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Immunological study of patients with herpetic stromal keratitis treated with Dialyzable Leukocyte Extracts

January 2007

Conference: 13th International Congress of Immunology – ICI.
Volume: Proceedings Immunology 2007, CD: ISBN:978-88-7587-380-6, Book: ISBN:978-88-7587-379-0

Authors:

Concepcion Santacruz

Instituto de Oftalmología Fundación Conde de Valenciana

Gustavo Aguilar Velazquez

National Autonomous University of Mexico

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The effective immunological response against herpes virus infections is mediated by Th1 cells. Dialyzable leukocyte extracts have the ability to modulate the immune response, driving a Th1 response. Therefore, we evaluate the cytokine production (Th1 vs Th2) in herpetic stromal keratitis patients treated with acyclovir and dialyzable leukocyte extracts. CD4+ T cells were analyzed in PBMC for the expression of intracellular IFN-g and IL-4 by flow cytometry. We observed an increase in the number of CD4+IFNg+ after treatment with dialyzable leukocyte extracts (p=0.01). Our findings suggest that dialyzable leukocyte extracts induce a Th1 response by increasing CD4+IFN-g+ T cells. Therapy with dialyzable leukocyte extracts could become an excellent therapeutic tool to improve the clinical outcome of patients with recurrent herpetic keratitis.

IFN-g and IL-4 expression in CD4+ T cells. Patients treated with DLE showed higher frequency of IFN-g and lower IL-4 expression. Asterisks (*) mean significant statistical differences (p<0.05).

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67H821C0625

Immunological Study of Patients with

Herpetic Stromal Keratitis Treated with

Dialyzable Leukocyte Extracts

G.A. Luna-Baca

, M. Linares

, C. Santacruz-Valdes

G. Aguilar-Velazquez

, R. Chavez

, M. Perez-Tapia

I. Estrada-Garcia

, S. Estrada-Parra

and M.C. Jimenez-Martinez

Research Unit and Cornea Department, Institute of Ophthalmology “Fundación Conde

de Valenciana” Mexico D.F., Mexico;

Department of Immunology, National School of Biological Sciences,

ENCB-IPN, México D.F., México;

Laboratorio de Inmunología, Departamento de Bioquímica, Facultad de Medicina,

Universidad Nacional Autónoma de México, México D.F., México

*mcjimenezm@investigacioniocv.com

Summary

The effective immunological response against herpes virus infections is

mediated by Th1 cells. Dialyzable leukocyte extracts have the ability to modulate

the immune response, driving a Th1 response. Therefore, we evaluate the

cytokine production (Th1 vs Th2) in herpetic stromal keratitis patients treated

with acyclovir and dialyzable leukocyte extracts. CD4+ T cells were analyzed

in PBMC for the expression of intracellular IFN-g and IL-4 by flow cytometry.

We observed an increase in the number of CD4+IFNg+ after treatment with

dialyzable leukocyte extracts (p=0.01). Our findings suggest that dialyzable

leukocyte extracts induce a Th1 response by increasing CD4+IFN-g+ T cells.

Therapy with dialyzable leukocyte extracts could become an excellent thera-

peutic tool to improve the clinical outcome of patients with recurrent herpetic

keratitis.

Introduction

The large diversity of recurrent viral diseases, especially due to herpes

simplex virus, have led to intensive research activities focused on the immu-

nological mechanisms that control the infection. One of the most significant

68 13th International Congress of Immunology – ICI

problems is herpetic stromal keratitis (HSK), which represents a serious health

problem due to its impact on vision and life quality of the patients

. Conven-

tional treatment of HSK has been based on the use of topic and/or systemic

antiviral drugs and topical steroids

2-6

. However, their use, although decreasing

notably the inflammatory process, generates a risk of viral reactivation, per-

petuating the symptoms and generating a torpid evolution. Therefore, it has

been suggested that the clinical evolution is related to the immunological

response of the patient

1, 7-8

. Dyalyzable Leukocyte Extracts (DLE) are ob-

tained from the lysate of human lymphoid cells

9, 10

. Because the effective

immunological response against herpes virus infections is of Th1 type

, and

because some experimental data suggest that DLE induce the cellular re-

sponse

11-12, 15

, the aim of this study was to evaluate the cytokine expression

(Th1 vs Th2) in patients with HSK treated with the conventional treatment

and DLE.

