Nitric Oxide on Extracorporeal Life Support-Circuit Modifications for a Safe Therapy.

Nitric oxide (NO) incorporation into the sweep gas of the extracorporeal life support (ECLS) circuit has been proposed as a strategy to ameliorate the insults caused by the systemic inflammatory response. This technical study describes circuit modifications allowing nitric oxide to be incorporated into the circuit and describing and validating the oxygenator sweep flow rates necessary to achieve consistent safe delivery of the therapy. For patients requiring sweep rates less than 2 L/min, a simplified setup, incorporating a pressure relief valve/low flow meter in the gas delivery line, was placed in line between the blender/NO injector module and the NO sampling port/oxygenator. This setup allows titration of sweep to low flows without the need to blend in CO2 while maintaining the manufacturer recommendation of a minimum 2 L/min of sweep gas to safely deliver NO without nitric dioxide (NO2) buildup. This setup was tested three times at three different FiO2 rates and eleven different desired low sweep flows to test for reproducibility and safety to build an easy-to-follow chart for making gas flow changes. For patients requiring oxygenator sweep rates greater than 2 L/min, the pressure relief valve/low flow meter apparatus is not needed. Maintaining consistent sweep rate and nitric oxide delivery is required in order to utilize this therapy in ECLS. We demonstrated gas delivery across all flow rates. There were no issues delivering 20 parts per million of NO and negligible NO2 detection. The results from testing this setup were used to provide the specialist a chart at which to set the low flow meter to produce the desired flow rate at which the patient needs. This has been used clinically on 15 ECLS patients with success.

HOXB13 suppresses de novo lipogenesis through HDAC3-mediated epigenetic reprogramming in prostate cancer.

HOXB13, a homeodomain transcription factor, critically regulates androgen receptor (AR) activities and androgen-dependent prostate cancer (PCa) growth. However, its functions in AR-independent contexts remain elusive. Here we report HOXB13 interaction with histone deacetylase HDAC3, which is disrupted by the HOXB13 G84E mutation that has been associated with early-onset PCa. Independently of AR, HOXB13 recruits HDAC3 to lipogenic enhancers to catalyze histone deacetylation and suppress lipogenic regulators such as fatty acid synthase. Analysis of human tissues reveals that the HOXB13 gene is hypermethylated and downregulated in approximately 30% of metastatic castration-resistant PCa. HOXB13 loss or G84E mutation leads to lipid accumulation in PCa cells, thereby promoting cell motility and xenograft tumor metastasis, which is mitigated by pharmaceutical inhibition of fatty acid synthase. In summary, we present evidence that HOXB13 recruits HDAC3 to suppress de novo lipogenesis and inhibit tumor metastasis and that lipogenic pathway inhibitors may be useful to treat HOXB13-low PCa.

Histopathologic findings in culture-positive secondary osteomyelitis.

As peripheral vascular disease and diabetes mellitus are increasingly common, chronic wounds are often seen. Bone biopsies, with imaging and microbial cultures, are often obtained to evaluate for osteomyelitis. Because much of the historical literature describing the histology of osteomyelitis pertains to primary osteomyelitis, this study characterizes the histologic findings and provides correlation with culture results in secondary osteomyelitis. The histologic features of bone biopsies were assessed over a 5 year period. Concurrent laboratory and radiographic data were obtained and these data were compared with culture results. This study included 163 cases, of which 104 were culture-positive osteomyelitis. All culture-positive cases had been present longer than 28 days and had at least one of the following histologic features: neutrophilic inflammation, plasmacytic inflammation, or eosinophilic fibrosis. However, none of these findings were restricted to culture-positive cases. Overall, plasmacytic and neutrophilic inflammation provided similar specificity, and positive predictive values for osteomyelitis. Medullary fibrosis gave a sensitivity of 95%, the highest for any single feature, and the combination of fibrosis and neutrophilic inflammation had the greatest specificity of 96%. Additionally, neutrophilic inflammation correlated often with isolation of Staphylococcus aureus, while plasma cell predominance was found more frequently with other infectious agents. This study describes histologic features in secondary osteomyelitis, which may challenge the widespread inclination to equate a neutrophilic inflammation with 'acute osteomyelitis' and 'chronic osteomyelitis' with one rich in plasma cells. We report an early correlation between common histopathologic findings and specific culture isolates, which can be further refined with additional research.

Application and Pitfalls of Immunohistochemistry in Diagnosis of Challenging Genitourinary Cases.

