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On the other hand (the second beneficial aspect of autoimmunity) the autoimmune system works to eliminate abnormal cell lines that emerge as a result of external (e.g. drugs, radiation, smoking, etc.) and internal (e.g. genetic) influences. Emerging cancer cells are recognized and eliminated by NK cells. In addition, cancer specific ags can stimulate a pathogenic lytic IgG aab response, particularly when presented to the system with an adjuvant. These aabs may lyse cancer cells in the presence of complement and eliminate

The two harmful aspects of autoimmunity, i.e., autoimmune disease and cancer, will manifest only if external (e.g. carcinogens, chemicals, infectious agents, UV irradiation, drugs, smoking) or internal (e.g. genetic) influences cause changes in the structural makeup of cells or cell products containing native ags. Such changes could result in harmful immune

Taking advantage of recent insight into the workings of the immune system, our MVT has proved itself to be effective in preventing the development of an experimental autoimmune kidney disease, and when the disease was in its progressive phase, in terminating it altogether, by halting immunopathological events that were causing the symptomatic,

The immune system has a natural ability to correct immunological mishaps and restore the body to normalcy, provided the right information is presented to it for processing. The MVT is a way of presenting that information, in the form of specific ICs, and triggering or enhancing the body's own ability to counteract autoimmune disease, cancer, and chronic infection. We have observed that by injecting ICs – made up of a given endogenous or exogenous ag and a specific ab against it at slight ag excess – into experimental animals, the recipient's immune system produced the same ab, with the same specificity against the

We have shown in experimental situations that corrective immune responses can be induced by the application of the MVT. We remain convinced that by the proper application of the MVT in humans, chronic diseases such as cancer, autoimmune disease, and chronic

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**29**

*Japan*

Yoshio Osada

**Parasitic Helminths as Potential Therapeutic**

In developed countries, the prevalence of both allergic disease (asthma) and autoimmune disorders such as Crohn's disease (CD), multiple sclerosis (MS), and type 1 diabetes (T1D) is increasing. This trend seems to be inversely correlated to prominent decreases in infectious diseases such as measles, tuberculosis, and hepatitis A (Bach, 2002). Based on such epidemiological observations, the "hygiene hypothesis", that exposure to infectious agents; i.e. bacteria, viruses, and parasites, especially in childhood, lowers the risk of later onset of immunological disorders, has attracted much attention. According to the hypothesis, a reduced exposure to infectious agents due to improved hygienic conditions in developed countries, urbanized areas, and/or a westernized lifestyle is responsible for the higher incidence of autoimmunity and allergies in modern society. A number of epidemiological and experimental studies have demonstrated the plausibility of this hypothesis. However, there is also considerable evidence that does not support or contradicts the hypothesis. In

Most studies trying to elucidate the mechanisms involved have been conducted using rodent models. In various models of autoimmune or allergic diseases, effects of bacterial, viral or parasitic infections were tested. Consequently, in many cases, preventive or ameliorating effects of infectious agents have been confirmed. In the main part of this chapter, the influence of parasitic helminths on autoimmune disorders will be introduced. Although the mechanisms underlying the amelioration and/or prevention of immunological disorders by infectious agents have been studied extensively, they are not yet fully understood. As both Th1-polarizing (bacteria and viruses) and Th2-polarizing (parasitic helminths) infectious agents have anti-autoimmune/anti-allergic activities (Zaccone & Cooke, 2011), the "Th1/Th2 paradigm" is not enough to explain the mechanisms involved. Additionally, some autoimmune diseases have been shown to be dependent on a pathogenic T helper subset (Th17), and not on the Th1 subset. The suppressive mechanisms of infectious agents have been attributed to various regulatory/suppressive cell populations, such as Treg cells, Breg cells, NKT cells and alternatively activated macrophages (AA MΦs). In addition, the involvement of suppressive cytokines (e.g. IL-4, IL-10 or TGF-β) has been studied and demonstrated. Focusing on helminth infections, the

Some autoimmune diseases are being treated successfully with recently developed biological products (mainly humanized monoclonal antibodies against pro-inflammatory

this chapter, conflicting reports are introduced and discussed.

possible involvement of regulatory cells and cytokines is discussed.

