Diversity of Matriptase Expression Level and Function in Breast Cancer

Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent

Differential stromal reprogramming in benign and malignant naturally occurring canine mammary tumours identifies disease-modulating stromal components

While cancer-associated stroma (CAS) in malignant tumours is well described, stromal changes in benign forms of naturally occurring tumours remain poorly characterized. Spontaneous canine mammary carcinomas (mCA) are viewed as excellent models of human mCA. We have recently reported highly conserved stromal reprogramming between canine and human mCA based on transcriptome analysis of laser-capture-microdissected FFPE specimen. To identify stromal changes between benign and malignant mammary tumours, we have analysed matched normal and adenoma-associated stroma (AAS) from 13 canine mammary adenomas and compared them to previous data from 15 canine mCA. Our analyses reveal distinct stromal reprogramming even in small benign tumours. While similarities between AAS and CAS exist, the stromal signature clearly distinguished adenomas from mCA. The distinction between AAS and CAS is further substantiated by differential enrichment in several hallmark signalling pathways as well as differential abundance in cellular composition. Finally, we identify COL11A1, VIT, CD74, HLA-DRA, STRA6, IGFBP4, PIGR, and TNIP1 as strongly discriminatory stromal genes between adenoma and mCA, and demonstrate their prognostic value for human breast cancer. Given the relevance of canine CAS as a model for the human disease, our approach identifies disease-modulating stromal components with implications for both human and canine breast cancer

Plant-Type Trehalose Synthetic Pathway in Cryptosporidium and Some Other Apicomplexans

The trehalose synthetic pathway is present in bacteria, fungi, plants and invertebrate animals, but is absent in vertebrates. This disaccharide mainly functions as a stress protectant against desiccation, heat, cold and oxidation. Genes involved in trehalose synthesis have been observed in apicomplexan parasites, but little was known about these enzymes. Study on trehalose synthesis in apicomplexans would not only shed new light into the evolution of this pathway, but also provide data for exploring this pathway as novel drug target.We have observed the presence of the trehalose synthetic pathway in Cryptosporidium and other apicomplexans and alveolates. Two key enzymes (trehalose 6-phosphate synthase [T6PS; EC 2.4.1.15] and trehalose phosphatase [TPase; EC 3.1.3.12] are present as Class II bifunctional proteins (T6PS-TPase) in the majority of apicomplexans with the exception of Plasmodium species. The enzyme for synthesizing the precursor (UDP-glucose) is homologous to dual-substrate UDP-galactose/glucose pyrophosphorylases (UGGPases), rather than the "classic" UDP-glucose pyrophosphorylase (UGPase). Phylogenetic recontructions indicate that both T6PS-TPases and UGGPases in apicomplexans and other alveolates are evolutionarily affiliated with stramenopiles and plants. The expression level of T6PS-TPase in C. parvum is highly elevated in the late intracellular developmental stage prior to or during the production of oocysts, implying that trehalose may be important in oocysts as a protectant against environmental stresses. Finally, trehalose has been detected in C. parvum oocysts, thus confirming the trehalose synthetic activity in this parasite.A trehalose synthetic pathway is described in the majority of apicomplexan parasites including Cryptosporidium and the presence of trehalose was confirmed in the C. parvum oocyst. Key enzymes in the pathway (i.e., T6PS-TPase and UGGPase) are plant-type and absent in humans and animals, and may potentially serve as novel drug targets in the apicomplexans

Evaluating the Impact of a Standardized Education Class on a Person Diagnosed with Chronic Kidney Disease, Stage IV

Background: Chronic kidney disease (CKD) is a complex, progressive, and costly disease impacting more than 26 million Americans. Providing effective education is necessary so the patient may actively participate in managing the disease process, but the effectiveness of the delivery of education to the patient with CKD is not well-known. Objective: To evaluate the effectiveness of a standardized education class on the basic kidney knowledge of persons diagnosed with CKD, stage IV. Method: This pilot study, using a one-group pre and post-test design, was conducted in an outpatient nephrology clinic located in Washington, D.C. The study participants, recruited using census sampling, completed a kidney disease-specific knowledge questionnaire prior to a standardized education class, and then completed the same questionnaire immediately after the class. A Wilcoxon Signed-Ranks test and descriptive statistics were utilized to examine the mean kidney knowledge and summarize the findings. Results: A total of 14 patients participated in this study. Participants’ mean age was 63 years, most were female (64%) and Black/African American (79%). Thirty-six percent were not married, 43% reported having a high school diploma or equivalent, and 43% rated their overall health status as good. The study results concluded that the post questionnaire scores were significantly higher than the pre questionnaire scores (Z = -3.299, p = 0.001). Conclusions: This pilot study showed that providing a standardized education class is associated with higher basic kidney knowledge. While compelling, further studies are needed to determine knowledge retention, and support these findings with a larger sample siz

