In situ detection of activated cytotoxic cells in follicular lymphomas.

The presence of cytotoxic cells and their activation status were analyzed in tissue sections of 26 follicular lymphomas. To this end, expression of the perforin and granzyme B genes was studied by in situ hybridization experiments, and expression of the TIA-1 antigen was analyzed by immunohistochemistry. Cells expressing the granzyme B gene and the perforin gene were detected in all cases. Their density was, however, highly heterogeneous from case to case, ranging from 160 to 7,040 positive cells/cm2 of tissue sections. TIA-1-positive cells were also evidenced in the 10 follicular lymphomas tested. Virtually all cytotoxic cells were located in interfollicular areas. Double labeling immunochemical experiments showed that most cytotoxic cells belonged to the CD8+ T lymphocyte population, although few CD4+ T lymphocytes and CD56+ natural killer cells also expressed the TIA-1 antigen. These findings show that development of a malignant B lymphocyte proliferation is associated with a host-derived immune response involving intratumoral cytotoxic T lymphocytes. Further studies comparing the density of such cells with the final outcome are required to determine whether the intensity of this immune response has a prognostic value.     

A method of immunofluorescent detection of antibody-producing cells and antigens in tissues

The authors have developed a simple technique for complete and selective suppression of the granulocytes nonspecific fluorescence. The method consists of an additional treatment of the histological sections in a medium for myeloperoxidase detection. Resulting staining of the granulocyte cytoplasm completely inhibits its fluorescence.     

In situ detection of rubella RNA and antigens in cultured cells

We developed in situ hybridization and immunofluorescent procedures to detect rubella RNA and antigens in tissue-cultured cells infected with rubella virus. cDNA fragments of the rubella virus E1 structural gene were used as probes for in situ hybridization to detect rubella RNA sequences in Vero cells infected with rubella virus. Using antibodies against rubella proteins, indirect immunofluorescence detected rubella virus structural proteins in Vero cells infected with rubella virus. The immunofluorescence method has also been applied to study the expression of rubella polypeptide E1 in transfected COS cells and may be applied to the detection and study of persistent rubella virus infection in human tissues.     

In situ immunocytochemical detection of cells containing antibodies specific for Eimeria tenella antigens.

A three-step immunocytochemical method for the in situ detection of antibodies specific for Eimeria tenella has been developed. The method is based on the binding of E. tenella antigens to antibodies in cryostat sections of chicken tissues and the recognition of these antigens by rabbit antiserum specific for E. tenella or mouse monoclonal antibodies specific for E. tenella. The rabbit antiserum and mouse monoclonal antibodies were revealed by the immunoperoxidase technique. Suspensions of sonicated sporulated oocysts, incubated with or without various concentrations of the non-ionic detergents Triton X-100 (TX-100) or Nonidet P-40 (NP-40), were used as antigen. Cells containing antibodies specific for E. tenella were detected only when detergent extracts of sonicated sporulated oocysts were used. After chickens were intravenously immunized with a suspension of sonicated sporulated oocyst antigen, cells containing antibodies specific for E. tenella antigens were detected in the red pulp of the spleen. After simultaneous immunoenzyme staining for isotype and antigen specificity, the E. tenella-specific antibody-containing cells were of the IgM isotype after the primary immunization and of the IgM and IgG isotype after the booster immunization. Immune complexes specific for E. tenella on the surfaces of follicular dendritic cells in the germinal centers were also stained. Chickens were also orally infected with sporulated oocysts. In these experiments, cells containing antibodies specific for E. tenella were detected in the lamina propria of the ceca and in the red pulp of the spleen. Specific immune complexes were also detected in the germinal centers of the cecal tonsils. When detergent extracts of sonicated sporulated oocysts were characterized by immunoblotting, rabbit antiserum specific for E. tenella reacted with proteins ranging in size from 16 kDa to 200 kDa, with major bands of 20 kDa, 24 kDa, 45 kDa, and 100 kDa. Monoclonal antibodies specific for E. tenella recognized only proteins of low molecular weight (20 kDa and 24 kDa) or high molecular weight (80-100 kDa). Immune chicken serum reacted with proteins of low and high molecular weight but especially with proteins of 100 kDa and 113 kDa. This method is the first by which immune complexes and cells containing antibodies specific for parasitic antigens can be detected in situ and may be of value for studies of the local humoral immune response to E. tenella in the mucosa of chickens.     

In situ detection of interleukin-1 mRNA in human monocytes

The interleukin-1 (IL-1) family of soluble pleiotropic immunoregulatory and proinflammatory peptides has at least two distinct members, alpha IL-1 and beta IL-1. Since beta IL-1 is the predominant species in human monocytes, this study was undertaken to identify its mRNA in monocytes using in situ hybridization with a 35S-dCTP labelled beta IL-1 cDNA probe. Grain count analysis demonstrated that adherent lipopolysaccharide-stimulated monocytes were positive, while unstimulated monocytes, lymphocytes and neutrophils, and cells probed with vector only (35S-labelled pBR322) were all negative. We have also shown that in situ hybridization is approx. 13-fold more sensitive than conventional hybridization and in addition this technique allows visualization of mRNA coding for IL-1 in individual cells with morphology preserved. We conclude that in situ hybridization is a specific and sensitive technique for the detection of beta IL-1 mRNA in individual human peripheral blood monocytes.     

