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Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas CHAPTER 7: CHARACTERIZATION OF CORNEAL MICROSPORIDIOSIS BY ELECTRON MICROSCOPIC STUDIES, CYTOKINE RESPONSES AND ASSESSMENT OF INFECTIVITY OF DONOR CORNEAS 7.1 ELECTRON MICROSCOPIC OBSERVATIONS OF MICROSPORIDIAL KERATITIS 7.1.1 Introduction Diagnostic electron microscopy (EM) can contribute decisively to the laboratory diagnosis of infectious diseases and is of particular importance if emerging infectious agents are to be characterized (Goldsmith et al. 2004, Morens et al. 2004). Definite genus identification of microsporidial ocular infections requires examination by electron microscope to demonstrate the pathognomonic coiled polar filament or tubule. Other features, such as arrangement and complexity of polar tubule, number and size of nuclei and relationship to the host cell cytoplasm allow more precise species diagnosis (Wittner 1999). Hence, transmission electron microscopy (TEM) is essential for viewing internal structures of microsporidian cells and till date remains the gold standard for the identification and speciation of these organisms (Franzen and Muller 1999). However, in recent years, studies of microsporidia have focused on the molecular biology, and detailed analysis of the cytology is often neglected. One reason for this might be that the detailed cytological investigation requires expertise and is time-consuming (Larsson 2005). Though EM has a great diagnostic potential, with the wide availability of alternative diagnostic methods, a change of paradigms in using diagnostic EM occurred. However, the “modern” techniques also show limitations: nucleic acid amplification techniques detect exclusively genomic sequences that are already known, and the Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 128 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas reactions may be inhibited by ingredients of the sample. Therefore, whenever a safe diagnosis appears urgent, the “open view” of diagnostic EM should be used in parallel to other methods (Hazelton and Gelderblom 2003). The objectives of this study were: - To perform TEM on corneal tissues as well as corneal scrapings to understand the pathobiology of microsporidia in the eye - To evaluate the TEM for the speciation of microsporidia associated with keratitis 7.1.2 Materials and Methods 7.1.2.1 Patients Five patients with microbiologically proven microsporidial keratitis and five patients with histopathologically proven microsporidial keratitis seen at the L.V. Prasad Eye Institute, Hyderabad, India between January 2002 and December 2005 were included in the study. The corneal scrapings from these patients were collected in 1 ml PBS and centrifuged at 1500g for 3 minutes. The pellet was washed twice using 5 ml of PBS buffer and sent to the electron microscopy laboratory (CCMB, Hyderabad and RUSKA Lab, College of Veterinary Sciences, ANGRAU, Hyderabad, India). In cases that underwent PK, the corneal button was bisected and sent for electron microscopic evaluation. 7.1.2.2 Transmission Electron Microscopy Tissue preparation of the clinical sample for identification, involves the fixation of the tissue, dehydration and infiltration with a medium that can be hardened to give a material suitable for thin sectioning. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 129 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas Fixation The tissue was fixed in 2.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) for 24 hours at 40C and post fixed in 2% aqueous Osmium tetroxide in the same buffer for 2 hours. Double fixation was adopted with glutaraldehyde as the primary fixative followed by OsO4. Glutaraldehyde stabilizes tissues by cross-linking proteins rendering them insoluble and immobile. Osmium tetroxide reacts with lipids and certain proteins but also provides electron density to the tissue. Therefore, OsO4 acts as both a second fixation step and an electron stain. Infiltration and Embedding This final process in tissue preparation is to infiltrate the specimens with a liquid embedding medium, which is then polymerized to produce a solid block. After dehydration, specimens are infiltrated with Spurr’s resin (Spur 1969) by passing them through a series of graded alcohol (50% - 99%) until the dehydrating agent has been completely replaced by the final embedding medium. This is done in small vials on a shaker at room temperature. Activator must be included in all dilutions, and after the tissues are transferred to capsules, filled with pure resins, and polymerized in an oven. Post-staining Procedure Both semithin and ultrathin sections were cut with a glass knife on a Leica Ultra cut UCT-GA_D/E –1/00 ultra microtome, semithin of 200-300 nm knife were stained with toludine blue and ultra thin sections (50-70 nm) were mounted on grids. Conventional double-staining of thin-sections of biological material involves staining first in