The efficacy, safety and acceptability of emergency embedded Psychiatry Assessment and Planning Units: An evaluation of Psychiatry Assessment and Planning Units in close proximity to their associated emergency departments.

OBJECTIVE: To determine the efficacy, safety and acceptability as well as the patient demographics of three newly developed emergency department-embedded Psychiatric Assessment and Planning Units located in Metropolitan Melbourne at Austin, Peninsula and Eastern Health Services. METHODS: The evaluation reviewed a 12-month period of service activity from 1 September 2017 to 31 August 2018, when all three Psychiatric Assessment and Planning Units services were operational. A 12-month period from 1 September 2014 to 31 August 2015 was compared as the pre-Psychiatric Assessment and Planning Units period. Mixed qualitative and quantitative methods were used. This included semi-structured interviews of 30 Psychiatric Assessment and Planning Units patients and 30 emergency department staff (10 of each for all 3 sites), patient survey, statistical analysis of Client Management Interface data for the emergency department and related Psychiatric Assessment and Planning Units as well as audit of RISKMAN registers. RESULTS: There were 365 Austin, 567 Eastern and 791 Peninsula Psychiatric Assessment and Planning Units admissions. Psychiatric Assessment and Planning Units were generally well accepted by patients and emergency department staff, relatively safe, operating within the Key Performance Indicators with mixed effect on emergency department flow. Austin emergency department processing times improved post-Psychiatric Assessment and Planning Units (4 hours 57 minutes to 4 hours 19 minutes; p < 0.001) while deteriorating at Eastern and Peninsula. Adjustment Disorder and Depression and Borderline Personality Disorder were the most common admission diagnoses. While the Psychiatric Assessment and Planning Units had mixed utility on emergency department processing times, they appear to serve a demographic not previously accommodated in traditional emergency department psychiatry models. CONCLUSION: The emergency department-embedded Psychiatric Assessment and Planning Unit model of care appears effective on some measures, safe and acceptable to patients and staff. The Psychiatric Assessment and Planning Units seem to service a group not previously accommodated in traditional emergency psychiatry models.

Timing of routine infant vaccinations and risk of food allergy and eczema at one year of age.

BACKGROUND: Epidemiological evidence suggests that routine vaccinations can have nontargeted effects on susceptibility to infections and allergic disease. Such effects may depend on age at vaccination, and a delay in pertussis vaccination has been linked to reduced risk of allergic disease. We aimed to test the hypothesis that delay in vaccines containing diphtheria-tetanus-acellular pertussis (DTaP) is associated with reduced risk of food allergy and other allergic diseases. METHODS: HealthNuts is a population-based cohort in Melbourne, Australia. Twelve-month-old infants were skin prick-tested to common food allergens, and sensitized infants were offered oral food challenges to determine food allergy status. In this data linkage study, vaccination data for children in the HealthNuts cohort were obtained from the Australian Childhood Immunisation Register. Associations were examined between age at the first dose of DTaP and allergic disease. RESULTS: Of 4433 children, 109 (2.5%) received the first dose of DTaP one month late (delayed DTaP). Overall, delayed DTaP was not associated with primary outcomes of food allergy (adjusted odds ratio (aOR) 0.77; 95% CI: 0.36-1.62, P = 0.49) or atopic sensitization (aOR: 0.66; 95% CI: 0.35-1.24, P = 0.19). Amongst secondary outcomes, delayed DTaP was associated with reduced eczema (aOR: 0.57; 95% CI: 0.34-0.97, P = 0.04) and reduced use of eczema medication (aOR: 0.45; 95% CI: 0.24-0.83, P = 0.01). CONCLUSIONS: There was no overall association between delayed DTaP and food allergy; however, children with delayed DTaP had less eczema and less use of eczema medication. Timing of routine infant immunizations may affect susceptibility to allergic disease.

Differences in HIV risk behavior of injection drug users in New York City by health care setting.

