**2. Novel therapies in infectious keratoconjunctivitis**

#### **2.1. Interferons (IFN) and adenoviral conjunctivitis**

Interferons were first described as the major effector cytokines of the host immune response against viral infections. IFN are well recognized by their potent antiviral properties, howev‐ er IFN production is also induced in response to bacterial ligands of innate immune recep‐ tors and/or bacterial infections, indicating a broader physiological role for these cytokines in host defence and homeostasis than was originally described.

© 2013 Santacruz et al.; licensee InTech. This is an open access article 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. © 2013 Santacruz et al.; licensee InTech. This is a paper 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.

Three main types of cytokines compose the IFN family: type I, type II and type III IFN. Type I IFN family is composed of 16 members, namely 12 IFNα subtypes, IFNβ, IFNε, IFNκ and IFNω. By contrast, the type II IFN family includes only one cytokine: IFNγ, which also exhibits antiviral activities. The third type of IFN is the IFNλ family, which in‐ cludes IFNλ1 (also known as IL-29), IFNλ2 (also known as IL-28A) and IFNλ3 (also known as IL-28B). On the basis of protein sequence and structure, type III IFN are markedly differ‐ ent from type I and type II IFN and are more similar to members of the interleukin-10 (IL-10) family; however, they provoke antiviral responses and induce the activation of IFNstimulated genes. [1]

Epidemic keratoconjunctivitis (EKC) is a severe ocular infection, caused by highly conta‐ gious adenoviruses Ad8, Ad19, and Ad37. Adenoviral infection of the eye induces keratitis and conjunctivitis, accompanied by pain, lacrimation, red and swollen eye, as well as de‐ creased vision that may last for months or even years. No specific antiviral drugs are cur‐ rently available for the treatment of EKC or any other infection caused by adenoviruses. Interestingly, it has been suggested that five strains of different serotypes of adenovirus, types 3 (AdV3; species B), 4 (species E), 8, 19a and 37 (species D) involved in acute kerato‐ conjunctivitis are highly inhibited by IFN-b and IFN-g in the A549 cell line, [2] However, IFN therapy in adenoviral keratoconjunctivitis has not been evaluated in clinical trials yet.

#### **2.2. Glycan interactions and EKC**

The initial event leading to EKC is binding of the viruses to glycans that contain sialic acid moieties on epithelial cells in the cornea or conjunctiva through trimeric fiber structures ex‐ tending from the viral particles. The receptor-binding domain is located at the C terminus of each fiber and contains three separate pockets that each can accommodate one sialic acid residue. Ad37 was recently shown to bind to cell-surface glycoproteins carrying a glycan structure named GD1a due to similitude to GD1a ganglioside. The GD1a glycan is a branched hexasaccharide with a terminal sialic acid residue on each of its two arms. Struc‐ tural studies showed that the two sialic acid moieties dock into two of three sialic acid bind‐ ing sites in the trimeric knob of the Ad37 fiber protein. Most likely, multiple fiber proteins simultaneously engage several host-cell epitopes containing terminal sialic acids; internali‐ zation and subsequent infection follow. In this context, the molecules named ME0322, ME0323, and ME0324 were synthetized as a tri- and tetravalent sialic acid compounds, and interestingly all of theses molecules inhibited the attachment of Ad37 virions to HCE cells in a dose-dependent manner and were at least two orders of magnitude more effective than sialic acid, suggesting a promissory inhibitor of Ad37 infection on corneal cells, composed by a multivalent sialic acid conjugate. If these compounds could be useful as a topical treat‐ ment is not known and needs further investigation. [3]

#### **2.3. Vaccines and Herpetic Stromal Keratitis (HSK)**

The disease course in herpetic stromal keratitis (HSK) begins with a primary infection by herpes simplex virus (HSV) followed by a period during which the virus enters latency in sensory and autonomic ganglia, after that a reactivation from the trigeminal ganglia follow‐ ing primary infection induce virus transportation to the ocular mucosa via antero-grade movement from the ganglia, ultimately causing herpetic keratitis, conjunctivitis and other ocular sequelae [4]

Many studies have shown that clinical disease is the result of a recruitment of inflammatory cells, mainly polymorphonuclear cells (PMN), macrophages, and T cells to the corneas of pa‐ tients with HSK. [5] Due to HSK could lead to a potentially blinding disease; several thera‐ peutical strategies are in development to control ocular damage at initial steps of inflammatory process, i.e. vaccination with different HSV epitopes.