In situ hybridization AT-tailing with catalyzed signal amplification for sensitive and specific in situ detection of human immunodeficiency virus-1 mRNA in formalin-fixed and paraffin-embedded tissues

In situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3' side. This (AT)10 of the probe is elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes.     

In situ hybridization demonstrates the stability of mRNA in post-mortem rat tissues.

In situ hybridization was used to detect messenger RNA (mRNA) in a variety of rat tissues which were fixed in formalin either immediately after death or after a 24 h period of storage at 5 degrees C. A synthetic polydeoxythymidine [poly d(T)] oligonucleotide probe was used to demonstrate polyadenylated [poly (A)] mRNA in the small intestine, pancreas, liver, cerebellum, and pituitary. Of these tissues, only the liver showed a small reproducible reduction in hybridization signal following delayed fixation. Synthetic oligonucleotide probes complementary to albumin and pro-opiomelanocortin (POMC) mRNAs were hybridized to liver and pituitary, respectively. There was no significant reduction in hybridization signal in post-mortem tissues. The results suggest that some mRNAs may be remarkably stable under certain post-mortem conditions and this should encourage the wider application of in situ hybridization techniques to post-mortem material.     

In situ visualization of antigen-specific T cells in cryopreserved human tissues

Tetrameric MHC/peptide complexes are important tools for analyzing antigen-specific T cells. The in situ use of tetrameric MHC/peptide complexes in viable tissue sections has several shortcomings: it does not allow the execution of multiple analyses on one single biopsy, the storage of the biopsies, and the co-staining of the tetramer-positive cells for various intracellular molecules. We have developed a novel approach using overnight pre-labeling of viable human tissues with MHC/peptide tetramers, followed by cryopreservation and labeling of the cryosections. The visualization of antigen-specific T cells, combined with detection of other membrane, cytoplasmic, or nuclear markers is now feasible.     

In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression.

We report a novel method for detecting intracellular messenger RNA by combining the techniques of in situ hybridization and polymerase chain reaction (PCR) (in situ cDNA PCR). The technique could detect low abundancy signals and distinguish different levels of gene expression. We examined the expression of the functional markers of activated cytotoxic T lymphocytes, granzyme A, and perforin in human lymphocytes from in vitro cultures. The amplification products were found in the cells and the supernatants, with the distribution critically affected by the protease digestion conditions. The specificity of amplification was confirmed by electrophoretic analysis and Southern blotting. We conclude that the in situ cDNA PCR technique offers a sensitive method of measuring the frequency of signal-expressing cells and has significant research and clinical applications.     

In situ mRNA hybridization technique for analysis of metastasis-related genes in human colon carcinoma cells.

The purpose of this study was to determine whether the expression level of several genes that regulate different steps of the metastatic process correlates with the metastatic potential of human colon carcinoma cells. The mRNA expression level for epidermal growth factor receptor (growth), basic fibroblast growth factor and interleukin-8 (angiogenesis), type IV collagenase (invasion), E-cadherin and carcinoembryonic antigen (adhesion), and the multidrug resistance gene mdr-1 (drug resistance) in the human KM12 colon carcinoma cell lines and clones with different metastatic potential was measured by Northern blot analysis and by in situ hybridization technique. Highly metastatic KM12SM and KM1214 cells growing in culture uniformly expressed high levels of epidermal growth factor receptor, basic fibroblast growth factor, and carcinoembryonic antigen mRNA, whereas cultures of low metastatic KM12C, clone 1, clone 3, and clone 6 cells displayed heterogeneous patterns of expression. KM12C (low metastatic) and KM12SM (highly metastatic) cells were implanted into the subcutis (ectopic) or the wall of the cecum (orthotopic) of nude mice. The mRNA expression level for epidermal growth factor receptor, basic fibroblast growth factor, interleukin-8, type IV collagenase, carcinoembryonic antigen, and mdr-1 was increased in the cecal wall tumors as compared with subcutaneous tumors or in vitro cultures. These data demonstrate a direct correlation between constitutive and inducible expression of several metastasis-related genes and the metastatic potential of human colon carcinoma cells.     

Distinct expression and localization of serine protease HtrA1 in human endometrium and first-trimester placenta.

