Antigen based therapies to prevent diabetes in NOD mice.

Interventional approaches that have been successful in delaying insulin-dependent diabetes mellitus (IDDM) using antigen-based immunotherapies include parenteral immunization. It has potential for clinical application provided that effective adjuvants suitable for human use can be found. We have previously shown that immunization with insulin and insulin B chain but not A chain in incomplete Freund's adjuvant (IFA) prevented diabetes by reducing IFN-gamma mRNA in the insulitis lesions. In this paper we show that the insulin B chain peptide (p9-23) contain the most protective epitope. Immunization with selected GAD peptides was ineffective. Immunization with B chain but not A chain using alum as adjuvant delayed diabetes onset (P = 0.012), whereas administration of alum alone was not protective. When Diphtheria-Tetanus toxoid-Acellular Pertussis (DTP) vaccine was used as the adjuvant vehicle, DTP itself induced significant protection (P < 0.003) which was associated with a Th2-like cytokine producing insulitis profile, IL-4 driven IgG1 antibody responses to insulin, GAD in the periphery and an augmentation of the autoimmune response to GAD. The anti-diabetic effect of DTP was enhanced when given with insulin B chain. These results encourage consideration of an approach using alum/DTP and insulin B chain immunization in clinical trials.

A method for detecting variability arising from errors in sample processing of paraffin-embedded tissue for DNA content analysis.

We present a method for controlling variability that may arise from inconsistencies in sample preparation for DNA content analysis of paraffin-embedded tissue. Human tonsil tissue obtained from routine surgical specimens was embedded in paraffin according to standard protocols. Fifty-micrometer sections were cut from the block and analyzed each day for 20 days to establish control ranges. One tonsil tissue section was processed in parallel with each run of clinical specimens. In this context, a run was defined as the simultaneous processing of 50-microns tissue sections for extraction of cell nuclei (dewaxing and rehydrating). If the tonsil G0/G1 peak coefficient of variation (CV) exceeded 2 SDs of the established mean, and optimum instrument performance and staining were verified, all samples prepared with the tonsil control were reprocessed. Instrument performance and staining were assessed by using the appropriate external controls. By using this rejection rule (12s), the frequency of sample reprocessing in our laboratory was approximately 6%. When the run was repeated and the tonsil control CV was within acceptable range, the G0/G1 peak CV of the corresponding clinical specimens improved 25% of the time. Because most investigators are willing to accept higher CVs for paraffin-embedded tissue than for fresh tissue, it is desirable to have a control to detect decreased peak resolution, resulting from errors in sample processing.

Quantitative morphologic assessment of nuclei extracted from paraffin for DNA flow cytometry.

We present a method for quality control of flow cytometric DNA content studies using nuclei extracted from paraffin-embedded tissue. This method is based on a quantitative morphologic assessment of extracted nuclei. Cell nuclei prepared from 22 paraffin-embedded tumors known to contain discrete diploid and aneuploid stemlines were deposited onto poly-L-lysine-coated glass slides. Nuclei were stained with Diff-quic and examined by light microscopy. Two hundred nuclei were counted and classified based on morphologic appearance into tumor and nontumor groups. Classification criteria included differences in nuclear size, nuclear chromatin structure, the degree of nuclear chromatin condensation, and the presence of nucleoli. Excellent agreement was found between two independent observers (R = 0.989) on the classification of nuclei. The relative number of tumor nuclei on the morphologic preparation was compared with the relative number of aneuploid cells in the DNA histogram. Good agreement was observed (R = 0.975) in all but three cases in which the relative number of tumor nuclei was underrepresented by the percentage of aneuploid nuclei in the DNA histogram. In each case, further analysis by image cytometry demonstrated a diploid and aneuploid component of the tumor cell population. This quantitative method of morphologic examination of the preparation ultimately analyzed by flow cytometry offers several distinct advantages, including: (a) identification of peaks in the DNA histogram, (b) assessment for selective loss of cell nuclei in the extraction process, and (c) identification of biologic heterogeneity in tumor populations.

Tissue section image analysis of breast neoplasms. Evidence of false aneuploidy.

Two methods have emerged for measuring the DNA content of paraffin-embedded tissue using image cytometry: (1) analysis of thin sections, and (2) analysis of nuclei extracted from thick sections. These methods were evaluated using 31 breast tumors for which paraffin-embedded material was available. Cases selected represented 11 diploid, 11 tetraploid, and 9 aneuploid tumors. Results generated using image cytometry methods were compared with those obtained using flow cytometry. For thin sections, the tissue correction feature of the CAS 200 Image Cytometer was used to estimate the DNA content of whole nuclei from measurements made on sectioned nuclei. DNA histograms were generated from tissue sections cut at the same microtome setting (5 microns) before and after software corrections of 4.5 microns, 5.0 microns, 5.5 microns, 6.0 microns. 6.5 microns, 7.0 microns, and 7.5 microns. A comparison of flow cytometry and thin-section image analysis in the absence of tissue correction showed 90% concordance for diploid, 27% concordance for tetraploid, and 77% concordance for aneuploid tumors. The ploidy estimated on thin sections by at least one of the correction values was discordant in 72% of diploid, 91% of tetraploid, and 78% of aneuploid tumors. For cell nuclei extracted from paraffin, excellent agreement was found between flow and image cytometry (r = 0.933). It was concluded that in most cases, cell nuclei extracted from paraffin are preferable to tissue sections for ploidy analysis of breast tumors using image cytometry.