Plasma cells in muscle in inclusion body myositis and polymyositis.

BACKGROUND: Previous immunohistochemical studies of muscle from patients with inclusion body myositis and polymyositis found many more T cells than B cells, suggesting a role for intramuscular cell-mediated immune mechanisms rather than humoral mechanisms. METHODS: Microarray studies were performed on muscle biopsy specimens from 40 patients with inclusion body myositis (IBM; n = 23), polymyositis (PM; n = 6), and without neuromuscular disease (n = 11). Reverse transcription PCR of selected immunoglobulin gene transcripts was performed on two patient samples. Qualitative immunohistochemical studies for B-cell lineage cell surface markers were performed on 28 muscle specimens and quantitative studies performed on a subset of 19 untreated patients with IBM or PM. CD138+ cells were isolated from muscle using laser capture microdissection, and immunoglobulin transcripts were PCR amplified to determine the presence or absence of immunoglobulin gene rearrangements unique to the B-cell lineage. RESULTS: Immunoglobulin gene transcripts accounted for 59% in IBM and 33% in PM of the most stringently defined highest differentially expressed muscle transcripts compared with normal. Plasma cells, terminally differentiated B cells expressing CD138 but not CD19 or CD20, are present in IBM and PM muscle in numbers several times higher than B cells. CONCLUSIONS: There are differentiated B cells in the form of CD138+ plasma cells within the muscle of patients with inclusion body myositis and polymyositis. The principle of linked recognition of B-cell activation predicts several strategies for autoantigen discovery that could not otherwise be pursued through the study of the infiltrating T-cell population alone.

Characterization of a murine high-affinity thiamine transporter, Slc19a2.

Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine Slc19a2 locus and characterized the resulting protein. Murine Slc19a2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans approximately 13kb with 6 exons, structurally identical to that of the human homolog. We localized the Slc19a2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of Slc19a2 in HEK293T cells resulted in specific uptake of [3H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of Slc19a2 protein. Immunohistochemistry studies indicate that Slc19a2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.

The value of thyroid transcription factor-1 in cytologic preparations as a marker for metastatic adenocarcinoma of lung origin.

In tissue sections, thyroid transcription factor-1 (TTF-1) is a sensitive marker for adenocarcinomas of lung and thyroid origin. This immunohistochemical study evaluates the effectiveness of TTF-1 as a marker for pulmonary adenocarcinomas in paraffin sections of cell block preparations derived from effusion and fine-needle aspiration specimens. We evaluated 122 cell blocks including 8 primary and 39 metastatic pulmonary adenocarcinomas, 11 pulmonary neoplasms of other types, 50 specimens with nonpulmonary metastatic tumors, and 14 mesotheliomas. TTF-1 was reactive in 42 (89%) of 47 pulmonary adenocarcinomas. Only 1 of 4 pulmonary small cell/neuroendocrine tumors was TTF-1 positive, while 1 of 7 squamous cell carcinomas was weakly reactive. Of 50 metastatic tumors of nonpulmonary origin, focal weak reactivity was noted only for 1 metastatic ovarian carcinoma. All mesotheliomas were nonreactive. In cytologic preparations, TTF-1 is a highly selective marker for pulmonary adenocarcinoma and also can have a role in the distinction between pulmonary adenocarcinoma and mesothelioma.

TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors.

Inflammatory myofibroblastic tumors (IMTs) are neoplastic mesenchymal proliferations featuring an inflammatory infiltrate composed primarily of lymphocytes and plasma cells. The myofibroblastic cells in some IMTs contain chromosomal rearrangements involving the ALK receptor tyrosine-kinase locus region (chromosome band 2p23). ALK-which is normally restricted in its expression to neural tissues-is expressed strikingly in the IMT cells with 2p23 rearrangements. We now report a recurrent oncogenic mechanism, in IMTs, in which tropomyosin (TPM) N-terminal coiled-coil domains are fused to the ALK C-terminal kinase domain. We have cloned two ALK fusion genes, TPM4-ALK and TPM3-ALK, which encode approximately 95-kd fusion oncoproteins characterized by constitutive kinase activity and tyrosylphosphorylation. Immunohistochemical and molecular correlations, in other IMTs, implicate non-TPM ALK oncoproteins that are predominantly cytoplasmic or pre- dominantly nuclear, presumably depending on the subcellular localization of the ALK fusion partner. Notably, a TPM3-ALK oncogene was reported recently in anaplastic lymphoma, and TPM3-ALK is thereby the first known fusion oncogene that transforms, in vivo, both mesenchymal and lymphoid human cell lineages.

