Molecular Pathology of Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell neoplasm of highly pleomorphic lymphoid cells. ATLL is usually widely disseminated, and it is caused by human T-cell leukemia virus type 1 (HTLV-1). It is a disease with a long latency, and affected individuals are usually exposed to the virus very early in life. The cumulative incidence of ATLL is estimated to be 2.5% among HTLV-1 carriers. ATLL cells express CD2, CD3, CD5, CD4, and CD25, as well as CCR4 and FoxP3 of the regulatory T-cell marker. HTLV-1 is causally linked to ATLL, but infection alone is not sufficient to result in neoplastic transformation. A significant finding in this connection is that the Tax viral protein leads to transcriptional activation of many genes, while the HTLV-1 basic leucine zipper factor is thought to be important for T-cell proliferation and oncogenesis. Half of ATLL cases retain the ability to express HTLV-1 Tax, which is a target of HTLV-1-specific cytotoxic T lymphocytes (CTL). An increase in HTLV-1-specific CTL responses is observed in some asymptomatic HTLV-1 carriers. Although HTLV-1-specific CTL are also present in the peripheral blood of ATLL patients, they do not expand sufficiently. We investigated the clinicopathological features and analyzed the staining of Tax-specific CTL and FoxP3. Tax-specific CTL correlated inversely with FoxP3, an increase in the ratio of CD163+ tumor-associated macrophages was associated with worse clinical prognosis, and ATLL cell lines proliferated significantly following direct co-culture with M2 macrophages. Several clinical variants of ATLL have been identified: acute, lymphomatous, chronic, and smoldering. Oligo-array comparative genomic hybridization revealed that genomic loss of 9p21.3 was a significant characteristic of acute-type, but not of chronic-type ATLL. Furthermore, we found that genomic alteration of CD58, which is implicated in immune escape, is more frequently observed in acute than in chronic ATLL. Interestingly, the chronic cases with cell cycle deregulation and disruption of immunosurveillance mechanism were associated with faster progression to acute ATLL. Immune evasion, microenvironment, and genetic alteration are therefore important in the multi-step progression of ATLL lymphomagenesis.

Purification, characterization and docking studies of the HIN domain of human myeloid nuclear differentiation antigen (MNDA).

The HIN domain of myeloid nuclear differentiation antigen (MNDA) was expressed and purified as a monomer using E. coli JM109 as host. The protein interacted with double-stranded DNA at a Kd of 3.15 µM and did not recognize the termini of double-stranded DNA. Isothermal titration calorimetry indicated that the interaction between the protein and double-stranded DNA is mainly mediated by electrostatic attractions and hydrogen bonding. We developed a model to analyze the potential DNA binding site of the MNDA HIN domain. Based on the model, molecular docking and mutation studies suggest that the double-stranded DNA binding site of the protein is different from other HIN-DNA structures. This work facilitates the design of specific drugs against pathogens detected by human MNDA.

Refolded recombinant Siglec5 for NMR investigation of complex carbohydrate binding.

Sialic-acid-binding immunoglobulin-like lectin (Siglec5) is a carbohydrate-binding surface receptor expressed on neutrophils, monocytes and B cells in human lymphoid and myeloid cell lineages. Existing structural and functional data fail to define the clear ligand specificity of Siglec5, though like other Siglec family members, it binds a variety of complex carbohydrates containing a sialic acid at the non-reducing terminus. Prokaryotic expression of this protein has proven challenging due to disulfide bonds and Asn-linked glycosylation. We developed an expression and purification protocol that uses an on-column strategy to refold Escherichia coli expressed protein that produced a high yield (2 mg/L) of the single N-terminal Siglec5 carbohydrate recognition domain (CRD). A 2D heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectrum showed this material was folded, and a secondary structure prediction based on the assigned chemical shifts of backbone atoms was consistent with a previously determined X-ray model. NMR chemical shift mapping of Siglec5 binding to three carbohydrate ligands revealed similarities in binding interfaces and affinities. In addition, the role of alternate protein conformations identified by NMR in ligand binding is discussed. These studies demonstrate the Siglec5 CRD alone is sufficient for binding sialylated carbohydrates and provide a foundation for further investigation of Siglec5 structure and function.