Materials and Methods

Patients. This study included a total of 7 individuals (mean age 41 years-

old, range 18-69), with HSK. HSK diagnosis was based on clinical history

and full ophthalmologic examination according to the diagnostics standards

of the American Academy of Ophthalmology. Healthy age and sex- matched

volunteers were used as controls. All patients received a 24-week treatment

period. Patients were treated with oral Acyclovir (AC) (400 mg 5 times daily)

and oral DLE as coadjuvant (1 unit daily for 10 days, 3 units weekly on

weeks 2 to 4, 2 units weekly on weeks 5 to 8, and 1 unit weekly on weeks

9 to 24). All patients also received a standard regimen of topical prednisolone

phosphate and trifluridine on the basis of previous reports

. Ophthalmologic

examinations were performed every 3 weeks during the 24-week course of

treatment.

Peripheral blood mononuclear cells (PBMC). Whole heparinized periph-

eral blood was diluted in phosphate buffered saline. PBMC were separated on

a ficoll density gradient by centrifugation at 1700 rpm for 30 min at 16°C.

After centrifugation, the interface cells were collected, washed twice, and

counted using a hemocytometer to assess viability by trypan blue dye exclu-

sion.

Cell cultures. PBMC were cultured in RPMI-1640 medium supplemented

with 1 mM sodium pyruvate, 2 mM L-glutamine, 50 mg/ml gentamicin and

0.5% heat-inactivated fetal calf serum, and incubated at 37

C in a 5% CO2

humidified chamber. After 24 h the culture medium was removed and fresh

culture medium supplemented with 10% heat-inactivated fetal calf serum,

PMA/ionomicyn (5 ng/ml-0.2 µg /ml) and brefeldin-A (10 µg/ml) were added.

Four hours later, the cells were harvested and processed to measure intracel-

lular protein expression by flow-cytometry.

Flow cytometric analysis and intracellular staining. Triple-colour staining

69Rio de Janeiro, Brazil, August 21-25, 2007

was performed on PBMC by direct immunofluorescence, using PE-Cy5-la-

belled mAbs against human CD4 After that the cells were processed to intra-

cellular staining with labeled mAbs (PE of FITC) against human intracellular

IFN-g and IL-4. Isotype matched controls were used. Then the cells were

analyzed by flow cytometry.

Statistical Analysis. T tests or Mann-Whitney U tests were used to detect

significant differences. P<0.05 was considered statistically significant.

Results

Intracellular cytokines in CD4+ T cells. We began by determining the

expression of CD4+IFN-g+ and CD4+IL-4+ T cells in the peripheral blood of

7 HSK patients, before and after DLE treatment, and in a control group. The

frequency of CD4+IFN-g+ T cells was 2.2 times higher in HSK patients

before DLE treatment than the control group (p=0.00006). After DLE treat-

ment we observed that the frequency of CD4+IFN-g+ T cells was 4.3 times

higher in patients with HSK than the control group (p=0.001) and 1.9 times

more than before DLE treatment (p=0.01). In contrast, we observed that the

frequency of CD4+IL-4+ T cells was 4.2 times higher in HSK patients than

the control group (p=0.005). Interestingly, the frequency of CD4+IL4+ T

cells was 2.6 times lower in HSK patients treated with DLE (p=0.01) (Figure

1).

Figure 1. IFN-g and IL-4 expression in CD4+ T cells. Patients treated with DLE showed

higher frequency of IFN-g and lower IL-4 expression. Asterisks (*) mean significant statis-

tical differences (p<0.05).

70 13th International Congress of Immunology – ICI

Conclusions

The immune response against herpes simplex virus (HSV) is mediated by

Th1 cells. Previous studies have shown that corneas infected with HSV attract

CD4+T cells to the site of lesion

. Interestingly, the cornea-derived CD4+ T

cells lines contain high numbers of HSV-specific T cells

and secreted IFN-

g, IL-4 and IL-5

. In humans and animal models, it is known that DLE are

able to modulate the immune response by increasing IFN-g production

11-12, 15

and probably other cytokines involved with immunoregulation. Therefore,

DLE have been used successfully in treating viral infections, such as herpes

simplex and herpes zoster virus

12, 16

. Thus, the aim of our study was to evalu-

ate the secretion profile in CD4+ T cells (IFN-g vs IL4) in HSK patients

before and after DLE treatment. Our findings suggest that DLE induce a Th1

response by increasing CD4+IFN-g+ T cells in patients with HSK. It has been

previously shown that CD4+ T-lymphocytes are critical mediators in HSK

In animal models there is evidence that IFN-gamma plays a protective role in

acute infection with herpes simplex virus type 1 infection

. In our study,

HSK patients showed higher frequency of CD4+IFN-g+ T cells after DLE

treatment. Comparison of this CD4+IFN-g+ T cells from HSK patients and

the control group, showed that the frequency of these cells was also higher

in HSK patients. In vitro and in vivo studies have shown that DLE increase

IFN-g production in herpes zoster infected patients

, which was coincident

with our results.

