Development of a cost-effective diagnostic algorithm incorporating transcription factor immunohistochemistry in the evaluation of pituitary tumours.

PURPOSE: To determine the utility of the 2022 WHO Classification of pituitary tumours in routine clinical practice and to develop an optimal diagnostic algorithm for evaluation of tumour type in a real-world setting. METHODS: Retrospective evaluation of pituitary tumour immunohistochemistry (IHC), operatively managed at St Vincent's Hospital Sydney, between 2019 and 2021. Routine IHC comprised evaluation of transcription factors [steroidogenic factor 1 (SF1), T-box transcription factor 19 (TPIT) and pituitary-specific positive transcription factor (PIT1)] and anterior pituitary hormones. Three tiered algorithms were tested, in which hormone IHC was performed selectively based on the initial transcription factor results. These were applied retrospectively and compared with current practice 'gold standard' comprising all transcription factor and hormone IHC. Diagnostic accuracy and cost were evaluated for each. RESULTS: There were 113 tumours included in the analysis. All three algorithms resulted in 100% concordance with the 'gold standard' in the characterisation of tumour lineage. While all three were associated with relative cost reduction, Algorithm #3, which omitted hormone IHC in the setting of positive SF1 or TPIT and performed IHC for growth hormone, prolactin and thyroid stimulating hormone only in the setting of PIT1 positivity, was the most cost-efficient. Additionally, there were 12/113 tumours with no distinct cell lineage. CONCLUSION: A diagnostic algorithm omitting hormone IHC except in cases of PIT1 positivity is an accurate and cost-effective approach to diagnose the type of pituitary tumour. A significant subgroup of pituitary tumours with no distinct cell lineage, frequently plurihormonal, remains difficult to classify with the new WHO criteria and requires further evaluation.

Retrospective comparison of outcomes following tibial plateau levelling osteotomy and lateral fabello-tibial suture stabilisation of cranial cruciate ligament disease in small dogs with high tibial plateau angles.

AIMS: To compare short and long-term outcomes after tibial plateau levelling osteotomy (TPLO) and lateral fabello-tibial suture (LFTS) techniques for the management of cranial cruciate ligament disease in small dogs with high tibial plateau angles (TPA). METHODS: In this retrospective study, the medical records of two veterinary specialist practices in the United Kingdom were searched for dogs (<20 kg) that underwent TPLO or LFTS between 2000 and 2010, and had a preoperative radiographic TPA >30° with either short-term (6 weeks) and/or long-term (>3 months) follow-up data. Data collected at a 6-week post-surgical re-examination was derived from orthopaedic examination and radiographic assessment and included the incidence of major and minor complications and scoring of the short-term outcome. Long-term outcome was scored based on results of a subjective owner questionnaire and veterinary orthopaedic examination. RESULTS: A total of 61 (84 stifles) dogs were included in the study: 24 (30 stilfes) and 37 (54 stifles) dogs underwent LFTS and TPLO, respectively. Long-term clinical outcome was different (p = 0.017) between groups; 15/15 stifles in the TPLO group had a good or excellent long-term clinical outcome, compared to 4/8 (50%) in the LFTS group. There was no evidence of a difference in short-term post-operative outcome or owner subjective long-term outcome between treatment groups.Stifles in the LFTS group were more likely (p = 0.027) to have palpable stifle pain at long-term follow-up. Owners reported that 5/16 (31.3%) dogs in the LFTS group required oral non-steroidal anti-inflammatory drug (NSAID) treatment at least monthly (4/5 required daily treatment), whereas no dogs in the TPLO group required treatment with NSAID more frequently than three times per year (p = 0.011).No correlation was found between short-term outcome and owner subjective long-term outcome but there was a positive correlation between short-term outcome and long-term clinical outcome.There was no evidence of a difference in overall major complication rates between treatment groups. The occurrence of complications was associated with heavier body weight at the time of surgery. No other variables were shown to be risk factors for complications. CONCLUSION AND CLINICAL RELEVANCE: Small breed dogs with high TPA that underwent TPLO had better long-term clinical outcomes and were less likely to require NSAID administration than those that underwent LFTS. The risk of complication increased with the weight of the dog at surgery. There was a positive correlation between short-term outcome and long-term clinical outcome.

