MALDI imaging–based identification of prognostically relevant signals in bladder cancer using large-scale tissue microarrays

ObjectiveAlthough most patients with urinary bladder cancer present with noninvasive and low-malignant stages of the disease, about 20% eventually develop life-threatening metastatic tumors. This study was designed to evaluate the potential of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to identify molecular markers predicting the clinical course of bladder cancer.Materials and methodsWe employed MALDI-MSI to a bladder cancer tissue microarray including paraffin-embedded tissue samples from 697 patients with clinical follow-up data to search for prognostically relevant associations.ResultsAnalysis of our MALDI imaging data revealed 40 signals in the mass spectra (m/z signals) associated with epithelial structures. The presence of numerous m/z signals was statistically related to one or several phenotypical findings including tumor aggressiveness (stage, grade, or nodal status; 30 signals), solid (5 signals) or papillary (3 signals) growth patterns, and increased (6 signals) or decreased (12 signals) cell proliferation, as determined by Ki-67 immunohistochemistry. Two signals were linked with tumor recurrence in noninvasive (pTa category) tumors, of which one was also related to progression from pTa-category to pT1-category disease. The absence of one m/z signal was linked with decreased survival in the subset of 102 muscle-invasive cancers.ConclusionOur data demonstrate the suitability of combining MSI and large-scale tissue microarrays to simultaneously identify and validate clinically useful molecular markers in urinary bladder cancer.     

Designing the epitope flanking regions for optimal generation of CTL epitopes

The flanking amino acids that surround epitopes are critical for effective antigen processing and maintenance of epitope integrity. In the present study, the frequency and characteristics of each amino acid that flanked the peptides generated from the proteasomal degradation of three different subtypes of HIV-1 Gag-p24 were determined. Synthetic flanking regions were designed based on the highest and the lowest frequencies of amino acid with the ideal characteristics at positions upstream and downstream of the proteasomal cleavage site. Peptides were synthesized that contained known CD8+ CTL-epitopes from HIV-1 Gag, CMV pp65, and vaccinia proteins HRP-2, and C16, flanked by amino acid sequences specifically designed to either generate or inhibit the known CD8+ CTL-epitopes. As predicted, the known CD8+ CTL-epitopes were effectively generated from the peptides with synthetic flanking regions specifically designed to promote epitope generation in a proteasome-dependent manner. The majority of the proteasome-generated epitopes were cleaved immediately after the C-terminal amino acid of the specific CTL-epitope. The synthetic peptide sequences containing known CD8+ CTL-epitopes with the flanking regions that promote epitope generation were effectively processed and presented to epitope specific CD8+ T-cells resulting in the production of IFN-γ. These results highlight the importance of flanking regions in promoting efficient antigen processing and presentation. This concept can have important implications in vaccine design and development strategies.     

Murine neutrophils treated with alphaB‐crystallin reduce IL‐12p40 production by dendritic cells

Neutrophils are essential in the fight against invading pathogens. They utilize antimicrobial effector mechanisms, such as phagocytosis, release of proteases and other antimicrobial products, robust oxidative bursts and neutrophil extracellular traps to combat infections. Neutrophils also modulate immune responses through the production of eicosanoids, cytokines and chemokines, as well as via direct communication with other immune cells. This system of high‐intensity offense against pathogens is exquisitely balanced through regulation to limit damage to host tissue. Unfortunately, the control of neutrophils is not failproof. In cases of sterile injury, autoimmunity and even during an infection, neutrophils can cause tissue destruction and become detrimental to the host. For that reason, there is a need to find means to regulate the aberrant activation of these cells. We found that alphaB‐crystallin (αBC), a heat‐shock protein known to have anti‐inflammatory abilities, affects certain properties of mouse neutrophils that subsequently influence the pro‐inflammatory state of antigen‐presenting cells (APCs). More specifically, αBC mediated small but significant increases in the levels of IL‐10 and matrix metalloproteinase 8, and altered hydrogen peroxide secretion by stimulated neutrophils. Further, the heat‐shock protein influenced the communication between neutrophils and dendritic cells by decreasing the production of pro‐inflammatory cytokines, specifically IL‐12p40, by the APCs. αBC could thus contribute to dampening neutrophil inflammatory responses by impacting the effect of neutrophils on other immune cells.     

Cost and unit cost calculations using step-down accounting

There is paucity of unit cost data from low- and middle-income countries, although recent initiatives have emerged to help rectify this. The limited budgets assigned to health care facilities mean that health planners and managers must be able to account for the resources used in health facilities as well as use them efficiently. Step-down cost accounting (SDCA) offers a relatively simple method for generating cost and unit cost data at the facility level. However, to the best of our knowledge, there is a lack of clear and concise guidance on how to undertake SDCA. Therefore, this paper, using a worked example, illustrates the different steps involved to generate cost and unit costs for a small hospital.     

Quantification of Human T-lymphotropic virus type I (HTLV-I) provirus load in a rural West African population: no enhancement of human immunodeficiency virus type 2 pathogenesis, but HTLV-I provirus load relates to mortality

Human T-lymphotropic virus type I (HTLV-I) provirus load was examined in a cohort of a population in Guinea-Bissau among whom human immunodeficiency virus (HIV) type 2 is endemic. Geometric mean of HIV-2 RNA load among HTLV-I-coinfected subjects was significantly lower than that in subjects infected with HIV-2 alone (212 vs. 724 copies/mL; P=.02). Adjusted for age, sex, and HIV status, the risk of death increased with HTLV-I provirus load; mortality hazard ratio was 1.59 for each log10 increase in HTLV-I provirus copies (P=.038). There is no enhancing effect of HTLV-I coinfection on HIV-2 disease, but high HTLV-I provirus loads may contribute to mortality.     

Antimicrobial Susceptibility and Resistance Patterns among Helicobacter pylori Strains from The Gambia,West Africa

Helicobacter pylori is a globally important and genetically diverse gastric pathogen that infects most people in developing countries. Eradication efforts are complicated by antibiotic resistance, which varies in frequency geographically. There are very few data on resistance in African strains. Sixty-four Gambian H. pylori strains were tested for antibiotic susceptibility. The role of rdxA in metronidazole (Mtz) susceptibility was tested by DNA transformation and sequencing; RdxA protein variants were interpreted in terms of RdxA structure. Forty-four strains (69%) were resistant to at least 8 μg of Mtz/ml. All six strains from infants, but only 24% of strains from adults, were sensitive (P = 0.0031). Representative Mtz-resistant (Mtz^r) strains were rendered Mtz susceptible (Mtz^s) by transformation with a functional rdxA gene; conversely, Mtz^s strains were rendered Mtz^r by rdxA inactivation. Many mutations were found by Gambian H. pylori rdxA sequencing; mutations that probably inactivated rdxA in Mtz^r strains were identified and explained using RdxA protein''s structure. All of the strains were sensitive to clarithromycin and erythromycin. Amoxicillin and tetracycline resistance was rare. Sequence analysis indicated that most tetracycline resistance, when found, was not due to 16S rRNA gene mutations. These data suggest caution in the use of Mtz-based therapies in The Gambia. The increasing use of macrolides against respiratory infections in The Gambia calls for continued antibiotic susceptibility monitoring. The rich variety of rdxA mutations that we found will be useful in further structure-function studies of RdxA, the enzyme responsible for Mtz susceptibility in this important pathogen.