Use of Immunotherapy in Ocular Infections
DAVID C. GRITZ and GILBERT SMOLIN
Table Of Contents
NONSTEROIDAL ANTI-INFLAMMATORY DRUGS
|Although antibiotic therapy still plays a crucial role in the medical armamentarium
against infection, the host immune response to infection
is extremely important. Even when antibiotics provide a bactericidal action, an
intact immune response is essential for “curing” the
infection. Without an intact immune response, it can be very difficult
to destroy an infectious agent. An example of this is the ability
of Pseudomonas keratitis to recur in the setting of corticosteroids despite apparently
adequate antibiotic treatment.1–3 With the emergence of increasing antibiotic resistance, even more emphasis
may be placed on the body's own ability to fight infection. If
the body's immune reaction to infection could be augmented, it
is possible that the infection could be cleared more quickly or more
In ocular infections, however, there is a delicate balance between the infection-fighting immune response and the tissue-damaging inflammatory response. Obviously these responses are interconnected. In ocular infections, however, we often attempt to limit the body's response to an infection in order to minimize the inflammatory reaction. An exuberant inflammatory reaction can cause more extensive damage than caused by the infectious organism.4–8 Optimally, we would like to utilize the body's infection fighting ability, while minimizing inflammatory damage. One of the challenges for ocular pharmacotherapy is to address this area of research. Our ability to modulate the immune response in ocular infections may play an important role in future treatment strategies.
Better infection-fighting treatments may also be realized through a better understanding of how the immune system naturally fights off infections. The use of interferon in fighting viral infection is one example. Other species' immune responses may yield additional weapons in our antimicrobial arsenal. Derived from the biological defense systems of insects, defensins and cecropins are two examples of antimicrobial peptides that hold promise.
This chapter will explore our current knowledge of immunotherapy for ocular infection. General aspects of ocular immunology will not be discussed in this chapter, since there are a number of excellent texts that discuss this broad topic in detail.9–11 This chapter focuses on (1) immune modulation to control the host response to infection, and (2) therapies that may augment or utilize the immune system's antimicrobial activities. Antimicrobial peptides derived from insect and animal immune responses to infection will also be discussed.
CORTICOSTEROIDS IN KERATITIS SECONDARY TO BACTERIA AND FUNGI
The use of topical steroids in the treatment of bacterial keratitis has been a controversial area since the introduction of steroids in the 1950s.12–19 As in other ocular infections, the goal of using steroids in this setting is to decrease the inflammatory response and associated scarring, thereby improving visual outcome. The controversy focuses on two points: (1) Can steroids actually decrease the scarring associated with infectious keratitis?; and (2) Can steroids be used in the setting of infectious keratitis without undue risk of additional morbidity, such as inducing perforation or aggravating the infectious process?
Some authors have suggested that corticosteroids can accomplish the goal of decreasing scarring and improving vision after an episode of infectious keratitis.20–23 Corticosteroids have a number of effects on the inflammatory response. Topical corticosteroids can decrease corneal inflammation, as measured by concentration of polymorphonuclear leukocytes in a noninfectious animal model of keratitis.24 These effects occur when steroids are given before or after the initiation of the inflammatory response. Prednisolone acetate was shown to be more effective in suppressing inflammation than a similar concentration of prednisolone phosphate, a fact that is now generally accepted. Inflammatory cells that have already invaded the cornea undergo an involutional response to corticosteroids.25 Not all studies evaluating the ability of corticosteroids to suppress inflammation in infectious keratitis have agreed with these findings. Bohigan and Foster26 were unable to demonstrate a reduction in inflammation when corticosteroids were used in the treatment of Pseudomonas keratitis.
In other noninfectious, nonocular models, steroids have been shown to inhibit the release of hydrolytic enzymes from inflammatory cells,27,28 which could prevent “irreversible structural damage to the cornea” and decrease the “chemotactic stimulus that attracts inflammatory cells to the area.”29 Therefore, the rationale for the use of steroids for infectious keratitis is understandable.
It is more difficult, however, to document improved outcome in infectious keratitis as a result of steroid use. Investigations aimed at answering this question have not been able to demonstrate an improvement in scarring with steroid treatment.30,31 In a guinea pig model, no differences were seen in the area of resultant scarring after treatment with antibiotics and steroids versus antibiotics alone.30 In this study, despite randomization, there appear to be differences in the lesion sizes at the onset of therapy. The statistical comparison of this initial clinical appearance was not described by the authors. They did comment that because of the relatively small sample sizes and the standard deviations in the measurements, statistical differences were not likely to have been demonstrated.