EndoCAM: a novel endothelial cell-cell adhesion molecule.

Cell-cell adhesion is controlled by many molecules found on the cell surface. In addition to the constituents of well-defined junctional structures, there are the molecules that are thought to play a role in the initial interactions of cells and that appear at precise times during development. These include the cadherins and cell adhesion molecules (CAMs). Representatives of these families of adhesion molecules have been isolated from most of the major tissues. The notable exception is the vascular endothelium. Here we report the identification of a cell surface molecule designated "endoCAM" (endothelial Cell Adhesion Molecule), which may function as an endothelial cell-cell adhesion molecule. EndoCAM is a 130-kD glycoprotein expressed on the surface of endothelial cells both in culture and in situ. It is localized to the borders of contiguous endothelial cells. It is also present on platelets and white blood cells. Antibodies against endoCAM prevent the initial formation of endothelial cell-cell contacts. Despite similarities in size and intercellular location, endoCAM does not appear to be a member of the cadherin family of adhesion receptors. The serologic and protease susceptibility characteristics of endoCAM are different from those of the known cadherins, including an endogenous endothelial cadherin. Although the precise biologic function of endoCAM has not been determined, it appears to be one of the molecules responsible for regulating endothelial cell-cell adhesion processes and may be involved in platelet and white blood cell interactions with the endothelium.

Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells.

The integrity of the endothelial layer, which lines the entire cavity of the vascular system, depends on tight adhesion of the cells to the underlying basement membrane as well as to each other. It has been previously shown that such interactions occur via membrane receptors that determine the specificity, topology, and mechanical properties of the surface adhesion. Cell-cell junctions between endothelial cells, in culture and in situ, involve both Ca(2+)-dependent and -independent mechanisms that are mediated by distinct adhesion molecules. Ca(2+)-dependent cell-cell adhesion occurs mostly via members of the cadherin family, which locally anchor the microfilament system to the plasma membrane, in adherens junctions. Ca(2+)-independent adhesions were reported to mainly involve members of the Ig superfamily. In this study, we performed three-dimensional microscopic analysis of the relative subcellular distributions of these two endothelial intercellular adhesion systems. We show that cadherins are located at adjacent (usually more apical), yet clearly distinct domains of the lateral plasma membrane, compared to PECAM-1. Moreover, cadherins were first organized in adherens junctions within 2 h after seeding of endothelial cells, forming multiple lateral patches which developed into an extensive belt-like structure over a period of 24 h. PECAM-1 became associated with surface adhesions significantly later and became progressively associated with the cadherin-containing adhesions. Cadherins and PECAM-1 also differed in their detergent extractability, reflecting differences in their mode of association with the cytoskeleton. Moreover, the two adhesion systems could be differentially modulated since short treatment with the Ca2+ chelator EGTA, disrupted the cadherin junctions leaving PECAM-1 apparently intact. These results confirm that endothelial cells possess distinct intercellular contact mechanisms that differ in their spatial and temporal organization as well as in their functional properties.

Heme carrier protein (HCP-1) spatially interacts with the CD163 hemoglobin uptake pathway and is a target of inflammatory macrophage activation.

Macrophages constitute the major cellular compartment for hemoglobin (Hb) degradation and subsequent recycling of heme-iron to erythropoiesis. Dysregulation of macrophage iron and heme metabolism is a major pathophysiologic determinant of anemia of chronic disease. In this study, we show that the heme transporter heme carrier protein 1 (HCP-1) is expressed in human macrophages. Within early endosomes, HCP-1 colocalizes with endocytosed Hb-haptoglobin (Hp) complexes, which are taken up via the CD163 scavenger receptor pathway. Hb-Hp passes the divalent metal transporter 1B/HCP-1-positive endosomal compartment on its route from the cell surface to lysosomes. HCP-1 mRNA and protein expression are down-regulated by stimulation of macrophages with various TLR agonists and IFN-gamma. The profound suppression of HCP-1 expression by inflammatory macrophage activation parallels the regulation of the iron exporter ferroportin. In contrast, dexamethasone enhanced HCP-1 expression significantly. Given the spatial relationship, we propose that the Hb scavenger receptor CD163 and HCP-1 constitute a linked pathway for Hb catabolism and heme-iron recycling in human macrophages.