The purpose of this study is to examine the HIV risk behaviors and demographic characteristics of injection drug users (IDUs) by type of health care setting, which can inform development of tailored structural interventions to increase access to HIV prevention and medical treatment services. IDU syringe customers were recruited from pharmacies as part of the "Pharmacist As Resources Making Links to Community Services" (PHARM-Link) study, a randomized community-based intervention in New York City (NYC) aimed at connecting IDUs to HIV prevention, medical, and social services. An ACASI survey ascertained demographics, risk behavior, health-care utilization, and location where health care services were received in the past year. Data were analyzed using logistic regression. Of 602 participants, 34% reported receiving health care at a community clinic, 46% a private medical office, 15% a mobile medical unit, and 59% an emergency room (ER). After adjustment, participants who attended a community clinic were significantly more likely to have health insurance, report syringe sharing, and be HIV positive. Whites, nondaily injectors, insured, and higher income IDUs were more likely to attend a private medical office. Participants who recently used a case manager and had multiple sexual partners were more likely to use a mobile medical unit. ER attendees were more likely to be homeless and report recent drug treatment use. These findings show that IDU demographics and risk behaviors differ by health care setting, suggesting that risk reduction interventions should be tailored to health care settings. Specifically, these data suggest that community clinics and mobile medical units serve high-risk IDUs, highlighting the need for more research to develop and test innovative prevention and care programs within these settings.

Stabilizing Effects of a Particulate Demineralized Bone Matrix in the L4 Interbody Space with and without PEEK Cage - A Literature Review and Preliminary Results of a Cadaveric Biomechanical Study.

We reviewed the biological elements supporting the usefulness of a specifically designed particulate form of demineralized bone matrix (DBM) with spinal fusion, and report some limitations of its use described in the medical literature and in the interbody space using a cadaveric biomechanical model. A literature review and description of the techniques used to augment spinal fusion are presented, including a more thorough review of recent findings of cadaveric biomechanical flexibility studies using DBM alone at different percentage fills of the existing disc space and DBM with a polyetheretherketone (PEEK) interbody cage. The need for DBM was established by reviewing limitations of autografts and allografts in spinal fusion. Demineralized bone matrix used alone did not increase stability post discectomy at L4-L5, but was demonstrated to exhibit satisfactory stability when used with a PEEK interbody cage. There may be a future role for DBM that hardens and fills disc space more rigidly, overcoming this limitation to its use.

Myocarditis following COVID-19 mRNA vaccinations: Twin and sibling case series.

ImportanceMyocarditis and myopericarditis are well described adverse events of special interest (AESI) following COVID-19 vaccinations. Whilst the aetiology is still being investigated; there is evidence that genetic predisposition may be a risk factor for the development of myocarditis. Furthermore, hormones are thought to contribute to sex-specific differences in myocarditis, skewed toward a larger risk in adolescent males. Objective: This unique sibling case series may help highlight potential mechanisms and prognostic factors in the development of myocarditis following COVID-19 vaccination in adolescent males. In this context, twin and familial studies provide a unique epidemiological perspective to investigate the interplay between genetic predisposition and other factors. Participants: Observational case series of all siblings reported to SAEFVIC with chest pain following COVID-19 vaccinations in Victoria, Australia. Exposure: mRNA vaccination (Comirnaty BNT162b2 COVID-19 (Pfizer-BioNTech) and Spikevax mRNA-1273 (Moderna). Findings: Our case series comprises 6 young males; two sets of monozygotic twins and one set of fraternal brothers following reports of chest pain associated with COVID-19 mRNA vaccination. Five patients were diagnosed with myocarditis as per Brighton Collaboration Criteria (Level 2). The remaining sibling, who did not have myocarditis, was subsequently diagnosed with pubertal delay. Conclusions: Understanding the genetic and hormonal risk factors and aetiology for myocarditis associated with COVID-19 vaccines is paramount. Further evaluation of specific genetic targets or biomarkers is required to understand the implications of population vaccine policy, particularly for adolescent and young adult males at highest risk for this AESI.

Australian immunisation registers: established foundations and opportunities for improvement.

The National Immunisation Program Schedule in Australia is formulated and funded nationally under the population-wide Medicare system. The policy is implemented by the eight state and territory jurisdictions. The national immunisation registers consist of the Australian Childhood Immunisation Register (ACIR), and, more recently, the National Human Papillomavirus (HPV) Vaccination Program Register. Moreover, a variety of jurisdiction-based registers and primary care practice software systems exist, which interact with the national registers. General practitioners can obtain reports listing patients under seven years attending their practice and recorded as 'not fully immunised', and immunisation coverage rates for their practice linked to government incentives through Medicare. A 2011 report documents national coverage of 91.8% fully immunised at 12 months, and 92.6% at 24 months. The HPV register provides information on vaccination coverage with the potential to link with a register of cervical cancer screening results. Limitations of current national register include inability to easily access immunisation histories beyond seven years of age, and issues of underreporting and timeliness, which impact significantly the immunisation coverage estimates. The linkage of these registers with healthcare outcome data will further enhance public health outcomes by enabling rapid, population-level vaccine safety and effectiveness investigations in a nation with a track record as an 'early adopter' of new childhood vaccines.