In a human clinical trial performed by Carmichael and associates,31 patients were randomized in an unmasked fashion to treatment or no treatment with steroids after 24 hours of intensive antibiotic treatment. If patients to receive steroids were stable or improved after 24 hours, 0.1% dexamethasone drops were started four times per day. If any deterioration was noted in a patient's clinical appearance, treatment was reassessed. The two groups were compared as to their initial clinical appearance. The initial and final best-corrected visual acuities were compared between the two groups. Ulcer size and epithelial healing rates were also assessed and compared. The steroid and control groups were compared for complications, including perforation, recurrence of infection, and persistent epithelial defects. Both groups showed a statistically significant improvement in vision during the course of the study. There were small differences in the mean initial and final visual acuities in the two groups; however there was no statistical difference in regard to visual change over the course of treatment. No differences in epithelial healing rates were seen.
The severity of the ulceration experienced by the subjects in this study is impressive. Six of the 15 steroid-treated patients and 7 of the 13 control patients had a final best-corrected visual acuity of 20/200 or less. One of the patients in the control group experienced further loss of vision after his initial presentation, deteriorating from being able to perceive hand motion to being unable to perceive light.
It is unclear from this study why there are six eyes missing from each group's analysis.31 Although one can surmise from the results section why some of the patients may have been excluded, the answer is never specifically addressed. The final number of subjects who completed the study is very small, only 15 and 13 patients in the steroid and control groups, respectively. To demonstrate a 30% difference in outcome between the two groups (as measured by change in visual acuity from presentation), 165 subjects would be needed in each group (assuming alpha error = 0.05, beta error = 0.80). One interesting side note is that no ulcers in the study were secondary to P. aeruginosa.
Are there unneeded risks incurred if steroids are used in the treatment of ulcerative keratitis? In the absence of appropriate antibiotic therapy, it has been well documented that steroids can cause a more rapid and worse clinical course.32,33 This is especially true if the ulceration is due to P. aeruginosa. In the presence of appropriate antibiotics, however, there does not seem to be any impairment in the bactericidal response of the organism.32–34 Infectious keratitis secondary to P. aeruginosa, however, has been reported to recur after apparently adequate treatment with appropriate antibiotics in combination with steroids.2,3 This phenomenon has also been demonstrated in an animal model.1 The ability of a Pseudomonas infection to recur in the presence of steroid therapy could be related to one of the following factors: (1) a species-specific ability; or (2) impairment of the host response combined with a species-specific ability.35
In the study discussed previously, Carmichael and associates31 did not find any differences in the number of complications between the steroid-treated patients versus the control patients. It is interesting that 8 complications were noted in the 21 steroid-treated patients and 10 complications in the 19 patients who did not receive steroids. This large percentage of patients in both groups experienced a variety of complications ranging from perforation and recurrence of infection to persistent epithelial defects and epithelial breakdown.
In the setting of infectious keratitis due to fungal organisms, steroids can be detrimental.15,16,36–38 This may be due to several factors. The available antifungal medications achieve only fungistatic concentrations in the cornea.36 With poor antimicrobial action and the lack of an intact host immune response due to steroids, the therapeutic effect of the antimicrobial drugs is reversed and a greater replication of organisms can take place compared with untreated controls. This enhancement of fungal growth may be related to the sterol components of the fungal cell wall and the similarity of the steroid molecules. Several clinical studies have demonstrated adverse effects of corticosteroids in the setting of fungal keratitis.15,16,37
Based on these data, we strongly recommend against using steroids in ulcerative keratitis caused by Pseudomonas species or fungi. The risk of accelerating an infection or predisposing a patient to recurrence of infection is too great in both of these etiologic settings. For other organisms, however, after the infectious organism and its antibiotic susceptibilities are determined and a patient is clinically improving, the use of steroids probably poses minimal increased risk and may provide some benefits for ulcerative keratitis patients. Steroid therapy could be instituted at this point. Patients must be monitored for clinical changes once steroids are added to the therapy. Should the clinical status of a patient begin to deteriorate after institution of steroid therapy, as evidenced by increased corneal infiltrate or thinning, steroids should be rapidly tapered or stopped. Antibiotics must be continued, often at a reduced level, for as long as steroid therapy is continued. One potential problem with this type of combination therapy with two or more topical medications is increased risk of toxicity from topical medications, which can cause slowed epithelial healing.