Langerhans cell histiocytosis of a metatarsal bone in an adult female.

Langerhans cell histiocytosis (LCH) commonly occurs in children. It mimics infection and many benign and malignant tumours. This disease mainly involves the spine, skull and long bones, and its incidence is sporadic in the small bones of the foot and hand. We could not find any case reports with the involvement of a metatarsal bone, and hence, awareness about its possibility is essential to suspect it as a differential diagnosis of lytic lesions in the foot bones and therefore treat it judiciously. We have reported a case of a 35-year-old woman with spontaneous onset of pain over her right foot for the last year. An extensive curettage was performed, where the histology confirmed the features of LCH. Awareness about this entity and its differential diagnosis may help to clinch and early diagnosis and to treat effectively.

Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N.

To determine the primary structure of CD13, a 150-kD cell surface glycoprotein originally identified on subsets of normal and malignant human myeloid cells, we isolated the complete sequences encoding the polypeptide in overlapping complementary DNA (cDNA) clones. The authenticity of our cDNA clones was demonstrated by the ability of the coding sequences, subcloned in a retroviral expression vector, to mediate expression of bona fide CD13 molecules at the surface of transfected mouse fibroblasts. The nucleotide sequence predicts a 967 amino acid integral membrane protein with a single, 24 amino acid hydrophobic segment near the amino terminus. Amino-terminal protein sequence analysis of CD13 molecules indicated that the hydrophobic segment is not cleaved, but rather serves as both a signal for membrane insertion and as a stable membrane-spanning segment. The remainder of the molecule consists of a large extracellular carboxyterminal domain, which contains a pentapeptide consensus sequence characteristic of members of the zinc-binding metalloprotease superfamily. Sequence comparisons with known enzymes of this class revealed that CD13 is identical to aminopeptidase N, a membrane-bound glycoprotein thought to be involved in the metabolism of regulatory peptides by diverse cell types, including small intestinal and renal tubular epithelial cells, macrophages, granulocytes, and synaptic membranes prepared from cells of the central nervous system.

Correlation of the topographical arrangement and the functional pattern of tissue-infiltrating macrophages in giant cell arteritis.

End organ ischemia, fragmentation of elastic membranes, and aneurysm formation in patients with giant cell vasculitis results from an inflammation destroying the mural layers of large and medium sized arteries. Although the inflammatory infiltrate extends through all layers of the affected blood vessel, the most pronounced changes involve the intima and the internal elastic lamina. Analysis of the functional profile of tissue infiltrating CD68+ cells demonstrates that different subsets of macrophages can be distinguished. TGFbeta1-expressing CD68+ cells coproduce IL-1beta and IL-6, are negative for inducible nitric oxide synthase (iNOS), and exhibit a strong preference for localization in the adventitia. The adventitial homing of TGFbeta1+ CD68+ cells places them in the vicinity of IFN-gamma secreting CD4+ T cells which also accumulate in the exterior layer of the artery. Conversely, iNOS expressing CD68+ cells are negative for TGFbeta and are almost exclusively found in the intimal layer of the inflamed artery. The intimal-medial junction is the preferred site for 72-kD collagenase expressing CD68+ cells. Thus, TGFbeta1-producing macrophages colocalize with activated CD4+ T cells and home to an area of inflammation which is distant from the site of tissue damage but critical in regulating cellular influx, suggesting that TGFbeta1 functions as a proinflammatory mediator in this disease. iNOS- and 72-kD collagenase-producing macrophages accumulate at the center of pathology implying a role of these products in tissue destruction. These data indicate that the microenvironment controls the topographical arrangement as well as the functional commitment of macrophages.

The receptor for advanced glycation end products (RAGE) is a central mediator of the interaction of AGE-beta2microglobulin with human mononuclear phagocytes via an oxidant-sensitive pathway. Implications for the pathogenesis of dialysis-related amyloidosis.