Acute disseminated encephalomyelitis and routine childhood vaccinations - a self-controlled case series.

Acute disseminated encephalomyelitis (ADEM) is an autoimmune, central nervous system demyelinating disorder that follows antecedent immunologic challenges, such as infection or vaccination. This study aimed to investigate the potential association between routine childhood vaccinations and ADEM. Children under 7 years of age admitted to the two tertiary level pediatric hospitals in Victoria, Australia with ADEM from 2000-2015 had their clinical information linked to vaccination records from the Australian Childhood Immunization Register. Chart review was undertaken utilizing the Brighton Collaboration ADEM criteria. The self-controlled case-series (SCCS) methodology was employed to determine the relative incidences of ADEM post-vaccination in two risk intervals: 5-28 days and 2-42 days. Forty-six cases were eligible for SCCS analysis with a median age of 3.2 years. Of the forty-six cases, three were vaccine proximate cases and received vaccinations 23, 25 and 28 days before ADEM onset. Two vaccine proximate cases received their 4-year-old scheduled vaccinations (MMR and DTPa-IPV) and one vaccine proximate case the 1-year old scheduled vaccinations (MMR and Hib-MenC). The relative incidence of ADEM during the narrow and broad risk intervals were 1.041 (95% CI 0.323-3.356, p = 0.946) and 0.585 (95% CI 0.182-1.886, p = 0.370) respectively. Sensitivity analyses did not yield any substantial deviations. These results do not provide evidence of an association between vaccinations routinely provided to children aged under 7 years in Australia and the incidence of ADEM. However, these results should be interpreted with caution as the number of ADEM cases identified was limited and further research is warranted.

Computed tomographic findings, surgical management and postoperative outcomes of large intermuscular lipomas in the hindlimb of 11 dogs.

OBJECTIVE: Lipomas are benign adipose tissue tumours of mesenchymal origin and can originate in various locations. Intermuscular lipomas in the thigh can cause substantial hindlimb expansion in the dog. We describe the computed tomography findings, surgical management and the outcomes of 11 dogs with large intermuscular lipomas of the hindlimb. DESIGN: Retrospective case series. METHODS: Medical records between 2009 and 2019 of dogs presenting to The Animal Hospital at Murdoch University were reviewed. Inclusion criteria included dogs with a histologically confirmed, large hindlimb lipoma that was surgically excised following preoperative computed tomography (CT) imaging. RESULTS: CT with intravenous contrast revealed a well-defined, smoothly marginated, fat attenuating mass with minimal vascularity, separating the muscle bellies of the caudal hindlimb. The mass was often in close proximity to the femoral artery and vein. All lipomas were marginally excised. At surgery, some lipomas were intimately associated with the sciatic nerve and some showed infiltration of, or attachment to, neighbouring muscle that could be excised en bloc with the lipoma. Postoperative closed-suction wound drainage was used in 6 of 11 dogs. One dog required revision surgery due to partial wound dehiscence. Long-term follow-up with owners reported good postoperative function of the affected hindlimb in all dogs. One dog developed an infiltrative lipoma in the same location 22 months post-excision. CONCLUSION: Preoperative CT allowed a presumptive diagnosis of intermuscular lipoma and facilitated surgical planning for marginal excision. Large intermuscular lipomas of the hindlimb can be safely excised with minimal short-term complications, good long-term functional outcome and low likelihood of recurrence.

Safety of meningococcal B vaccine (4CMenB) in adolescents in Australia.