The systemic immunological changes observed in this study suggest that

DLE could act as a systemic immunological booster, inducing Th1 response

by CD4+IFN-g+ T cells. DLE could be used as an adjuvant treatment in

herpetic stromal keratitis. However, additional clinical and immunological

studies are needed to confirm our results.

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Immunofluorescence, histological, and electron microscopic observations were made on rabbit corneas from animals with experimentally induced stromal keratitis following intracorneal injection with the RE strain of herpes simplex virus. Electron microscopic observations were also made on human corneas obtained from patients with a history of herpetic stromal disease. Viral antigens were demonstrated by immunofluorescence in keratocytes of rabbit corneas with herpetic stromal keratitis. Electron microscopic observations and viral culture failed to reveal the presence of viral particles in these tissues. Lymphocytes, a major infiltrating cell type found in both the rabbit and human corneas, were often found in intimate contact with degenerating keratocytes.

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To evaluate the efficacy of topical corticosteroids in treating herpes simplex stromal keratitis. The authors performed a randomized, double-masked, placebo-controlled, multicenter clinical trial of 106 patients with active herpes simplex stromal keratitis who had not received any corticosteroids for at least 10 days before study enrollment. Patients were assigned to the placebo group (n = 49) or the steroid group (topical prednisolone phosphate; n = 57); both regimens were tapered over 10 weeks. Both groups received topical trifluridine. Visual acuity assessment and slit-lamp biomicroscopy were performed weekly for 10 weeks, every other week for an additional 6 weeks or until removal from the trial, and at 6 months after randomization. The time to treatment failure (defined by specific criteria as persistent or progressive stromal keratouveitis or an adverse event) was significantly longer in the steroid group compared with the placebo group. Compared with placebo, corticosteroid therapy reduced the risk of persistent or progressive stromal keratouveitis by 68%. The time from randomization to resolution of stromal keratitis and uveitis was significantly shorter in the steroid group compared with the placebo group even though both groups included patients who were removed from the study and treated with topical corticosteroids according to best medical judgment. Nineteen (33%) of the steroid-treated patients and 11 (22%) of the placebo-treated patients completed the 10 weeks of protocol therapy and had stable, noninflamed corneas after 16 weeks. At 6 months after randomization, no clinically or statistically significant differences in visual outcome or recurrent herpetic eye disease were identified between the steroid and placebo groups. The topical corticosteroid regimen used in this study was significantly better than placebo in reducing persistence or progression of stromal inflammation and in shortening the duration of herpes simplex stromal keratitis. Postponing steroids during careful observation for a few weeks delayed resolution of stromal keratitis but had no detrimental effect as assessed by visual outcome at 6 months.

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To evaluate the efficacy of oral acyclovir in treating stromal keratitis caused by herpes simplex virus (HSV) in patients receiving concomitant topical corticosteroids and trifluridine. The authors performed a randomized, double-masked, placebo-controlled, multicenter trial in 104 patients with HSV stromal keratitis without accompanying HSV epithelial keratitis. Sample size was chosen so that a 5%, one-tailed test would have an 80% chance of detecting a doubling of the median time to treatment failure. Patients were randomized to receive a 10-week course of either oral acyclovir (400 mg 5 times daily, n = 51) or placebo (n = 53). All patients also received a standard regimen of topical prednisolone phosphate and trifluridine. Ophthalmologic examinations were performed weekly during the 10-week treatment period, every 2 weeks for an additional 6 weeks, and at 6 months after entry into the trial. The median time to treatment failure (defined as worsening or no improvement of stromal keratitis or an adverse event) was 84 days (95% confidence interval, 69-93 days) for the acyclovir group and 62 days (95% confidence interval, 57-90 days) for the placebo group. By 16 weeks, 38 patients (75%) in the acyclovir group and 39 patients (74%) in the placebo group had failed treatment. Also by that time, the keratitis had resolved with trial medications, and there was no subsequent worsening in nine patients (18%) in the acyclovir group and ten (19%) in the placebo group. None of these results were significantly different between the two groups. However, visual acuity improved over 6 months in significantly more patients in the acyclovir group than in the placebo group. There was no statistically or clinically significant beneficial effect of oral acyclovir in treating HSV stromal keratitis in patients receiving concomitant topical corticosteroids and trifluridine with regard to time to treatment failure, proportion of patients who failed treatment, proportion of patients whose keratitis resolved, time to resolution, or 6-month best-corrected visual acuity. Visual acuity improved over 6 months in more patients in the acyclovir group than in the placebo group.

