therapies for patients. The harmonized implementation of national and ultimately global patient registries has been central to the success of TREAT-NMD. For the DMD registries within TREAT-NMD, individual countries have chosen to collect patient information in the form of standardized patient registries to increase the overall patient population on which clinical outcomes and new technologies can be assessed. The registries comprise more than 13,500 patients from 31 different countries. Here, we describe how the TREAT-NMD national patient registries for DMD were established. We look at their continued growth and assess how successful they have been at fostering collaboration between academia, patient organizations, and industry., (© 2013 WILEY PERIODICALS, INC.)

Rare disease registries (RDRs) are an essential tool to improve knowledge and monitor interventions for rare diseases. If designed appropriately, patient and disease related information captured within them can become the cornerstone for effective diagnosis and new therapies. Surprisingly however, registries possess a diverse range of functionality, operate in different, often-times incompatible, software environments and serve various, and sometimes incongruous, purposes. Given the ambitious goals of the International Rare Diseases Research Consortium (IRDiRC) by 2020 and beyond, RDRs must be designed with the agility to evolve and efficiently interoperate in an ever changing rare disease landscape, as well as to cater for rapid changes in Information Communication Technologies. In this paper, we contend that RDR requirements will also evolve in response to a number of factors such as changing disease definitions and diagnostic criteria, the requirement to integrate patient/disease information from advances in either biotechnology and/or phenotypying approaches, as well as the need to adapt dynamically to security and privacy concerns. We dispel a number of myths in RDR development, outline key criteria for robust and sustainable RDR implementation and introduce the concept of a RDR Checklist to guide future RDR development.

Background: Deliberative public engagement has been proposed for policy development, where issues are complex and there are diverse public perspectives and low awareness of competing issues. Scholars suggest a range of potential outcomes for citizens and government agencies from involvement in such processes. Few studies have examined outcomes from the perspective of citizen participants in deliberative processes., Aims: To examine participant perceptions of their involvement in and outcomes of a deliberative engagement exercise., Method: A case study using semistructured interviews was conducted with participants following a deliberative forum on biobanking., Results: From their involvement in the deliberative exercise, participants described transformations in their knowledge and beliefs about the policy issues. They reported being more informed to the extent of having confidence to educate others and effectively contribute to public policy development. They had developed greater trust in government policymakers who they believed would take reasonable account of their recommendations., Conclusion: We conclude that the participants were satisfied with the outcomes of the deliberative public engagement process and viewed it as an effective means of citizen involvement in public policy development. Particularly for citizens who participate in deliberative processes, such processes may promote active citizenship, empower citizens to undertake representative and educative roles, and improve relations between citizens and government agencies. Actions taken by policymakers subsequent to the deliberative exercise, whereby the majority of citizen recommendations were incorporated in the policy developed, may have contributed to participants holding sustained levels of trust in the commissioning government agency.

Objective: In light of prior inconsistent findings, this study revisits the relationship between weight suppression and treatment outcome in bulimia nervosa. Aside from differences in methodology, we propose that moderator effects may assist the field in interpreting previous inconsistency. In this study, we considered moderators that might place individuals at risk of broad cognitive and biobehavioral mechanisms implicated in weight (dys)regulation and binge eating, and that within the context of a history of weight suppression, might be associated with especially poor outcomes., Method: Participants were 117 female outpatients aged 16-54 years (M = 25.5) with bulimic disorders treated with enhanced cognitive behavioral therapy., Results: Logistic regression indicated that higher pretreatment weight suppression did not predict drop-out or poor treatment outcome (nonabstinence from binging and purging). Moderators of parental history of overweight, childhood body shape, pretreatment body mass index, and the difference between highest and lowest ever adult body weight were analyzed, but no moderator effects were apparent., Discussion: This study, along with other negative studies, calls into question the association between weight suppression and treatment outcome. We maintain that moderators may account for inconsistencies, but no candidates were identified in this study. Moderator models could assist us to refine conceptualizations of why some patients high in weight suppression may be vulnerable to poor treatment adherence and outcome and to establish clinical interventions that enhance prognosis., (Copyright © 2013 Wiley Periodicals, Inc.)

With advances in therapeutics for rare, genetic and syndromic diseases, there is an increasing need for objective assessments of phenotypic endpoints. These assessments will preferentially be high precision, non-invasive, non-irradiating, and relatively inexpensive and portable. We report a case of a child with an extensive lymphatic vascular malformation of the head and neck, treated with an mammalian target of Rapamycin (mTOR) inhibitor that was assessed using 3D facial analysis. This case illustrates that this technology is prospectively a cost-effective modality for treatment monitoring, and it supports that it may also be used for novel explorations of disease biology for conditions associated with disturbances in the mTOR, and interrelated, pathways.

Background: Public deliberation is recommended for obtaining citizen input to policy development when policies involve contested ethical dimensions, diverse perspectives on how to trade-off competing public interests and low public awareness of these perspectives. Several norms have been proposed for the design of deliberative methods. Evidence is scarce regarding whether such norms are achievable in practice., Purpose: This paper refers to principles of deliberative democracy theory to describe a deliberative public forum on biobanking. Practical challenges and contextual facilitators of achieving deliberative ideals are discussed, along with factors that influenced use of the forum output in policy development., Method: The forum ran for 4 days over two weekends in Perth, Western Australia. Key methodological features were socio-demographic stratification to randomly recruit a mini-public of citizens for discursive representation, provision of information inclusive of diverse perspectives and framed for difference, provision of a fair way for reasoning and collective decision making and adoption of processes to achieve publicity, accountability and independence from undue institutional influence., Results: Most design principles were achieved in practice, with the fundamental exception of representativeness. Factors influencing these outcomes, and the use of deliberated outputs to develop policy, included institutional characteristics, the design involvement of deliberative experts and quality of the outputs when compared to other consultation methods., Conclusions: Public deliberations can achieve design ideals and influence (ethics-based) public health policy. The representation of 'hard to reach' citizens and their views needs further consideration, particularly as this relates to the procedural legitimacy of ethical analyses and the just inclusion of deliberative citizen advice within the broader policy-making process., (© 2011 John Wiley & Sons Ltd.)