Kaposi's sarcoma-associated herpesvirus gene sequences are detectable at low copy number in primary amyloidosis.

Primary amyloidosis (AL), like multiple myeloma (MM), results from a clonal proliferation of plasma cells. Recent detection of Kaposi's sarcoma-associated herpesvirus (KSHV) gene sequences in MM patients, although controversial, suggested that KSHV may also be present in AL. In the present study, we assayed for KSHV gene sequences in patients with primary AL independently in 2 laboratories. Nested polymerase chain reaction (PCR) was performed on DNA isolated from 21 bone marrow (BM) core biopsy samples to amplify orf26 and orf72, 2 regions of the KSHV genome. Eighteen of 21 (86%) BM core biopsy samples were KSHV PCR positive. BM aspirates from 16 of these 21 AL patients were cultured for 4-6 weeks to generate long term bone marrow stromal cells (LT-BMSCs), and 13 of 16 (81%) LT-BMSCs were also KSHV PCR positive. Results in all but 1 sample were consistent in the 2 laboratories. Sequencing of the PCR products in the 2 laboratories confirmed 94-98% and 95-98% homology to the published orf 26 and orf 72 KSHV gene sequences respectively, with interpatient base pair differences. Despite the presence of KSHV gene sequences, only 4/18 (22%) KSHV PCR positive patients demonstrated KSHV lytic antibodies by immunoblot assay. A sensitive assay performed on the BCBL-1 cell line confirmed the presence of KSHV at a very low copy number in AL. PCR using patient specific light chain gene primers also amplified DNA isolated from 2 AL BM core biopsies and 3 AL LT-BMSCs which were KSHV PCR positive, suggesting the presence of clonotypic cells. Our results therefore demonstrate KSHV gene sequences albeit at a very low copy number in the majority of BM core biopsies and LT-BMSCs from AL patients, and serological responses in only a minority of cases. Ongoing studies to identify viral transcripts and gene products will determine the biological relevance of KSHV in AL disease pathogenesis.

Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMTi. A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

Detection of Kaposi's sarcoma herpesvirus DNA sequences in multiple myeloma bone marrow stromal cells.

Whether Kaposi's sarcoma herpesvirus (KSHV) is associated with multiple myeloma (MM) remains controversial. We assayed for KSHV DNA sequences in long-term bone marrow stromal cells (BMSCs) from 26 patients with MM and 4 normal donors. Polymerase chain reaction (PCR) using primers which amplify a KSHV gene sequence to yield a 233-bp fragment (KS330233 within open reading frame 26) was negative in all cases. Aliquots of these PCR products were used as templates in subsequent nested PCR, with primers that amplify a 186-bp product internal to KS330233. BMSCs from 24 of 26 (92%) patients with MM and 1 of 4 normal donors were KSHV PCR+. DNA sequence analyses showed interpatient specific mutations (2 to 3 bp). Both Southern blot and sequence analyses confirmed the specificity of PCR results. The presence of the KSHV gene sequences was further confirmed by amplifying T 1.1 (open reading frame [ORF] K7) and viral cyclin D (ORF 72), two other domains within the KSHV genome. Immunohistochemical studies of KSHV PCR+ MM BMSCs demonstrate expression of dendritic cell (DC) lineage markers (CD68, CD83, and fascin). Serological studies for the presence of KSHV lytic or latent antibodies were performed using sera from 53 MM patients, 12 normal donors, and 5 human immunodeficiency virus (HIV)/KSHV+ patients. No lytic or latent antibodies were present in sera from either MM patients or normal donors. Taken together, these findings show that KSHV DNA sequences are detectable in BMSCs from the majority of MM patients, but that serologic responses to KSHV are not present. Ongoing studies are defining whether the lack of antibody response is caused by the absence of ongoing infection, the presence of a novel viral strain associated with MM, or underlying immunodeficiency in these patients.

Alterations in fascin-expressing germinal center dendritic cells in neoplastic follicles of B-cell lymphomas.

