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.     

Functional and clinical relevance of CD44 variant isoform expression on B-cell chronic lymphocytic leukemia cells

BACKGROUND AND OBJECTIVE: Recent studies have shown that expression of adhesion molecules of the Ig superfamily, of integrins and of selectins allows definition of high vs low risk B-cell chronic lymphocytic leukemia (B-CLL). The proteoglycan CD44 is an adhesion molecule that may be expressed as a standard form of 85-95 KD or as several variant isoforms. The presence of certain CD44 variant (v) isoforms on neoplastic cells indicates poor prognosis in epithelial and lymphoid malignancies, as it is associated with tumor progression and metastasis. DESIGN AND METHODS: The expression of CD44 v3, 4, 5, 6, 7, 9 and 10 was analyzed in cells from 85 B-CLL patients. Indirect immunofluorescence and flow cytometry were used to identify CD44v. Functional studies were performed by analysis of adhesion to hyaluronate (HA), one CD44 ligand, and HA-induced Ca2+ influx. A variety of statistical methods were used to define phenotypic and functional differences between the various clones, to calculate survival curves, and for multivariate analyses. RESULTS: In 17/85 B-CLL (20%), one or more CD44v were detectable by indirect immunofluorescence, whereas in 68/85 cases (80%) this technique yielded negative results. However, moAb "mixes" against CD44v and patching of surface molecules on B-CLL cells have shown that all B-CLL clones express CD44v. This has been confirmed by Western blot in a number of cases. Thus, two groups of patients whose cells bear CD44v at high or low density, are distinguished. Functions of the two clonotypes were investigated, namely their adhesion to a CD44 ligand and hyaluronate (HA), and effect on HA-induced Ca2+ influx. Cells expressing high density CD44v adhere to HA-coated substrates more efficiently than cells with low density CD44v. In all clones, HA-signaling via CD44 yields Ca2+ influx. This indicates that CD44 mediates activatory signals following interaction with the ligand. INTERPRETATION AND CONCLUSIONS: The clinical relevance of these findings has been ascertained. The 17/85 cases whose cells bore high density CD44v had significantly worse prognostic features than those of patients with low density CD44v, namely more advanced disease stage, LDT < 12 months and therapy requirement. Moreover, the median survival in the former group of patients was < 5 years as opposed to > 12 years in the latter. Therefore, analysis of CD44v expression provides indications of biological and clinical relevance also in low grade lymphoproliferative disorders.     

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.     

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.     

Trans-acting factors regulate the expression of CD44 splice variants

Variant isoforms of the cell surface glycoprotein CD44 (CD44v) are expressed during development, in selected adult tissues and in certain metastatic tumor cells. CD44v differ from the standard isoform (CD44s) by up to ten additional exon sequences included by alternative splicing. By cell fusion experiments, we have obtained evidence for the existence of cell-type specific trans-acting factors recruiting CD44 variant exon sequences. Stable cell hybrids of CD44s and CD44v expressing cells indicated a dominant mechanism for variant-exon inclusion. In transient interspecies heterokaryons of human keratinocytes and rat fibroblasts, the ability of the keratinocytes to include all variant exon sequences in CD44 was conferred completely on the rat fibroblast nucleus. Fusions of cells with complex CD44 splice patterns do not permit interpretation of splice control by the relative abundance of a single trans-acting factor, but rather by (a) positively acting factor(s) recruiting variant exon sequences in the 3' to 5' direction and additional factors selecting individual exons. Since the pancreatic carcinoma cell line BSp73ASML (in contrast to the cervix carcinoma cell lines SiHa and ME180) could not transfer its specific splice pattern in cell fusions, we conclude that in some tumors, splicing is also controlled by mutation of cis-acting recognition sites.     

