Preparation of purified lymphoma cells suitable for therapy

BACKGROUND: Very few tumoral Ags have yet been isolated in NHL B cells. It is nevertheless possible to use whole tumor cells as a source of tumor Ags. We describe the purification of large numbers of human NHL B cells directly from lymph node or spleen biopsies, and different preparations allowing their use in a clinical setting. METHODS: The purification procedure consists of the negative selection of tumor B cells: cells to be eliminated are opsonized by CD2 Abs, and then coupled to magnetic beads for separation by the Isolex 300 magnetic separator. RESULTS: The mean yield of the purification was 74% for CD19+ cells, with a mean purity of 87%, dependent on the initial fraction of tumor cells in the biopsy. Using this procedure, a large number of purified tumor cells can be recovered from a biopsy in sterile conditions. We also describe treatments of B cells that can enhance their uptake by APCs, a critical step in anti-tumor immunotherapy strategies. Cells were opsonized by rituximab, or induced in apoptosis by irradiation, or necrosis by heating. Cell lysates were directly prepared from purified tumor cells. DISCUSSION: These procedures were reproducible on every lymphoma cell, and treated cells were phagocytosed by APCs. The methodology described here allows the evaluation of the immunological potential of apoptotic, necrotic, opsonized lymphoma cells, or their lysates, in a clinical setting.     

Adding denture cleanser to microwave disinfection regimen to reduce the irradiation time and the exposure of dentures to high temperatures

Background: The microwave energy is an efficient disinfection method; however, it can generate high temperatures that can result in distortion of the dentures. objectives: To evaluate whether the addition of an enzymatic cleanser to microwave disinfection regimen would disinfect dentures with shorter irradiation time. Materials and methods: Seven resin discs colonized with Candida albicans biofilm were placed on the palatal surface of sterile dentures to be randomly assigned to the following treatments: immersion in distilled water for 3 min with 0 (DW), 1 (DW + M1), 2 (DW + M2), or 3 min (DW + M3) of microwave irradiation; or immersion in denture cleanser for 3 min with 0 (DC), 1 (DC + M1), 2 (DC + M2) or 3 min (DC + M3) of irradiation. After the treatments, the viable cells were counted by a blinded examiner. The temperature was measured immediately after irradiation. The data were analyzed by ANOVA and Tukey post hoc tests (α = 0.05). Results: No viable cells were found after DC + M2, DC + M3, and DW + M3 treatments, of which DC + M2 achieved the lowest temperature. No significant difference was found between the effectiveness of DW, DW + M1 and DC treatments (p > 0.05). Conclusion: Within the limits of this study, the association of a denture cleanser and microwave energy is efficient to disinfect dentures in lower irradiation time and temperature.     

Virulence of Klebsiella pneumoniae Isolates Harboring bla KPC-2 Carbapenemase Gene in a Caenorhabditis elegans Model

Klebsiella pneumoniae carbapenemase (KPC) is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i) five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii) seven transformants obtained after electroporation of either these natural KPC plasmids or a recombinant plasmid harboring only the bla _KPC-2 gene into reference strains K. pneumoniae ATCC10031/{"type":"entrez-protein","attrs":{"text":"CIP53153","term_id":"878514309","term_text":"CIP53153"}}CIP53153; and (iii) five clinical strains cured of plasmids. The virulence of K. pneumoniae isolates was evaluated in the Caenorhabditis elegans model. The clinical KPC producers and transformants were significantly less virulent (LT50: 5.5 days) than K. pneumoniae reference strain (LT50: 4.3 days) (p<0.01). However, the worldwide spread KPC-2 positive K. pneumoniae ST258 strains and reference strains containing plasmids extracted from K. pneumoniae ST258 strains had a higher virulence than KPC-2 strains belonging to other ST types (LT50: 5 days vs. 6 days, p<0.01). The increased virulence observed in cured strains confirmed this trend. The bla _KPC-2 gene itself was not associated to increased virulence.     

In Situ Immune Response in Human Chromoblastomycosis – A Possible Role for Regulatory and Th17 T Cells

Background

Chromoblastomycosis is a chronic fungal infection that affects skin and subcutaneous tissue. Lesions can be classified in tumorous, verrucous, cicatricial and plaque type. The cellular immune response in the severe form of the disease seems to correlate with a Th2 pattern of cytokines. The humoral immune response also seems to play a role. We intended to explore the populations of regulatory T cells and the Th17 pattern.

Methodology

Twenty-three biopsies of verrucous form were obtained from patients with clinical, culture and histopathological diagnostic of chromoblastomycosis, without treatment. It was performed an immunohistochemistry method to detect Foxp3, CD25, TGF-β, IL-6, IL-17 and IL-23.

Principal findings

IL-17 was the only cytokine with high expression in CBM when compared to normal skin. The expression of Treg cells, TGF- β, IL-6 and IL-23 were similar to normal skin.

Conclusions/Significance

The constitution of a local immune response with high expression of IL-17 and low expression of other cytokines could be at least in part, an attempt to help the immune system against fungal infection. On the other hand, high levels of local immune response mediated by Th17 profile could overcome the role of Treg cells. The inefficient immunomodulation as a consequence of the unbalance by Treg/Th17 cells seems to corroborate with the less effective immune response against fungi.