Improved two-dimensional electrophoretic mapping of Japanese human hair proteins; application to curved and straight Japanese human hairs; and protein identification by MALDI MS and MS/MS quadrupole time-of-flight mass spectrometry.

OBJECTIVES: To evaluate improved protein extraction and two-dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women's human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. METHODS: Hair keratin and keratin-associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2-carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric-focused on first-dimensional pH gradient gel strips, then separated by molecular weight on laboratory-made, second-dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty-eight proteins from a JRH 2DE gel were enzyme-cleaved for MALDI-TOF-MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in-house human hair protein database incorporating ninety-eight annotated protein sequences assisted MS analysis. RESULTS: 2DE gels of tris(2-carboxyethyl) phosphine-extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol-extracted JRH and published commercially made second-dimensional gels. Silver-stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over-staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty-nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG-stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. CONCLUSION: Root-end human hair extraction with tris(2-carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight.

OBJECTIFS: évaluer l'amélioration de l'extraction de protéines et les méthodes de séparation bidimensionnelle par électrophorèse (2DE) avec des cheveux humains de référence Japonais (JRH), déterminer si la courbure de la fibre est liée à la composition protéique dans les échantillons de cheveux humains des Japonaises (JHH) bouclés et raides et identifier les protéines issues des cartes JRH 2DE et les différences d'expression entre les JHH bouclés et raides. MÉTHODES: la kératine des cheveux et les protéines associées à la kératine (KAP) ont été extraites intactes avec du dithiothréitol ou du tris (2-carboxyéthyl) phosphine des JRH ou des JHH bouclés ou raides. Les protéines extraites ont subi une focalisation isoélectrique sur des bandes de gel à gradient de pH unidimensionnelles, puis ont été séparées par poids moléculaire sur des gels bidimensionnels de grand format, fabriqués en laboratoire. Le logiciel a comparé l'abondance des protéines entre les deux duplicatas de gels 2DE des JHH bouclés et raides. Trente-huit protéines provenant d'un gel 2DE JRH ont été clivés par enzyme pour l'analyse MALDI-TOF-MS afin de déterminer la composition des peptides, et dans la mesure du possible, un séquençage de novo a donné des données de séquence des peptides. Une base de données interne des protéines capillaires humaines incorporant 98 séquences de protéines annotées a aidé l'analyse MS. RÉSULTATS: les gels 2DE de JRH extraits par le tris (2-carboxyéthyl) ont amélioré la résolution et le nombre de la kératine et du KAP par rapport à ceux du JRH extrait par le dithiothréitol et des gels bidimensionnels fabriqués commercialement. Les gels 2DE à coloration argentée des ensembles de JHH raides ou bouclés étaient remarquablement similaires. La sur-coloration pour révéler les protéines de base a provoqué une mauvaise résolution des principales classes de protéines acides. Les comparaisons logicielles des 59 protéines résolues ont révélé que deux présentaient une différence significative d'abondance entre les cheveux bouclés et raides, mais en quantités insuffisantes pour une analyse MS. La MS a identifié douze protéines provenant d'un gel 2DE coloré CBBG JRH : six kératines de type II, trois kératines de type I et trois protéines à forte teneur en soufre. Huit autres étaient des isoformes conformationnels potentiels et des variantes isoélectriques des protéines identifiées, ramenant le total à 20 protéines identifiées ou partiellement identifiées. CONCLUSION: l'extraction des cheveux humains à la racine avec du tris (2-carboxyéthyl) phosphine améliore la résolution des protéines et permet de visualiser plus de protéines sur les gels 2DE grand format. Les deux différences de protéines mineures entre les duplicatas des gels 2DE JHH raides ou bouclés étaient peu susceptibles de changer la structure des fibres de cheveux raides à bouclés. Les résultats de la MS ont confirmé qu'il existe plusieurs isoformes de diverses protéines capillaires. Une faible couverture de séquence a empêché la distinction entre les protéines homologues de poids moléculaire similaire.

Aiming for elimination: Outcomes of a consultation pathway supporting regional general practitioners to prescribe direct-acting antiviral therapy for hepatitis C.

