Augmented expression of CD44 splice variants in lymphoproliferative disorder of the lacrimal gland in Sj?gren's syndrome

We explored the involvement of CD44 isoforms in lymphoproliferative disorder (LPD) of the lacrimal gland of a Sj?gren's syndrome patient with unilateral LPD. The CD44 variant with the v6 exon was selectively detected from infiltrating lymphocytes in the gland with LPD, but not from infiltrating lymphocytes in the normal lacrimal gland, suggesting that the CD44 v6 variant exon may be closely associated with the development of LPD in Sj?gren's syndrome.     

Expression of CD44 standard and CD44 variant 6 in human lung cancer

We immunohistochemically examined the expression of CD44 standard (CD44 st) and CD44 variant 6 (CD44 v6) in 112 cases of primary lung cancer, and their relationship to the clinical milieu, including the clinical stage. In 46 cases of squamous cell carcinoma, expression of CD44 st was observed in 45.7% of the cases, and expression of CD44 v6 was observed in 60.9%. In 43 cases of adenocarcinoma, positive staining of CD44 st and CD44 v6 was seen in 2.3% and 4.7% of the cases, respectively. None of 21 small cell carcinomas was positive for CD44 st or CD44 v6. In squamous cell carcinomas, the expression of CD44 st and CD44 v6 was observed at a rate significantly higher than in other histologic type. Most specimens positive for CD44 st stained positively for CD44 v6. Therefore, it seems likely that the CD44 expression observed in squamous cell carcinoma of the lung was a variant CD44 containing the domain encoded by variant exon 6. The expression of CD44 v6 was not related to the clinical stage. Significant association between CD44 v6 and differentiation of squamous cell carcinoma was seen; 2/7 (28.6%) for poorly differentiated, 19/31 (61.3%) for moderately differentiated, and 7/8 (87.5%) for well differentiated squamous cell carcinomas (p = 0.02 by trend test). It was previously reported that CD44 st and CD44 v6 were expressed in both normal bronchial epithelium and squamous cell metaplasia. These results suggest that the expression of CD44 v6 in squamous cell carcinoma of the lung may reflect the immunohistochemical characteristics of the tissue from which such carcinoma emerge.     

CD44 expression in the developing human retina

OBJECTIVE: This study was conducted to evaluate the cosmetic use of botulinum toxin type A (Botox), which blocks the release of acetylcholine at the presynaptic neuromuscular junction leading to an irreversible, but temporary chemical denervation muscular paralysis and weakness. This produces a significant cosmetic improvement of wrinkling in the upper face due to hyperfunctional animation. METHOD: A prospective clinical study representing our experience with this new technique is presented. Patient selection and evaluation, classification of animation lines, techniques, results and complications are discussed. In a 15-month period, 23 patients with seven anatomic sites were injected. Twenty-three patients had the lateral aspect and the inferior aspect of their squint lines injected, and 26 patients had their glabellar frownlines injected. RESULTS: Significant improvement occurred to the average depth and length of the glabellar frownlines. The subjective improvement by the patients was also significant. Regarding the crow's feet, the lateral canthal lines showed more improvement than the inferior lateral canthal lines because the latter has a greater component of zygomaticus major and minor muscle, which contributes to the inferior lateral squint line. CONCLUSION: Botox is a safe, easy-to-use, effective modality for the temporary elimination of hyperfunctioning upper-facial muscles.     

Physical association between CD155 and CD44 in human monocytes

Regulation of CD44-mediated binding to hyaluronan is critical in normal and diseased immune cell function. In earlier work by others (Shepley and Racaniello, J. Virol., 68, 1301 1309), anti-CD44 mAb blocked poliovirus binding to CD155 (the poliovirus receptor) in HeLa cells, suggesting that CD155 and CD44 may be physically associated. Here, we present evidence that CD155 and CD44 are physically associated in human monocytes. In co-modulation experiments in U937 monocytic cells, CD155 and CD44 reciprocally co-modulated. In primary human monocytes, CD 155 syn-capped with CD44. In immunofluorescence flow cytometric experiments, anti CD44 mAb inhibited up to 94% of binding by anti-CD155 mAb which blocks poliovirus binding to CD155. This inhibition was specific for CD155. Culturing monocytes increased the extent of inhibition. In addition, mAb against PRR2, a novel molecule that is related to CD 155, was inhibited by anti-CD44 in a dose-dependent manner, but not by anti-CD14. These data support the interpretation that CD155 (and related proteins) are physically associated with CD44 on monocyte cell surfaces. Although the current study does not address functional significance, we speculate that this interaction may have a role in regulating monocyte CD44 ligand binding which may be critical in pathological processes such as tumor metastasis and arthritis.     

