Active secretion of S100B from astrocytes during metabolic stress.

In patients suffering from cerebrovascular diseases and traumatic brain damage, increases in serum levels of protein S100B are positively correlated with the severity of the insult. Since high concentrations of S100B have been shown to exert neurotoxic effects, the objective of this study was to characterize the regulatory mechanisms underlying control of S100B release from astrocytes. To that end, we analyzed the kinetics and amount of S100B release in correlation with regulation of S100B gene expression in an in vitro ischemia model. Astrocyte cultures were treated with combined oxygen, serum and glucose deprivation, serum and glucose deprivation or hypoxia alone for 6, 12 and 24 h, respectively. While oxygen, serum and glucose deprivation triggered the most rapid release of S100B, serum and glucose deprivation provoked comparable levels of released S100B at the later time points. In contrast to oxygen, serum and glucose deprivation and serum and glucose deprivation, hypoxia alone elicited only marginal increases in secreted S100B. Parallel analysis of extracellular lactate dehydrogenase and the number of viable cells revealed only moderate cell death in the cultures, indicating that S100B was actively secreted during in vitro ischemia. Interestingly, S100B mRNA expression was potently downregulated after 12 and 24 h of oxygen, serum and glucose deprivation, and prolonged oxygen, serum and glucose deprivation for 48 h was associated with a significant reduction of S100B release at later time intervals, whereas lactate dehydrogenase levels remained constant. Our data suggest that secretion of S100B during the glial response to metabolic injury is an early and active process.

Down-regulation of BOB.1/OBF.1 and Oct2 in classical Hodgkin disease but not in lymphocyte predominant Hodgkin disease correlates with immunoglobulin transcription.

In contrast to the tumor cells (L&H cells) of lymphocyte predominant Hodgkin disease (LPHD), Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD) are unable to transcribe immunoglobulin, despite the presence of rearranged immunoglobulin genes. Although initial studies have suggested crippling immunoglobulin gene mutations to be the cause of absent immunoglobulin expression in cHD, recent work of our group has demonstrated an impaired activation of the immunoglobulin promoter as a superior mechanism. As immunoglobulin transcription is mainly regulated by the B-cell transcription factors Oct2 and BOB.1/OBF.1, we analyzed 35 cases of LPHD, 32 cases of cHD, and 2 Hodgkin disease cell lines for the expression of these transcription factors and also in parallel for immunoglobulin expression. Our results demonstrate an absence of Oct2 and/or BOB.1/OBF.1 in cHD and a striking overexpression of Oct2 in LPHD. Immunoglobulin expression was lacking in cHD but present in LPHD. Furthermore, the reintroduction of BOB.1/OBF.1 and Oct2 into cultured HRS cells restored the activity of cotransduced immunoglobulin promoter constructs. Our findings dismiss the concept that the different immunoglobulin expression in cHD and LPHD is due to disrupting mutations of immunoglobulin V genes in cHD but is most likely due to a down-regulation of Oct2 and/or BOB.1/OBF.1. This study further revealed Oct2 as a new and valuable marker for the identification of L&H cells and their distinction from HRS cells. The impairment of immunoglobulin transcription with a down-regulated synthesis of Oct2 and BOB.1/OBF.1 is the first established general recurrent defect found in HRS cells.

Telomerase expression in uveal melanoma.

BACKGROUND/AIMS: Accumulating evidence indicates that telomerase activity is repressed in normal human somatic cells but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity has a role in carcinogenesis and immortalisation. To date, telomerase in uveal melanoma and, whether, it may have a role in the development or progression of these tumours has not been described. The expression patterns and the activity of telomerase were investigated in 14 uveal melanoma and these results were correlated with histological and immunohistological features of these tumours. METHODS: A modified PCR based telomeric repeat amplification protocol (TRAP) assay was used to demonstrate telomerase activity in 14 uveal melanomas. In addition, in situ hybridisation was used to demonstrate the expression pattern of the telomerase RNA component (hTR) at the single cell level in eight of these globes. RESULTS: The TRAP assay revealed moderate telomerase activity in all uveal melanomas examined. In situ hybridisation visualised a moderate to high upregulation of hTR in the melanoma cells but not in the admixed reactive cells. There was no correlation among tumour location, cell type, or growth fraction and the amount of telomerase activity. In addition, the cells of the germinative zone of the lens demonstrated a strong hTR expression. CONCLUSION: Telomerase activity is upregulated in uveal melanomas. The expression of hTR was located to the tumour cells and not the reactive tumour infiltrating cells. Strong telomerase expression was also demonstrated in cells of the germinative zone of the lens.