To increase access to treatment, the Australian government enabled general practitioners (GPs) to prescribe direct-acting antivirals (DAAs) to treat hepatitis C virus (HCV)-in consultation with a specialist if inexperienced in HCV management. This study describes the establishment and outcomes of a remote consultation pathway supporting GPs to treat HCV. Key stakeholders from primary and tertiary healthcare services in the Barwon South Western region developed and implemented an HCV remote consultation pathway. Pharmaceutical Benefits Schedule prescription data were used to evaluate GP DAA prescription 12 months pre-and post- pathway implementation. A retrospective review of patients referred for remote consultation for 12 months post- pathway inception was undertaken to determine the care cascade. HCV treatment initiation by GPs increased after implementation of the remote consultation pathway. In the 12-month study period, 74 GPs referred 169 people for remote consultation; 114 (67%) were approved for GP DAA treatment; 48 (28%) were referred for specialist assessment. In total, 119 (71%) patients commenced DAA; 69 were eligible for SVR12 assessment. Post-treatment HCV RNA data were available for 52 (75%) people; 37 achieved SVR12; 15 achieved SVR ranging from week 5 to 11 post-treatment. No treatment failure was detected. Collaborative development and implementation of a remote consultation pathway has engaged regional GPs in managing HCV. Follow-up post-treatment could be improved; however, no treatment failure has been documented. To eliminate HCV as a public health threat, it is vital that specialists support GPs to prescribe DAA.

Optimizing experimental design for genome sequencing and assembly with Oxford Nanopore Technologies.

High quality reference genome sequences are the core of modern genomics. Oxford Nanopore Technologies (ONT) produces inexpensive DNA sequences, but has high error rates, which make sequence assembly and analysis difficult as genome size and complexity increases. Robust experimental design is necessary for ONT genome sequencing and assembly, but few studies have addressed eukaryotic organisms. Here, we present novel results using simulated and empirical ONT and DNA libraries to identify best practices for sequencing and assembly for several model species. We find that the unique error structure of ONT libraries causes errors to accumulate and assembly statistics plateau as sequence depth increases. High-quality assembled eukaryotic sequences require high-molecular-weight DNA extractions that increase sequence read length, and computational protocols that reduce error through pre-assembly correction and read selection. Our quantitative results will be helpful for researchers seeking guidance for de novo assembly projects.

Lack of effect of MICA antibodies on graft survival following heart transplantation.

Little is known about the effect of MICA antibodies (Abs) on cardiac allograft function and survival. Pretransplant and posttransplant serum from 491 and 196 adult cardiac allograft recipients, respectively, has been investigated for MICA Abs, donor specificity and the effect of MICA Abs on graft survival, acute rejection episodes (AR) and cardiac allograft vasculopathy (CAV). Patients with HLA Abs (11.6%) were excluded from the analysis. A total of 11.8% of patients had MICA Abs, without HLA Abs, before their transplant. Actuarial graft survival demonstrated slightly better survival of patients with donor-specific MICA Abs at 1 and 5 years (88.9% and 83.3%) than patients negative for MICA Abs (72% and 63.7%, p = 0.051). After transplantation, 15.8% of patients produced MICA Abs, and in 17 patients these were produced de novo. There was no effect of pretransplant or posttransplant production of MICA Abs on numbers of AR episodes in year 1, or CAV assessed at years 3 and 5. Immunocytochemistry of cardiac biopsies from 11 patients did not demonstrate a presence of MICA. Sera from only 4/69 patients with MICA Abs fixed complement prior to transplantation and from 7/38 patients following transplantation. In conclusion, this study suggests that MICA Abs do not adversely affect the outcome of cardiac transplantation.

Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast.

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.

Vimentin autoantibodies induce platelet activation and formation of platelet-leukocyte conjugates via platelet-activating factor.

