**3. Carbohydrate binding modules**

Many of the enzymes involved in the pathway of polysaccharide biosynthesis present a carbohydrate binding domain in its structure. The first carbohydrate binding domain described was a cellulose-binding domain [45-47]; but later it has been found other modules in related enzymes that bind polysaccharides other than cellulose. These findings compelled to redefine the nomenclature of these domains, and now are called CBM (carbohydrate binding module). To date over 300 putative sequences in more than 50 different species have been identified, and binding domains have been classified into 64 families based on amino acid sequence, the substrate binding specificity and structure [48] (see Carbohydrate-Binding Module Family Server, http://afmb.cnrsmrs. fr/~pedro/CAZY/cbm.html).

 Plant Biotechnology for the Development of Design Starches 497 CBMs have been found in several non-hydrolytic and hydrolytic proteins. Those with hydrolytic activity, such as cellulases, have a complex molecular structure comprised of discrete modules (one catalytic domain and one or more CBMs) that are normally linked by unstructured sequences. The CBMs increase the speed of enzymatic reactions by conducting the catalysis in a close and prolonged physical association with substrates [48] The CBMs present in non-hydrolytic proteins constitute a subunit of the catalytic domain hosts generating cohesive multienzyme complex, which lose enzymatic activity when the CBMs are removed from the structure [48]. Therefore, the CBMs have three general roles with respect to the function of their cognate catalytic modules: (i) a proximity effect, (ii) a targeting function and (iii) a disruptive function [49].

496 The Complex World of Polysaccharides

is in leaf or endosperm (Figure 1).

**Starch Synthases SSI SSIIa (E) SSIIb (L) SSIII SSIV (L)**

**Branching Enzymes BEI BEIIa BEIIb (E)**

> **Isoamylase Isa1 Isa2 (L)**

[10,43].

crucial for the generation of longer, clustered linear segments in the amylopectin molecule that can crystallize and increase the density of the polysaccharide [42]. Plants contain four DBE genes, three of which are classified as isoamylases on the basis of their sequence homologies and substrate specificities, and one pullulanase-type debranching enzyme

While the steps leading to the synthesis of starch are common in most cereals, there are differences in the location and engagement of enzymes, depending on whether the synthesis

**Glc1P**

**ADPGlc - PPase cytosolic form (E) plastidic form**

**ATP**

**PPi**

**Figure 1.** Starch biosynthesis pathway in plants from Glc1P. The scheme indicates the involvement of different isoforms in cereal leaf (L) or endosperm (E). When not specified, the enzymes are dual

**Granular Starch**

ADP

**ADP Amylopectin Amylose**

**Granule Bound Starch Synthase GBSSI (E) GBSSII (L)**

**ADPGlc**

Many of the enzymes involved in the pathway of polysaccharide biosynthesis present a carbohydrate binding domain in its structure. The first carbohydrate binding domain described was a cellulose-binding domain [45-47]; but later it has been found other modules in related enzymes that bind polysaccharides other than cellulose. These findings compelled to redefine the nomenclature of these domains, and now are called CBM (carbohydrate binding module). To date over 300 putative sequences in more than 50 different species have been identified, and binding domains have been classified into 64 families based on amino acid sequence, the substrate binding specificity and structure [48] (see Carbohydrate-Binding Module Family Server, http://afmb.cnrsmrs.

localized in both compartments (adapted from [10] and [44]).

**3. Carbohydrate binding modules**

fr/~pedro/CAZY/cbm.html).

The SBD (starch binding domain) is usually a distinct sequence-structural module that improves the efficiency of an amylolytic enzyme, improving the binding to starch and its hydrolysis. Because this module was first recognised in amylases and thus revealed to cope with raw starch, it was named the raw (granular) starch binding site [50]. At the present, due to the occurrence of SBDs in a wide spectrum of non-amylolytic enzymes, it has become logical to expect a more variable function of these modules. However, there is little evidence that SBD could bind polysaccharides different to starch, although the ability of pure starch binding and degrading seems to be reserved for microorganisms [50,51].

The CBMs have been clasified in ten families based on sequence comparison: (i) CBM20, such as the C-terminal SBD from *Aspergillus niger* glucoamylase; (ii) CBM21, located at the N-terminal domain in amylase proteins; (iii) CBM25, containing one (i.e. β-amylase from *Bacillus circulans*) or two (i.e. *Bacillus sp*. α-amylase) domains; (iv) CBM26, mainly organized in tandem repeats (i.e. C-terminal domains from *Lactobacillus manihotivorans* α-amylase); (v) CBM34, present in the N-terminal domains of neopullulanase, maltogenic amylase and cyclomaltodextrinase; (vi) CBM41, N-terminal SBD, present mostly in bacterial pullulanases; (vii) CBM45, originating from eukaryotic proteins from the plant kingdom (i.e. N-terminal modules of α-glucan water dikinases and α-amylases); (viii) CBM48, which display glycogen-binding properties (including SBD from the GH13 pullulanase and regulatory modules of mammalian AMP-activated protein kinase); (ix) CBM53, SBD modules from SSIII and (x) CBM58, find in α-amylase/neopullulanase of *Bacteroides thetaiotaomicron* showing maltoheptaose binding [52-54] (http://www.cazy.org). This modules becomes important in breaking down the structure of the substrate due to the presence of two polysaccharide-binding sites [55].

Using bioinformatics techniques several SBDs and several sequences exhibiting similarities to SBDs have also been recognised in enzymes and proteins that are not necessarily amylases such as dual-specific phosphatases. These enzymes should deserve special attention because of their participation in various important physiological processes in plants and mammals. It is worth mentioning that in plants these processes concern starch metabolism, whereas in mammals they participate in the metabolism of glycogen [56,57]. The presence of an SBD motif in protein phosphatases reflects their regulatory function since they are involved in polysaccharide metabolism indirectly via modulation of activity of degradative enzymes (i.e. also amylases), such as isoamylase, β-amylase and disproportionating enzyme [85]. In particular, the initial steps of starch degradation at the granule surface are regulated mainly by phosphorylation [50,51]. Furthermore, a starch biosynthetic enzyme, the starch synthase III (SSIII) from *Arabidopsis thaliana* (AtSSIII) has been reported by our group to have a regulatory role in the synthesis of transient starch [33]. This enzyme contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization followed by three in tandem starch-binding domains (SBD D1, D2 and D3, residues 22-591), which bind to raw starch and its individual components, amylose or amylopectin [53,54,58,59]. The adsorption experiments show that the SBD123 region binds preferentially to amylose, and that the D1 domain is mainly responsible for this selective binding. The D2 domain contains two binding sites including amino acid residues Y394 (binding site 1) and W366 (binding site 2) which act in cooperation with the D1 domain in the binding activity while G335 and W340 have a minor role [54]. It is worth mentioning that our work was the first report on the existence of an SBD in a synthesizing enzyme (AtSSIII) and the first experimental evidence of its starch binding capacity.