CD44H expression by human neuroblastoma cells: relation to MYCN amplification and lineage differentiation

The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment, and tumor metastasis. Due to the alternative splicing of the single gene, a large family of different variants or isoforms is generated. Several reports have indicated an up-regulation of CD44 variant (v) isoforms in malignant process, conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumor characterized by an aggressive and metastatic behavior in advanced stages with amplification of the MYCN protooncogene. In this report we show that the CD44 standard molecule is highly expressed in 100% of stage I-III, IVs neuroblastomas and ganglioneuromas but only in a subset of stage IV tumors. In contrast, no expression of CD44 was detected on MYCN amplified stage IV tumors, thus demonstrating a highly significant negative relationship between MYCN amplification and CD44 expression in neuroblastoma. The expression of CD44 on neuroblastoma cultured cell lines was not shown to be related to MYCN amplification but rather linked to the S-type, schwann/glial differentiation lineage. Immunochemical analysis of tumor samples with anti-CD44v3 and -v6 antibodies and Northern blot analysis of mRNA from cell lines with probes spanning exons 4-10 did not reveal any expression of splice variants on neuroblastomas of all stages and cell lines, thus ruling out a major role of these isoforms in neuroblastoma progression and metastasis.     

The prognostic value of CD44 isoform expression in endometrial cancer

Elite sport requires high training volumes. However, little is known about the relationship between training volume and performance development. This relationship appears to have an inverted U-shape. Short-term overtraining or overreaching is probably associated with insufficient metabolic recovery, resulting in a decline in ATP levels. Systemic overtraining or staleness is attributed to failure of the hypothalamus to cope with the total amount of stress. Clinically, a parasympathetic and sympathetic form has been distinguished. It is assumed that these two forms express different stages of staleness. No specific, simple, and reliable parameters are known to diagnose overreaching and overtraining in the earliest stage.     

Localization of glycosaminoglycans and CD44 in the human lacrimal gland

Recent analyses of tears indicate the presence of glycosaminoglycans as their components, but their origin remains unknown. To further understand the origin of these tear components, we investigated by immunohistochemical techniques the localization of glycosaminoglycans and CD44 human lacrimal glands obtained from 20 cadavers at autopsy. Monoclonal antibodies to CD44, a receptor for hyaluronic acid, dermatan sulfate, chondroitin sulfate, and keratan sulfate were applied to the tissue. Hyaluronic acid binding region was also used for the staining of hyaluronic acid. By light microscopy, immunoreactivity for CD44 was mostly detected on the baso-lateral membrane of acinar and ductal cells, and the vascular endothelium in the interstitium. Positive staining of hyaluronic acid was associated intensely with the basal membrane of acinar and ductal cells and weakly, faintly or not at all with their lateral membrane. Positive staining of hyaluronic acid and immunoreactivity for dermatan sulfate were detected in interstitial fibrous structures; particularly, the former was intense in the perivascular fibrous structures, and the latter along the periparenchimal fibrous structures. Immunoreactivity for chondroitin sulfate and keratan sulfate was seen in some acinar cells and the acinar and ductal lumen. By electron microscopy, immunogold particles indication chondroitin sulfate sulfate or keratan sulfate labeled secretory granules of the acinar cells. Considering the fact that CD44 is a receptor molecule for hyaluronic acid, the association of hyaluronic acid with the basal membrane and weakly or faintly with the lateral membrane of acinar and ductal cells may be attributed to the expression of CD44 on the baso-lateral membrane of the cells. Moreover, the presence of immunoreactivity for chondroitin sulfate and keratan sulfate in secretory granules of acinar cells and their lumens suggests that tears from the lacrimal gland contain these glycosaminoglycans.     