Thyrotropin receptor-specific lymphocytes in adenovirus-TSHR-immunized native and human leukocyte antigen-DR3-transgenic mice and in Graves' disease patient blood

BACKGROUND: Antigen-specific lymphocytes are increasingly investigated in autoimmune diseases and immune therapies. We sought to identify thyrotropin receptor (TSHR)-specific lymphocytes in mouse models of Graves' disease, including Graves' patient-specific immunotype human leukocyte antigen (HLA)-DR3, and in frozen and thawed Graves' patient blood samples. METHODS AND RESULTS: Splenic lymphocytes of adenovirus (Ad)-TSHR-immunized BALB/c mice were stimulated with TSHR-specific peptides C, D, or J. Furthermore, CD154-expressing cells were enriched, expanded in vitro, and analyzed for binding of peptide-major histocompatibility complex (MHC) II multimers ("tetramers," immunotype H2-IA(d)). Only peptides C and J were able to elicit increased expression/secretion of CD154 and interferon-γ, and tetramers which were loaded with peptide C resulted in antigen-specific signals in splenic lymphocytes from Ad-TSHR-immunized mice. Accordingly, TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 specifically bound to human HLA-DR3-(allele B1*03:01)-transgenic Bl/6 mouse splenic T lymphocytes. In addition, we fine-tuned a protocol to reliably measure thawed human peripheral blood mononuclear cells (PBMCs), which resulted in reliable recovery after freezing and thawing with regard to vitality and B and T cell subpopulation markers including regulatory T cells (CD3, CD4, CD25, FoxP3, CD25(high), CD127(low)). TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 identified antigen-specific T cells in HLA-DR3-positive Graves' patients' thawed PBMCs. Moreover, stimulation-dependent release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha from thawed PBMCs occurred at the expected levels. CONCLUSIONS: Novel MHC II tetramers identified TSHR-specific T lymphocytes in Ad-TSHR-immunized hyperthyroid BALB/c or HLA-DR3-transgenic mice and in thawed human PBMCs from patients with Graves' disease. These assays may contribute to measure both disease severity and effects of novel immune therapies in future animal studies and clinical investigations of Graves' disease

Determinants of translocation and folding of TreF, a trehalase of Escherichia coli

One isoform of trehalase, TreF, is present in the cytoplasm and a second, TreA, in the periplasm. To study the questions of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular compartment, we fused the signal sequence of periplasmic TreA to cytoplasmic TreF. Even though this TreF construct was exported efficiently to the periplasm, it was not active. It was insoluble and degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre+ revertants of periplasmic TreF. To further characterize periplasmic TreF, we used a genetic selection to isolate functional TreA-TreF hybrids, which were analyzed with respect to solubility and function. These data suggested that a domain located between residues 255 and 350 of TreF is sufficient to cause folding problems in the periplasm. In contrast to TreF, periplasmic TreA could fold into the active conformation in its nonnative cellular compartment, the cytoplasm, after removal of its signal sequence

Direct Detection of Antibody Concentration and Affinity in Human Serum Using Microscale Thermophoresis

The direct quantification of both the binding affinity and absolute concentration of disease-related biomarkers in biological fluids is particularly beneficial for differential diagnosis and therapy monitoring. Here, we extend microscale thermophoresis to target immunological questions. Optically generated thermal gradients were used to deplete fluorescently marked antigens in 2- and 10-fold-diluted human serum. We devised and validated an autocompetitive strategy to independently fit the concentration and dissociation constant of autoimmune antibodies against the cardiac beta 1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen, the peptide CORI was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibody concentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding in its native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix and probe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine