**Biomimetic Materials as Potential Medical Adhesives – Composition and Adhesive Properties of the Material Coating the Cuvierian Tubules Expelled by** *Holothuria dofleinii*

 1Yong Y. Peng1, Veronica Glattauer1, Timothy D. Skewes2, Jacinta F. White1, Kate M. Nairn1, Andrew N. McDevitt3, Christopher M. Elvin3, Jerome A. Werkmeister1, Lloyd D. Graham4 and John A.M. Ramshaw1

### **1. Introduction**

244 Biomaterials – Physics and Chemistry

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Switching of Biopolymer Layers. Insights from Combined Electrokinetics and

Novel, distinct adhesive systems have been described for a wide range of marine species (Kamino, 2008). These highly effective, natural materials provide a link between biological science and material science, and can serve as models on which new, bioinspired synthetic materials could be based. These various adhesive systems have developed independently, on many occasions, and provide a wide range of opportunities for the development of new, biologically-inspired adhesives. The natural adhesives include, for example, the marine mussel (*Mytilus sp.*) (Lin et al., 2007), barnacle (Nakano et al., 2007) and stickleback (Jones et al., 2001) adhesives, which are protein-based, as well as sponge, certain algal and marine bacterial adhesives (Mancuso-Nichols et al., 2009) that are polysaccharide-based.

In the present paper, we examine the adhesive system found associated with the Cuvierian tubules of a holothurian species (sea cucumber), *Holothuria dofleinii*. This is an example of the particularly rapid marine adhesive that is found on the surface of Cuvierian tubules when they are expelled (DeMoor et al., 2003; Müller et al., 1972; VandenSpiegel & Jangoux, 1987). The unique nature of this natural adhesive system, especially its rapid action under water, has suggested that if the mechanism can be understood, then it may prove to be possible to mimic the adhesive through biotechnology and/or synthetic chemistry. An adhesive that functions readily in an aqueous environment would be particularly valuable, especially in medical applications, as the majority of existing adhesives bind to dry surfaces more strongly than the same surfaces when wet.

Cuvierian tubules provide a host defence mechanism for certain species of holothurians (Lawrence, 2001; VandenSpiegel & Jangoux, 1987). It has long been known that, on expulsion, the Cuvierian tubules fill with liquid and lengthen, become sticky and rapidly

*1CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, VIC 3169, Australia 2CSIRO Marine and Atmospheric Research, Middle Street, Cleveland, QLD 4163, Australia 3CSIRO Livestock Industries, Carmody Road, St Lucia, QLD 4067, Australia*

*4CSIRO Food and Nutritional Sciences, Julius Ave, North Ryde, NSW 2113 ,Australia1*

Biomimetic Materials as Potential Medical Adhesives – Composition and Adhesive

euthanised by freezing at minus 20 °C.

pass filters on an Olympus BX61 fluorescence microscope.

XL30 FESEM microscope at an accelerating voltage of 2 kV.

**2.4 Gel electrophoresis analysis**

**2.5 Amino acid analysis**

glass slides. After air drying, the tubules were examined by microscopy.

insoluble material, although the yield of adhesive proteins was less.

**2.3 Microscopy**

Properties of the Material Coating the Cuvierian Tubules Expelled by *Holothuria dofleinii* 247

to drain briefly (<20 sec), but not by squeezing as had been proposed by others (Zahn et al., 1973). Intact tubules prior to expulsion were obtained by dissection of animals that had been

To look for the presence of glycoprotein on the surface of expelled tubules, samples were treated with fluorescently-labelled lectins; fluorescein isothiocyanate (FITC)-labelled concanavilin A (ConA), FITC-labelled *Datura stramonium* agglutinin (DSA), and FITClabelled *Lycopersicon esculentum* agglutinin (LEA) (all from Sigma, St Louis). All FITClabelled lectins were applied as 20 μg/mL solutions in Tris-buffered saline (TBS) for 60 min, followed by 3 × 5 min washes in TBS. Samples were examined using appropriate narrow

To examine the distribution of adhesiveness on tubules, individual freshly expelled tubules after draining (see above) were transferred to a wash solution in a plastic trough which contained a suspension of 0.5% w/v Bio-Gel P2 (45-90 m particle size) in 3.5% w/v NaCl, 10 mM sodium phosphate, pH 7.6. After 5 sec immersion, the tubules were washed 3 times in 3.5% NaCl, 10 mM sodium phosphate, pH 7.6 and were then drained and placed onto

For scanning electron microscopy (SEM) expelled tubules were examined using a Philips

Freshly expelled and drained tubules were allowed to adhere to a glass plate and were air dried. The tubules on glass plates were removed by peeling, leaving the layer of adhesive, and potentially other components of the tubule wall as a print on the glass (DeMoor et al., 2003). This material was collected by removal with a sharp razor blade and was then extracted in electrophoresis sample buffer, containing 2-mercaptoethanol. SDSpolyacrylamide gel electrophoresis (SDS-PAGE) was based on the method of Laemmli (1970) using Invitrogen NuPAGE Novex 4-14% Bis-Tris Gel with MES running gel buffer, at 180V for 60 min. Molecular weights were determined by comparison to globular protein standards (BioRad) using BioRad Quantity One v.4.4.0 software. For protein identification, gels were stained by Coomassie Blue R-250. Samples that had not been dried completely, but only sufficient to remove excess liquid, appeared to give samples that contained less

Protein extracts were separated by SDS-PAGE, followed by transfer of the protein bands to PVDF membrane using Invitrogen NuPAGE Transfer buffer (NP0006-1). Amino acid analysis of PVDF membrane pieces used vapour-phase hydrolysis (5.8 M HCl at 108 °C for 18 h), followed by precolumn derivatisation with 6-aminoquinolyl-*N*-hydroxysuccinimidyl carbamate (Cohen & DeAntonis, 1994). Derivatives were separated and quantified by reversed phase (C18 Waters AccQTag) HPLC at 37 C (Cohen, 2001) (Australian Proteome Analysis Facility), using a Waters Alliance 2695 Separation Module, a Waters 474

Fluorescence Detector and a Waters 2487 Dual Absorbance Detector in series.

immobilise most organisms with which they come into contact (VandenSpiegel & Jangoux, 1987). The tubules, once expelled, are immediately adhesive on contact with a solid surface (VandenSpiegel & Jangoux, 1987), such as the exoskeleton or skin of a predator. Crabs, molluscs and sea stars can stimulate tubule expulsion, and the tubules stick to these species. This adhesion happens entirely under water, and does not need the mixed environment of the intertidal zone where many of the other potential adhesives are sourced.

Sticky tubules are found only within the family Holothuridae within the order Aspidochirotida, and mostly in the genus Bohadschia and the genus Holothuria. Various authors have described the ultrastructure of the tubules, especially for *H. forskåli* (Lawrence, 2001; VandenSpiegel & Jangoux, 1987; VandenSpiegel et al., 2000), as well as their expulsion and release (Flammang et al., 2002) and the timeframe of regeneration (Flammang et al., 2002; VandenSpiegel et al., 2000).

Flammang and Jangoux (2004) suggested, from the differences in the surface (adhesive) protein types and compositions in *H. forskåli* and *H. maculosa*, that the adhesion proteins and mechanism may differ between species. Other studies showed that adhesive strengths varied between species, with the adhesion in *H. leucospilota* being several times greater than for six other species (Flammang et al., 2002). A limited number of studies have probed the mechanism of adhesion, focusing on *H. forskåli* and *H. leucospilota* (De Moor et al., 2003; Müller et al., 1972; Zahn et al., 1973). These studies have shown that best adhesion is found at temperatures, salinity and pH similar to those found in the marine environment in which the organism flourishes, and is most effective with hydrophilic surfaces (Flammang et al., 2002; Müller et al., 1972; Zahn et al., 1973). Increasing concentrations of urea led to a loss of adhesion, suggesting that native protein structure(s) or interctions(s) may be required for effective bonding (Müller et al., 1972). Later biochemical studies have also suggested that the adhesive mechanism involves protein components (DeMoor et al., 2003).

In the present study, we have extended the information on Cuvierian tubule adhesion. In this study we examined the tubules of a different species, *H. dofleinii* Augustin, 1908. We have examined the distribution of the adhesive substance on the surface of expelled tubules, along with the molecular weights and amino acid compositions of its main protein components. We have estimated the strength of adhesion of *H. dofleinii* tubules to different substrata, and examined the effects of salinity, pH, ionic strength and denaturants on the adhesive properties.


**3. Discussion**

The need for tissue replacement materials, e.g., in the head and neck area, has increased in the last years, particularly with regard to the increasing amount of cancerous diseases [14]. The gold standard

of using autologous material has the disadvantage of being limited, and patients have to undergo two operations. Allografts, which are also common, have to be treated with strong chemicals to reduce the immunogenic potential. At the same time, the matrix integrity is lost [15].

Synthetic materials, which are not limited in availability, often cannot withstand the biomechanical strain and are resorbed quickly, which makes them unsuitable for use in major bone defects [5]. The treatment of allogenic material with high hydrostatic pressure (HHP) could be a gentle and effective alternative to already existing methods for providing decellularized tissue replacement materials without negative e ffects on the tissue matrix [12]. The aim of this study was to evaluate an optimal HHP protocol for di fferent cell types (osteoblasts and chondrocytes) regarding devitalization and induction of apoptosis or necrosis. Rivalain et al. already described the e ffect of di fferent HHPs on mammalian cells and observed, that after treatment of around 200 MPa, cells underwent apoptosis whereas a treatment higher than 300 MPa resulted in necrosis [12].

We were able to characterize the influence of high hydrostatic pressure on cell survival and cell death in human osteoblasts and chondrocytes. We further aimed to distinguish di ffering HHP-e ffects when cells were exposed as cell pellets. The first observation was that the lowest HHP range of 100–150 MPa had no significant e ffect on the metabolic activity of all tested cell types. In contrast, treatment of cells with HHP ranging from either 250–300 MPa or 450–500 MPa had the same e ffect regarding the significant reduction of metabolic activity. This observation was a first hint that HHP treatment of 250 MPa and higher destroyed cells and resulted in devitalization. The previous work of Hiemer et al. [8] showed that an HHP of 480 MPa led to successful devitalization of hyaline cartilage cells. However, the limitation of the work was that this devitalizing e ffect with respect to apoptosis and necrosis was not investigated. As mentioned before, this point is of high relevance because cell death is decisive for the induction of immunological processes. The requirement for tissue replacement materials is low to negligible immunological potential for the acceptor. Therefore, the devitalization process should not have a necrotic e ffect because this would lead to inflammatory responses regarding the release of, e.g., damage-associated molecular patterns (DAMPS) which further stimulate the pro-inflammatory response [13]. To analyze the type of cell death induced by HHP, necrosis and apoptosis were first detected by ELISA. The collected data showed that the untreated control groups also underwent apoptosis and necrosis partly, which can be attributed to the fact that the controls were exposed to non-standard conditions (e.g., room temperature) during this time that the treated groups underwent the HHP treatment. Aside from this finding, HHD treatment of 100–150 MPa had no significant e ffect in comparison to the control group. A balanced apoptosis- and necrosis-inducing effect on osteoblasts was observed at 250–300 MPa, whereas an HHP treatment of 450–500 MPa induced primarily necrosis. In contrast, an HHP range of 100–150 MPa induced apoptosis in chondrocytes derived from hyaline cartilage whereby necrosis was already induced by a range from 250–300 MPa. The most e ffective HHP treatment should first and foremost lead to metabolically inactive cells that have, ideally, not yet undergone necrosis. Thus, taking the results of both, the WST-1 assay and ELISA-derived apoptosis and necrosis analysis into consideration, these findings suggest that an HHP of 200–300 MPa may be the most e ffective for gentle and noninflammatory cell devitalization. Analysis via flow cytometry, providing a more precise separation of vital, necrotic, apoptotic, and late apoptotic/necrotic cells, revealed that most osteoblasts were vital after HHP treatment of 100–150 MPa, while exposure to 250–300 MPa led to necrosis and apoptosis only in a small number of cells. Moreover, most of these cells were detected as being in a late apoptotic/necrotic condition. Identical observations were made for chondrocytes. We assumed that cells treated with the higher HHP range were apoptotic at an earlier time point and underwent necrosis. This result can be explained, among other things, by the fact that there was a time delay between the HHP treatment and the subsequent FACS analysis due to the availability of equipment at di fferent locations. Therefore, it cannot be excluded that necrosis was induced in the cells after a short time. Additionally, the absence of any phagocytic cells in the experimental setting led to the absence of cell debris removal. Consequently, the membrane of the treated cells became permeable over time, and intracellular contents could be released, which might further stimulate the immunological response [13]. Additionally, permeable and destroyed cell membranes as well as cellular components were found in TEM analysis of osteoblasts treated with 250–300 MPa. In summary, the results support the idea that in the early phase after HHP, apoptosis was induced following a pressure of 250–300 MPa, but the initiation of intracellular material packaging was incomplete or aborted. As confirmed by FACS analyses, the cells were in a late apoptotic/necrotic state, which led to an uncontrolled destruction of cell components in the intracellular cavity, which was additionally supported by TEM. Interestingly, these results were not observed for the lower HHP range of 150–200 MPa. Therefore, we assumed that either a reduction of the HHP level or a shortening of the HHP exposure time could initiate apoptotic devitalization. This aspect has to be confirmed in future studies.

The aim of this study was to examine the cell-typical reactions to various HHP treatments to assess the tissue-specific reactions to HHP in the next step. Furthermore, our results on devitalization efficiency and cell death detection will help to better evaluate the results of a previous animal study on devitalized autografts [10]. Applied pressure of 480 MPa to osteochondral cylinders led to an extensive tissue remodeling process following tissue re-transplantation. However, excellent integration and revitalization were detected [10]. Possible reasons for these findings might be the autograft transplantation in the experimental setting and the animal-specific immune system, which plays an important role in tissue integration. For better understanding, it is even more important to perform targeted analysis of HHP-mediated cell death which is of high relevance for the subsequent translation of specific tissue types into the clinical use of allografts. It is also important to study additional tissue-specific cells to prove whether the respective high pressure successfully devitalizes all resident cells in the tissue through apoptosis. Taking late apoptosis/necrosis into account, rapid purification of the HHP-processed tissue is still mandatory so that cell residues can be removed gently and in sufficient time. These aspects will be analyzed in more detail in further studies.

## **4. Materials and Methods**


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*3.10. Microgrid Operating Modes*

A considerable number of papers have been published on interconnected microgrids, while discussing various modes of microgrid operation. On the other hand, the stand-alone mode is considered by many authors as an alternative supply measure mainly in the rural areas or regions with no conventional grids [122]. Thus, both the on- and off-grid operating modes are a feasible alternative. Table 4 summarizes the above considerations.


**3. Transplantation tolerance**

The alloimmune response can be divided into central and peripheral tolerance, according to the mechanisms that induce a tolerance state. These are related and not exclusive [27] (Figure 2).

#### **3.1. Central tolerance**

Central tolerance is the most important means by which T and B autoreactive lymphocytes are eliminated in a process termed clonal deletion. T and B cells mature and are educated in the thymus and the bone marrow, respectively (Figure 2B).

Immature T lineage cells emerge from hematopoietic progenitors in the bone marrow and enter the thymus without expressing either the TCR or coreceptors. Since they lack CD4 and CD8 antigens, these cells are called double-negative (DN) cells or thymocytes. T cell selection begins after DN cells have undergone a TCR-mediated rearrangement process and up-regulated both CD4 and CD8 antigens, thus becoming double-positive (DP) cells [28]. From here, the thymo‐ cyte's fate is determined by the nature of its interaction with self-peptides that are presented on the self-HLA molecules of thymic stromal cells. This process is called "the affinity-avidity model". If a T cell reacts too strongly with self-antigens presented on bone marrow–derived APCs, it is eliminated by apoptosis or negative selection in the thymus [29]. Thymocytes with TCRs that interact with self HLA/peptides with lesser avidity, are positively selected and evolve into mature T cells that express either the CD4 or CD8 receptor (single positive T cells).  At the end of the process, only 3% of the total number of CD4+ CD8+ DP cells are exported from the thymus, having developed into single positive CD4+ or CD8+ cells [30].

Currently, it is not completely understood how many peripheral tissue-specific antigens are expressed and presented in the thymus to ensure central T-cell tolerance to antigens that will be encountered in the periphery eventually. The expression of peripheral pro‐ teins in the thymus (such as insulin, thyroglobulin, and renal autoantigens) is driven in part by a gene called AIRE (autoimmune regulator). Mutations in the AIRE gene result in a disease known as autoimmune polyglandular syndrome type I. Interestingly, only cer‐ tain organs and systems are involved, and within these, only particular parts of the organ tend to be affected, confirming that additional mechanisms must be involved to maintain systemic tolerance [31].

B cells undergo a similar process, as they are tested for reactivity to self-antigens before they enter the periphery. Immature B cells, developing in the bone marrow, test antigen through their antigen receptor, a surface IgM called the B cell receptor (BCR). If signaling through the BCR is sufficiently weak, immature B cells can be rendered permanently unresponsive or anergic. However, if immature B cells are strongly self-reactive, there are two possible scenarios to ensure tolerance. The first is deletion of these self-reactive B cells. The second is receptor editing, a process by which a new receptor with altered specificity is generated through another sequence of B cell receptor gene rearrangements [32].

#### **3.2. Peripheral tolerance**

including HLA class I and IL-2 [26]. There are other important events implicated in the activation of T cells, including leukocyte migration and the interaction of chemokines with

The alloimmune response can be divided into central and peripheral tolerance, according to the mechanisms that induce a tolerance state. These are related and not exclusive [27] (Figure 2).

Central tolerance is the most important means by which T and B autoreactive lymphocytes are eliminated in a process termed clonal deletion. T and B cells mature and are educated in the

Immature T lineage cells emerge from hematopoietic progenitors in the bone marrow and enter the thymus without expressing either the TCR or coreceptors. Since they lack CD4 and CD8 antigens, these cells are called double-negative (DN) cells or thymocytes. T cell selection begins after DN cells have undergone a TCR-mediated rearrangement process and up-regulated both CD4 and CD8 antigens, thus becoming double-positive (DP) cells [28]. From here, the thymo‐ cyte's fate is determined by the nature of its interaction with self-peptides that are presented on the self-HLA molecules of thymic stromal cells. This process is called "the affinity-avidity model". If a T cell reacts too strongly with self-antigens presented on bone marrow–derived APCs, it is eliminated by apoptosis or negative selection in the thymus [29]. Thymocytes with TCRs that interact with self HLA/peptides with lesser avidity, are positively selected and evolve into mature T cells that express either the CD4 or CD8 receptor (single positive T cells). The cells with very low avidity interactions fail to induce survival signals and die within the

Currently, it is not completely understood how many peripheral tissue-specific antigens are expressed and presented in the thymus to ensure central T-cell tolerance to antigens that will be encountered in the periphery eventually. The expression of peripheral pro‐ teins in the thymus (such as insulin, thyroglobulin, and renal autoantigens) is driven in part by a gene called AIRE (autoimmune regulator). Mutations in the AIRE gene result in a disease known as autoimmune polyglandular syndrome type I. Interestingly, only cer‐ tain organs and systems are involved, and within these, only particular parts of the organ tend to be affected, confirming that additional mechanisms must be involved to maintain

B cells undergo a similar process, as they are tested for reactivity to self-antigens before they enter the periphery. Immature B cells, developing in the bone marrow, test antigen through their antigen receptor, a surface IgM called the B cell receptor (BCR). If signaling through the BCR is sufficiently weak, immature B cells can be rendered permanently unresponsive or

thymus. At the end of the process, only 3% of the total number of CD4+

exported from the thymus, having developed into single positive CD4+

their receptors.