**Belt Road Initiative in Times of 'Synchronized Downturn': Issues, Challenges, Opportunities**

Editors

**Anna Visvizi Miltiadis D. Lytras**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Anna Visvizi Effat College of Business Effat University Jeddah Saudi Arabia

Miltiadis D. Lytras Effat College of Engineering Effat University Jeddah Saudi Arabia

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

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**4. Discussion**

We found that on the reefs at Gazi Bay coral genus richness was negatively correlated with the abundance of macroalgae, whereas coral cover was positively correlated with the abundance of herbivorous invertebrates (effectively sea urchins) and with fish family richness. Fish family richness was mainly correlated with coral cover and invertebrate abundance, but no determinants of fish abundance could be convincingly identified. However, caution is required before assuming these trends apply more widely, because, for example, the abundance of fishes and the extent of live coral cover observed in Gazi Bay appeared noticeably low compared to protected reef sites on the Kenyan coast visited by the authors in the same year (e.g., in the Kisite-Mpunguti Marine National Park and the Malindi National Park).

*Acanthuridae* (surgeon fishes) were the most abundant family across the sites, followed by the *Chaetodontidae* (butterflyfishes). As specific butterflyfishes are facultative or obligate corallivores they are generally associated with high live coral cover [47,51–53], even though in some cases this correlation was found to be quite low [54,55]. We found that there are enough living corals for at least some butterflyfish species to inhabit the reefs of Gazi Bay. Nevertheless, the benthic composition was dominated by macroalgae and turf algae. This may be because of the near absence of some other herbivorous fishes, notably species of parrotfish [10,25]. Their intensive feeding promotes the settlement, growth, and survivorship of corals due to the removal of competitive macroalgae. In this way herbivores

are considered important for coral reef resilience, because they keep macroalgae at low levels after any disturbance affecting corals, thus allowing the corals to recover [3,56,57]. The fact that during the study we observed no groupers and only a few parrotfishes, rabbitfishes, and snappers, is likely a consequence of overfishing, because all three families are important food-fish on the Kenyan coast.

In addition to fish, we found sea urchin abundance to be positively related to coral cover. This relationship is likely explained by the negative impact of sea urchins on algae, which otherwise compete with corals for space and light. Various field and computer studies have shown that urchin domination can help maintain coral cover by reducing competitive algae cover [32,58–62]. The study by Nozawa et al. (2020) showed that diadematid sea urchins enhance coral recruitment on Taiwanese reefs [32]. Another study in Kenya found that areas that were inaccessible to urchin grazing retained a high cover of algal turf and so were not suitable for coral recruitment, whereas in stable reef systems grazing fishes graze down the turf algae, facilitating coral recruitment [58]. The areas that were less accessible to echinoids were, in the absence of grazing fishes, dominated by large, fleshy algae which smothered corals [58]. However, the effects of sea urchins on the dynamics of phase shifts on coral reefs are density dependent, because they can play two different roles. In low abundances, reef echinoids promote coral reef resilience by controlling algal cover [56,63–65]. In high abundances, echinoids may erode large parts of both living corals and the dead coral framework (in addition to the algae growing on the latter), resulting in loss in live coral cover, reduced coral recruitment, reduced calcium carbonate accretion, and reduced associated faunal diversity [9,10,56,63–65]. Our results, however, showed a positive effect of herbivorous invertebrate/sea urchin abundances on corals, indicating that at Gazi Bay sea urchin population density is not (yet?) harmful to the coral community.

Our results also showed that, in our study area, coral genus richness was negatively related to macroalgae abundance. This is likely also because of the negative effect (just discussed) of macroalgae on hard coral cover, as overgrowth of corals by macroalgae can suppress the coral fecundity, hinder coral growth, and smother coral recruits [7,58,66,67]. Algal turfs, by comparison with macroalgae, are less successful competitors of corals [66,68]. Hopkins (2009) also found this correlation between coral and macroalgae cover, with the hard-coral cover being low when algae cover is high (and vice versa). Coral-macroalgae phase shifts have been reported from many countries, including, notably, Jamaica [69], and also East Africa [44], a phenomenon generally believed to be triggered by various impacts, such as the loss of herbivores (either urchins or fish), increased nutrient input, hurricane damage, or crown-of-thorns-starfish outbreak [63].

In our study, seagrass abundance was negatively related to coral genus richness, although this relationship was only marginally significant in the best supported model. A negative relationship between seagrass cover and coral genus richness may be largely explained by corals and seagrasses colonizing different types of substrates, but there may also be a degree of competition for space on some substrates. A previous study described seagrass colonizing the reef substratum after it was eroded by sea urchins [44]. However, other studies have shown that seagrass meadows may enhance coral reef resilience against ocean acidification by modifying the pH of the water column [68,70].

We found the cover of corals to be positively related to both fish abundance and fish family richness. However, this could be the result either of fish activity, such as grazing on algae, favoring the health of corals, or of corals providing a better habitat for fish [10,71,72]. Herbivorous fishes, which made up a large proportion in the counted fish, may clear space for coral settlement [73] by ingesting seagrass, macroalgae, or algal turfs. In particular, by reducing the amount of competitively superior macroalgae, herbivorous fishes allow corals and cementing coralline algae to survive [3,72]. Exclusion experiments such as those described by Hughes et al. [61] have repeatedly found that after excluding herbivores from parts of a reef, the coverage of macroalgae will quickly increase but will decline rapidly again when the area is made newly accessible to grazers. With increasing biomass

of grazers/detritivores, macroalgal cover decreases, thereby increasing the cover of live coral [7,25,66].

Although in our study the fact that herbivorous surgeonfishes (*Acanthuridae*) were the most abundant family makes it plausible that in our location it is reef fish diversity and abundance that are influencing coral cover, a positive relationship between coral cover and fish density and diversity has also been interpreted in many studies as inferring that it is coral cover which influences fish abundance [41,42]. In particular, increasing topographic or architectural complexity, substratum diversity, and live coral cover has been associated with increasing fish abundance and diversity [25,42,56,74,75]. Because of the difficulties in coral identification, the relationship between species richness of the fish assemblage and the diversity or cover of coral is less apparent in the literature than the effects of habitat complexity [42,76]. Coral reefs can provide fish with physical refuges from predators, in addition to breeding, nursery, and feeding sites, and thus enhance fish diversity. In contrast, coral mortality has been predicted to reduce reef fish abundance and diversity and so have long-term consequences on the community [19,56]. In fact, a recent study by Darling et al. suggests that reefs without corals will no longer support diverse fish faunas but be numerically dominated by a small subset of species that prefer algal or rubble substrata [77].

In our study, the abundance of sea urchins, besides being positively related to coral cover, was negatively related to fish species diversity. As we have said, echinoids are important grazers in shallow reef ecosystems and compete for resources with herbivorous fish and other benthic organisms [58,78]. Other studies have also revealed a negative impact of sea urchin abundance on the number of fish species and their abundance [58,78,79]. McClanahan et al. (1994) found that in heavily fished Kenyan reef lagoons, sea urchins are the most abundant grazers with herbivorous fishes showing a contrasting pattern of distribution. It appears that, as the fish density is reduced by fishing, echinoid populations expand to use the newly available resources [79]. Once an urchin-dominated community is established, it can be hard for herbivorous fishes to re-establish themselves [73]; high sea urchin abundances may even suppress fish growth and recruitment and hence recovery of the fish populations [58].

Due to the correlative nature of our study, it is not possible to determine the true causal relationship between fishes and corals or fishes and sea urchins, e.g., whether low abundances of fishes cause an increase in sea urchins or if high abundances of sea urchins cause a decline in fish species populations. Nevertheless, for the persistence of herbivorous fishes, it may be important to protect sea urchin predators, such as triggerfish (*Balistidae*) [21,28,80], which prevent sea urchins from becoming dominant, because otherwise these effects are not expected to be reversible [73,81,82].


**2.5 Specific competences of a food and nutrition engineer, an example of updated curriculum implemented in Slovenia**

Education for food and nutrition professions in Slovenia comprises 3-year vocational training (most common are baker, confectioner, dairy farmer and butcher), 4-year technical secondary education (e.g. food and nutrition technician), 2-year post-secondary vocational education (e.g. food and nutrition engineer) and 3-year higher education studies (e.g. Bachelor's degree in food and nutrition engineering, manager in food and nutrition). There are also upgrades at Master's level (Master of Food and Nutrition, Master of Food Safety) and PhD level.

The occupation-specific competences of food and nutrition engineers/technologists do not differ drastically between European countries. An example of the training objectives and expected competences for the Food and Nutrition Engineer VET programme is given below (summarised from the Centre for Vocational Education, Slovenia [35]). The duration of the study programme is 2 years and it is assessed with 120 ECTS credits.

The main objectives of the training programme for the profession of food and nutrition engineer after the revision in 2023 are:

#### **Table 1.**

*Generic and occupation-specific competences of a food and nutrition engineer, summarised from [38].*

*Perspective Chapter: Key Issues of Curriculum Revision and Integration of Novelties... DOI: http://dx.doi.org/10.5772/intechopen.112017*


**6. Conclusion**

AS as molecular target for drug development will be important for the treatment of a wide variety of diseases associated with deficiency of NO production, and also could transform *Bj*-PROs or their synthetic analogous into blockbuster medicine, as happened in the 80s with Captopril [90, 91]. The properties of *Bj*-PROs enhancing AS activity [14, 17], provides a pre‐ cise pharmacological tool for controlling pathophysiological mechanisms with advantages of uncontrolled application of exogenous L-arginine or NO donors [92]. For instance, the ef‐ fect of exogenous NO donors is not subject to physiological control, thus being more suscep‐ tible of generating undesired ROS [93, 94]. For all these reasons, keeping NO production in a safe level, so that a deleterious threshold would not be reached, is of particular interest. In this way, *Bj*-PROs should serve as structural models for the development of therapeutic agents for the treatment of various diseases related to NO deficiency, as cause or effect, as well AS deficiency.

Chemical properties of *Bj*-PROs make these peptides even more attractive potential lead compounds for drug development. For instance, *Bj*-PRO-10c is able to penetrate cells, where it remains as an intact molecule for hours [14]. Moreover, *Bj*-PROs contain a notable high proline content [13], which gives them some resistance to hydrolysis by aminopeptidases, carboxypeptidases and endopeptidases [95]. Nevertheless, cyclodextrins or nanocompounds could provide carriers for *Bj*-PROs, since drug release could be controlled in accordance with therapeutic propose [96, 97].


**8. Reconstruction techniques**

Different reconstruction techniques have been known and well documented for bony defects in the oral and maxillofacial area. Distraction osteogenesis and guided bone regeneration techniques, grafting procedures and especially autogenous bone grafting still are the treat‐ ments of choice in most alveolar bony defects. Soft tissue consideration and management should be borne in mind for successful stable results.

#### **8.1. Bone grafting**

"Any implanted material that promotes bone healing" is defined as a bone graft [24]. Ideally it must be: osteoconductive, osteoinductive and osteogenic.

**An osteoconductive capacity** means allowing or directing the new bone to form within the material structure.

**An osteoinductive capacity** describes supplying recruitment and/or differentiation factors for bone-forming cells by the grafting material.

**An osteogenic graft material** provides induced or inducible bone-forming cells.

Bone grafts are used not only for a defect facilitating healing but also for contour augmenta‐ tions. For this purpose more attention is directed towards the amount and rate of graft resorption. Graft incorporation is proportional to amount of graft resistance to resorption [24].

Bone grafts can be classified as:

**Autografts** (transferring bone in one human(,

**Allografts** (transferring inter-humans), and

**Xenografts** (transferring from other species, synthetic materials and any combination of them).

Autografts can be cancellous, cortical, corticocancellous, vascularized bone or aspirated bone marrow. The main advantage of autogenous bone is retention of at least some osteogenic cells without triggering the immune system. On the other hand donor site morbidity and limited amount are basic disadvantages. Ideally, the bone graft should be incorporated into the recipient bed; the space that the bone graft occupies should finally become viable bone with physiological remodeling mechanisms. Many factors are involved in the incorporation process namely the graft type, graft bed (recipient site), and interface in between. Graft related factors including the type of graft, porosity and mechanism of incorporation. Recipient site viability and vascularity are very important in any autogenous grafting procedures. Graft incorporation has been summarized by Bauer and Muschler in five steps [24[

**7.4. Rib graft**

nowadays [27].

**8.1. Bone grafting**

material structure.

overgrowth of the graft [27, 28].

**8. Reconstruction techniques**

522 A Textbook of Advanced Oral and Maxillofacial Surgery Volume 2

should be borne in mind for successful stable results.

bone-forming cells by the grafting material.

**Autografts** (transferring bone in one human(,

**Allografts** (transferring inter-humans), and

Bone grafts can be classified as:

it must be: osteoconductive, osteoinductive and osteogenic.

Free rib bone was one of the first autogenous bone grafts used for reconstruction of mandibular defects. Osseous or osseochondral grafts can be harvested from fifth to seventh ribs. Although costochondral grafts remain popular for the treatment of mandibular ramus and condylar defects, the quality and quantity of rib bone make it less popular for jaw defect reconstruction

**Complications.** Postoperative chest wall pain, pleural injury leading to pneumothorax, and

Different reconstruction techniques have been known and well documented for bony defects in the oral and maxillofacial area. Distraction osteogenesis and guided bone regeneration techniques, grafting procedures and especially autogenous bone grafting still are the treat‐ ments of choice in most alveolar bony defects. Soft tissue consideration and management

"Any implanted material that promotes bone healing" is defined as a bone graft [24]. Ideally

**An osteoconductive capacity** means allowing or directing the new bone to form within the

**An osteoinductive capacity** describes supplying recruitment and/or differentiation factors for

Bone grafts are used not only for a defect facilitating healing but also for contour augmenta‐ tions. For this purpose more attention is directed towards the amount and rate of graft resorption. Graft incorporation is proportional to amount of graft resistance to resorption [24].

**Xenografts** (transferring from other species, synthetic materials and any combination of them).

Autografts can be cancellous, cortical, corticocancellous, vascularized bone or aspirated bone marrow. The main advantage of autogenous bone is retention of at least some osteogenic cells without triggering the immune system. On the other hand donor site morbidity and limited amount are basic disadvantages. Ideally, the bone graft should be incorporated into the

**An osteogenic graft material** provides induced or inducible bone-forming cells.

Graft stabilization is other critical issue in bone graft incorporation and vascularization. Instability leads to bone resorption and infection. Cancellous bone grafts can be packed in defect cavities. In these cases more graft material transfer, leads to more vital cells and increase in osteogenesis. Cortical or corticocancellous block grafts should be stabilized using fixation devices.

