Pax5 immunostaining in paraffin-embedded sections of canine non-Hodgkin lymphoma: a novel canine pan pre-B- and B-cell marker.

The Pax5 gene encodes the B-cell specific activator protein (BSAP), a member of the highly conserved paired box (PAX)-domain family of transcription factors and a key regulator in the development and differentiation of B-cells. Pax5 serves as a valuable B-cell marker in the classification of human lymphoma patients as it is restricted to lymphomas of B-cell lineage. In dogs, detection of Pax5 protein in lymphoma tissue has not been reported. Therefore, we have investigated the expression and detection of BSAP using a monoclonal anti-Pax5 antibody (anti-BSAP, clone 24) in canine lymphoma tissue samples to evaluate its diagnostic relevance as a B-cell marker. A series of 25 lymph nodes from 23 canine non-Hodgkin lymphoma patients, a reactive canine lymph node, and a normal non-reactive canine lymph node, were evaluated. All B-cell non-Hodgkin lymphomas (15) were found to express Pax5 protein. In addition, there was a strong correlation between Pax5 and CD79a expression. Three CD3 positive and five CD3 and CD79a positive lymphomas were immunophenotypically negative for anti-Pax5, indicating a T-cell lineage. In conclusion, anti-Pax5 antibody may offer an excellent B-cell marker in canine lymphomas.

Livin and Bcl-2 expression in high-grade osteosarcoma.

AIMS: The evaluation of prognosis in patients with osteosarcoma is limited to clinical parameters. Although numerous molecular markers have been studied, none are currently in routine clinical use. The aim of this study was to determine if Livin and Bcl-2, acting as antiapoptotic proteins through different mechanisms, are expressed in osteosarcoma, and whether they can be used as prognostic markers in human osteosarcoma. METHODS: Tumor specimens of 29 patients with high-grade central osteosarcoma, with complete clinical follow-up for a minimum of 5 years, were studied. The localization and distribution of Livin and Bcl-2 were investigated using immunohistochemistry. Results were correlated with the histological response to chemotherapy, 5-year disease-free and 5-year overall survival. RESULTS: Bcl-2 was expressed only in the cytoplasm of 16/29 cases and there was no statistically significant correlation between expression and any of the studied parameters. Livin was detected in 17/29 cases, in the cytoplasm of all 17 and in the nucleus of only 3 cases. Nuclear expression was significantly correlated with a decreased overall survival (P < 0.0002) compared with those patients without nuclear expression. CONCLUSIONS: The results of this study indicate that Bc1-2 and Livin are highly expressed in osteosarcoma cells and that possibly, the evaluation of nuclear Livin expression might be a useful prognostic marker in osteosarcoma.

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7.

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.

Cdc25A phosphatase suppresses apoptosis induced by serum deprivation.

The phosphatase Cdc25A was shown to be a target of the transcription factor c-Myc. Myc-induced apoptosis appeared dependent on Cdc25A expression and Cdc25A over-expression could substitute for Myc-triggered apoptosis. These findings suggested that an important downstream component of Myc-mediated apoptosis was identified. However and in contrast, we recently reported that during TNFalpha-induced apoptosis, which required c-Myc function, Cdc25A was down-regulated in a human carcinoma cell line. We now provide evidence that Cdc25A rendered the non-transformed rat embryonic cell line 423 refractory to apoptosis, which was induced by serum deprivation and in absence of detectable c-myc levels. The survival promoting activity of cdc25A was abolished upon infection of cells with a full-length cdc25A antisense construct. To identify the signaling proteins mediating the survival function of the phosphatase, cdc25A- and akt- over-expressing pooled clones were exposed to selected chemicals, which inhibit or activate key proteins in signaling pathways. Inhibition of apoptosis by SU4984, NF023 and Rapamycin placed Cdc25A and Akt function downstream of FGF.R, PDGF.R, and compensated G-protein- and PP2A- activity. Interestingly, upon treatment with LY-294002, cdc25A- and akt- over-expressing clones exhibited similar apoptotic patterns as control cells, which indicates that neither Akt- nor Cdc25A-mediated survival functions are dependent on PI.3 kinase activity in rat 423 cells. In cdc25A-overexpressing cells increased levels of serine 473 phosphorylated Akt were found, which co-precipitated with Cdc25A and Raf1. Since activation of proteins requires dephosphorylation of particular residues in addition to site-specific phosphorylation, the anti-apoptotic effect of Cdc25A might derive from its participation in a multimeric protein complex with phosphoAkt and Raf1, two prominent components of survival pathways.

