Digitally synthesized high purity, high-voltage radio frequency drive electronics for mass spectrometry.

Reported herein is development of a quadrupole mass spectrometer controller (MSC) with integrated radio frequency (rf) power supply and mass spectrometer drive electronics. Advances have been made in terms of the physical size and power consumption of the MSC, while simultaneously making improvements in frequency stability, total harmonic distortion, and spectral purity. The rf power supply portion of the MSC is based on a series-resonant LC tank, where the capacitive load is the mass spectrometer itself, and the inductor is a solenoid or toroid, with various core materials. The MSC drive electronics is based on a field programmable gate array (FPGA), with serial peripheral interface for analog-to-digital and digital-to-analog converter support, and RS232/RS422 communications interfaces. The MSC offers spectral quality comparable to, or exceeding, that of conventional rf power supplies used in commercially available mass spectrometers; and as well an inherent flexibility, via the FPGA implementation, for a variety of tasks that includes proportional-integral derivative closed-loop feedback and control of rf, rf amplitude, and mass spectrometer sensitivity. Also provided are dc offsets and resonant dipole excitation for mass selective accumulation in applications involving quadrupole ion traps; rf phase locking and phase shifting for external loading of a quadrupole ion trap; and multichannel scaling of acquired mass spectra. The functionality of the MSC is task specific, and is easily modified by simply loading FPGA registers or reprogramming FPGA firmware.

Immune mechanisms in C57B1 mice genetically resistant to Trypanosoma congolense infection. II. Aspects of the humoral response.

Some aspects of the humoral response in trypanotolerant C57B1 mice and susceptible A/J mice were investigated to determine the possible basis of trypanotolerance. When the hepatic uptake of 75Se-labelled T. congolense by infected mice was measured as an index of antibody production, it was found that only C57B1 mice could remove circulating labelled parasites, this ability persisting for several weeks after infection. Estimation of the immunoglobulin concentrations in both strains of mice showed that C57B1 mice developed a pronounced IgM response during the first parasitaemic wave, while A/J mice did not. Over the same period the IgM concentrations in C57B1 mice initially fell, but recovered at the time of peak parasitaemia. In contrast, A/J mice showed a continual fall in total IgG concentrations in the circulation until death 10 days after infection. Finally, it was shown that during the initial rising parasitaemia, the plaque forming cell responses of both strains of mice to sheep red blood cells were normal indicating that neither strain of mice was immunosuppressed. Also, A/J mice vaccinated with irradiated T. brucei on day 4 and C57B1 mice vaccinated either on day 4 or day 60 of a T. congolense infection were able to mount an effective immune response to the vaccine, as judged by the hepatic uptake of radiolabelled parasites. All of the results indicate that the trypanotolerance of C57B1 mice depends, at least in part, on their more efficient antibody response.

The use of 75-seleno-methionine labelled Trypanosoma brucei to measure parasite replication in vivo.

The suitability of 75-Se-labelled trypanosomes for the measurement of trypanosome replication rates in vivo was investigated. The principle used to estimate the doubling time of the circulating parasites was the decrease in specific activity of 10(-7) parasites and this was determined both for parasites contained in a whole blood sample and for parasites separated from the blood sample by DEAE-cellulose chromatography. When two stocks of T. brucei (TrEU 226, TrEU 667) were compared it was found that the parasitemic profiles of radio-labelled trypanosomes transfered into naive mice were essentially the same as those of unlabelled parasites of each stock. Furthermore it was found that in an acute infection (TrEU 226) there was a rapid and continuous fall in the specific activity implying a constant doubling time. However, in the more chronic relapsing infection (TrEU 667) a biphasic decrease in the specific activity occurred which although initially similar to that of the acute infection changed to a much slower rate of replication at the time of peak parasitemia. The results indicate that radiolabelled trypanosomes can be used to measure parasite replication rates in vivo, and may therefore be a valuable method for investigating the factors governing parasite growth in the circulation.

Immune mechanisms in C57Bl mice genetically resistant to Trypanosoma congolense infection. I. Effects of immune modulation.

The effect of immune modulation on the pattern and course of infection with T. congolense was investigated in a strain of mice (C57Bl) which is known to possess a significant degree of trypanotolerance, and a susceptible strain (CFLP) which rapidly succumbs to infection. Immunosuppression of C57Bl mice by splenectomy, cyclophosphamide treatment or gamma irradiation reduced their survival to near that of susceptible strains of mice. In contrast, attempts to enhance the immune response of susceptible CFLP mice using either a variety of immunostimulants, simultaneous vaccination with irradiated parasites at the time of infection, passive immunization or reducing the number of parasites used for infection, failed to confer a level of protection comparable to that of C57Bl mice. It was concluded that the basis of trypanotolerance, although immunological in nature, is associated with, as yet, undetermined factors.