**3.1. Central tolerance**

systemic tolerance [31].

**3. Transplantation tolerance**

468 Current Issues and Future Direction in Kidney Transplantation

thymus and the bone marrow, respectively (Figure 2B).

Besides the deletion process of autoreactive cells occurring during central tolerance, some T or B cells with self-reactivity may escape from the thymus or bone marrow, making the loss of self-tolerance easier. However, several mechanisms, collectively named peripheral toler‐ ance, can control or eliminate such cells. Peripheral tolerance involves deletion and apoptosis, anergy, and regulation or suppression (Figure 2C).

### *3.2.1. Deletion and apoptosis*

This mechanism is used to eliminate activated T cells specific for self-antigen. The programmed cell death, or apoptosis, is also termed activation-induced cell death (AICD). This process is mediated by the interaction of Fas (CD95) with its ligand (Fas-L or CD95L) on T cells, and can occur in developing thymocytes as well as mature T cells [33]. IL-2 can activate the STAT 5 sig‐ naling pathway through the IL-2 receptor (IL-2R), which in turn potentiates the up-regulation of Fas-L and the down-regulation of Bcl2 expression on T cells, thus promoting AICD. Con‐ versely, IL-15 acts as a growth and survival factor for T cells [34, 35]. Since augmented AICD can induce tolerance through elimination of populations of reactive lymphocytes [36], certain tol‐ erogenic models which use IL-15 antagonists and IL-2 agonists during transplantation havere‐ sulted in donor-specific tolerance [37]. Further research on this topic is needed before considering this peripheral mechanism as a therapeutic approach.

#### *3.2.2. Anergy*

CD8+ DP cells are

cells [30].

or CD8+

The hyporesponsiveness of T or B cells to further antigenic stimulation, also called anergy, is a process that can result from antigenic stimulation in the absence of costimulation. In the case of T cells, complete activation requires the presentation of peptide on the HLA molecule to the TCR (first signal), and costimulatory signals, such as the B7-CD28 and CD40-CD154 interac‐ tions (second signal). The second signal is required to induce the multiple pathways that will lead to the activation of IL-2 gene transcription, ultimately inducing T cell activation and proliferation. However, it has been shown that IL-2 production and subsequent signaling through its receptor, IL-2R, is necessary for T cells to escape anergy, since blocking IL-2/IL-2R engagement even after stimulation through the TCR and CD28 still results in induction of T cell anergy [38].

As with T cell activation, B cell activation requires two signals. In this context, naïve B cells can be anergized if their surface immunoglobulins bind to self-antigens (first signal) in the absence of the additional necessary T cell signals (second or costimulatory signal) [39].

#### *3.2.3. Regulation or suppression*

A third mechanism of peripheral tolerance is regulation or suppression of immune responses to self or foreign antigens. Perhaps, the regulatory T cells (Treg cells) are the most important and well documented effectors of this mechanism to date. These cells control the type and magni‐ tude of the immune response to foreign antigen to ensure that the host remains undamaged. Treg cells are also integral to maintaining a lack of response to self-antigens or tolerance [40].

were not compliant with their immunosuppressive regimens or in individuals who had previously received a bone marrow transplant for hematological disorders [52]. At the present time, in looking for tolerance in renal transplantation, physicians in clinical prac‐ tice have implemented protocols and surgical procedures in which tolerance was the

Tolerance in Renal Transplantation http://dx.doi.org/10.5772/54734 471

Protocols in which tolerance in renal transplantation was the planned objective before the transplant may be divided into three subgroups, namely molecule-based, cell-based, and total