An important component of amyloid fibrils in dialysis-related amyloidosis is a form of beta2microglobulin modified with advanced glycation end products (AGEs) of the Maillard reaction, known as AGE-beta2M. We demonstrate here that the interaction of AGE-beta2M with mononuclear phagocytes (MPs), cells important in the pathogenesis of the inflammatory arthropathy of dialysis-related amyloidosis, is mediated by the receptor for AGEs, or RAGE. 125I-AGE-beta2M bound to immobilized RAGE or to MPs in a specific, dose-dependent manner (Kd approximately 53.5 and approximately 81.6 nM, respectively), a process inhibited in the presence of RAGE blockade. AGE-beta2M-mediated monocyte chemotaxis was prevented by excess sRAGE or anti-RAGE IgG. Induction of tumor necrosis factor-alpha (TNF) expression by MPs exposed to AGE-beta2M resulted from engagement of RAGE, as appearances of TNF transcripts and TNF antigen release into culture supernatants were prevented by addition of sRAGE, a process mediated, at least in part, by oxidant stress. AGE-beta2M reduced cytochrome c and the elaboration of TNF by MPs was inhibited by N-acetylcysteine. Consistent with these data, immunohistochemical studies of AGE-laden amyloid deposits of a long-term hemodialysis patient revealed positive staining for RAGE in the MPs infiltrating these lesions. These data indicate that RAGE is a central binding site for AGEs formed in vivo and suggest that AGE-beta2M-MP-RAGE interaction likely contributes to the initiation of an inflammatory response in amyloid deposits of long-term hemodialysis patients, a process which may ultimately lead to bone and joint destruction.

Phenotypic and functional characterization of porcine granulocyte developmental stages using two new markers.

Here, we describe two new surface antigens, named 6D10 and 2B2, whose expression is restricted to porcine granulocytes. 6D10 is only detected in neutrophils and its expression decreases from promyelocytes to mature cells. By contrast, 2B2 antigen is selectively expressed in mature neutrophils, eosinophils and basophils. The expression of these antigens along granulocyte maturation allows the discrimination of several developmental stages of granulocytes based on phenotypic, morphological and functional characteristics previously established. Moreover, these new markers are useful tools to easily characterize the different granulocytes lineages (neutrophils, eosinophils and basophils). By using multiparameter flow cytometric analysis, we have performed a phenotypic and functional characterization of the granulocyte subsets identified by the combination of 6D10 and 2B2 antigens.

Phenotypic variation in myocardial macrophage populations suggests a role for macrophage activation in SIV-associated cardiac disease.

Cardiac abnormalities are common in HIV-infected individuals, and have been especially well documented as contributors to mortality in HIV-infected children. Underlying pathogenetic mechanisms responsible for myocardial disease in HIV-infection remain imperfectly understood. SIV-infected rhesus monkeys develop a spectrum of cardiac lesions similar to those seen in HIV-infected people, providing an important model for pathogenesis studies. Retrospective analysis of cardiac tissue collected at necropsy from SIV-infected rhesus monkeys was performed to evaluate myocardial macrophage and dendritic cell populations as a function of previously quantitated lymphocytic inflammatory infiltrates and cardiomyocyte degeneration or necrosis. Variations in the size and phenotype of macrophage and dendritic cell populations were examined as possible contributors to the pathogenesis of SIV-associated inflammatory lesions. Macrophages labeling immunohistochemically for CD163 differed substantially from macrophages labeling for HAM56 in overall number, distribution across groups, involvement in inflammatory clusters, correlation with the DC-SIGN(+) subpopulation of macrophages, and correlation with numbers of SIV-infected cells. CD163(+) macrophages occurred in significantly higher numbers in uninflamed hearts from SIV-infected animals than in hearts from SIV-infected animals with myocarditis or uninfected controls (p < 0.01). Numbers of CD163(+) cells correlated positively with numbers of SIV-infected cells (p < 0.05) suggesting that the CD163(+) population was associated with decreased inflammatory infiltration and reduced control of virus within the heart. As CD163 has been associated with nonclassical macrophage activation and an antiinflammatory phenotype, these results suggest that a balance between classical and nonclassical activation may affect levels of inflammatory infiltration and of myocardial virus burden.

Inhibition of murine hematopoiesis by CAMAL, an inhibitor of human hematopoiesis.