Germinal center dendritic cells (GCDCs) have essential functions in retention of immune complexes within secondary follicles, B-lymphocyte antigenic stimulation, B-cell activation, homing of B-cells via adhesion molecules such as ICAM-1 and VCAM-1, and B-cell survival via apoptosis. The neoplastic cells of follicular lymphomas (FLs) are thought to derive from follicular B lymphocytes, but their relationship to GCDCs remains unclear. This study used immunohistochemical staining for fascin, a 55-kDa actin-bundling protein strongly expressed in GCDCs and their processes, to evaluate their distribution in neoplastic follicles. Forty-two cases of B-cell FL were evaluated, and immunoreactive staining for fascin was compared with six cases of Castleman's disease (CD) and six cases of follicular hyperplasia. FLs generally revealed decreased or absent fascin-staining GCDCs, suggesting loss of fascin expression by dendritic cells in neoplastic follicles compared with hyperplastic follicular centers. In some follicles, there was partial retention of dendritic architecture with islands of residual syncytial network. Interdigitating reticulum cells in the parafollicular regions revealed normal fascin expression with intense staining of dendritic processes. In contrast with FLs, cases of follicular hyperplasia revealed normal or increased fascin-positive follicular dendritic cells, and in cases of hyaline vascular CD, follicular dendritic cells revealed tight syncytial networks. These results suggest that GCDCs are deficient in neoplastic follicles, compared with benign reactive or hyperplastic follicles. This alterations in the germinal center microenvironment might explain the inability of FL cells to present antigen to T lymphocytes and to mount an effective antitumor immune response.

Localization of Kaposi's sarcoma-associated herpesvirus in bone marrow biopsy samples from patients with multiple myeloma.

We have recently demonstrated the presence of Kaposi's sarcoma-associated herpesvirus (KSHV) in cultured bone marrow (BM) stromal dendritic cells from all patients with myeloma studied. To show that these findings were not an artifact of tissue culture, we performed in situ hybridization (ISH) and polymerase chain reaction (PCR) to detect KSHV in BM core biopsies. Using ISH to open reading frame-72 (ORF 72), we localized KSHV to BM dendritic cells in 17 of 20 patients with myeloma, 2 patients with plasmacytosis associated with the acquired immunodeficiency syndrome, and 1 case of aplastic anemia. In contrast, BM from normal subjects (n = 4) and patients with lymphoma and leukemia (n = 21) did not contain KSHV. PCR amplification with KSHV primers demonstrated product in fresh BM biopsy samples from 6 of 7 myeloma patients, whereas three normal marrows contained no amplified product. These findings suggest that KSHV, possibly through alterations in the BM microenvironment and production of viral interleukin-6 (vIL-6), may stimulate and maintain abnormal plasma cell proliferation in myeloma and related disorders.

The role of follicular and interdigitating dendritic cells in HIV-related lymphoid hyperplasia: localization of fascin.

Fascin is a 55-kDa, actin-bundling protein expressed in follicular and interdigitating dendritic cells (DCs). Because these cells play a pivotal role in the pathogenesis of human immunodeficiency virus-related lymphoid hyperplasias (HLP), we evaluated them in 49 cases by immunohistochemical localization of fascin, and we related the findings to the histologic stage of disease. Fifteen cases of early HLP revealed strong localization of fascin within DCs of hyperplastic follicles and intense staining in interdigitating DCs and their processes in the interfollicular zones. Some follicles revealed focal disruption of the fascin-positive dendritic framework. DCs were also aligned beneath the peripheral sinuses of the node. In 15 cases of progressive HLP with partial follicular involution, 3 cases revealed findings similar to those described above. Twelve cases revealed loss of follicular dendritic staining, which was moderate in five cases and marked in seven. Residual clusters of DCs remained after partial dissolution. Interfollicular dendritic staining was reduced in 13 cases, and perisinusoidal staining was reduced in 7. In late-stage HLP, staining of both follicular and interdigitating DCs was reduced or absent. Nine cases of diffuse immunoblastic or angioimmunoblastic lymphadenopathy-like proliferation had markedly reduced or absent follicular DCs, but prominent staining was present in interfollicular DCs, which formed a meshwork throughout the node in eight cases. Two cases of hyaline-vascular lymphoid hyperplasia had increased follicular DCs forming a tight syncytial network. In most cases, there was reduction of dendritic fascin-expressing cells with advanced disease and destruction of the dendritic framework of the node. Diffuse hyperplasia of fascin-positive interdigitating cells occurred in cases with angioimmunoblastic morphologic features, and cases of hyaline-vascular lymphoid hyperplasia had increased follicular dendritic histiocytes forming a tight syncytial network. Destruction of DCs and, in some cases, interdigitating cell hyperplasia might be associated with the variable clinical course of HLP.

Fascin, a sensitive new marker for Reed-Sternberg cells of hodgkin's disease. Evidence for a dendritic or B cell derivation?

Immunohistochemical localization of human fascin, a distinct 55-kd actin-bundling protein, was determined for a wide variety of lymphoid tissues (364 specimens total). In non-neoplastic tissues, reactivity was highly selective and localized predominantly in dendritic cells. In the thymus, this protein was distinctly localized to medullary dendritic cells. In reactive nodes, interdigitating reticulum cells of T zones, cells in subcapsular areas, and cells of the reticular network were reactive, with variable reactivity observed for follicular dendritic cells. Splenic dendritic cells of the white pulp and sinus-lining cells of the red pulp were reactive. Endothelial cells of all tissues exhibited variable reactivity. Lymphoid cells, myeloid cells, and plasma cells were uniformly nonreactive. In the peripheral blood, only dendritic (veiled) cells were reactive for fascin. A striking finding was observed for cases of Hodgkin's disease (total 187 cases). In all cases of nodular sclerosis (132), mixed cellularity (34), lymphocyte depletion (2), and unclassified types (5), all or nearly all Reed-Sternberg cells and variants were immunoreactive for fascin. Neoplastic cells exhibited strong diffuse cytoplasmic staining and frequently assumed dendritic shapes, particularly in the nodular sclerosis type, producing an interdigitating meshwork or syncytial network of cells. In cases of mixed cellularity type, neoplastic cells generally appeared more discrete. In all 14 cases of nodular lymphocyte predominance type, L&H variants were nonreactive. By contrast, neoplastic lymphoid cells of only 24 of 156 (15%) other lymphoid neoplasms (127 B cell, 27 T cell, and two null cell evaluated) were reactive for fascin. Fascin represents a highly effective marker for detection of certain dendritic cells in normal and neoplastic tissues, is an extremely consistent marker for Reed-Sternberg cells and variants of Hodgkin's disease (except L&H types), and may be helpful to distinguish between Hodgkin's disease and non-Hodgkin's lymphoma in difficult cases. The staining profile for fascin raises the possibility of a dendritic cell derivation, particularly an interdigitating reticulum cell, for the neoplastic cells of Hodgkin's disease, notably in nodular sclerosis type. However, as fascin expression may be induced by Epstein-Barr virus infection of B cells, the possibility that viral induction of fascin in lymphoid or other cell types must also be considered in Epstein-Barr virus-positive cases.

Lineage-restricted chromosome translocation in a benign fibrous tumor of the breast.

We describe a clonal chromosome rearrangement, t(4;14)(q24-25;q24.3), in a benign fibrous tumor of the breast. This is the first reported cytogenetic analysis of a fibrous tumor of the breast and the first evidence for clonality in these lesions. A combined immunohistochemical/cytogenetic study demonstrated that cells with the clonal translocation were mesenchymal. Epithelial cells, in contrast, had normal chromosomes.

Clonal rearrangement of chromosome band 6p21 in the mesenchymal component of pulmonary chondroid hamartoma.

Pulmonary chondroid hamartomas (PCH) are biphasic benign tumors that contain both mesenchymal and epithelial populations. In this report we describe two PCH in which clonal translocations at chromosome band 6p21 were demonstrated in mesenchymal cells. One of these had a unique translocation, t(6;14)(p21;q24), that was also found in one of two PCH karyotyped previously. The t(6;14) has not been described in other varieties of benign or malignant neoplasia. The 6p21 aberrations are of particular interest because break points in this chromosomal region appear to be characteristic of endometrial polyps. Endometrial polyps, like PCH, are biphasic benign tumors in which mesenchymal clonality has been demonstrated.

Clonal 6p21 rearrangement is restricted to the mesenchymal component of an endometrial polyp.

Previous cytogenetic analyses revealed t(6;20)(p21;q13) in two endometrial polyps. We karyotyped a large endometrial polyp in which 19 of 25 metaphase cells contained a t(1;6;4)(q21;p21;q13). Subsequent combined immunohistochemical/cytogenetic analysis showed all aberrant metaphase cells to be of mesenchymal derivation, whereas epithelial cells from the polyp were diploid. These studies indicate that rearrangement of chromosome band 6p21 is a characteristic cytogenetic aberration in the stromal component of endometrial polyps.

Myeloperoxidase: a specific marker for myeloid cells in paraffin sections.

Immunohistochemical detection of intracellular myeloperoxidase, a major constituent of primary granules of neutrophilic myeloid cells, was determined in paraffin sections of 161 specimens using a rabbit polyclonal antibody to human myeloperoxidase and an indirect immunoperoxidase technique. In normal tissues and in a variety of myeloproliferative disorders, myeloid cells of both neutrophilic and eosinophilic types, at all stages of maturation, exhibited strong cytoplasmic reactivity for myeloperoxidase. Myeloperoxidase was readily detected in myeloblasts and immature myeloid cells of acute myelogenous leukemia, progranulocytic leukemia, monomyelocytic leukemia, erythroleukemia, myeloblastomas, and other hematopoietic disorders. Erythroid precursors, megakaryocytes. other hematopoietic disorders. Erythroid precursors, megakaryocytes, lymphoid cells, mast cells, and plasma cells were nonreactive. Cells of monocytic derivation revealed variable reactivity and were typically weakly positive or nonreactive. In a few specimens, rare histiocytes were reactive, some possibly due to phagocytosed material. Cells comprising the infiltrate of a spectrum of lymphoid malignancies, e.q., lymphoblastic lymphoma or leukemia, chronic lymphocytic leukemia, hairy cell leukemia, non-Hodgkin's lymphomas of T- or B-cell type, and Hodgkin's disease, were nonreactive, as were the non-neoplastic tissues present in these specimens, except for occasional cells of myeloid derivation. Myeloperoxidase was not observed in the neoplastic cells of a wide variety of epithelial tumors and sarcomas, or in the contiguous non-neoplastic tissues. Immunoreactivity for myeloperoxidase was well preserved following fixation in a variety of fixatives, including Zenker's-acetic acid solution (employed for processing bone marrow biopsies), B5 solution, and formalin. Immunohistochemical detection of myeloperoxidase represents a sensitive and highly specific technique for identification of mature and immature myeloid cells in paraffin-embedded tissue.

Deuterium-labeled steroids for study in humans. II. Preliminary studies on estrogen production rates in pre- and post-menopausal women.

6,7-Dideuterio-3-hydroxy-1,3,5(10)-estratrien-17-one (dideuterio-estrone) and 4-deuterio-1,3,5(10)-estratriene-3,17 beta-diol (monodeuterio-17 beta-estradiol) were used for the estimation of estrogen production rates in pre- and post-menopausal women. The results are concordant with those obtained by radioisotope administration as reported in the literature. This preliminary study suggests that one or more steroids labeled with one or multiple deuterium and/or other stable isotopes may be employed for the measurement of production rates of steroid hormones which are derived from multiple precursors.

The significance of urinary free cortisol and progesterone in normal and anencephalic pregnancy.

Urinary free cortisol and progesterone were determined by radioimmunoassay in 18 normal and 16 anencephalic pregnancies and urinary free cortisol levels in 9 nonpregnant women. In normal pregnancy the urinary free cortisol (46.89 +/- 34.02 mug per 24 hours) was significantly higher (P less than 0.001) than that found with anencephaly (17.19 +/- 13.20 mug per 24 hours) and 2 1/2 times (P less than 0.001) the nonpregnant value (18.47 +/- 5.44 mug per 24 hours). In 12 of the anencephalic pregnancies, urinary free cortisol levels (11.05 +/- 5.56 mug per 24 hours) were significantly lower than in nonpregnant women (P less than 0.001). Urinary progesterone levels in normal pregnancy (15.57 +/- 9.66 mug per 24 hours) and anencephaly (18.54 +/- 12.69 mug per 24 hours) were comparable. The cortisol excretion values associated with anencephaly indicate that the normal fetus contributes substantially to the maternal plasma cortisol pool. Urinary free cortisol determinations may be a useful index of fetal adrenal dysfunction.