Since the early nineties many attempts have been made to develop a vaccine that would be effective in preventing HSK. Most of these vaccines were useful to prevent primary HSK when given prior to HSV infection however failed to prevent recurrent HSK lesions. [6, 7, 8] Recently, a novel construct with a DNA vaccine expressing herpes simplex virus type 1gD and IL-21, appears to be effective in protect from primary lesions, and also ameliorates her‐ pes keratitis severity and time course after corneal infection with HSV-1 in the animal model [9] Nevertheles, future studies are needed in humans HSK to study efficacy of this vaccine.

#### **2.4. Lipids mediators and HSK**

Three main types of cytokines compose the IFN family: type I, type II and type III IFN. Type I IFN family is composed of 16 members, namely 12 IFNα subtypes, IFNβ, IFNε, IFNκ and IFNω. By contrast, the type II IFN family includes only one cytokine: IFNγ, which also exhibits antiviral activities. The third type of IFN is the IFNλ family, which in‐ cludes IFNλ1 (also known as IL-29), IFNλ2 (also known as IL-28A) and IFNλ3 (also known as IL-28B). On the basis of protein sequence and structure, type III IFN are markedly differ‐ ent from type I and type II IFN and are more similar to members of the interleukin-10 (IL-10) family; however, they provoke antiviral responses and induce the activation of IFN-

Epidemic keratoconjunctivitis (EKC) is a severe ocular infection, caused by highly conta‐ gious adenoviruses Ad8, Ad19, and Ad37. Adenoviral infection of the eye induces keratitis and conjunctivitis, accompanied by pain, lacrimation, red and swollen eye, as well as de‐ creased vision that may last for months or even years. No specific antiviral drugs are cur‐ rently available for the treatment of EKC or any other infection caused by adenoviruses. Interestingly, it has been suggested that five strains of different serotypes of adenovirus, types 3 (AdV3; species B), 4 (species E), 8, 19a and 37 (species D) involved in acute kerato‐ conjunctivitis are highly inhibited by IFN-b and IFN-g in the A549 cell line, [2] However, IFN therapy in adenoviral keratoconjunctivitis has not been evaluated in clinical trials yet.

The initial event leading to EKC is binding of the viruses to glycans that contain sialic acid moieties on epithelial cells in the cornea or conjunctiva through trimeric fiber structures ex‐ tending from the viral particles. The receptor-binding domain is located at the C terminus of each fiber and contains three separate pockets that each can accommodate one sialic acid residue. Ad37 was recently shown to bind to cell-surface glycoproteins carrying a glycan structure named GD1a due to similitude to GD1a ganglioside. The GD1a glycan is a branched hexasaccharide with a terminal sialic acid residue on each of its two arms. Struc‐ tural studies showed that the two sialic acid moieties dock into two of three sialic acid bind‐ ing sites in the trimeric knob of the Ad37 fiber protein. Most likely, multiple fiber proteins simultaneously engage several host-cell epitopes containing terminal sialic acids; internali‐ zation and subsequent infection follow. In this context, the molecules named ME0322, ME0323, and ME0324 were synthetized as a tri- and tetravalent sialic acid compounds, and interestingly all of theses molecules inhibited the attachment of Ad37 virions to HCE cells in a dose-dependent manner and were at least two orders of magnitude more effective than sialic acid, suggesting a promissory inhibitor of Ad37 infection on corneal cells, composed by a multivalent sialic acid conjugate. If these compounds could be useful as a topical treat‐

The disease course in herpetic stromal keratitis (HSK) begins with a primary infection by herpes simplex virus (HSV) followed by a period during which the virus enters latency in sensory and autonomic ganglia, after that a reactivation from the trigeminal ganglia follow‐

stimulated genes. [1]

46 Common Eye Infections

**2.2. Glycan interactions and EKC**

ment is not known and needs further investigation. [3]

**2.3. Vaccines and Herpetic Stromal Keratitis (HSK)**

Resolvins are lipid mediators that are derived from the v-3 polyunsaturated fatty acids eico‐ sapentaenoic acid and do- cosahexaenoic acid [10] The name of these lipid mediators is re‐ lated to their main function, control of inflammation. Resolvins are involved in prevention of diapedesis, regulation of dendritic cell costimulatory factors, [11], increased macrophage phagocytosis of apoptotic neutrophils, inhibition of host tissue inflammatory responses, with the release of chemokines and cytokines, [12] promotion of tissue repair, and preven‐ tion of host tissue cell death during stress. [13] Interestingly, topical therapy with resolvins in corneas infected with HSV showed a diminished lesion severity and corneal neovasculari‐ zation when compared with non-treated eyes. Therapy with resolvins, induced a decreased influx of effector CD4+ T cells and neutrophils to corneal tissue; a diminished production of proinflammatory cytokines and molecules involved in ocular neovascularization were also observed during this treatment in the animal model, suggesting resolvins as promissory molecules in the treatment of HSK.