Three-dimensional (3D) facial analysis is ideal for high-resolution, nonionizing, noninvasive objective, high-throughput phenotypic, and phenomic studies. It is a natural complement to (epi)genetic technologies to facilitate advances in the understanding of rare and common diseases. The face is uniquely reflective of the primordial tissues, and there is evidence supporting the application of 3D facial analysis to the investigation of variation and disease including studies showing that the face can reflect systemic health, provides diagnostic clues to disorders, and that facial variation reflects biological pathways. In addition, facial variation has been related to evolutionary factors. The purpose of this review is to look backward to suggest that knowledge of human evolution supports, and may instruct, the application and interpretation of studies of facial morphology for documentation of human variation and investigation of its relationships with health and disease. Furthermore, in the context of advances of deep phenotyping and data integration, to look forward to suggest approaches to scalable implementation of facial analysis, and to suggest avenues for future research and clinical application of this technology., (© 2012 WILEY PERIODICALS, INC.)

The development of effective treatments for neuromuscular diseases is a significant challenge due to difficulties in identifying adequate numbers of patients for clinical trials. Low patient numbers in these rare diseases also has an effect when establishing sound clinical practices based on experience gained from patients with similar diagnosis. The Muscular Dystrophy Association of New Zealand (MDA), working in consort with interested clinicians has established the New Zealand Neuromuscular Disease (NZ NMD) Registry in order to help address these problems. The NZ NMD Registry is exceptional in that it comprises one registry for all neuromuscular conditions and will significantly benefit both patients with neuromuscular disease and their clinicians., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

There is a need to develop Internet-based rare disease registries to support health care stakeholders to deliver improved quality patient outcomes. Such systems should be architected to enable multiple-level access by a range of user groups within a region or across regional/country borders in a secure and private way. However, this functionality is currently not available in many existing systems. A new approach to the design of an Internet-based architecture for disease registries has been developed for patients with clinical and genetic data in geographical disparate locations. The system addresses issues of multiple-level access by key stakeholders, security and privacy. The system has been successfully adopted for specific rare diseases in Australia and is open source. The results of this work demonstrate that it is feasible to design an open source Internet-based disease registry system in a scalable and customizable fashion and designed to facilitate interoperability with other systems., (© 2012 Wiley Periodicals, Inc.)

Background: Calls have been made for governments to adopt a cohesive approach to rare diseases through the development of national plans. At present, Australia does not have a national plan for rare diseases. To progress such a plan an inaugural Australian Rare Diseases Symposium was held in Western Australia in April 2011. This paper describes the key issues identified by symposium attendees for the development of a national plan, compares these to the content of EUROPLAN and national plans elsewhere and discusses how the outcomes might be integrated for national planning., Methods: The symposium was comprised of a series of plenary sessions followed by workshops. The topics covered were; 1) Development of national plans for rare diseases; 2) Patient empowerment; 3) Patient care, support and management; 4) Research and translation; 5) Networks, partnerships and collaboration. All stakeholders within the rare diseases community were invited to participate, including: people affected by rare diseases such as patients, carers, and families; clinicians and allied health practitioners; social and disability services; researchers; patient support groups; industry (e.g. pharmaceutical, biotechnology and medical device companies); regulators and policy-makers., Results: All of these stakeholder groups were represented at the symposium. Workshop participants indicated the need for a national plan, a national peak body, a standard definition of 'rare diseases', education campaigns, lobbying of government, research infrastructure, streamlined whole-of-lifetime service provision, case co-ordination, early diagnosis, support for health professionals and dedicated funding., Conclusions: These findings are consistent with frameworks and initiatives being undertaken internationally (such as EUROPLAN), and with national plans in other countries. This implies that the development of an Australian national plan could plausibly draw on frameworks for plan development that have been proposed for use in other jurisdictions. The translation of the symposium outcomes to government policy (i.e. a national plan) requires the consideration of several factors such as the under-representation of some stakeholder groups (e.g. clinicians) and the current lack of evidence required to translate some of the symposium outcomes to policy options. The acquisition of evidence provides a necessary first step in a comprehensive planning approach.

Background: Deliberative public engagement is recommended for policy development in contested ethical areas. Scholars provide little guidance on how deliberative outputs can be translated to policy. This paper describes the processes we undertook to design a deliberative public forum for citizens to develop recommendations on biobanking that were adopted as health policy., Method: The 4-day forum, held in 2008 in Perth, Western Australia, was designed in collaboration with academic experts. Deliberant recommendations were recorded in a formal report presented to policy-makers. Deliberations were audio-taped and transcribed. Translation involved transcript analyses, comparison of recommendations to other stakeholder views and post-forum consultations., Results: Sixteen citizens made recommendations on ethical, legal and social issues related to biobanking. Most recommendations were translated into biobanking guidelines, with which Western Australia government health agencies must comply. The value of deliberative public participation in policy-making was most evident when trade-offs in competing interests, hopes and concerns were required. Translation issues included the impact of a small number of participants with limited socio-demographic diversity on procedural and policy legitimacy., Conclusions: Assessing the sufficiency of diversity in citizen representation was central to the deliberation-to-translation process. Institutional context facilitated the uptake of deliberation and translation processes. The use of these processes influenced policy substance and credibility among stakeholders and contributed to the state government directive that policy compliance be mandatory. We urge others to publish deliberation-to-translation processes so that best-practices may be identified., (Copyright © 2011 S. Karger AG, Basel.)

There are two objectives of this work. The first objective is to study the hydrolysis behavior of monetite cements formed in the presence and absence of the chitosan in cell culture media (α-MEM) and simulated body fluid (SBF) solutions at 37°C. During hydrolysis, monetite transformed to carbonated apatite. Therefore, the second objective is to examine how addition of chitosan affects on the formation of carbonated apatite phases. The changes in the phase structure of monetite after hydrolysis reactions were characterized using XRD, FTIR and SEM. Pure monetite and monetite/chitosan composite were soaked in α-MEM and SBF solution for 4 and 7 days. In α-MEM solution, the monetite particles started to transform into carbonated apatite with a slow rate. However, in SBF, the rate of monetite transformation to carbonated apatite was more rapid. The presence of the chitosan had no significant effect on the precipitation of carbonated apatite on the monetite particles.