Immunohistochemical properties of bone marrow mast cells in systemic mastocytosis: evidence for expression of CD2, CD117/Kit, and bcl-x(L).

In an attempt to identify novel diagnostic markers for mast cell (MC)-proliferative disorders, serial bone marrow (bm) sections of 22 patients with mastocytosis (systemic indolent mastocytosis, n = 19; mast cell leukemia [MCL], n = 1; isolated bm mastocytosis, n = 2) were analyzed by immunohistochemistry using antibodies against CD2, CD15, CD29, CD30, CD31, CD34, CD45, CD51, CD56, CD68R, CD117, HLA-DR, bcl-2, bcl-x(L), myeloperoxidase (MPO), and tryptase. Staining results revealed expression of bcl-x(L), CD68R, and tryptase in neoplastic MCs (focal dense infiltrates) in all patients. Mastocytosis infiltrates were also immunoreactive for CD45, CD117 (Kit), and HLA-DR. In most cases, the CD2 antibody produced reactivity with bm MCs in mastocytosis, whereas in control cases (reactive bm, immunocytoma, myelodysplastic syndrome), MCs were consistently CD2 negative. Expression of bcl-2 was detectable in a subset of MCs in the patient with MCL, whereas no reactivity was seen in patients with SIM or bm mastocytosis. Mastocytosis infiltrates did not react with antibodies against CD15, CD30, CD31, CD34, or MPO. In summary, our data confirm the diagnostic value of staining for tryptase, Kit, and CD68R in mastocytosis. Apart from these, CD2 may be a novel useful marker because MCs in mastocytosis frequently express this antigen, whereas MCs in other pathologic conditions are CD2 negative.

Platelet-derived growth factor-AA and -alpha receptor expression suggests an autocrine and/or paracrine loop in osteosarcoma.

Platelet-derived growth factor (PDGF) is a major mitogen and chemotactic factor for mesenchymal cells such as fibroblasts, smooth muscle cells, and osteoblasts. PDGF exists as disulfide-linked homo- or heterodimers composed of two polypeptide chains encoded by distinct genes, designated PDGF-A and PDGF-B. Upon binding to its tyrosine kinase receptor PDGF-alpha, especially PDGF-AA stimulates the proliferation of osteoblastic cells and may exert autocrine and paracrine effects in regulating bone-forming processes. The purpose of this immunohistochemical study was to determine the expression of PDGF-AA and PDGF-alpha receptor in benign and malignant neoplastic bone lesions. Polyclonal antibodies to PDGF-AA and PDGF-alpha receptor were used on paraffin sections of 23 osteosarcomas and 17 osteoblastomas. Immunostaining was assessed quantitatively by evaluating the percentage of reactive tumor cells. In osteosarcomas, the mean expression of PDGF-AA and PDGF-alpha receptor was 33.97% (range, 2 to 80%; SD, 24.26%) and 27.13% (range, 3.2 to 72%; SD, 18.38%), respectively. Osteoblastomas showed significantly lower expression of PDGF-AA than osteosarcomas (mean, 15.71%; range, 5 to 34%; SD, 9.43%; P = .019). Although the mean expression of PDGF-alpha receptor in osteoblastomas was much lower than in osteosarcomas (mean, 17.55%; range, 3.6 to 26.8%; SD, 6.47%), the difference was not significant (P = .122). For osteosarcomas, Spearman correlation coefficient (two-tailed) revealed a significant correlation between the expression of PDGF-AA and PDGF-alpha receptor (r = .688), which was not the case for osteoblastomas (r = .267). These data suggest that in contrast to osteoblastoma, the growth of osteosarcoma may be supported by the coordinate expression of the potent mitogenic growth factor and its receptor that exert their functions by autocrine and paracrine mechanisms.

Fas ligand is expressed in normal breast epithelial cells and is frequently up-regulated in breast cancer.