#### **8.2. Bone Grafting with intra oral donor sites (localized bone augmentation)**

#### *8.2.1. Symphysis block harvesting*

There are three basic approaches to access the mandibular symphysis for bone graft harvesting: 1) sulcular, 2) attached gingiva, or 3) vestibular. The advantages of sulcular and attached gingiva approaches are reductions in wound dehiscence and bleeding compared to the vestibular approach. Use of the sulcular approach is not advocated in pre-existing periodontal diseases or crowns. The vestibular approach is done through the mucosa 5 to 10 mm below the mucogingival junction; first by partial thickness dissection apically for 3mm to maintain 3mm of periosteum and mentalis muscle fibers on the bone side, which will be used to reattach the muscle fiber [29]. Below this level a full thickness incision is made and full thickness flap reflection is used. Careful attention must be paid to prevent trans-section of the mental nerve at the distal extent of the incision bilaterally (Figure 10).

It is suggested that at least 5 mm bone is maintain below the teeth apices, inferior border and bilaterally anterior to mental foramina. When a large bone block is needed, the anterior most portion of the symphysis the mental protuberance must be retained. If it is necessary to harvest two graft blocks from each side, leaving a 3mm midline connection to maintain support for the chin profile is necessary [30].The block graft can be osteotomized by a rotary bur, recipro‐ cating saw, or piezo instrument. Using rotary burs has disadvantages of losing some amount of bone in comparison to two other methods. Osteotomies should enter the inter-cortical layer, giving close attention not violate the lingual cortex. A fine osteotome or chisel can be used to

**Figure 10.** Full mucoperiosteal flap retracted to expose the donor site for harvesting symphyseal bone. Two osteotomy sites are determined on both sides and 3mm bone is maintained in between to support the chin profile.

reflect block bone graft from its bed. After block removal a hemostatic agent can be used in the donor site. Some clinicians prefer to fill the donor site with Freeze Dried Bone Allograft (FDBA), especially when a large block has been harvested. In the vestibular approach, when closing, a resorbable suture is first used to attach the mentalis muscle to the 3mm periosteal muscle layer left on the bone side.

#### *8.2.2. Lateral ramus block harvesting*

The approach to harvest bone graft from the lateral ramus can proceed two different ways: 1) Vestibular or 2) Sulcular. The vestibular approach has access through the area through vestibular incision on external oblique ridge. Advantage of this approach is lack of disturbing the periodontium of the adjacent teeth. The indication of sulcular approach is when recipient site is located nearby. The distal extent of the incision should not be more than occlusal plane to minimize the risk of facial nerve damage, bleeding and exposing buccal fat. Osteotomy is suggested to be performed in a defined sequence; superior cut, then anterior, then posterior and finally inferior cut (Figure 11).

The superior cut length and thickness is important. This cut is usually made approximately 4 mm medial to the external oblique ridge but can be performed up to 6 mm depending on the regional anatomy. It may be extended anteriorly to the distal area of the first molar, depending on the anatomy. The anterior and posterior vertical cuts are made in parallel to the predicted length and width of the bone graft block, and are limited by anatomic position of the man‐ dibular canal, which determines the harvesting block width. Complete cortical penetration of inferior osteotomy cut is avoided due to its proximity to the mandibular canal in many cases. An osteotome or a chisel can be used to remove the bone graft from its bed avoiding penetrating excessively to damage mandibular canal. Closing the incision usually is done without applying any graft or hemostatic agent.

**Figure 11. Intraoral approach to harvest the lateral ramus bone block. The .osteotomy line of the superior cut is seen Figure 11.** Intraoral approach to harvest the lateral ramus bone block. The osteotomy line of the superior cut is seen.

#### **8.3. Anterior iliac crest bone grafting** The superior cut length and thickness is important. This cut is usually made

reflect block bone graft from its bed. After block removal a hemostatic agent can be used in the donor site. Some clinicians prefer to fill the donor site with Freeze Dried Bone Allograft (FDBA), especially when a large block has been harvested. In the vestibular approach, when closing, a resorbable suture is first used to attach the mentalis muscle to the 3mm periosteal

**Figure 10.** Full mucoperiosteal flap retracted to expose the donor site for harvesting symphyseal bone. Two osteotomy

sites are determined on both sides and 3mm bone is maintained in between to support the chin profile.

The approach to harvest bone graft from the lateral ramus can proceed two different ways: 1) Vestibular or 2) Sulcular. The vestibular approach has access through the area through vestibular incision on external oblique ridge. Advantage of this approach is lack of disturbing the periodontium of the adjacent teeth. The indication of sulcular approach is when recipient site is located nearby. The distal extent of the incision should not be more than occlusal plane to minimize the risk of facial nerve damage, bleeding and exposing buccal fat. Osteotomy is suggested to be performed in a defined sequence; superior cut, then anterior, then posterior

The superior cut length and thickness is important. This cut is usually made approximately 4 mm medial to the external oblique ridge but can be performed up to 6 mm depending on the regional anatomy. It may be extended anteriorly to the distal area of the first molar, depending on the anatomy. The anterior and posterior vertical cuts are made in parallel to the predicted length and width of the bone graft block, and are limited by anatomic position of the man‐ dibular canal, which determines the harvesting block width. Complete cortical penetration of inferior osteotomy cut is avoided due to its proximity to the mandibular canal in many cases. An osteotome or a chisel can be used to remove the bone graft from its bed avoiding penetrating excessively to damage mandibular canal. Closing the incision usually is done without applying

muscle layer left on the bone side.

524 A Textbook of Advanced Oral and Maxillofacial Surgery Volume 2

*8.2.2. Lateral ramus block harvesting*

and finally inferior cut (Figure 11).

any graft or hemostatic agent.

Anterior iliac crest bone grafts are common used grafts not only in maxillofacial surgery but also in orthopedic surgery. The iliac crest is almost subcutaneous and cortical or corticocan‐ cellous grafts in different shapes and size can be taken from this region simply and safety. The *anterior superior iliac spine (ASIS)*, is easily palpable which is located in the most anterior and superior portion of the crest. Posteriorly along the crest of the ilium in the widest portion is the iliac tubercle. The incision starts 2 cm posterior to ASIS and continues up to 8 cm along the crest. The neural branches, which are in risk of damage, are iliohypogastric, subcostal branches and lateral femoral cutaneous nerves. Retracting the skin medially and avoiding extending the incision posteriorly are suggested to decrease this risk. Dissecting laterally and violating iliotibial fascia is not recommended. Harvesting bone from iliac crest can be performed via different approaches including using a trephine device, monocortically and bicortically with different techniques (Figure 12). approximately 4 mm medial to the external oblique ridge but can be performed up to 6 mm depending on the regional anatomy. It may be extended anteriorly to the distal area of the first molar, depending on the anatomy. The anterior and posterior vertical cuts are made in parallel to the predicted length and width of the bone graft block, and are limited by anatomic position of the mandibular canal, which determines the harvesting block width. Complete cortical penetration of inferior osteotomy cut is avoided due to its proximity to the mandibular canal in many cases. An osteotome or a chisel can be used to remove the bone graft from its bed

Usually monocortical bone blocks are harvested from the medial surface with osteotomes or a saw. In young ages, the border portion of the iliac crest consists of chondral struc‐ ture which should be bypassed in the harvesting procedure. Closing the donor site is done in three layers, and a vacuum drain usually is placed. Minor complications of this bone graft harvesting included superficial infections, superficial seromas, and minor hemato‐ avoiding penetrating excessively to damage mandibular canal. Closing the .incision usually is done without applying any graft or hemostatic agent **Anterior iliac crest bone grafting** Anterior iliac crest bone grafts are common used grafts not only in

maxillofacial surgery but also in orthopedic surgery. The iliac crest is almost subcutaneous and cortical or corticocancellous grafts in different shapes and size can be taken from this region simply and safety. The *anterior superior iliac spine (ASIS)*, is easily palpable which is located in the most anterior and superior portion of the crest. Posteriorly along the crest of the ilium in the widest portion is the iliac tubercle. The incision starts 2 cm posterior to ASIS and continues up to 8 cm along the crest. The neural

**Figure 12.** Iliac bone graft harvesting procedure.

mas. Major complications are herniation of abdominal contents, vascular injuries, deep infections at the donor site, neurologic injuries, deep hematoma formation requiring surgical drainage, and iliac fractures [31].

