Gamma interferon production by cytotoxic T lymphocytes is required for resolution of Chlamydia trachomatis infection

In this study, we used mice in which the gene for gamma interferon (IFN-gamma) has been disrupted (IFN-gamma-/- mice) to study the role of this cytokine in the resolution of Chlamydia trachomatis infection. We show that IFN-gamma-/- mice are impaired in the ability to clear infection with C. trachomatis compared to IFN-gamma+/+ control mice. Activated CD8(+) cytotoxic T lymphocytes (CTL) secrete IFN-gamma in response to intracellular infection, and we have shown previously that a Chlamydia-specific CTL line can reduce C. trachomatis infection when adoptively transferred into infected mice. In the present study, we found that when these IFN-gamma+/+ CTL lines are transferred into Chlamydia-infected IFN-gamma-/- mice, the transferred CTL cannot overcome the immune defect seen in the IFN-gamma-/- mice. We also show that Chlamydia-specific CTL can be cultured from IFN-gamma-deficient mice infected with C. trachomatis; however, the adoptive transfer of IFN-gamma-/- CTL into infected IFN-gamma+/+ mice does not reduce the level of infection. These results suggest that IFN-gamma production by CTL is not sufficient to overcome the defect that IFN-gamma-/- mice have in the resolution of Chlamydia infection, yet IFN-gamma production by CTL is required for the protective effect seen upon adoptive transfer of CTL into IFN-gamma+/+ mice.     

Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells

Electroporation is a widely used method for introducing macromolecules into cells. We developed an electroporation device that requires only 1 microl of sample to load adherent cells in a 10-mm2 surface area while retaining greater than 90% cell survivability. To better understand this device, field-induced permeabilization of adherent rat basophilic leukemia and neocortical neuroblastoma cells was investigated by using fluorescent calcium and voltage indicators. Rectangular field pulses led to the formation of only a few calcium entry sites, preferentially in the hyperpolarized parts of the cell body and processes. Individual entry sites were formed at the same locations when field pulses were repeated. Before calcium entry, a partial breakdown of the membrane potential was observed in both polar regions. Based on our results, a model is proposed for the formation and closure of macromolecule entry sites in adherent cells. First, the rapid formation of a large number of small pores leads to a partial membrane potential breakdown in both polar regions of the cell. Second, over tens of milliseconds, a few entry sites for macromolecules are formed, preferentially in the hyperpolarized part of cell body and processes, at locations defined by the local membrane structure. These entry sites reseal on a time scale of 50 ms to several seconds, with residual small pores remaining open for several minutes.     

Computational neural networks for predictive microbiology: I. Methodology

Artificial neural networks are mathematical tools inspired by what is known about the physical structure and mechanism of the biological cognition and learning. Neural networks have attracted considerable attention due to their efficacy to model wide spectrum of challenging problems. In this paper, we present one of the most popular networks, the backpropagation, and discuss its learning algorithm and analyze several issues necessary for designating optimal networks that can generalize after being trained on examples. As an application in the area of predictive microbiology, modeling of microorganism growth by neural networks will be presented in a second paper of this series.     

Continuous glucose monitoring in the free-moving rat

The aim of this work was to set up an experimental model of glycemic fluctuations for assessing in the conscious freely moving rat, the performance of a continuous glucose-monitoring system, using a pocket-calculator-size electronic control unit and a miniaturized subcutaneous glucose sensor. The well-known triphasic glycemic pattern following streptozotocin injection (initial peak and secondary hypoglycemia preceding the establishment of permanent hyperglycemia) was used as a way to obtain spontaneous changes in blood glucose level over a wide concentration range. This report demonstrates that streptozotocin injection produced highly reproducible changes in the current generated by the sensor: an initial peak and a secondary nadir, during which blood sampling provided the evidence of hyperglycemia associated with immunoreactive hypoinsulinemia, and of hypoglycemia associated with hyperinsulinemia, respectively. This reproducible experimental model should be valuable for the assessment of a continuous glucose-monitoring system.     

A mathematical model of the regulation of the G1 phase of Rb+/+ and Rb-/- mouse embryonic fibroblasts and an osteosarcoma cell line

A mathematical model integrating the roles of cyclin D, cdk4, cyclin E, cdk2, E2F and RB in control of the G1 phase of the cell cycle is described. Experimental results described with murine embryo fibroblasts (MEFs), either Rb+/+ or Rb-/-, and with the RB-deficient osteosarcoma cell line, Saos-2, served as the basis for the formulation of this mathematical model. A model employing the known interactions of these six proteins does not reproduce the experimental observations described in the MEFs. The appropriate modelling of G1 requires the inclusion of a sensing mechanism which adjusts the activity of cyclin E/cdk2 in response to both RB concentration and growth factors. Incorporation of this sensing mechanism into the model allows it to reproduce most of the experimental results observed in Saos-2 cells, Rb-/- MEFS, and Rb+/+ MEFs. The model also makes specific predictions which have not been tested experimentally.     

Posttransplant T-cell lymphoproliferative disorders--an aggressive, late complication of solid-organ transplantation

T-cell non-Hodgkin's lymphomas are an uncommon occurrence after solid-organ transplantation. We describe a morphologically and immunophenotypically distinct group of T-cell lymphoproliferative disorders that occurred late in the course of six patients with solid-organ transplants. The patients ranged in age from 31 to 56 years (median, 43). Three were male; all were splenectomized. The interval from transplant to the diagnosis of lymphoma ranged from 4 to 26 years (median, 15). Symptoms at presentation were related to sites of involvement. Pulmonary, marrow, and CNS involvement were present in five, four, and one case, respectively. No patient had lymphadenopathy. Five patients had an elevated lactate dehydrogenase level (range, 226 to 4,880 IU/L; median, 1,220 IU/L). Five of six patients had a leukoerythroblastic reaction. All cases had large-cell histology and frequently contained cytoplasmic granules. Those cases tested expressed CD2, CD3, and CD8 and were negative for B-cell antigens. T-cell receptor beta- and gamma-chain genes were clonally rearranged in three of three and one of three cases, respectively. All T-cell posttransplant lymphoproliferative disorders (T-PTLDs) studied were negative for Epstein-Barr virus (EBV), human T-cell leukemia/lymphoma virus type 1 (HTLV-1), human T-cell leukemia/lymphoma virus type 2 (HTLV-2), and human herpes virus type 8 (HHV-8) genomes. Treatment with acyclovir (three patients) or chemotherapy (three patients) resulted in two responses. All patients had an aggressive course, with a median survival duration of 5 weeks. In conclusion, a clinically aggressive T-PTLD may be a late complication of solid-organ transplantation and does not appear to be related to EBV, HTLV-1, HTLV-2, or HHV-8 infection.