CONTEXT.­: Immunohistochemistry (IHC) has become increasingly important in the evaluation of pathologic conditions in the genitourinary (GU) organs. In addition to careful evaluation of hematoxylin-eosin sections and generation of a differential diagnosis, choosing the optimal panel of IHC markers becomes even more important when the biopsy material is very limited. The following summary of our experience supplemented with relevant literature review exemplifies how to use IHC to facilitate pathologic diagnosis in the GU system. OBJECTIVE.­: To describe our experience with the most common immunohistochemical markers used in GU pathology. DATA SOURCES.­: Institutional experience and literature search comprise our data sources. CONCLUSIONS.­: Application of IHC provides enormous benefits to the interpretation of GU pathologic conditions, including benign and malignant lesions. However, both insufficient and excessive types of use of IHC, as well as incorrect interpretations in common and rare GU conditions, could present pitfalls in diagnosis.

Rare case of atypical crescentic glomerulonephritis and interstitial lung disease with negative anti-GBM antibody and positive anti-MPO antibody.

A 70-year-old man presented with 1 month of haematuria and mild right-sided flank pain with no other symptoms. Diagnostic workup included serum studies which showed the presence of antimyeloperoxidase antibodies, a kidney biopsy which demonstrated necrotising crescentic glomerulonephritis with linear immunofluorescence of the basement membrane, and electron microscopy which exhibited thickening of the glomerular basement membrane. Incidentally, the patient was discovered to have a latent hepatitis B infection, which complicated immunosuppressive therapy. He was treated with a course of plasmapheresis and methylprednisolone, followed by entecavir for hepatitis B prophylaxis, and finally by rituximab. This case of glomerulonephritis was notable for its resemblance to the better known Goodpasture's disease. Typically, Goodpasture's syndrome exists on a spectrum from seronegative disease to double-positive disease that presents with both anti-glomerular basement membrane (anti-GBM) and cytoplasmic-antineutrophil cytoplasmic antibodies/antiproteinase 3 antibodies (c-ANCA/anti-PR3). However, this patient's glomerulonephritis was unique because he presented negative for anti-GBM antibodies and positive for perinuclear-antineutrophil cytoplasmic antibodies/antimyeloperoxidase antibodies (p-ANCA/anti-MPO).

The Radiologic and Pathologic Diagnosis of Biphasic Pulmonary Blastoma.

Pulmonary blastomas are rare malignancies, representing 0.25% to 0.5% of all primary lung neoplasms with often aggressive progression and poor prognosis. Clinical management of pulmonary blastomas depends on histologic subtype, staging, and presentation, and may consist of surgery, chemotherapy, and radiation. Biphasic pulmonary blastoma is a subtype of pulmonary blastoma that exhibits biphasic histology, with both epithelial and mesenchymal malignant elements. We report a case of biphasic pulmonary blastoma in a 33-year-old female with 1 pack per day history of smoking for approximately 16 years, who presented with left-sided pleuritic chest pain on deep inspiration without otherwise significant pat medical history. Imaging evaluation using chest radiography, computed tomography, and magnetic resonance imaging identified a heterogenous, well-circumscribed, left lower lobe mass with extensive necrosis and hemorrhage. No lymphadenopathy or distant metastasis was detected through imaging evaluation. Surgical resection of the tumor followed by histopathological analysis confirmed a biphasic pulmonary blastoma.

Studying T Cell Development in Thymic Slices.

Recently, tissue slices have been adapted to study both mouse and human T cell development. Thymic slices combine and complement the strengths of existing organotypic culture systems to study thymocyte differentiation. Specifically, the thymic slice system allows for high throughput experiments and the ability to introduce homogenous developmental intermediate populations into an environment with a well-established cortex and medulla. These qualities make thymic slices a highly versatile and technically accessible model to study thymocyte development. Here we describe methods to prepare, embed, and slice thymic lobes to study T cell development in situ.

Stable interactions and sustained TCR signaling characterize thymocyte-thymocyte interactions that support negative selection.

Negative selection is one of the primary mechanisms that render T cells tolerant to self. Thymic dendritic cells play an important role in negative selection, in line with their ability to induce migratory arrest and sustained TCR signals. Thymocytes themselves display self-peptide/MHC class I complexes, and although there is evidence that they can support clonal deletion, it is not clear whether they do so directly via stable cell-cell contacts and sustained TCR signals. In this study, we show that murine thymocytes can support surprisingly efficient negative selection of Ag-specific thymocytes. Furthermore, we observe that agonist-dependent thymocyte-thymocyte interactions occurred as stable, motile conjugates led by the peptide-presenting thymocyte and in which the trailing peptide-specific thymocyte exhibited persistent elevations in intracellular calcium concentration. These data confirm that self-Ag presentation by thymocytes is an additional mechanism to ensure T cell tolerance and further strengthen the correlation between stable cellular contacts, sustained TCR signals, and efficient negative selection.