BACKGROUND: Four-component meningococcal B (4CMenB) vaccine is licensed in many countries but has had limited use in adolescents despite this age group being at increased risk of meningococcal disease. OBJECTIVES: To assess the safety profile of two doses of 4CMenB in adolescents. METHODS: Cluster randomised controlled trial of senior school students in South Australia (SA) with participating schools randomised to intervention (4CMenB) or control. Vaccine safety was monitored using the South Australian Vaccine Safety Surveillance System (SAVSS), a spontaneous reporting system for adverse events following immunisation (AEFI) with enhanced follow-up of AEFI. RESULTS: 58,637 doses of 4CMenB vaccine were administered to 30,522 students (median age 16 years) during 2017-2018. Of 18,348 and 12,174 students vaccinated in 2017 and 2018, 97.3% and 84.3%, respectively, received both scheduled doses (N = 28,115). 193 AEFI in 187 students were reported with a reporting rate of 0.32% (95%CI: 0.28-0.39%). Seventy individuals sought medical review, including nine serious adverse events. 98% (166/169) of those who were contactable for AEFI follow-up (87.6% 169/193) reported resolution of the event. Most common AEFI were injection site reaction (126/193), headache (99/193) and nausea (61/193). AEFI were more frequently reported in females (aOR = 1.409 (95%CI: 1.002, 1.980)), schools with high level of educational advantage (adjusted Odds Ratio (aOR) = 1.515 (95%CI: 1.005, 2.284)), following first dose (aOR = 1.619 (95%CI: 1.168, 2.244)), and in 2017 (aOR = 1.437 (95%CI: 1.001, 2.064)). Reported AEFI declined with increasing age (aOR = 0.771 (95%CI: 0.673, 0.883)). CONCLUSION: In this largest post-licensure use of 4CMenB in adolescents, the low AEFI reporting rate provides real-world evidence of 4CMenB safety in this age group. (ClinicalTrials.gov number: NCT03089086).

DR5-targeted, chemotherapeutic drug-loaded nanoparticles induce apoptosis and tumor regression in pancreatic cancer in vivo models.

Pancreatic cancer is usually advanced and drug resistant at diagnosis. A potential therapeutic approach outlined here uses nanoparticle (NP)-based drug carriers, which have unique properties that enhance intra-tumor drug exposure and reduce systemic toxicity of encapsulated drugs. Here we report that patients whose pancreatic cancers express elevated levels of Death Receptor 5 (DR5) and its downstream regulators/effectors FLIP, Caspase-8, and FADD had particularly poor prognoses. To take advantage of elevated expression of this pathway, we designed drug-loaded NPs with a surface-conjugated αDR5 antibody (AMG 655). Binding and clustering of the DR5 is a prerequisite for efficient apoptosis initiation, and the αDR5-NPs were indeed found to activate apoptosis in multiple pancreatic cancer models, whereas the free antibody did not. The extent of apoptosis induced by αDR5-NPs was enhanced by down-regulating FLIP, a key modulator of death receptor-mediated activation of caspase-8. Moreover, the DNA topoisomerase-1 inhibitor camptothecin (CPT) down-regulated FLIP in pancreatic cancer models and enhanced apoptosis induced by αDR5-NPs. CPT-loaded αDR5-NPs significantly increased apoptosis and decreased cell viability in vitro in a caspase-8- and FADD-dependent manner consistent with their expected mechanism-of-action. Importantly, CPT-loaded αDR5-NPs markedly reduced tumor growth rates in vivo in established pancreatic tumor models, inducing regressions in one model. These proof-of-concept studies indicate that αDR5-NPs loaded with agents that downregulate or inhibit FLIP are promising candidate agents for the treatment of pancreatic cancer.

Identification of a mobile endogenous transposon in Arabidopsis thaliana.

A mobile endogenous transposable element, Tag1, has been identified in the plant Arabidopsis thaliana. Tag1 was found in the nitrate transporter gene, CHL1, of a chlorate-resistant mutant present in a population of plants containing an active maize Ac transposon. Tag1 excises from the chl1 gene producing chlorate-sensitive revertants with Tag1 or Tag1-related elements at different loci. Tag1 and related elements are present in the Landsberg but not Columbia or Wassilewskija ecotypes of Arabidopsis. Thus, Tag1 provides a tool for the insertional mutagenesis of plant genes essential for biological processes of agronomic importance.

Polymicrogyria in standard poodles.