Transfer factor 1993: New frontiers

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Feb 1994
Prog Rech Pharm

Adoptive transfer of antigen-specific cell-mediated immunity in humans, first demonstrated by Lawrence [1] in 1949, opened a new avenue of research that has led both to increased understanding of basic immune mechanisms and to the development of many forms of immunomodulant therapy, alone or in combination with other immu-notherapeutic or chemotherapeutic agents. Lawrence originally showed that transfer of intact viable lymphocytes from a normal tuberculin skin test-positive donor for a given antigen to a normal recipient, skin test-negative for that antigen, resulted in a conversion (“transfer”) of the recipient to skin-test positivity; however, this observation created little interest in the immunologic community for several reasons. In 1955, the lymphocyte was mentioned for the first time as an immunologic organ. (Before this, the lymphocyte had been studied in hematologic rather than in immunologic terms.)

Randomised trial of ganciclovir and acyclovir in the treatment of herpes simplex dendritic keratitis: A multicentre study

Article

Mar 1996

This study was designed to assess the relative efficacy of topical ganciclovir 0.15% gel and acyclovir 3% ointment in the treatment of herpes simplex dendritic keratitis. Both treatment modalities were administered on a five times daily basis to patients suffering from herpes simplex keratitis. Patients were assigned randomly to one of the two treatment groups for the purpose of the trial. They were then examined on days 2, 7, 10, and 14 to assess the rate of healing of the dendritic ulceration. There was no statistically significant difference detected in the rate of healing between the two treatment groups over the course of the trial. Review of the relative efficacy of topical ganciclovir and acyclovir in the treatment of herpes simplex dendritic keratitis showed that both treatment modalities were equally effective in their ability to heal the viral induced corneal ulceration. There were no significant side effects or adverse effects reported for either treatment modality.

Profiles of cytokine production in recipients of transfer factors

Article

Feb 1996

Transfer factors (TF) are proteins that transfer the ability to express cell-mediated immunity from immune donors to non-immune recipients. The mechanisms of these effects have not been defined. The experiments described in this report were undertaken to test the hypothesis that a mechanism through which the beneficial effects of TF are expressed in clinical situation is through “education” of the immune system to produce certain cytokines in response to antigenic stimulation. BALB/c mice were sensitized to Herpes simplexvirus (HSV) either by sublethal systemic or cutaneous infections by administration of a HSV-specific TF. One week later their spleen cells were collected and single cell suspensions were stimulated in vitro with irradiated HSV or concanavalin A. Culture supernatants were collected and assayed for content of IL-2, IL-4, IL-10 and IFN-g. Spleen cells from infected mice responded to concanavalin A and to HSV by secreting large amounts of IL-2 and IFN-g, modest amounts of IL-10, and no IL-4. Transfer factor recipients produced similar cytokine profiles in response to concavalin A. These mice, however, responded to HSV by secreting IFN-g, but no IL-2. Thus, TF treatment selectively affects cytokine production in response to antigenic stimulation.

Herpes Simplex Virus Epidemiology and Ocular Importance

Article

Feb 2001
CORNEA

Thomas J. Liesegang

To review the changing epidemiology of herpes simplex disease and correlate it with the epidemiology of ocular herpes simplex disease. A review of pertinent reports in the world literature about the epidemiology of herpes simplex and specifically about ocular herpes simplex. In developed countries, many individuals are reaching adolescence and adulthood without prior herpesvirus infection. Herpes simplex genital infection is increasing at a rapid rate in sexually active adolescents and adults, with about one in six adults now infected in the United States. Similar statistics are confirmatory worldwide in developed countries. Active herpes simplex infection is a risk factor for acquisition of human immunodeficiency virus. The Herpetic Eye Disease Study, as well as prior studies from Moorfields Eye Hospital and the Mayo Clinic in Rochester, Minnesota, provides us with the epidemiology of ocular herpes simplex. Recent studies suggest an older age of onset and perhaps overall more severe ocular disease as compared with the older literature. Herpes simplex is a significant health concern at present with genital infections increasing in epidemic proportions. This is also reflected in a rise in the incidence of neonatal herpes. Herpes simplex virus type 1 (HSV-1) infection is being acquired for the first time in an older age group. A significant and increasing proportion of genital herpes is caused by HSV-1. Serologic studies are no longer as useful in distinguishing orofacial herpes from genital herpes. More acute retinal necrosis syndrome cases are associated with HSV-2. Speculation about the future of ocular herpes is made based on this changing epidemiology.