#### **8.4. Placing the bone graft into the recipient site**

A moist environment with saline is suggested as a reservoir for the autogenous bone graft. Cortical or corticocancellous block grafts can be adjusted for recipient site with burs, saws or discs. The block should be prepared so that when placed in the recipient site it does not rock and fits snuggly and is in intimate contact with the underlying host bone bed. Fixation of the block graft is a principle issue. Screws and plates are devices, which can be used to achieve sufficient stability. Applying two screws is recommended and using the lag screw technique is suggested. The recipient bed and block graft may be penetrated to facilitate vascular ingrowths. Applying particulate bone graft around the bone block is usually advocated to maintain space for more osteogenesis. The graft structure is then covered with a barrier membrane to prevent soft tissue ingrowth into the integrating new bone especially when particulate materials are added. Tension free closure of the grafted site is critical to success.

#### **8.5. Anatomic repositioning**

#### *8.5.1. Distraction osteogenesis*

Distraction osteogenesis (DO) is a contemporary method that has been used in oral and maxillofacial defects. DO is a method to generate new bone by gradual separation of bone segments. In this procedure a distractor device is placed on two sides of an osteotomy site (Figure 13).

After a latency period the device is gradually activated and makes a gap between two bone segments. The new immature bone is generated between these two segments in the created gap. Then the device will not be activated for a period to give the new bone a time to mineralize

mas. Major complications are herniation of abdominal contents, vascular injuries, deep infections at the donor site, neurologic injuries, deep hematoma formation requiring surgical

A moist environment with saline is suggested as a reservoir for the autogenous bone graft. Cortical or corticocancellous block grafts can be adjusted for recipient site with burs, saws or discs. The block should be prepared so that when placed in the recipient site it does not rock and fits snuggly and is in intimate contact with the underlying host bone bed. Fixation of the block graft is a principle issue. Screws and plates are devices, which can be used to achieve sufficient stability. Applying two screws is recommended and using the lag screw technique is suggested. The recipient bed and block graft may be penetrated to facilitate vascular ingrowths. Applying particulate bone graft around the bone block is usually advocated to maintain space for more osteogenesis. The graft structure is then covered with a barrier membrane to prevent soft tissue ingrowth into the integrating new bone especially when particulate materials are added. Tension free closure of the grafted site is critical to success.

Distraction osteogenesis (DO) is a contemporary method that has been used in oral and maxillofacial defects. DO is a method to generate new bone by gradual separation of bone segments. In this procedure a distractor device is placed on two sides of an osteotomy site

After a latency period the device is gradually activated and makes a gap between two bone segments. The new immature bone is generated between these two segments in the created gap. Then the device will not be activated for a period to give the new bone a time to mineralize

drainage, and iliac fractures [31].

**Figure 12.** Iliac bone graft harvesting procedure.

526 A Textbook of Advanced Oral and Maxillofacial Surgery Volume 2

**8.5. Anatomic repositioning**

*8.5.1. Distraction osteogenesis*

(Figure 13).

**8.4. Placing the bone graft into the recipient site**

**Figure 13.** A. Patient with premaxillary deficiency. B. The patient has lost his anterior incisal teeth due to an anterior maxillary defect. C. Intra-oral view of the premaxilla defect. D. DO device is inserted in the surgery phase.

and turn into mature bone. This is called the consolidation phase and is usually twice the activation period. After the consolidation period the device is removed. During the activation period the surrounding soft tissue grows simultaneously with the bone formation (Figure 14). This is why the DO is also called distraction histogenesis. DO devices are divided into two groups of intraoral and extraoral types each of which have certain indications.

**Indications.** DO was generally used in orthopedics years before being used in maxillofacial surgery. The most popular indication of DO is in hemifacial or hemimandibular microsomia. Actually DO was used in a case of hemicraniofacial microsomia successfully for the first time by McCarthy et al. in 1992 [32]. The most important indication of DO is in syndromes associated with congenital anomalies like cerebral palsy, hemifacial microsomia, Treacher–Collins syndrome, Pierre–Robin sequence, Nager syndrome and others. Investigations have shown the successful results of DO in such cases [33].

**DO in vertical dimension** is another important indication. Although new methods of bone grafting like fibular microvascular graft have been broadly used in these defects sometimes their use is restricted by the large size of the defect. In these large defects DO is a better technique to regenerate new bone and reconstruct the defect [34]. Sometimes the combination of microvascularized grafts with DO procedure is an ideal technique to reconstruct large defects especially defects caused by resection of pathologic lesions [7].

**Figure 14.** Inserted DO device is shown to generate new bone for reconstruction of the maxilla. The distractor device has been activated for months. The alveolar bone height has increased.

**DO in transverse dimension** is an interesting method being used in patients with arch constriction or an alveolar cleft. Reviews of the clinical studies about the use of DO in maxillary hypoplasia in patients with cleft lip and palate have shown the benefits of this technique as an alternative to orthognathic surgeries [35]. The important advantage of DO in these patients is unchanged or better velopharyngeal function. This method can be used in the mixed dentition period which is an advantage of this procedure comparing to orthognathic surgery procedures.

DO has been recently used in patients with midface hypoplasia in craniosynostosis like Crouzon, Apert, and Pfeiffer syndromes. Several investigations have evaluated this technique and compared it to LeFort III osteotomy [36, 37]. Although LeFort III osteotomy has been widely used to correct the maxillary retrusion, it is not possible to advance the midface a large amount. Lefort III-DO technique has been suggested in patients with great discrepancy; however trials have shown higher relapse of this method compared to the usual LeFort III osteotomy procedure. The advantage of LeFort III-DO technique is the lower risk for severe complications like cerebrospinal fluid leakage, meningitis, and infection.

**Advantages.**Simultaneous distraction of the soft tissue is a great advantage of this technique. The quality or quantity of the soft tissue bed makes the results of bone grafting unpredictable and reduces the success of the bone graft. In most cases DO obviates the need for bone grafting in the future. Morbidity of a donor site is also eliminated. The process of inserting and removing the device is less extensive as well.

**Disadvantages.**DO is a technique sensitive procedure and should be performed by an expert surgeon. The quality of the device is an important factor in success rate of the DO results. Loosening of the screws and displacement of the device may occur in some cases. DO proce‐ dures consist of two operations: one for insertion of the device and a second surgery to remove it. Sometimes a third surgery is needed in the future to achieve the perfect outcome especially when DO has been performed in a young patient. Unpredictable outcomes or malocclusion are inadvertent results. Motor and sensory nerve dysfunction is an untoward complication of DO. This complication is especially seen in DO of the mandible which may lead to permanent or transient weakness of marginal branch of facial nerve or hypoesthesia of inferior alveolar nerve. Scar formation and infection should be considered a more usual complication of DO.