BACKGROUND: Polymicrogyria is a disorder of cerebrocortical migration resulting in increased numbers of small, disorganized gyri. This disorder occurs in Standard Poodles and in cattle. OBJECTIVES: To describe the clinical, electroencephalographic, imaging, and histopathologic features in poodles with polymicrogyria. ANIMALS: Five Standard Poodles with histologically confirmed polymicrogyria. METHODS: Retrospective case series. Cases were obtained by personal communication with 1 of 2 authors (TJVW, DPO). RESULTS: All dogs had cortical blindness and other neurologic abnormalities including gait and behavioral changes. Magnetic resonance imaging of 3 dogs showed multiple disorganized gyri, which were especially apparent on T2-weighted dorsal plane images. Electroencephalogram (EEG) of 1 dog revealed epileptiform discharges, including both spike and spike and wave discharges with voltage maximum potentials over the parietal/occipital region. The EEG supported that the repetitive behavior displayed by the dog was a complex partial motor seizure. One dog had concurrent hydrocephalus. All dogs had occipital lobe involvement and 2 dogs had involvement of other lobes. CLINICAL IMPORTANCE: The cases presented here demonstrate a larger age range (7 weeks to 5 years) and a decreased frequency of associated hydrocephalus when compared with the previous report.

TBX2 interacts with heterochromatin protein 1 to recruit a novel repression complex to EGR1-targeted promoters to drive the proliferation of breast cancer cells.

Early Growth Response 1 (EGR1) is a stress response transcription factor with multiple tumour suppressor roles in breast tissue, whose expression is often lost in breast cancers. We have previously shown that the breast cancer oncogene TBX2 (T-BOX2) interacts with EGR1 to co-repress EGR1-target genes including the breast tumour suppressor NDRG1. Here, we show the mechanistic basis of this TBX2 repression complex. We show that siRNA knockdown of TBX2, EGR1, Heterochromatin Protein 1 (HP1) isoforms and the generic HP1-associated corepressor protein KAP1 all resulted in growth inhibition of TBX2-expressing breast cancer cells. We show that TBX2 interacts with HP1 through a conserved HP1-binding motif in its N-terminus, which in turn leads to the recruitment of KAP1 and other associated proteins. Mutation of the TBX2 HP1 binding domain abrogates the TBX2-HP1 interaction and loss of repression of target genes such as NDRG1. Chromatin-immunoprecipitation (ChIP) assays showed that TBX2 establishes a repressive chromatin mark, specifically H3K9me3, around the NDRG1 proximal promoter coincident with the recruitment of the DNA methyltransferase DNMT3B and histone methyltransferase (HMT) complex components (G9A, Enhancer of Zeste 2 (EZH2) and Suppressor of Zeste 12 (SUZ12)). Knockdown of G9A, EZH2 or SUZ12 resulted in upregulation of TBX2/EGR1 co-regulated targets accompanied by a dramatic inhibition of cell proliferation. We show that a generic inhibitor of HMT activity, DzNep, phenocopies expression of an inducible dominant negative TBX2. Knockdown of TBX2, KAP1 or HP1 inhibited NDRG1 promoter decoration specifically with the H3K9me3 repression mark. Correspondingly, treatment with a G9A inhibitor effectively reversed TBX2 repression of NDRG1 and synergistically downregulated cell proliferation following TBX2 functional inhibition. These data demonstrate that TBX2 promotes suppression of normal growth control mechanisms through recruitment of a large repression complex to EGR1-responsive promoters leading to the uncontrolled proliferation of breast cancer cells.

Defining the Influence of Germline Variation on Metastasis Using Systems Genetics Approaches.

Cancer is estimated to be responsible for 8 million deaths worldwide and over half a million deaths every year in the United States. The majority of cancer-related deaths in solid tumors is directly associated with the effects of metastasis. While the influence of germline factors on cancer risk and development has long been recognized, the contribution of hereditary variation to tumor progression and metastasis has only gained acceptance more recently. A variety of approaches have been used to define how hereditary variation influences tumor progression and metastasis. One approach that garnered much early attention was epidemiological studies of cohorts of cancer patients, which demonstrated that specific loci within the human genome are associated with a differential propensity for aggressive tumor development. However, a powerful, and somewhat underutilized approach has been the use of systems genetics approaches in transgenic mouse models of human cancer. Such approaches are typically multifaceted, and involve integration of multiple lines of evidence derived, for example, from genetic and transcriptomic screens of genetically diverse mouse models of cancer, coupled with bioinformatics analysis of human cancer datasets, and functional analysis of candidate genes. These methodologies have allowed for the identification of multiple hereditary metastasis susceptibility genes, with wide-ranging cellular functions including regulation of gene transcription, cell proliferation, and cell-cell adhesion. In this chapter, we review how each of these approaches have facilitated the identification of these hereditary metastasis modifiers, the molecular functions of these metastasis-associated genes, and the implications of these findings upon patient survival.

