ImmunoMax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens.

BACKGROUND: Since the introduction of mAb, immunohistochemistry has become an important tool in research and in surgical pathology. The most widely used fixative in routine histopathology is formaldehyde, and it has become the gold standard for morphologic tissue preservation. Although the molecular mechanism underlying the tissue fixation is not well understood, it has become clear that available immunoreactive Ag are progressively lost during the fixation process. For a long time, it was thought that formalin-sensitive Ag might be irreversibly destroyed during the fixation process. Although monoclonal anti-Ig Ab frequently worked inadequately, polyclonal anti-Ig Ab were shown to produce reproducible staining results. It thus appeared possible that most cellular Ag might not be irreversibly destroyed but only masked. EXPERIMENTAL DESIGN: Although some Ag may be retrieved under appropriate conditions, there might still be many for which available antigenic epitopes are still too sparse to be visualized, as observed for a large number of leukocyte differentiation Ag. One reliable approach to resolve this dilemma is the use of a combination of an optimized Ag retrieval system and a powerful immunohistochemical staining protocol introducing a biotin amplification step, in which signal amplification is accomplished by covalent deposition of biotin molecules. RESULTS: Cryostat and paraffin sections were stained with the avidin-biotin complex technique and, for comparison, with the new maximized immunohistochemical staining protocol, termed the ImmunoMax method. Each step was monitored to establish how effectively it enhanced the overall sensitivity. Although pretreatment with detergent, protease, a chaotropic substance, or microwave heating resulted in only moderately improved immunostaining, the biotinylated tyramine enhancement step proved to be the most efficient one, although the latter is not sufficient for many Ag when used without pretreatment steps. The combination of an Ag retrieval step with the biotinylated tyramine enhancement step resulted in a 100 to 10,000-fold boost in sensitivity without loss of specificity. CONCLUSIONS: With the ImmunoMax method, defined Ag can be reproducibly detected in formalin-fixed, paraffin-embedded tissues, and the sensitivity of the method is tremendously enhanced. Moreover, it also allows many previously unreactive or unsatisfactorily reactive Ag to be detected, as shown here for IgD, IgM, and CD7 with the use of mAb.

Large-cell anaplastic lymphoma-specific translocation (t[2;5] [p23;q35]) in Hodgkin's disease: indication of a common pathogenesis?

Chromosomal aberrations are characteristic and specific events; the detection of chromosomal abnormalities often provides information on diagnosis and prognosis of disease. Some patients with large-cell anaplastic lymphoma (Ki 1 lymphoma) have the translocation t(2;5) (p23; q35), involving a possible growth-regulating tyrosine kinase. We found this translocation in 11 patients with Hodgkin's disease of nodular sclerosis and mixed-cellularity types. This finding has implications for the understanding of the relation between large-cell anaplastic lymphoma and Hodgkin's disease, diseases with morphological and immunophenotypical similarities. Study of this translocation may help understanding of the origins of cancer and cancer growth. It also allows a more precise definition of Hodgkin's disease and may be used as an indicator for clonality--which has long been sought.

An immortalized cell line with features of human follicular dendritic cells. Antigen and cytokine expression analysis.

Follicular dendritic cells (FDC) are specialized cells residing primarily within lymphoid follicles. They bind immunocomplexes and play an important role in the presentation of antigen to follicular B cells. Isolation of FDC for in vitro studies, however, is difficult because they constitute only about 1% of the cells in lymphoid tissue and form tight clusters entrapping lymphocytes within their dendritic processes. The monoclonal antibody (mAb) Ki-M4, which is highly restricted in its binding to FDC, is used to identify these cells. In order to establish a new immortalized cell line with features of FDC, we applied a modified procedure to isolate and enrich FDC from human tonsils and fused them with the myeloma cell line SP2/0-Ag14. The new hybrid cell line, designated FDC-H1, is of both mouse lymphoid and human FDC origin. FDC-H1 was found to have unlimited growth potential and to consistently express the Ki-M4 antigen and other surface antigens of human FDC. Semiquantitative reverse-transcribed polymerase chain reaction (RT-PCR) of enriched FDC and FDC-H1 revealed the same highly restricted cytokine/mRNA profile for both, with detectable levels of interleukin (IL)-1 alpha and surface CD23 and a lack of mRNA for IL-1 beta, IL-2, IL-3, IL-4, IL-7, IL-9, IL-10, interferon-gamma, tumor necrosis factor-alpha, transforming growth factor-beta and granulocyte/macrophage-colony-stimulating factor. Additionally a weak but constant IL-6 mRNA expression was found in the cell line FDC-H1 by RT-PCR. In situ hybridization experiments in tonsils revealed IL-6 transcripts in cells with a staining pattern characteristic of a dendritic cell only in a few germinal centers. To our knowledge, FDC-H1 is the first cell line that constantly expresses surface antigens and a cytokine profile characteristic of FDC. It is, therefore, well suited for studying the biology of FDC and the functional relationship between FDC and normal or neoplastic lymphatic cells.

Interleukin 7 as interleukin 9 drives phytohemagglutinin-activated T cells through several cell cycles; no synergism between interleukin 7, interleukin 9 and interleukin 4.

The effects of the interleukins IL-7 and IL-9 on cell cycle progression were investigated by conventional [3H]thymidine incorporation and by the bivariate BrdU/Hoechst technique. Both IL-7 and IL-9 drive phytohemagglutinin-activated T cells through more than one cell cycle, but IL-7 was more potent on cell cycle progression than IL-9. Neither synergistic nor inhibitory effects were seen between various combinations of the lymphokines IL-7, IL-9 and IL-4 compared to each lymphokine alone. When T cells are activated with phytohemagglutinin for 3 days, all or most IL-4 responsive cells respond to IL-7 as well, whereas only a part of IL-7 responders are IL-4 responders. In contrast, when T cells are activated with phytohemagglutinin for 7 days, the quantitative data of the cell cycle distribution suggest that the population of IL-7 responders is at least an overlapping, if not a real subset of the population of the IL-4 responders.

Constant detection of surface and cytoplasmic immunoglobulin heavy and light chain expression in formalin-fixed and paraffin-embedded material.

The introduction of microwave detection systems has significantly enhanced the sensitivity of immunohistochemistry in formalin-fixed, paraffin-embedded tissue. The combination of protease digestion and microwave treatment including the use of heavy metal salts or urea gives an Ig heavy and light chain detection system in formalin-fixed and paraffin-embedded material as good as that achieved using fresh frozen material, with the advantage of a well-preserved morphology. The sensitivity of this method depends on the heavy metal salt used and its concentration. The use of urea yields equally sensitive results, while avoiding the toxicity of heavy metal salts. Heating times in the microwave oven also have an important influence on the results. The microwave-based technique may allow for the improved accessibility of formalin-masked antigens, as was shown here for cytoplasmic and surface immunoglobulins. It was demonstrated that formalin-fixed specimens of Burkitt's lymphoma when subjected to the new protocol almost always showed surface Ig expression, giving results comparable to those obtained in frozen sections. Moreover, it could be shown that in cases of B-chronic lymphocytic lymphoma (B-CLL) or immunocytoma, surface and cytoplasmic Ig could be detected, so that the difference may be just a quantitative one and thus may no longer be safely used as a criterion in differential diagnosis. In future, the wide use of this technique will allow for the study of Ig expression at the single cell level in morphologically well-preserved material.

Cytokine expression in T-cell lymphomas and Hodgkin's disease. Its possible implication in autocrine or paracrine production as a potential basis for neoplastic growth.

The detection of an increasing number of cytokines and the demonstration of autocrine and paracrine mechanisms perpetuating tumor growth prompted the investigation of the expression of the cytokines IL-2, IL-3, IL-4, IL-5, IL-6, IFN gamma, Tac, and GMCSF in primary lymph-node biopsies of patients with peripheral T-cell lymphoma (n = 11), Hodgkin's disease (n = 13), and large-cell anaplastic lymphoma (n = 6) by means of Northern blot analysis and in situ hybridization (ISH); 15 of 28 cases had IL-6 message, predominantly in cases of Hodgkin's disease (HD) and large-cell anaplastic lymphomas (LCAL). Interferon gamma was found in about 50% of the cases among all entities. Other cytokine expression was rare except two cases of HD with high amounts of IL-4 mRNA. These results indicate that large amounts of growth factor transcripts are present in a variety of malignant lymphomas. The meaning of this expression is still unclear. It may be a loss of physiologic regulation within the cytokine network which may thus influence neoplastic cell growth as some cases have a quantity of cytokine expression which is similar or even above that of stimulated T cells. ISH demonstrates in individual cases that the expression is at least in part due to malignant cells.

Interleukin-9 expression in human malignant lymphomas: unique association with Hodgkin's disease and large cell anaplastic lymphoma.

To test the possibility that interleukin-9 (IL-9), the human homologue of the mouse T-cell growth factor P40, may be involved in the pathogenesis of human lymphomas, we examined IL-9 expression in a variety of tumors both by Northern blot analysis and by in situ hybridization. Of 18 B-cell non-Hodgkin's lymphomas and 11 peripheral T-cell lymphomas, none expressed IL-9 message. By contrast, IL-9 message was found in two of six cases of large cell anaplastic lymphoma (LCAL) and in 6 of 13 cases of Hodgkin's disease (HD). In HD the strongest signals were observed in Hodgkin (H) and Sternberg-Reed (SR) cells, but IL-9 mRNA was also detected in small lymphocytic cells. A search for IL-9 message in a panel of 20 cell lines derived both from hematopoietic and nonhematopoietic tumors confirmed the unique association of IL-9 expression with HD and LCAL in as much as the only two cell lines with IL-9 message were derived from cases of HD and LCAL. These results suggest that IL-9 is not involved as an autocrine growth factor in the pathogenesis of most B- and T-cell lymphomas, but that it may play a role in HD and LCAL.