CAMAL (common antigen of myelogenous acute leukemia) is an antigenic preparation isolated in this laboratory from the bone marrow or peripheral blood leucocytes of persons with myeloid leukemias and shown using an immunoperoxidase slide test to be diagnostic of these leukemias. Material further purified from CAMAL preparations, which migrates in the range of 30-35 kilodaltons (kDa) by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE), which is referred to as P30-35 CAMAL, and was previously shown to be inhibitory to colony formation by progenitor cells from normal healthy human donors in vitro. This inhibitory activity was directed toward neutrophilic granulocyte colonies (CFU-G) in particular. We now report that P30-35 CAMAL is inhibitory to colony formation by murine progenitor cells in vitro. Colonies from P30-35 CAMAL-treated cultures of murine bone marrow cells were reduced in number and in size, an effect similar to that seen in cultures of human cells. As in assays using human cells, murine CFU-G appeared to be preferentially targeted by the inhibitory activity of P30-35 CAMAL. In addition, day 10 spleen colony formation was inhibited by P30-35 CAMAL in an ex vivo assay. Hence, the effects of P30-35 CAMAL on murine progenitor cells appear to parallel the effects observed using human cells. These observations support the possibility that CAMAL might be a regulatory protein in hematopoiesis which is conserved between species.

Purification of an in vitro inhibitor of normal myelopoiesis using a monoclonal antibody directed to a common antigen of myelogenous leukemia (CAMAL).

A monoclonal antibody, CAMAL-1, has been previously shown to react specifically and at high frequencies with cells of patients with acute myelogenous leukemia (AML) and chronic granulocytic leukemia (CGL). High expression of this antigen in remission patients' bone marrow cells was shown to correlate with both relapse and lower survival times. Preliminary studies showed that material extracted from leukemic cells and eluted from CAMAL-1 immunoadsorbent columns profoundly inhibited the formation of normal colony-forming units (CFU) but had no effect on formation of such colonies from cells of patients with CGL. We have used CAMAL-1 affinity chromatography in combination with FPLC gel filtration to purify the inhibitory material from leukemic cell extracts. We have successfully isolated a 30-kd component (P30) that functions as an inhibitor of normal myelopoiesis in vitro; when P30 was added to normal progenitor cell assays it significantly inhibited the growth of normal CFU but had no inhibitory effect on the growth of CGL progenitor cells at equivalent concentrations. The inhibitory effect is preferentially directed to granulocytic progenitors. Antibodies raised to P30 reacted in Western blot analyses with affinity purified material from patients' cells as well as with a 30-kd component in the cell lysates and supernatants of the leukemic cell lines HL60 and K562; this finding suggested that the P30 inhibitory component might be produced by leukemic cells.

Only the soluble form of the scavenger receptor CD163 acts inhibitory on phorbol ester-activated T-lymphocytes, whereas membrane-bound protein has no effect.

The extracellular moiety of the hemoglobin/haptoglobin scavenger receptor CD163 (RM3/1 antigen) can be shed from monocytes and is a normal plasma component. We found that in a dose-dependent manner soluble CD163 induces a decrease in CD69 expression, a reduced [(3)H]thymidine uptake and a down-regulated matrix metalloproteinase-9 RNA expression in phorbol myristate acetate-stimulated T-cells. Co-culturing T-cells on transgenic fibroblasts, expressing membrane-bound CD163, yielded no differences compared to culture on non-transfected cells. We conclude that CD163 has at least two distinct functions: the clearance of hemoglobin in its cell-bound form and participation in anti-inflammation as a soluble factor, exhibiting cytokine-like functions.

Behavior in immunoblotting of dimeric and monomeric forms of nonspecific cross-reacting antigen (NCA-50) from normal lung.

The dimeric and monomeric forms of NCA-50 from normal lung showed differences in binding to nitrocellulose in an immunoblotting procedure. The NCA dimer showed weaker binding, it passed through nitrocellulose and was more easily washed out from the blot. The NCA monomer was bound strongly and was detected with higher sensitivity following immunoblotting.

Expression of a leukemia-associated antigen (CAMAL) in four myeloid leukemia cell lines.

