RS Cells Research Articles

Absence of a prevalent laminar distribution of IPSPs in association cortical neurons of cat.

Absence of a prevalent laminar distribution of IPSPs in association cortical neurons of cat. J. Neurophysiol. 78: 2742-2753, 1997. The depth distribution of inhibitory postsynaptic potentials (IPSPs) was studied in cat suprasylvian (association) cortex in vivo. Single and dual simultaneous intracellular recordings from cortical neurons were performed in the anterior part of suprasylvian gyrus (area 5). Synaptic responses were obtained by stimulating the suprasylvian cortex, 2-3 mm anterior to the recording site, as well as the thalamic lateral posterior (LP) nucleus. Neurons were recorded from layers 2 to 6 and were classified as regular spiking (RS, n = 132), intrinsically bursting (IB, n = 24), and fast spiking (FS, n = 4). Most IB cells were located in deep layers (below 0.7 mm, n = 19), but we also found some IB cells more superficially (between 0.2 and 0.5 mm, n = 5). Deeply lying corticothalamic neurons were identified by their antidromic invasion on thalamic stimulation. Neurons responded with a combination of excitatory postsynaptic potentials (EPSPs) and IPSPs to both cortical and thalamic stimulation. No consistent relation was found between cell type or cell depth and the amplitude or duration of the IPSPs. In response to thalamic stimulation, RS cells had IPSPs of 7.9 +/- 0.9 (SE) mV amplitude and 88.9 +/- 6.4 ms duration. In IB cells, IPSPs elicited by thalamic stimulation had 7.4 +/- 1.3 mV amplitude and 84.7 +/- 14.3 ms duration. The differences between the two (RS and IB) groups were not statistically significant. Compared with thalamically elicited inhibitory responses, cortical stimulation evoked IPSPs with higher amplitude (12.3 +/- 1.7 mV) and longer duration (117 +/- 17.3 ms) at all depths. Both cortically and thalamically evoked IPSPs were predominantly monophasic. Injections of Cl- fully reversed thalamically as well as cortically evoked IPSPs and revealed additional late synaptic components in response to cortical stimulation. These data show that the amount of feed forward and feedback inhibition to cat's cortical association cells is not orderly distributed to distinct layers. Thus local cortical microcircuitry goes beyond the simplified structure determined by cortical layers.

Reappraisal of the Relationship Between Immunoglobulin Heavy Chain Gene Rearrangement and Epstein-Barr Virus Infection in Reed-Sternberg Cells of Hodgkin's Disease

We investigated 44 cases of Hodgkin's disease for Epstein-Barr virus genome with EBER-1 in situ hybridization. Twenty of 44 (45.5%) were positive for EBV. Simultaneously, immunoglobulin gene rearrangements were assessed in 32 of these 44 cases with PCR on DNA extracted from Reed-Sternberg cell (RS-cell) -rich areas microdissected from paraffin sections. Clonally rearranged immunoglobulin (IgH) gene was observed in 15 cases (46.9%). EBV-negative cases showed more frequent IgH rearrangement than EBV-positive cases (10 and 5 cases, respectively). In 9 cases, the RS cells were CD20-positive immunohistochemically and these were all EBV negative and the IgH gene was rearranged in all except one. These findings may suggest that EBV infection has occurred before the immunoglobulin gene rearrangement or that EBV infection has influenced the rearrangement of the immunoglobulin gene. The results may also hint towards the obscure B-cell nature of the RS cells.

Variable expression of Epstein-Barr virus latent membrane protein I in Reed-Sternberg cells of Hodgkin's disease.

Reed-Sternberg cells (RS cells) of Hodgkin's disease (HD) are frequently infected with Epstein-Barr virus (EBV) and express EBV-encoded nonpolyadenylated RNA transcripts (EBER)-1. EBV latency has been classified into three distinct forms: Latency I, expressing only one of the latent proteins, EBV nuclear antigen (EBNA)-1, latency II, coexpressing EBNA-1 and LMPs, and latency III, expressing all latent viral proteins. RS cells express LMP-1 in addition to EBNA-1 and are considered to be EBV latency II frequently encountered in nasopharyngeal carcinoma. We examined 13 cases of EBV-infected HD by combined EBER-1 in situ hybridization and immunostaining for LMP-1. All of the RS cells expressed EBER-1, but a substantial number of EBER-1+ RS, cells were negative for LMP-1. The percentage of LMP-1+ RS cells out of EBER-1+ RS cells varied from 7% to 100% (average 69%). In this study, we showed that all EBV-infected RS cells were not restricted to latency II, and some belonged to latency I.