uranyl Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 130 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas acetate followed by lead citrate. A drop of uranyl acetate stain is placed onto a clean surface in a chamber (petri dish). The grid with sections is floated on the drop, section side down (Fig. 7.1B). After the desired time (normally five minutes), the grid is washed three times by immersion in a rinse made of the same solution as the stain (for example, if 50% methanolic uranyl acetate is the stain, the rinse should be 50% methanol) and then air dried. A drop of lead citrate stain is placed onto a clean surface in a petri dish and the grid placed into the drop with the section side up and stained for 5 minutes. The grid is washed three times by immersion in a rinse made of boiled, distilled water and then air dried. Then the ultra thin sections were observed at various magnifications under transmission electron microscope (Model: Hitachi, H-7500, Fig. 7.1A) A B Figure 7.1 : (A) A picture of the transmission electron microscope (Model: Hitachi, H-7500) used in the study. (B) Petri dish containing ultra thin sections mounted on grids after staining with uranyl acetate. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 131 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas 7.1.3 Results Thick sections of corneal scrapings showed many extracellular spores with scattered degenerated structures with a vacuolated appearance and were surrounded by a thick capsule. The degenerated spores had a thick wall, but no internal structures were identified. The electron microscopic observations from the keratoplasty specimens revealed masses of round to oval sporoblasts, measuring 3.5 - 5.0 m in length by 2.5 to 3.0 m in width, dissecting amongst the corneal lamellae. Vegetative and spore stages of the organisms can be found in the host epithelial cells as the parasite undergoes merogony and sporogony, resulting in the production of the infective spore stage of the parasite. These stages together gave an understanding into the development of microsporidia in the eye. 7.1.3.1 Ultrastructure of development of microsporidia in the eye After entry of microsporidia on the surface of the eye, the polar tubule of the spore is extruded (Fig 7.2 A-B) penetrating the epithelial cell in patients with keratoconjunctivitis. The empty spore coat can be visualized on the surface of the epithelial cells after discharge of sporoplasm inside the host epithelial cells (Fig 7.2 C). In patients with stromal keratitis, the spores due to trauma are directly lodged onto the stromal cells of the cornea, where they begin their development by extruding their polar tubule, penetrating the host stromal cells (Fig 7.2 D). Contact of the end of the tubule with a host cell membrane allows the spore to transfer its contents (sporoplasm) to initiate infection, where in some cells the parasites lay in direct contact with the host cell cytoplasm, in Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 132 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas others, the parasites are situated within a clearly defined parasitophorous vacuole (PV) or lay in direct contact with the PV membrane within the new host cell. The earliest developmental stage seen are the meronts which are irregular in outline and bounded by a plasma membrane, and occur randomly in the host cell cytoplasm (Fig 7.2 E-F). The meronts divide by binary fission and the resultant daughter cells appear to be thickened with dense particles external to the limiting membrane. The daughter cells transform into sporonts (Fig 7.3 A-B), which in more advanced stages are easily recognized by their elongated appearance and their wrinkled surface. The boundary layer of sporonts are thickened and electron-dense and appear to have been formed by the deposition of dense material between the two membranes seen in the daughter cells. Sporonts contained a nucleus and numerous free ribosomes. Chains of sporonts appear to form by repeated fission, later separate to form sporoblasts. Sporoblasts are bounded by a distinct wall composed of two membranes, the outermost of which is covered by a flocculent dense layer (Fig 7.3 C-D). Each sporoblast has a nucleus, ribosomes, and accumulation of dense, membranous Golgi like material, the latter of which appear to contribute to the formation of the polar tubule (Fig 7.3 E-F). In mature spores the polar tubule is arranged into six to twelve coils in the posterior region of the spore (Fig 7.4 A - D). Closely packed ribosomes impart a dense appearance to the cytoplasm of the mature spores. The wall of mature spores is thickened. External to the plasma membrane of the parasite’s cytoplasm is a broad, electron-lucent layer, the endospore. This layer is overlain by the outer layer of the spore wall, the exospore, which is covered with a dense flocculent material. The surface of mature spores are smooth. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 133 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas A B C D E F Fig 7.2: Electron micrograph of ‘‘early stages’’ of microsporidial infection in human corneal epithelial and stromal cells. (A) An activated spore whose polar filament has started the process of translocation inside the epithelial cell from patient (L2096/05) diagnosed as microsporidial keratoconjunctivitis (B) Polar tube arising from an anchoring disk, being extruded through the disrupted spore wall found in patient (L1835/05) diagnosed as microsporidial keratoconjunctivitis (C)The remnants of membranes, still remain in the empty spore found on the surface of the epithelial cell of a patient (L1820/03) diagnosed as microsporidial keratoconjunctivitis (D) An activated spore whose polar filament has started the process of translocation inside the stromal cell of a patient (H490/02) diagnosed as microsporidial stromal keratitis (E) The parasite is in direct contact with the host cell cytoplasm and several meronts are interspersed in the epithelial cells in case (L1841/03) (F) Meronts are interspersed in the stromal cells in (H 490/02) Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 134 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas A B C D E F Figure 7.3: Electron micrograph of intracellular events and development of microsporidia (A) Meronts transformed to sporonts in epithelial cells of patient (L1835/05) (B) Phagocytized sporonts in stromal cells of a patient (L 2457/05) (C) Sporoblasts have a "thick" dense surface coat and a more complex and dense cytoplasm found in patient (L1786/05) diagnosed as microsporidial keratoconjunctivitis (D) Sporoblast containing portions of the developing polar tube, nucleus and a "thick" surface coat within a phagosome extending into the remains of the cytoplasm of the infected stromal cell of patient (L 2457/05) (E) Characteristic electron-dense outer and electron-lucent inner layers of the wall of a mature spore, as well as various sections of coils of the polar tube The spore (S) contains a polar tube, Note the irregular shape of the cell and the more complex cytoplasm found in a patient (L 2631/05) diagnosed as microsporidial keratoconjunctivitis (F)Maturation of spores in the stromal cells showing coils of the polar tubule and two nucleus found in a patient (H 374/02) diagnosed as microsporidial stromal keratitis. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 135 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas A B C D Figure 7.4: Electron micrograph of (A) a mature spore enclosed by a wide electron-lucent endospore, which is in turn covered by a dense, thick exospore and displaying approx 10 coils of the polar tubule giving a possible identification of Vittaforma corneae from a patient (V 367/05) with stromal keratitis. (B) Transverse section of coils of polar tubule of a spore present in the stromal cells of the corneal tissue from a patient (H 374/02) with microsporidial stromal keratits (C) A mature spore showing electron dense cytoplasm with polar tubule and ribosomes and nucleus from a patient (L 2828/05) with keratoconjunctivitis (D) a mature spore enclosed by a wide electron-lucent endospore, which is in turn covered by a dense, thick exospore and displaying approx 4-7 coils of the polar tubule giving a possible identification of Encephalitozoon spp. from a patient (L 2631/05) with keratoconjunctivitis. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 136 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas 7.1.3.2 Speciation of Microsporidial keratitis Electron microscopy confirmed microsporidial spores within the stromal cells for the keratoplasty specimens and on the epithelial cells for the corneal scrapings specimens. Only the spore stage of development was easily recognizable for species identification. Among the five cases of keratoconjunctivitis, the electron micrograph of only one case showed the polar tube having a single row of 4-7 coils, a feature that is consistent with the genus Encephalitozoon spp. Speciation of this genus was not possible as the distinctive parasitophorous vacuole, characteristic of E. intestinalis could not be visualized with certainty in the sample. Further sectioning was not possible due to the small amount of material. Similarly, among the five cases of stromal keratitis, the electron micrograph of only one case showed the polar tube having a single row of 10-14 coils, a feature that is consistent with Vittaforma corneae. 