Influence of ischaemia-reperfusion injury on CD44 expression in rat small intestine

BACKGROUND: CD44 is an adhesion molecule expressed by neutrophils and lymphocytes which is involved in cell-cell and cell-matrix binding. In this study, the effect of ischaemia-reperfusion injury on CD44 messenger RNA (mRNA) and cell surface immunohistochemical expression of CD44 in the rat small intestine was evaluated. METHODS: Wistar rats (n=16) were randomized to either serve as controls (sham surgery) or to be subjected to a standardized ischaemia-reperfusion injury (suprarenal aorta occluded for 1 h followed by 1 h of reperfusion). Standardized segments of jejunum were harvested after ischaemia-reperfusion injury (ischaemic and reperfused samples) to measure the mucosal protein and DNA content, mRNA expression of CD44 and the immunohistochemical expression of CD44. RESULTS: Reperfusion significantly damaged the jejunal mucosa, e.g. mucosal protein content was lower after reperfusion compared with that in the control group (z=-2.31, P=0.02) and the ischaemic samples (z=-2.52, P=001). The expression of cell surface CD44 protein was also significantly decreased after ischaemic injury (z=-1.99, P=0.04); this coincided with a decrease in the amount of cytoplasmic CD44 mRNA within isolated enterocytes (z=-2.31, P=0.02). CONCLUSION: Ischaemia-reperfusion injury decreases the expression of CD44 within the jejunal mucosa. This may contribute to the failure of the gut barrier after such injury.     

Heterogeneity of human platelet density subpopulations in aggregation, secretion of ATP, and cytosolic-free calcium concentration

AIM: To investigate thrombin (500 U.L-1)-, ADP (0.1-30 mumol.L-1)-, and 5-hydroxytryptamine (5-HT, 3 mumol.L-1)-induced aggregation, secretion of ATP and cytosolic-free calcium mobilization in density subpopulations of human washed platelets. METHODS: Using Percoll discontinuous gradient. RESULTS: The human platelets were separated into high density (HD), intermediate density (ID), and low density (LD) subpopulations, and their sizes were diminished with decreasing density (r = 0.978, P < 0.01). The magnitude of aggregations by thrombin, ADP, and 5-HT was more significant in HD platelets than that in LD platelets (P < 0.01). The amount of secretion of ATP induced by thrombin and ADP in HD platelets was also much higher than that in LD platelets (P < 0.01), except for 5-HT which did not cause the ensuring release reaction in any subpopulation of human platelets. Thrombin (1500 U.L-1)-, ADP (mumol.L-1)-, and 5-HT (3 mumol.L-1)-induced cytosolic-free calcium mobilization was evaluated as well. Results showed that the resting level of cytosolic-free calcium concentration ([Ca2+]i) was the same in all subpopulations, about 80-90 nmol.L-1. However, the level of [Ca2+]i mobilization was entirely different, heightened with increasing density. CONCLUSION: The function of HD platelets was much stronger and more active than that of LD platelets in human.     

CD44 isoform expression follows two alternative splicing pathways in breast tissue

PURPOSE: To investigate the three-point Dixon technique as a method for obtaining fat-nulled images of contrast material-enhancing breast lesions with a 0.5-T open magnetic resonance (MR) imager. MATERIALS AND METHODS: Real and imaginary source images were obtained with an interleaved gradient-echo sequence with a repetition time of 550 msec and echo times of 12.8, 19.8, and 26.8 msec. Twenty-four to 28 sections were obtained in the sagittal plane with a 90 degrees flip angle, 256 x 192 matrix, 3-4.5-mm section thickness, and acquisition time of 10 minutes 54 seconds. A three-point Dixon reconstruction algorithm was used to generate water-specific, fat-specific, and combined images from the raw image data. Twelve breasts in 10 patients and one healthy volunteer were imaged. RESULTS: Three-point Dixon images were superior to extended two-point Dixon and fat-suppressed images and to images generated by means of subtraction of three-dimensional fast spoiled gradient-echo images obtained before contrast material injection from those obtained after. CONCLUSION: Three-point Dixon imaging provides a robust method for creating fat-nulled images of enhancing breast lesions in the 0.5-T open MR environment. Water-specific three-point Dixon images are successful in regions of B0 heterogeneity and are superior to fat-suppressed images. They are much less susceptible to motion artifact than are subtraction images.     

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.