MHC II on transfused murine blood is not required for alloimmunization against MHC I.

BACKGROUND AND OBJECTIVES: Transfusion of allogeneic platelet products can result in antibodies against donor major histocompatibility complex (MHC) I antigens, leading to a refractory state to subsequent platelet transfusions. However, there is disagreement in the field regarding the molecular mechanisms of humoral alloimmunization. One hypothesis states that donor MHC II is a requirement for alloimmunization. However, other studies have suggested that donor MHC I is alone sufficient and MHC II is not required. MATERIALS AND METHODS: We utilized a mouse model of anti-MHC I alloimmunization to transfused blood, which employed donors with a complete deletion of all MHC II genes. BALB/c (H-2(d)) recipients were transfused with blood from either C57BL/6 (H-2(b)) or MHC II null donors on a C57BL/6 background. Anti-MHC I alloimmunization was monitored by indirect immunofluorescence. RESULTS: Recipients of either wild type or MHC II null blood produced equivalent humoral responses against donor MHC I antigens. However, there was variation in the relative amounts of IgG subclasses. CONCLUSION: These data reject the hypothesis that donor MHC II expression is required for alloimmunization to MHC I antigens.

Cross-linking induces non-haemolytic antigen-loss from transfused red blood cells: a potential role for rheumatoid factor.

Transfusion of cross-match incompatible blood can lead to haemolysis. However, in some cases, incompatible transfused red blood cells are bound by antibody and then converted to being negative for both the incompatible antigen and the direct antiglobulin test. Using a murine model of this phenomenon, we have recently reported that antibodies binding to multiple epitopes are required. Herein, we report that antibodies against one epitope can induce antigen-loss if an anti-immunoglobulin G (IgG) antibody is also present. These findings support a model of cross-linking being required, and raise the possibility that naturally occurring anti-IgG, such as rheumatoid factor, may contribute to antigen-loss.

The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3.

Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein (W. Jiang, K. Middleton, H.-J. Yoon, C. Fouquet, and J. Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolar-cytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperature-sensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5delta null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.

The effects of wild-type p53 tumor suppressor activity and mutant p53 gain-of-function on cell growth.

The tumor suppressor p53 plays a central role in the protection against DNA damage and other forms of physiological stress primarily by inducing cell cycle arrest or apoptosis. Mutation of p53, which is the most frequent genetic alteration detected in human cancers, inactivates these growth regulatory functions and causes a loss of tumor suppressor activity. In some cases, mutation also confers tumor-promoting functions, such as the transcriptional activation of genes involved in cell proliferation, cell survival and angiogenesis. Consequently, cells expressing some forms of mutant p53 show enhanced tumorigenic potential with increased resistance to chemotherapy and radiation. Our current understanding of these activities is summarized in this review. By dissecting out mechanistic differences between wild-type and mutant p53 activities, it may be possible to develop therapeutics that restore tumor suppressor function to mutant p53 or that selectively inactivate mutant p53 tumor-promoting functions.

Intra-aortic balloon counterpulsation timing.

Intra-aortic balloon counterpulsation is a product of an endeavor begun more than 3 decades ago. First the concept, then applications of inflating a nonthrombogenic balloon within the aorta during diastole were explored. The intra-aortic balloon is timed to inflate with aortic valve closure. Two options are available for deflation timing: (1) conventional timing, where deflation is estimated to be completed at some point during isovolumetric contraction, prior to ejection; and (2) R wave deflation, also known as real timing, where deflation is triggered by each QRS complex to occur during isovolumetric contraction or early systole. Although clinical implementation of both conventional and real timing was introduced in 1968, limited information about these methods of deflation is currently available. This article elucidates the differences between these models and suggests implications for clinical practice and further research.

Clinical observations with real timing.

This article describes the hemodynamic effects of IABC therapy on patients supported with real timing. Criteria used to assess "proper" timing are discussed and challenged as they relate to the limited research available on the subject and to the cases in this article. It is important that clinicians who time the IAB consoles and assess therapeutic benefit of this therapy are aware of the different timing modalities and their potential benefits or ill effects in certain patient populations. The observations made by the authors strongly suggest the need for clinical research, case reports, and further evidence of valid assessment criteria to develop a research-based theory for practice of IABC timing and to provide criteria for selecting timing modalities for specific patient populations.

Ablation of Stat3 by siRNA alters gene expression profiles in JEG-3 cells: a systems biology approach.