**1. Introduction**

**Agents Against Autoimmune Disorders**

*University of Occupational and Environmental Health, Japan*


*3.4. Institutions Active in Relevant Research*

In Table 3, the 10 organizations with the highest rates of publication in the field of social networks related to health and young people are presented. Additionally, the top three keywords used in each of these institutions are included in this table.

**Table 3.** Publications and keywords utilized by the top ten international institutions.

The University of Toronto is in first position, with 158 documents, which is not surprising, as the *Journal of Medical Internet Research* is based at this location. Next is the University of Sydney, in second position with 157 documents, and the University of Michigan in third position with 155. In positions four–six are the University of North Carolina at Chapel Hill with 152, the University of Washington with 143, and the University of Melbourne with 140 documents published. Finally, Harvard Medical School has 132, University of California has 131, Johns Hopkins Bloomberg School of Public Health has 126, and the University of California has 123. It should be highlighted that the keyword used most often by these institutions is "human/s", ranking in first place in all cases.

The increase of publications and the ranking of affiliations might be related to collaboration between authors. These collaborations have been previously studied by other authors, showing that, since 1997, collaborations in the United States or Canada have increased by 20% [51]. Moreover, these factors have been linked to collaborations between the United States and the other countries, showing a possible node of union [51].

Regarding the type of study implemented by each institution, according to Scopus, the results showed that all of the institutions focused on articles in the area of medicine, followed by the area of social sciences. The central countries with a higher number of publications were the United States, United Kingdom, and Canada. Finally, the most common keywords used according to Scopus were "human/s", "female", "articles", "male", and "social media."


**1. Introduction**

Cell encapsulation is the process of entrapping cells into a matrix. In general, the matrix is spherical in shape and in the form of a polymeric hydrogel. Cell encapsulation technology has shown great promise for immunoisolation and controlled release of therapeutic products towards gene therapy. Figure 1 demonstrates the mechanism of encapsulated transgenic cells for gene therapy.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1.1. Encapsulation materials**

Both natural and synthetic polymers have been utilized for cell encapsulation. Natural polymers that have been used include alginate, agarose, collagen, and hyaluronic acid, while synthetic polymers, including poly(vinyl alcohol), poly(lactic-co-glycolic acid), polyacrylates, HEMA-MMA-MAA, polyphosphazines, and polyepoxides, have been studied.[1] Natural polymers are more commonly used because of their biocompatibility and are easily accepted by the public. However, their product quality and characteristics can vary greatly between companies and batches compared to synthetic polymers. Alginate, agarose, and polylactideco-glycolide (PLGA), the most commonly used encapsulation materials, are introduced here.

#### *1.1.1. Alginate*

Alginates, polysaccharides, are linear block polymers consisting of α-l-guluronic acid (G) and β-d-manuronic acid (M) blocks (Figure 2). Divalent cations, such as Ca2+, Ba2+, and Sr2+, can link alginate molecules together (i.e. through ionic cross-linking) forming alginate hydrogel capsules while encapsulating cells inside. The G and M contents of the alginate molecules can affect the gel properties including mechanical strength, biocompatibility, and permeability.[2– 6] Recently, it has also been shown that oligochitosan could be used as a cross-linker for polysaccharide-based gel formations.[7]

**Figure 1.** A conceptual schematic demonstrating cell encapsulation for gene therapy.

**Figure 2.** Chemical structure of alginate (A) and alginate-based hydrogel formation mechanism (B).

#### *1.1.2. Agarose*

**1.1. Encapsulation materials**

192 Gene Therapy - Principles and Challenges

polysaccharide-based gel formations.[7]

**Figure 1.** A conceptual schematic demonstrating cell encapsulation for gene therapy.

*1.1.1. Alginate*

Both natural and synthetic polymers have been utilized for cell encapsulation. Natural polymers that have been used include alginate, agarose, collagen, and hyaluronic acid, while synthetic polymers, including poly(vinyl alcohol), poly(lactic-co-glycolic acid), polyacrylates, HEMA-MMA-MAA, polyphosphazines, and polyepoxides, have been studied.[1] Natural polymers are more commonly used because of their biocompatibility and are easily accepted by the public. However, their product quality and characteristics can vary greatly between companies and batches compared to synthetic polymers. Alginate, agarose, and polylactideco-glycolide (PLGA), the most commonly used encapsulation materials, are introduced here.

