Genetic and genetic expression analyses of clear cell sarcoma of the kidney

Clear cell sarcoma of the kidney (CCSK) represents a significant diagnostic and clinical challenge. In search of diagnostically useful or biologically significant genetic abnormalities, we screened 30 CCSKs from the National Wilms Tumor Study Group. Genetic gains and losses were analyzed using comparative genomic hybridization; loss of heterozygosity at 11p15 was studied using microsatellite analysis. Loss of imprinting (LOI) was studied using allele-specific expression or methylation analysis at the ApaI polymorphic site for IGF2, AluI and RsaI sites for H19, and Cfo I site for SNRPN. Comparative genomic hybridization analysis revealed quantitative abnormalities in only 4 of 30 CCSKs. Two showed gain of 1q, one also showed loss of 10q, and the other also showed loss of terminal 4p. The other two cases demonstrated chromosome 19 loss and chromosome 19p gain, respectively. All 22 cases informative for 11p15 showed retention of both alleles. Of 14 CCSKs informative for IGF2, six showed biallelic expression; all three CCSKs informative for H19 exhibited monoallelic expression. The normal imprint pattern was present in all six CCSKs analyzed for SNRPN methylation. These data demonstrate an absence of consistent genetic gains or losses in CCSKs using these methods. The high frequency of LOI for IGF2 in CCSKs (43%) is comparable to that reported in Wilms tumors. The retention of imprinting at the SNRPN and H19 loci confirm that LOI is not a ubiquitous epigenetic change. This suggests that IGF2, a potent growth factor, may play a role in the development or progression of CCSK.     

Cross-reactive trinitrophenylated peptides as antigens for class II major histocompatibility complex-restricted T cells and inducers of contact sensitivity in mice. Limited T cell receptor repertoire

The induction of contact sensitivity in mice by hapten reagents such as trinitrochlorobenzene (TNCB) involves the activation of class II major histocompatibility complex (MHC)-restricted, hapten-specific, CD4⁺ T cells. Reports from different laboratories have indicated that the relevant antigenic epitopes in such reactions might include hapten-conjugated, MHC class II-associated peptides. This study for the first time directly demonstrates that hapten-peptides account for the majority of determinants recognized by trinitrophenyl (TNP)-specific CD4⁺ T lymphocytes. The sequences of those TNP carrier peptides do not have to be related to mouse proteins. Thus, we show that TNP-modified peptides derived from mouse IgG, pigeon cytochrome c or staphylococcal nuclease known to bind to I-A^b or from λ represser with specificity to I-A^d as well as TNP-proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin all create class II-restricted hapten determinants for a number of TNP-specific T cell clones and hybridomas. All of these cells were induced with cells modified by trinitrobenzene sulfonic acid (TNBS). In addition, we present arguments indicating that individual TNP-specific helper T cells may cross-react with different TNP-peptides bound to identical class II molecules. Chemical treatment of antigen-presenting cells with TNCB or TNBS may thus result in a limited number of particularly repetitive immunodominant hapten epitopes. Immunodominant epitopes were also indicated by an overrepresentation of the TCR elements Vβ2 and Vα10 in I-A^b/TNP-specific T cells. Most importantly, however, we demonstrate that TNP attached to lysine 97 in the staphylococcal nuclease peptide 93–105 (i.e. a clearly “non-self” sequence) is able to prime mice for subsequent elicitation of contact sensitivity by TNCB in the absence of foreign protein. We take this to indicate that those TNP-peptide determinants defined by us as immuno-dominant are responsible for the induction of contact sensitivity to haptens.     

Decreased anti-donor major histocompatibility complex class I and increased class II alloantibody response in allograft tolerance in adult rats

