Comparison of diffusion MRI and CLARITY fiber orientation estimates in both gray and white matter regions of human and primate brain.

Diffusion MRI (dMRI) represents one of the few methods for mapping brain fiber orientations non-invasively. Unfortunately, dMRI fiber mapping is an indirect method that relies on inference from measured diffusion patterns. Comparing dMRI results with other modalities is a way to improve the interpretation of dMRI data and help advance dMRI technologies. Here, we present methods for comparing dMRI fiber orientation estimates with optical imaging of fluorescently labeled neurofilaments and vasculature in 3D human and primate brain tissue cuboids cleared using CLARITY. The recent advancements in tissue clearing provide a new opportunity to histologically map fibers projecting in 3D, which represents a captivating complement to dMRI measurements. In this work, we demonstrate the capability to directly compare dMRI and CLARITY in the same human brain tissue and assess multiple approaches for extracting fiber orientation estimates from CLARITY data. We estimate the three-dimensional neuronal fiber and vasculature orientations from neurofilament and vasculature stained CLARITY images by calculating the tertiary eigenvector of structure tensors. We then extend CLARITY orientation estimates to an orientation distribution function (ODF) formalism by summing multiple sub-voxel structure tensor orientation estimates. In a sample containing part of the human thalamus, there is a mean angular difference of 19o±15o between the primary eigenvectors of the dMRI tensors and the tertiary eigenvectors from the CLARITY neurofilament stain. We also demonstrate evidence that vascular compartments do not affect the dMRI orientation estimates by showing an apparent lack of correspondence (mean angular difference = 49o±23o) between the orientation of the dMRI tensors and the structure tensors in the vasculature stained CLARITY images. In a macaque brain dataset, we examine how the CLARITY feature extraction depends on the chosen feature extraction parameters. By varying the volume of tissue over which the structure tensor estimates are derived, we show that orientation estimates are noisier with more spurious ODF peaks for sub-voxels below 30 µm3 and that, for our data, the optimal gray matter sub-voxel size is between 62.5 µm3 and 125 µm3. The example experiments presented here represent an important advancement towards robust multi-modal MRI-CLARITY comparisons.

Reduction of Motion Artifacts and Noise Using Independent Component Analysis in Task-Based Functional MRI for Preoperative Planning in Patients with Brain Tumor.

BACKGROUND AND PURPOSE: Although it is a potentially powerful presurgical tool, fMRI can be fraught with artifacts, leading to interpretive errors, many of which are not fully accounted for in routinely applied correction methods. The purpose of this investigation was to evaluate the effects of data denoising by independent component analysis in patients undergoing preoperative evaluation for glioma resection compared with more routinely applied correction methods such as realignment or motion scrubbing. MATERIALS AND METHODS: Thirty-five functional runs (both motor and language) in 12 consecutive patients with glioma were analyzed retrospectively by double-blind review. Data were processed and compared with the following: 1) realignment alone, 2) motion scrubbing, 3) independent component analysis denoising, and 4) both independent component analysis denoising and motion scrubbing. Primary outcome measures included a change in false-positives, false-negatives, z score, and diagnostic rating. RESULTS: Independent component analysis denoising reduced false-positives in 63% of studies versus realignment alone. There was also an increase in the z score in areas of true activation in 71.4% of studies. Areas of new expected activation (previous false-negatives) were revealed in 34.4% of cases with independent component analysis denoising versus motion scrubbing or realignment alone. Of studies deemed nondiagnostic with realignment or motion scrubbing alone, 65% were considered diagnostic after independent component analysis denoising. CONCLUSIONS: The addition of independent component analysis denoising of fMRI data in preoperative patients with glioma has a significant impact on data quality, resulting in reduced false-positives and an increase in true-positives compared with more commonly applied motion scrubbing or simple realignment methods.

Sonography for the investigation of a child with a limp.

Hip pain is a common paediatric presentation and is potentially serious. While hip pain can be attributed to primary hip pathology, the hip area is also a common site for referred pain. This often poses a diagnostic challenge particularly in the young child who may not verbalise the point of pain and may not report an injury. Differential diagnoses for paediatric hip joint pain range from fracture, transient synovitis, septic arthritis with or without osteomyelitis, juvenile idiopathic arthritis (JIA, previously juvenile rheumatoid arthritis JRA), Legg-Calve-Perthes' disease (LCP), slipped capital femoral epiphysis (SCFE) to haemarthrosis in patient with a clotting disorder. Referred pain from abdominal pathology, for example; appendicitis, psoas abscess or haematoma should also be considered. The evaluation and management of hip pain requires a thorough history and physical examination. A radiograph is usually indicated to rule out any bony injury. Septic arthritis is a medical emergency requiring urgent surgical as well as medical treatment. Ultrasound of the hip joint plays a role in helping to differentiate which hips require early intervention and to guide a needle aspiration of joint fluid if indicated. In this article, we aim to review the anatomy of the hip, techniques in ultrasonographic examination and some common pathologies in the paediatric hip.

New insight into the mechanism of action of IVIg: the role of dendritic cells.

Intravenous immunoglobulin (IVIg) is used to treat an ever-increasing number of autoimmune diseases. While the exact mechanism of action of IVIg has remained elusive, many theories have been suggested, including mononuclear phagocytic system blockade, autoantibody neutralization by anti-idiotype antibodies, accelerated pathogenic autoantibody clearance by saturation of the neonatal Fc receptor, cytokine modulation and complement neutralization. More recently, a key role for dendritic cells (DC) in the amelioration of autoimmunity by IVIg has been suggested. Here we will focus on the role that DC may play in IVIg function using data from both mouse and human studies.

The role of ResA in type II cytochrome c maturation.

Numerous bacterial proteins involved in the nitrogen cycle, and other processes, require c-type haem as a cofactor. c-type cytochromes are formed by covalent attachment of haem to the conserved CXXCH motif. Here, we briefly review what is presently known about cytochrome c maturation in Bacillus subtilis with particular emphasis on the crystal structures of ResA.

IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity.

Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody-dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region-reactive (anti-idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet-specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre-established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti-idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody-sensitized red blood cells. F(ab')2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti-idiotype interactions are not required.

Monoclonal antibody-mediated inhibition of the human HLA alloimmune response to platelet transfusion is antigen specific and independent of Fcgamma receptor-mediated immune suppression.

Presensitization of donor platelets with allo-specific immunoglobulin (Ig)G results in a diminished immune response against subsequent transfusions of platelets. To understand better the mechanism of how alloantibody presensitization results in a decreased alloimmune response, we have used murine monoclonal antibodies directed to polymorphic and non-polymorphic regions of human leucocyte antigen (HLA) as well as platelet-specific molecules. Here, we demonstrated that presensitization with anti-human HLA class I antibodies, as well as beta2-microglobulin-specific antibody, protected against alloantibody production to five subsequent untreated platelet challenges. Use of complement fixing, non-fixing or F(ab')2 fragments of HLA-specific antibody also resulted in complete inhibition of alloantibody production. This protection was not seen when the platelets were presensitized with monoclonal antibodies to CD42a (GPIX), CD32 (low-affinity IgG/Fcgamma receptor) or murine IgG and was thus independent of B-cell FcgammaRII-mediated immune suppression. The inhibition observed was independent of HLA alloantigenic specificity as antibodies directed at the beta2-microglobulin portion of HLA class I were as effective as antibodies against any of the HLA-alpha regions (either polymorphic or non-polymorphic) of class I. This work demonstrates that monoclonal antibody-mediated suppression of the human HLA alloimmune response to platelet transfusion is antigen specific and is independent of FcgammaRII-mediated immune regulation, complement fixing or HLA alloantigenic specificity.

Clinical and biopsy specimen features in black and white men with clinically localized prostate cancer.

BACKGROUND: The incidence of prostate cancer in black men is estimated to be 30% to 50% higher than among age-matched white men, and black men have a twofold higher mortality rate. To determine whether racial differences exist in men with similarly staged disease, we compared clinical and pathologic features in black and white men with clinical stage T1-T2 prostate cancer. METHODS: We retrospectively reviewed the records of all men who had prostate biopsy at our facilities. Men were included in this study if they were of black or white race, if the clinical stage of their prostate cancer was T1-T2, and if detailed information regarding their prostate biopsy was available. RESULTS: Black men were found to have significantly higher serum prostate-specific antigen (PSA) levels, but no other differences were noted in the other clinical or pathologic features studied. CONCLUSION: While racial differences in the incidence and mortality of prostate cancer are well known, differences in the clinical and pathologic features between black and white men with similarly staged disease have been poorly studied. Our results suggest that serum PSA levels are higher in black men, but no other differences were noted. This suggests that early detection programs for prostate cancer in black men can potentially decrease prostate cancer mortality.

Comparison of platelet immunity in patients with SLE and with ITP.