Background: Basiliximab is an anti-CD25 monoclonal antibody used as induction therapy in solid-organ transplantation. In this study we aim to determine whether pre-operative administration of basiliximab is beneficial in preventing early heart allograft rejection., Methods: In this investigation we assess the effect of pre-implantation basiliximab on CD25 count and on acute rejection in children undergoing cardiothoracic transplantation. The notes of all children undergoing cardiothoracic transplantation at the Great Ormond Street Hospital between January 2000 and June 2007 were retrospectively reviewed. One hundred twenty-one heart transplant recipients were included: 29 patients did not receive basiliximab; 33 patients received basiliximab after coming off cardiopulmonary bypass (CPB); and 59 patients received basiliximab prior to organ implantation., Results: All patients receiving basiliximab had an effectively suppressed CD25 count (<0.2%) on Days 1 and 10 post-transplant. Freedom from Grade 3A or greater rejection in the first year was significantly greater in the pre-implantation basiliximab group than in the post-implantation and no-basiliximab groups (95%, 70% and 72%, respectively; p = 0.02). Induction regimen was the only significant explanatory variable after multivariate Cox regression., Conclusions: The results of this study confirm that basiliximab is effective at suppressing CD25 count whether given pre- or post-CPB. Basiliximab before transplantation appeared to reduce acute rejection, whereas post-CPB administration did not suggest similar effects. These findings require independent validation in randomized trials and further studies should seek to mechanistically delineate these observations.

Due to concerns over the side effects of cyclosporine, tacrolimus is widely used in pediatric heart transplantation. However, tacrolimus therapy is also accompanied by potentially serious side effects. This paper examines the side effect profile of tacrolimus in a large group of pediatric heart recipients. Data on renal function, diabetes, hyperlipidemia and hypertension were collected by case-note review of 100 patients who had received . OR = 12 months treatment with tacrolimus. Forty-two patients received tacrolimus from the time of transplant (de novo), and 58 were initially treated with cyclosporine (switch). Mean estimated glomerular filtration rate improved in the first six months post transplant in the de novo group (66.7-84.6 mL/min/1.73 m2, p = 0.002). Conversely, it decreased in those initially treated with cyclosporine (82.1-68.8, p = 0.032), but improved after switch to tacrolimus (77.3-85.6, p = 0.006). Twenty-one percent exhibited glucose intolerance, and 2% had diabetes. Borderline or elevated fasting cholesterol levels were present in 4.4%. Hypertension was seen in 67% at the point of switch from cyclosporine, which fell to 36% at latest follow-up (p = 0.001). These results present an encouraging outlook for this cohort of patients. The relatively low levels of complications shown may be due to early weaning of steroids, and concomitant statin therapy.

With growing numbers of children with complex congenital heart disease surviving initial surgical procedures, more patients are presenting in later childhood or early adulthood in cardiac failure. This presents an obvious increased burden on transplant centres, and a further strain on a limited donor pool. Historically, results for heart transplant following congenital heart disease (CHD) have been worse than those following cardiomyopathy. With increased surgical experience and intensive care expertise, the gap between the two aetiologies in our practice is decreasing. This article reviews the current protocols for transplantation in this setting, presenting a large single-centre experience over 20 years, and speculates on possible future advancements in this very challenging field.

Sarcomatoid renal cell carcinoma (SRCC) is an aggressive tumor variant thought to arise predominantly from dedifferentiation of clear cell carcinoma. A few reports of SRCC associated with non-clear cell tumors led to the presumption that SRCC may arise from any renal cell carcinoma, although direct evidence of this is lacking. Cytogenetic studies on 3 previously documented SRCCs associated with papillary renal cancers showed either 3p deletions or absence of trisomy 7, 17 in the sarcomatoid tumors, suggesting origin from a coexistent clear cell tumor. The present case represents the first conclusive evidence of direct progression of non-clear cell carcinoma to SRCC with both tumor components containing multiple copies of chromosomes 7 and 17. Many genetic anomalies, including p53 mutations, frequently recognized in SRCC were not recognized in this case, highlighting the importance of cytogenetic evaluation of all SRCC. The patient is well and without evidence of tumor progression 1 year after surgery, and the sinister outlook of SRCC in association with clear cell carcinoma may not apply in SRCC of non-clear cell origin.

Background: Prostate ducts and acini whose lumens are filled with malignant cells represent a well-recognized histological pattern recently termed intraductal carcinoma of the prostate (IDC-P). These tumors are often associated with rapid disease progression, and most recur after radical surgery. Controversy exists as to whether IDC-P should be recognized as a separate entity, an extension of high-grade dysplasia (PIN) or invasive carcinoma as described by the Gleason grading system. This study investigates the use of molecular markers in defining the position of IDC-P in the evolutionary hierarchy of prostate cancer progression., Methods: IDC-P, high-grade dysplasia, and invasive cancers from a cohort of 20 selected radical prostatectomy specimens were screened for loss of heterozygosity (LOH), using 12 polymorphic microsatellite markers frequently lost in prostate cancer., Results: LOH was absent in Gleason grade 3 cancer, infrequent in high-grade dysplasia (9%) and Gleason grade 4 cancer (29%), but common in IDC-P (60%). In IDC-P, and to a lesser extent Gleason grade 4 cancers, multiple sites of allelic loss in individual cases were usual., Conclusions: Allelic instability provides further evidence that IDC-P is not a simple extension of dysplasia, nor does it represent invasion of Gleason grade 3 cancers into the ductal/acinar system. IDC-P and Gleason grade 4 cancer represent late but possibly separate events in prostate cancer evolution., (Copyright 2000 Wiley-Liss, Inc.)

Background: Prostate secretory granules (PSG) represent the basic secretory unit of the prostate gland, containing many of its exocrine proteases. Recent analysis of intraluminal corpora amylacea, a proposed by-product of PSG secretion, detected sulfated glycosaminoglycans (GAG) possibly keratan sulfate (KS), indicating a secretory mechanism for GAG in the human prostate surface epithelial cell., Methods: Immunostains using anti-KS and anti-prostate-specific antigen (PSA) were evaluated on 10 sequential radical prostatectomy specimens, three of which had received neoadjuvant antiandrogen therapy. Extracts of normal secretory tissue as well as a sample composed almost entirely of prostatic stroma were subjected to Western blot analysis, using the same antibody panel., Results: Keratan sulfate secretion from the normal prostate epithelial cell has been confirmed and correlates, as does PSA, with the presence of cytoplasmic PSG. No such correlation exists in most adenocarcinomas or in benign epithelium after androgen ablation. Western blot analyses confirmed tissue immunostains and demonstrated a secretory proteoglycan of 70-95 kDa., Conclusions: Recognition of PSG heralds a novel secretory mechanism within the human prostate gland that is linked to the secretion of KS. The role of KS in normal prostate secretion remains unknown, although it appears downregulated in neoplastic and androgen-ablated cells., (Copyright 2000 Wiley-Liss, Inc.)