We agree with the conservative guidelines for corticosteroid use in infectious settings as recommended by Stern and Buttross.39 They suggested withholding steroids in the presence of any of the following conditions:
No organism or antibiotic susceptibilities are identified.
They suggested that corticosteroids are most useful in eyes that have received several days of appropriate antibiotic therapy, but that continue to have persistent inflammation.
CORTICOSTEROIDS AND KERATITIS SECONDARY TO HERPES SIMPLEX VIRUS
In the past, the use of corticosteroids for the treatment of stromal keratitis due to herpes simplex virus (HSV) was very controversial.40 The Herpetic Eye Disease Study examined this issue and has helped to settle some of the controversy.41 The study was a multicenter, randomized, double-masked, placebo-controlled clinical trial of prednisolone in the therapy of herpetic stromal keratitis. All subjects in the study received antiviral therapy, and some received concomitant steroid therapy. The results demonstrated significant differences with steroid treatment in regard to a number of parameters. Risk of persistent or progressive stromal keratouveitis was decreased by 68% with steroid treatment. Resolution of stromal keratitis and uveitis was significantly shortened by steroid therapy. No clinically or statistically significant differences were observed in the visual outcome or recurrence of herpetic disease at 6 months' follow-up.
One area was not addressed in the published results of this study. Some clinicians believe that past steroid treatment will alter the inflammatory course of future recurrences.42 None of the patients included in the study had received steroid treatment within 10 days of study enrollment. Relatively similar percentages of patients had received steroid therapy in the interval between 14 days and 2 years before study enrollment. No comments were made as to whether prior treatment with steroids affected the tendency for treatment failure in the placebo group. We do know that lack of a prior history of herpetic eye disease was prognostic of successful completion of the study treatment for placebo patients. Although patients had thus not been treated for herpetic eye disease in the past, no conclusions can be drawn because of the tendency for a less severe course during a primary episode of infection and the possibility that these patients were misclassified in regard to their diagnosis of HSV infection.
There is general agreement that epithelial herpetic keratitis should never be treated with corticosteroids. The clinical course can be significantly worsened in epithelial disease, particularly in the absence of antiviral therapy.
CORTICOSTEROIDS AND INFECTIOUS ENDOPHTHALMITIS
The management of infectious endophthalmitis is another controversial area concerning the role of corticosteroid therapy. Endophthalmitis is almost inevitably an emergent situation, requiring immediate therapy for the best possible visual outcome. At the time of initial diagnosis, treatment is complicated by the fact that the responsible organism and its antibiotic sensitivities cannot be identified. The Endophthalmitis Vitrectomy Study has helped to answer questions regarding antibiotic therapy and the utility of vitrectomy in the study of this problem, but it did not address the use of steroids.43
This infectious process of endophthalmitis can progress very rapidly. Even when the infection is treated with appropriate antibiotics, the inflammatory response to both live and dead organisms continues, having the potential to destroy the retina and any chance for useful vision.44,45 In fact, the bactericidal action of antibiotics may produce additional destructive inflammation as bacteria lyse and release endotoxin and other cell components.46 Some type of therapy to suppress this exuberant and destructive inflammatory response would likely be of use in improving outcome.
Several studies have examined the use of corticosteroids in animal models of endophthalmitis.45,47–49 All of these studies showed that the addition of corticosteroids improved the outcome of endophthalmitis. The outcome measured in each of these studies did vary from clinical appearance to histopathology to electrophysiology. Particularly in aggressive types of infection, Graham and Peyman47 showed that corticosteroid treatment should be instituted early in the clinical course for the best result. The use of steroids in the treatment of infectious endophthalmitis, however, is not without risks. In the absence of appropriate antimicrobial agents, corticosteroids can result in a more rapid progression of infection. This situation is similar to that in the cornea, but without the risk of thinning. Partially because of the low incidence of infectious endophthalmitis, no well-performed, controlled clinical trials have addressed the use of corticosteroids in this setting. So far, only case series of patients treated with intravitreal corticosteroids have been reported.50,51 A multicenter, controlled trial similar to the Endophthalmitis Vitrectomy Study is needed to provide a definitive answer as to the role of corticosteroids in the treatment of infectious endophthalmitis.