Hodgkin and reed-sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription.

Single cell studies aimed at clarifying the nature and clonality of Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin's disease (HD) have so far produced conflicting results. Using an improved single cell procedure, the HRS cells of 25 patients with nodular sclerosing HD lacking B- and T-cell antigens, with and without Epstein-Barr virus infection, were analyzed for the presence of immunoglobulin (Ig) gene rearrangements. One patient with HD developed follicular lymphoma 2 years later. Both lymphomas originated from a common precursor identified as a germinal center B cell. The data show that all but one of the investigated cases harbored rearranged Ig genes, which were clonal in all instances and carried a high load of somatic mutations. The Ig coding capacity was preserved in 18 of the 24 cases (75%) with rearrangements. However, expression of Ig messenger RNA was not detectable in the HRS cells with the exception of Ig kappa light chain expression in some tumor cells of 1 case. The lack of Ig gene transcription in HRS cells was confirmed by analyzing the HD cell lines L428 and KM-H2 in transient transfection experiments. An Ig promoter/enhancer reporter construct showed virtually no activity in these cells compared to 5 control B-cell lines. We conclude that (1) classical HD is a B-cell lymphoma in most instances, (2) HRS cells are clonal without any exception, (3) they are derived from germinal center B-cells that (4) mostly lack crippling mutations but (5) have consistently lost their Ig gene transcription ability, due to functional defects in the Ig gene regulatory elements. (Blood. 2000;95:1443-1450)

Frequent expression of the B-cell-specific activator protein in Reed-Sternberg cells of classical Hodgkin's disease provides further evidence for its B-cell origin.

The neoplastic cells of classical Hodgkin's disease (cHD), ie, Hodgkin and Reed-Sternberg cells (HRS cells), contain clonally rearranged Ig genes, but are dissimilar to normal B cells in that they mostly do not display B-cell antigens such as CD20 or CD19. The transcription factor B-cell-specific activator protein (BSAP) influences numerous B-cell functions such as B-cell antigen expression, Ig expression, and class switch. We analyzed the expression of BSAP in cHD and control tissues by isotopic in situ hybridization and immunohistochemistry to determine whether BSAP is expressed in HRS cells and, if so, whether it may be involved in the genesis of the abnormal phenotype of these cells. Both in normal lymphoid tissue and non-Hodgkin lymphomas, BSAP transcripts and protein were almost exclusively found in B cells and B-cell lymphomas (40 cases), but were absent from the tumor cells of T-cell neoplasms (41 cases), including 19 cases of anaplastic large cell lymphoma of T- and null-cell type. Among cHD, variable numbers of HRS cells exhibited BSAP transcripts (22 of 25 cases) and protein (28 of 31 cases). Our findings show that BSAP is sufficiently specific to serve as B-cell marker. BSAP expression in HRS cells provides further strong evidence for a frequent B-cell origin of cHD and helps distinguish this disease from anaplastic large cell lymphoma of T- and null-cell type. Because BSAP is much more frequently expressed in HRS cells than the conventional B-cell antigens, the abnormal immunophenotype of HRS cells with frequent absence of B-cell antigens does not appear to be due to absent BSAP expression.

Overexpression of I kappa B alpha without inhibition of NF-kappaB activity and mutations in the I kappa B alpha gene in Reed-Sternberg cells.