#### **8.6. Nerve repositioning**

**DO in transverse dimension** is an interesting method being used in patients with arch constriction or an alveolar cleft. Reviews of the clinical studies about the use of DO in maxillary hypoplasia in patients with cleft lip and palate have shown the benefits of this technique as an alternative to orthognathic surgeries [35]. The important advantage of DO in these patients is unchanged or better velopharyngeal function. This method can be used in the mixed dentition period which is an advantage of this procedure comparing to orthognathic surgery procedures.

**Figure 14.** Inserted DO device is shown to generate new bone for reconstruction of the maxilla. The distractor device

DO has been recently used in patients with midface hypoplasia in craniosynostosis like Crouzon, Apert, and Pfeiffer syndromes. Several investigations have evaluated this technique and compared it to LeFort III osteotomy [36, 37]. Although LeFort III osteotomy has been widely used to correct the maxillary retrusion, it is not possible to advance the midface a large amount. Lefort III-DO technique has been suggested in patients with great discrepancy; however trials have shown higher relapse of this method compared to the usual LeFort III osteotomy procedure. The advantage of LeFort III-DO technique is the lower risk for severe

**Advantages.**Simultaneous distraction of the soft tissue is a great advantage of this technique. The quality or quantity of the soft tissue bed makes the results of bone grafting unpredictable and reduces the success of the bone graft. In most cases DO obviates the need for bone grafting in the future. Morbidity of a donor site is also eliminated. The process of inserting and

**Disadvantages.**DO is a technique sensitive procedure and should be performed by an expert surgeon. The quality of the device is an important factor in success rate of the DO results. Loosening of the screws and displacement of the device may occur in some cases. DO proce‐ dures consist of two operations: one for insertion of the device and a second surgery to remove it. Sometimes a third surgery is needed in the future to achieve the perfect outcome especially

complications like cerebrospinal fluid leakage, meningitis, and infection.

removing the device is less extensive as well.

has been activated for months. The alveolar bone height has increased.

528 A Textbook of Advanced Oral and Maxillofacial Surgery Volume 2

Rehabilitation of edentulous patients is often complicated and requires special consideration. In edentulous patients with atrophic bone above the mandibular canal is insufficient; reposi‐ tioning the inferior alveolar nerve (IAN) is a treatment option. This treatment is done if the overall bone height is enough to place implant fixtures, but the IAN interferes with this procedure. Repositioning of IAN is done to move the nerve from the canal placing it in a new position (outside the bone).

**Nerve lateralization** is a procedure in which the IAN is exposed and retracted laterally while the surgeon is inserting the fixtures. Then the nerve is left to fall back against the inserted fixtures or the lateral cortex.  Then the IAN is transected from its junction with the incisive nerve. In this way the nerve is freed and its retraction is much easier. The IAN is replaced posteriorly after cutting the incisive nerve. The surgeon is able to install the implant fixture after distalization of the IAN (Figure 15).

**Figure 15.** A, Nerve lateralization in an atrophic mandible to eliminate the nerve interfering with implant surgery. B, The IAN is transposed from the mandibular canal to make space for installation the implants. C, Simultaneous implant installation is also possible in this technique.

**Indications**.The actual indication of IAN transposition or lateralization is in atrophic posterior mandible where remaining bone above the mandibular canal is less than 10 mm [38,39]. There is no actual contraindication of IAN transposition reported in the literature.

**Advantages.** The risk of damage to IAN during the installation of fixtures is reduced by retracting and repositioning the nerve. The surgeon is able to use a longer fixture which may engage the inferior cortex of the mandible. The fixtures have more stability due to their bicortical insertion. This procedure is performed simultaneously with implant fixture instal‐ lation with or without bone grafting.

**Disadvantages.** The risk of damage to the IAN is a prominent disadvantage of nerve trans‐ positioning; Traction on the nerve usually causes temporary sensory loss [40]. Mandibular fracture, implant loss, hemorrhage, and osteomyelitis are other possible complications in long implant installation, associated with the transposition and lateralization of the IAN [38, 41, 42].


*3.1. Modeling of Uncertainties*

The forecast error of the RES and load is the main source of uncertainty, which is the deviation of the forecast value from the actual value. The forecast error can be seen as a continuous random variable and described by a continuous probability distribution model. Therefore, the actual power injection of RES and loads can be modeled as a forecast value plus a continuous random variable that represents forecast error:

$$\begin{cases} \quad \frac{P\_{Ri} = P\_{Ri} + \delta\_{Ri}}{P\_{Di} = P\_{Di} + \delta\_{Di}} & \forall i\\ \quad \frac{P\_{Di} = P\_{Di} + \delta\_{Di}}{P\_{Di} + \delta\_{Di}} & \end{cases} \tag{6}$$

where *P Ri* and *P Di* are the actual power injection of the *i*th RES and load, respectively, and δ*Ri* and δ*Di* are random variables that represent the forecast error of the power injection of the *i*th RES and load, respectively.

The proper distribution model to describe forecast error depends on the type of power injection, forecasting scale [27], etc. For instance, the forecast error of a load is usually assumed to follow a Gaussian distribution, while the beta distribution [28] is an appropriate choice to describe the short-term forecast error of wind power. Although there are many distribution models that can be used to describe a forecast error, the optimization model proposed in this paper is not sensitive to the distribution used. The first four order moments of a distribution model is the only information that is required and this can be obtained from historical data This moment information is used to approximate the CDF of a random variable, which is discussed in the following section.

The line flow under a single system topology is linearly dependent on power injections; when considering uncertainty, its expression is:

*Pk l* = *N G Gi*=1 *AkGi PGi* + *N R Ri*=1 *AkRi P Ri* − *N D Di*=1 *AkDi P Di* = ( *N G Gi*=1 *AkGi PGi* + *N R Ri*=1 *AkRi PRi* − *N D Di*=1 *AkDi PDi*)+( *N R Ri*=1 *AkRi*δ*Ri* − *N D Di*=1 *AkDi*δ*Di*) ∀*l*, ∀*k* (7)

where *Pk l* is the *l*th line flow under the *k*th system topology. The term in the first bracket of Equation (7) is the line flow part formed by forecast power injections, which is consistent with the traditional PSCOPF. The term in the second bracket of Equation (7) is the uncertainty part of a line flow, which is the linear combination of the forecast error of power injections of RES and load.

The overall line flow probability distribution, which comprehensively considers the influence of the uncertainty of power injection and system topology is our concern. Therefore, the occurrence probability of a system topology, or the so-called contingency probability, should be obtained.

The Poisson distribution [30] is used in this paper to describe the occurrence probability of a system topology:

$$P\_{\mathbf{r}}(s\_k) = 1 - e^{-\lambda\_l} \tag{8}$$

where *sk* is the *k*th contingency's system topology, Pr(*sk*) is the corresponding occurrence probability, *e* is the base of the natural logarithm and λ*t* is the failure rate of the component. Note that the failure rate can be modified according to the external weather conditions. An approach to calculate the failure rate under different weather conditions (normal, adverse and major adverse) is provided in [31].