Immunosuppression in bovine trypanosomiasis: response of cattle infected with Trypanosoma congolense to foot-and-mouth disease vaccination and subsequent live virus challenge.

The primary and secondary antibody responses to foot-and-mouth disease virus vaccine were examined in cattle infected with Trypanosoma congolense and the response of some of these animals to live foot-and-mouth disease virus challenge was assessed. Infected groups of cattle had rather lower antibody responses than uninfected control cattle after primary vaccination but the antibody titres were not significantly depressed until after secondary vaccination. These levels remained depressed for the duration of the experiment, ie, 183 days. Trypanocidal therapy with diminazene aceturate of infected cattle at the time of vaccination did not significantly improve the antibody response to primary vaccination. Their subsequent response to live virus challenge was somewhat equivocal in that the number of animals protected was not significantly different in comparison to the untreated infected and uninfected controls. It was concluded that trypanosome-infected cattle do not produce optimal responses to foot-and-mouth disease vaccination. Nevertheless, the antibody titres are generally above those considered adequate to confer 95 per cent protection against needle challenge.

Immunological clearance of 75Se-labelled Trypanosoma brucei in mice. III. Studies in animals with acute infections.

Using trypanosomes labelled with [75Se]-methionine a series of experiments was conducted to investigate antibody production in mice with acute fulminating T. brucei infections. As measured by the hepatic uptake of radiolabelled parasites, we were unable to demonstrate any evidence of antibody-mediated uptake by the liver in such mice. It was concluded that this was not due to impaired macrophage function but was caused by the inability of antibody production to cope with the massive parasitaemias produced by rapidly-replicating infections so that effective opsonization of the parasites did not occur. In contrast, a train of trypanosome which causes a more chronic infection, although initially having a similar replication, although initially having a similar replication rate, subsequently switched t a slower one and thereby allowed antibody to reach levels which permitted effective opsonization. There was no evidence that the parasite caused any significant suppression of antibody responses in these acute infections since inoculation with trypanosomes of one stock at the same time as vaccination with irradiated organisms of a second stock did not prevent the development of antibody to the latter, as measured by the hepatic uptake of radiolabelled parasites.

Immunological clearance of 75Se-labelled Trypanosoma brucei in mice. II. Mechanisms in immune animals.

Using trypanosomes labelled with [75Se]-methionine a series of experiments was conducted to investigate the respective roles of antibody, macrophage activtion and complement in the removal of trypanosomes from the circulation of immune mice. It was found that clearance in such animals is largely accomplished by antibody-mediated hepatic phagocytosis, which, at least in passively immunized animals, is dependent on opsonization involving C3. No evidence was found to suggest that intravascular lysis or activated macrophages are involved in immune clearance.

Genetic resistance to Trypanosoma congolense infections in mice.

The mechanisms of genetic resistance or "trypanotolerance" to infection with Trypanosoma congolense were investigated in two strains of mice. One strain C57BL, is outstandingly resistant to most stabilates of T. congolense and can survive for over 80 days, whereas CFLP, in common with most other strains, generally succumbs in less than 20 days. Evaluation of several pathophysiological and immunological parameters showed that after infection both strains initially developed similar levels of parasitemia, anemia, biochemical derangement, and immunosuppression. The most outstanding difference was after day 8 postinfection, when the susceptible strain (CFLP) sustained high levels of parasitemia (10(9) trypanosomes per ml) until death 2 to 4 days later, whereas the resistant strain (C57BL) showed a marked decrease to less than 10(6) trypanosomes per ml by day 10 postinfection. Clear evidence that this was associated with the presence of trypanocidal antibody in the resistant mice was provided by the results of an infectivity neutralization test on serum collected from each strain at 10 days postinfection. Chronically infected C57BL mice showed declining waves of parasitemia and a slow restoration of most hematological and biochemical indexes to near normal levels by 80 days postinfection, although at this stage they remained immunosuppressed.

Immunological clearance of 75Se-labelled Trypanosoma brucei in mice. I. Aspects of the radiolabelling technique.

A reliable and simple technique for the in vivo labelling of Trypanosoma brucei with [75Se]-methionine was developed. Between 97 and 99% of the radioactivity was protein bound in the trypanosomes and spontaneous elution in vitro was less than 10% over 4 h. The fate of the labelled trypanosomes after i.v. injection into normal and immune mice was studied. Whilst the vast majority of parasites remained in the circulation of normal animals they rapidly disappeared from the blood of immune animals. In the latter the liver was found to be the principal site of phagocytosis removing over 50% of the radiolabelled parasites within 15 min of injection.