Trypanosoma cruzi: nitrogenous-base-containing phosphatides in trypomastigote forms--isolation and chemical analysis

Increasingly clinicians attempt to base decisions regarding patient management on the results of clinical studies in addition to expert opinion and their own practical experience. In this article, the author reviews the published studies available to assist clinicians to make evidence-based decisions in three topics related to small volume red blood cell (RBC) transfusions for preterm infants; namely, studies examining the effects of RBC transfusions on possible symptoms of anemia such as tachypnea, apnea or other cardiorespiratory irregularities, studies investigating the collection and transfusion of umbilical cord blood and finally studies addressing the duration of storage and use of additive solutions for RBCs for transfusion to neonates. Based on the review of these studies, guidelines for small volume RBC transfusions in preterm infants are suggested.     

CD28-induced cytokine production and proliferation by thymocytes are differentially regulated by the p59fyn tyrosine kinase

CD28 is a 44-kDa homodimeric receptor that is expressed on the majority of T cells. Engagement of the CD28 receptor by soluble anti-CD28 mAb in conjunction with phorbol ester (PMA) induces the production of cytokines and the proliferation of resting T cells via signal transduction pathways independent of the TCR. Evidence is provided herein that CD28 signals leading to cytokine production do not require the p59fyn (Fyn) tyrosine kinase, whereas CD28-mediated proliferation is dependent on the presence of the Fyn kinase in thymic, but not lymph node, cells. The defect in proliferation is not due to failure of IL-2R signaling, since addition of high concentrations of exogenous IL-2 can overcome the proliferative defect. Analysis of CD28-directed induction of the IL-2R alpha (CD25)-chain, which confers high affinity binding to IL-2, showed that Fyn-deficient thymocytes, but not lymph node cells, failed to up-regulate CD25 expression following anti-CD28 and PMA stimulation. Thus, the Fyn tyrosine kinase is critically required for thymic CD28-mediated CD25 expression and proliferation but not for CD28-mediated cytokine production.     

HLA-DRB1*08 influences the development of disease in Mexican Mestizo with spondyloarthropathy

OBJECTIVE: HLA class II encoded factors may influence the phenotype of ankylosing spondylitis (AS). These include HLA DRB1*07 for peripheral arthritis, and polymorphism of the HLA-linked LMP2 locus and HLA DRB1*08 for acute anterior uveitis (AAU). We studied the relationship between DRB1*08 and disease phenotype in additional populations of individuals with AS. METHODS: The patient population included 385 unrelated HLA-B27 positive individuals with AS. These included 204 Caucasians and 2 populations of Mexican Mestizo with AS: 106 with predominately adult onset disease from Guadalajara and 75 with predominately juvenile onset disease from Mexico City. The control population of 428 individuals included 210 random and 36 HLA-B27 positive unrelated Canadian Caucasians and 173 random and 9 HLA-B27 positive Mexican Mestizo from Mexico City. DRB1*08 typing was by sequence specific polymerase chain reaction. RESULTS: A significantly higher prevalence of DRB1*08 was observed in Mexican patients with juvenile onset disease (44.9%) and especially those with undifferentiated spondyloarthropathy (55.6%) compared to normal unrelated Mexican Mestizo (25.4%) (p < 0.01 for both) and in patients with undifferentiated spondyloarthropathy versus B27 controls (11.1%) (p = 0.03), although no significant differences were observed in within patient group comparisons based on phenotypic features of disease such as AAU and age at onset. No significant relationship between DRB1*08 and disease phenotype was evident in Caucasian individuals. CONCLUSION: Our data suggest DRB1*08 may influence the phenotype of spondyloarthritis in Mexican Mestizo, but do not support the view that DRB1*08 influences the development of AAU, as reported in a Japanese population.     

NMR studies of caldesmon-calmodulin interactions

The binding of the calcium-regulatory protein calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. Two regions of caldesmon have been identified as putative calmodulin-binding domains. We have earlier reported on the binding of one of these domains to calmodulin (Zhang & Vogel (1994) Biochemistry 33, 1163-1171). Here we have studied the binding of CaM to synthetic peptides of CaD which contain: (1) both the first and second CaM-binding domains; (2) the second CaM-binding domain; and (3) the sequence between the first and second CaM-binding domains. Two-dimensional transferred nuclear Overhauser enhancement proton NMR measurements as well as circular dichroism studies of a 22-residue peptide NKETAGLKVGVSSRINEWLTK, which contains the second CaM-binding domain, show that only the C-terminal half of the peptide becomes alpha-helical upon binding to CaM. Somewhat surprisingly, the shorter 9-residue peptide SRINEWLTK was sufficient to form a 1:1 complex with CaM; this peptide appears to bind as a 3(10)-helix. Proton-carbon-13 correlation NMR titration studies with specifically labeled [methyl-13C]methionine CaM were used to study the participation of the hydrophobic regions in both domains of the dumbbell shaped CaM in peptide binding. Binding of a 54-residue CaD peptide containing both CaM-binding domains affects all the 8 Met residues in the two hydrophobic domains of CaM (only Met 76 in the linker region of CaM is not involved), while binding of the second CaM-binding domain of CaD influences principally Met 51, 71, and Met 124, 144. Simultaneous binding to CaM of two peptides comprising the first and the second CaM-binding domains also caused changes to all Met residues except Met 76. Taken together, these data demonstrate that both CaM-binding domains of CaD can bind simultaneously to the two hydrophobic regions of CaM.     