Heritabilities and genetic correlations of pulmonary arterial pressure and performance traits in Angus cattle at high altitude.

Pulmonary arterial pressure (PAP) is an indicator trait for pulmonary hypertension and for the risk of developing high-altitude disease (HAD) in cattle. Pulmonary arterial pressures provide a tool for selection of breeding cattle for tolerance to high altitude in mountainous regions of the United States. The objective of this study was to evaluate relationships between growth performance traits and yearling PAP (42.4 ± 9.9 mmHg; = 5,776; elevation 2,150 m) using data from 1993 to 2014 in the John E. Rouse Colorado State University Beef Improvement Center (CSU-BIC) Angus herd. The breeding program used sires ( = 299) from both low- and high-elevation environments. We hypothesized that little to no genetic relationship exists between PAP and birth weight (BWT; direct and maternal), weaning weight (WW; direct and maternal), yearling weight (YW; direct and maternal), and postweaning gain (PWG). Historic selection of natural service sires from within the herd required a PAP of ≤ 42 mmHg. Outside AI sires ( = 156) used in this breeding program were not PAP tested and therefore were used with little knowledge of these sires' high-altitude adaptability. Performance traits (± SD) routinely recorded included BWT (36.2 ± 5.1 kg; = 8,695), WW (213.5 ± 31.8 kg; = 8,010), YW (345.6 ± 83.8 kg; = 5,580), and PWG (122.0 ± 63.7 kg; = 5,449), where PWG represented the total weight gained from weaning to yearling age. Four-trait analyses using REML were conducted with an animal model. The heritability estimates (± SE) for PAP (0.26 ± 0.03), BWT direct (0.42 ± 0.04) and maternal (0.14 ± 0.02), WW direct (0.29 ± 0.04) and maternal (0.19 ± 0.03), YW direct (0.45 ± 0.04) and maternal (0.23 ± 0.03), and PWG (0.14 ± 0.02) were in the range of those reported in previous literature. Estimates of genetic correlations (± SE) revealed weak relationships between PAP and direct and maternal BWT, direct and maternal WW, direct and maternal YW, and PWG of 0.15 ± 0.09, 0.14 ± 0.10, 0.23 ± 0.09, -0.01 ± 0.10, 0.12 ± 0.08, 0.00 ± 0.09, and -0.10 ± 0.10, respectively. The results of this study suggest that selection for lower PAP measures should have minimal influence on the growth performance of yearling Angus bulls and heifers at the CSU-BIC, supporting our hypothesis.

Electrokinetic, analytical and functional heterogeneity of circulating human platelets: separation of subpopulations by continuous flow electrophoresis after taxol stabilization.

A continuous flow electrophoresis procedure has been developed to study platelet subpopulation heterogeneity with separations based upon surface electrical charge differences. Taxol at low concentrations has been used to transiently stabilize the cells during the separations. At a concentration of 10(-5) M taxol has no effect upon a wide range of physical, analytical and enzymatic properties and does not compromise agonist-induced activation responses (aggregation and secretion). A typical normal platelet subpopulation profile extends over 15-20 fractions with mobilities from -0.97 to -0.78 microns per s per volt per cm. Platelet size (resistive particle counter volumes) differed significantly across the profile, the most electronegative cells being the larger, and the least electronegative the smaller platelets. Total platelet sialic acid content and surface neuraminidase-labile sialic acid correlated positively with electronegativity, but the surface -SH group status had an inverse relationship with the least electronegative smaller platelets, having twice as many surface DTNB-titratable - SH groups as the most electrophoretically mobile and larger cells. Normalisation of analytical and enzymatic data to cell volumes revealed that the smaller less electronegative platelets were substantially richer in all constituents and properties than the larger more electronegative platelets. These smaller cells showed higher activities for lysosomal enzymes, and their functions (capacity to transport 5-hydroxytryptamine and adenosine across the plasma membrane and responsiveness to thrombin expressed by synthesis of thromboxane B2 (TXB2) or release of 5HT) were greater than the larger more electronegative cells. No significant differences were observed, however, in the subpopulations by optical aggregometry using six different agonists each at three different concentrations. This free flow electrophoresis separation of platelets, which can be carried out on a preparative scale, may have some advantages over the conventional density gradient separations of subpopulations for investigating clinical states affecting thrombopoietic regulation or platelet losses from the circulation due to vessel wall disease, prosthetic implants or during extracorporeal circuitry.