The transcription factor NF kappa B (NF-kappaB) mediates the expression of numerous genes involved in diverse functions such as inflammation, immune response, apoptosis, and cell proliferation. We recently identified constitutive activation of NF-kappaB (p50/p65) as a common feature of Hodgkin/Reed-Sternberg (HRS) cells preventing these cells from undergoing apoptosis and triggering proliferation. To examine possible alterations in the NF-kappaB/IkappaB system, which might be responsible for constitutive NF-kappaB activity, we have analyzed the inhibitor I kappa B alpha (IkappaBalpha) in primary and cultured HRS cells on protein, mRNA, and genomic levels. In lymph node biopsy samples from Hodgkin's disease patients, IkappaBalpha mRNA proved to be strongly overexpressed in the HRS cells. In 2 cell lines (L428 and KM-H2), we detected mutations in the IkappaBalpha gene, resulting in C-terminally truncated proteins, which are presumably not able to inhibit NF-kappaB-DNA binding activity. Furthermore, an analysis of the IkappaBalpha gene in single HRS cells micromanipulated from frozen tissue sections showed a monoallelic mutation in 1 of 10 patients coding for a comparable C-terminally truncated IkappaBalpha protein. We suggest that the observed IkappaBalpha mutations contribute to constitutive NF-kappaB activity in cultured and primary HRS cells and are therefore involved in the pathogenesis of these Hodgkin's disease (HD) patients. The demonstrated constitutive overexpression of IkappaBalpha in HRS cells evidences a deregulation of the NF-kappaB/IkappaB system also in the remaining cases, probably due to defects in other members of the IkappaB family.

Hodgkin and Reed-Sternberg cells of classical Hodgkin's disease overexpress the telomerase RNA template (hTR).

There is accumulating evidence to suggest that Hodgkin and Reed-Sternberg (HRS) cells represent the malignant cell population in Hodgkin's disease (HD). A recent report that HD tissue is in most instances devoid of telomerase activity was therefore unexpected. Since telomerase activity was determined in whole tissue extracts and HRS cells comprise only a small minority of the cells in the affected tissue, the telomerase activity of the HRS cells might have escaped detection. To test this possibility and to clarify whether HRS cells contain the enzyme telomerase, 13 cases of classical HD were analysed by three different methods. The presence of telomerase was studied at the single cell level by a sensitive radioactive in situ hybridization method employing a probe specific for the telomerase RNA template (hTR). In addition, tissue extracts were studied for telomerase activity by a modified TRAP assay and for hTR by reverse transcription-polymerase chain reaction (RT-PCR). The extractive methods revealed telomerase activity in eight and hTR in all of the 13 HD cases studied. In situ hybridization located large amounts of hTR in the HRS cells of all 13 HD cases and low to medium amounts in some of the non-malignant lymphoid bystander cells. These results indicate that HRS cells constitutively overexpress telomerase and thus use this enzyme for stabilizing their telomeres. This substantiates the malignant nature of HRS cells. Furthermore, the results confirm that normal lymphoid cells can express telomerase. In consequence, methods of measuring telomerase in tissue extracts are not suitable for determining the presence of this molecule in lymphoma cells, since the vast majority of lymphoid neoplasms contain significant amounts of non-neoplastic lymphoid cells.

FasL is more frequently expressed in liver metastases of colorectal cancer than in matched primary carcinomas.

Colorectal carcinoma cells have recently been shown to express Fas ligand (FasL). This ligand could allow the tumour cells to evade activated tumour-infiltrating lymphocytes (TILs) by inducing their apoptosis and would thus promote tumour survival and possibly metastasis formation. To test this hypothesis in vivo we analysed the expression of FasL mRNA and protein in paired tissue samples of normal colonic mucosa (N), primary colorectal carcinomas (T) and their metastases (M) from a total of 21 patients by four different methods. Additionally, the presence and activation status of infiltrating lymphocytes, which might contribute to the total amount of FasL in the tissue, was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in the same samples. The frequency of FasL detection was 30-40% in T and was 60-100% in M, depending on the sensitivity of the method. Simultaneously, the amount of CD25 mRNA, used as a measure of the number of activated TILs, was in 90% of patients lower in M than in T. The increased frequency of FasL detection in liver metastases was therefore not due to the presence of activated TILs. We conclude that metastasizing subpopulations of colorectal tumour cells express FasL more frequently than the primary carcinomas and may be able to eliminate activated TILs in vivo via Fas/FasL-induced apoptosis or other hitherto unknown mechanisms.