Alginates, polysaccharides, are linear block polymers consisting of α-l-guluronic acid (G) and β-d-manuronic acid (M) blocks (Figure 2). Divalent cations, such as Ca2+, Ba2+, and Sr2+, can link alginate molecules together (i.e. through ionic cross-linking) forming alginate hydrogel capsules while encapsulating cells inside. The G and M contents of the alginate molecules can affect the gel properties including mechanical strength, biocompatibility, and permeability.[2– 6] Recently, it has also been shown that oligochitosan could be used as a cross-linker for

> Agarose, a thermal-responsive polymer, consists of β-d-galactopyranose and 3,6-anhydro-αl-galactopyranose units which can undergo a sol–gel transition upon cooling (i.e. through thermal cross-linking) (Figure 3). Some agarose products have a transition temperature close to body temperature, making it a good candidate for cell encapsulation.[8]

#### *1.1.3. Polylactide-co-Glycolide (PLGA)*

PLGA polymers belong to aliphatic polyesters and are biodegradable (Figure 4). To prepare the capsules, PLGA is dissolved in methylene chloride, and then a second component is added to precipitate the polymer molecules (interfacial precipitation).[1,9]

#### **1.2. Encapsulation technologies**

Different technologies have been used for preparing macro/microcapsules, which include airjet encapsulation, electrostatic spray, laminar jet breakup, and microfluidic channel/nozzle. Among them, electrostatic spray and microfluidic channel/nozzle are two of the most fre‐ quently used encapsulation approaches.[10]

#### *1.2.1. Electrostatic spray method*

The electrostatic spray method has a significant appeal due to its ease of operation, scale-up capabilities, negligible damage to cells, and allowance for sterile operation conditions.[10] The mechanism of cell encapsulation by using the electrostatic pray method is shown in Figure

**Figure 3.** Chemical structure of agarose (A) and agarose-based hydrogel formation mechanism (B).

**Figure 4.** Chemical structure of PLGA.

5A. In general, a cell polymer mixture is extruded through a nozzle by using a pump or compressed air. The droplets are broken down into smaller ones under electrostatic force and/ or other introduced forces (e.g. vibration). Once the droplets reach the gelling bath containing the cross-linkers, the cell-loaded hydrogel capsules form immediately through various forces, such as ionotropic reaction between divalent ions and alginate molecules. Moreover, the system could be modified to prepare the core-shell structure hydrogel capsules, as depicted in Figure 5B.[11]

#### *1.2.2. Microfluidics channel/nozzle method*

Microfluidics devices can be used to generate micrometer-scale droplets with a narrow size distribution and controlled morphology.[12–14] This method shows great promise for cell encapsulation, especially for single cell encapsulation.[15] In general, capsules are formed by allowing a core fluid to be surrounded by a flowing sheath stream.[16] Recently, these devices have also been successfully applied for the generation of cell-loaded core-shell capsules (Figure 6).[14] Besides the relatively low encapsulation efficiency, a significant drawback of the current microfluidic technologies is that the oil used for shearing may leave a residual adhesive oil layer on the capsule which affects subsequent coating processes.[10,17] a residual adhesive oil layer on the capsule which affects subsequent coating processes.[10,17]

 Figure 5. A sketch of the electrostatic spray device used for generating polymeric hydrogel capsules (A).[10] *Reproduced by permission of The American Society of Mechanical Engineering* **Figure 5.** A sketch of the electrostatic spray device used for generating polymeric hydrogel capsules (A).[10] *Repro‐ duced by permission of The American Society of Mechanical Engineering (ASME)*; A modified electrostatic spray setup for fabricating the core-shell structure hydrogel capsules (B).[11] *Reproduced by permission of The Royal Society of Chemistry.*

*(ASME)*; A modified electrostatic spray setup for fabricating the core‐shell structure hydrogel capsules (B).[11] *Reproduced by permission of The Royal Society of Chemistry.*

