Bacterial adhesion to different termite flagellates: Ultrastructural and functional evidence for distinct molecular attachment modes

Summary The attachment modes of rodlike ectobiotic bacteria to the surface of two different termite flagellates were studied.Devescovina glabra was covered by laterally attached bacteria. Treatment with chemicals that disturb hydrophobic interactions and solubilize proteins removed the ectobionts. Freeze-fracture and freeze-etching electron microscopy revealed rows of intramembrane particles that occurred exclusively along the attachment sites. The adhering Gram-negative bacteria possessed an S-layer (surface layer) composed of globular protein particles. The S-layer could be removed by protein-solubilizing chemicals, e.g., urea, as shown by ultrathin-section electron microscopy. Therefore, it seems plausible that the attachment was mediated by hydrophobic interactions between the flagellate's plasma membrane and the S-layer of the bacteria. The bacteria of the second flagellate,Joenia annectens, adhered by their tips. The attachment was extremely strong. Chemicals disturbing ionic or hydrophobic bindings or solubilizing proteins did not detach the ectobionts. Globular intramembrane protein particles were preferentially found in a ringlike array at the external fracture face of the flagellate's contact sites.Abbreviations DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - TEM transmission electron microscope - Tween 20 polyoxyethylenesorbitan     

Pathogenesis of prosthesis-related infection

In spite of its incidence decreasing to 1% nowadays, prosthesis-related infection remains a research, diagnostic, therapeutic and cost-related problem. It can be defined as a presence of bacteria in the artificial joint space, which is significantly associated with evident laboratory and/or tissue markers, and clinical signs of running infection. We believe that the more precise understanding of pathogenesis, the more effective preventative and therapeutic measures, and the lower infection rate. The implants are colonized by airborne, skin-, and/ or surgeon-related bacteria during surgery despite being operated in closely respected operating regime. Some prosthetic characteristics are advantageous and may play important roles in the process of bacterial adherence. After successful attachment on the biomaterial surface bacteria multiply and physiologically transform into a "biofilm" community, making them much more resistant to antibiotic therapy and host immunity. Bacterial resistance is a complex phenomenon influenced by intrinsic and extrinsic factors, including the cell configuration in the biofilm community. So the cure of periprosthetic sepsis without removing of all foreign bodies and necrotic bone fragments is often ineffective. Acute hematogenous sepsis is suggestive of a distortion of a previously aseptic joint space by invasion of bacteria through the vessels.     

Generation of functional dendritic cells for potential use in the treatment of acute lymphoblastic leukemia

Immunotherapy of malignant diseases mediated by dendritic cells (DC) pulsed with tumor antigens ex vivo is a promising new tool in the individual treatment of malignant diseases. The present study focuses on the problem of how to optimize in vitro culture conditions and induce the maturation of DC with the capacity to induce antitumor immunity toward leukemic cells. DC were generated from peripheral mononuclear cells by co-cultivation with granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Tumor antigens were added for 2 h after 7 days in culture. Irradiated leukemic blasts, blast lysate, apoptotic cells from the Jurkat cell line (T ALL) and their lysate were used in various concentrations for antigen pulsing. Harvested DC were phenotyped by flow cytometry, and viability was assessed using trypan blue exclusion (Annexin test). After the cells had been pulsed with tumor antigens and co-cultured with autologous lymphocytes, the production of interferon-gamma (IFN-gamma) and IL-12 was analyzed, and lymphocyte proliferative response and cytotoxicity against the target tumor cell line were assessed. The cultivation of monocytes under the described conditions led to the expression of surface markers typical of DC (i.e. CD83, CD86, HLA-DR, CD11c and CD40). Pulsation by antigens from leukemic cells further increased the cell populations expressing these markers. Antigen pulsation decreased the viability of generated DC depending on the increase in concentration of tumor antigens. Pulsed DC-lymphocyte interaction increased the proliferative response of lymphocytes and IFN-gamma production depending on the type of tumor antigens used for pulsation. The highest proliferative response was detected with DC pulsed with Jurkat cell-line lysate. Similarly to the proliferation assay, cytotoxic testing showed the highest efficiency of DC pulsed with Jurkat cell-line lysate in killing the target malignant cells. Our results show that an appropriate antigen concentration used for DC pulsing is one of the crucial factors in an effective treatment strategy, as high concentrations of tumor antigens induce apoptosis of DC, thereby rendering them non-functional. Under optimal conditions, pulsation by lysate from leukemic blasts induced the maturation of DC and led to an increase in the proliferation of autologous lymphocytes, to the production of Th1-cytokines and to the induction of cytotoxicity toward the leukemic cell line. These results are encouraging for the possible application of pulsed DC in the therapy of acute lymphoblastic leukemia.     

