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.

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.

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.

European Society of Endocrinology Clinical Practice Guidelines for the management of aggressive pituitary tumours and carcinomas

BACKGROUND: Pituitary tumours are common and easily treated by surgery or medical treatment in most cases. However, a small subset of pituitary tumours does not respond to standard medical treatment and presents with multiple local recurrences (aggressive pituitary tumours) and in rare occasion with metastases (pituitary carcinoma). The present European Society of Endocrinology (ESE) guideline aims to provide clinical guidance on diagnosis, treatment and follow-up in aggressive pituitary tumours and carcinomas. METHODS: We decided upfront, while acknowledging that literature on aggressive pituitary tumours and carcinomas is scarce, to systematically review the literature according to the GRADE (Grading of Recommendations Assessment, Development and Evaluation) system. The review focused primarily on first- and second-line treatment in aggressive pituitary tumours and carcinomas. We included 14 single-arm cohort studies (total number of patients = 116) most on temozolomide treatment (n = 11 studies, total number of patients = 106). A positive treatment effect was seen in 47% (95% CI: 36-58%) of temozolomide treated. Data from the recently performed ESE survey on aggressive pituitary tumours and carcinomas (165 patients) were also used as backbone for the guideline. SELECTED RECOMMENDATION: (i) Patients with aggressive pituitary tumours should be managed by a multidisciplinary expert team. (ii) Histopathological analyses including pituitary hormones and proliferative markers are needed for correct tumour classification. (iii) Temozolomide monotherapy is the first-line chemotherapy for aggressive pituitary tumours and pituitary carcinomas after failure of standard therapies; treatment evaluation after 3 cycles allows identification of responder and non-responder patients. (iv) In patients responding to first-line temozolomide, we suggest continuing treatment for at least 6 months in total. Furthermore, the guideline offers recommendations for patients who recurred after temozolomide treatment, for those who did not respond to temozolomide and for patients with systemic metastasis.

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.

Increased α-synuclein phosphorylation and nitration in the aging primate substantia nigra.

Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson's disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes.

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.

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 and characterization of lens specific calpain (Lp82) from bovine lens.

Ubiquitous type m-calpain and lens specific Lp82 calpain were separated and partially purified from fetal bovine lens and the enzymatic characteristics were compared. Lens m-calpain required 200 microM calcium for 1/2 maximal activity, while Lp82 required 30 microM. Both types of calpains were inhibited by 0.1 mM E64, and 5 mM iodoacetamide, but not by 1 mM phenylmethylsulfonyl fluoride. Lp82 was insensitive to 1 microM calpastatin peptide while m-calpain was effectively inhibited. In the presence of calcium, m-calpain lost most of its activity within 2 hr, while Lp82 was continually active for 18 hr. Both calpains cleaved the natural substrates betaA3 and alphaB crystallins in a similar manner. However, incubation of alphaA crystallin with m-calpain removed ten amino acid residues from its C-terminus, while incubation with Lp82 removed only five residues. The latter truncation product of alphaA was also found in vivo. These data suggested that Lp82 may have a more important role than m-calpain in modification of crystallins during lens maturation.

Effects of an aerobic exercise program on median nerve conduction and symptoms associated with carpal tunnel syndrome.

Carpal tunnel syndrome is associated with greater body mass index and less physical activity. To determine the effect of aerobic exercise on median nerve conduction and symptoms suggestive of carpal tunnel syndrome, 30 symptomatic volunteers (30 to 64 years old) with abnormal median nerve conduction studies participated in a 10-month program of supervised aerobic exercise. Changes in percentage of body fat, body mass index, peak oxygen consumption, 14-cm median sensory latency, and hand/wrist symptoms were assessed. A decrease in 14-cm sensory median latency correlated with a decrease in percentage of body fat (R = 0.52, P = 0.004) and was predicted by an increase in peak oxygen utilization (partial R = 0.52, P = 0.005) and a decrease in body mass index (partial R = 0.47, P = 0.014). There was also a tendency for a set of symptoms sometimes associated with carpal tunnel syndrome (pain, tightness, and clumsiness) to be relieved by the exercise program. These results suggest that an aerobic exercise program can be beneficial to median nerve function and may be associated with a reduction in hand symptoms.

Lack of nigral pathology in transgenic mice expressing human alpha-synuclein driven by the tyrosine hydroxylase promoter.

alpha-Synuclein has been identified as a major component of Lewy body inclusions, which are one of the pathologic hallmarks of idiopathic Parkinson's disease. Mutations in alpha-synuclein have been found to be responsible for rare familial cases of Parkinsonism. To test whether overexpression of human alpha-synuclein leads to inclusion formation and neuronal loss of dopaminergic cells in the substantia nigra, we made transgenic mice in which the expression of wild-type or mutant (A30P and A53T) human alpha-synuclein protein was driven by the promoter from the tyrosine hydroxylase gene. Even though high levels of human alpha-synuclein accumulated in dopaminergic cell bodies, Lewy-type-positive inclusions did not develop in the nigrostriatal system. In addition, the number of nigral neurons and the levels of striatal dopamine were unchanged relative to non-transgenic littermates, in mice up to one year of age. These findings suggest that overexpression of alpha-synuclein within nigrostriatal dopaminergic neurons is not in itself sufficient to cause aggregation into Lewy body-like inclusions, nor does it trigger overt neurodegenerative changes.

In vivo MHC class II presentation of cytosolic proteins revealed by rapid automated tandem mass spectrometry and functional analyses.

We report a strategy for high through-put sequence analyses of large MHC class II-bound peptide repertoires which combines automated electrospray ionization tandem mass-spectrometry with computer-assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I-Ab in activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H-2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen-presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II-bound self peptides derived from cytosolic proteins exists in vivo.

Valve interstitial cells induce donor-specific T-cell anergy.

OBJECTIVES: Valve allografts produce an immune response, which can influence their performance. The exact role of the interaction between recipient T cells and the different cellular components of the donor valve in stimulating an immune response is not known. Therefore the T-cell response to valve endothelial and interstitial cells was investigated in vitro. METHODS: Valve endothelial and interstitial cells were characterized for cell-surface molecules before and after interferon gamma treatment by means of a panel of specific monoclonal antibodies and flow cytometry. The proliferative response of highly purified T lymphocytes was used to assess the immunogenicity of cultured valve endothelial and interstitial cells. This was further investigated by using a 2-step tolerance-induction protocol. RESULTS: Valve endothelial and interstitial cells express similar levels of human leukocyte antigens and adhesion and costimulatory molecules, which are either induced or upregulated after interferon gamma treatment. T-cell responses to endothelial cells were detected after interferon gamma treatment, but responses to interferon gamma-treated interstitial cells were not detected. This lack of response resulted in the induction of T-cell anergy, which was reversed by the presence of the costimulatory molecule B7-1. CONCLUSIONS: Although valve endothelial and interstitial cells express a similar range of cell-surface molecules, it is only the endothelial cells that are immunogenic. In addition, we have shown that these 2 cell types interact in a donor-specific manner to orchestrate the immune response and therefore may have clinical relevance in the allogeneic response of the heart valve recipients.

Biomechanics of procedures used in adult flatfoot deformity.

A flatfoot deformity can occur secondary to fairly obvious causes, or more subtle and less definable entities. Complicating the situation further, it is likely that the cause of an acquired flatfoot deformity in an adult is multifactorial. This likelihood makes the definition, diagnosis, and appropriate treatment of this condition a daunting task. More research is needed to define further the biomechanics of the foot and to understand the significance of the forces that combine to create flatfoot deformity.