Idiopathic thrombocytopenic purpura (ITP) is characterized by the development of a specific anti-platelet autoantibody immune response mediating the development of thrombocytopenia. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of a wide variety of autoantibodies. In 15-20% of SLE cases, patients develop thrombocytopenia which appears to be autoimmune in nature (SLE-TP). To better understand the pathogenesis of the thrombocytopenia associated with SLE, we investigated the overlapping platelet and cellular immune features between SLE and ITP. Thirty-one patients with SLE, eight with SLE-TP, and 17 with ITP, were studied and compared to 60 healthy controls. We evaluated platelet-associated IgG, platelet microparticles, reticulated platelets, platelet HLA-DR expression, in vivo cytokine levels, lymphocyte proliferation, and the T lymphocyte anti-platelet immune response in these patients. Patients with SLE-TP and those with ITP had increased platelet-associated IgG, an increased percentage of platelet microparticles, a higher percentage of reticulated platelets and larger platelets, suggesting antibody-mediated platelet destruction and increased platelet production. More than 50% of patients with ITP had increased HLA-DR on their platelet surface whereas subjects with SLE-TP did not. Analysis of serum cytokines demonstrated increased levels of IL-10, IL-15 and TNF-alpha in patients with SLE, but in those with ITP, only increased levels of IL-15 were seen, no increases in any of these cytokines were observed in patients with in SLE-TP. The ability of lymphocytes to proliferate in response to phorbol myristate acetate (PMA) stimulation was increased in SLE-TP, but was normal in both SLE and ITP. Lymphocytes from subjects with ITP displayed an increased ability to proliferate on exposure to platelets, in contrast, those with SLE-TP did not. While the number of subjects evaluated with SLE-TP was small, these data reveal a number of differences in the immunopathogenesis between SLE-TP and ITP.

Inhibition of a secondary human alloimmune response via the soluble active component of CD154 (CD40L) in severe combined immune-deficient mice engrafted with human lymphocytes.

BACKGROUND: Alloimmunization requires a process known as co-stimulation. An important co-stimulatory pathway for most immune responses is mediated by the interaction of CD40 on antigen-presenting cells with CD154 (CD40L) on host T cells. Blockade of this co-stimulatory pathway simultaneous with exposure to challenge with HLA-incompatible cells is hypothesized to inhibit alloimmunization. STUDY DESIGN AND METHODS: Severe combined immune-deficient (SCID) mice were reconstituted with human peripheral blood lymphocytes (Hu-PBL-SCID mice) from a subject primed to HLA antigens and challenged with HLA-incompatible lymphocytes. Mice were challenged in the presence or absence of an 18-kDa soluble recombinant active form of human CD154 (18-kDa CD154). Human IgG production, alloimmunization, and in vitro T-cell responsiveness were assessed. RESULTS: There was no significant effect of 18-kDa CD154 on human IgG levels in these mice, but it inhibited the development of HLA-specific alloantibody in this model to five subsequent untreated white cell challenges. In vitro T-cell proliferation in a mixed lymphocyte culture was also prevented by 18-kDa CD154. CONCLUSION: The recombinant protein 18-kDa CD154 inhibited the ability of the Hu-PBL-SCID mice to mount a secondary immune response to allostimulation. This implies that transfusion-induced alloimmunization utilizes CD40-CD154 co-stimulation and that blockade of this pathway can inhibit T-cell function and interfere with the development of alloimmunization.

Antibody-mediated inhibition of the human alloimmune response to platelet transfusion in Hu-PBL-SCID mice.

Severe combined immune deficient (SCID) mice were engrafted with human (Hu) peripheral blood lymphocytes (PBL) from a previously alloimmunized donor and transfused with HLA-mismatched platelets. We have previously shown this to be a useful model for platelet transfusion. These engrafted mice (Hu-PBL-SCID mice) produced high levels of alloantibody in response to standard platelet preparations. However, when the first platelet challenge was presensitized with anti-HLA antibody and then transfused there was a marked reduction in the amount of alloantibody produced to five subsequent untreated platelet transfusions. Platelets pretreated with platelet-specific anti-HPA-1a (PL(A1)) sera did not induce a decrease in the anti-HLA alloantibody response. This demonstrated that platelet-induced HLA alloimmunization can be blocked by anti-HLA antibody-sensitized cells.

Analysis of transmembrane signalling and T cell defects associated with idiopathic thrombocytopenic purpura (ITP).