#### **2.5. Dialyzable Leuckocyte Extracts (DLE) and HSK**

DLE were described by Lawrence in 1955, who proved that the extract obtained from a dia‐ lyzed of viable leukocytes from a health donor presenting a positive percutaneous tubercu‐ lin test was able to transfer to a healthy receptor the ability to respond to this test [14] DLE are constituted by a group of numerous molecules all of them with a molecular weight be‐ tween 1-12 KDa. DLE have been widely used as adjuvant for treating patients with infec‐ tious diseases, and deficient cell-mediated immune response. [15]

The most consistent effects of DLE on the immune system are expression of delayed-type hypersensitivity (DTH) and production of cytokines. [16] Despite DLE have been extensive‐ ly studied in worldwide, in our country, only Transferon® has been approved for human

use by the federal regulatory authorities of health (COFEPRIS), this clarification is relevant, since the following immunological activities correspond exclusively to preclinical and clini‐ cal research related to Transferon®. Immunomodulation by Transferon® has been demon‐ strated by restoration of iNOS expression in a mouse model of tuberculosis, provoking inhibition of bacterial proliferation and significant increase of DTH [17] Transferon® also in‐ duces mRNA expression and IFN-γ secretion in peripheral blood mononuclear cells (PBMC) in animals with experimental glioma when compared with non-treated animals. [18] Due to Transferon® induces a Th1 response a clinical study comparing acyclovir treatment and Transferon® during human herpes virus infection was conducted; in that study patients treated with Transferon® had low incidence of clinical complications, better pain control, and also IFN-g was significant increased in serum when compared with patients treated on‐ ly with acyclovir. [19] Then, our group conducted a second clinical trial to evaluate immu‐ nological data and clinical outcome of patients with HSK treated with acyclovir or acyclovir and Transferon® as adjuvant therapy in patients with herpetic keratitis. Interestingly, pa‐ tients treated with acyclovir and Transferon® showed higher frequency of circulating CD4+IFN-g+ T cells and lower frequency of circulating CD4+IL4+ T cells after treatment; [20], when clinical outcome was evaluated, patients who received acyclovir and Transfer‐ on® as adjuvant showed a significant better clinical outcome than patients treated only with Acyclovir after three months of treatment. (Figure 1)

Despite conclusion of this study was that Transferon® could be used as therapeutical tool as adjuvant treatment in herpetic keratitis, additional clinical studies with more number of pa‐ tients are needed to confirm these results.

#### **2.6. Amniotic membrane as immunomodulator in infectious keratitis**

Amniotic membrane (AM) is the inner layer of the fetal membranes that is in contact with the fetus. An avascular stroma and single epithelial cells constitute the amniotic membrane [21] It has been documented in various clinical trials that transplantation of amniotic mem‐ brane is therapeutically useful in different superficial ocular pathologies [22, 23, 24, 25] Its beneficial effects for transplantation are due to the following characteristics: amniotic mem‐ brane promotes epithelialization, [26] inhibits angiogenesis [27] and has been used as a car‐ rier for ex-vivo expansion of corneal epithelial [28] and endothelial cells [29] Recently, we demonstrated that AM is able to induce apoptosis, inhibit cell proliferation of human PBMC, and abolish the synthesis and the secretion of pro-inflammatory cytokines even when they are LPS stimulated in vitro. [30] Similarly to us, Bauer *et. al.* demonstrated that amniotic membrane transplantation (AMT) in a mouse model of necrotizing HSK, induced an increased rate of local macrophages apoptosis, with decrement in proinflammatory cyto‐ kines IL-6, IL-10, IL-12, TNF-α. Nevertheless, in this animal model, the authors suggest that corneas treated with AMT induced peroxisome proliferator-activated receptor-γ (PPAR- γ) which is associated to phenotypical change in macrophages, turning them from classically activated into alternatively activated macrophages or macrophage cell death, through lipid metabolism and PPAR-γ pathway. [31] In the other hand, animal models of *Staphylococcus aureus* keratitis treated with AMT, have suggested that AM improved the healing process, resulting in decreased corneal haze and less neovascularization.[32] however the exact mo‐ lecular mechanism remains unknown and needs investigation. Due to a lack in this molecu‐ lar aspects clinical use of AM is limited and only in certain cases immunomodulation function of AM could be exploited, i.e. keratitis with secondary ocular surface damage. (Figure 2)