Separation of renal cell tumors into different prognostic groups is an imperative function of the diagnostic pathologist. Recently, chromophobe renal carcinoma has been described as a tumor that is morphologically distinct from conventional "clear cell" carcinoma and that has a low metastatic potential. Identification is based on routine light microscopic features and is confirmed by special stains, immunohistochemistry, and electron microscopy. We present a variant of chromophobe renal carcinoma that did not show the typical cytomorphologic features on light microscopy after formaldehyde fixation. After fixation in Solufix (a commercial fixative), these features were recognized and the diagnosis was confirmed. The tumor also showed an unusual form of calcification and psammoma body formation not previously recognized in chromophobe tumors. Molecular biological assessment was inconclusive, but excluded a chromosome 3p deletion usually found in conventional renal carcinoma. The use of a different primary fixative may provide a cost-effective screening tool to detect variant renal tumors and may have important prognostic implications.

Recent changes in tissue fixation strategy, using glutaraldehyde, have clarified the secretory mechanisms of the normal prostate identifying cytoplasmic prostatic secretory granules, structures not preserved by formalin fixation. This normal secretory mechanism was absent in most adenocarcinomas, depicting an important metabolic change in transformed prostate cells. The current study further investigates differences between benign and malignant prostate secretion and relates them to the production of corpora amylacea by benign glands and crystalloids or mucin by cancer. In all normal prostate cells examined (6 cases), prostate secretory granules (PSG) were approximately 1-microm, brightly eosinophilic granules filling the cytoplasm of secretory cells and released in packets by a specialized apocrine cell structure. After apocrine decapitation and luminal dispersal, some of the cytoplasmic and PSG remnants condensed to form eosinophilic bodies (EB) with a glycoprotein rim and central protein core. EB were observed adsorbing and layering onto the surface of prostatic corpora amylacea representing their chief mode of enlargement. Biochemical analysis and x-ray diffraction studies confirmed sulfated glycosaminoglycans of similar structure as the main constituent of both PSG and corpora amylacea. Peripheral zone amphiphilic "dark cell" carcinoma (9 cases) contained almost no PSG, and showed neither apical decapitation nor EB formation, but mucin secretion was frequently detected. Crystalloids that share the same staining characteristics and sulfur content as PSG and corpora amylacea were identified in 3 selected "clear cell" carcinomas, all of which showed at least focal PSG secretion. The recognition of these differing secretory mechanisms and their deviation from normal further defines the histological criteria and spectrum of prostate malignancy.

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)

Cytoplasmic clarity is a histological feature of normal prostatic secretory cells, but in this study, tissue fixation in strong (>2.5%) glutaraldehyde dramatically altered cytological staining. Secretory cytoplasm appeared red and granular on routine stains because of myriad intensely staining eosinophilic granules (PSG). Immunostaining for prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) showed their exclusive localization to the PSG. Electron microscopy confirmed these findings and also showed that after fixation in many agents, including formaldehyde, PSG appeared empty, accounting for the artefactual "clear cell" appearance on light microscopy. PSG were most densely concentrated apically in a bud-shaped luminal compartment in which cytokeratin was selectively absent. Normal exocrine secretion was visualized as detachment of apocrine buds or their in situ disintegration. Distinctively in dysplasia and almost all carcinomas, PSG were rare to absent, and proteases were free in the cytoplasm, often concentrated beneath the apical membrane. The apocrine compartment was absent, with no observed secretory mechanism. Tumor cells had dark amphiphilic cytoplasm after all fixatives. This provided a reliable method of distinguishing malignant from benign glands in tissues fixed in strong glutaraldehyde. Clear cell carcinomas, whose cytoplasm mimicked routinely fixed normal secretory cells, surprisingly had almost no PSG. Instead, their "granules" were lipid-filled vacuoles reflecting a secretory pathway not seen in normal cells, dysplasia, or the common "dark cell" carcinomas. These observations may define two distinctive biological pathways of prostate cancer evolution and may facilitate diagnostic decisions on needle biopsy samples.

Human prostatic secretion and seminal plasma contain certain protein kinase activities. Protein kinases play important roles in regulating a vast variety of cellular functions. The objective of this study was to determine whether one of these protein kinase activities in human prostatic secretion and seminal plasma is due to CK2, a messenger-independent, serine/threonine protein kinase that has considerable potential as a regulatory enzyme. By employing an anti-CK2 antibody and a CK2-specific peptide substrate, we have established that CK2 is present in these secretions. Approximately 70% of the CK2 activity present in seminal plasma of normozoospermic men (n = 49) is correlated to the number of sperm originally present in the semen. Further, both the prostate gland and the seminal vesicles are sources of CK2 activity in the seminal plasma of vasectomized men (n = 38). Although there was considerable variation between individuals in CK2 activity, the variation in repeat semen samples of the same vasectomized men (n = 6) was within 21%. There was no correlation of CK2 activity in seminal plasma with age for vasectomized (27-48 years, n = 38), oligozoospermic (28-43 years, n = 24), or normozoospermic men (26-48 years, n = 49). These data suggest that the majority of CK2 activity in the seminal plasma of normozoospermic men originates from sperm but that the prostate and seminal vesicles are accessory sex-gland sources of this enzyme.