|NONSTEROIDAL ANTI-INFLAMMATORY DRUGS|
|Nonsteroidal anti-inflammatory drugs (NSAIDs) were developed in the hope
that the goal of suppressing inflammation without increasing risks associated
with steroids and infection would be achieved. The mechanism
of action of NSAIDs differs from that of corticosteroids (Fig. 1).52–61 Corticosteroids, which block the action of phospholipase A2, prevent the production of products for both the cyclooxygenase and the
lipoxygenase pathways. For the most part, all of the NSAIDs presently
available selectively block one of these two pathways. In evaluating
the possible use of these types of agents in infectious keratitis, we
must ask the same questions that were asked when evaluating corticosteroids: (1) Can
these agents decrease the inflammation and scarring associated
with infectious keratitis?; and (2) Can NSAIDs be used in the
setting of infectious keratitis without undue risk of additional morbidity?|
Initial studies into immunogenic models of keratitis provided encouraging results. Belfort and co-workers62 found that indomethacin, a cyclooxygenase inhibitor, was capable of decreasing the clinical inflammatory response to intracorneal injection of bovine gamma globulin. Despite this decrease in inflammation, there were no statistically significant differences in immune response. Antibody titers in the aqueous, vitreous, and serum were very similar in both the indomethacin-treated eyes and the untreated eyes. Antibody production by cells in the uveal tissues and homolateral lymph nodes was also similar between the two groups.
Verbey and colleagues63 examined the effect of suprofen (a cyclooxygenase inhibitor), Rev 5901 (a lipoxygenase inhibitor), and Bay 08276 (both a lipoxygenase inhibitor and a mild cyclooxygenase inhibitor) in an immunogenic keratitis model utilizing intracorneal injection of human serum albumin. All animals were examined by slit lamp and pachymetry. All untreated animals developed corneal edema and clouding, Wessely's ring formation, and neovascularization. All of these changes resolved after 30 days. Treatment with 0.1% fluorometholone (a corticosteroid) prevented all of these inflammatory and neovascular signs. Treatment with Rev 5901 resulted in a decrease in the period of corneal opacification, less intense opacification, and less intense Wessely's ring formation. Neovascularization was significantly decreased. Bay 08276 treatment resulted in a slightly greater inhibition in adverse clinical findings compared with Rev 5901, but these differences were not statistically significant. Treatment with suprofen decreased the intensity of corneal edema, but not the duration of corneal opacification. The duration of the neovascular response was shortened significantly. Combination treatment with suprofen and Bay 08276 produced the same changes in clinical course as produced by Bay 08276 treatment alone (shorter duration of corneal opacification and neovascularization and inhibition of corneal edema compared with controls).
Verbey and associates63 concluded that cyclooxygenase inhibition does not affect leukocyte infiltration and neovascularization, but does decrease corneal edema through its action on prostaglandin formation. The efficiency of leukotriene B4 inhibition by lipoxygenase inhibitors is proposed as the mechanism for inhibition of inflammation. These authors did not present data on the inhibition of these products in the same animal model; however, they did calculate the inhibitory effects based on the work of other authors who used whole-cell assays. Clinical corneal opacification that was not associated with thickening of the cornea by pachymetry was interpreted as leukocyte infiltration; however, no histologic evaluation or radiolabeling of leukocytes was used to evaluate the actual number of inflammatory cells present.
Leibowitz and co-workers21 evaluated suprofen's ability to suppress inflammation in an animal model of chemical keratitis using clove oil. They measured inflammation using radioactively labeled leukocytes. They found that if suprofen was initiated at the same time or 24 hours after induction of inflammation, there was no effect on corneal inflammation. However, if pretreatment with suprofen was begun 48 hours before clove oil injection and continued after injection, there was a significant decrease in leukocyte infiltration into the cornea. In the setting of a clinical infection, obviously pretreatment with an anti-inflammatory agent is not possible.
Other authors have investigated the effect of NSAIDs in animal models of infectious keratitis.30,33,64 Gritz and associates33 investigated the course of infectious keratitis in a rabbit model treated with vehicle, prednisolone, and flurbiprofen (a cyclooxygenase inhibitor). In the absence of antibiotic therapy for Pseudomonas keratitis, there was a more rapid progression of disease in the prednisolone-treated animals and further progression with flurbiprofen treatment. The progression to descemetocele was more rapid for the flurbiprofen group when compared with either the vehicle- or prednisolone-treated groups (p < 0.01). With antibiotic treatment for Pseudomonas keratitis, there were no differences among the groups in regard to clinical examination or antibiotic action as measured by residual viable bacteria after 24 hours of antibiotic treatment. Infectious keratitis secondary to Streptococcus pneumoniae did not demonstrate a clinical worsening when treated with anti-inflammatory agents in the absence of appropriate antibiotic therapy. There were no differences among the three groups in terms of the number of residual viable bacteria after 24 hours of antibiotic therapy.