Autologous lymphokine-activated killer cell therapy of Epstein-Barr virus-positive and -negative lymphoproliferative disorders arising in organ transplant recipients

Lymphoreticular malignancies, collectively called posttransplant lymphoproliferative disorders (PTLD), eventually develop in 2-5% of organ transplant recipients. They frequently undergo regression when immunosuppression is reduced or stopped. This feature has been associated with a previous or de novo Epstein-Barr virus (EBV) infection. We herein describe immunotherapy with autologous lymphokine-activated killer (LAK) cells in seven patients with PTLD (four EBV-positive patients and three EBV-negative patients). Autologous peripheral blood mononuclear cells were obtained by leukapheresis, depleted of monocytes, and cultured in the presence of interleukin 2 for 10 to 11 days. A single dose of 5.2 x 10(9) to 5.6 x 10(10) LAK cells was given intravenously. Systemic interleukin 2 was not administered. The four patients with EBV+ PTLD had complete tumor regression; two of them developed controllable rejection. Three patients are well 13-16 months after treatment; the fourth patient died of pneumonia 41 days after infusion. Three patients with EBV- lymphomas had no response despite prior evidence that their tumors also were subject to immune surveillance. Two of these three patients died after being given other treatment, and the third patient has persistent tumor. In conclusion, autologous LAK cell infusion was effective for treatment of four EBV+ organ transplant recipients. LAK cell efficacy for three patients with EBV- PTLD was not evaluable under the management circumstances in which this treatment was utilized.     

Temporal filter of the motion sensor in glaucoma

Glaucoma reportedly affects motion perception. As an initial step in characterizing glaucoma-induced changes in the motion system, we determined the range of temporal frequencies that the motion system could process. A noise-masking paradigm was used to measure contrast energy thresholds of 26 glaucoma patients at various stages of the disease and 16 age-similar subjects with normal vision. Using a sinusoidal stimulus, thresholds were measured for the discrimination of motion direction and for the stimulus embedded within a pattern of dynamic spatial noise. The noise was filtered to contain only low spatial frequencies, and the temporal-frequency spectrum of the noise was manipulated across conditions to derive the temporal filter shape of the most efficient motion sensor. The results show that the range of temporal frequencies processed by the motion system is diminished in the glaucoma group. The filters of the glaucoma subjects have reduced bandwidths compared with the normal-vision group. In addition, the upper cut-off frequency of the filters of the glaucoma subjects is correlated with stage of disease as indexed by the mean deviation of the Humphrey Visual Field Analyzer program 24-2, as well as the cup-to-disk ratio.     

Activation of auditory cortex during silent lipreading

Watching a speaker's lips during face-to-face conversation (lipreading) markedly improves speech perception, particularly in noisy conditions. With functional magnetic resonance imaging it was found that these linguistic visual cues are sufficient to activate auditory cortex in normal hearing individuals in the absence of auditory speech sounds. Two further experiments suggest that these auditory cortical areas are not engaged when an individual is viewing nonlinguistic facial movements but appear to be activated by silent meaningless speechlike movements (pseudospeech). This supports psycholinguistic evidence that seen speech influences the perception of heard speech at a prelexical stage.     

Serum levels of sCD23, interleukin-10 and interferon-gamma in patients with coeliac disease

The role of cellular and humoral immunity coeliac disease was investigated by the measurement of serum levels of interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and soluble CD23 (sCD23). Coeliac disease was diagnosed by duodenal biopsy and response to a gluten-free diet (GFD). The results were compared with age and sex-matched patients with non-specific upper gastrointestinal symptoms and normal duodenal histology. While the levels of serum IL-10 were significantly elevated (P < 0.01) in patients with coeliac disease taken as a whole, the levels of serum IFN-gamma were normal and sCD23 significantly decreased (P < 0.002). The median serum sCD23 was significantly lower in the coeliac disease patients not on a GFD compared with those asymptomatic on a GFD (P < 0.03) and the control group (P < 0.0004). The coeliac disease patients on a GFD also had significantly lower serum sCD23 and higher IL-10 compared with the control group (P < 0.01 and P < 0.015). There was no significant difference in the serum IL-10 between the coeliac disease patients on a GFD and those not on a GFD and between the latter and the control group. The low levels of serum sCD23 in coeliac disease suggest diminished humoral immunity and, conversely, exaggerated cellular immunity. The aetiology of the raised levels of IL-10 in coeliac disease is unclear and similar to that observed in patients with inflammatory bowel disease. However, this may represent a regulatory response to the elevated levels of proinflammatory cytokines described in coeliac disease. A combination of diminished sCD23 and raised IL-10 is clearly unusual as both are associated with Th2-type functions. The possible causes of this finding are discussed.     

The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal protein

Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.