Variant exons v6 and v7 together expand the repertoire of glycosaminoglycans bound by CD44

Isoforms of the glycoprotein CD44 are cell surface receptors for the glycosaminoglycan hyaluronate. They have been implicated in many biological processes, but their function in these is poorly understood and cannot be explained solely by hyaluronate binding. In the present work we examine the ligand binding properties of alternatively spliced CD44 variant isoforms which are functionally involved in the immune system, embryonic development, and tumor behavior. We show that these isoforms bind directly to the purified glycosaminoglycans chondroitin sulfate, heparin, and heparin sulfate, in addition to being able to bind to hyaluronate. Binding to this extended repertoire of glycosaminoglycans by CD44 depends on the inclusion of peptide sequences encoded by the alternatively spliced exons v6 and v7, and occurs both when the CD44 is solubilized from the plasma membrane and when it is expressed on intact cells. A single point mutation in the most N-terminal hyaluronate binding motif of CD44 ablates both hyaluronate and chondroitin sulfate binding, suggesting that glycosaminoglycans are bound through a common motif, and that only one of the hyaluronate binding motifs is responsible for the majority of glycosaminoglycan binding by CD44 on the cell surface. Taken together, these observations indicate that alternative splicing regulates the ligand binding specificity of CD44 and suggest that structural changes in the CD44 protein have a profound effect on the range of ligands to which this molecule can bind with potentially wide-ranging functional consequences.     

Primary ductal adenocarcinoma of the lacrimal gland

The morphological similarity of salivary and lacrimal gland tumors is known. Many clinicopathological studies and characteristics of salivary duct carcinoma, which bears histological similarities to mammary duct carcinoma, have been recently reported; however, only one case of lacrimal duct carcinoma is reported. A second case of lacrimal duct carcinoma is presented. A 67-year-old male with a painless mass in the right upper eyelid underwent total removal of the tumor mass. Microscopic examination of the tumor mass revealed ductal adenocarcinoma of the lacrimal gland, which was the equivalent of salivary duct carcinoma. The immunohistological studies of the lacrimal duct carcinoma showed similar results to those reported for salivary duct carcinoma. The recurrent tumor in the subdural spaces was removed 2 years after the initial surgery and the patient is followed as an outpatient.     

Tumors arising in the palpebral lobe of the lacrimal gland

PURPOSE: Tumors arising in the palpebral lobe of the lacrimal gland are uncommon. There have been only eight reported patients, all of whom had pleomorphic adenomas. The authors present another six patients with primary palpebral lobe tumors. METHODS: The clinical presentation, computed tomographic, and pathologic findings of these six patients were reviewed. RESULTS: Tumor types include three pleomorphic adenomas, one well-differentiated adenoid cystic carcinoma, one carcinoma in pleomorphic adenoma, and one carcinoma ex pleomorphic adenoma. All six patients presented with a painless mass in the upper outer eyelid without significant displacement of the globe, but the superficial location of these lesions led to early detection. The tumors were localized to the lacrimal gland, predominantly anterior to the orbital rim. In all six patients, the involved palpebral lobe was removed, five via a direct anterior subcutaneous approach and one by superolateral orbitotomy. In two patients (adenoid cystic carcinoma and carcinoma ex pleomorphic adenoma), repeat surgery via superolateral orbitotomy was necessary to achieve complete removal of the lacrimal gland and surrounding tissue. No recurrences were detected during follow-up (range, 6 months to 7 years; mean, 26 months). CONCLUSION: Palpebral lobe tumors of the lacrimal gland are more common and consist of a greater variety of histologic types than previously described. In our series, these tumors comprise 17% of all epithelial lacrimal gland lesions. These are the first reported cases of malignancy in this location. A longer follow-up period is necessary to determine the true recurrence rate of these tumors.     

Heterogeneity of CD44 expression among human B-cell subpopulations

CD44 is a widely distributed cell surface glycoprotein that participates in a number of cellular adhesion and signal transduction processes. Germinal center B cells express very low levels of CD44, whereas their precursors and differentiation products express much higher levels. In immunofluorescence studies comparing 20 antibodies classified as being against the hematopoietic isoform of CD44, one antibody, A1G3, was unreactive with germinal center B cells, whereas the other antibodies showed low intensity but definite reactivity. Western blotting and sequential immunoprecipitation studies of lysates from density-separated lymphocyte fractions showed two bands that were differentially expressed and reacted differently with A1G3 compared with the other CD44 antibodies. These results suggest that germinal center B cells and non-germinal center B cells express different forms of CD44. Of 21 malignant B-cell populations examined, 5 showed reactivity with a "standard" CD44 reagent and significantly reduced reactivity with A1G3, while one sample showed the opposite pattern and the remainder were positive for both reagents. Of 10 cell lines studied, 5 were differentially stained by A1G3 and a standard CD44 antibody. PCR amplification of reverse transcribed mRNA from sorted human tonsil B-cell subpopulations and Southern blotting showed that B cells express a number of splice isoforms of CD44. These results demonstrate that B cells express multiple forms of CD44; both splice insert isoforms and variants distinguished by A1G3; the form of CD44 expressed depends on B-cell differentiation state.     