Ohadi and colleagues30 investigated the effects of prednisolone, flurbiprofen, nordihydroguariatic acid (NGDA; a lipoxygenase inhibitor), and SKF104353 (a lipoxygenase antagonist) in a guinea pig model of Pseudomonas keratitis. No statistical differences were seen in the progression of clinical disease in the absence of antibiotic therapy in this model. Flurbiprofen-treated animals did tend to have a larger area of corneal involvement compared with the control group (p = 0.22). Concomitant treatment with both an antibiotic and each of the anti-inflammatory agents did not demonstrate any differences in the clearing of bacteria as measured by quantitative culture. NDGA-treated animals exhibited greater conjunctival swelling compared with the other treatment groups. This difference was statistically significant between the NDGA- versus the prednisolone-treated animals (p < 0.05). This difference could have been due to NGDA toxicity or the effects of NDGA on the inflammatory and disease processes. Conjunctival discharge was significantly decreased in the flurbiprofen-treated group, compared with the animals treated with either vehicle or one of the leukotriene antagonists (p < 0.01). No differences were seen among the groups in terms of resultant corneal scarring. Small sample sizes relative to the standard deviation of the measurements were a problem with this study.
Hobden and co-workers64 stratified rabbits by age in their investigation of flurbiprofen and ciprofloxacin in the treatment of Pseudomonas keratitis. Animals were divided into three treatment groups and received saline, 0.3% ciprofloxacin, or a combination of 0.3% ciprofloxacin, 1% prednisolone acetate, and 0.03% flurbiprofen. They began treatment of the animals at a very early stage in the model, before significant stromal edema or infiltrate occurred. Slit-lamp grading of inflammation showed significantly less inflammation among animals in the same age group treated with flurbiprofen; however, there was no statistical difference in this group in comparison with the other groups in terms of the number of polymorphonuclear leukocytes present, as measured by myeloperoxidase activity. Younger animals had significantly worse inflammation and more leukocyte infiltration compared with older animals in the same treatment group.
The animals in this study had a much milder keratitis than those in previous studies. In a model like this, some animals may not develop infectious keratitis clinically, despite intrastromal injection of bacteria.1,33 Because the only treatment group that received anti-inflammatory treatment received a combination of corticosteroids and a cyclooxygenase inhibitor, it is impossible to differentiate between the individual effects caused by each of these anti-inflammatory medications.
The effect of flurbiprofen on epithelial HSV keratitis was investigated by Trousdale and co-workers.65 Animals were treated with placebo, 0.1% dexamethasone, or 0.1% flurbiprofen. Treatment with either the corticosteroid or the cyclooxygenase inhibitor caused a significant worsening in corneal opacity, area of corneal involvement, and conjunctivitis compared with the placebo group. There was no difference in the degree of worsening between the corticosteroid and the cyclooxygenase inhibitor. No antiviral medications were available at that time, and thus these animals did not receive antiviral treatment.
Hendricks and colleagues66 investigated the effect of flurbiprofen on an animal model of stromal HSV keratitis. Animals were treated with dexamethasone or flurbiprofen and no antiviral agent. Dexamethasone had a variable effect, ranging from significant improvement in the degree of inflammation to significant exacerbation of disease. Flurbiprofen did not exacerbate disease in any of the animals and resulted in reduction or no change in the stromal opacity. In vitro studies in cell culture demonstrated a virucidal effect for flurbiprofen, but none for dexamethasone. The authors suggested that the improved stromal opacity could be due to the virucidal effect. There is a lack of consistent results in this animal model, and the results of this study therefore should be viewed with this fact in mind.
There have been fewer investigations into the use of NSAIDs compared with corticosteroids in the treatment of ocular infections. Additional agents in the NSAID class are being developed. There is a possibility of increased morbidity when these agents are used in the absence of appropriate antimicrobial therapy. Therefore, the guidelines that we recommend for NSAID use are similar to those given for steroid use in ulcerative keratitis (see above). From the studies performed thus far, this class of therapeutics does not seem to have any advantages over corticosteroids in regard to decreasing inflammation or subsequent corneal scar formation. In fact, the cyclooxygenase inhibitors may increase leukocyte infiltration when used after initiation of inflammatory insult and thus may be detrimental.
