Patellofemoral joint: kinematic MR imaging to assess tracking abnormalities

The patellofemoral joint was imaged with magnetic resonance (MR) in the axial plane while the knee was positioned from 0 degrees to 32 degrees of flexion (nine positions). These multiple sequential images obtained within the early phases of flexion of the knee were viewed in a "cine-loop" format, producing a kinematic study that clearly demonstrated the relationship of the patella to the trochlear groove. Four healthy subjects and one patient with known bilateral subluxing patellae were studied. The preliminary results suggest that kinematic MR imaging of the patellofemoral joint is potentially useful for the evaluation of patellar tracking abnormalities.     

Phenotypic markers associated with gastrointestinal Aeromonas hydrophila isolates from symptomatic children.

Aeromonas hydrophila gastroenteritis was detected in 12 pediatric patients during a 5-month period. Chief complaints included bloody diarrhea, fever, vomiting, and abdominal pain. Severe symptoms in two patients necessitated hospitalization and supportive care. Phenotypic characteristics associated with enterotoxigenicity of A. hydrophila strains demonstrated that all 12 isolates were cytotoxic to HeLa cells and most were lysine decarboxylase positive (75%). A correlation existed between the presence of the five virulence-associated markers of two isolates of A. hydrophila and the severity of disease. Although the length and symptoms of gastroenteritis varied among all 12 patients, most had self-limiting diarrhea. The frequent occurrence of A. hydrophila gastroenteritis in pediatric patients warrants a greater appreciation of this agent as a significant cause of diarrhea, especially in summer.     

Comparative studies and laboratory diagnosis of Vibrio vulnificus, an invasive Vibrio sp

Vibrio vulnificus was isolated from a bacteremic patient. This strain, together with other isolates of V. vulnificus, was compared with V. alginolyticus, V. fluvialis, and V. parahaemolyticus with regard to growth characteristics on enteric agar media (enabling isolation and identification) and production of exoenzymes which could correlate with invasive potential. V. vulnificus grew well on MacConkey. Endo, xylose-lysine deoxycholate, and Hektoen enteric agar plates. Because V. vulnificus colonies resembled those of lactose-fermenting strains of the family Enterobacteriaceae, however, isolation of this vibrio from mixed specimens or stools may require the use of thiosulfate-citrate-bile salts-sucrose agar. V. vulnificus produced numerous exoenzymes (protease, DNase, lipase, and esterase) but not elastase or lecithinase. Although differences in exoenzyme production were observed among the four vibrio species, no single exoenzyme could be linked to the invasive potential of V. vulnificus.     

Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85

Lymphoproliferation of healthy donors was tested against mycobacterial antigens (PPD, Ag85, Ag85 peptides). All PPD responders recognized the secretory antigen Ag85 and the peptide specificity for Ag85B was defined. Peptide 91-108 was recognized by 85% of donors. In addition, all CD4 T cell lines generated from 12 donors against PPD or Ag85 responded to 91-108. When this peptide was used to generate T cell lines, the cells responded also to tuberculins from atypical mycobacterial species. Thus the cross-reactive peptide behaved as quasi-universal. The analysis of TCR-BV gene usage by cell lines showed that most Ag85-specific T cells correspond to 91-108-specific clonotypes. Intracytoplasmic staining of cell lines after phorbol myristate acetate stimulation resulted in dominance of interferon-gamma (IFN-gamma)-IL-4 double-positive cells, whereas antigen stimulation resulted in production of IFN-gamma only. The data show that peptide 91-108 is the major focus of the CD4 response to mycobacterial antigens in peripheral blood mononuclear cells and in T cell lines from PPD responders.     