Anti-vimentin antibodies (AVA) are associated with autoimmunity and solid organ transplantation, conditions associated with vascular disease, but their contribution to disease pathogenesis is unknown. Here, we have examined interactions between AVA (mAb and serum from patients) and various leukocyte populations using whole blood and flow cytometry. Normal blood treated with patient sera containing high AVA-IgM titers or with a vimentin-specific monoclonal IgM led to activation of platelets and other leukocytes, as demonstrated by induced expression of P-selectin, fibrinogen, tissue factor, and formation of platelet:leukocyte (P:L) conjugates and a reduction in platelet counts. This activity was antigen (vimentin)-specific and was not mediated by irrelevant IgM antibodies. Flow cytometry demonstrated that AVA do not bind directly to resting platelets in whole blood, but they bind to approximately 10% of leukocytes. Supernatant, derived from AVA-treated leukocytes, induced platelet activation, as measured by the generation of platelet microparticles, when added to platelet-rich plasma. When AVA were added to whole blood in the presence of CV-6209, a platelet-activating factor (PAF) receptor inhibitor, platelet depletion was inhibited. This suggests that PAF is one of the mediators released from AVA-activated leukocytes that leads to P:L conjugation formation and platelet activation. In summary, AVA bind to leukocytes, resulting in release of a PAF and prothrombotic factor that exert a paracrine-activating effect on platelets. Overall, this proposed mechanism may explain the pathogenesis of thrombotic events in autoimmune diseases associated with AVA.

Decreased susceptibility to oxidative stress underlies the resistance of specific dopaminergic cell populations to paraquat-induced degeneration.

The vulnerability of different dopaminergic cell populations to damage caused by the herbicide paraquat was assessed by stereological counts of tyrosine hydroxylase-positive and calbindin-D28k-immunoreactive neurons in A9 (substantia nigra pars compacta) and A10 (ventral tegmental area and other cell groups). In saline-treated control mice, tyrosine hydroxylase-immunoreactive neurons represented 80% and 45% of the total neuronal population in A9 and A10, respectively, and the number of calbindin-D28k-positive neurons was five times greater in A10 than A9. Sequential injections with paraquat resulted in a significant loss of dopaminergic neurons in A9. In contrast, tyrosine hydroxylase-positive cells in A10 were spared from paraquat-induced degeneration. Furthermore, expression of calbindin-D28k was consistently associated with neuronal resistance to the herbicide in both A9 and A10. Paraquat exposure also induced oxidative stress as indicated by an increase in the number of midbrain cells positive for 4-hydroxy-2-nonenal, a marker of lipid peroxidation. Co-localization studies revealed that calbindin-D28k immunoreactivity overlapped with tyrosine hydroxylase labeling and that, after paraquat administration, (i) the vast majority of midbrain 4-hydroxy-2-nonenal-immunoreactive cells were dopaminergic (tyrosine hydroxylase-immunoreactive), (ii) tyrosine hydroxylase/4-hydroxy-2-nonenal-positive neurons were much more prevalent in A9 than A10, and (iii) all calbindin-D28k-containing neurons were characterized by lack of lipid peroxidation (4-hydroxy-2-nonenal immunoreactivity). Results in this paraquat model emphasize that, despite sharing a similar dopaminergic phenotype, different groups of midbrain neurons vary dramatically in their vulnerability to injury. Data also indicate that these differences are attributable, at least in part, to a varying susceptibility of dopaminergic cell populations to oxidative stress.

Purification of α-synuclein containing inclusions from human post mortem brain tissue.

UNLABELLED: Comparison with existing methods. BACKGROUND: Neurodegenerative disorders affect a large proportion of the elderly population. A group of disorders, known as the α-synucleinopathies, are characterised by the presence of α-synuclein-containing protein inclusions, such as Lewy Bodies (LBs) found in neurons from Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB), and Glial Cytoplasmic Inclusions (GCIs) found in oligodendrocytes from Multiple System Atrophy (MSA). The analysis of the protein composition of inclusions has been hindered by limitations of methods for isolating the inclusions from the surrounding tissue. METHOD: Four modifications were made to the published method for GCI purification by Gai et al. (1999) which were: collecting the entire inclusion-containing part of the Percoll gradient; lysis of nuclei prior to DNAse digestion; limited tryptic digestion to release inclusions from the cytoskeletal meshwork; and increased antibody and magnetic bead concentrations/volumes to capture the larger amounts of inclusions. RESULTS: The optimised method gave a 28-fold increase in yield compared to the published method of Gai et al. (1999). A 2D-DIGE comparison revealed a 3.8-fold increase in α-synuclein enrichment and a corresponding 5.2-fold reduction in tubulin contamination. This method was also successfully adapted to the purification of LBs from DLB tissue. A 2D-DIGE comparison of purified GCIs (n=2) revealed that GCIs consist of 11.7% α-synuclein, 1.9% α-ß-crystallin and 2.3% 14-3-3 proteins compared to 8.5%, 2.0% and 1.5% in LBs, respectively. CONCLUSIONS: This study has generated an improved method for the purification of α-synuclein-containing inclusions with a yield sufficient for multiple forms of analysis.

Ultraviolet light-induced G2 phase cell cycle checkpoint blocks cdc25-dependent progression into mitosis.

In response to low doses of ultraviolet (U.V.) radiation, cells undergo a G2 delay. In this study we have shown that the G2 delay results in the accumulation of inactive forms of cyclin B1/cdc2 and both the G2 and mitotic complexes of cyclin A/cdk. This appears to be through a block in the cdc25-dependent activation of these complexes. The expression and localisation of cyclin A and cyclin B1/cdk complexes are similar in U.V.-induced G2 delay and normal early G2 phase cells. Cdc25B and cdc25C also accumulate to normal G2 levels in U.V. irradiated cells, but the mitotic phosphorylation associated with increased activity of both cdc25B and cdc25C is absent. The cdc25B accumulates in the nucleus of U.V. irradiated cells and in normal G2 phase cells. Thus the block in cyclin B/cdc2 activation is in part due to the physical separation of cyclin B/cdc2, localised in the cytoplasm, from the cdc25B and cdc25C phosphatases localised in the nucleus. The data positions the U.V.-induced G2 checkpoint at either the S/G2 transition or early G2 phase, prior to the activation of cyclin A/cdk2.

Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells: induction of VCAM-1 but no proliferation.

OBJECTIVE: Intercellular adhesion molecule (ICAM)-1 is an immunoglobulin-like cell adhesion molecule expressed by several cell types, including proliferating vascular smooth muscle cells (VSMC). Cross-linking ICAM-1 on the surface of different cell types has previously been shown to cause an increase in cellular activation within the cytoplasm. Here, our objective was to examine events following ligation of ICAM-1 on the surface of human VSMC. METHODS: VSMC were isolated by explant from human pulmonary arteries or aortic tissue from cardiac transplant donors. ICAM-1 was ligated with monoclonal antibodies, followed by cross-linking with a secondary antibody. Activation of signalling pathways, proliferation and expression of a second adhesion molecule, vascular cell adhesion molecule (VCAM)-1 were investigated. RESULTS: ICAM-1 cross-linking caused an increase in activation of extracellular regulated kinase (Erk)-1/-2 and Jun N-terminal kinase (JNK)-1/-2. mRNA and protein for VCAM-1 was observed after ICAM-1 cross-linking, and this was abrogated by addition of an upstream inhibitor of Erk-1/-2, PD98059. No increase in cell proliferation was observed. CONCLUSIONS: Ligation of ICAM-1 on the surface of vascular smooth muscle cells in vitro, leads to the expression of adhesion molecules associated with monocyte infiltration, but does not contribute to smooth muscle cell proliferation. In vivo, this might lead to prolongation of the inflammatory response within diseased blood vessels, by arresting monocytes within atherosclerotic plaques.

Statistical issues in the analysis of neuroimages.

Statistical issues in the analysis of neuroimages are reviewed. These include biological questions of interest, basic problems of measurement and experimental design, normalisation, standardisation and transformation, and statistical methodology. Finally, three data sets are reviewed to illustrate some of the issues raised in the report.

Abused to abuser: antecedents of socially deviant behaviors.

The authors interviewed 34 young people who had been sexually abused as children 6 or 8 years after the abuse had occurred and compared them with 34 control subjects who had not been abused. They also compared subjects who had been abused for less than 1 year with those who had been abused for more than 1 year. The findings suggest a link between childhood sexual abuse and later drug abuse, juvenile delinquency, and criminal behavior. The authors explore the effects of pretrauma factors of previous childhood physical abuse and parental modeling of aggression and the postdisclosure factors of social and family blaming.

Distribution of the voltage-dependent calcium channel alpha(1A) subunit throughout the mature rat brain and its relationship to neurotransmitter pathways.