5A. In general, a cell polymer mixture is extruded through a nozzle by using a pump or compressed air. The droplets are broken down into smaller ones under electrostatic force and/ or other introduced forces (e.g. vibration). Once the droplets reach the gelling bath containing the cross-linkers, the cell-loaded hydrogel capsules form immediately through various forces, such as ionotropic reaction between divalent ions and alginate molecules. Moreover, the system could be modified to prepare the core-shell structure hydrogel capsules, as depicted

**Figure 3.** Chemical structure of agarose (A) and agarose-based hydrogel formation mechanism (B).

Microfluidics devices can be used to generate micrometer-scale droplets with a narrow size distribution and controlled morphology.[12–14] This method shows great promise for cell

in Figure 5B.[11]

*1.2.2. Microfluidics channel/nozzle method*

**Figure 4.** Chemical structure of PLGA.

194 Gene Therapy - Principles and Challenges

Figure 6. A sketch of the **Figure 6.** A sketch of the microfluidics device for generating core-shell hydrogel capsules. The core channel height (H1) is the lowest. H: height and W: width.[14] *Reproduced by permission of The Royal Society of Chemistry.*

microfluidics device for generating core‐shell hydrogel capsules. The core channel height (H1) is the lowest. H: height and W: width.[14] *Reproduced by permission of The Royal Society of*

*Chemistry.*

5

### **2. Recent progress on transgenic cell encapsulation for gene therapy**

Encapsulation of genetically modified cells has been conducted for the treatment of central nervous system diseases, cardiovascular disorders, mucopolysaccharidosis type VII (MPSVII) disease, wounds, bone fractures, and cancer.[18–30] Considering most genetically engineered cells are from allogeneic or xenogeneic sources, immunoisolation is a critical factor when using these cells.[5]

#### **2.1. Bone-related diseases**

Bone morphogenic protein-2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) superfamily and has been widely reported to have osteoinductive activity. Ding *et al.* [31] studied the behaviour of BMP-2 gene-transfected bone marrow-derived mesenchy‐ mal stem cells in alginate-poly-l-lysine-alginate (APA) microcapsules. The results showed that encapsulated transfected cells could secrete BMP-2 proteins for at least 30 days and the APA microcapsules could be used for immunoisolation. Olabisi *et al.* [28] investigated microencapsulation of AdBMP-2-transduced MRC-5 cells (human diploid fetal lung fibroblasts) in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. After injecting the encapsulated cells intramuscularly, the volume of the bone formed was about twice that of the control group (unencapsulated cells). Recently, rapid heterotrophic ossification by using cryopreserved PEGDA encapsulated BMP-2 expressing mesenchymal stem cells (MSCs) was also observed (as shown in Figure 7).[32] Additionally, human calcitonin delivered by microencapsulated recombinant myoblasts showed potential for allergenic gene therapy for postmenopausal osteoporosis. [33] Furthermore, transplantation of fibrin glue-compound‐ ing hepatocyte growth factor-transgenic MSCs is a promising novel method for avascular necrosis of the femoral head (ANFH) therapy.[34]

#### **2.2. Cancer**

Both mouse myoblasts (C2C12 cells) and human embryonic kidney 293 (HEK293) cells were engineered to continuously secrete angiostatin, and were encapsulated into alginate-based microcapsules for cancer treatment. The *in vivo* experimental results demonstrated the potential for angiostatin-mediated cancer therapy by using an encapsulated transgenic cellbased approach.[35,36] Considering immunotherapies have been proven to be alternative strategies for malignancy treatment[37], combined immunotherapy (an interleukin 2 fusion protein, sFvIL-2) and antiangiogenic therapy (angiostatin) were tested. It was shown that transplantation of angiostatin expression and sFvIL-2-expressing C2C12 cells encapsulated in APA microcapsules improved the survival rate of experimental animals.[38] Recently, microencapsulation of therapeutic antibodies producing cells in APA microcapsules was tested for cancer treatment. [39] Additionally, with the advancement of stem cell research, there is an increased potential for cancer therapy by using encapsulated stem cells.[40]

**Figure 7.** Microencapsulated BMP2-transduced MSCs in a mouse model for heterotopic ossification. X-ray and Mi‐ croCT images of the resulting heterotopic ossification for freshly prepared BMP2 microencapsulated MSCs (a and b) and for cryopreserved BMP2 microencapsulated MSCs (d and e).[32]

#### **2.3. Neural diseases**

**2. Recent progress on transgenic cell encapsulation for gene therapy**

these cells.[5]

**2.2. Cancer**

**2.1. Bone-related diseases**

196 Gene Therapy - Principles and Challenges

necrosis of the femoral head (ANFH) therapy.[34]

Encapsulation of genetically modified cells has been conducted for the treatment of central nervous system diseases, cardiovascular disorders, mucopolysaccharidosis type VII (MPSVII) disease, wounds, bone fractures, and cancer.[18–30] Considering most genetically engineered cells are from allogeneic or xenogeneic sources, immunoisolation is a critical factor when using

Bone morphogenic protein-2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) superfamily and has been widely reported to have osteoinductive activity. Ding *et al.* [31] studied the behaviour of BMP-2 gene-transfected bone marrow-derived mesenchy‐ mal stem cells in alginate-poly-l-lysine-alginate (APA) microcapsules. The results showed that encapsulated transfected cells could secrete BMP-2 proteins for at least 30 days and the APA microcapsules could be used for immunoisolation. Olabisi *et al.* [28] investigated microencapsulation of AdBMP-2-transduced MRC-5 cells (human diploid fetal lung fibroblasts) in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. After injecting the encapsulated cells intramuscularly, the volume of the bone formed was about twice that of the control group (unencapsulated cells). Recently, rapid heterotrophic ossification by using cryopreserved PEGDA encapsulated BMP-2 expressing mesenchymal stem cells (MSCs) was also observed (as shown in Figure 7).[32] Additionally, human calcitonin delivered by microencapsulated recombinant myoblasts showed potential for allergenic gene therapy for postmenopausal osteoporosis. [33] Furthermore, transplantation of fibrin glue-compound‐ ing hepatocyte growth factor-transgenic MSCs is a promising novel method for avascular

Both mouse myoblasts (C2C12 cells) and human embryonic kidney 293 (HEK293) cells were engineered to continuously secrete angiostatin, and were encapsulated into alginate-based microcapsules for cancer treatment. The *in vivo* experimental results demonstrated the potential for angiostatin-mediated cancer therapy by using an encapsulated transgenic cellbased approach.[35,36] Considering immunotherapies have been proven to be alternative strategies for malignancy treatment[37], combined immunotherapy (an interleukin 2 fusion protein, sFvIL-2) and antiangiogenic therapy (angiostatin) were tested. It was shown that transplantation of angiostatin expression and sFvIL-2-expressing C2C12 cells encapsulated in APA microcapsules improved the survival rate of experimental animals.[38] Recently, microencapsulation of therapeutic antibodies producing cells in APA microcapsules was tested for cancer treatment. [39] Additionally, with the advancement of stem cell research, there is an increased potential for cancer therapy by using encapsulated stem cells.[40]

Parkinson's disease (PD) belongs to a group of conditions called motor system disorders, resulting from the loss of dopamine-producing brain cells.[41] This disease could be amenable to gene product replacement strategies including implantation of encapsulated transgenic cells.[42] There are several publications regarding encapsulated cell biodelivery of glial cell line-derived neurotrophic factor (GDNF) for PD treatment; GDNF has been proven to have neuroprotective and neurotrophic properties on dopaminergic neurons.[26,43,44] Further‐ more, encapsulated transgenic cells could be utilized in brain tumour treatment.[45,46]

Small capsules (<200 µm) have been developed for the delivery of gene products, secreted by encapsulated transgenic cells, to the brain, bypassing the blood–brain barrier (BBB). To date, several alginate-based microcapsule systems, Ca-alginate, APA, and alginate-chitosanalginate (ACA), have been reported.[10,47,48] Encapsulation of transgenic cells has also been used for other disease treatments, such as mucopolysaccharidosis VII and myocardial infarction. Table 1 summarizes the recent gene therapy studies based on encapsulated transgenic cells, with the exception of bone-related and neural diseases and cancer treatment.

**Table 1.** Recent gene therapy studies by using encapsulated transgenic cells