E. Filley

A simple new method for using antigens separated by polyacrylamide gel electrophoresis to stimulate lymphocytes in vitro after converting bands cut from Western blots into antigen-bearing particles

The individual antigenic components present in microgram quantities of complex mixtures can be separated reproducibly by polyacrylamide gel electrophoresis and transferred onto nitrocellulose blots. We report that the ng quantities of antigen present in single lines cut from such Western blots can be used to induce maximal lymphoproliferative responses in 30-60 microtitre wells. In order to achieve this the excised lines of antigen-bearing nitrocellulose sheet must be converted into antigen-bearing particles small enough to be engulfed by macrophages. We describe optimal conditions and discuss the applications of this technique.

Hiv infection alters the production of both type 1 and 2 cytokines but does not induce a polarized type 1 or 2 state

Objective:To test the T-helper (TH)1/TH2 cytokine paradigm in HIV infection. Design and methods:Cytokine profiles in two separate studies of HIV patients and controls are presented: (i) a longitudinal study of HIV patients with CD4 counts > 500 × 106/l tested at three timepoints compared with controls; (ii) a blinded cross-sectional study of controls and patients with high (> 500 × 10/6l) and low (< 500 × 106/l) CD4 counts. Peripheral blood mononuclear cells (PBMC) from patients and controls were tested for the production of two type 1 [interleukin (IL)-2, interferon (IFN)-γ] and two type 2 (IL-4, IL-10) cytokines by enzyme-linked immunosorbent assay. Both spontaneous and mitogen-induced cytokine production was measured. Results:HIV infection was noted to have the following effects on cytokine production: (i) it led to the in vivo activation of type 2 cytokines in a small group of individuals with high CD4 numbers characterized by the spontaneous release of IL-4 and IL-10. Longitudinal data showed high spontaneous IL-4 and IL-10 to be a consistent feature of the patient group (at each timepoint some patients were high producers) but to be variable in a given individual; (ii) HIV infection impaired the ability of PBMC to respond to stimuli (selected for their ability to optimally induce each cytokine) in terms of IL-2, IL-4 and IL-10 production in patients with both high and low CD4 cell counts; and (iii) conversely, HIV infection led to an overproduction of IFN-γ in patients with high CD4 counts; patients with low CD4 produced normal levels of IFN-γ. Conclusions:Our observations did not suggest polarization of the type 1/type 2 cytokine profile in HIV patients. Instead, the data suggested more complex changes to type 1/type 2 cytokine patterns in HIV infection than originally proposed by the TH1/TH2 dichtomy.

Mycobacteria as immunogens: Development of expression vectors for use in multiple mycobacterial species

The development of gene transfer systems that are applicable to a wide variety of mycobacterial hosts will provide an important tool for the generation of recombinant mycobacterial candidate vaccines.

New Insights into the Immunopathology of Tuberculosis

Tuberculosis is characterised by fever, weight loss and necrosis in both lesions and tuberculin skin test sites (Koch phenomenon), although the antigens of Mycobacterium tuberculosis are not directly toxic to the tissues. The tissue damage appears to be due to several interacting factors. First, M. tuberculosis induces an immunoregulatory disorder of which a raised percentage of agalactosyl IgG is a marker. This is seen also in rheumatoid arthritis and Crohns disease and is associated with tissue-damaging inflammation. Subsequently, several properties of M. tuberculosis exacerbate this disorder by triggering cytokine release, and rendering tissues sensitive to the toxicity of tumor necrosis factor (TNF). Moreover, M. tuberculosis, but not bacillus Calmette-Guérin or several Mycobacterium avium strains, produces a factor which increases the toxicity of TNF for individual cells. Thus, M. tuberculosis may distort the normal protective role of TNF so that this cytokine becomes toxic to the host. The immunoregulatory disorder associated with agalactosyl IgG appears to be susceptible to immunotherapy, so novel types of treatment for the immunopathological component of tuberculosis are being explored.