CD44 is a ubiquitous multistructural and multifunctional cells surface adhesion molecule involved in cell-cell and cell-matrix interactions. Twenty exons are involved in the genomic organization of this molecule. The first five and the last 5 exons are constant, whereas the 10 exons located between these regions are subjected to alternative splicing, resulting in the generation of a variable region. Differential utilization of the 10 variable region exons, as well as variations in N-glycosylation, O-glycosylation, and glycosaminoglycanation (by heparan sulfate or chondroitin sulfate), generate multiple isoforms (at least 20 are known) of different molecular sizes (85-230 kDa). The smallest CD44 molecule (85-95 kDa), which lacks the entire variable region, is standard CD44 (CD44s). As it is expressed mainly on cells of lymphohematopoietic origin, CD44s is also known as hematopoietic CD44 (CD44H). CD44s is a single-chain molecule composed of a distal extracellular domain (containing, the ligand-binding sites), a membrane-proximal region, a transmembrane-spanning domain, and a cytoplasmic tail. The molecular sequence (with the exception of the membrane-proximal region) displays high interspecies homology. After immunological activation, T lymphocytes and other leukocytes transiently upregulate CD44 isoforms expressing variant exons (designated CD44v). A CD44 isform containing the last 3 exon products of the variable region (CD44V8-10, also known as epithelial CD44 or CD44E), is preferentially expressed on epithelial cells. The longest CD44 isoform expressing in tandem eight exons of the variable region (CD44V3-10) was detected in keratinocytes. Hyaluronic acid (HA), an important component of the extracellular matrix (ECM), is the principal, but by no means the only, ligand of CD44. Other CD44 ligands include the ECM components collagen, fibronectin, laminin, and chondroitin sulfate. Mucosal addressin, serglycin, osteopontin, and the class II invariant chain (Ii) are additional, ECM-unrelated, ligands of the molecule. In many, but not in all cases, CD44 does not bind HA unless it is stimulated by phorbol esters, activated by agonistic anti-CD44 antibody, or deglycosylated (e.g., by tunicamycin). CD44 is a multifunctional receptor involved in cell-cell and cell-ECM interactions, cell traffic, lymph node homing, presentation of chemokines and growth factors to traveling cells, and transmission of growth signals. CD44 also participates in the uptake and intracellular degradation of HA, as well as in transmission of signals mediating hematopoiesis and apoptosis. Many cancer cell types as well as their metastases express high levels of CD44. Whereas some tumors, such as gliomas, exclusively express standard CD44, other neoplasms, including gastrointestinal cancer, bladder cancer, uterine cervical cancer, breast cancer and non-Hodgkin's lymphomas, also express CD44 variants. Hence CD44, particularly its variants, may be used as diagnostic or prognostic markers of at least some human malignant diseases. Furthermore, it has been shown in animal models that injection of reagents interfering with CD44-ligand interaction (e.g., CD44s- or CD44v-specific antibodies) inhibit local tumor growth and metastatic spread. These findings suggest that CD44 may confer a growth advantage on some neoplastic cells and, therefore, could be used as a target for cancer therapy. It is hoped that identification of CD44 variants expressed on cancer but not on normal cells will lead to the development of anti-CD44 reagents restricted to the neoplastic growth.

CD44: Structure, Function and Association with the Malignant Process

https://www.eng.tau.ac.il/~meitalz/Articles/E4.pdf

Antibiotic-eluting medical devices for various applications

Soluble proteins that serve as mediators of cell function and are produced by various cell types, such as structural and inflammatory cells, are collectively called cytokines. Several lines of evidence have revealed that cytokines play important roles not only in tissue homeostasis but also in the pathogenesis of many infectious diseases. Recent research on biological activities in normal periodontium and the pathogenesis of periodontal diseases has clarified the involvement of various cytokines in the biological activities observed in the sites. Cytokines play crucial roles in the maintenance of tissue homeostasis, a process which requires a delicate balance between anabolic and catabolic activities. In particular, growth factors--such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), transforming growth factor-beta (TGF-beta)--are thought to play important roles in modulating the proliferation and/or migration of structural cells in the periodontium and the production of various extracellular matrices by these cells. On the other hand, there is little doubt that excessive and/or continuous production of cytokines in inflamed periodontal tissues is responsible for the progress of periodontitis and periodontal tissue destruction. Particularly, inflammatory cytokines--such as IL-1 alpha, IL-1 beta, IL-6, and IL-8--are present in the diseased periodontal tissues, and their unrestricted production seems to play a role in chronic leukocyte recruitment and tissue destruction. It is possible that monitoring cytokine production or its profile may allow us to diagnose an individual's periodontal disease status and/or susceptibility to the disease. In addition, although the hypothesis is still controversial, it has been suggested that discrete T-cell subsets (Th1 and Th2) with different cytokine profiles play specific roles in the immunopathogenesis of periodontal diseases.