Effect of whole-body vibration therapy on performance recovery.

PURPOSE: To compare whole-body vibration (WBV) with traditional recovery protocols after a high-intensity training bout. METHODS: In a randomized crossover study, 16 athletes performed 6 × 30-s Wingate sprints before completing either an active recovery (10 min of cycling and stretching) or WBV for 10 min in a series of exercises on a vibration platform. Muscle hemodynamics (assessed via near-infrared spectroscopy) were measured before and during exercise and into the 10-min recovery period. Blood lactate concentration, vertical jump, quadriceps strength, flexibility, rating of perceived exertion (RPE), muscle soreness, and performance during a single 30-s Wingate test were assessed at baseline and 30 and 60 min postexercise. A subset of participants (n = 6) completed a 3rd identical trial (1 wk later) using a passive 10-min recovery period (sitting). RESULTS: There were no clear effects between the recovery protocols for blood lactate concentration, quadriceps strength, jump height, flexibility, RPE, muscle soreness, or single Wingate performance across all measured recovery time points. However, the WBV recovery protocol substantially increased the tissue-oxygenation index compared with the active (11.2% ± 2.4% [mean ± 95% CI], effect size [ES] = 3.1, and -7.3% ± 4.1%, ES = -2.1 for the 10 min postexercise and postrecovery, respectively) and passive recovery conditions (4.1% ± 2.2%, ES = 1.3, 10 min postexercise only). CONCLUSION: Although WBV during recovery increased muscle oxygenation, it had little effect in improving subsequent performance compared with a normal active recovery.

Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development.

The catalytic activity of Zap70 is crucial for T cell antigen receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap70 catalytic activity in a model of synchronized thymic selection, we showed that CD4(+)CD8(+) thymocytes integrate multiple, transient, Zap70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas 1 h of signaling was sufficient for negative selection. Titration of Zap70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, which revealed heterogeneity, even among CD4(+)CD8(+) thymocytes expressing identical TCRs undergoing positive selection.

Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns.

Positive selection of CD8 T cells in the thymus is thought to be a multistep process lasting 3-4 d; however, the discrete steps involved are poorly understood. Here, we examine phenotypic changes, calcium signaling, and intrathymic migration in a synchronized cohort of MHC class I-specific thymocytes undergoing positive selection in situ. Transient elevations in intracellular calcium concentration ([Ca(2+)]i) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca(2+)]i over time. Changes in chemokine-receptor expression and relocalization from the cortex to medulla occurred between 12 and 24 h after the initial encounter with positive-selecting ligands, a time frame at which the majority of thymocytes retain CD4 and CD8 expression and still require T-cell receptor (TCR) signaling to efficiently complete positive selection. Our results identify distinct phases in the positive selection of MHC class I-specific thymocytes that are distinguished by their TCR-signaling pattern and intrathymic location and provide a framework for understanding the multistep process of positive selection in the thymus.

Long-term effect of whole body vibration training on jump height: meta-analysis.

Whole body vibration (WBV) is widely promoted as a means of improving muscle strength, but the evidence of a performance benefit is unclear with some reporting improvements and others finding none. The objective of this study was to analyze the current evidence for the effectiveness of WBV on jump height. We included randomized controlled trials or matched design studies comparing the effect of WBV training on countermovement and squat jump (SJ) height, which were gathered from MEDLINE, Web of Knowledge, Sciencedirect, Proquest, Scopus, Google Scholar, and SPORTDiscus databases. The overall effect of WBV training (from the 15 studies included) compared with having no additional exercise on countermovement jump height yielded a positive standardized mean difference of 0.77 (95% confidence interval, 0.55-0.99). The effect of WBV training on SJ height was 0.68 (0.08-1.11). Vibration exercise consisting of a higher frequency (>30 Hz, 0.86, 0.62-1.10), higher amplitude (>3 mm, 0.84, 0.52-1.17), longer exposure duration (>10 minutes per session, 0.92, 0.48-1.36), longer training period (>12 weeks, 0.87, 0.56-1.19) and among nonathletes (0.96, 0.63-1.30) had greater benefit for jump height improvement than a lower frequency (≤ 30 Hz, 0.56, 0.13-0.99), lower amplitude (≤ 3 mm, 0.66, 0.35-0.98), shorter exposure duration (≤ 10 minutes per session, 0.68, 0.45-0.92), intermediate training period (4-12 weeks, 0.72, 0.35-1.09), shorter training period (<4 weeks, 0.58, -0.08 to -1.23) and in athletes (0.59, 0.31-0.88). The effect of WBV training compared with a standard cardiovascular-type exercise group from 4 studies was 0.63 (0.10-1.15). In conclusion, WBV training produces a moderate-to-large effect on jump height. Vibration training protocols with higher frequencies, higher amplitudes, longer exposures per session, and longer training periods are more likely to enhance muscle power.