Adult chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by production of autoreactive antibodies to platelet antigens. It is now becoming clear that autoantibody production, in general, is regulated by T helper (Th) cells. Several recent studies have examined potential defects in T cell function in this disease and have demonstrated that patients with ITP possess abnormal lymphocyte activation and Th1/Th2-mediated cytokine production. Although the underlying cause(s) of aberrant T cell function in this disease are not known, studies from other models of autoimmune disease indicate that defects in T cell transmembrane signalling can be causally linked to abnormal T cell activation and cytokine production. This review will present some of the major T cell signalling pathways and discuss how altered T cell signalling may be linked to autoimmunity with an emphasis on ITP. Recent preliminary findings of a potential defect in the signal transduction apparatus in lymphocytes from three patients with ITP will also be presented.

Clinical characteristics and biopsy specimen features in African-American and white men without prostate cancer.

BACKGROUND: The reported incidence of prostate cancer is higher among African-American men than among white men. We conducted a study of African-American and white men without prostate cancer to determine whether clinical and histologic findings might be associated with racial differences in serum prostate-specific antigen (PSA) levels. METHODS: From January 1990 through March 1997, 493 (59.5%) of 829 African-American men and 736 (74.1%) of 993 white men who had elevated serum PSA levels (> or = 4.0 ng/mL) and/or abnormal digital rectal examinations and who underwent transrectal ultrasound-guided prostate biopsies were found to be without prostate cancer. Also reviewed were patients' age and race, indication for biopsy, histologic features of the prostate biopsy specimen, ultrasound-measured prostate volume, PSA density (i.e., the PSA level divided by the prostate volume), and (in some cases) serum testosterone levels. RESULTS: Among these men without prostate cancer, there were no statistically significant differences by race in the ages of the patients, their prostate volumes, or their serum testosterone levels; however, the mean serum PSA levels and PSA densities were significantly higher in African-American men than in white men (two-sided P values of .00003 and .000009, respectively). A higher proportion of African-American men than white men had inflammation in their prostate biopsy specimen, and men of both races with prostate inflammation had higher PSA values than those without inflammation. African-American men without inflammation had higher PSA values than white men without inflammation. CONCLUSIONS: In this study, African-American men without histologic evidence of prostate cancer had significantly higher PSA levels and PSA densities than similarly aged white men. This finding was not accounted for by racial differences in patients' age, serum testosterone level, or prostate volume.

Induction of a secondary human anti-HLA alloimmune response in severe combined immunodeficient mice engrafted with human lymphocytes.

BACKGROUND: Experimental manipulation of transfusion-induced alloimmunization is limited in humans by ethical considerations. Conversely, studies of alloimmunization in animal models may not reflect the human immune system closely enough to be of optimal benefit. The development of an in vivo model of human alloimmunization that is amenable to experimental manipulation is thus desirable. STUDY DESIGN AND METHODS: An in vivo model of human alloimmunization was evaluated by using mice with severe combined immunodeficiency (SCID). SCID mice underwent gamma-radiation (200 cGy) and received an intraperitoneal injection of human peripheral blood lymphocytes (PBLs) from donors immunized to HLA antigens by prior pregnancy (reconstitution). These Hu [human]-PBL-SCID mice were then challenged with HLA-mismatched PBLs. Alloantibodies were evaluated by flow cytometry and a standard two-stage microlymphocytotoxicity assay. RESULTS: Hu-PBL-SCID mice (n = 22) that were challenged with PBLs expressing the HLA antigens to which the donors had previously been immunized, made significantly more IgM and IgG alloantibodies than did the unchallenged mice. Responses were measurable by 1 week after reconstitution and challenge. Prior treatment of SCID mice with anti-asialo GM1, which depletes murine natural killer cells and macrophages, further increased the alloantibody response of challenged mice. The human alloantibodies generated were specific to the challenge HLA antigens as assessed by microlymphocytotoxicity assay. CONCLUSION: Hu-PBL-SCID mice are a useful model system in which to study and manipulate the induction of secondary human alloimmune responses against cellular HLA class I antigens. This model will be valuable for testing the in vivo effect of novel immunotherapies on the inhibition of the human alloantibody response.

Arm vein reconstruction for limb salvage: long-term outcome.