Mammalian embryos cannot survive without the placenta. Development of the human placenta requires trophoblast proliferation, differentiation, and invasion as well as highly coordinated modulation of the maternal uterus. HtrA1 is a member of the recently identified mammalian HtrA (high temperature requirement factor A) serine protease family with a high level of expression in the placenta. In this study, we examined whether HtrA1 expression (mRNA and protein) is associated with placental development in the human. HtrA1 is up-regulated in both endometrial glands and decidual cells during endometrial preparation for embryo implantation and during first-trimester pregnancy at placentation. HtrA1 expression was also detected in certain trophoblast subtypes during early pregnancy. The villous syncytiotrophoblast and cytotrophoblast showed the strongest expression while the interstitial extravillous trophoblast showed the lowest or no expression of HtrA1. The distinct distribution of HtrA1 at the maternal-trophoblast interface suggests that HtrA1 may play a role in placental development.     

A novel platform for in situ investigation of cells and tissues under mechanical strain

The mechanical micro-environment influences cellular responses such as migration, proliferation, differentiation and apoptosis. Cells are subjected to mechanical stretching in vivo, e.g., epithelial cells during embryogenesis. Current methodologies do not allow high-resolution in situ observation of cells and tissues under applied strain, which may reveal intracellular dynamics and the origin of cell mechanosensitivity. A novel polydimethylsiloxane substrate was developed, capable of applying tensile and compressive strain (up to 45%) to cells and tissues while allowing in situ observation with high-resolution optics. The strain field of the substrate was characterized experimentally using digital image correlation, and the deformation was modeled by the finite element method, using a Mooney–Rivlin hyperelastic constitutive relation. The substrate strain was found to be uniform for >95% of the substrate area. As a demonstration of the system, mechanical strain was applied to single fibroblasts transfected with GFP-actin and whole transgenic Drosophila embryos expressing GFP in all neurons during live imaging. Three observations of biological responses due to applied strain are reported: (1) dynamic rotation of intact actin stress fibers in fibroblasts; (2) lamellipodia activity and actin polymerization in fibroblasts; (3) active axonal contraction in Drosophila embryo motor neurons. The novel platform may serve as an important tool in studying the mechanoresponse of cells and tissues, including whole embryos.     

Rapid detection of xenotransplanted human tissues using in situ hybridization.

A rapid and sensitive method for the identification of human tissues xenotransplanted in nude mice was developed. An in situ hybridization technique made it possible to distinguish between cells of human origin and cells of murine origin in formalin-fixed paraffin sections. High-molecular-weight DNAs extracted from human or mouse tissues were sonicated, nick-translated with 32P-dCTP, and used as hybridization probes. Dot blot hybridization of 32P-labeled probes revealed clear species-specific signals. Formalin-fixed paraffin-embedded tissue samples from repopulated tracheal transplants, containing either human tracheal epithelial cells or human renal tubular cells, were used. Cells of human and murine origin were distinguishable by in situ hybridization with sonicated DNA probes. This method has several advantages; simple preparation of probes, high sensitivity, and applicability to formalin-fixed paraffin-embedded tissue sections. In situ hybridization with sonicated DNA probes should provide a powerful tool for verifying the human origin of xenotransplanted tissues in nude mice.     

Cellular localization of simian immunodeficiency virus in lymphoid tissues. II. In situ hybridization

Lymph nodes and spleens were collected at autopsy and by biopsy from 29 rhesus monkeys infected with simian immunodeficiency virus (SIV). Lymph nodes were classified morphologically into stages of follicular hyperplasia, follicular involution, follicular depletion with normal or expanded paracortices, follicular and paracortical depletion, granulomatous lymphadenitis, or normal. The distribution of SIV RNA was determined by in situ hybridization using a nick translated, 35S labeled, SIVmac DNA probe. Numbers of SIV-infected cells were rare during follicular hyperplasia, numerous during follicular and paracortical expansion, and rare during follicular and paracortical depletion. The splenic morphology reflected that of the lymph nodes; however, the numbers of SIV-positive cells were uniformly lower. SIV RNA was frequently restricted to a single nucleus within multinucleate syncytial cells in two cases of granulomatous lymphadenitis. These results, combined with those of a previous study, provide evidence for antigen trapping in SIV-infected hyperplastic lymph nodes and for widespread viral infection of macrophages and lymphocytes during paracortical expansion.     

In situ analysis of LKB1/STK11 mRNA expression in human normal tissues and tumours

The LKB1/STK11 serine/threonine kinase is mutated in Peutz-Jeghers' syndrome and acts as a tumour suppressor. Using northern blotting and RT-PCR, LKB1 has been reported to be expressed widely in human adult tissues, although in Xenopus the expression of its homologue, XEEK1, is apparently restricted to early embryogenesis. In situ hybridization has been used to detect and localize LKB1 mRNA in a variety of adult and fetal tissues and tumours. The results show that LKB1 expression is widespread, but predominant in epithelia and in the seminiferous tubules of the testis. Expression is higher in fetal than in adult tissues. Expression also appears to be higher in many malignant tumours than in normal tissues or benign lesions, although some cancers have lost LKB1 expression, quite possibly as part of the process of tumourigenesis. These data are consistent with a widespread functional role for LKB1 in tissues of most types, and with a role for LKB1 in the pathogenesis of some sporadic cancers. LKB1 expression may primarily be related to the rate of cell replication.