Distinct temporal patterns of T cell receptor signaling during positive versus negative selection in situ.

The recognition by the T cell receptor (TCR) of self-peptides presented by the major histocompatibility complex (MHC) on antigen-presenting cells, such as dendritic cells and thymic epithelial cells, controls T cell fate in the thymus, with weak TCR signals inducing survival (positive selection) and stronger signals inducing death (negative selection). In vitro studies indicate that peptide ligands that induce positive selection stimulate a low, but sustained, pattern of TCR signaling; however, the temporal pattern of TCR signaling in MHC class I-restricted thymocytes (thymocytes that are presented with peptides by MHC class I) in the thymus, under conditions that support positive selection, is unknown. We addressed this question by examining intracellular Ca(2+) dynamics and migratory changes in thymocytes undergoing positive and negative selection in thymic slices. Brief, serial signaling events that were separated by migratory periods and low cytosolic Ca(2+) concentrations correlated with the positive selection of MHC class I-restricted thymocytes, whereas sustained Ca(2+) signaling and the arrest of thymocytes were associated with negative selection. Low-avidity peptides and the presentation of peptides by cortical thymic epithelial cells, rather than dendritic cells, failed to induce strong migratory arrest of thymocytes, which led to transient TCR signaling. Thus, we provide a comparison of positive and negative selection signals in situ and suggest that the absence of strong stop signals distinguishes between positive and negative selection.

Opposing chemokine gradients control human thymocyte migration in situ.

The ordered migration of thymocytes from the cortex to the medulla is critical for the appropriate selection of the mature T cell repertoire. Most studies of thymocyte migration rely on mouse models, but we know relatively little about how human thymocytes find their appropriate anatomical niches within the thymus. Moreover, the signals that retain CD4+CD8+ double-positive (DP) thymocytes in the cortex and prevent them from entering the medulla prior to positive selection have not been identified in mice or humans. Here, we examined the intrathymic migration of human thymocytes in both mouse and human thymic stroma and found that human thymocyte subsets localized appropriately to the cortex on mouse thymic stroma and that MHC-dependent interactions between human thymocytes and mouse stroma could maintain the activation and motility of DP cells. We also showed that CXCR4 was required to retain human DP thymocytes in the cortex, whereas CCR7 promoted migration of mature human thymocytes to the medulla. Thus, 2 opposing chemokine gradients control the migration of thymocytes from the cortex to the medulla. These findings point to significant interspecies conservation in thymocyte-stroma interactions and provide the first evidence that chemokines not only attract mature thymocytes to the medulla, but also play an active role in retaining DP thymocytes in the cortex prior to positive selection.

Effects of low-load resistance training combined with blood flow restriction or hypoxia on muscle function and performance in netball athletes.

OBJECTIVES: To investigate the effect of blood flow restriction or normobaric hypoxic exposure combined with low-load resistant exercise (LRE), on muscular strength and endurance. DESIGN: A randomised controlled trial. METHODS: Well-trained netball players (n=30) took part in a 5 weeks training of knee flexor and extensor muscles in which LRE (20% of one repetition maximum) was combined with (1) an occlusion pressure of approximately 230mmHg around the upper thigh (KT, n=10), (2) hypoxic air to generate blood oxyhaemoglobin levels of approximately 80% (HT, n=10) or (3) with no additional stimulus (CT, n=10). The training was of the same intensity and amount in all groups. One to five days before and after training, participants performed a series of strength and endurance tests of the lower limbs (3-s maximal voluntary contraction [MVC3], area under 30-s force curve [MVC30], number of repetitions at 20% 1RM [Reps201RM]). In addition, the cross-sectional area (CSA) of the quadriceps and hamstrings were measured. RESULTS: Relative to CT, KT and HT increased MVC3 (11.0±11.9% and 15.0±13.1%), MVC30 (10.2±9.0% and 18.3±17.4%) and Reps201RM (28.9±23.7% and 23.3±24.0%, mean±90% confidence interval) after training. CSA increased by 6.6±4.5%, 6.1±5.1% and 2.9±2.7% in the KT, HT and CT groups respectively. CONCLUSIONS: LRE in conjunction with KT or HT can provide substantial improvements in muscle strength and endurance and may be useful alternatives to traditional training practices.