Purpose: A number of peptidases which can metabolize certain bioactive peptides and growth factors have been identified in seminal plasma. Our goal in this study was to determine molecular properties and the tissue source(s) for one of these peptidases, dipeptidylpeptidase IV (DPP IV), in human seminal plasma., Materials and Methods: We measured the activities of DPP IV with the dipeptide glycylprolyl-p-nitroanalide and its molecular forms using immunoblotting of seminal plasmas of men who were vasectomized or with different sperm concentrations, and in prostatic and seminal vesicle secretions of men undergoing prostatic surgery., Results: DPP IV in seminal plasma of vasectomized men was a membrane associated dimer comprised of subunits of approximately 110 kDa. Its activity did not differ in seminal plasmas of vasectomized, azoospermic, oligozoospermic and normozoospermic men indicating no correlation with the concentration of sperm originally present in the semen. The DPP IV antigen (CD -26) and enzymic activity were present in prostatic secretion, but absent from that of the seminal vesicles. These data indicate that the prostate gland is the primary source of DPP IV activity in seminal plasma. There was little variation in its activities in repeat seminal plasma samples from the same individual, and there was no change in its activity with age to 50 years., Conclusions: DPP IV in seminal plasma was derived from the prostate gland and it may be useful as a bioindicator of prostate function and/or disease with age in men.

Genomic subtractive hybridization of closely related Pasteurella multocida isolates has generated clones useful in distinguishing hemorrhagic septicemia-causing type B strains from other P. multocida serotypes. Oligonucleotide primers designed during the sequencing of these clones have proved valuable in the development of PCR assays for rapid species- and type-specific detection of P. multocida and of type B:2 in particular. This study demonstrated that the primer pair designed from the sequence of the clone 6b (KTT72 and KTSP61) specifically amplified a DNA fragment from types B:2, B:5, and B:2,5 P. multocida and that the primers KMT1T7 and KMT1SP6 produced an amplification product unique to all P. multocida isolates analyzed. It was also shown that PCR amplification performed directly on bacterial colonies or cultures represents an extremely rapid, sensitive method of P. multocida identification.

The mouse mammary gland undergoes rapid proliferation during puberty, then cyclical proliferation and involution during adulthood within a 5-day estrous cycle. Although proliferation of mammary epithelial cells is directed by elevated serum levels of estrogen acting via the estrogen receptor (ER), the ER status of the proliferating cells remains unknown. We examined the ER expression of proliferating epithelial cell types during pubertal development and normal adult growth using simultaneous immunohistochemistry for ER and tritiated thymidine (3H-Tdr) autoradiography. These studies demonstrate that during pubertal growth (4-6 weeks) ER-negative cells comprise more than 50% of the epithelial cell populations in the terminal end buds (TEBs) and ducts. Furthermore, the majority of proliferating cells in both TEBs and ducts are ER-negative. These findings indicate that proliferation of cells within both the TEBs and the mammary ducts contribute to pubertal growth of the mammary gland and that the greater proportion of dividing cells are ER-negative. Similar patterns of cell growth were observed in the normal estrous cycle when the majority of dividing cells were ER-negative during both pro-estrous and estrous. Intensive labelling of cells with 3H-Tdr was used to identify long-lived mammary epithelial cells which retained 3H-Tdr 2 weeks following labelling (i.e., following 3 estrous cycles). Of the small number of mammary epithelial cells retaining 3H-Tdr label, most were ER-positive luminal cells and only a few were ER-negative basal cells. This study indicates that pubertal growth of the mammary gland comprises division of ER-negative cap cells and of both ER-negative and ER-positive cells in the body of the TEBs and elongating mammary ducts. Similarly, estrogen-driven proliferation of ER-negative and ER-positive luminal cells and ER-negative basal cells maintains the differentiated mammary gland in the adult mouse.

Case-control methodology was used to evaluate the significance of vascularity in small breast carcinomas with regard to the presence or absence of axillary lymph node metastases. Vascularity was assessed in 32 axillary node positive primary breast tumours (LN+ve) less than 2 cm in size and compared with 56 control axillary node negative primary tumours (LN-ve), which were matched for histological type and grade and tumour size. This study design employed computer-assisted video analysis (CAVA) to assess the total blood vessel perimeter (BVP), total blood vessel area (BVA), and total blood vessel density (BVD) throughout a tissue section that encompassed an entire cross section of the tumour and its immediate periphery. The BVA and BVD in these tumours were not significantly different between LN+ve and LN-ve groups. The LN-ve carcinomas had, on average, a significantly (P<0.05) higher total BVP (3355 microm/mm2) than LN+ve tumours (2771 microm/mm2). 'Hot spot' areas were also independently assessed by two pathologists and the same areas measured by CAVA. A strong correlation (P<0.001) between the two methods of assessment of BVD of the neovascular 'hot spots' was found; however, no association with axillary lymph node metastasis was found using either method of assessment. In conclusion, vascularity assessed by either blood vessel density or blood vessel size in primary invasive breast cancers less than 2 cm in diameter showed no association with axillary lymph node metastasis; in fact a negative association was found with total BVP of whole tumour sections and BVD in 'hot spots' using CAVA. Further, this study has established a computer-assisted method of quantifying vascularity in solid neoplasms and is a positive step towards a standardised approach to this diverse and methodologically variable area.

We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non-isotopic single-strand conformation polymorphism (SSCP) method we screened for mutations in exons 4-10 of the p53 gene in 375 primary breast cancers from patients with a median follow-up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S-phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node-negative and hormone receptor-positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S-phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction-SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information.

Objective: To assess the effects of three types of apical dissection on urinary continence after radical retropubic prostatectomy and to evaluate possible contributing factors, e.g. preservation of the bladder neck and preprostatic sphincter, age, anastomotic strictures, previous transurethral resection and nerve-sparing surgery., Patients and Methods: Having undergone one of three types of apical dissection, 280 patients were evaluated: in Group 1 (sphincter-damaging) 134 patients underwent the original technique of ligating and transecting the venous complex; in Group 2 (sphincter-repairing), 76 patients had the venous complex with part of striated sphincter incorporated within anastomotic suture(s); and in Group 3 (sphincter-preserving), 70 patients had the venous complex alone ligated using the 'bunching' technique of Myers. The outcome was analysed for the number becoming continent and the time to continence., Results: Continence was achieved in 93% overall, with 90%, 93% and 99% achieving continence in Groups 1, 2 and 3, respectively. The mean time to continence was 68 days overall, taking 100, 52 and 30 days for the respective groups. Twenty patients (7%) did not achieve full continence; 15 had minor incontinence and five severe, with none of the latter being in Group 3. The group (preservation of external sphincter), age and freedom from development of anastomotic strictures were the most important factors both in regaining continence and decreasing the time to continence., Conclusions: Preservation of as much as possible of the normal anatomy of the sphincter mechanisms and their nerve supplies results in an excellent return to continence after radical retropubic prostatectomy.