For each contingency, an occurrence probability can be obtained from Equation (8). We assume that contingencies outside the contingency set will have little impact on system operation, so the probability of the normal state is approximated and expressed as:

$$\Pr\_{\mathbf{r}}(\mathbf{s}\_0) = 1 - \sum\_{k=1}^{N\_k} \Pr\_{\mathbf{r}}(\mathbf{s}\_k) \tag{9}$$

Considering the uncertainty of power injections and the probability of contingency, the overall line flow *Pl* can be obtained through the law of total probability theory, and it is expressed as:

$$\begin{array}{rcl}P\_{l} &=& \operatorname{\mathbf{P}\_{\mathbf{r}}}(\mathbf{s}\_{0})P\_{l}^{0} + \operatorname{\mathbf{P}\_{\mathbf{r}}}(\mathbf{s}\_{1})P\_{l}^{1} + \cdots \operatorname{\mathbf{P}\_{\mathbf{r}}}(\mathbf{s}\_{N\_{k}})P\_{l}^{N\_{k}} & \forall l\\ &=& \sum\_{k=0}^{N\_{k}}{\sum\limits\_{G=1}^{k}{A\_{G}^{k}}P\_{G}P\_{\mathbf{r}}}(\mathbf{s}\_{k}) + \sum\_{k=0}^{N\_{k}}{\sum\limits\_{Ri=1}^{k}{A\_{Ri}^{k}}P\_{Ri}P\_{\mathbf{r}}}(\mathbf{s}\_{k}) -\\ & \sum\_{k=0}^{N\_{k}}{\sum\limits\_{Ri=1}^{k}{A\_{Di}^{k}}P\_{Dl}P\_{\mathbf{r}}}(\mathbf{s}\_{k}) + \sum\_{k=0}^{N\_{k}}{\sum\limits\_{Ri=1}^{k}{A\_{Ri}^{k}}P\_{Ri}P\_{\mathbf{r}}}(\mathbf{s}\_{k}) - \sum\_{k=0}^{N\_{k}}{\sum\limits\_{Ri=1}^{k}{A\_{Di}^{k}}P\_{Di}P\_{\mathbf{r}}}(\mathbf{s}\_{k}) \end{array} \qquad \forall l \tag{10}$$

Obviously, the probability distribution of the overall line flow *Pl* can be regarded as the weighted average of the line flow probability distribution under each system topology.


**1. Introduction**

Psoriasis is a chronic inflammatory skin disorder characterized by hyper-proliferation of basal keratinocytes, thickened and scaly epidermis, and recruitment of inflammatory cells to the skin. It affects approximately 2% of the world's population. The disease follows a pathogenic pathway involving various immunocytes and immune molecules. Activated T cells have been shown to trigger a chain of cellular and molecular reactions leading to the formation of psoriatic lesions. Fusion proteins that can block T cell activation or the function of anergized T cells, and cytokines and biologics that can inhibit T cell migration are effective in the treatment of psoriasis. Intradermal injection of T cells from psoriatic patients into human skin/severe combined immunodeficient (SCID) mice can induce spontaneous psoriatic conversion of skins from healthy human or non-lesional skin from psoriatic patients. Thus, it is widely accepted that psoriasis is a T lymphocyte-mediated autoimmune disease. Although the roles of T cells in psoriasis have been confirmed, the exact mechanisms of psoriasis and the origin of abnormal T cells are still unclear.

Beside T cells, psoriatic patients have a wide variety of other immune abnormalities such as B cells, monocytes, neutrophils and erythrocytes. As the precursor of immune cells, bone marrow hematopoietic stem cells have been suggested to be responsible for immune dysregulation of T cells in psoriasis. Several recent studies have indicated that abnormal T cells may be closely related to anomalous hematopoietic stem cells (HSCs) determined by psoriatic hereditary background. In addition to HSCs, aberrant bone marrow mesenchymal stem cells have also been demonstrated in patients with psoriasis.

#### **2. The clinical cue of relationship between psoriasis and bone marrow cells**

Although various exogenous and endogenous factors are believed to activate the immune system leading to imbalance of the system and initiation of psoriasis, increasing evidences suggest that inherent and intrinsic rather than extrinsic factors are more important in psoriasis pathogenesis. These intrinsic factors may be involved in spontaneous T-cell activation or proliferation, regulation of cytokine production, hematopoietic cell development, and T-cell development in the thymus. Allogeneic bone marrow transplantations (BMT) have been reported to either eliminate or aggravate psoriasis.

Psoriasis and Stem Cells 5

enhanced in psoriatic CD34+ bone marrow cells compared to normal controls. Beside *HES*  genes, another transcription factor RUNX-1, which is essential for hematopoietic cell development, has long been suspected to be involved in the pathogenesis of psoriasis because loss of RUNX1 binding site located between the SLC9A3R1 and NAT9 genes at 17q25 has been found increased expression in the psoriatic CD34+ cells. These studies suggest that the dysfunction of immune cells in psoriatic patients can be traced back to the

**4. T cells differentiated from bone marrow derived hematopoietic cells of**

Since T cells are derived from bone marrow hematopoietic cells, it is suggested that hematopoietic cells are partly relevant to the dysfunction of T cells in psoriasis. To demonstrate whether T cells are produced inherently dysfunctional from the immune system, Zhang et al. cultured bone marrow CD34+ cells from psoriatic patients and induced them to differentiate into T cells and CD4+CD25+ regulatory T cells *in vitro*. A further functional study revealed abnormal characters of these cells compared to normal bone

The main hallmark of CD4+CD25+ T cells is their immune regulatory function by interacting with effector T cells. Several studies have reported that the CD4+CD25+ T-lymphocyte subpopulation in peripheral blood and lesional skin demonstrates a less inhibitory effect on effector T cells, leading to accelerated proliferation of pathogenic/effector T-cells in autoimmune diseases, especially in psoriasis. Although the proportion of CD4+CD25+ T cells and FOXP3 gene expression are comparable in both psoriatic and healthy samples, proliferation of psoriatic bone marrow derived CD4+CD25+ T cells is significantly attenuated and secretion of cytokines IL-2 and IL-10 is decreased compared to normal controls in response to streptococcal superantigen (Strep-A). In particular, CD4+CD25+ T cells differentiated from psoriatic CD34+ cells are functionally insufficient to restrain proliferation of activated effector T-cells. That is to say, the function of CD4+CD25+ T cells derived from psoriatic bone marrow CD34+ cells *in vitro* is similar to that of peripheral CD4+CD25+ T-

In another study, bone marrow CD34+ hematopoietic cells from psoriatic patients with family history were induced into effector T cells and their functions such as in vitro proliferation ability, secretion of cytokines IL-4, IL-8 and IFN–γ, and their ability to induce human keratinocytes producing C-myc, Bcl-xL, and Ki67 proteins were compared with their counterpart from healthy objects. The differentiated T cells from CD34+ cells of psoriatic patients showed higher proliferative activity and stronger capacity to secret Th1 cytokines in response to streptococcal superantigen and could induce expression of C-myc and Ki67, but not Bcl-XL in keratinocytes co-cultured with psoriatic differentiated T

These studies show that regulatory as well as effector T cells differentiated from CD34+ cells of psoriatic patients, but not normal controls, are functionally similar to those psoriatic circulating T cells and suggest that dysfunctional activity of T cells in psoriatic patients can

be traced back to the early development of hematopoietic cells.

**psoriatic patients are functionally different from normal T cells**

early development of hematopoietic cells.

lymphocytes of psoriatic patient *in vivo.*

marrow derived ones.

cells.

Leukemia patients with psoriasis reportedly have long-term psoriasis remission or amelioration. On the other hand, non-psoriatic leukemia patients can develop psoriasis after transplanting bone marrow from psoriatic donors. These clinical reports indicate that psoriatic immune abnormalities transferred by BMT may have originated from bone marrow HSC.