How oxymonads lost their groove: an ultrastructural comparison of Monocercomonoides and excavate taxa

Despite being amongst the more familiar groups of heterotrophic flagellates, the evolutionary affinities of oxymonads remain poorly understood. A re-interpretation of the cytoskeleton of the oxymonad Monocercomonoides hausmanni suggests that this organism has a similar ultrastructural organisation to members of the informal assemblage 'excavate taxa'. The preaxostyle, 'R1' root, and 'R2' root of M. hausmanni are proposed to be homologous to the right, left, and anterior roots respectively of excavate taxa. The 'paracrystalline' portion of the preaxostyle, previously treated as unique to oxymonads, is proposed to be homologous to the I fibre of excavate taxa. Other non-microtubular fibres are identified that have both positional and substructural similarity to the distinctive B and C fibres of excavate taxa. A homologue to the 'singlet root', otherwise distinctive for excavate taxa, is also proposed. The preaxostyle and C fibre homologue in Monocercomonoides are most similar to the homologous structures in Trimastix. suggesting a particularly close relationship. This supports and extends recent molecular phylogenetic findings that Trimastix and oxymonads form a clade. We conclude that oxymonads have an excavate ancestry, and that the 'excavate taxa' sensu stricto form a paraphyletic assemblage.     

Chromogranin A in pituitary adenomas: immunohistochemical detection and plasma concentrations

Forty one pituitary adenomas excised surgically were immunostained to reveal pituitary hormones and chromogranin A (CgA). In 23 patients, plasma CgA concentration was determined before surgery by ELISA method. The CgA immunopositivity was found in 70.7% of investigated tumors. It was observed in all tumors of gonadotropinoma type and in the majority of null cell adenomas. Elevated (>18 U/L) plasma CgA concentration was observed in approx. a half of the examined patients, being more frequent in gonadotropinomas and null cell adenomas. It may have some, although limited, diagnostic value in these types of pituitary tumors.     

Gliadin fragments induce phenotypic and functional maturation of human dendritic cells

Celiac disease is a chronic inflammatory disease developing in genetically predisposed individuals. Ingested gliadin, the triggering agent of the disease, can cross the epithelial barrier and elicit a harmful T cell-mediated immune response. Dendritic cells (DC) are supposed to play a pivotal role in shaping the immune response. The direction of the immune response toward immunity or tolerance depends on the stage of maturation and the functional properties of the DC. DC become fully functional APC upon maturation by various stimuli. We investigated the effect of a peptic digest of gliadin on the maturation of human monocyte-derived DC. Stimulation of cells with gliadin, in contrast with other tested food proteins, led to enhanced expression of maturation markers (CD80, CD83, CD86, and HLA-DR molecules) and increased secretion of chemokines and cytokines (mainly of IL-6, IL-8, IL-10, TNF-alpha, growth-related oncogene, MCP-1, MCP-2, macrophage-derived chemokine, and RANTES). Maturation was accompanied by a greater capacity to stimulate proliferation of allogeneic T cells and significantly reduced endocytic activity. Furthermore, gliadin-induced phosphorylation of members of three MAPK families (ERK1/2, JNK, and p38 MAPK) was demonstrated. The largest contribution of p38 MAPK was confirmed using its inhibitor SB203580, which markedly down-regulated the gliadin-triggered up-regulation of maturation markers and cytokine production. Gliadin treatment also resulted in increased NF-kappaB/DNA binding activity of p50 and p65 subunits. Taken together, gliadin peptides can contribute to overcoming the stage of unresponsiveness of immature DC by inducing phenotypic and functional DC maturation, resulting in more efficient processing and presentation of gliadin peptides to specific T lymphocytes.     

Sentinel lymph node in esophageal cancer before neoadjuvant therapy

The technique of sentinel lymph node identification and biopsy has become a new popular technique for surgeon to improve staging of malignant diseases. It may also reduce the risk of complication related to standard lymphadenectomy. The method is still in experimental phase in case of esophageal cancer. A possible complication for employment of the method in this tumor is neoadjuvant therapy. The authors developed the technique for identifying and obtaining the sentinel lymph node in esophageal cancer using minimally invasive surgical technique before neoadjuvant therapy. The sentinel lymph node is detected using 99mTc-labelled nanocolloid. The authors report and discuss possible difficulties of the method in the case of a patient with detected sentinel lymph node in this way. Conclusion: It is possible to identify and obtain a sentinel lymph node before neoadjuvant therapy in esophageal cancer. On the other hand, the clinical significance and applicability of the method of sentinel lymph node still remains controversial in this kind of a tumor.