Amplification of multiple P multocida genomic DNA fragments by outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence, generated complex profiles in a PCR-based fingerprinting method known as REP-PCR. Polymorphisms within REP-PCR profiles were used to characterise 38 isolates of P multocida. The high degree of homogeneity observed among haemorrhagic septicaemia (HS) strains of serotype B and E provided evidence of a disease-associated REP profile that may serve as a novel method for the identification of HS strains regardless of serotype. REP-PCR profiles of other P multocida serotypes were highly variable, illustrating the potential of this technique for the molecular fingerprinting of fowl cholera or atrophic rhinitis isolates. A specific amplified REP fragment was isolated and used to probe membrane-bound digested P multocida genomic DNA. Hybridisation patterns not only distinguished HS-causing isolates from non-HS P multocida, but also demonstrated a degree of relatedness between HS and HS-like strains.

Two Carter type B Pasteurella multocida isolates, Izatnagar 52 and 25, isolated from cases of haemorrhagic septicaemia (HS), were used in a modified subtractive hybridisation technique with the specific aim of cloning unique DNA sequences related to the pathogenesis of HS. Biochemical and protein analyses have shown these isolates to be similar, but reports indicate that they have differences in pathogenicity. The subtracted inserts were screened against genomic DNA from a wide range of P multocida isolates, with two distinct fragments demonstrating specific hybridisation with Carter type B isolates that cause HS. No identity was observed with either Carter type E isolates or non-HS type B strains. The clones were sequenced and a search of the GenBank database revealed significant identity of the clone A3b (296 nt) to P haemolytica lipoprotein, whereas there was no significant identity with 6b (956 nt). Both these fragments had a high level of identity (72.8 to 76.9 per cent) to the H influenzae Rd genome.

Routine axillary dissection is primarily used as a means of assessing prognosis to establish appropriate treatment plans for patients with primary breast carcinoma. However, axillary dissection offers no therapeutic benefit to node negative patients and patients may incur unnecessary morbidity, including mild to severe impairment of arm motion and lymphedema, as a result. This paper outlines a method of evaluating the probability of harbouring lymph node metastases at the time of initial surgery by assessment of tumour based parameters, in order to provide an objective basis for further selection of patients for treatment or investigation. The novel aspect of this study is the use of Maximum Entropy Estimation (MEE) to construct probabilistic models of the relationship between the risk factors and the outcome. Two hundred and seventeen patients with invasive breast carcinoma were studied. Surgical treatment included axillary clearance in all cases, so that the pathologic status of the nodes was known. Tumour size was found to be significantly correlated (P < 0.001) to the axillary lymph node status in the multivariate anlaysis with age (P = 0.089) and vascular invasion (P = 0.08) marginally correlated. Using the multivariate model constructed, 38 patients were predicted to have risk of nodal metastases lower than 20%, of these only 4 (10%) patients had lymph node metastases. A comparison with the Multivariate Logistic Regression (MLR) was carried out. It was found that the predictive quality of the MEE model was better than that of the MLR model. In view of the small sample size, further verification of this model is required in assessing its practical application to a larger population.

Describes two artificial neural network architectures for constructing maximum entropy models using multinomial distributions. The architectures presented maximize entropy in two ways: by the use of the partition function (which involves the solution of simultaneous polynomial equations), and by constrained gradient ascent. Results comparing the convergence properties of these two architectures are presented. The practical use of these two architectures as a method of inference is illustrated by an application to the prediction of metastases in early breast cancer patients. To assess the predictive accuracy of the maximum entropy models, we compared the results with those obtained by the use of the multilayer perceptron and the probabilistic neural network.

Basal cell carcinomas (BCCs) are the most common sporadic cancers worldwide. They are also a cardinal manifestation of a familial cancer predisposition syndrome, naevoid BCC syndrome (NBCCS). The gene responsible for NBCCS is likely to be a tumour suppressor gene and has been genetically mapped to a 2cM region between microsatellite markers, D9S196 and D9S180 at 9q22.3-q31. 101 BCCs (63 sporadic and 38 familial) were examined for loss of heterozygosity (LOH) in the candidate region of the NBCCS gene. Deletions were found in 46% and all LOH is consistent with genetic mapping of the NBCC locus. These findings strongly support the hypothesis that inactivation of the putative tumour suppressor, the NBCCS gene, is important in the formation of sporadic BCCs. One sporadic tumour indicates that the smallest region of overlap of these deletions is within the interval between D9S287 and D9S180. If this is confirmed in additional tumours, it would further narrow down the NBCCS region and exclude one candidate gene, that for the C complementation group of Fanconi anaemia, which maps proximally to D9S287. However, it would not exclude another candidate, the gene for the A complementation group of xeroderma pigmentosum (XPAC). Evidence of imprinting was also sought but preliminary data indicate that it is unlikely to occur at the NBCCS locus.

Expression levels of nm23-H1 were evaluated in a variety of normal benign and malignant breast tissues by Northern and slot blot. Tissues from 153 patients presenting with palpable breast lesions were studied: 132 primary infiltrating breast cancers, 9 pure duct carcinoma in situ lesions, a phyllodes tumor, 9 benign lesions and 2 local recurrences of carcinoma. In addition to lesional tissue, 49 samples of macroscopically normal breast tissue, 37 axillary lymph nodes and 9 samples from patients undergoing cosmetic reduction mammoplasty were studied. Sets of normal breast tissue, primary tumor and lymph node tissue from individual patients were available for comparison in 37 cases. A wide range of gene expression was detected in the various tissue types. The highest levels of expression were detected in malignant samples with in situ carcinomas being associated with the highest levels of gene expression. The expression levels of nm23-H1 in normal breast tissue were lower than the corresponding tumors from the same patients (p < 0.0005). Benign breast lesions (including 6 fibroadenomas) had levels of gene expression approximating those of the normal tissue samples. Normal axillary lymph nodes had significantly lower levels of nm23-H1 expression than nodes with metastatic deposits (p < 0.03). No significant association was observed between nm23-H1 expression levels and axillary node status in patients with infiltrating carcinoma, although there was a slight trend toward lower nm23-H1 mRNA levels in the node negative group.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological functions proposed for the ST3 and nm23 genes in tumour development and progression seem to be directly opposed. Stromelysin-3 (ST3) is a putative member of the matrix metalloproteinase family. ST3 has been implicated in the progression of epithelial malignancies, specifically with regard to an invasive (and therefore potentially metastasizing) phenotype. The nm23 gene, on the other hand, encodes a nucleoside diphosphate kinase which allegedly has a metastasis-suppressor-type function. It was therefore of interest to compare the expression of ST3 and nm23 in various surgically excised normal and neoplastic breast tissues. RNA was isolated from over 200 surgical specimens and studied by Northern blots. Normal breast tissues did not express ST3, and ST3 expression was detected in only 1 of 20 normal axillary lymph nodes. None of 7 fibroadenomas expressed ST3. In contrast, 60% of primary and metastatic breast carcinomas contained ST3-mRNA. The expression of ST3 was mainly confined to invasive carcinomas and was observed less frequently in pure ductal carcinoma in situ (DCIS) lesions. Our results support the suggestion that ST3 expression is related to the malignant process in breast cancer. The role of nm23 is far less clear-cut.