Tumor necrosis factor receptor-associated factor 1 is overexpressed in Reed-Sternberg cells of Hodgkin's disease and Epstein-Barr virus-transformed lymphoid cells.

The tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) is a member of the recently defined TRAF family. It takes part in the signal transduction of the TNF receptor 2 (TNFR2), the lymphotoxin-beta receptor (LT-betaR), CD40, CD30, and LMP1; is induced by LMP1 in vitro; and protects lymphoid cells from apoptosis. To identify the cells in which TRAF1 is active in vivo, we studied TRAF1 transcripts in normal lymphoid tissue, in Epstein-Barr virus (EBV)-induced lymphoproliferations, and in malignant lymphomas with special reference to those that overexpress the cytokine receptor CD30 and CD40 of the TNF receptor family at the single-cell level using a radioactive in situ hybridization. In normal lymphoid tissue, TRAF1 message proved to be absent from all resting B and T cells as well as from macrophages and accessory cells (follicular dendritic cells and interdigitating cells) and present in few perifollicular and intrafollicular lymphoid blasts. In contrast, there was a high and consistent TRAF1 overexpression in EBV-induced lymphoproliferations and Hodgkin's disease. Nearly all non-Hodgkin's lymphoma show low or no TRAF1 expression. Only some cases of diffuse large B-cell lymphoma showed a moderate to high TRAF1 signal. Several of the latter cases were EBV+. These data confirm that TRAF1 is an inducible molecule and indicates its deregulation in the mentioned disorders with the potential of a blockage of the apoptotic pathway.

Demonstration of constant upregulation of the telomerase RNA component in human gastric carcinomas using in situ hybridization.

Upregulation of the ribonucleoprotein telomerase seems to be a prerequisite for immortality, a feature of malignant cells. Using a polymerase chain reaction (PCR)-based assay, it is possible to demonstrate telomerase activity (TA) in specimens of most human malignancies, whereas it is absent from most normal tissues. It remains unclear, however, why between 5 and 50 per cent of various malignant tumour samples give negative results when TA is measured by the telomeric repeat amplification protocol (TRAP). The expectation that reverse transcription (RT)-PCR for detection of the telomerase RNA component (hTR) would be able to complement or to replace the TRAP assay failed, since malignant as well as non-malignant tissue samples gave positive results in most instances. In the present study, in situ hybridization (ISH) was developed to demonstrate the RNA component of human telomerase at the single cell level. With this method, 13 specimens of fresh frozen gastric carcinoma and four of normal, dysplastic, or inflamed gastric mucosa were investigated and the results were compared with those obtained by RT-PCR and the TRAP assay. In addition, ISH was performed on formalin-fixed sections of the same cases. The TRAP assay revealed positive results in 8 out of 13 gastric carcinomas and was negative in all non-malignant tissues. RT-PCR led to amplification of the telomerase RNA component in all specimens tested, irrespective of the presence or absence of malignant cells. By ISH, all gastric carcinomas showed strong telomerase RNA component-specific signals over malignant cells, whereas only a few grains were detectable over some types of normal somatic cells, including activated lymphocytes. In conclusion, high expression of the telomerase RNA component was restricted to the malignant cells of all the gastric carcinomas investigated, as shown by ISH. This indicates that the absence of TA in a proportion of carcinomas is due to methodological problems of the TRAP assay and is not caused by biological factors. The detection of high levels of the telomerase RNA component by ISH is thus a useful technique for demonstrating malignant cells in frozen and formalin-fixed pathological specimens.

Expression of vascular endothelial growth factor in lymphomas and Castleman's disease.

Vascular endothelial growth factor (VEGF) is one of the main angiogenic cytokines in human solid tumours and inhibition of VEGF-induced angiogenesis suppresses tumour growth. Some groups of malignant lymphoma, including peripheral T-cell lymphomas and Hodgkin's disease, are characterized by a conspicuous proliferation of small vessels. To test the hypothesis that VEGF may also be involved in the angiogenesis in lymphomas and other lesions of the lymphoid system, VEGF expression was analysed in tissues, employing in situ hybridization with a 35S-labelled RNA probe specific for this cytokine. Significant expression of VEGF transcripts was observed in Hodgkin's disease and peripheral T-cell lymphomas, particularly of the angioimmunoblastic type. In contrast, expression of this cytokine was minimal or absent in follicle centre lymphoma and chronic lymphocytic leukemia of B-cell type. VEGF was mainly observed in reactive non-lymphoid CD68-negative cells, which probably represent fibroblasts or myofibroblasts. In normal and ulcerated tonsils, VEGF was expressed in the squamous epithelium but only rarely found in the lymphoid tissue. Although infectious mononucleosis tonsils contained high numbers of VEGF-positive cells in the interfollicular zone, expression of this cytokine was not found in Epstein-Barr virus (EBV)-infected cells, as determined by simultaneous in situ hybridization for VEGF and EBV-encoded small nuclear RNAs (EBER). In 5/8 cases of Castleman's disease, germinal centres containing small vessels also showed expression of VEGF, in contrast to normal tonsillar germinal centres which are devoid of both vessels and VEGF transcripts. It is concluded that VEGF may be involved in the induction of the angiogenesis of both peripheral T-cell lymphomas and Hodgkin's disease, but not in low-grade B-cell lymphomas. In contradistinction to solid tumours, in which this cytokine is commonly secreted by the tumour cells themselves, in malignant lymphoma VEGF is not a product of neoplastic cells. Vascularization of germinal centres in Castleman's disease may also be a consequence of abnormal local expression of VEGF.

Expression of cytotoxic molecules in intestinal T-cell lymphomas. The German Study Group on Intestinal Non-Hodgkin Lymphoma.

Intestinal T-cell lymphoma (ITCL) represents a subgroup of peripheral T-cell lymphomas which is thought to arise from alpha beta intraepithelial T-lymphocytes. Since these lymphocytes may contain cytotoxic molecules, the question of whether this also holds true for ITCL arises. Twenty ITCL cases were examined for the presence of granzyme B, perforin, and T-cell-restricted intracellular antigen (TIA-1)/granule membrane protein of 17 kD (GMP-17). Two molecules with restricted expression in cytotoxic cells, granzyme B and perforin, were detected by immunocytochemistry and by in situ hybridization with an isotopically labelled RNA probe, respectively. Immunocytochemistry was also performed with the antibody 2G9, which recognizes two molecules, one expressed by cytotoxic cells (TIA-1) and the other found in granulocytes and cytotoxic cells (GMP-17). Granzyme B, TIA-1/GMP-17, and perforin were found in the neoplastic cells of 16/19 cases, 19/20 cases, and 16/17 cases, respectively, of ITCL, but not in the tumour cells of the control group, which consisted of intestinal B-cell lymphomas (five cases) and CD8-negative peripheral nodal T-cell lymphomas (six cases). At least one of these molecules was expressed in the tumour cells of all ITCL cases. 2G9 proved to be the most sensitive immunohistological marker, since reactivity with this antibody was not only observed in the highest number of cases, but also found in high numbers of neoplastic cells in positive cases. In conclusion, ITCL appears to show cytotoxic differentiation in all cases. In conjunction with immunophenotypic and genotypic data, our results support a uniform derivation of this tumour from intraepithelial alpha beta cytotoxic T-lymphocytes.

Uniform expression of cytotoxic molecules in anaplastic large cell lymphoma of null/T cell phenotype and in cell lines derived from anaplastic large cell lymphoma.

We recently provided ample evidence that anaplastic large cell lymphomas of T/null phenotype (T-/null-ALCL) genotypically represent peripheral T cell lymphomas which in up to 90% have a phenotype of cytotoxic cells with expression of granzyme B protein and perforin transcripts. However, the issue of granzyme B expression in T-/null-ALCL is still controversial due to differing results from another laboratory. To verify our earlier immunohistochemical stainings for granzyme B, we looked for granzyme B transcripts by in situ hybridization (ISH). In addition, we investigated our previously analyzed cases by immunohistology (IH) with another antibody (2G9), which reacts with two molecules known to be expressed in cytotoxic cells: T-cell-restricted intracellular antigen (TIA)-1 and granule membrane protein-17 (GMP-17). We also extended our studies to homogenous tumor cell populations provided by ALCL-derived cell lines. As evidenced by ISH, transcripts for perforin, TIA-1 and granzyme B were found in all ALCL-derived cell lines. Similarly, proteins of TIA-1/GMP-17, granzyme B and perforin were expressed in all of these lines as shown by IH. In biopsy specimens, TIA-1/GMP-17 were detected by IH in 14/16 cases of T-/null-ALCL, and granzyme B transcripts were found in 13/13 T-/null-ALCL cases, but not in 6 B-ALCL cases. The detection of granzyme B transcripts yielded results largely identical to those of IH for granzyme B protein, thus confirming our earlier data and suggesting that the regulation of the expression of this molecule largely occurs at the transcriptional level. Our data further confirm the almost uniform expression of cytotoxic molecules in both primary ALCL cases and ALCL-derived cell lines and therefore suggest that the derivation from cytotoxic T cells may be the unifying characteristic for T-/null-ALCL.

[Telomerase as a tumor marker?]. / Die Telomerase als Krebsmarker?

The maintenance of the length of chromosomes (telomeres) is the prerequisite of unlimited malignant growth of cells. In normal cells the telomeres will be shortened by each cell division finally leading to cell senescence. Unlimited growing cells possess an enzyme, the telomerase, which is able to maintain the length of the telomeres by de novo synthesis of the telomeric repeats. In consequence the telomerase is detectable in nearly all malignant tumours and cell lines. However, in addition to malignant cells some type of non-malignant cells such as germ cells, activated lymphocytes and basal epithelial cells contain significant amounts of telomerase activity. Therefore, the detection of telomerase activity by extractive procedures such as TRAP assay is not sufficient to determine malignant nature of tissue samples.

Anaplastic large-cell lymphomas of T-cell and null-cell phenotype express cytotoxic molecules.

To further specify the cellular origin and nature of anaplastic large-cell lymphoma (ALCL) and its relationship to other lymphoid neoplasms, particularly Hodgkin's disease (HD), we investigated the presence of cytotoxic molecules in a large well-characterized series of these tumors. For expression of the cytotoxic molecules perforin and granzyme B, in situ hybridization (ISH) and immunohistology were used, respectively. Overall, 23 of 25 ALCLs of T/null phenotype and five (three mixed cellularity and two nodular sclerosis) of 57 HD cases showed the presence of perforin transcripts and/or granzyme B molecules in neoplastic cells. Polymerase chain reaction (PCR) analysis of ALCLs showed that most (10 of 11) cases of null-cell ALCL (null-ALCL) contained a clonal rearrangement of T-cell receptor beta-chain genes, as did T-cell ALCL (T-ALCL; 9 of 10 cases). However, both cytotoxic molecules and clonally rearranged T-cell receptor beta-chain genes were absent in seven of seven and eight of nine cases of B-cell ALCL (B-ALCL), respectively. These data show that all or nearly all T-ALCLs, irrespective of the clinical subform or the lack of T-cell-associated molecules, are derived from activated cytotoxic T cells. The same appears to be true for the neoplastic cells of rare HD cases. These findings indicate that T-ALCLs are different from B-ALCLs and the majority of HD cases, and suggest that some HD cases, especially those with T-cell antigen-positive tumor cells, may be closely related to T-ALCL, at least in terms of cellular origin.

Expression of Epstein-Barr virus-gene products in Burkitt's lymphoma in Northeast Brazil.

The Epstein-Barr Virus (EBV) is consistently found in tumor cells of Burkitt's lymphoma (BL) endemic in central Africa and malaria is considered a pathogenic cofactor. In contrast, fewer than 20% of BL cases occurring in Western countries are EBV-associated. We have investigated 54 BL cases from Bahia, a tropical region of Northeast Brazil, for expression of EBV gene products by in situ hybridization and immunohistology and performed typing of the EBV by polymerase chain reaction. Ten pediatric BL cases from Germany served as controls. New cases of malaria were not observed in the period and area of our study. Small nuclear EBV encoded transcripts, EBER, were found in tumor cells of 47 of 54 Brazilian cases (87%) but in only 2 of 10 German cases (20%). Type I latency of the EBV infection with absence of EBV-encoded proteins LMP1 and EBNA2 was found in 45 of 47 of the EBER-positive Brazilian cases. In two cases, occasional LMP1-containing tumor cells were found in the neighborhood of small Schistosoma mansoni granulomas and scars. BHLF1 transcripts associated with lytic EBV infection could be detected in few cells in 3 of the 40 EBER-positive Brazilian cases investigated. EBV type A was found in the majority of Brazilian BL cases (20 of 30 A-type, 7 of 30 B-type, and 3 of 30 not amplifiable). Our results indicate that the association of Bahian BL with EBV, but not the regional prevalence of malaria, is similar to endemic African BL. In two cases, type II latency was found in association with schistosomiasis, suggesting a role of this parasitosis in the induction of an EBV expression pattern that is unusual for BL. Because chronic schistosomiasis is associated with elevated Th2 cytokine expression resulting in reduced cell-mediated cytotoxicity, it seems possible that altered local immunity is responsible for this peculiar phenotype.

Interleukin-8 in Hodgkin's disease. Preferential expression by reactive cells and association with neutrophil density.

Hodgkin's disease (HD) shows rare neoplastic Hodgkin and Reed-Sternberg cells embedded in an abundant reactive infiltrate containing, among other cell types, neutrophilic granulocytes. Interleukin (IL)-8 is chemotactic for neutrophils. The expression of IL-8 was tested by in situ hybridization with 35S-labeled IL-8-specific RNA probes on 38 cases of HD. Reactive lesions, non-Hodgkin's lymphomas of B and T phenotype, and Langerhans cell histiocytosis served as controls. IL-8 expression was observed in Hodgkin and Reed-Sternberg cells in 3 of 33 cases of classical HD and in reactive cells in 20 of 33 HD cases as evidenced by combined isotopic in situ hybridization and immunohistology for the demonstration of cell-type-characteristic antigens or enzyme histochemistry for chloroacetate esterase. IL-8-positive cells were more numerous in cases of nodular sclerosing HD as compared with the mixed cellularity histotype (P = 0.01). The number of IL-8-positive cells and the density of neutrophils were positively correlated (P < 0. 01). In 5 cases of lymphocyte-predominant HD, IL-8 expression was not displayed. Non-Hodgkin's lymphoma cases contained IL-8 transcripts only in 1 of 23 cases in sparse reactive cells. In 4 of 7 cases of Langerhans cell histiocytosis, IL-8-specific signals were displayed in S100-negative cells. In conclusion, IL-8 expression in HD is largely confined to reactive cells and associated with infiltration by neutrophils. Elaboration of other cytokines by Hodgkin and Reed-Sternberg cells and reactive cells may explain the frequent expression of this cytokine in HD, particularly in the nodular sclerosing type.

Identification of genes encoding for peptide synthetases in the gram-negative bacterium Lysobacter sp. ATCC 53042 and the fungus Cylindrotrichum oligospermum.

Genes encoding for the multifunctional peptide synthetases lysobactin synthetase and peptolide SDZ 214-103 synthetase were identified by hybridization of genomic libraries with oligonucleotides derived from consensus motifs of various genes encoding for delta-(L-alpha amino-adipoyl)-L-cysteinyl-D-valine (ACV) synthetases and gramicidin S synthetase. The sequence of subcloned gene fragments revealed core motifs and a modular structure typical for the family of peptide synthetase genes. A fragment of 4.6 kb of the lysobactin synthetase gene was sequenced and one amino acid activating module was localized. The cloning of lysobactin synthetase was verified by marker-exchange mutagenesis and the lysobactin minus phenotype of the mutant. The sequenced 3.1 kb fragment of peptolide SDZ 214-103 synthetase contained parts of two modules and was highly homologous to corresponding regions of module 6 and 7 of cyclosporin synthetase. Therefore, the localized modules may activate the amino acids threonine and glycine.