Pattern of distribution of neuropeptides in the camel lacrimal gland

The pattern of distribution of neuropeptides, including neuropeptide-Y (NPY), vasoactive intestinal polypeptide (VIP), neurotensin (NT), serotonin (5-HT), galanin (GAL), leucine-enkephalin (LEU-ENK) and calcitoningene-related-peptide (CGRP), in the nerves of the camel lacrimal gland was investigated using immunohistochemical techniques. Fresh lacrimal gland segments, obtained from adult camels slaughtered in the local abattoir, were used for the immunohistochemical techniques. NPY and LEU-ENK immunoreactivity was observed in the nerve cell bodies and nerve fibers of the camel lacrimal gland. VIP, GAL and CGRP were demonstrated predominantly in fine varicose nerve fibers lying on the basolateral surfaces of the lacrimal acinar cells. NT and 5-HT were identified mainly in neurons situated in the periacinar regions, close to the basal surfaces of the acinar cells. It is concluded that the camel lacrimal nerves contain several neuropeptides including NPY, VIP, NT, 5-HT, GAL, LEU-ENK and CGRP which may modulate lacrimal fluid and protein secretion.     

Adenoid cystic carcinoma of the lacrimal gland in a six-year-old girl

A 6-year-old white female was found to have an adenoid cystic carcinoma originating in a lacrimal gland. Eighteen months following diagnosis, the tumor recurred. Conservative surgery has been the sole mode of therapy. To date, after four operations and quadrimenstral imaging surveillance, there is no sign of disease progression. Our purpose is to record the unusual occurrence of an adenoid cystic carcinoma of the lacrimal gland in a child. An interim report, 32 months after diagnosis, is presented.     

Involvement of CD44 in matrix metalloproteinase-2 regulation in human melanoma cells

CD44 is a family of cell-surface-adhesion proteins that are thought to play an important role in cancer invasion and metastasis. However, the specific mechanisms by which CD44 expression modulates invasion or metastasis are not well understood. In the current study, we have demonstrated that treatment of human melanoma cells with a CD44 MAb, F10-44-2, induces up-regulation of matrix metalloproteinase-2 (MMP-2) protein and mRNA. Moreover, treatment of melanoma cells with MAb F10-44-2 enhances their migration through gelatin-coated membranes and invasion through reconstituted basement membranes. Treatment of melanoma cells with several known CD44 ligands, including hyaluronate, extracellular-matrix proteins, and osteopontin, did not induce MMP-2 production. CD44 binding by F10-44-2 MAb results in induction of MMP-2 expression, which is associated with enhanced cell migration and invasion. These findings have several implications for investigations into tumor metastasis, development, and lymphocyte function.     

Structural variability of CD44v molecules and reliability of immunodetection of CD44 isoforms using mAbs specific for CD44 variant exon products

CD44 can be considered structurally and functionally one of the most variable surface molecules. Alternative splicing of variant exons as well as posttranslational modifications of the molecule (differences in glycosylation) generate a rich repertoire of CD44 isoforms (CD44v), some of which seem to play a key role in tumor growth and progression. Immunodetection of CD44 isoforms in vivo, using mAbs specific for CD44 variant exon products, is largely used to identify those CD44 molecules involved in tumor growth and progression and to interfere with CD44-mediated processes. In the present work we demonstrate that the immunoreactivity of some mAbs directed to CD44 exon-specific epitopes can be impaired by the structural variability of the molecule. Our findings demonstrate that (1) specific exon assortment and/or posttranslational modifications of CD44v molecules can mask CD44 exon-specific epitopes; (2) glycosaminoglycan side chains, carried by some CD44v isoforms of high molecular weight, may play a critical role in determining the exact conformation of the molecule, which is necessary for the detection of CD44 variant epitopes by specific mAbs; and (3) in a panel of stable transfectants expressing CD44 N-glycosylation site-specific mutants, generated in the constant region of CD44 extracellular domain, asparagine-isoleucine substitution is sufficient per se to impair the immunoreactivity of several mAbs to pan-CD44. Thus, conformational changes due to the alternative splicing of CD44 variant exons and/or posttranslational modifications of the molecule (different degree of glycosylation), which are cell type-specific, are likely to generate CD44 variants that elude immunodetection. These findings strongly suggest that immunohistochemical analysis of CD44 expression in vitro and in vivo, using mAbs specific for CD44 variant exon epitopes, can potentially be impaired by a large number of false negative results.     