We describe a sensitive and practical in situ hybridization method, using a digoxigenin-labeled probe, for the detection of c-erbB-2 amplification in breast cancer in formalin-fixed, paraffin-embedded tissue sections. Forty-six primary breast carcinomas were studied. Nuclear hybridization signal was observed in 36 of 46 carcinomas. Signal was confined to malignant cells. Normal breast epithelium and stromal and inflammatory cells were uniformally negative. DNase predigestion, no-probe preparations, and competitive hybridization confirmed the specificity of the reaction. The hybridization reaction was localized to multiple discrete foci in tumor cell nuclei, suggesting multiple sites of gene copy and transcriptional activity in the nucleus. Considerable cell-to-cell variation in hybridization signal was evident within individual tumors and positive reactions were observed in several cases in which amplification could not be detected by either Southern or slot blot analysis. The high sensitivity and specificity of the reaction and its use in a tissue-based system will allow the study of a range of possible precursor lesions of breast cancer for evidence of c-erbB-2 amplification.

Total tumor cathepsin D (TCD) levels were determined prospectively by a radioimmunometric assay in tumor cytosol of 858 primary breast cancer patients diagnosed between 1989-1991. In 581 of these patients, tumor HER-2/neu oncogene amplification was simultaneously determined. In a "training-set" of 313 patients, "high" TCD was associated with significantly shorter disease-free survival (DFS). For the whole group, there was no correlation between TCD and pathologic stage, number of axillary nodes with tumor deposits, tumor size, histologic type and grade, or hormone receptor levels. In the node-positive group, high TCD level was associated with HER-2/neu amplification. After a median follow-up duration of 31 months, univariate analysis indicated that high TCD level was significantly associated with shorter DFS only in node-positive patients. The shorter DFS in association with high TCD levels was observed in both estrogen-receptor-positive and -negative patients. Cox multivariate analysis of DFS confirmed that high TCD level was predictive of shorter DFS in node-positive patients only. Because of the short duration of follow-up, the significance of TCD in overall survival was not determined. We conclude that high tumor TCD in node-positive patients is predictive of shorter DFS, and is often associated with HER-2/neu amplification. The possibility exists that high tumor TCD may act in combination with HER-2/neu amplification to promote dissemination of metastases.

The oncogene c-erbB-2 is frequently amplified in human breast carcinoma. The c-erbB-2 gene is present as a single copy in normal cells, and has been mapped to chromosome 17 in the region 17q 12-21.32. c-erbB-2 encodes a transmembrane glycoprotein known as p185. The intracellular component of p185 has tyrosine kinase activity; the extracellular domain has a structure resembling a growth factor receptor. c-erbB-2 amplification, p185 overexpression and levels of transcribed c-erbB-2 specific messenger RNA have been studied in a large number of breast carcinomas using a variety of techniques. In general, overexpression of p185 oncoprotein reflects various levels of DNA amplification, though in some cases amplification can be detected in the absence of overexpression of p185 and similarly overexpression of p185 can be present without detectable levels of c-erbB-2 amplification. This findings suggests that multiple mechanisms may be responsible for overexpression. c-erbB-2 amplification and/or overexpression occurs in almost all cases of high grade duct carcinoma in-situ, but has been reported in only 10%-40% of infiltrating duct carcinoma. c-erbB-2 amplification or overexpression occurs rarely in invasive lobular carcinoma, and has not been detected in ductal or lobular epithelial hyperplasia, or in atypical ductal or atypical lobular hyperplasia. It is generally believed that c-erbB-2 amplification/overexpression is an important independent prognostic indicator in breast carcinoma, identifying a subset of patients with poor prognosis tumours, particularly if axillary node metasases are present. However, many unanswered questions remain regarding c-erbB-2 and its role in breast cancer development and progression. The causes of c-erbB-2 amplification are unknown. There is no evidence of mutations in the human gene which might cause amplification or overexpression. The significance of the differences in levels of c-erbB-2 amplification/overexpression in in-situ duct carcinoma and associated invasive duct carcinoma has not been established. Amplification or overexpression have not been reported in atypical duct hyperplasia, a proposed precursor of duct carcinoma in-situ, yet overexpression occurs almost always in high grade duct carcinoma in-situ. c-erbB-2 may play a critical role in the development of a clonal in-situ, proliferation of high histological grade, yet does not obviously influence the acquisition of an invasive phenotype. We would postulated that this instability in amplification/overexpression is of biological significance, and if better understood may aid in the study of progression of human breast carcinoma.

Since the description of the original technique of field alternation gel electrophoresis (FAGE) about ten years ago there have been significant developments in the area. Between 1983 and early 1987 dramatic improvements in the technique and apparatus resulted in a 500- to 600-fold increase in the functional separation capacity of conventional agarose gel electrophoresis. Details of the improvements in technique and equipment was the subject of an earlier review [H. J. S. Dawkins, J. Chromatogr., 492 (1989) 615]. This review concentrates on the application of FAGE technology. The FAGE technique is no longer restricted to simply separating large DNA fragments. This method is presently being used for electrophoretic karyotyping, long-range genomic mapping, cloning of large DNA fragments into new vectors, the study of pathogenic chromosomal alterations and the structural analysis of chromosomes. The applications of FAGE in molecular biology and genetics is constantly expanding, with the full potential of this technique still to be realised.