CLASSICAL HODGKIN AND REED-STERNBERG CELLS DEMONSTRATE A NON-CLONAL IMMATURE B LYMPHOID LINEAGE: EVIDENCE FROM A SINGLE CELL ASSAY ANDIN SITU HYBRIDIZATION

Hodgkin and Reed-Sternberg (H & RS) cells are generally considered to be the neoplastic cells of Hodgkin's disease (HD), however such cells are only found in a minority of the lesions. Recently in a few studies on HD, the clonality of H & RS cells was examined, using a single-cell polymerase chain reaction (PCR) examination. To clarify the lineage and clonality of H & RS cells, we performed single cell PCR and in situ hybridization (ISH), and nine cases of classical HD were thus studied. By ISH, the immunoglobulin J chain, and the kappa and lambda light chain were rarely expressed in the H & RS cells, however, no T-cell markers could be detected. The expression of the recombination activating genes (RAG-1, 2) could be determined in the H & RS cells. We isolated CD30+ H & RS cells, CD3 + T cells and CD20 + B cells from suspended materials using a mechanical sorter. We performed single cell PCR in a sorted individual cell, to amplify the complementarity determining region of the Ig heavy chain (IgH) gene and T-cell receptor gamma chain (TCR gamma) gene. In all cases, TCR gamma could be frequently amplified in the T cells, but was only rarely amplified in the H & RS and B cells. In contrast, the IgH was frequently amplified in the H & RS and B cells, but not in the T cells. In addition, the PCR production of the H & RS cells all showed different lengths. The results therefore support the polyclonal nature and immature B lymphoid cell origin of H & RS cells.

3 Immunology of Hodgkin's disease

Hodgkin's disease is characterized by an immune response in the involved tissues that is predominantly CD4 mediated. The CD4+ T-cells are CD45RO+ and CD45RBdim, they express several activation markers but lack CD26, and in vitro can be stimulated to produce gamma-interferon and IL-4, but not IL-2. This is not the usual immunophenotype and cytokine production pattern of Th1, Th2 or Th0 cells and may be a reflection of anergy. The cause of such an anergic reaction is not clear since RS cells express HLA class II as well as the co-stimulator molecules CD80 and CD86. It is possible that a (hypothetical) super antigen expressed on the RS cells may play a role. The absence of IL-2 production however explains the absence of a CD8 mediated response. In addition to that, RS cells generally do not express HLA class I, which allows them to escape CD8 mediated responses. The link between the ineffective immune response in the tissue and the generalized immune deficiency in Hodgkin's disease may consist of several components. These include the influx of mature T-cells into the affected tissues, the secretion of inhibitory molecules by the neoplastic cells and the spill-over of the anergic T-cell response into the general circulation by either the Hodgkin related antigen or also as a result of an IL-4 dominated response. The latter possibility may also be related to the hyper-gamma-globulinaemia and the frequently observed high IgE levels.

The effect of early exposure of Coho salmon ( Oncorhynchus kisutch) eggs to the p57 protein of Renibacterium salmoninarumon the development of immunity to the pathogen

Bacterial kidney disease (BKD) in salmonids is caused by Renibacterium salmoninarum(Rs) which is transmitted vertically through salmonid eggs. This study investigated the effect of vertical transmission of one protein of Rs, p57, on the development of the immune system of coho salmon ( Oncorhynchus kisutch) and on the susceptibility of the fish to subsequent challenge with Rs. It was shown that exposure of coho salmon at the egg stage to the p57 protein of Rs induces at least partial immunosuppression. Coho salmon eggs were injected with varying amounts of p57. The eggs were fertilized, the progeny were vaccinated against Rs, and then challenged with live, virulent Rs. Fish that had been injected as eggs with 100 ng of p57 demonstrated a significantly higher cumulative percent mortality (50 %) than those from the saline-injected eggs (controls) (14 %). The same fish also produced significantly lower levels of antibodies against p57, although not against whole Rs cells, than those derived from saline-injected eggs. It appeared, therefore, that there was some specificity to the immune suppression. A decreased proportion of phagocytic cells isolated from fish exposed as eggs to 100 ng p57, showed respiratory burst activity (0 ·4 %) compared with the proportion of cells isolated from the saline-injected (control) group (16 ·5 %). These data suggest that early (egg stage) exposure of coho salmon to p57 results in long-term immunosuppression and a decreased ability in the animal to resist subsequent challenges with Rs.

Correlation between presence of clonal rearrangements of immunoglobulin heavy chain genes and B-cell antigen in patients with nodular sclerosis and mixed cellularity Hodgkin's disease

Correlation between presence of clonal rearrangements of immunoglobulin heavy chain genes and B-cell antigen in patients with nodular sclerosis and mixed cellularity Hodgkin's disease

RAG-1 and RAG-2 expression in Hodgkin's and non-Hodgkin's lymphoma

to further characterize genes that are predominantly expressed in RS cells. Three single RS cells from a nodular-sclerosing and mixed cellularity HD were used as a source for the driver cDNA. No Tor B-cell specific transcripts but those for CD30, TNF[3, and IL2 receptor were present. The biotinylated tracers for subtractive hybridization were from single T and B cells of the same HD node, and from peripheral blood leukocytes. So far, 11 subtraction libraries have been screened. Sequence information of the 3' untraslated region was obtained from 59 clones which were considered tracer-specific since they did not hybridize with driver cDNA. Six different clones, previously hybridized to a variety of HD-derived and other hematopoietic cell line cDNAs, were used to recover full-length cDNA clones from the HD cell line KMH2-derived library. The molecular genetical characteristic of these SR cell-derived clones, their specificity and expression profiles in hematopoietic cell lineages will be presented.

Molecular findings in HD — distinctive gene expression in isolated RS cells

Molecular findings in HD — distinctive gene expression in isolated RS cells

Expression and function of CD40 on Hodgkin and Reed-Sternberg cells and the possible relevance for Hodgkin's disease

CD40 was originally described as a B-cell-restricted antigen and was subsequently found to be a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is also expressed on dendritic cells, thymic epithelium, monocytes, and some carcinoma cell lines, and plays a critical role in cell contact-dependent activation. Primary and cultured Hodgkin and Reed-Sternberg (H-RS) cells, the presumed malignant cells of Hodgkin's disease (HD); were found to express high levels of cell surface CD40. We found that recombinant CD40 ligand (CD40L) induced interleukin-8 (IL-8) secretion and enhanced IL-6, TNF, and lymphotoxin-alpha (LT-alpha/TNF-beta) release from cultured H-RS cells. These cytokines play a significant role in the clinical presentation and pathology of HD, a tumor of cytokine-producing cells. CD40L had no mitogenic activity for HD-derived cell lines. In contrast, CD40L enhanced expression of costimulatory molecules intracellular adhesion molecule-T and B7–1 on cultured H-RS cells, both of which are overexpressed on primary H-RS cells. In addition, CD40L induced a 40% to 60% reduction of the expression of the HD-associated CD30 antigen, another member of the TNF receptor superfamily. Primary and cultured H- RS cells express not only CD30, but also CD40. CD40L has pleiotropic biologic activities on H-RS cells, and the CD40-CD40L interaction might be a critical element in the deregulated cytokine network and cell contact-dependent activation cascade typical for HD.