Cytokine Expression in Periodontal Health and Disease

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix (ECM) and occurs transiently in both the cell nucleus and cytoplasm. It has been shown to promote cell motility, adhesion, and proliferation and thus it has an important role in such processes as morphogenesis, wound repair, inflammation, and metastasis. These processes require massive cell movement and tissue reorganization and are always accompanied by elevated levels of HA. Many of the effects of HA are mediated through cell surface receptors, three of which have been molecularly characterized, namely CD44, RHAMM, and ICAM-1. Binding of the HA ligand to its receptors triggers signal transduction events which, in concert with other ECM and cytoskeletal components, can direct cell trafficking during physiological and pathological events. The HA mediated signals are transmitted, at least in part, by the activation of protein phosphorylation cascades, cytokine release, and the stimulation of cell cycle proteins. A variety of extracellular signals regulate the expression of both HA and the receptors necessitating that HA-receptor signalling is a tightly controlled process. Regulated production of soluble forms of the receptors, alternately spliced cell surface isoforms, and glycosylation variants of these receptors can dramatically modulate HA binding, ligand specificity, and stimulation of the signalling pathway. When these processes are deregulated cell behaviour becomes uncontrolled leading to developmental abnormalities, abnormal physiological responses, and tumorigenesis. The elucidation of the molecular mechanisms regulating HA-mediated events will not only contribute greatly to our understanding of a variety of disease processes but will also offer many new avenues of therapeutic intervention.

HA receptors: regulators of signalling to the cytoskeleton.

Background: Platelet-rich plasma (PRP) promotes regeneration of bone, presumably through the action of concentrated growth factors. However, it is not clear how PRP affects the inflammatory response. The purpose of this study was to analyze the growth factors in PRP and to study the effects of PRP on monocyte cytokine release and lipoxin A4 (LXA4) generation.Methods: PRP was prepared from healthy donors. Platelet-derived growth factor (PDGF)-AB, PDGF-BB, transforming growth factor-β1, insulin-like growth factor-I, fibroblast growth factor-basic (FGF-b), epidermal growth factor (EGF), vascular endothelial growth factor, interleukin-12 (p40/70), and regulated on activation, normal T-cell expressed and secreted (RANTES) levels were evaluated by enzyme-linked immunosorbent assay and bead-based multiplexing. Peripheral blood monocytes were isolated and cultured with or without PRP. Cytokine, chemokine, and LXA4 levels as well as monocyte chemotactic migration were analyzed.Results: Growth factors were increase...

Platelet-Rich Plasma: Growth Factors and Pro- and Anti-Inflammatory Properties

In order to clarify the regulatory mechanisms of periodontal regeneration by basic fibroblast growth factor (bFGF), effects of bFGF on proliferation, alkaline phosphatase activity, calcified nodule formation and extracellular matrix synthesis of human periodontal ligament (PDL) cells were examined in this study. bFGF enhanced the proliferative responses of PDL cells in a dose-dependent manner. The maximum mitogenic effect of bFGF on PDL cells was observed at the concentration of 10 ng/ml. In contrast, bFGF inhibited the induction of alkaline phosphatase activity and the mineralized nodule formation by PDL cells. Moreover, employing the reverse transcription-polymerase chain reaction (RT-PCR) technique, we observed that the levels of laminin mRNA of human PDL cells was specifically upregulated by bFGF stimulation, but that of type I collagen mRNA was downregulated. On the other hand, the expression of type III collagen and fibronectin mRNA were not altered even when the cells were activated by bFGF. These results suggest that suppressing cytodifferentiation of PDL cells into mineralized tissue forming cells, bFGF may play a role in wound healing by inducing growth of immature PDL cells and that in turn accelerates periodontal regeneration.

Effects of basic fibroblast growth factor on human periodontal ligament cells.

Cementum forms the interface through which soft connective tissue of the periodontium is attached to the root surface. The interactions between cementum and connective tissue are not completely understood and whether cementum influences periodontal connective tissue formation and regeneration is not clear. We have examined the effect of cementum components on the attachment of gingival fibroblasts. Cementum was harvested from healthy human and bovine teeth and extracted sequentially in 0.5 M CH3COOH, 4 M guanidine and bacterial collagenase. Fibroblast attachment was measured using 51Cr-labelled human gingival fibroblasts on tissue culture plates previously incubated with cementum components. Results showed that all three extracts mediated fibroblast attachment and attachment was dependent on concentration and incubation time. The attachment activity was not destroyed by digestion with bacterial collagenase or by antibodies to fibronectin and laminin. However, it was inhibited by a peptide containing the amino acid sequence RGD. By gel filtration or HPLC using a DEAE-cellulose column several proteins with attachment activity were fractionated. SDS-polyacrylamide gel electrophoresis revealed that HPLC fraction eluted by 0.2–0.3 M NaCl contained a protein with molecular weight 55 kDa as a major component. This protein was isolated and shown to promote fibroblast attachment, and optimal attachment occurred at a concentration of 2 μg/ml. We conclude that cementum contains substances capable of mediating fibroblast attachment and that these substances play an important role in periodontal connective tissue formation and regeneration by facilitating fibroblast attachment to root surfaces.