A series of 42 consecutive patients undergoing infrainguinal vascular reconstruction for limb salvage using vein harvested from the arm were followed prospectively to determine the long-term graft patency and stenosis rates. Vein harvested from the arm ('arm vein') was used for secondary or tertiary reconstruction in 22 patients (52.4%). The outflow was to a single calf vessel in 37 grafts (88.1%). The cumulative primary, primary assisted and secondary graft patency rates were 35.6%, 49.6% and 59.5% at 2 years, respectively, and the limb salvage rate was 69.0% at 2 years. Eight grafts developed stenoses detected by a graft surveillance programme. Six stenoses were dilated successfully with percutaneous transluminal angioplasty (PTA) and one was treated with an interposition vein graft. Bypass using arm vein is time-consuming and technically demanding as multiple anastomoses are often required. Arm vein grafts, however, have no greater incidence of stenosis than long saphenous vein grafts and these stenoses may be dilated with PTA with good results. The long-term outcome suggests that an arm vein graft is an important treatment option in the absence of the long saphenous vein.

Thromboxane and neutrophil changes following intermittent claudication suggest ischaemia-reperfusion injury.

OBJECTIVES: It has been postulated that ischaemia-reperfusion occurs in intermittent claudication resulting in neutrophil activation and release of soluble mediators, increasing systemic vascular permeability and enhancing atherogenesis. METHODS: We measured neutrophil deformability, plasma thromboxane levels, and urinary microalbumin excretion in 30 male claudicants, and 10 age- and sex-matched controls, before and after exercise to maximum walking distance. Blood was taken from an antecubital vein. RESULTS: There was an increase in urinary microalbumin excretion after exercise in claudicants. Statistically significant increases in the median and 90th percentile transit times (markers of neutrophil deformability) for isolated neutrophils from blood drawn 5 min after exercise in the claudicants were observed with no change in control subjects. Plasma thromboxane concentrations in claudicants increased within 10 min post-exercise. Plasma concentrations in controls were significantly lower throughout the study period. In the claudicant group, a positive correlation between the percentage change in the median transit time for neutrophils, and the percentage change in plasma thromboxane at 60 min post-exercise was found. CONCLUSIONS: The results lend further support to the concept of ischaemia-reperfusion events in patients with intermittent claudication, leading to a systemic increase in vascular permeability as a result of endothelial injury or dysfunction (a crucial step in atherogenesis), associated with thromboxane production and neutrophil activation. We suggest that the above changes may contribute to the increased mortality seen in such patients.

A mammalian activity required for the second step of pre-messenger RNA splicing.

Splicing of precursors to messenger RNAs occurs via a two-step mechanism. In the first step, the 5'-exon is released concomitant with the production of a lariat intermediate, and in the second step, the exons are joined, releasing the intron in the form of a lariat product. Several gene products of the yeast Saccharomyces cerevisiae have been shown to be required exclusively for the second step. Although mammalian proteins have been implicated in the second step of splicing, none have been shown to act only at this step. We identify here the first mammalian activity shown to be exclusively required for the second step. The activity was shown to increase by 5-fold the rate for this splicing step, whereas it had no effect on the rate of the first step. The activity was not affected by treatment with micrococcal nuclease, whereas it is sensitive to heating to 55 degrees C, suggesting that it is not dependent on an RNA, but more likely is a protein. The second step activity was separated from other factors required for the first step and from PSF, a splicing factor thought to have a second step activity. The activity does not require ATP hydrolysis, suggesting that it acts at a late stage of the second step of splicing.

Chronic muscle stimulation improves ischaemic muscle performance in patients with peripheral vascular disease.

There is currently no established treatment for intermittent claudication with proven long term benefit. Exercise classes have been shown to improve walking distance. Chronic electromyostimulation (CEMS) a method of stimulating skeletal muscle has effects on normal muscle which may also benefit claudicants. We investigated the effects of one month of CEMS on claudicants in a single blind placebo controlled study. Patients were randomised to either CEMS (treatment) or transcutaneous nerve stimulation (TENS) placebo. The effects of the two modalities were assessed using the conventional measures of claudicating distance (CD), maximum walking distance (MWD), ankle-brachial pressure index (ABPI) and pressure recovery time (PRT). Muscle performance was assessed by the fatigue index (FI) a technique determining the decrease in ischaemic muscle response to repeated contraction. After 4 weeks treatment the CEMS group showed significant improvements in their median CD (88 to 111) and MWD (118 to 158); this was not seen in the control group. Muscle performance also increased significantly during the 4 weeks of treatment in the CEMS group but not in the control group. These changes were not maintained after CEMS was stopped. This pilot study suggests that CEMS may well have a role to play in the treatment of intermittent claudication though a number of further studies need to be undertaken.