Control of inflammation at the maternal-fetal interface is a critical element in mammalian pregnancy. Previous work from our laboratory has shown that Stat3 may be a placental mediator involved in maintaining immunologic homeostasis at the maternal-fetal interface. The aim of the current study is to further elucidate the role of Stat3 in response to inflammation. As ablation of Stat3 in mice results in embryonic lethality, we evaluated the role of Stat3 in vitro using an siRNA approach. Trophoblast-like JEG-3 cells were transfected with an siRNA construct specific to Stat3. Experimental and control cells were exposed to conditioned medium from PHA-activated peripheral blood mononuclear cells and incubated for 45 min. Cells were then collected and RNA isolated for transcriptional profiling using human Affymetrix U133 plus 2.0 GeneChips. Differences in gene expression between control and Stat3-ablated cells were evaluated using conventional statistical methods. Fifty-two genes were detected as up-regulated in conditioned medium in both mock transfected and in Stat3 siRNA transfected JEG-3 cells. Two genes (EPAS1 and RASGEF1B) were up-regulated only in cells transfected with negative control siRNA, while 36 genes were up-regulated only in cells transfected with Stat3 siRNA. Sixty genes were differentially expressed between Stat3 siRNA transfected cells relative to mock transfected cells both in basal and conditioned medium. These included 31 genes up-regulated with Stat3 siRNA transfected cells and 29 genes down-regulated with Stat3 siRNA. Eleven genes were differentially expressed only in basal medium. Seven of these were up-regulated in the presence of Stat3 siRNA and four were down-regulated. Nine genes were differentially expressed only in conditioned medium. Six of these were up-regulated and three down-regulated in the presence of Stat3 siRNA. Off-target effects were excluded in a second set of experiments in which Stat3 mRNA was targeted at a different site and quantitative real-time PCR performed on selected genes derived from the microarray analysis. While some of the genes that showed differential expression between Stat3-ablated cells and mock transfected controls were genes typically associated with immune response (e.g., CCR7 and IRAK1), in silico modeling of the microarray data also revealed complex networks of signaling molecules and molecules associated with cellular metabolism previously seen in transcription factor ablation in model organisms. We conclude thus: Stat3 controls a specific gene set in trophoblast-like JEG-3 cells. While some differentially expressed genes and in silico models of their functions are consistent with the hypothesis that Stat3 plays a role in regulating inflammation, Stat3-mediated response to inflammation appears to also involve complex homeostatic adaptations of a non-immunologic nature.

Template-driven gene selection procedure.

The hierarchical clustering and statistical techniques usually used to analyse microarray data do not inherently represent the underlying biology. Herein, a hybrid approach involving characteristics of both supervised and unsupervised learning is presented. This approach is based on template matching in which the interaction of the variables of inherent malignancy and the ability to express the malignant phenotype are modelled. Immortalised normal urothelial cells and bladder cancer cells of different malignancy were grown in conventional two-dimensional tissue culture and in three dimensions on extracellular matrices (ECMs) that were either permissive or restrictive for expression of the malignant phenotype. The transcriptome represents the effects of two variables--inherent malignancy and the modulatory effect of ECM. By assigning values to each of the biological variables of inherent malignancy and the ability to express the malignant phenotype, a template was constructed, which encapsulated the interaction between them. Gene expression correlating both positively and negatively with the template was observed, but when iterative correlations were carried out, the different models for the template converged on the same actual template. A subset of 21 genes was identified, which correlated with two a priori models or an optimised model above the 95% confidence limits identified in a bootstrap resampling with 5000 permutations of the data set. The correlation coefficients of expression of several genes were > 0.8. Analysis of upstream transcriptional regulatory elements (TREs) confirmed that these genes were not a randomly selected set of genes. Several TREs were identified as significantly over-expressed in the sample of 20 genes for which TREs were identified, and the high correlations of several genes were consistent with transcriptional co-regulation. The authors suggest that the template method can be used to identify a unique set of genes for further investigation.

A genome-scale assessment of peripheral blood B-cell molecular homeostasis in patients with rheumatoid arthritis.

OBJECTIVE: While rheumatoid arthritis (RA) is considered a prototypical autoimmune disease, the specific roles of B-cells in RA pathogenesis is not fully delineated. METHODS: We performed microarray expression profiling of peripheral blood B-cells from RA patients and controls. Data were analysed using differential gene expression analysis and 'gene networking' analysis (characterizing clusters of functionally inter-relelated genes) to identify both regulatory genes and the pathways in which they participate. Results were confirmed by quantitative real-time polymerase chain reaction and by measuring the levels of 10 serum cytokines involved in the pathways identified. RESULTS: Genes regulating and effecting the cell-cycle, proliferation, apoptosis, autoimmunity, cytokine networks, angiogenesis and neuro-immune regulation were differentially expressed in RA B-cells. Moreover, the serum levels of several soluble factors that modulate these pathways, including IL-1beta, IL-5, IL-6, IL-10, IL-12p40, IL-17 and VEGF were significantly increased in this cohort of RA patients. CONCLUSIONS: These results outline aspects of the multifaceted role B-cells play in RA pathogenesis in which immune dysregulation in RA modulates B-cell biology and thereby contributes to the induction and perpetuation of a pathogenic humoral immune response.

The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast.

The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and chf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophila melanogaster homolog, Nop60B, was identified. The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases. Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis. Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast chf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila.

An inherited p53 mutation that contributes in a tissue-specific manner to pediatric adrenal cortical carcinoma.

The incidence of pediatric adrenal cortical carcinoma (ACC) in southern Brazil is 10-15 times higher than that of pediatric ACC worldwide. Because childhood ACC is associated with Li-Fraumeni syndrome, we examined the cancer history and p53 status of 36 Brazilian patients and their families. Remarkably, 35 of 36 patients had an identical germ-line point mutation of p53 encoding an R337H amino acid substitution. Differences within intragenic polymorphic markers demonstrated that at least some mutant alleles arose independently, thus eliminating a founder effect. In tumor cells, the wild-type allele was deleted, and mutant p53 protein accumulated within the nuclei. Although these features are consistent with Li-Fraumeni syndrome-associated adrenal tumors, there was no history of increased cancer incidence among family members. Therefore, this inherited R337H p53 mutation represents a low-penetrance p53 allele that contributes in a tissue-specific manner to the development of pediatric ACC.