Interaction between CD44 and hyaluronic acid regulates human prostate cancer development

PURPOSE: This study was designed to investigate the significance of the interaction between CD44 and hyaluronic acid in the development of human prostate cancer. MATERIALS AND METHODS: We transfected the standard CD44 isoform (CD44s) cDNA into PC3, a human prostate cancer cell line that barely expresses CD44s protein. The effects of the reintroduction of CD44s into PC3 cells on the ability to bind hyaluronic acid (HA) were analyzed by the cell adhesion assay and by the cell migration assay. The in vitro growth rate of CD44s transfected PC3 was measured by using the MTT assay. We then evaluated the in vivo tumor development of CD44s transfected PC3 cells by subcutaneous, intravenous, and intraperitoneal injection models in athymic nude mice. RESULTS: The introduction of CD44s in PC3 cells markedly enhanced the binding and migration of these cells to HA, but not to other extracellular matrix molecules. In vitro growth of CD44s-transfected PC3 was found to be significantly decreased. In addition, the CD44s-transfected PC3 cells also demonstrated reduced tumorigenicity and metastatic potential in in vivo experimental models. CONCLUSIONS: These findings suggest that the CD44s downregulation plays an important role in the development of human prostate cancer, in part through reduction of the ability to bind HA.     

Neoplastic human tissue mast cells express the adhesion molecule CD44/HCAM

Expression of the homing-associated cell adhesion molecule/HCAM (CD44) in normal/reactive and neoplastic human tissue mast cells (TMC) was determined immunohistochemically using the antibody DAKO-DF1485, which detects all isoforms of CD44. Studies were performed on 30 routinely processed specimens. Twenty of these, from bone marrow, skin, spleen, liver, lymph node and jejunal mucosa, contained infiltrates of TMC. These represented various types of generalized mastocytosis/systemic mast cell disease, including benign systemic mastocytosis. Ten specimens consisted of tissue with a marked reactive increase in TMC; most of these were lymph nodes with chronic nonspecific lymphadenitis and benign or malignant solid tumours. In all 30 specimens TMC exhibited an annular pattern of immunostaining, which was usually very strong. Both normal/reactive and neoplastic TMC exhibited consistent immunoreactivity with the antibody DAKO-DF1485, and this antibody may be of diagnostic value in the detection of atypical TMC associated with malignant mastocytosis. TMC and their neoplastic derivatives belong to a large family of mesenchymal and epithelial cells containing the principal surface receptor for hyaluronan.     

N-terminal and central regions of the human CD44 extracellular domain participate in cell surface hyaluronan binding

CD44 molecules are cell surface receptors for hyaluronan (HA). To define regions of the extracellular domain of CD44 that are important for HA binding, we have studied the ability of HA-blocking CD44 mAbs to bind to CD44 from a variety of sources. Five CD44 mAbs (5F12, BRIC235, 3F12, BU-75, and HP2/9) of 21 studied were identified that at least partially blocked FITC-labeled HA (HA-FITC) binding to the standard form of CD44 (CD44S) in CD44-transfected Jurkat cells. Analysis of reactivity of HA-blocking CD44 mAbs defined three distinct epitopes. Lack of reactivity of mAb 5F12 with a CD44 fusion protein (CD44-Rg) containing an N-terminal truncation of 20 amino acids (aa), as well as reactivity of mAb 5F12 with an N-terminal CD44 synthetic peptide (CD44-9A), demonstrated that the N-terminal proximal region of CD44 (aa 1 to 20) was involved in mAb 5F12 binding. A mutant cell line, CEM-NKR, derived from the T-ALL cell line, CEM, did not bind mAb 5F12 nor bind HA, whereas wild-type CEM did bind mAb 5F12 and HA. Sequence analysis of wild-type CEM and CEM-NKR CD44 cDNA demonstrated a G to A point mutation at position 575 in the CD44 cDNA of CEM-NKR, resulting in an arginine to histidine mutation at aa position 154. Taken together, our studies demonstrated that there are three epitopes to which HA-blocking mAbs bind in the extracellular domain of CD44, and that the CD44 N-terminal proximal and central regions are two regions in the extracellular domain of CD44 that may interact and either mediate or regulate HA binding to cell surface CD44.