Isolation of a fibroblast attachment protein from cementum

We have examined molecular mechanisms of the PMA-inducible HA binding ability of human lymphocytes. Newly established OS/6 and OS/37, specific for human CD44, specifically inhibited PMA-induced HA binding of several human cell lines, suggesting that both mAb detect HA binding epitope(s) on CD44. Sequential staining revealed that these mAb cross-blocked each other's binding to Molt-4, T lymphoblast lines, and that neither of them interfered with staining of Molt-4 by other anti-CD44 mAb which induced significant homotypic cell aggregation. Biochemical and PCR analyses did not provide any evidence that PMA stimulation induced dramatic changes in molecular weight or molecular isoforms of CD44. Interestingly, HA binding was not affected and rather slightly increased by cytochalasin B which disrupts F-actin microfilament integrity. This suggests that the ability of CD44 to bind to HA does not correlate with the association of CD44 with the cytoskeleton. On the other hand, protein synthesis inhibitors, cycloheximide and anisomycin clearly inhibited the induction of HA binding of PMA-activated Molt-4 without affecting the expression of CD44 at the same time after stimulation. The same treatment had no effect on PMA-induced FN binding of the cells, which was mediated by VLA integrins. These results suggest that the adhesion functions of CD44 and integrins are differently regulated despite the fact that both are induced by PMA stimulation, and that new protein synthesis is essential for the PMA-induced HA binding by CD44.

Inducible binding of human lymphocytes to hyaluronate via CD44 does not require cytoskeleton association but does require new protein synthesis.

The recognition that destructive periodontal diseases may be caused by specific microorganisms in periodontal pockets has led to an increased interest in and usage of antimicrobial agents in periodontal therapy. Recently, a new controlledrelease insert containing ofloxacin, a synthetic antibiotic, has been developed. In this study, the controlled-release insert (PT-01) was microbiologically evaluated in combination with or without subgingival mechanical debridement. PT-01 was applied in the periodontal pockets of 27 patients with chronic periodontitis. Three sites with a deep probing pocket depth (≥5 mm) were randomly selected in different quadrants of each patient, and were assigned into three groups, i.e., PT01 applied (T), placebo applied (P) and control sites (C). Periodontal treatments consisted of supragingival scaling with oral hygiene instruction for the first 2 weeks followed by root planing and subgingival scaling. PT-01 was applied weekly on day 0 to 35, and the subgingival plaque samples from each site were collected on d 0, 14, 21 and 42. The dynamics of the subgingival microflora was investigated by dark field microscopy and by anaerobic and aerobic cultivation. In the supragingival scaling period, significant reduction in percentages of spirochetes and motile rods and significant increase of the percentage of coccoid cells were observed only at T sites. In addition, the total viable counts of bacteria, black-pigmented Bacteroides and Fusobacterium species were significantly reduced at T sites. After mechanical subgingival debridement, significant shifts in the proportion and reduction of the viable counts in the subgingival microflora were found at all sites. Although PT-01 applied sites showed some further shift in the proportion and reduction in subgingival microorganisms, statistically no significant differences in the microbiological results between the three groups of experimental sites were found. These results suggested that weekly application of PT-01 in the periodontal pocket could have significant effects on both qualitative and quantitative changes in the subgingival microflora. However, the mechanical subgingival debridement, consisting of root planing and subgingival scaling, was very effective in eliminating the subgingival microflora by itself. Then, in the combination with mechanical subgingival debridement, statistically no additional effects could be found. Consequently, it was suggested that the application of PT-01 might have an ameliorating effect as an adjunct in conventional periodontal therapy.

Yasuo Miki

Papers

Effects of basic fibroblast growth factor on human periodontal ligament cells.

Isolation of a fibroblast attachment protein from cementum

Inducible binding of human lymphocytes to hyaluronate via CD44 does not require cytoskeleton association but does require new protein synthesis.

Topical chemotherapy in human periodontitis using a new controlled-release insert containing ofloxacin. I. Microbiological observation.

Isolation and partial characterization of a growth factor from human cementum.