use by the federal regulatory authorities of health (COFEPRIS), this clarification is relevant, since the following immunological activities correspond exclusively to preclinical and clini‐ cal research related to Transferon®. Immunomodulation by Transferon® has been demon‐ strated by restoration of iNOS expression in a mouse model of tuberculosis, provoking inhibition of bacterial proliferation and significant increase of DTH [17] Transferon® also in‐ duces mRNA expression and IFN-γ secretion in peripheral blood mononuclear cells (PBMC) in animals with experimental glioma when compared with non-treated animals. [18] Due to Transferon® induces a Th1 response a clinical study comparing acyclovir treatment and Transferon® during human herpes virus infection was conducted; in that study patients treated with Transferon® had low incidence of clinical complications, better pain control, and also IFN-g was significant increased in serum when compared with patients treated on‐ ly with acyclovir. [19] Then, our group conducted a second clinical trial to evaluate immu‐ nological data and clinical outcome of patients with HSK treated with acyclovir or acyclovir and Transferon® as adjuvant therapy in patients with herpetic keratitis. Interestingly, pa‐ tients treated with acyclovir and Transferon® showed higher frequency of circulating CD4+IFN-g+ T cells and lower frequency of circulating CD4+IL4+ T cells after treatment; [20], when clinical outcome was evaluated, patients who received acyclovir and Transfer‐ on® as adjuvant showed a significant better clinical outcome than patients treated only with

Despite conclusion of this study was that Transferon® could be used as therapeutical tool as adjuvant treatment in herpetic keratitis, additional clinical studies with more number of pa‐

Amniotic membrane (AM) is the inner layer of the fetal membranes that is in contact with the fetus. An avascular stroma and single epithelial cells constitute the amniotic membrane [21] It has been documented in various clinical trials that transplantation of amniotic mem‐ brane is therapeutically useful in different superficial ocular pathologies [22, 23, 24, 25] Its beneficial effects for transplantation are due to the following characteristics: amniotic mem‐ brane promotes epithelialization, [26] inhibits angiogenesis [27] and has been used as a car‐ rier for ex-vivo expansion of corneal epithelial [28] and endothelial cells [29] Recently, we demonstrated that AM is able to induce apoptosis, inhibit cell proliferation of human PBMC, and abolish the synthesis and the secretion of pro-inflammatory cytokines even when they are LPS stimulated in vitro. [30] Similarly to us, Bauer *et. al.* demonstrated that amniotic membrane transplantation (AMT) in a mouse model of necrotizing HSK, induced an increased rate of local macrophages apoptosis, with decrement in proinflammatory cyto‐ kines IL-6, IL-10, IL-12, TNF-α. Nevertheless, in this animal model, the authors suggest that corneas treated with AMT induced peroxisome proliferator-activated receptor-γ (PPAR- γ) which is associated to phenotypical change in macrophages, turning them from classically activated into alternatively activated macrophages or macrophage cell death, through lipid metabolism and PPAR-γ pathway. [31] In the other hand, animal models of *Staphylococcus aureus* keratitis treated with AMT, have suggested that AM improved the healing process,

Acyclovir after three months of treatment. (Figure 1)

**2.6. Amniotic membrane as immunomodulator in infectious keratitis**

tients are needed to confirm these results.

48 Common Eye Infections

**Figure 1.** Representative clinical photographs of patients with herpetic keratitis treated with Acyclovir or treated with acyclovir and Transferon®. Upper left, Before treatment; Upper right, Same patient, at 3 months of treatment with acy‐ clovir; Low left, Before treatment; Low right, Same patient, at 3 months of treatment with acyclovir and Transferon®

**Figure 2.** Clinical photographs of AMT in 67 year old female patient with a history of peripheral infectious keratitis secondary to trichiasis. Left, AMT covering the lower peripheral corneal defect. Amniotic membrane was folded sever‐ al times over the cornea to increase their anti-inflammatory properties. Right, Same patient, 15 days after AMT, clinical photograph showing apparent control of hyperaemia and inflammation

#### **2.7. MIF-CD74 blockade in** *Pseudomona aeurginosa* **keratitis**

Macrophage migration inhibitory factor (MIF) is an integral component of inflammatory re‐ sponses. MIF induces and sustains expression of several pro-inflammatory cytokines.[33] trough interaction with a receptor complex composed by CD74/CD44 [34] CD74 was first described as class II invariant chain, while CD44 is an adhesion molecule that binds hyalur‐ onic acid and other matrix metalloproteinases. Interaction of MIF with CD74/CD44 results in activation of Mitogen-Activated Protein Kinase (MAPK), production of PGE214 and further induction of inflammatory mediators [35]

Corneal infections by *Pseudomonas aeruginosa* are more difficult to treat and result in worse visual outcome than other bacterial corneal ulcers. Unfortunately the existing therapies fail to control the inflammation secondary to P. aeruginosa keratitis and novel interventions are needed to alleviate tissue damage resulting from local inflammation, recently two studies suggest that blockade of MIF-CD74 ligation ameliorate the disease-associated pathology by decreased proinflammatory mediators and reduced bacterial presence in the cornea [36, 37]