Improvements in the Direct Fluorescent Antibody Technique for the Detection, Identification, and Quantification ofRenibacterium salmoninarumin Salmonid Kidney Smears

Staining trials were conducted over a 5-year period to explore ways of improving the direct fluorescent antibody technique for Renibacterium salmoninarum (Rs). In most salmonid kidney tissue smears that were pretreated with the organic solvent xylene, acetone, or methanol for 2 min, Rs had substantially higher fluorescence intensity and better uniformity of fluorescence than Rs in non-pretreated controls. Only in smears prepared with cultured Rs cells were staining responses of Rs cells similar regardless of pretreatment. Increasing fluorescent antibody staining time greatly increased fluorescence intensity in most trials, but did not affect uniformity of fluorescence; 30 min was considered minimum or adequate staining time and 60 min was found to be optimal. Improved staining facilitated Rs detection and identification, allowing more reliable quantification in standardized smear preparations.

Presence of Epstein‐Barr virus harbouring small and intermediate‐sized cells in Hodgkin'S disease. Is there a relationship with Reed‐Sternberg cells?

Forty-four cases of Hodgkin's disease (HD), mostly of the nodular sclerosing type, were investigated for the presence of Epstein-Barr virus (EBV) by polymerase chain reaction (PCR) and DNA and RNA in situ hybridization (DISH, RISH), as well as by immunohistochemistry for the detection of latent membrane protein-1 (LMP-1) of EBV. In situ hybridization (ISH) was combined with immunohistochemistry to correlate the presence and activity of the virus at the cellular level. In 18/34 (53 per cent) cases, EBV-DNA sequences could be detected with the PCR method. In 12/18 positive cases, DISH and RISH were also positive. In the remaining six EBV-PCR positive cases, two were also positive with RISH and LMP-1, whereas no positive signal with DISH could be obtained. All DISH and/or RISH positive cases were also positive for LMP-1. With RISH, not only the Reed-Sternberg cells and their mononuclear variants (RS cells) stained positive, but also small and intermediate cells frequently reacted with the EBV-specific probes (EBER-1 and -2). Double staining with cellular markers (CD3, CD20, CD45, CD45RO, CD68, and the lectin PNA) revealed that most of the smaller EBER-positive cells frequently did not express T, B, or histiocytic markers, but that they, as well as the RS cells, showed cytoplasmic and membranous staining with PNA. These smaller EBER-positive cells were not found in EBV-PCR negative HD. EBER-positive RS cells were almost always LMP-1 positive, as well as a substantial proportion of the intermediate-sized cells, whereas the majority of the small EBER-positive cells remained LMP-1 negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Single-Cell Analysis of Hodgkin and Reed-Sternberg Cells: Molecular Heterogeneity of Gene Expression and p53 Mutations

We have used a single-cell based polymerase chain reaction (PCR) amplification technique to examine the gene expression pattern in single Hodgkin's and Reed-Sternberg (H&RS) cells from seven patients with Hodgkin's disease. Single cells were isolated from lymph nodes obtained at diagnosis (5 of 7 patients) or in first or second relapse (2 of 7 patients). Gene expression was examined by hybridization to a panel of 22 cDNA probes. Forty-nine H&RS cells (and 23 CD3+ or CD20+ lymphocytes as controls) from four patients with nodular sclerosing Hodgkin's disease (HD) and one patient each with lymphocyte predominant and mixed-cellularity HD were successfully analyzed by PCR. This analysis provides evidence that single H&RS cells can coexpress genes characteristic of several hematopoietic lineages (monocytes and lymphocytes). Genes characteristic of activated lymphoid cells are expressed in most H&RS cells. Heterogeneity of expression for certain genes between different cases was found and may eventually define molecular subgroups of HD. These findings indicate that H&RS cells of HD resemble activated hematopoietic cells. Phenotypically similar cells from different cases exhibit characteristic molecular differences. In one patient, 5 of 7 single RS cells showed identical p53 cDNA mutations at codon 246 on specific reverse transcriptase [RT]-PCR and sequencing of exons 5 through 8. The novel experimental approach may provide a valuable tool for understanding the molecular events in newly diagnosed Hodgkin's disease and progression of the disease.

Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations

We have used a single-cell based polymerase chain reaction (PCR) amplification technique to examine the gene expression pattern in single Hodgkin's and Reed-Sternberg (H&RS) cells from seven patients with Hodgkin's disease. Single cells were isolated from lymph nodes obtained at diagnosis (5 of 7 patients) or in first or second relapse (2 of 7 patients). Gene expression was examined by hybridization to a panel of 22 cDNA probes. Forty-nine H&RS cells (and 23 CD3+ or CD20+ lymphocytes as controls) from four patients with nodular sclerosing Hodgkin's disease (HD) and one patient each with lymphocyte predominant and mixed-cellularity HD were successfully analyzed by PCR. This analysis provides evidence that single H&RS cells can coexpress genes characteristic of several hematopoietic lineages (monocytes and lymphocytes). Genes characteristic of activated lymphoid cells are expressed in most H&RS cells. Heterogeneity of expression for certain genes between different cases was found and may eventually define molecular subgroups of HD. These findings indicate that H&RS cells of HD resemble activated hematopoietic cells. Phenotypically similar cells from different cases exhibit characteristic molecular differences. In one patient, 5 of 7 single RS cells showed identical p53 cDNA mutations at codon 246 on specific reverse transcriptase [RT]-PCR and sequencing of exons 5 through 8. The novel experimental approach may provide a valuable tool for understanding the molecular events in newly diagnosed Hodgkin's disease and progression of the disease.

Increased levels of circulating interleukin‐6 in patients with hodgkin's disease

Expression of interleukin-6 (IL-6) and IL-6 receptors has been demonstrated in Hodgkin and Reed-Sternberg (H and RS) cells in vitro and in vivo. In order to evaluate the clinical significance of IL-6 serum levels in patients with Hodgkin's disease (HD), we tested the sera of 56 untreated patients with HD by means of a sensitive sandwich ELISA. While IL-6 was only rarely detectable in healthy controls or patients with non-Hodgkin's lymphoma, 32 of 56 patients (57 per cent) had detectable IL-6 levels (range 12-32 pg/ml). The rates of detectable IL-6 levels and the median levels were not correlated with age, sex, histological subtype, stage or the presence of B-symptoms, nor with any of a wide spectrum of laboratory parameters tested, including erythrocyte sedimentation rate, total leukocyte and lymphocyte counts, serum levels of soluble CD8, CD25 or CD30. The rates of complete remissions and freedom from treatment failure were not different in IL-6-negative and IL-6-positive patients. Except in one of 23 follow-up sera taken after therapy, IL-6 was no longer detectable even for patients who suffered from progressing disease, suggesting that the neoplastic H and RS cells are not the major source of circulating IL-6.

Relationship between cellular accumulation of rhodamine 123 (R123) and cytotoxicity in B16 melanoma cells

Rhodamine 123 (R123) is a mitochondria-specific prototype anticancer agent because its target is the energy-producing mechanism of the cell. The goal of this study was to investigate the relationship between intracellular R123 accumulation and cytotoxicity in a R123-sensitive cell line (RS) and a R123-resistant subline (RR) that we developed. Cytotoxicity after exposure to R123 (0–60 μg/ml) was assessed using the clonogenic assay. Intracellular R123 was extracted with acid-alcohol and measured by fluorimetry. The rate of R123 accumulation over 1 hr was significantly higher ( P < 0.0001) for RS cells (4.65 ± 0.39 μg/min/10 6 cells) than for RR cells (1.29 ± 0.24 μg/min/10 6 cells). R123 accumulation in RS cells was strongly correlated ( r = 0.80; P < 0.0001) with cytotoxicity. Treatment of RR cells with verapamil (100 μM) reversed R123 resistance. The resulting dose-survival curve was identical to the dose-response curve of RS cells treated with R123 alone. Cellular content of R123 in RR cells treated with verapamil increased to a level similar to that of RS cells and correlated with cytotoxicity. These data suggest that cytotoxicity of R123 in B16 cells results from increased cellular accumulation of R123.

Pathology of Hodgkin's disease: anything new?