Multiparametric assessment of the cell-cycle effects of tamoxifen on mcf-7 human breast-cancer cells

Tamoxifen (TAM)-induced changes in proliferation kinetics of the human breast cancer cell line MCF-7 were investigated using dual parameter flow cytometry (FCM) of bromodeoxyuridine (BrdU) immunolabelling and of the expression of cell-cycle related proteins (the proliferating cell nuclear antigen, PCNA, and the non proliferation-specific protein, Statin), versus the DNA content. Single-parameter FCM DNA histograms confirmed that after 96 hours of treatment with 10(-7) M TAM the fraction of S-phase cells decreased significantly, with a simultaneous accumulation of cells in the G(0)/G(1) range of DNA content. In dual-parameter FCM cytograms, the fraction of BrdU-positive cells after TAM exposure was significantly lower than in the controls, and no unlabeled S-phase cells were found. The TAM-induced block in G(0)/G(1) phase was paralleled by a decrease in the number of cells with a DNA content typical of the S-phase expressing PCNA, and by an increase in Statin-positive (G(0)) cells. Upon readdition of 10(-9) M 17 beta-estradiol (E2) to the TAM-treated cultures, BrdU-labelling as well as PCNA expression levels increased significantly, whereas the fraction of Statin-positive cells remained higher than in the controls. The results obtained confirm that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide experimental evidence that two main mechanisms are operating: the accumulation of cells in G(1), before the onset of S-phase, and the exit of some cells from the cycling compartment; however, some of those that are blocked at G(0); cannot be totally reversed by estrogens and may be permanent; These data should be taken into account in the attempt to combine the antiestrogen treatment with chemotherapy more effectively.     

Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector

Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)     

High throughput functional microdissection of pathogen-specific T-cell immunity using antigen and lymphocyte arrays

The analysis of the human T-cell response specific for relevant pathogens is useful for diagnostic purposes and for research. Several methods enumerate antigen specific T-cells and measure their functions. Since screening of numerous antigens from pathogens is often needed to evaluate immunocompetence, lymphocytes, labor and cost are limiting factors. To examine pathogen-specific T-cell immunity, we have miniaturized the analysis of T-cell responses using an array approach in 384- and 1536-well plates with as few as 10 x 10(3) PBMC per well instead of the 500 x 10(3) PBMC used for current assays. Secreted cytokines were detected in the same wells used for lymphocyte cultures. The method can detect about ten CMV specific T-cells diluted into 50 x 10(3) PBMC (0.02%), and can quantify secreted cytokines. The microarray approach allowed evaluation of T-cell immunity in children with a sensitivity higher than current methods. When applied to CMV epitope mapping, the data obtained with conventional methods were confirmed. The assay could be automated, allowing high throughput processing. The assay provides quantitative information on cytokines induced by antigen stimulation and can be applied in a simplified format as a field test to monitor T-cell immunity in vaccine trials or in veterinary medicine.     

Helper function of cytolytic lymphocytes: switching roles in the immune response

T helper (Th) cells and cytolytic T lymphocytes (CTL) play defined roles in the cellular immune response. This distinction wavered when Th lymphocytes were shown to kill antigen-presenting cells displaying the relevant antigen. Here we demonstrate that also the opposite can be true: CTL can exert helper functions. We noticed that certain CMV-specific CTL lines grew after antigen activation also without exogenous IL-2. These lines produced their own IL-2, which supported the expansion of other CTL and Th cell lines. High levels of helper cytokines like IL-4, IL-5 and IL-6 were detected in the culture supernatants. Thus, we set up a helper assay to study the functional interactions between T cells (or their supernatants) and B cells. Conditioned media from helper CTL lines induced secretion of antigen-specific antibodies by B cells pulsed with antigen as first signal. We conclude that it is possible to isolate CTL lines that exhibit helper functions for T cells and B cells. If this possibility is proven also in vivo, we should revise some of our views on the pathogenesis of diseases in which CD8 cells are key players, such as in viral infections, graft rejection and GVHD.     

Myelosuppressive conditioning improves autologous engraftment of genetically marked hematopoietic repopulating cells in dogs

We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells.