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Figure 1.
Western blot of Staphylococcus aureus culture supernatant (pooled toxin) and purifiedexotoxins. Nitrocellulose membranes were probed and then visualized with theEnhanced Chemiluminescence detection system (Amersham Biosciences, Piscataway,NJ). Primary antibody: 1:10 000 dilution of 10% Gamimune N (Bayer Corporation,Pittsburgh, Pa). Secondary antibody: donkey antihuman. Lanes 1 and 2: toxicshock syndrome toxin-1 (TSST-1), 0.2 µg and 0.5 µg. Lanes 3 and4: β-hemolysin, 0.2 µg and 1.25 µg. Lanes 5 and 6: Beef heartextract medium. Lanes 7 and 8: concentrated culture supernatant (pooled toxin),0.5 µg and 1.0 µg. Molecular weight markers (Bio-Rad, Hercules,Calif) indicated on the right are serum albumin (66 kDa), ovalbumin (45 kDa),carbonic anhydrase (31 kDa), trypsin inhibitor (21 kDa), and lysozyme (14kDa). Solid arrow indicates β-hemolysin; open arrow, TSST-1.

Western blot of Staphylococcus aureus culture supernatant (pooled toxin) and purifiedexotoxins. Nitrocellulose membranes were probed and then visualized with theEnhanced Chemiluminescence detection system (Amersham Biosciences, Piscataway,NJ). Primary antibody: 1:10 000 dilution of 10% Gamimune N (Bayer Corporation,Pittsburgh, Pa). Secondary antibody: donkey antihuman. Lanes 1 and 2: toxicshock syndrome toxin-1 (TSST-1), 0.2 µg and 0.5 µg. Lanes 3 and4: β-hemolysin, 0.2 µg and 1.25 µg. Lanes 5 and 6: Beef heartextract medium. Lanes 7 and 8: concentrated culture supernatant (pooled toxin),0.5 µg and 1.0 µg. Molecular weight markers (Bio-Rad, Hercules,Calif) indicated on the right are serum albumin (66 kDa), ovalbumin (45 kDa),carbonic anhydrase (31 kDa), trypsin inhibitor (21 kDa), and lysozyme (14kDa). Solid arrow indicates β-hemolysin; open arrow, TSST-1.

Figure 2.
Fundus reflex of rabbit eyes 7days after pooled toxin injection, without (A) or with (B) simultaneous immunoglobulin.

Fundus reflex of rabbit eyes 7days after pooled toxin injection, without (A) or with (B) simultaneous immunoglobulin.

Figure 3.
Course of fundus scores followinginjection of pooled toxin, without or with immunoglobulin. A, Pooled toxinand simultaneous injection of balanced salt solution (BSS) or immunoglobulin.B, Pooled toxin and sequential injection of BSS or immunoglobulin. C, Pooledtoxin and delayed injection of BSS or immunoglobulin. The sample size was6 eyes per data point except for postinjection day 8 in the simultaneous injectiongroup (A), in which there were 3 eyes per data point. The y-axis indicatesmean ± SE fundus score; broken line, pooled toxin and BSS; solid line,pooled toxin and immunoglobulin; and asterisk, a significant difference existsat P<.05, calculated using the Mann-Whitney test.

Course of fundus scores followinginjection of pooled toxin, without or with immunoglobulin. A, Pooled toxinand simultaneous injection of balanced salt solution (BSS) or immunoglobulin.B, Pooled toxin and sequential injection of BSS or immunoglobulin. C, Pooledtoxin and delayed injection of BSS or immunoglobulin. The sample size was6 eyes per data point except for postinjection day 8 in the simultaneous injectiongroup (A), in which there were 3 eyes per data point. The y-axis indicatesmean ± SE fundus score; broken line, pooled toxin and BSS; solid line,pooled toxin and immunoglobulin; and asterisk, a significant difference existsat P<.05, calculated using the Mann-Whitney test.

Figure 4.
Histologic score following injectionof pooled toxin, without or with immunoglobulin. A, Pooled toxin and simultaneousinjection of balanced salt solution (BSS) or immunoglobulin. B, Pooled toxinand sequential injection of BSS or immunoglobulin. C, Pooled toxin and delayedinjection of BSS or immunoglobulin. The sample size was 6 eyes per data point.All eyes were evaluated on postinjection day 9 except for 6 eyes (3, pooledtoxin and BSS; 3, pooled toxin and immunoglobulin) in the simultaneous injectiongroup (A), which were evaluated on postinjection day 7. The y-axis representsmean ± SE fundus score; gray bars, pooled toxin and BSS; black bars,pooled toxin and immunoglobulin; and asterisk, a significant difference existsat P<.05, calculated using the Mann-Whitney test.

Histologic score following injectionof pooled toxin, without or with immunoglobulin. A, Pooled toxin and simultaneousinjection of balanced salt solution (BSS) or immunoglobulin. B, Pooled toxinand sequential injection of BSS or immunoglobulin. C, Pooled toxin and delayedinjection of BSS or immunoglobulin. The sample size was 6 eyes per data point.All eyes were evaluated on postinjection day 9 except for 6 eyes (3, pooledtoxin and BSS; 3, pooled toxin and immunoglobulin) in the simultaneous injectiongroup (A), which were evaluated on postinjection day 7. The y-axis representsmean ± SE fundus score; gray bars, pooled toxin and BSS; black bars,pooled toxin and immunoglobulin; and asterisk, a significant difference existsat P<.05, calculated using the Mann-Whitney test.

Figure 5.
Histopathologic features of rabbiteyes 9 days after pooled toxin injection, without (A) or with (B) immunoglobulin(hematoxylin-eosin, original magnification ×125 [A] or ×82.5 [B]).

Histopathologic features of rabbiteyes 9 days after pooled toxin injection, without (A) or with (B) immunoglobulin(hematoxylin-eosin, original magnification ×125 [A] or ×82.5 [B]).

Table 1. 
Clinical Grading Scale for Severity of Ocular Inflammation*
Clinical Grading Scale for Severity of Ocular Inflammation*
Table 2. 
Histologic Grading Scale for Severity of Ocular Inflammation*
Histologic Grading Scale for Severity of Ocular Inflammation*
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Laboratory Sciences
October 2004

Intravitreally Injected Human Immunoglobulin Attenuates the Effectsof Staphylococcus aureus Culture Supernatant in aRabbit Model of Toxin-Mediated Endophthalmitis

Author Affiliations

From the Eye Institute, Medical College of Wisconsin, Milwaukee (DrsPerkins, Han, Burke, Wirostko, and Tarasewicz and Ms Skumatz); and the Departmentof Microbiology, University of Minnesota School of Medicine, Minneapolis (DrSchlievert). The authors have no relevant financial interest in this article.

Arch Ophthalmol. 2004;122(10):1499-1506. doi:10.1001/archopht.122.10.1499
Abstract

Objective  To determine whether human immunoglobulin attenuates the toxic effectsof Staphylococcus aureus culture supernatant in arabbit model of endophthalmitis.

Methods  Immunoglobulin binding to products of S aureus strainRN4220 was tested by Western blot analysis using known toxins (β-hemolysinand toxic shock syndrome toxin-1) and a concentrated culture supernatant containing S aureus exotoxins (pooled toxin). To induce endophthalmitis,pooled toxin was injected into the rabbit vitreous. For immunoglobulin treatment,immunoglobulin and pooled toxin were either mixed and injected simultaneouslyor immunoglobulin was injected immediately after or 6 hours after pooled toxininjection. Severity of endophthalmitis was graded according to a 9-day coursewith clinical examination (slitlamp biomicroscopy or indirect ophthalmoscopy)and evaluation of histologic sections.

Results  The toxic effects of pooled toxin were markedly reduced when immunoglobulinwas mixed with pooled toxin and injected simultaneously. Delayed injectionof immunoglobulin diminished its ability to reduce toxicity. Clinical andhistologic signs of toxicity were partially attenuated when immunoglobulinwas injected immediately after pooled toxin, but only minimal clinically detectablereductions in toxicity were observed when immunoglobulin injection was delayedfor 6 hours.

Conclusion  Pooled human immunoglobulin can attenuate the toxic intravitreal effectsof a concentrated culture supernatant containing S aureus exotoxins.

Clinical Relevance  Immunoglobulin may represent a novel adjuvant in the treatment of bacterialendophthalmitis. To optimize the potential therapeutic benefit, maximizingthe mixture of immunoglobulin with bacterial products and early interventionare likely to be important.

Infectious endophthalmitis can be a devastating complication of ocularsurgery or trauma. Despite treatment parameters established by the EndophthalmitisVitrectomy Study,1 25% of patients in thatstudy experienced permanent visual loss to a level of 20/200 or worse in theaffected eye. Infections caused by Staphylococcus aureus, Streptococcus species, and gram-negativeorganisms were associated with poor visual outcomes.2S aureus constituted 10% of isolates in the EndophthalmitisVitrectomy Study and is an infrequent, albeit important, cause of bleb-relatedand posttraumatic endophthalmitis.36

Poor visual outcomes and organism virulence appear to be strongly related.For some bacteria, exotoxins are a critical component of virulence becausethey enhance bacterial propagation through host tissue destruction.710 Tissuedestruction in S aureus endophthalmitis partly resultsfrom the combined effects of several exotoxins.1115

Attempts to mitigate inflammatory tissue destruction with steroids havebeen unsuccessful in treating experimental S aureus endophthalmitis.1619 Improvementin the treatment of S aureus endophthalmitis maybe achieved by targeting secreted toxins and has been suggested for otherbacteria.20 Experimentally, staphylococcaltoxins can be neutralized with monoclonal and polyclonal antibodies.21 Clinically, intravenous immunoglobulins containingantibodies capable of neutralizing toxins have been used to help treat somestaphylococcal toxin–mediated illnesses.22

The intravitreal use of toxin-specific antibodies represents a novelapproach to the treatment of endophthalmitis. In a preliminary study, we foundthat pooled human immunoglobulin injected into the vitreous penetrates theretina and persists in the vitreous and retina for up to 5 days without producingclinically detectable inflammation (Rajeev Buddi, MD, D.P.H., S.L.P., andJ.M.B., unpublished data, 2003). We conducted a "proof of principle" investigationto determine whether pooled human immunoglobulin binds proteins in S aureus culture supernatant and 2 purified S aureus exotoxins and whether immunoglobulin injected into the vitreous reducesthe tissue destruction and inflammatory effects produced by an intravitrealinjection of S aureus culture supernatant.

METHODS
POOLED BACTERIAL TOXIN PREPARATION

Staphylococcus aureus strain RN4220 is a derivativeof strain 8325-4 modified for genetic manipulation, which primarily produces β-hemolysin(molecular weight, approximately 35 kDa).23 Wechose RN4220 because of our extensive laboratory experience with this lineand the relative ease with which we can manipulate some aspects of toxin production.The particular strain of RN4220 used in this study also produces toxic shocksyndrome toxin-1 (TSST-1) (molecular weight, approximately 22 kDa), α-hemolysin(molecular weight, approximately 32 kDa), and δ-hemolysin (molecularweight, approximately 3 kDa) in small amounts (P.M.S., oral communication,October 2000). The technique for collecting culture supernatant has been describedpreviously.24 Bacteria were propagated overnightwith aeration at 37°C in 1200 mL of beef heart extract medium.25 Briefly, this medium is prepared from a tryptic digestof beef hearts that is dialyzed using dialysis tubing with a molecular weightcutoff of 4000 to 6000 Da. The insoluble residue is discarded, and the dialysateis sterilized, supplemented with glucose buffer, and inoculated with RN4220.After bacterial growth, extracellular proteins were precipitated from 100mL of culture medium with 4 volumes of ethanol chilled overnight at 4°C.The precipitate was resuspended in 10 mL of double-distilled water. The suspensionwas then centrifuged at 10 000g for 20 minutesto remove cellular debris. The supernatant was removed and dialyzed (molecularweight cutoff, 12 000-14 000 Da) against 2 L of water overnightat 4°C.24 This concentrated culture supernatant(pooled toxin) contained a protein concentration of approximately 60 mg/mL.Bacteria-free beef heart extract medium was similarly precipitated to producea toxin-free control solution. Sterility was maintained during the entireprocedure.

PURIFIED BACTERIAL TOXIN PREPARATION

The β-hemolysin was purified from RN4220 culture supernatant followingthe initial steps outlined previously. Once the culture supernatant was collected, β-hemolysinwas separated using an isoelectric focusing technique.23 TheTSST-1 was derived from an RN4220 line containing the vector pCE107. Followingthe collection of culture fluids, TSST-1 was separated with isoelectric focusingusing successive gradients of pH 3.5 to 10 and pH 6 to 8.26

WESTERN BLOT ANALYSIS

The immunoglobulin preparation used in this study is a sterile, preservative-freesolution of γ-globulin prepared from large pools of human plasma (GamimuneN, 10%; Bayer Corporation, Pittsburgh, Pa). It is approved for intravenoususe in humans for the prevention or attenuation of a variety of infectiousdiseases. Western blot analysis was performed to determine whether immunoglobulinbinds to known toxins produced by S aureus (β-hemolysinand TSST-1; source: laboratory of P.M.S.) and to proteins, including toxins,in pooled toxin. Known toxins, pooled toxin, and control beef heart extractmedium were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresisin reducing conditions (5mM β-mercaptoethanol) using 12.5% separatinggels. Protein loading was empirically determined and is reported in the "Results"section. Separated proteins were transferred to nitrocellulose membranes andblotted overnight with a 1:10 000 dilution of immunoglobulin. Bands werevisualized with a detection system (Enhanced Chemiluminescence; Amersham Biosciences,Piscataway, NJ) after exposure to a donkey antihuman secondary antibody.

INTRAOCULAR INJECTIONS AND CLINICAL EXAMINATION

Approval from the Institutional Animal Care and Use Committee of theMedical College of Wisconsin (Milwaukee) was obtained prior to initiatinganimal experiments. New Zealand white rabbits (weight, 2-3 kg) were housedand handled in accordance with the Association for Research in Vision andOphthalmology's Statement for the use of Animals in Ophthalmic and VisualResearch. Animals were anesthetized prior to intravitreal injection with intramuscularketamine (Phoenix Scientific Inc, St Joseph, Mo) (20 mg/kg of body weight)and xylazine (Bayer Corporation)(1 mg/kg of body weight) and 0.5% topicalproparacaine hydrochloride (Allergan, Hormigueros, Puerto Rico). Pupils weredilated with 2.5% phenylephrine hydrochloride (Akorn Inc, Buffalo Grove, Ill)and mydriacyl (Akorn Inc), and 5% povidine iodine (Alcon Laboratories, Inc,Fort Worth, Tex) was used for asepsis. Preliminary studies indicated thatan intravitreal injection of pooled toxin containing 330 µg of proteinat concentrations of 6.6 g/L or 66 g/L created reproducible intraocular inflammationwith a red reflex but no ophthalmoscopically detectable fundus details. Wechose this pathologic outcome because it closely mirrors the degree of inflammationreported in a rabbit model of S aureus endophthalmitisinvolving live bacteria.12 We also wanted tocreate a model of intraocular inflammation similar to what is typically seenin a clinical case of S aureus endophthalmitis inwhich the visual acuity is counting fingers to hand motions.

Rabbits were divided into 3 groups, each with 6 experimental and 6 controlanimals, in which the timing of the intravitreal injections was varied. Ingroup 1 (simultaneous), pooled toxin and immunoglobulin were mixed and deliveredsimultaneously; in group 2 (sequential), pooled toxin and immunoglobulin weredelivered sequentially with immunoglobulin injected immediately after pooledtoxin; and in group 3 (delayed), pooled toxin and immunoglobulin were deliveredsequentially with immunoglobulin injected 6 hours after pooled toxin. Group1 data consisted of 2 sets of 3 experimental and 3 control rabbits evaluatedat separate times. The entire protocol was identical except that rabbits fromthe first set were euthanized on postinjection day 7 and those from the secondset on postinjection day 9. Groups 2 and 3 consisted of 6 experimental and6 control animals evaluated concurrently, and all were euthanized on postinjectionday 9.

The volume of immunoglobulin used in this study was determined by 2competing factors regarding the nonvitrectomized eye. We wanted to injectthe largest volume of immunoglobulin possible to maximize the probabilityof immunoglobulin and toxin interaction yet were limited to volumes less than250 µL because of associated intraocular pressure elevation. We determinedthat 200 µL of aqueous humor could be safely aspirated from the anteriorchamber without a risk of lens or iris damage. We therefore chose an immunoglobulinvolume of 145 µL and adjusted our other parameters.

Prior to intravitreal injection, 150 µL (group 1) or 200 µL(groups 2 and 3) of aqueous humor was aspirated with a tuberculin syringeto relieve intraocular pressure and reduce the risk of vascular occlusion.For group 1, 5 µL of the concentrated pooled toxin (containing 330 µgof protein [66 g/L]) was mixed with 145 µL of immunoglobulin (14.5 µgof protein [0.1 g/L]) and injected, within 10 minutes of mixing, through thepars plana into the mid vitreous using a 30-gauge needle and a tuberculinsyringe. For the other groups, 5 µL of the concentrated pooled toxinwas diluted to 50 µL with balanced salt solution and injected into themid vitreous. (It was not possible to reliably inject into the vitreous lessthan 50 µL using a tuberculin syringe. Increasing the 5-µL volumeto 50 µL allowed for reliable toxin injection into the mid vitreouscavity.) Immunoglobulin (145 µL) was then injected at a site 90°away either immediately after (group 2) or 6 hours after (group 3) pooledtoxin injection. Control animals in all groups received 145 µL of balancedsalt solution in lieu of immunoglobulin. Injectable materials were preparedin sterile conditions, and sterile surgical technique was used during theinjections. For all animals, only 1 eye was used. Immediately following injection,all eyes were examined for intraocular complications; none were seen.

For all animal groups, slitlamp biomicroscopy and indirect ophthalmoscopywere performed 4 times during the 9 postinjection days by 1 investigator (S.L.P.)and 2 masked vitreoretinal surgeons (W.J.W. and D.G.T.) experienced in thetreatment of clinical endophthalmitis. In group 1 (simultaneous) and group2 (sequential), examinations were performed on postinjection days 1, 3, 5,and 8. Because of limitations in examiner availability, examinations for group3 (delayed) were performed on postinjection days 2, 4, 6, and 9. At each timepoint, we examined 6 experimental and 6 control eyes except for postinjectionday 8 in group 1, for which there were only 3 experimental and 3 control eyes.

The anterior chamber reaction and fundus reflex were graded for evidenceof ocular inflammation using an adaptation of a grading scale of 0 to 4, asreported by others (Table 1).The mean score for each parameter was determined for the 6 eyes in each group,and control and experimental groups were analyzed for statistically significantdifferences (P<.05) using the Mann-Whitney testat each time point except for the final day in group 1 (simultaneous), forwhich there were only 3 eyes in both the experimental and control groups.

HISTOPATHOLOGIC FEATURES

Animals were euthanized with an intracardiac injection of pentobarbitalsodium (25 mg/kg) on postinjection day 7 or 9, and the treated eyes were enucleatedand fixed in 10% formalin for histopathologic analysis. Eyes were embeddedin paraffin, sectioned, and stained with hematoxylin-eosin according to standardprotocols. Sections were examined and scored by an investigator masked tothe identity of the treatment group. Each eye received scores for 4 tissuesusing grading scales for severity changes adapted from studies by other investigators.10,27 The cornea, anterior chamber, andvitreous each received a single score of 0 to 3 (Table 2). For the retina, a template was used to divide each retinalsection into 6 regions, each of which was graded separately using a scaleof 0 to 4 (Table 2). A singleretinal score per eye was obtained by taking the mean of the 6 regional scores.The Mann-Whitney test was used to determine statistically significant differences(P<.05) between the experimental and control groups.

RESULTS
REACTIVITY OF IMMUNOGLOBULIN WITH S AUREUS PRODUCTS

When immunoglobulin was used as a primary antibody for Western blotanalysis, we observed reactivity with numerous proteins in the pooled toxin,including a prominent unidentified high-molecular-weight protein (Figure 1). The absence of comigrating bandsin the control beef heart extract medium suggests that the immunoreactiveproteins in the pooled toxin are bacterial products. To determine whetherthe immunoglobulin could react with any known S aureus toxins,which may constitute a small fraction of the protein in the total pooled toxinand therefore contribute little to the blotting signal, β-hemolysin andTSST-1 were purified from bacterial supernatants and blotted with immunoglobulin.As shown in Figure 1, immunoglobulindemonstrates reactivity with both β-hemolysin and TSST-1.

CLINICAL EXAMINATION
Pooled Toxin Alone

Intravitreal injection of pooled toxin alone produced a grade 3 to 4anterior chamber reaction on postinjection day 1. This reaction declined untilit resolved by days 5 to 7. The fundus reflex was diminished in all eyes bypostinjection day 1. Despite the obscuration of most retinal details in alleyes on postinjection day 1, intraretinal hemorrhages could be observed insome. During the first 5 postinjection days, the fundus reflex worsened inmost eyes, at which point it plateaued and remained diminished at approximatelythe same level on postinjection days 5 through 9. The initial decline in thereflex was attributed to vitreal inflammation. To a variable extent, a whitemembrane would form on the posterior surface of the lens by postinjectionday 2, continue to proliferate from postinjection days 2 through 4, and thenstabilize. This membrane would continue to mature even though anterior segmentinflammation was resolving, and it prevented adequate evaluation of the posteriorsegment. The fundus reflex appearance on postinjection day 7 is illustratedin Figure 2A, and scores for thefundus reflex during the 8- to 9-day course are shown in Figure 3 (broken lines).

Pooled Toxin and Simultaneous Injection of Immunoglobulin

When pooled toxin was mixed with immunoglobulin prior to injection,an anterior chamber reaction was seen in only 3 of 6 eyes and was limitedto grade 1. The fundus reflex (Figure 2B)was essentially normal throughout the course, and retinal details were easilyseen. Fundus reflex scores are graphed in Figure 3A. Compared with eyes receiving pooled toxin and balancedsalt solution, the mean fundus reflex scores for eyes receiving immunoglobulinwere significantly lower (Figure 3A)at all time points.

Pooled Toxin and Sequential Injection of Immunoglobulin

When immunoglobulin was injected immediately following pooled toxin,the expected inflammatory response was attenuated compared with eyes receivingpooled toxin alone (Figure 3B).The difference in the fundus reflex scores between the 2 groups became greaterduring the 9-day course and could be attributed to a worsening of the fundusreflex in the eyes not receiving immunoglobulin. The differences were statisticallysignificant at all but the second time point. Compared with eyes receivingsimultaneous injections of pooled toxin and immunoglobulin, the fundus scoresfor eyes receiving sequential injections were higher (ie, more diminished),particularly at earlier time points (Figure3A and B).

Pooled Toxin and Delayed Injection of Immunoglobulin

When the injection of immunoglobulin was delayed 6 hours, only a slightattenuation of the expected inflammatory response was observed. The fundusreflex was diminished more than in eyes receiving simultaneous or sequentialinjections of immunoglobulin (Figure 3A,B, and C). Despite the diminished reflex, fundus scores were significantlylower at all but the first time point when compared with eyes not receivingimmunoglobulin (Figure 3C). Retinaldetails were obscured throughout the course.

HISTOPATHOLOGIC EXAMINATION
Pooled Toxin Alone

We found essentially no inflammatory response in the cornea and anteriorchamber of these eyes (Figure 4).The vitreous cavity was partially filled with inflammatory cells. Some eyeshad abscesses, whereas others did not. Full-thickness retinal disruption wasevident with ganglion cell loss, increased vacuolation of the inner nuclearlayers, complete loss of photoreceptors, and choroidal thickening observedin most sections (Figure 5A). Wenoted interspersed areas of focal retinal thinning due to the loss of cellularelements.

Pooled Toxin and Simultaneous Injection of Immunoglobulin

In these eyes, we noted a mild inflammatory response in the vitreouscavity. Compared with eyes receiving pooled toxin alone, we observed a markedpreservation of retinal architecture with the presence of distinct layerscomposed of intact cells (Figure 5B).In some sections, vacuolation of the inner nuclear layers and choroidal thickeningwere observed.

Pooled Toxin and Sequential Injection of Immunoglobulin

When immunoglobulin was injected immediately following pooled toxin,we noted an attenuation of the expected histologic response in the retinabut not in the vitreous (Figure 4B).Compared with eyes receiving pooled toxin simultaneously with immunoglobulin,the retinal architecture was slightly more disrupted but significantly lessthan in eyes not receiving immunoglobulin. Focal areas of inner nuclear layervacuolation, disruption of the ganglion cell layer, retinal edema, photoreceptorloss, and choroidal thickening were noted. We observed other sections withpreserved retinal architecture.

Pooled Toxin and Delayed Injection of Immunoglobulin

When immunoglobulin was injected 6 hours after pooled toxin, the histologicappearance of all tissues was indistinguishable from eyes receiving pooledtoxin alone (Figure 4C). We observedfull-thickness retinal disorganization in most sections with ganglion cellloss, increased vacuolation of the inner nuclear layers, complete loss ofphotoreceptors, and choroidal thickening. As in eyes receiving pooled toxinalone, some sections contained focal areas of retinal thinning due to cellloss.

COMMENT

Staphylococcus aureus endophthalmitis oftenresults in poor visual outcomes with only 50% of patients achieving a finalvisual acuity of 20/100 or better in the affected eye.2 Animalstudies suggest that poor outcomes are related to secreted bacterial productsthat are toxic to the retina, are highly inflammatory, and can induce damagesimilar to that seen with a natural infection.7 Althoughcell wall components produce significant intraocular inflammation in thissame model, retinal function does not appear to be significantly altered.7 The fact that retinal damage appears to be secondaryto direct toxin effects instead of a host inflammatory response likely explainsthe lack of a beneficial effect from the adjuvant use of corticosteroids inthe treatment of experimental S aureus endophthalmitis.1619 Incontrast to the rapid tissue destruction that occurs with intraocular Bacillus cereus infections, the natural history of S aureus endophthalmitis may allow the timely introductionof appropriate antitoxin therapy.7

The potential role of antibody therapy in the treatment of ocular diseasehas been evaluated in the case of a monoclonal antibody as adjuvant therapyfor cytomegaloviral retinitis as well as for the neovascular form of maculardegeneration.28,29 Because thepathogenicity of S aureus endophthalmitis is complex,likely involving numerous exotoxins and regulatory genes and gene products,attempting to treat endophthalmitis by targeting specific bacterial productsmay prove elusive. We hypothesized that a commercially available pooled humanimmunoglobulin product might prove to be an effective adjuvant therapy inthe treatment of S aureus endophthalmitis. A pooledproduct offers the potential benefits of targeting numerous exoproteins simultaneously,being widely available, and being approved for human use.

Our model of using pooled toxins extracted from Saureus culture supernatant to induce toxin-mediated endophthalmitiseliminated several experimental variables that would likely occur in a modelinvolving the inoculation of live bacteria, such as the rate and quantityof toxin elaboration, bacterial growth, and antibiotic effect. Such variableswould potentially make the evaluation of toxin neutralization by immunoglobulindifficult. The validity of our model as it relates to clinical endophthalmitisis supported by substantial evidence that toxins isolated from complex growthmedia are biologically active and are functionally involved in many clinicaldisease states.15,30

The mixing of pooled immunoglobulin with pooled toxin appears to conferneutralization of its toxic effects. In this study, we found that if a volumeof immunoglobulin that could be used clinically was allowed to preincubatefor as little as 5 minutes with a volume of pooled toxin known to induce significantintraocular inflammation and retinal damage, the pooled toxin was renderedessentially free of such effects according to clinical and histologic measures.The mechanism whereby immunoglobulin is able to render pooled toxin ineffectiveis unknown, but we suspect that it is related to the direct binding of antibodyto toxin. Similar findings are noted in the case of experimental B cereus endophthalmitis, in which other researchers have demonstratedthat specific antibodies against hemolysin, a known B cereus exotoxin, can attenuate the toxin's effects.27

In our study using Western blot technique, we were able to demonstratethat immunoglobulin could bind purified toxins as well as numerous proteinsfrom S aureus strain RN4220 culture supernatant.These findings suggest that specific antibodies present in commercially availablepooled immunoglobulin are capable of binding bacterial products, thus providinga biochemical basis for the attenuated clinical and histologic effect observedwhen immunoglobulin and pooled toxin interact. Although S aureus RN4220 is a modified laboratory strain that may prove to bemore or less virulent than other S aureus strains,it has been well studied and can be manipulated to produce different toxins,thus providing a framework for further study of the concept discussed in thisarticle.

The pharmacokinetics of immunoglobulin remain unknown but certainlyinfluence the therapeutic effects of immunoglobulin in this model. When immunoglobulinwas injected immediately following pooled toxin, the toxic effects of thelatter substance were attenuated clinically and histologically but less sowhen compared with eyes receiving pooled toxin simultaneously injected withimmunoglobulin. This observation would suggest that optimizing the mixingof the two in vivo might facilitate the treatment effect.

The rapidity with which immunoglobulin is capable of inactivating thebiological activity of pooled toxin would suggest that its administrationduring a critical phase of bacterial toxin production could ameliorate thedestructive effects of the toxins. An opportunity for intervention in S aureus endophthalmitis stems from the fact that significanttoxin production typically does not occur until the postexponential bacterialgrowth phase.15 In a rabbit model of endophthalmitisproduced by the intravitreal injection of S aureus,bacteria grew exponentially during the first 24 hours.12 Intraocularinflammation was observed at 24 to 48 hours postinjection, whereas attenuationof the electroretinographic recording did not begin until 48 hours postinjection.12 Thus, treatment that occurs within or near this timeframe may favorably influence the course of endophthalmitis. The importanceof timely intervention prior to the bacterial release of increasingly toxicdoses of exotoxins is also suggested by our study. We saw little differenceclinically and none histologically when immunoglobulin injection was delayed6 hours following pooled toxin injection, most likely indicative of the rapidityof tissue destruction from abrupt exposure to a suprathreshold dose of toxin.Because clinical endophthalmitis is probably associated with a gradually increasingconcentration of a variety of toxins rather than a bolus dose, we believethat the concept of toxin neutralization early in the course of endophthalmitisretains merit.

The role of immunoglobulin in the clinical treatment of endophthalmitisneeds further investigation examining factors such as the interaction of oculartissues with immunoglobulin-bound toxin, efficacy of immunoglobulin in bacterialmodels of endophthalmitis, ocular immune host response, immune complex formation,interaction of immunoglobulin with antibiotics, effect of vitrectomy, timingof intervention, and role of hyperimmune immunoglobulin (toxin-specific antibody).Our study provides preliminary support of antibody-mediated antitoxin therapyfor bacterial endophthalmitis. We believe that pooled human immunoglobulinmay represent a novel adjunctive therapy in the treatment of S aureus endophthalmitis.

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Article Information

Submitted for publication December 24, 2002; final revision receivedJune 21, 2004; accepted June 21, 2004.

This study was supported in part by an unrestricted grant from Researchto Prevent Blindness, Inc, New York, NY; core grant EY01931 from the NationalEye Institute, Bethesda, Md; The Thomas M. Aaberg Retina Research Fund, Milwaukee(Medical College of Wisconsin); and grant HL 36611 from the National Heart,Lung and Blood Institute, Bethesda (University of Minnesota School of Medicine).

This study was presented in part at the Annual Meeting of the Associationfor Research in Vision and Ophthalmology; May 9, 2002; Ft Lauderdale, Fla.

This article is based on a thesis prepared in partial fulfillment ofthe requirements for membership in the American Ophthalmological Society.

Correspondence: Dennis P. Han, MD, Medical College of Wisconsin EyeInstitute, 925 N 87th St, Milwaukee, WI 53226-4812 (dhan@mcw.edu).

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