Epitope mapping of neutralizing TSST-1 specific antibodies induced by immunization with toxin or toxoids

Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced by Staphylococcus aureus, is a potent stimulator of the immune system. T-cells are activated by crosslinking of MHC class II molecules on antigen presenting cells with T-cell receptors (TCR). TSST-1 is associated with the majority of the cases of menstrual staphylococcal toxic shock, a severe and life-threatening multisystem disorder. Even though antibody mediated protection has been studied, information on antibody specificity directed to individual antigenic determinants of the protein is incomplete.To obtain immunogens with low toxicity, we generated a double-site mutant (dmTSST-1), modified at solvent-exposed residues predicted to be important for both MHC class II and TCR binding, and detoxified recombinantly expressed TSST-1 (rTSST-1) as well as native TSST-1 (nTSST-1) isolated from Staphylococcus aureus by treatment with formaldehyde. Rabbits were immunized with rTSST-1, nTSST-1, dmTSST-1, and formaldehyde inactivated toxoids. The sera obtained were used to map the antigen-reactive regions of the molecule and to identify specificities of antibodies induced by immunization with the different antigens. To detect linear antigenic epitopes of TSST-1 the reactivity of the sera with 11-meric peptides having an overhang of four residues, covering the entire molecule of TSST-1, have been studied. We found that sera of TSST-1 immunized rabbits predominantly reacted with N-terminal residues 1-15, while sera generated with formaldehyde inactivated toxoid recognized a total of 7 regions located at the N- and C-terminus and internal sites of TSST-1. Despite different specificities all sera were able to inhibit TSST-1 induced proliferation of human mononuclear cells.     

Immunity to mycobacteria: possible role of alpha/beta and gamma/delta T lymphocytes

Immunity to pathogenic mycobacteria is mediated by T lymphocytes. The possible contribution of CD4 alpha/beta T cells, CD8 alpha/beta T cells and gamma/delta T cells as well as the possible role of interleukin-mediated macrophage activation and target cell lysis through direct cell contact is discussed. Furthermore, attempts to define mycobacterial antigens for T lymphocytes with particular emphasis on heat shock proteins are described. The data currently available suggest complex interactions between different T-cell types in immunity to mycobacteria.     

Primary responses of human T cells to mycobacteria: a frequent set of gamma/delta T cells are stimulated by protease-resistant ligands

T lymphocyte subsets expressing either T cell receptor alpha/beta or gamma/delta were selected from human peripheral blood T cells and proliferative responses to molecular mass-fractionated mycobacterial lysates were determined. alpha/beta T cells primarily responded to fractions greater than 30 kDa whereas gamma/delta T cells preferentially reacted to fractions less than 3 kDa. Protease digestion abolished the stimulating activities for alpha/beta T cells, confirming that alpha/beta T cells respond to protein components. In contrast, components recognized by gamma/delta T cells proved resistant to protease digestion. In limiting dilution studies, frequencies of proliferating gamma/delta T cells remained virtually unaltered by protease treatment of stimulating lysates, while those of alpha/beta T cells became almost undetectable. Furthermore, only few gamma/delta T cells responded to the 65-kDa heat-shock protein. Our data indicate that, unlike alpha/beta T cells, gamma/delta T cells respond to mycobacterial components which are resistant to vigorous protease digestion.     

High thrombin concentrations in fibrin sealants induce apoptosis in human keratinocytes

Over the last century many studies have been performed to assess the impact of fibrin sealant (FS) components on cells. Because of the noncovalent bonding of thrombin to fibrin during fibrin clot formation, we wanted to further evaluate the impact of fibrin bound thrombin on cell viability. Initially, we quantified the activity of thrombin in three different, commercially available FS. This information was used to prepare fibrin clots covering a range of thrombin concentrations from 4 to 820 IU mL(-1), but which were identical with respect to all other constituents. Although these fibrin clots did not differ in their three-dimensional structure, clots prepared with highly concentrated thrombin (820 IU mL(-1)) failed to support adhesion and spreading of primary human keratinocytes (NHEK). The number of attached cells was also significantly reduced on high thrombin activity clots. We hypothesized that these observations are not only the consequence of decreased proliferation but of apoptotic mechanisms, since the expression of cleaved caspase 3 and 7 was strongly enhanced on fibrin clots with high thrombin activity. This was accompanied by an induction of expression of Trail-R2 which is a receptor known to mediate apoptosis signals. Blocking of thrombin activity by hirudin led to an improvement of cell morphology and to an increase in number of attached cells. In addition, the induction of caspase 3 and 7 was also reduced. Thus, here we report for the first time that fibrin bound thrombin does not only decrease proliferation (as already published by others), it also does induce NHEK apoptosis when present at high concentrations.     