Fas (CD95/Apo-1) is a cell membrane receptor that upon binding by its ligand (FasL), triggers a signal resulting in apoptotic cell death. Fas is produced by breast epithelial cells, but its contribution to breast tissue homeostasis is unknown. This study investigated whether FasL is synthesized in the breast. By reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry, FasL expression was analysed in normal and malignant human breast epithelial cell lines, normal breast tissue, benign breast disease (fibrocystic changes, fibroadenoma), and breast cancer (ductal carcinoma in situ; invasive ductal, lobular, mucinous and medullary carcinomas). The results demonstrate expression of FasL by normal breast epithelial cells and show a marked increase of FasL protein in the majority of breast carcinomas, compared with normal breast tissue and benign breast disease. By western blot analysis, soluble FasL was detected in culture supernatants of one of three normal breast epithelial cell lines and in all four breast cancer cell lines tested. The expression of Fas protein was more heterogeneous in benign and malignant breast tissue, with expression levels ranging from weak to strong, but breast cancer cells frequently exhibited a weaker Fas expression than surrounding residual normal breast epithelial cells. In vitro, two out of three normal breast epithelial cell lines were sensitive to cell death induction by an agonistic anti-Fas antibody. Co-treatment with cycloheximide, an inhibitor of protein translation, rendered the resistant cell line sensitive. In contrast, two out of four breast cancer cell lines were resistant to the anti-Fas antibody and this resistance could not be reversed by cycloheximide. These results suggest that increased expression of FasL may confer an advantage on breast cancer cells, possibly by eliminating tumour-infiltrating immune cells, and/or by facilitating tissue destruction during invasion.

Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type 1 insulin-like growth factor receptor.

An important biological feature of prostate cancer (PCa) is its marked preference for bone marrow as a metastatic site. To identify factors that may support the growth of PCa in bone marrow, expression of receptor and nonreceptor tyrosine kinases by androgen-independent PCa bone marrow metastases was assessed. Bone marrow biopsies largely replaced by PCa were analyzed using reverse transcriptase-polymerase chain reaction amplification with degenerate primers that amplified the conserved kinase domain. Sequence analyses of the cloned products demonstrated expression of multiple kinases. Expression of the receptor and nonreceptor tyrosine kinases, alpha platelet-derived growth factor receptor and Jak 1, respectively, was confirmed by immunohistochemistry. In contrast, the type 1 insulin-like growth factor receptor, thought to play a role in PCa development, was lost in metastatic PCa. These results implicate several specific growth factors and signaling pathways in metastatic androgen-independent PCa and indicate that loss of the type 1 insulin-like growth factor receptor contributes to PCa progression.

Immunophenotypic characterization of human bone marrow endosteal cells.

In order to determine the relationship between bone marrow (bm) endosteal cells (EDC) and hemopoietic progenitors, we have analyzed the immunophenotype of EDC using various antibodies (Ab) against mesenchymal antigens. The Ab were applied on paraffin sections of normal bm (iliac crest, n=17; talus, n=1; phalanx, n=1), myeloregenerative bm (after chemotherapy), and hematologic disorders (acute myeloid leukemia (AML), n=8; chronic myeloid leukemia (CML), n=6; myelodysplastic syndromes (MDS), n=14; severe aplastic anemia (SAA), n=4; essential thrombocythemia (ET), n=2; idiopathic (primary) osteomyelo-fibrosis (IMF), n=1; polycythemia vera (PV), n=1). In normal bm, EDC were found to react with Ab against vimentin, tenascin, alpha-smooth muscle actin, osteocalcin, CD51, and CD56, but did not react with Ab against CD3, CD15, CD20, CD34, CD45, CD68, or CD117. An identical phenotype of EDC was found in AML, MDS, SAA, ET, IMF, PV, myeloregenerative bm, and peripheral bones lacking active hemopoiesis (talus, phalanx). In patients with CML, EDC reacted with Ab to CD51, but did not react with Ab to CD56. Based on their unique antigen profile, EDC were enriched from normal bm by enzyme digestion and cell sorting. However, these enriched cells (CD56+, CD45-, CD34-) did not give rise to hemopoietic cells under the culture conditions used, i.e. in the presence of the growth factors IGF-1, bFGF, SCF, IL-3, and GM-CSF Together, our data do not support the hypothesis that EDC are totipotent mesenchymal progenitors giving rise to hemopoietic cells.

Classification of intestinal T-cell neoplasms and their differential diagnosis.

We studied 42 intestinal T-cell lymphomas (ITLs) to establish a simple and reproducible classification. The ITLs were classified into pleomorphic small cell (n = 3), monomorphic medium-sized cell (n = 10), pleomorphic medium and large cell (n = 17), immunoblastic (n = 1), and anaplastic large cell (n = 9) lymphomas. Two cases were unclassifiable. Overlapping histologic features were noted between some cases and also within the same tumor and between multiple lesions of the same patient. Histologic evidence of enteropathy was present in most cases. The ITLs expressed cytoplasmic CD3 and antigens associated with cytolytic function (TIA-1, granzyme B), but not CD4 and CD5. Six of the 10 monomorphic medium-sized cell lymphomas were CD56+ T-cell lymphomas. Because of the histologic heterogeneity in some ITLs, we suggest a classification system with 2 main categories: (1) small to medium-sized cell, comprising pleomorphic small and monomorphic medium-sized cell lymphomas; and (2) large cell, comprising the remaining subtypes. The differential diagnosis includes B-cell lymphomas, tumors of histiocytic origin, anaplastic carcinoma, and malignant melanoma.

Most CD56+ intestinal lymphomas are CD8+CD5-T-cell lymphomas of monomorphic small to medium size histology.

The expression of the natural killer (NK) cell marker CD56 has been reported to occur in NK cell lymphomas/leukemias and a small group of peripheral T-cell lymphomas but has not been studied extensively in primary intestinal non-B-cell lymphomas. Normal human jejunal intraepithelial lymphocytes (IELs) are mainly T-cell receptor (TCR)-alphabeta+CD3+CD8+CD5low and include an approximately 15% fraction of CD56+ cells that could be the cells of origin for CD56+ intestinal T-cell lymphoma (ITL). To test this hypothesis, 70 cases diagnosed as ITL were immunophenotyped, and 15 CD56+ cases (21%) were identified. The majority of the CD56+ lymphomas was of monomorphic small to medium-sized histology, shared the common phenotype betaF1+/-CD3epsilon/cyt+CD8+CD4-CD5-CD57-TIA-1+ and had clonally rearranged TCR gamma-chain genes. In contrast, the CD56- lymphomas were mainly composed of pleomorphic medium and large cells or had a morphology most consistent with anaplastic large-cell lymphoma and were mostly CD8-. These findings suggest that the majority of CD56+ intestinal lymphomas are morphologically and phenotypically distinct T-cell lymphomas most likely derived from activated cytotoxic CD56+CD8+ IELs. Some overlapping histological and clinical features between CD56+ and CD56- ITLs indicate that the former belong to the clinicopathological entity of ITL. The consistent expression of cytotoxic-granule-associated proteins introduces ITL (both CD56+ and CD56-) into the growing family of usually aggressive extranodal lymphomas of cytotoxic T-cell and NK-cell derivation. In contrast to putative NK-cell lymphoma of the sinonasal region, intestinal NK-cell lymphoma seems to be very rare.

Fas ligand expression in nodal non-Hodgkin's lymphoma.

Fas (Apo-1/CD95) ligand (FasL) is a cytotoxic molecule used by T lymphocytes and natural killer cells for target-cell killing and by nonmalignant and malignant cells in the suppression of immune responses. In this study, FasL expression in B- and T-cell non-Hodgkin's lymphomas was investigated by paraffin immunohistochemical analysis. FasL expression was found to be weak in nonaggressive lymphomas (chronic lymphocytic leukemia/small lymphocytic lymphoma, lymphoplasmacytoid lymphoma, Grade 1 follicular center cell lymphoma) and mantle cell lymphoma but strong in aggressive B-cell lymphomas (diffuse large B-cell lymphoma, Burkitt's-lymphoma). Precursor B-lymphoblastic lymphomas were more heterogeneous, with expression varying from weak to strong. In T-cell lymphomas (anaplastic large-cell lymphoma; peripheral T-cell lymphoma, unspecified), strong FasL expression was observed. Apparently, FasL expression is not limited to neoplasms derived from T cells or natural killer cells, and it might play a supporting role in the progression of non-Hodgkin's lymphomas.

High proportion of granzyme B-positive (activated) intraepithelial and lamina propria lymphocytes in lymphocytic gastritis.

Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LpLs) have not been well studied in gastric mucosa, particularly in lymphocytic gastritis. Therefore, they were immunohistologically characterized with antibodies recognizing CD3, CD8, CD57, T cell-restricted intracellular antigen (TIA-1), and granzyme B (GrB). The TIA-1 labels cytotoxic granules of resting and activated T-cells, whereas GrB decorates activated cytotoxic T cells. Thirty patients with celiac disease, including 20 taking gluten and 10 on a gluten-free diet, 15 patients with nonceliac disease-associated lymphocytic gastritis, and 20 controls were studied. Stained cells were counted and results were given as IELs/100 epithelial cells or percentage of lamina propria cells. Sixty percent to 90% of CD3+ IELs and up to 12% of lamina propria cells contained TIA-1-positive cytotoxic granules. The number of GrB+ IELs and LpLs was increased in Helicobacter pylori-positive controls (p < 0.03 vs. H pylori-negative controls) and celiac disease patients taking gluten (p < 0.05 vs. controls). The highest number of GrB+ IELs and LpLs was found in nonceliac disease-associated lymphocytic gastritis (p < 0.009 vs. controls, p < 0.05 vs. celiac disease). This study shows that a high proportion of gastric IELs and LpLs is potentially cytotoxic in nature. Through stimuli not yet identified, a proportion of them becomes activated after H pylori infestation and in lymphocytic gastritis.

Characterization of the inflammatory infiltrate in autoimmune cholangitis. A morphological and immunhistochemical study.

Autoimmune cholangitis (AIC) is characterised by clinical and/or laboratory features of cholestasis, the presence of antinuclear antibodies and the lack of antimitochondrial antibodies. Histologically, changes largely identical to those found in primary biliary cirrhosis (PBC) are typically found. It is not possible to differentiate between AIC and PBC on conventional morphological grounds, and we therefore wished to find whether there is a difference between these entities in the composition of the inflammatory infiltrate leading to bile duct destruction. In liver biopsies from ten patients with confirmed AIC and ten patients with PBC the inflammatory infiltrate was characterised with antibodies against CD 3, OPD 4 CD 8, GB 7, L 26, CD 56 and CD 57. In AIC, T cells were predominant in the portal inflammatory infiltrate in nine cases. Granzyme B-positive activated cytolytic T lymphocytes were found in the bile duct epithelium in five cases. All these five cases showed inflammatory bile duct destruction. No significant differences between the immunohistochemical findings in AIC and in PBC were found. We suggest that AIC is a subgroup of PBC, antimitochondrial antibody-negative type.

Intraepithelial lymphocytes in normal human intestine do not express proteins associated with cytolytic function.

Human small intestine contains a very large population of intraepithelial T lymphocytes (IELs) that are oligoclonal, appear functionally to be cytolytic T cells, and may contribute to the normal and pathological turnover of intestinal epithelial cells. This report addresses the cytolytic function of IELs in normal small intestine by examining their expression of molecules that carry out cell-mediated cytolysis. Immunohistochemical analyses of granzyme B, perforin, Fas ligand, and tumor necrosis factor-alpha demonstrated these proteins were not expressed by small intestinal IELs in situ. These proteins also were not expressed by colonic IELs or by lamina propria lymphocytes in the small or large intestine. Granzyme A, however, was expressed by a large fraction of IELs. In contrast to these in situ results, isolated and in vitro activated IELs were shown to express effector proteins consistent with cytolytic T cells, including granzyme B, Fas ligand, tumor necrosis factor-alpha, and interferon-gamma. These results are most consistent with the vast majority of IELs in normal human small intestine being resting cytolytic T cells and suggest that these cells do not contribute to the apoptotic cell death of epithelial cells in normal intestine.

Blood groups Lewis(b) and ABH expression in gastric mucosa: lack of inter-relation with Helicobacter pylori colonisation and occurrence of gastric MALT lymphoma.

BACKGROUND: Blood group Lewis(b) antigens mediate Helicobacter pylori attachment to gastric mucosa with attachment being particularly strong in subjects with ABH blood group O. AIMS: To determine whether H pylori colonisation or the occurrence of gastric mucosa associated lymphoid tissue (MALT) lymphomas might be related to gastric Lewis(b) expression or occurrence of particular ABH blood groups on gastric mucosa. PATIENTS: Gastric resection specimens from 89 cases with gastric MALT lymphoma and gastric mucosal biopsy specimens from 95 patients undergoing upper endoscopy due to upper gastrointestinal complaints, including five cases with gastric MALT lymphoma, were studied. METHODS: H pylori was visualised with the Warthin-Starry stain. Immunostaining (Lewis(b), Lewis(a), A, B) was performed by applying a three step immunoperoxidase technique and indirect immunofluorescence staining on formalin fixed and paraffin wax embedded tissue. In 40 patients red blood cell Lewis phenotype and ABH blood groups were additionally determined by haemagglutination assay. RESULTS: Gastric surface epithelial cells showed an immunoreactivity to blood groups A, B, and AB in 80 (43.5%), 22 (12%), and 11 (6%) cases respectively and no immunoreactivity to any of these blood group substances (blood group O) in 71 (38.5%) patients. Lewis(b) expression of all gastric surface epithelial cells (secretor status) was found in 130 (70.7%) cases. Lewis(a) expression of all gastric surface epithelial cells (non-secretor status) was found in 36 (19.6%) cases, secretor status remained unclassified in 18 (9.8%) patients. Colonisation with H pylori was found in 134 (72.8%) cases. The occurrence of H pylori was neither significantly associated with secretor status nor with certain ABH blood groups. The infiltration of gastric mucosa with MALT lymphoma was highly significantly associated with H pylori colonisation (p < 0.0003) but neither with secretor status nor with certain ABH blood groups. There was no inter-relation between secretor status or ABH blood groups and type, stage, grade of, and survival after MALT lymphoma. CONCLUSION: This study failed to show an inter-relation between secretor status or particular ABH blood groups and either H pylori infection or the occurrence of gastric MALT lymphomas.

Variability of nm23-H1/NDPK-A expression in human lymphomas and its relation to tumour aggressiveness.

The nm23-H1 gene is a putative metastasis-suppressor gene encoding a 17 kDa protein with nucleoside diphosphate kinase activity. Expression of nm23-H1/NDPK-A correlates inversely with the metastasising potential of some human tumours and experimental animal cells. No nm23 expression studies exist for human malignant lymphomas so far. In this study, we examined nm23-H1 expression by Northern and immunohistochemical analysis in 106 primary lymphoma samples from patients with Hodgkin's disease (HD) (n = 15), high-grade non-Hodgkin's lymphoma (NHL) from different lineages (n = 71) and low-grade NHL (n = 20). Both inter- and intra-subtype variations in nm23-H1/NDPK-A expression levels were demonstrated by all disease subtypes. Besides this heterogeneity, a general trend towards highly malignant samples expressing higher nm23-H1/NDPK-A, levels than the low-grade lymphomas was observed. Both adult and childhood HD and high-grade NHL samples exhibited significantly higher NDPK-A expression than the low-grade NHL found only in adults. High nm23-H1/NDPK-A levels in lymphoma samples did not always reflect proliferative activity of tumour cells as monitored by Ki-67 antigen staining. Fifty samples were further investigated for possible mutations in the nm23-H1 coding sequence by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and single-strand conformation polymorphism (SSCP) analysis. No mutation was found by this screening. Our results suggest a role for nm23-H1 expression in the disease aggressiveness of lymphomas.

Expression of the monoclonal antibody HECA-452 defined E-selectin ligands in Langerhans cell histiocytosis.

The cutaneous lymphocyte associated antigen (CLA) recognized by the monoclonal antibody (moAb) HECA-452 plays a major role in the homing of lymphocyte subpopulations to the skin by binding to E-selectin on dermal microvessels. The factors responsible for the immigration of Langerhans cells (LC) and their precursors into the skin are still unknown, but because normal resting LC are also capable of expressing CLA, the antigen was proposed as a candidate LC-homing structure. To gain insight into these mechanisms, the expression of HECA-452 on neoplastic LC within and outside the skin was investigated in paraffin-embedded sections from 44 patients with localized and disseminated forms of Langerhans cell histiocytosis (LCH) presenting with proliferating cells positive for CD45, CD1a, S100 and HLA-DR. Irrespective of the clinical presentation or the type of organ involved, HECA-452 positive LC were detected in all biopsies tested (range 5->90%). The most prominent HECA-452 reactivity was observed in skin lesions and in areas with accumulations of eosinophilic granulocytes. Our data provide evidence for heterogeneous expression of sLex/sLea structures in various stages of activated and/or differentiated LCH cells. Remarkably, CLA-antigen expression on LCH-cells was not restricted to cutaneous sites. In view of recent findings on the expression of HECA-452 on resting epidermal LC, our data are compatible with the view that local cytokine production by keratinocytes or cells from the surrounding infiltrate induce and/or modulate CLA expression on LC in both cutaneous and extra-cutaneous sites.

[Characterization of inflammatory infiltrates in autoimmune cholangitis: an immunohistochemical study]. / Charakterisierung des Entzündungszellinfiltrats bei Autoimmun-Cholangitis: Eine Immunhistochemische Studie.

Autoimmune cholangitis is clinically characterized by cholestasis and serologically by the presence of antinuclear antibodies (ANA) and a lack of antimitochondrial antibodies (AMA). Morphologic characteristics are non suppurative bile duct destruction (NSBDD), ductopenia and occasionally granulomas. The aim of the study was to characterize the inflammatory infiltrate to find about the possible mechanism of bile duct destruction in AIC. Liver biopsy material of ten patients (9 female/1 male, mean age of 45,5 a) with the clinical and serologic features of AIC were evaluated morphologically for the presence of NSBDD, ductopenia and granulomas. The inflammatory infiltrate was characterized immunohistochemically using Ab against CD3 (pan T lymphocytes), OPD 4 (helper T lymphocytes and memory T lymphocytes), CD8 (cytotoxic T lymphocytes), GB4 (activated cytotoxic T lymphocytes), L26 (pan B lymphocytes), CD56 and CD57 (NK/K cells). Lymphocyte subsets were investigated with regard to quantity and distribution within liver tissue. For control ten cases of PBC were also investigated simultaneously. NSBDD was found in five, granulomas in three cases. Portal inflammation was seen in all cases. In 9 cases T cells outweighed B cells. GB4 positive activated cytotoxic T lymphocytes were found within bile duct epithelium in five cases. All these cases showed associated NSBDD. NK/K cells were seen infrequently. Similar findings were observed in the ten cases of PBC. We therefore conclude that bile duct destruction in AIC is mediated by activated cytotoxic T lymphocytes. AIC cannot be differentiated from PBC histologically or by immunomorphology.

Expression of monoclonal antibody HML-1-defined alpha E beta 7 integrin in cutaneous T cell lymphoma.

Considering that integrins may play a major role in the localization in distinct biological features as well as in the dissemination of several types of lymphomas, we studied the expression of the monoclonal antibody HML-1-defined alpha E beta 7 integrin (CD103) in the clinically and histologically determined stages of 53 mycosis fungoides (MF) skin biopsies and in 16 affected lymph nodes. Immunoperoxidase staining revealed HML-1 immunoreactivity with T cells in the early stages of disease (patch and plaque stage MF). HML-1 expression was more pronounced on infiltrating epidermal than on dermal T cells. In contrast to early stages, tumor stage MF and lymph nodes affected in the course of cutaneous T cell lymphoma were HML-1 negative. We found a strong association between HML-1 expression, epidermotropism of infiltrating T cells, and the stage of disease. We provide evidence that: 1) the loss of the HML-1 antigen on T cells in MF is a marker of poor prognosis and 2) because the HML-1 antigen is selectively expressed on T lymphocytes of epithelial sites such as gut and skin, our results are compatible with the view that alpha E beta 7 integrins perform homing receptor functions for epitheliotropic T cells.