The oncogene c-erbB-2 has been shown to be amplified in 17-30% of breast cancers, with similar levels of overexpression of the oncogene product p185, a transmembrane growth factor receptor glycoprotein. Amplification of c-erbB-2 is now generally considered to be a significant prognostic indicator in patients with breast cancer. A series of 74 consecutive breast carcinomas were analysed for c-erbB-2 amplification and p185 overexpression. The procedures of Southern blotting and slot blot were used for the analysis of oncogene amplification, while immunoperoxidase (IPOX) staining and enzyme-linked immunosorbent assay (ELISA) were used for the analysis of p185 overexpression. Detection of c-erbB-2 oncogene amplification by both the conventional Southern blotting technique and by the slot blot technique showed complete accord, with the amplified c-erbB-2 oncogene being detected in 14 of the 74 patients (18.9%). The c-erbB-2 oncoprotein, as measured by IPOX and ELISA, was found to be overexpressed in 21% and 19% of patients, respectively. Comparison was made between the results attained by all four methods, and further comparison of the techniques was made from the point of view of ease of use, expense and ease of introduction into routine diagnostic laboratories. Immunocytochemistry in combination with slot blotting procedures were considered to be the most cost effective methods for evaluation of overexpression and amplification in routine pathology laboratories.

We determined that the protein profiles of 14 isolates from animals with hemorrhagic septicemia were relatively homogeneous and could be placed in 2 distinct groups on the basis of their country of origin. Such differences correlated with the serotypic properties of the individual isolates; hemorrhagic septicemia isolates of Asian and North American origin (Carter B) had a major protein band with an apparent molecular mass of 32 kDa, whereas those of African origins (Carter E) had a major protein band with an apparent molecular mass of 37 kDa. The possession of a major 32-kDa protein band appeared to be unique to Carter B isolates, suggesting that electrophoresis may be a useful nonserologic technique for the identification of organisms of this serotype. Other major bands with apparent molecular masses of 27, 45, and 47 kDa were shared by all strains, regardless of their serotype. The lipopolysaccharides were of low molecular mass and relatively uniform from 1 isolate to the next.

Thirteen of 100 fallow deer, aged between 6 months and 10 years, died over a 5 week period. The deaths occurred in 2 outbreaks 3 weeks apart. Both outbreaks were preceded by at least 3 days of cold wet and windy weather, and were associated with water-logged pastures. Affected animals were usually found dead, with a frothy blood-stained nasal discharge. In the 8 deer necropsied, gross lesions included widespread subserosal petechial haemorrhages, severe pulmonary congestion and oedema with froth-filled airways, and fibrinous pneumonia and pleurisy in 4 deer. Two deer, also, had extensive subcutaneous petechial and ecchymotic haemorrhages and oedema of skeletal musculature. Histologically, the most significant lesions were present in the lungs. Moderate to severe pulmonary congestion and oedema, with fibrinous exudation into alveoli and septal oedema, were present in all deer. In some deer these changes were accompanied by a diffuse infiltration with polymorphonuclear leucocytes. Pasteurella multocida was isolated from a range of tissues from 7 of 8 deer examined. The remaining animal had been treated with antibiotics 8 hours before death. The isolates had identical polyacrylamide gel electrophoresis patterns and were of the same antigenic type-Carter group A, Heddleston type 3,4.

This study examined the protection induced by oil adjuvant vaccine and broth bacterin in mice. Protective immunity was induced by both oil adjuvant and bacterin vaccination procedures. Oil adjuvant vaccination induced a 10(5)-fold increase for lethal challenge over control mice, while secondary vaccination induced a further 10-fold increase in resistance to lethal challenge. Broth bacterin induced a slightly weaker protective response with 10(4)- and 10(5)-fold increases in resistance to lethal challenge following primary and secondary vaccination, respectively. There was a significant relationship between IgG antibody levels and resistance to challenge (P = 0.026). Protection lasted for at least 20 weeks after a primary oil adjuvant vaccination. There was also a strong and significant relationship between IgG antibody levels and the passive protection afforded by serum transfer in each experiment within this study and the overall correlation was highly significant (P = 0.00001). There appeared to be a relationship between protection and the antibody response to major protein bands with the apparent molecular mass Mr. 94,000; 80,000; 67,000; 35,000 and 32,000 as well as to the bands in the region of the lipopolysaccharide components of P. multocida (approximately Mr, 14-15,000). Whether protection resulted from recognition of specific antigens or was a result of both antibody levels and antibody specificity remains to be defined.

Haemorrhagic septicaemia-causing strains of Pasteurella multocida were identified by a disease-specific ELISA. Some strains, however, were of the same serotype as those which cause haemorrhagic septicaemia (HS) but were negative when tested in the disease specific ELISA. The suspect false negative isolates were passaged in mice and retested in the HS ELISA with the same result. Immunoelectron microscopy was used to examine further these suspect HS-causing strains. Monoclonal antibodies and protein A-gold showed that the suspect negative organisms were a mixture of phenotypes with less than 10 per cent, and usually less than 2 per cent, of the population expressing HS-associated epitopes. The degree of staining on the organisms expressing the HS-epitopes was of the same intensity as the positive control organism. Expression of the HS-associated epitopes is presumably too low to allow detection in the current HS ELISA.

Haemorrhagic septicaemia (HS) is caused by specific serotypes of Pasteurella multocida and is one of the major economic diseases of cattle and buffalo in South East Asia. Definitive diagnosis of the disease-causing organism with the available methods is labour intensive and not totally reliable, consequently, an ELISA system to identify P multocida organisms which cause HS was developed. One hundred and twenty-four P multocida isolates were tested, 58 were type strains and 66 were field isolates. Analysis of these strains indicated the assay had a specificity of 99 per cent and sensitivity of at least 86 per cent. The sensitivity could be an underestimate, as five isolates assumed to be false negative reactions may not all be HS-causing strains. The HS ELISA provides a rapid, simple, accurate and inexpensive diagnostic assay for identification of HS causing organisms but does not represent a new typing system for P multocida. This assay will also enable countries to assess the impact of HS more accurately.