Permanent tolerance to allografts can be induced in adult rats by donor-specific transfusions (DST) prior to transplantation. We have previously reported, in a model of heart allograft, the presence of a heavy leukocyte infiltrate, in the allograft which displayed a strong allospecific cytotoxicity when tested in vitro against donor cells, and a strong accumulation of mRNA for granzyme A and perforin in vivo. In contrast, there was a major decrease in the accumulation of mRNA for interleukin-2 and interferon-γ. These results suggested that the DST-induced tolerance was associated with a decrease in type-1 T helper (Th1) cell function. The major role of preformed antibodies in xeno and allorejection is clearly established. Nevertheless, the consequences of alloantibody production in acute rejection and tolerance induction remains to be elucidated. We here analyze the alloantibody response in rejecting and DST-treated recipients. We show that, after transplantation, tolerant recipients, in contrast to rejecting ones, mount a low IgM alloresponse that switches to low IgG production. Detailed analysis of IgG alloantibodies in DST-treated recipients revealed that their production decrease was not equally distributed. Whereas rejecting animals mounted a strong anti-class I and II IgG alloantibody response, DST-treated recipients produced anti-class II and low titers of anti-class I IgG alloantibodies. Furthermore, among IgG subclasses, tolerant recipients predominantly produced IgG2a, a profile which, in the rat, is compatible with a Th2-controlled response. Finally, the passive transfer of immune serum from rejecting animals to DST-treated recipients could abrogate the tolerance. We suggest that the absence of anti-class I alloantibodies combined with preserved and/or increased anti-class II production plays a major role in graft tolerance in this model. These results reinforced the role of alloantibodies in rejection and in induction of tolerance.     

Role of Tris-CaEDTA as an adjuvant with nebulised tobramycin in cystic fibrosis patients with Pseudomonas aeruginosa lung infections: A randomised controlled trial

BackgroundWe tested if disrupting iron utilisation by P. aeruginosa by adding the Tris-buffered chelating agent CaEDTA to nebulised tobramycin would enhance bacterial clearance and improve lung function in CF patients.MethodsIn this double-blind, randomised controlled trial, 26 episodes (25 patients) with P. aeruginosa infection admitted to two CF centres for treatment of an acute pulmonary exacerbation were randomly assigned to receive either 75 mg CaEDTA in Tris-buffered saline or placebo (Tris-buffered saline) nebulised in combination with 250 mg tobramycin twice daily for six weeks followed with four week safety follow-up. Primary endpoints were safety, tolerability, and bacterial density of P. aeruginosa. A secondary endpoint was lung function.ResultsThe study drug was well tolerated with adverse events comparable in both groups. The mean (SD) reduction in sputum P. aeruginosa count (log₁₀ CFU/g) in the CaEDTA vs placebo group was 2·05 (2·57) vs 0·82 (2·71) at two weeks relative to admission (p = 0·39). The mean improvement in ppFEV₁ was 16 vs 5 (p = 0·16); 11 vs 2 (p = 0·28); and 6 vs 2 percentage points (p = 0·47) at two, six, and ten weeks in CaEDTA and placebo groups, respectively.ConclusionsIn this pilot study in CF patients, an increase in the reduction of sputum density of P. aeruginosa and an increase in ppFEV₁ was observed in the group of patients who received Tris-CaEDTA added to inhaled tobramycin compared to the group who received inhaled tobramycin alone, although these differences were not statistically significant. The treatment was also shown to be safe.     

Urinary excretion of adenosine deaminase binding protein in neonates treated with tobramycin

The potential tubulotoxicity of tobramycin and cefotaxim were assessed in neonates by measuring the urinary level of adenosine deaminase binding protein (ABP) and urinary ₁-microglobulin and ₂-microglobulin. In a prospective study, 33 neonates who received tobramycin and cefotaxim for suspected neonatal sepsis were compared with 48 untreated newborns during the first 10 days of life. The urinary concentrations of ABP and its excretion rates, corrected for body weight and body surface area, were significantly increased from the 1st day of treatment. Urinary ₁-microglobulin and ₂-microglobulin were not elevated under tobramycin and cefotaxim during the first 2 days of treatment. We conclude that ABP may be a sensitive marker for the detection of proximal renal tubular injury during tobramycin and cefotaxim treatments of neonates. The increase in urinary ABP which occurs before an elevation of urinary ₁-microglobulin and ₂-microglobulin may reflect earlier structural than functional alterations. However, since none of the treated infants had signs of electrolyte disorders or glomerular dysfunction, the clinical relevance of ABP measurement should be reevaluated.     

Amacrine cells,bipolar cells and ganglion cells of the cat retina: A Golgi study

Neuronal types contributing to the inner plexiform layer of the cat retina are described based primarily on light microscopy of Golgi-impregnated retinal whole-mounts. Cells have been characterized on morphological criteria that include dendritic branching patterns, dendritic tree sizes, cell body sizes and stratification of processes in the inner plexiform layer. Nine different types of bipolar cell, 22 different types of amacrine cell and 23 different types of ganglion cell can be distinguished using one or more of these morphological criteria. The significance of the different morphological types of cells is discussed, particularly in relationship to the functional bisublamination of the cat inner plexiform layer.