We have previously described the detection and partial characterization of a common myelogenous leukemia-associated antigen (CAMAL), in CGL and ANLL patients. Both polyclonal and monoclonal (CAMAL-1) antibodies have been raised to p70 (CAMAL) and have been shown to react with both p70 and myeloid leukemia cell preparations. p70 (CAMAL) has been shown to be a monomeric protein of Mr 70,000 and pI 7.2 and was also detectable in the myeloid leukemia cell lines HL60, KG1, K562 and U937, but not in the lymphocytic cell lines Molt-4, Hut-78 and CEM by immunoprecipitation from iodinated cell samples. Using [35S] methionine-labeled cell lines and immunoprecipitation, we have demonstrated the constitutive expression of p70 as well as a major component at p58 and a number at lower molecular weights in the myeloid leukemia cell lines HL60, KG1, K562 and U937, but not in the lymphocytic leukemia cell lines Molt-4, Hut-78 and CEM. The implications of these observations are discussed.

CD33 as a target for selective ablation of acute myeloid leukemia.

The CD33 antigen is a 67-kd glycosylated transmembrane protein of the sialic acid-binding immunoglobulinlike lectin (siglec) family with immunoreceptor tyrosine-based inhibitory motifs. It is expressed on the surface of normal mature and immature myeloid cells, including colony-forming progenitor cells, and on leukemic blasts from the majority of patients with acute myeloid leukemia (AML). CD33 is not expressed by the normal stem cells, suggesting that in vivo ablation of CD33-bearing normal and leukemic myeloid cells might lead to the establishment of normal hematopoiesis by the remaining normal stem cells. However, whether there are significant numbers of CD33- leukemic stem cells is controversial. Therapeutic trials using unmodified anti-CD33 antibodies have, thus far, met with limited success. Studies with a radiolabeled anti-CD33 antibody have demonstrated rapid saturation of, and internalization by, leukemic blast cells after intravenous administration, suggesting the possibility of using an anti-CD33 antibody to deliver a cytotoxic drug. Using gemtuzumab ozogamicin (Mylotarg, a humanized anti-CD33 antibody conjugated with calicheamicin, the effectiveness of in vivo ablation of CD33+ cells to treat patients with AML was borne out by the portion of patients who achieved remission. To what extent CD33- leukemic precursors are responsible for failure to respond or for relapse following gemtuzumab ozogamicin therapy remains to be determined.

Characterization of the human myeloid cell nuclear differentiation antigen: relationship to interferon-inducible proteins.

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed specifically in cells of the granulocyte/monocyte lineage. The MNDA has been isolated by using a monoclonal antibody affinity matrix and reversed-phase high performance liquid chromatography. Its NH2-terminal sequence has been obtained, as well as additional sequence information derived from peptides produced by cyanogen bromide and SV8 protease cleavages. Meaningful similarities were observed in extended regions between the MNDA and the reported beta interferon-inducible proteins, 202 and 204, from Ehrlich ascites mouse tumor cells. An amphipathic, basic alpha-helical region, showing no similarity to the 202 and 204 proteins, exhibited close similarity to a region in the interferon response factor-2, a protein which binds the interferon stimulated response element. The relatively high number of S(T)PXX motifs present in the partial amino acid sequence of the MNDA, described herein, suggests that the MNDA binds DNA and is a transcription factor.

Isolation of a cDNA encoding CD33, a differentiation antigen of myeloid progenitor cells.

A cDNA clone encoding the human myeloid Ag CD33 was isolated from a U937 cDNA library after three rounds of transient expression in COS cells and enrichment by panning. COS cells transfected with the isolated clone expressed a surface protein recognized by the anti-CD33 mAb MY9, L1B2, and L4F3, having mass similar to but slightly smaller than the mass of CD33 expressed on myeloid cells. CD33 transcripts were found constitutively expressed in several myeloid progenitor cell lines. The cDNA sequence predicts a 40-kDa polypeptide with the typical features of a glycosylated integral membrane protein. The extracellular part of CD33 contains two Ig-like domains which are highly related to the first two domains of the neural cell myelin-associated glycoprotein and the B cell Ag CD22.