The molecule-based group includes all cases in which the induction of tolerance was attempted through administration of presumed tolerogenic drugs. These tolerogenic drugs include polyclonal antithymocyte globulin antibodies and anti-CD25 monoclonal antibodies. Anti-CD25 monoclonal antibodies competitively inhibit IL-2R-dependent T cell activation, while the polyclonal antithymocyte globulin antibodies are directed against lymphocyte antigens. The goal of the induction treatment was the nonspecific removal of clones of immune cells responsible for rejection before contact with foreign donor antigens occured. Once the donor antigens were in place after implantation of the new kidney, repletion of immune cells occured, favored by the homeostatic expansion triggered by leukocyte depletion. In addition, minimi‐ zation of maintenance immunosuppression was implemented to further reduce the anti donor response with just enough treatment to prevent irreversible immune damage to the graft, but not with such heavy treatment that the donor specific clonal exhaustion-deletion process was

In the cell-based group, patients received a donor-cell infusion of highly enriched CD34+ hematopoietic progenitor cells mixed with CD3+ T cells, [54] ie, patients received heavy conditioning regimens in association with the perioperative infusion of immunomodulatory cells, such as transplant-acceptance inducing cells. Afterward, maintenance immunosuppres‐ sion was given for a few months until complete withdrawal, when possible. Overall, although these trials demonstrated that the infusion of transplant-acceptance inducing cells is feasible, major concerns remain regarding the efficacy and safety of such an approach. Whether this approach confers any benefit in the establishment of minimal immunosuppression in renal transplantation patients when compared with the protocols currently in use is unclear. Lastly, the optimal dose and timing of cell infusions, along with the most appropriate concomitant

Patients who received renal transplantation after bone marrow transplantation from the same donor are also included in this group. Bone marrow transplantation, when success‐ ful, generally results in the total replacement of the recipient's bone marrow with the do‐

immunosuppression regimen, remains to be determined [55,56].

planned objective before the transplant.

**4.1. Strategies and protocols**

*4.1.1. Molecule-based protocols*

lymphoid irradiation.

precluded [53].

*4.1.2. Cell-based protocols*

There are two subsets of Treg cells."Natural" Treg cells, are a thymus-derived population that constitute about 10% of the CD4 population. Natural Treg cells express CD4, CD25, CTLA4, and GITR on their surface [41], and express transcription factor Foxp3 intracellularly [42]. The importance of Foxp3 as the orchestrator of the molecular programs involved in mediating Treg function has been highlighted by diseases such as IPEX syndrome (immune dysfunction, polyendocrinopathy, enteropathy and X-linked inheritance), in which a mutation in the Foxp3 gene has been described [43].

The other subset of Treg cells, commonly termed "adaptative" Treg cells, develops in the periphery, in a thymic-independent manner, following antigen encounter under particular circumstances, namely exposure to transforming growth factor-β (TGF-β). This leads to the expression of Foxp3; the hallmark of Treg cells [44]. Data suggesting the role of these cells in immunologic tolerance has been obtained from different studies in which patients with normal graft function reportedly possess a smaller Treg population compared with patients having chronic allograft rejection, suggesting that Treg cells may prevent damage and graft loss [45]. Other groups have shown that certain immunosuppressive protocols are more permissive than others in generating these populations [46].

The mechanisms by which Treg cells exert their effects are not completely understood. There have been two main mechanisms proposed. One mechanism requires cell contact between CD4+CD25+ Treg and responder cells and interaction between CTLA-4 and GITR molecules [47], while the other mechanism involves the induction of suppression or regulation by newly generated suppressor T cells in a cytokine-dependent manner through IL-10 and/or TGF β [48, 49]. Although promising, there is still too much to learn, before using this subset of cells for tolerance induction in renal transplantation.

In addition to Treg cells, there are other cell phenotypes with regulatory properties, such as CD8+ T cells and certain NK populations [50]. CD8+ T cells with regulatory/suppressive properties have been named "veto cells". Such cells maintain peripheral tolerance by attacking alloreactive T cells which are present in bone marrow with increased frequency, and may be responsible in part for the reduction in graft versus host disease and the induction of chimerism seen in some bone marrow transplant models [51].