The alpha(1) subunit provides both the voltage-sensing mechanism and the ion pore of voltage-dependent calcium channels. Of the six classes of alpha(1) subunit cloned to date, alpha)1A) is the subject of debate in terms of its functional correlate, although it is generally thought to encode voltage-dependent calcium channels of the omega-agatoxin IVA-sensitive, P/Q type. In the present study, an alpha(1A)-specific riboprobe and antibody were used with in situ hybridisation and immunohistochemical techniques to localise alpha(1A) messenger ribonucleic acid transcripts and subunit protein throughout the mature rat brain. Dual localisation of alpha(1A) protein and markers for acetylcholine, catecholamines, and 5-hydroxytryptamine have also been performed in a number of discrete areas. Abundant and widespread distribution of alpha(1A) protein was found, with immunoreactivity occurring both in cell bodies and as punctate staining in areas of neuronal processes. Several associations were noted across alpha(1A) localisation, defined neuroanatomical regions, and neurotransmitter systems. However, alpha(1A) expression was not confined to loci corresponding to any one neurotransmitter type, although a high level of expression was observed in cholinergic neurones. The distribution of the alpha(1A) subunit in the rat corresponded well with the limited human mapping data that are available.

Affinity purification of rat cortical and chicken forebrain synaptosomes using a biotinylated derivative of omega-CgTx GVIA.

We describe a magnetophoretic method for the affinity purification of synaptosomes expressing omega-CgTx GVIA-sensitive, N-type voltage-sensitive calcium channels (VSCCs). The method utilizes a biotinylated derivative of omega-CgTx GVIA which retains its ability to displace [125I] omega-CgTx GVIA from its binding sites on rat synaptic membranes. When coupled to streptavidin coated magnetizable beads, the hexanoyl spacer between omega-CgTx GVIA and the biotin:streptavidin bead complex is sufficiently long to allow flexibility of the toxin to bind to its receptor on synaptosomes. We have used this ligand successfully to isolate deaggregated synaptosomes from the parent fractions of chicken forebrain and rat cortex. In the chicken synaptosome parent fraction, omega-CgTx GVIA (1 nM-1 microM) produced a concentration-dependent block of the KCl-induced intracellular free Ca2+, [Ca2+]i, elevation with an IC50 of 28 nM. After affinity magnetophoresis no increase in [Ca2+]i elevation was observed in either the bound or unbound fractions. In the rat synaptosome parent fraction, the KCl-induced increase in free intracellular Ca2+ ([Ca2+]i) elevation was partially blocked by omega-CgTx GVIA (17 +/- 2% / 1 microM) and to a greater extent by omega-Aga IVA (55 +/- / 1 microM): a combination of the two toxins was additive (72 +/- 4% / 1 microM). The block obtained by omega-CgTx GVIA (1 microM) in the unbound fraction was reduced to 3 +/- 2%, whereas that by omega-Aga IVA (1 microM) increased to 82 +/- 3%. The block obtained by a combination of both toxins (83 +/- 2%) was the same as that with omega-Aga IVA alone (82 +/- 3%). No increase in free [Ca2+]i elevation was observed in the bound fraction although single synaptosome-like structures, displaying synaptophysin immunoreactivity, were detected on the beads. We conclude that omega-CgTx GVIA-sensitive N-type calcium channels are present on all chicken forebrain synaptosomes but only a subset of rat cortical synaptosomes.

Mediation of acute but not chronic rejection of MHC-incompatible rat kidney grafts by alloreactive CD4 T cells activated by the direct pathway of sensitization.

It has been previously postulated that there are two pathways of sensitization of MHC-incompatible kidney allografts: a direct pathway in which the host responder T cells are activated by MHC-incompatible passenger dendritic cells of the graft, and an indirect pathway, in which graft alloantigens are processed like "nominal" T cell antigens by host accessory cells, and presented as self-MHC restricted moieties. We show here that a rat AS anti-August alloreactive CD4+ T cell line, and a presumptive clone, activated through the direct pathway are capable in an adoptive transfer model of initiating rejection of normal August kidney grafts. However, neither the T cell line nor the presumptive clone initiates rejection of passenger cell-depleted August kidneys. The results support the hypothesis that direct pathway--sensitized T cells play a dominant role in early acute rejection, but not in chronic rejection.