Reversible electropermeabilisation of human and rat blood platelets: evaluation of morphological and functional integrity 'in vitro' and 'in vivo'.

A high-voltage discharge procedure has been developed for permeabilising the plasma membranes of both human and rat blood platelets. The cells can be resealed by incubation at 37 degrees C, show less than 4% loss of lactate dehydrogenase (LDH) implying minimal cell lysis and also have well maintained morphological and functional integrity. The prototype apparatus used at field strengths between 6 and 8 kV/cm produces membrane pores which allow free diffusion of low molecular weight substances such as adenine nucleotides, inositol phosphate and fluorescent dyes. Two properties, namely Ca2+-induced secretion of granule stored 5-hydroxytryptamine (5HT) and inositol 1,4,5-trisphosphate (IP3)-induced release of intracellularly sequestered 45Ca, which are both well expressed immediately after permeabilisation, are essentially abolished after resealing. The efficiency of permeabilisation and resealing can be simply monitored by shifts in 'apparent platelet volume' using a resistive particle counter (Coulter). Permeabilised platelets show a shift in modal volumes from a control range 4-7 fl to 10-15 fl. Resealing restores these modal volumes to the original control range. Encapsulation of the fluorochrome, Lucifer yellow (Mr 550), during permeabilisation revealed that after resealing greater than 85% of rat platelets, and close to 100% human platelets, contained the encapsulated dye. The initial rates and % aggregation responses of both human and rat platelets to collagen, thrombin and the thromboxane A2-mimetic U46619 remained essentially normal after permeabilisation and resealing further illustrating the maintenance of functional competence following treatment. Resealed rat platelets reinfused into the circulation after labelling with [111In]indium oxine gave survival curves similar to those of control platelets. Therefore, this reversible permeabilisation procedure may allow the use of autologous or heterologous platelets as carrier vehicles for the delivery of drugs and other agents 'in vivo'.

The isolation and characterisation of guinea-pig polymorphonuclear leucocyte actin and myosin.

The contractile proteins actin and myosin have been isolated from the soluble phase of guinea-pig polymorphonuclear leucocytes and partially characterised. Two forms of actin have been identified, designated 'Mg-actin' and 'KCl-actin'. They have different polymerising properties but their propensity to form synthetic homologous and heterologous actomyosins and to inhibit DNAase-1 does not significantly differ. Both show beta and gamma isoelectric forms in focusing gels and the Mg-actin accounts for about 5% of the soluble-phase protein and te KCl-actin around 2%. Leucocyte myosin has been isolated by affinity chromatography on N6-ADP-Sepharose with a good enrichment of both Ca2+-ATPase and the ATPase activity measured in the absence of Ca2+ or Mg2+ and in the presence of EDTA. This protein, too, has the capacity to form synthetic homologous and hybrid actomyosins with enhancement of the basal Mg2+-ATPase activity. The ratio of actin to myosin in the leucocyte calculated on a molar basis is well in excess of 100, a figure consistent with the findings from other non-muscle cells.

Ecto-protein kinase activity in rabbit peritoneal polymorphonuclear leucocytes.

An ectoprotein kinase activity has been identified on intact rabbit peritoneal polymorphonuclear leucocytes and the time course of phosphate incorporation into proteins has been followed at different ATP levels. Saturation is reached at around 3 mM ATP and the activity is inhibited by p-chloromercuribenzoate. The possibility that the observed protein phosphorylation arises through the action of a membrane ATPase liberating phosphate for transfer into the cell, incorporation into ATP and its utilisation by endogenous kinases, has been excluded by studying both enzymes concomitantly and measuring the rate of [32P]orthophosphate uptake. Lactate dehydrogenase measurements in the extracellular media also exclude the possibility of kinase liberation from lysed cells. Moreover, the pattern of 32P-labelling of polypeptides when intact cells are exposed to [32P]ATP is quite different from that when homogenates are incubated with [32P]ATP or intact cells with [32P]-orthophosphate. We have been unable to demonstrate any cAMP dependency for this ectokinase activity.