This presentation deals with the gross and microscopic pathology of HD. Recent advances in immunohistochemistry, gene rearrangement studies and cell culture are not discussed, except where they shed light on the pathology. Clinical and pathological experience, over the past 2 decades, suggests that HD should be divided into six subtypes, as originally proposed by Lukes et al. (1966b), rather than the four subtypes included in the Rye classification. Nodular lymphocyte/histiocyte predominant HD forms a clinicopathological entity separate from the other subtypes. It most frequently presents at a single nodal site and, even without therapy, progresses only slowly over a period of many years. A proportion of the patients (in the region of 10%) develop large cell NHL and a smaller number develop other types of Hodgkin's disease. This progression is not due to therapy since it most frequently occurs in untreated patients. Characteristic polylobated RS cell variants are seen in NLPHD. These differ from classic RS cells in that they have a B-cell phenotype, they do not show light chain restriction and, therefore, they do not appear to be a clonal proliferation. Although current dogma states that classic RS cells must be identified before a diagnosis of HD, including NLPHD, is made, it is the author's contention, supported by immunohistochemistry, that this type of RS cell does not occur in NLPHD. Polylobated RS cell variants in the appropriate cellular setting are, in themselves, diagnostic of NLPHD. They also serve to differentiate NLPHD from progressive transformation of germinal centres, an unusual proliferative expansion that may occur in association with HD but which, in itself, appears to be an entirely benign, reactive process. Diffuse lymphocyte/histiocyte predominant HD (DLPHD) differs from NLPHD in its diffuse growth pattern and the frequent presence of larger numbers of histiocytes. Polylobated RS cells are characteristic of both diseases. In some biopsies nodular and diffuse areas are seen in the same lymph node. Despite these similarities, the two diseases differ clinically (NLPHD is usually stage 1, DLPHD is frequently of a higher stage) and in their immunohistochemistry (fewer RS cell variants in DLPHD contain J-chain than in NLPHD). The reasons for these differences are not apparent and require further investigation. It may be due, at least in part, to the inclusion of cases of MCHD and NHL in the DLPHD category. Nodular sclerosis is the commonest category of HD in most reported series.(ABSTRACT TRUNCATED AT 400 WORDS)

The lectin binding affinity of Reed-Sternberg cells.

Reed-Sternberg cells from ten cases of Hodgkin's disease were examined by the direct immunofluorescence technique, for their affinity for nine lectins. The surrounding lymphocytes and monocytes of HD tissue were also assessed for their ability to bind lectins. RS cells showed considerable heterogeneity of reaction. Overall, there was a marked decrease in the binding of most of the lectins studied in HD cases as compared to normal peripheral blood mononuclear cells. This was particularly evident for RCA, PHA and PNA binding. It is suggested that there is a defect in carbohydrate metabolism, with fewer lectin-binding sites on both RS cells and on the mononuclear cell populations in Hodgkin's disease. Further quantitative work is required to verify this.

Immunoglobulin in Reed-Sternberg and Hodgkin cells.

In eight cases of Hodgkin's disease of various types, Ig was found within approximately one-third of the RS and HD cells. The Ig within each RS and HD cell consisted of gamma heavy chains and both k and lambda light chains. The most probable origin of the Ig would appear to be uptake of IgG-containing immune complexes. The presence of both types of light chain in individual RS and HD cells is inconsistent with an origin of these types of cell from B lymphocytes.

Restricted Replication of Herpes Simplex Virus in Neural Cells

SummaryThe relationship between the replication of HSV-1 in C6 cells and an interferon-mediated state of viral refractoriness was studied. The cycles of HSV-1 replication in RS and C6 cells reflected the restrictive nature of the HSV-1 C6 interaction and indicated that the restrictive event(s) occurred intracellularly. However, interferon activity could not be demonstrated in the composite of medium mixed with the total intracellular contents from HSV-1 infected C6 cells from 0 through 72 hpi. The lack of a viral refractory state was also reflected in only a slight inhibition at 10 hpi of the replication of poliovirus RNA which was used to superinfect HSV-1 infected C6 cells. Furthermore, the exogenous addition of 5 units/ml of interferon supplied either before or after HSV-1 adsorption as well as 50 units/ml added after virus adsorption failed to affect the production HSV-1 in C6 cells. These observations suggest that interferon is not mediating the restrictive replication of HSV-1 in C6 cells.T. L. Z. i...