Cyclosporin A (CsA) is a immunosuppressant with very specific, targeted actions focusing around the basic events surrounding T-cell lymphocyte activation. Normally the activation of noncommitted, resting T-cells requires both specific and nonspecific signals.67 Activation of the T-cells leads to expression of receptors on the cell membrane of the T-cell, including the receptor for the lymphokine interleukin 2 (IL-2). This receptor is believed to be the primary mechanism for recruitment and activation of new T-cells.68 CsA appears to block the formation of IL-2 receptors, the stimulation of IL-2, and its ongoing production. There is conflicting evidence as to the population of T-cells that are affected by CsA.68 There is general agreement that there is prevention of induction of cytotoxic T-cells, but the effect on suppressor T-cells is less clear. This subset of cells may be spared from suppression or may be induced by CsA administration, depending on the investigation and the animal model used for investigation.
Because of the mechanism of action of CsA, the applications most extensively investigated regard control and prevention of corneal graft rejection. Systemic and topical administration has been investigated regarding corneal graft rejection, intermediate and posterior uveitis, and noninfectious, immune-related corneal ulcers.68–73 Animal models have shown topical CsA to be of use in corneal graft rejection, but adequate placebo-controlled studies in humans are lacking. A large study regarding systemic CsA in high-risk patients is underway. Certain uveitis patients appear to achieve better control when systemic CsA is used. In human case series, marginal corneal ulceration related to autoimmune disease can be improved through the use of topical and systemic CsA.69–72
No investigations have been published regarding the use of CsA in immunomodulation therapy in infectious ocular disease. CsA has been shown to worsen the course of herpetic keratitis in the absence of antiviral therapy. CsA could possibly be used in the therapy of stromal HSV keratitis. Patients who experience a steroid-responsive glaucoma constitute one group that could benefit from CsA therapy. No studies have been done to investigate this potential use. A multicenter study would be required to recruit adequate numbers of patients to address this question properly. In light of the Herpetic Eye Disease Study, it would be more difficult to design an ethical study to evaluate the use of CsA for stromal HSV keratitis. Because of its effect on cell-mediated immunity, CsA could be of use in the control of inflammation seen with Toxoplasma infection of the eye. These studies also have yet to be performed.
Interferons are a group of glycoproteins secreted by mammalian cells in response to viral and parasitic infections and other stimuli. This multifunctional group is part of the host's defenses against viral and parasitic infections and certain tumors.74 Through induction and synthesis of a number of proteins, they exert a number of effects on the immune system and also affect cell proliferation and differentiation. Interferons are classified according to the animal of origin (e.g., human, murine) and the antigenic specificities (designated alpha, beta, and gamma). Biosynthetic interferon and interferon purified from cell preparations have been found to have similar clinical antiviral activity.75 The different subtypes of interferon have been shown to have different levels of activity, both in vitro and in vivo.
Most of the investigations into ophthalmic applications of interferon have dealt with viral infection, mainly HSV. Smolin75 reported an improvement in stromal and epithelial HSV keratitis with topical or intramuscular interferon therapy compared with placebo in an animal model. This improvement was observed with interferon treatment alone (no concomitant treatment with antiviral agents), regardless of whether therapy was begun before or up to 2 days after inoculation with HSV. In additional experiments, Smolin reported that combined treatment with acyclovir and interferon was significantly better than either therapy alone.
Neumann-Haefelin and associates76 used an African green monkey model to investigate the effect of interferon therapy in HSV keratitis. They found that prophylactic and simultaneous administration of interferon resulted in inhibition of keratitis. When interferon was administered after inoculation with virus, only a slight moderation was observed in the course of keratitis.
Three studies have reported human trials of combined therapy with topical acyclovir and alpha interferon for dendritic HSV keratitis.77–79 Colin and co-workers78 used “human leukocyte interferon,” but there was no further description of the source (human cells or recombinant DNA) or interferon subtype. Subjects in the study received 3% acyclovir ointment five times per day and either interferon or placebo once per day. Both treatment groups were comparable at the initiation of treatment. Interferon treatment resulted in a much faster healing of the corneal epithelium over the ulcer. De Koning and co-workers77 performed a similar trial using topical acyclovir and alpha interferon derived from human leukocytes. The groups were similar before therapy was begun and, once again, more rapid healing was observed with interferon treatment. In both studies, the time needed for epithelial healing was almost twice as fast in the treated group when compared with control patients.43
Sundmacher and associates79 examined recombinant human interferon alpha and gamma. They reproduced the same degree of improved epithelial healing when the same concentrations of either type of interferon (30 million IU/mL) were used alone. When interferon alpha and gamma were used in combination, however, a very low concentration (1.5 million IU/mL) of each type produced the same degree of improvement. Similarly low concentrations of a preparation containing only one type of either interferon did not produce more rapid healing.
