SUMMARYWe have demonstrated that a single intravenous bolus of rat atiti-CD4 MoAb caused a small but prolonged increase in apoptosis in murine lymph nodes. We have quantified this process using the novel Highly Optimized Microscope Environment (HOME) interactive itnage analysis system and shown that the increase in apoptosis was sufficient to account for the observed depiction of the peripheral CD4' T cell subset. This occurred in the absence of any other exogenous signal. Furthermore, there was no evidence of an inflammatory or tiecrotic response in the tissues, indicating that this was unlikely to be He or complement-mediated antibody killing. The anti-CD4-induced depiction selectively removed CD44" T cells. Using mice previously immunized with yeast-derived HIV-1 p24 recombinant protein there was sparing of memory Tcell function after in viro afiti-CD4 treatment, except during a window of less than 24 h duration, when simultaneous exposure to antigen and anti-CD4 antibody resulted in the depiction of specific memory T lymphocyte function. This indicated that a very minor alteration in the frequency of apoptosis had a marked effect on cell number over time, and suggested that opportunistic infection associated with CD4' Tcell depletion may be explained by lossofmcmory cells when there is antigenic stimulation at the same time as CD4 ligation. These results have implications for the pathology of HlV-a.ssociated disease which is associated with ligation of CD4 molecules In vivo.
Summary A major practical advantage of the HOME (highly optimized microscope environment) computerized microscope is the facility for relocating cells or other microscopic objects. Features can be marked directly on the microscope image using a mouse‐driven cursor, and an interactive finder can then be used to relocate the marked features. Tests on a prototype HOME microscope have shown that positions can be relocated with an accuracy of standard deviation (SD) < 7 μm. The marked features could also be relocated on a second HOME microscope, although with somewhat reduced accuracy (standard deviations of < 17 μm). The system provides a very user‐friendly environment for tasks requiring relocation of microscopic objects.
IL-17A+ CD8+ T-cells, termed Tc17 cells, have been identified at sites of inflammation in several immune-mediated inflammatory diseases. However, the biological function of human IL-17A+ CD8+ T-cells is not well-characterised, likely due in part to the relative scarcity of these cells. Here we expanded IL-17A+ CD8+ T-cells from healthy donor PBMC or bulk CD8+ T-cell populations using an in vitro polarisation protocol. We show that T-cell activation in the presence of IL-1β and IL-23 significantly increased the frequencies of IL-17A+ CD8+ T-cells, which was not further enhanced by IL-6, IL-2 or anti-IFNγ mAb addition. In vitro-generated IL-17A+ CD8+ T-cells displayed a distinct type-17 profile compared with IL-17A- CD8+ T-cells, as defined by transcriptional signature (IL17A, IL17F, RORC, RORA, MAF, IL23R, CCR6); high surface expression of CCR6 and CD161; and polyfunctional production of IL-17A, IL-17F, IL-22, IFNγ, TNFα and GM-CSF. A significant proportion of in vitro-induced IL-17A+ CD8+ T-cells expressed TCRVα7.2 and bound MR1 tetramers indicative of MAIT cells, indicating that our protocol expanded both conventional and unconventional IL-17A+ CD8+ T-cells. Using an IL-17A secretion assay, we sorted the in vitro-generated IL-17A+ CD8+ T-cells for functional analysis. Both conventional and unconventional IL-17A+ CD8+ T-cells were able to induce pro-inflammatory IL-6 and IL-8 production by synovial fibroblasts from patients with psoriatic arthritis, which was reduced upon addition of anti-TNFα and anti-IL-17A neutralising antibodies. Collectively, these data demonstrate that human in vitro-generated IL-17A+ CD8+ T-cells are biologically functional and that their pro-inflammatory function can be targeted, at least in vitro, using existing immunotherapy.
Aims-To describe a systematic investigation of interobserver differences in interpretation of nuclear morphology in preparations of small cell lung cancer (SCLC). Methods-The screening/reviewing facility on the highly optimised microscope environment was used to individually tag 127 nuclei, chosen to reflect the spectrum of morphological appearances in nuclear preparations from three biopsy specimens of SCLC. Each nucleus was reviewed and labelled as control (lymphocyte), malignant or unsatisfactory by each of four observers. DNA histograms were plotted for each specimen using the nuclei identified as malignant by each participant. The histograms were compared in terms of identification of DNA stemlines and by calculation of a 5c exceeding rate (5cER).Results-Interobserver variation in assessment ofmorphology was seen in 55-1% of nuclei. Disagreement occurred most frequently in the malignant/unsatisfactory category. Differences in morphological classification had little influence on histogram assessment by means of visual inspection but did show an effect on 5cER. Conclusions-There are significant interobserver differences in subjective assessment of nuclear morphology in cytometric preparations. This effect may seriously influence cytometric meas- The highly optimised microscope environment (HOME) system is a new concept in microscopy and image analysis involving projection of a computer overlay on to microscope slides.3 An observer can "interact" with the visual image using a mouse attachment. The HOME has a screening/reviewing program which permits highly accurate cell marking and relocation. A variety of computer generated markers (symbols) may be attached to a given object and subsequently relocated by another observer. We decided to use this system to test interobserver variation in nuclear categorisation in image cytometry and the effect of any variation on tumour DNA ploidy estimation. MethodsIn our laboratory we have an interest in cytometric measurement in lung cancer. We therefore decided to assess the problem of nuclear identification in SCLC. Nuclear preparations were made from paraffin wax embedded blocks of three cases of SCLC using the protocol of Hedley et al. 4 The nuclei were spun on to slides and stained in a standard Feulgen reaction with acid hydrolysis in 4N HCI at 27-5°C for 55 minutes. The slides were initially screened by a single observer (FAC) on the HOME microscope (AxioHOME, Zeiss, Germany) and a sample of approximately 40 nuclei per case were marked and stored on the computer. The sample was chosen so as to be representative of the spectrum of nuclear appearances present in each specimen and so that no more than a single nucleus was selected from any one high power ( x 400) field. The slides were subsequently reviewed by each of four observers (two experienced pathologists, a trainee pathologist and a clinician with a research interest in cytometry). All had some experience in cytometry, were familiar with the cytological features of SCLC and appreciated the importance of e...
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