Low back pain: an assessment using positional MRI and MDT.

Current guidelines advise against the use of routine imaging for low back pain. Positional MRI can provide enhanced assessment of the lumbar spine in functionally loaded positions which are often relevant to the presenting clinical symptoms. The purpose of this case report is to highlight the use of positional MRI in the assessment and classification of a subject with low back pain. A low back pain subject underwent a Mechanical Diagnosis and Therapy (MDT) assessment and positional MRI scan of the lumbar spine. The MDT assessment classified the subject as "other" since the subjective history indicated a possible posterior derangement whilst the objective assessment indicated a possible anterior derangement. Positional MRI scanning in flexed, upright and extended sitting postures confirmed the MDT assessment findings to reveal a dynamic spinal stenosis which reduced in flexion and increased in extension.

The frequency of thrombotic events among adults given antifibrinolytic drugs for spontaneous bleeding: systematic review and meta-analysis of observational studies and randomized trials.

AIMS: The antifibrinolytic drug tranexamic acid (TXA) improves survival after trauma. Antifibrinolytic drugs may also improve outcome after spontaneous bleeding, so we conducted a systematic review of the frequency of thrombotic events associated with their use after spontaneous bleeding, to help design future randomized controlled trials. METHODS: We sought trials or observational studies of ≥20 adults involving any antifibrinolytic drug (TXA, epsilonaminocaproic acid (EACA) or aprotinin) for spontaneous (non-traumatic, non-surgical/iatrogenic), non-heamophiliac bleeding. We searched the Cochrane Central Register of Controlled Trials, OVID Medline from 1966, EMBASE from 1980, and the bibliographies of relevant articles in October 2009. We meta-analysed proportions of patients with thrombotic events, using a random effects model. RESULTS: We found 57 studies involving 5,049 patients, 3,616 (72%) of whom had spontaneous subarachnoid haemorrhage. 3,414 (68%) patients received TXA-based treatment and 1,635 (32%) received EACA. The frequencies of limb ischaemia and myocardial infarction were <1% for TXA and EACA. The frequency of deep vein thrombosis or pulmonary embolism was 1.9% (95% confidence interval (CI) 1.1 to 2.9) for TXA and 3.0% (95% CI 1.8 to 4.6) for EACA. The occurrence of cerebral infarction was restricted to studies of subarachnoid haemorrhage when compared to other indications, both for TXA (9.7% [95% CI 5.5 to 14.8] versus 0% [95% CI 0 to 0.5]) and for EACA (7.7% [95% CI 1.8 to 17.4] versus 0% [95% CI 0 to 2.1]). CONCLUSIONS: Thrombotic events have occurred infrequently with antifibrinolytic drugs after spontaneous bleeding apart from subarachnoid haemorrhage, so further exploration of their safety and efficacy after spontaneous bleeding is justified in randomized trials.

Two-photon imaging of the immune system.

Two-photon microscopy is a powerful method for visualizing biological processes as they occur in their native environment in real time. The immune system uniquely benefits from this technology as most of its constituent cells are highly motile and interact extensively with each other and with the environment. Two-photon microscopy has provided many novel insights into the dynamics of the development and function of the immune system that could not have been deduced by other methods and has become an indispensible tool in the arsenal of immunologists. In this unit, we provide several protocols for preparation of various organs for imaging by two-photon microscopy that are intended to introduce the new user to some basic aspects of this method.

Comparative study of hematopoietic differentiation between human embryonic stem cell lines.

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34(+) or CD34(+)CD45(+) hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34(+) precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34(+) hematopoietic precursors generated in vitro versus in vivo.

SMN deficiency disrupts brain development in a mouse model of severe spinal muscular atrophy.

Reduced expression of the survival motor neuron (SMN) gene causes the childhood motor neuron disease spinal muscular atrophy (SMA). Low levels of ubiquitously expressed SMN protein result in the degeneration of lower motor neurons, but it remains unclear whether other regions of the nervous system are also affected. Here we show that reduced levels of SMN lead to impaired perinatal brain development in a mouse model of severe SMA. Regionally selective changes in brain morphology were apparent in areas normally associated with higher SMN levels in the healthy postnatal brain, including the hippocampus, and were associated with decreased cell density, reduced cell proliferation and impaired hippocampal neurogenesis. A comparative proteomics analysis of the hippocampus from SMA and wild-type littermate mice revealed widespread modifications in expression levels of proteins regulating cellular proliferation, migration and development when SMN levels were reduced. This study reveals novel roles for SMN protein in brain development and maintenance and provides the first insights into cellular and molecular pathways disrupted in the brain in a severe form of SMA.