An interesting case of topical recombinant interferon alpha-2a treatment for HSV keratitis was reported, in which ulcerative HSV keratitis developed in a renal transplant patient who was on chronic immunosuppressive and acyclovir treatment.80 The HSV strain was resistant to multiple antiviral agents, both in vitro and in vivo. After 7 weeks of multiple therapies, including trifluridine, vidarabine, debridement, and topical and systemic acyclovir, interferon was added to topical acyclovir therapy. The keratitis quickly resolved. Moderate epithelial toxicity was noted after 11 weeks of therapy, and interferon and acyclovir were discontinued. A dendritic ulcer recurred 5 weeks later. Topical antiviral agents were, once again, ineffective alone and the addition of topical interferon resulted in resolution of the keratitis.
Interferon therapy has been reported to be of help in patients with another viral-related ocular disease, hepatitis C.81,82 Three patients with chronic hepatitis C infection presented with severe corneal ulceration resembling Mooren's ulcer, which are painful, crescentic peripheral corneal ulcers.81–83 Cultures of these ulcers were negative for bacteria, fungi, and viruses. Despite treatment with topical antibiotics, topical and systemic corticosteroids, NSAIDs, and topical cyclosporine drops, the ulcers continued to progress. Because these patients had a positive serology for hepatitis C, they were given systemic interferon alpha-2b treatment, which has been shown to be effective in suppressing the associated hepatic inflammation. All three patients improved with this therapy, and each received 6 months of continued interferon therapy. All patients were doing well at 1 year of follow-up.
Clinical investigations into the use of interferon therapy for adenoviral conjunctivitis have been less promising. In two clinical trials of interferon alpha-2, there were no significant effects on the duration of clinical disease, virus shedding, occurrence of bilateral infection, and subepithelial infiltrates.83,84 Adams and co-workers83 found that, although there was no difference in the incidence of bilateral infection between the control and treatment groups, symptoms in the second eye were less severe in the interferon-treated group. Unfortunately, both of these studies involved relatively few patients. Despite the negative findings of these two studies, a larger scale study involving more patients may reveal advantages of topical interferon in patients with adenoviral keratoconjunctivitis.
Interferon alpha has been shown to inhibit endothelial cell migration and proliferation. Because of these properties, it has been successfully used to treat pediatric pulmonary hemangioma and hairy cell leukemia. When topical and systemic interferon was used in an animal model of corneal neovascularization, however, no inhibition in corneal vascular growth was observed.85 Topical prednisolone acetate did inhibit neovascularization in this study, but no additive effect was seen when interferon was added to prednisolone treatment. This study used silk sutures to induce corneal neovascularization. It is possible that the mechanisms inducing neovascularization after infection-related inflammation may be different than is present in this model. It is also possible that other dosages of interferon may effect corneal neovascularization.
Defensins are a group of antimicrobial and cytotoxic peptides ranging from 3 to 4 kd in molecular weight and containing six disulfide-paired cysteines. There are three distinct peptide families of defensins: “classic” defensins, β-defensins, and insect defensins. The classic defensins are contained within mammalian (e.g., human, rabbit, guinea pig, rat) neutrophil granules, alveolar macrophages (rabbit), and intestinal Paneth cells (mouse and human).86–92 In humans, these peptides make up about 5% of the total protein in granulocytes, being the most abundant in the densest, azurophilic granules. They demonstrate in vitro activity against a number of organisms, including gram-negative and -positive bacteria, mycobacteria, fungi, and viruses such as HIV and HSV.89,91,93–102 β-Defensins are found in bovine trachea and neutrophils and in chicken leukocytes. They are effective against gram-positive and -negative organisms in vitro.103,104 Insect defensins are found in the hemolymph.105,106 They are predominantly active against gram-positive bacteria.