### **3. Bone marrow derived hematopoietic stem / progenitor cells from psoriatic patients are anomalously proliferative**

Based on the above evidence, many researchers begin to pay close attention to bone marrow abnormality of psoriatic patients. *In vitro* studies have shown that monocytopoietic activity is enhanced in psoriasis and functional bone marrow scintigraphy using 99Tcm-labelled human serum albumin millimicrospheres has shown hyperplasia of phagocytes in psoriasis.

Bone marrow, with its rapidly renewing cell populations, is one of the most sensitive tissues to various stimulations of exogenous or endogenous factors. Once activated the pathogenic peripheral immunocytes in psoriasis and their released soluble factors, such as gammainterferon (IFN-γ), interleukin-2 (IL-2), IL-8 and tumor necrosis factor-alpha (TNF-α), may influence hematopoietic microenvironment, and even hematopoiesis. *In vitro* assessment using high proliferative potential colony-forming cell (HPP-CFCs) and colony formation units (CFU) is used as a surrogate marker of hematopoietic activity and can play a key role in linking hematopoiesis to psoriasis. Supernatant of *in vitro* cultured psoriatic peripheral blood mononuclear cells was found to suppress the proliferative activity of normal bone marrow HPP-CFCs, CFU-GM (granulocyte-macrophage colony-forming units) and CFU-E (erythroid colony-forming units). These results support the hypothesis that aberrant psoriatic peripheral immunocytes and cytokines can influence hematopoiesis.

Recently, besides the influence of aberrant peripheral immunocytes and cytokines, researchers began to pay attention to the intrinsic deficiency of psoriatic bone marrow hematopoietic cells. Zhang *et al.* cultured psoriatic bone marrow mononuclear cells in methylcellulose semisolid medium and observed their colony formation ability in the presence of exogenous cytokine combinations. These studies show a decreased colony formation ability of HPP-CFC, CFU-GM but not CFU-E, implying that the proliferative activity of HSCs in patients with psoriasis may be intrinsically decreased. They further investigated the molecular mechanisms of abnormal proliferative activity of HSCs in psoriasis and found that promoter methylation of p15, p16 and p21 genes is significantly decreased and transcription levels of these genes are enhanced in *ex vivo* cultured bone marrow HPP-CFCs from psoriatic patients in comparison to those from healthy volunteers. The P15, P16 and P21 proteins belong to the INK4 kinase family of cyclin-dependent kinase inhibitors and can negatively regulate the cell cycle through competitive inhibition of cyclindependent kinases 2, 4 and 6. Higher expression of these genes may contribute to the low proliferative activity of psoriatic hematopoietic cells.

Expression of Notch receptors and their ligands in hematopoietic system has been widely reported, and Notch signaling has been shown to influence hematopoietic cell proliferation and differentiation at several stages. The activation of Notch signaling results in transcriptional activation of *E(spl)/HES* genes, which function as negative regulators of cell proliferation and differentiation. Moreover, Notch1 and Hes-1 expression is significantly

Leukemia patients with psoriasis reportedly have long-term psoriasis remission or amelioration. On the other hand, non-psoriatic leukemia patients can develop psoriasis after transplanting bone marrow from psoriatic donors. These clinical reports indicate that psoriatic immune abnormalities transferred by BMT may have originated from bone

**3. Bone marrow derived hematopoietic stem / progenitor cells from psoriatic**

Based on the above evidence, many researchers begin to pay close attention to bone marrow abnormality of psoriatic patients. *In vitro* studies have shown that monocytopoietic activity is enhanced in psoriasis and functional bone marrow scintigraphy using 99Tcm-labelled human serum albumin millimicrospheres has shown hyperplasia of phagocytes in psoriasis. Bone marrow, with its rapidly renewing cell populations, is one of the most sensitive tissues to various stimulations of exogenous or endogenous factors. Once activated the pathogenic peripheral immunocytes in psoriasis and their released soluble factors, such as gammainterferon (IFN-γ), interleukin-2 (IL-2), IL-8 and tumor necrosis factor-alpha (TNF-α), may influence hematopoietic microenvironment, and even hematopoiesis. *In vitro* assessment using high proliferative potential colony-forming cell (HPP-CFCs) and colony formation units (CFU) is used as a surrogate marker of hematopoietic activity and can play a key role in linking hematopoiesis to psoriasis. Supernatant of *in vitro* cultured psoriatic peripheral blood mononuclear cells was found to suppress the proliferative activity of normal bone marrow HPP-CFCs, CFU-GM (granulocyte-macrophage colony-forming units) and CFU-E (erythroid colony-forming units). These results support the hypothesis that aberrant

psoriatic peripheral immunocytes and cytokines can influence hematopoiesis.

proliferative activity of psoriatic hematopoietic cells.

Recently, besides the influence of aberrant peripheral immunocytes and cytokines, researchers began to pay attention to the intrinsic deficiency of psoriatic bone marrow hematopoietic cells. Zhang *et al.* cultured psoriatic bone marrow mononuclear cells in methylcellulose semisolid medium and observed their colony formation ability in the presence of exogenous cytokine combinations. These studies show a decreased colony formation ability of HPP-CFC, CFU-GM but not CFU-E, implying that the proliferative activity of HSCs in patients with psoriasis may be intrinsically decreased. They further investigated the molecular mechanisms of abnormal proliferative activity of HSCs in psoriasis and found that promoter methylation of p15, p16 and p21 genes is significantly decreased and transcription levels of these genes are enhanced in *ex vivo* cultured bone marrow HPP-CFCs from psoriatic patients in comparison to those from healthy volunteers. The P15, P16 and P21 proteins belong to the INK4 kinase family of cyclin-dependent kinase inhibitors and can negatively regulate the cell cycle through competitive inhibition of cyclindependent kinases 2, 4 and 6. Higher expression of these genes may contribute to the low

Expression of Notch receptors and their ligands in hematopoietic system has been widely reported, and Notch signaling has been shown to influence hematopoietic cell proliferation and differentiation at several stages. The activation of Notch signaling results in transcriptional activation of *E(spl)/HES* genes, which function as negative regulators of cell proliferation and differentiation. Moreover, Notch1 and Hes-1 expression is significantly

marrow HSC.

**patients are anomalously proliferative**

enhanced in psoriatic CD34+ bone marrow cells compared to normal controls. Beside *HES*  genes, another transcription factor RUNX-1, which is essential for hematopoietic cell development, has long been suspected to be involved in the pathogenesis of psoriasis because loss of RUNX1 binding site located between the SLC9A3R1 and NAT9 genes at 17q25 has been found increased expression in the psoriatic CD34+ cells. These studies suggest that the dysfunction of immune cells in psoriatic patients can be traced back to the early development of hematopoietic cells.


**9. Screening examination**

The initial non-contact ocular examination was conducted by trained non-ophthalmologists and included measurement of refraction and keratometry (Topcon KR-8100PA, Tokyo, Japan), IOP by noncontact pneumotonometry (Topcon CT-90A, Tokyo, Japan), angle width (Scanning Peripheral Anterior Chamber Analyzer, Takagi Seiko, Nagano, Japan), non-mydriatic optic disc photography by stereoscopic fundus camera (30o angle, 3-DX/NM, Nidek, Gamagori, Japan), and confocal laser scanning tomography (Heidelberg Retina Tomograph II, software version 3.0, Heidelberg Instruments, Heidelberg, Germany). IOP was measured three times, and the mean value was adopted.