Axial vascularization of a large volume calcium phosphate ceramic bone substitute in the sheep AV loop model

Vascularization still remains an obstacle to engineering of bone tissue with clinically relevant dimensions. Our aim was to induce axial vascularization in a large volume of a clinically approved biphasic calcium phosphate ceramic by transferring the arteriovenous (AV) loop approach to a large animal model. HA/β‐TCP granula were mixed with fibrin gel for a total volume of 16 cm³, followed by incorporation into an isolation chamber together with an AV loop. The chambers were implanted into the groins of merino sheep and the development of vascularization was monitored by sequential non‐invasive magnetic resonance imaging (MRI). The chambers were explanted after 6 and 12 weeks, the pedicle was perfused with contrast agent and specimens were subjected to micro‐computed tomography (µ‐CT) scan and histological analysis. Sequential MRI demonstrated a significantly increased perfusion in the HA/β‐TCP matrices over time. Micro‐CT scans and histology confirmed successful axial vascularization of HA/β‐TCP constructs. This study demonstrates, for the first time, successful axial vascularization of a clinically approved bone substitute with a significant volume in a large animal model by means of a microsurgically created AV loop, thus paving the way for the first microsurgical transplantation of a tissue‐engineered, axially vascularized bone with clinically relevant dimensions. Copyright © 2009 John Wiley & Sons, Ltd.     

Delayed Onset of Brown–Sequard Syndrome Involving Upper Extremity Pain

Report of a case: A 60-year-old white male with a history of C3–C4 spinal cord injury with subsequent C3–C4 fusion complained of right upper extremity painful spasms of 2 years duration with associated hyperspasticity, motor weakness and poor positional and vibrational sense. The patient was diagnosed with Brown–Sequard syndrome (BSS) and treated with botulinum toxin type A injections distributed into the affected muscle groups that provided substantial and lasting relief. This case is unique in that the patient's trauma occurred 28 years before the development of the BSS suggesting a slow evolution of the condition.     

Quality of life analysis of renal donors

OBJECTIVE: The aim of this study was to investigate the quality of life of renal donors during long-term follow-up. PATIENTS AND METHODS: The short form health survey (SF-36) questionnaire was compared between renal donors and the general population. We evaluated the relationship to postoperative complications and preoperative information with the quality of life. RESULTS: Fifty renal donors of mean age 55.8 +/- 12 years (range, 29-70 years) had a mean follow-up of 55.1 +/- 47.2 months (range, 12-168 months). Complications after donor nephrectomy were related with physical function loss (r = -.397; P < .05) and vitality (r = -.463; P = .01). Renal donor candidates who did not have satisfactory information before the operation experienced difficulty with decision making (r = -.555; P = .0001). Physical function, limitation of physical role and limitation of emotional role were comparable to the general population. Pain scale was worse among donors compared with the general population (P = .001). Educational status of renal donors was related to the pain scale and vitality (r = .369; P < .05 and r = .523; P < .05, respectively). General health perception, vitality, mental health, and social functioning were worse compared with the general population (P = .0001, P = .002, P = .0001, and P = .001, respectively). Health problems occurring after donation were related to negation of interfamily relations (r = .695; P = .0001). CONCLUSIONS: Reducing complications after nephrectomy will directly increase the quality of the donor's life. Informing renal donor candidates and their families about the postoperative course with consideration of the candidate's and his or her family's educational status is a sociological approach which helps to increase the donor's quality of life. In addition to good patient selection/preparation, meticulous surgery, and follow-up.     

Sleep quality and depression–anxiety in mothers of children with two chronic respiratory diseases: Asthma and cystic fibrosis

BackgroundSleep quality and psychological well being of parents are expected to be influenced by the child's health and disease status. The aim of this study was to compare sleep quality and depression–anxiety parameters in mothers of children with cystic fibrosis (CF) asthma and healthy controls.MethodsThe study included mothers of 62 children with asthma, 21 children with CF and 35 healthy children. All mothers filled in the Pittsburgh Sleep Quality Index (PSQI) questionnaire and hospital anxiety depression scale (HADS).ResultsComparison of the three groups with Kruskall Wallis analysis demonstrated that subjective sleep, sleep efficiency and total PSQI scores were significantly different between the groups (p = 0.02, p = 0.01 and p = 0.04 respectively). Comparisons of the groups in pairs with Mann Whitney U test with Bonferroni correction revealed that subjective sleep quality scores in mothers of children with asthma were significantly higher than the ones in the control group (1.0 ± 0.9 vs 0.6 ± 0.7, p = 0.015). The other PSQI scores as well as the anxiety and depression scores were higher in CF and asthma groups when compared to the control group but did not reach statistical significance. Anxiety and depression scores were significantly correlated with PSQI total score in CF (rho = 0.54 and 0.49 respectively) and asthma groups (rho = 0.45 and 0.60 respectively) but not in the control group.ConclusionIn conclusion, presence of a chronic respiratory disease in a child may be associated with disturbed sleep quality and increased depression and anxiety in mothers.