Classic and insect defensins (and probably β-defensins) have both bactericidal and biologic effects. Their bactericidal effects occur by action on the bacterial cell membranes.93,98,107,108 They form channels in the membranes, increasing the membrane permeability in a voltage-dependent manner. This results in leakage of potassium and limited membrane depolarization, with subsequent depletion of adenosine triphosphate and inhibition of cellular respiration. Defensins also cause an inhibition of corticosteroid production in both rat and rabbit adrenal cells.87
An in vitro trial of two defensins, rabbit neutrophil peptide-1 and -5 (NP-1 and NP-5), against human isolates from ulcerative keratitis yielded promising results.96 The following were among the organisms tested: Candida albicans, an alpha-hemolytic Streptococcus, S. pneumoniae, P. aeruginosa, and Morganella morganii. The effect of defensin NP-1 was particularly impressive. At a concentration of 10 μg/mL, it produced a marked bactericidal effect within a 60-minute exposure. All of the isolates experienced a 2 to 3 log10 decrease in colony-forming units.
Another study examined the possible application of NP-1 as a microbicide in corneal storage media.97 Growth curves were established for Staphylococcus aureus, S. pneumoniae, and P. aeruginosa at different concentrations (NP-1 at 10 and 100 μg/mL; NP-5 at 25 and 50 μg/mL) and different temperatures (4°C, 23°C, and 37°C). A concentration of 200 μg/mL successfully killed 99.9% of all three organisms within 30 minutes at all three temperatures tested. The assay used in this study could not detect killing of greater than 99.9%. This bactericidal effect is impressive, especially when the rapidity and low temperatures are considered. Conventional antibiotics do not have a significant effect at lower temperatures. Because defensins can have a cytopathic effect at some concentrations, the effect of defensins on the cornea, especially the endothelial cells, will need to be investigated.
Cecropins are antimicrobial peptides (30 to 35 amino acids), originally isolated from the hemolymph of the giant silk moth (Hyalophora cecropia). They are produced in response to a bacterial challenge.109,110 They have also been isolated from other silkworm moths, and synthetic analogs have been produced.111,112 Of these analogs, Shiva-11 has shown the most promise for antimicrobial applications.
Like defensins, the cecropins act on cell membranes, but the exact mechanism of action is different. Cecropins are small molecules, highly cationic, and have a secondary helical structure. This helical structure is essential to the lytic action of these peptides by facilitating the disruption of the cell membranes.113,114 In vitro antimicrobial activity against gram-positive and -negative bacteria, fungi, enveloped viruses, and protozoa has been demonstrated.113,114
One study examined the antimicrobial effect of Shiva-11 against pathogens isolated from human cases of severe ulcerative keratitis.115 The organisms including C. albicans, S. pneumoniae, P. aeruginosa, and S. aureus. Shiva-11 yielded a greater than 3 log10 bactericidal effect against all of the isolates of C. albicans, P. aeruginosa, and S. aureus after only a 30-minute exposure at 37°C. S. pneumoniae required a 60-minute exposure to undergo such a dramatic decrease in the number of viable bacteria. Studies into the possible toxicity of the cecropins and their clinical potential as antimicrobial agents have yet to be performed.
Another group of antimicrobial peptides are the magainins, which were first isolated from the skin of the frog Xenopus laevis.116 This group of peptides are linear amphipathic, cationic peptides, 21 to 27 residues in length. Their bactericidal action is accomplished through disruption of membrane permeability. They have demonstrated broadspectrum activity against a wide variety of organisms, including gram-positive and -negative bacteria, viruses, fungi, and protozoa.117–121 Initial investigations into the antimicrobial activity of this group of peptides are very promising. No studies have yet been published to investigate the application of this class of antimicrobial agents to ocular infections.
|In the past 20 years, there has been an explosion of knowledge regarding
the immune system. Some of the agents we use to modulate the immune
and inflammatory response, such as corticosteroids, have been in use for
a very long time. The appropriate use of corticosteroids, however, is
still controversial. Through additional, well-performed studies, we
perhaps will be able to answer some of the questions that remain. Other, newer
anti-inflammatory and immunomodulating agents may provide a
way to control the ocular inflammatory response without presenting additional
risks to the patient.|
A number of other agents offer exciting possibilities for the future. The use of interferon and development of other cytokines that may have therapeutic applications will perhaps be better understood and more widespread in the years to come. The immune systems of both humans and other animals have yielded promising new antimicrobial agents. The future holds great possibilities for the immunotherapy of ocular infections.
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99. Kohashi O, Ono T, Ohki K et al: Bactericidal activities of rat defensins and synthetic rabbit defensins on Staphylococci, Klebsiella pneumoniae (Chedid, 277, and 8N3), Pseudomonas aeruginosa (mucoid and nonmucoid strains), Salmonella typhimurium (Ra, Rc, Rd, and Re of LPS mutants) and Escherichia coli. Microbiol Immunol 36:369, 1992
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