7.1.4 Discussion The most familiar stage in the microsporidial developmental cycle is the spore, as it is more easily detected than the intracellular proliferative stages. Following infection, a two stage developmental cycle follows – an initial proliferative phase (merogony) followed by a spore-forming phase (sporogony). Despite cumulative number of reports on corneal infections due to microsporidia, few studies have given conclusive evidence of the development of these organisms in the eye. This study has addressed this issue using ultra structural studies and demonstrated the difference in their biology during infection on both epithelial or stromal cells. The results suggest that different species of microsporidia Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 137 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas could possess a range of pathogenicity, tissue predilection and virulence to human host. Species known to cause keratitis include E. intestinalis, E. hellem, E. cuniculi, Vittaforma corneae, Nosema oculorum, Nosema connori, and Trachipleistophora hominis (Franzen and Muller 1999). There remains a possibility that more species are yet to be identified if detailed studies using electron microscopy coupled with molecular characterization are employed. In some circumstances, inadequate information obtained from limited material may be delineated by in vitro cultivation using appropriate cell lines that provides more detail description and identification of microsporidial species. Undoubtedly, definite species identification of microsporidia remains an important issue in order to know the range of pathogenic species, the extent of disease profiles and appropriate management of human microsporidiosis. The differentiation of Vittaforma corneae species from Encephalitozoon is based on several electron microscopic features. First, Vittaforma are larger than Encephalitozoon (Vittaforma organisms measure approximately 3.5–5.0 mm in length versus 2.0 –3.0 mm in length for Encephalatozoon organisms). Second, the absence of a parasitophorous vacuole surrounding the organism within the host cell is more consistent with Vittaforma. Third, the coils of the filament range from 10 to 13 in Vittaforma corneae versus 4 to 7 in Encephalitozoon (Pinnolis et al. 1981). The electron microscopy findings in our study are consistent with Vittaforma corneae species in another case of stromal keratitis. However, in one case of keratoconjunctivitis, though we could make a possible genus identification of Encephalitozoon, speciation was not possible as E. hellem and E. cuniculi are morphologically indistinguishable, whereas E. intestinalis has the distinctive Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 138 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas parasitophorous vacuole (Chu and West 1996) which could not be visualized with certainty in this study. Comparison of speciation by EM with the PCR and sequencing results, described in Chapter 6, showed that while V. corneae was identified by both methods, E. cuniculi could be identified by PCR alone as EM only suggested presence of Encephalitozoon spp. In addition, PCR could also determine the species for the remaining eight cases included in this study. Moreover, evaluation by EM is time-consuming, expensive and preparation and evaluation cannot be performed in an automated way, they are dependent on experienced staff. Another limitation is the relatively low sensitivity (105-6 particles/ml) of diagnostic EM compared to other diagnostic methods (Hazelton and Gelderblom 2003). Conclusions Though it was possible to identify the species involved in two of the ten cases in this study, the recent impression is that EM is not very useful for speciation of MK as corneal scrapings / corneal biopsies reveal more often the developing stages and not mature spores which is necessary for proper identification of the species causing keratitis. This suggests that the accurate speciation of an infecting parasite is possible only by means of a molecular biology technique and EM may have limited application as a diagnostic tool in clinical studies. Nevertheless, this method can be useful to study the development and pathogenicity of these parasites, as well as for the characterization of new species of this rare entity. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 139 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas 7.2 INVESTIGATION OF THE CYTOKINES RESPONSE OF HUMAN CORNEAL EPITHELIAL CELLS TO MICROSPORIDIAL INFECTION 7.2.1 Introduction Microsporidial keratitis is a disease initiated by infection of the epithelial and stromal layers of the cornea with spores of microsporidia, resulting in infiltration of neutrophils and mononuclear lymphocytes (Wittner 1999). Studies by Didier (1995) have shown that cytokines released by sensitized T cells activate macrophages to kill E. cuniculi in vitro. These findings suggest that a protective immune response to E. cuniculi is likely dependent on cytokine-producing immune T cells. Studies with E. intestinalis, have shown that mice lacking the IFN-γ gene are unable to clear the infection (Achbarou et al. 1996). In a previous study (Khan et al. 1999), it was observed that treatment of E. cuniculi-infected mice with neutralizing antibody against IFN-γ or IL-12 resulted in increased mortality for these animals. Also, IL-10 has been reported to be involved in the regulation of Th1 immune response in other infectious disease models (Gazzinelli et al. 1994, Trinchieri 1997), it is possible that it plays a similar role in infection caused by microsporidia too. Most of what is known about mammalian microsporidiosis is based on studies using E. cuniculi (Didier 1995), because this was the first microsporidian that could be grown continuously on tissue culture (Shadduck 1969) and most of these in vitro studies used murine culture cell systems (Didier 1995, Khan and Moretto 1999). However in addition to E. cuniculi, Vittaforma corneae was also found to be an important pathogen, in our series, causing microsporidial keratitis. Although the inflammatory response is necessary to clear the organism from the infected tissue, it can also be destructive to the host cornea, leading to scar formation and vision loss. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 140 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas Corneal epithelial cells constitute the first line of defense against microbial pathogens and therefore should possess the ability to respond to their presence. It is reasonable to propose that, in response to microsporidial infection, the epithelial cells send initial signals, such as the release of proinflammatory cytokines, to recruit neutrophils and mononuclear lymphocytes into the cornea. Epithelial cells could also produce factors to enhance the antimicrobial activity of residential cells of the cornea. Hence, we hypothesize that human corneal epithelial cells, which constitute the outermost layer of the cornea, can be infected with microsporidia, and that the infection leads to the activation of proinflammatory macromolecules. In the present study, we examined the cytokine responses of human corneal epithelial (HCE) cells to Vittaforma corneae, isolated from a patient with microsporidial keratoconjunctivitis. 7.2.2 Materials and Methods 7.2.2.1 Parasites Microsporidial spores isolated from corneal scrapings of a patient with microsporidial keratoconjunctivitis was collected in MEM, thawed, vortexed vigorously for 30 seconds and an equal volume (0.5 ml/ vial) of the sample was inoculated into monkey kidney cells (Vero cells) maintained on Eagle's minimal essential medium (EMEM) fortified with 2 mM glutamine and 10% fetal bovine serum (FBS). The culture medium from each flask (Nunc, 25 cm2) was removed daily for the first week and twice weekly thereafter and replaced with fresh medium containing the antibiotics. The cultures were incubated at 37°C. Spores that were extruded into the culture medium were harvested by centrifugation at 1500g for 20 min at 4 °C. The supernatant was removed and the spore Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 141 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas sediment was washed once with 0.25% sodium dodecyl sulfate in PBS and then washed once more in PBS. Spores were purified by centrifugation over 50% Percoll (Amersham Pharmacia Biotech, Piscataway, N.J) at 500g for 30 minutes at 4 °C. The purified sediment was washed once again in PBS, counted in a hemocytometer (Neubauer-ruled Bright Line counting chambers; Hausser Scientific, Horsham, Pa.), suspended in PBS to obtain 108 spores/mL, and stored at 4 °C until use. 7.2.2.2 HCE Infection Human corneal epithelial (HCE) cell line (Kind gift from Dr. Araki-Sasaki K, Osaka University Medical School, Osaka, Japan) was established by immortalizing primary cultured human corneal epithelial cells (obtained from a donor cornea) with a recombinant SV40-adenovirus vector and cloned three times to obtain a continuously growing cell line. This cell line has been shown to have properties similar to normal human corneal epithelial cells (Araki-Sasaki 1995). HCE cells were grown using the supplemented hormonal epithelial medium consisting of equal volumes of MEM and Ham's nutrient mixture F-12 supplemented with 5% (vol./vol.) heat inactivated fetal bovine serum (FBS), 5 μg/ml insulin, 10 ng/ml human epidermal growth factor, 0.5% dimethyl sulfoxide and 40 μg/ml gentamicin. All the reagents were obtained from Sigma, St. Louis. To infect cells, confluent epithelial cultures grown in Nunc 25 cm2 flasks were inoculated with 1x108 spores/mL of microsporidia. Simultaneously, an uninoculated HCE culture was also stored for analysis. At 0 and 24 hrs the culture supernatants of infected culture as well as the control, were collected in duplicates for cytokine determination and stored at -70°C for cytokine determination. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 142 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas 7.2.2.3 Cytokine assays The cytokine analysis was measured by the evidence investigator™ using a biochip, according to the manufacturer’s directions (Randox laboratories ltd, United Kingdom) which included Interleukin-1 alpha (IL-1), Interleukin-1 beta (IL-1), Interleukin-2 (IL2), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Vascular Endothelial Growth Factor (VEGF), Tumour Necrosis Factor-alpha (TNF-) Interferon-gamma (IFN-), Epidermal Growth Factor (EGF), Monocyte Chemotactic Protein-1 (MCP-1) . Conventional immunoassay techniques are utilized for the measurement of analytes on the surface of a biochip, which results in the specific and simultaneous profiling of markers. Rather than having to divide and separately test a patient sample to obtain each test result, evidence investigator™ offers a means for simultaneous testing of a sample and thus provides a more complete diagnostic profile for each patient. The cytokine and growth factors array measures 12 analytes from a single sample. The cytokines and growth factors array of tests are presented on a single biochip. All twelve tests are performed simultaneously using just one assay diluent, one panel-specific conjugate solution and one signal reagent. All reagents required to run assays are provided in a single kit – assay buffer, multi-analyte conjugate, nine levels of multi-analyte calibrator, wash buffer and signal reagent. Multi-analyte controls are available as a separate kit. The evidence investigator™ CVs are below 10% thus providing the user with confidence in results obtained. There is reduced variation as all results are determined at the exact same time point. One vial of multi-analyte calibrator is all that is required for each of the nine levels. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 143 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas Figure 7.5 : Picture of the evidence investigator™ (Randox Laboratories) which offers a means for simultaneous testing of a sample and thus provides a more complete diagnostic profile. Insert showing the cytokines and growth factors array of tests presented on a single biochip. 7.2.3 Results The microsporidial spores inoculated in human corneal epithelial cells were identified as Vittaforma corneae after sequencing the PCR product obtained using pan-microsporidial primers based polymerase chain reaction described in Chapter 6. The levels of cytokines and growth factors at 0 hour and after 24 hours of inoculation of microsporidial spores in HCE culture supernatants are shown in Table 7.1. The levels of cytokines in both test sample and control sample at 0 hour were comparable. Among the 12 cytokines screened, six (IL-6, IL-8 , VEGF, IFN-γ, EGF, MCP-1) were induced after 24 hours of infection with microsporidia. The levels of TNF-α, IL-10, IL-1 and IL-1 remained unchanged in the control during the course of the study. Levels of IL-2 and IL-4 were not elevated above control levels 24 hours post infection. IL-6 and VEGF were significantly elevated in test sample infected with microsporidial spores (approximately two fold higher) compared to uninfected HCE cells. Similarly, IL-8 and MCP-1 were significantly Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 144 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas elevated (approximately 10-fold) in infected HCE cells compared to uninfected cells. Infection of HCE cells by microsporidia did not increase levels of Th1 type cytokines like TNF-α and IL-2 (Table 7.1) above baseline levels. Table 7.1: Levels of cytokines in culture supernatants of HCE cell line at 0 hour and 24 hours after infection. Cytokines IL2 (pg/ml) IL4 (pg/ml) IL6 (pg/ml) IL8 (pg/ml) IL10 (pg/ml) VEGF (pg/ml) IFN (pg/ml) TNF (pg/ml) IL1 (pg/ml) IL1 (pg/ml) MCP1 (pg/ml) EGF (pg/ml) 0 hr control 0 hr test 24 hr control 24 hr test <0.000 2.792 1.705 0.868 3.493 2.689 2.55 1.828 <0.000 <0.000 122.412 >275.000 <0.000 <0.000 178.241 1530.388 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 428.45 925.587 <0.000 <0.000 <0.000 9.261 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 <0.000 >1200.000 <0.000 <0.000 <0.000 1.438 IL-2 : Interleukin 2, IL-4: Interleukin 4, IL-6: Interleukin 6, IL-8: Interleukin 8, IL-10: Interleukin 10, VEGF : Vascular Endothelial Growth Factor, IFN : Interferon – gamma , TNF - Tumour Necrosis Factor-alpha, EGF: Epidermal Growth Factor, MCP-1 : Monocyte Chemotactic Protein1, IL-1 : Interleukin-1 alpha, IL-1 : Interleukin-1 beta 7.2.4 Discussion In the immunobiology of microsporidial infections the role of both humoral and cellular immunity has been demonstrated (Braunfuchsona et al. 1999). As microsporidia are Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 145 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas intracellular parasites, the role of cell-mediated immunity is probably more significant (Schmidt and Shadduck 1983). To understand the role of epithelial cells in the innate response to microsporidial infection of the cornea, an in vitro infection model of human corneal epithelial cells (HCE) grown in the laboratory was used. This study reveal that corneal epithelial cells respond to the presence of microsporidial parasites and initiate a rapid innate immune response, leading to the production of pro-inflammatory cytokines. The most striking result in this study was the abundant expression of MCP-1 and IFN-γ which is a strong activator of monocytes and macrophages suggesting that these mediators contribute to the cellular infiltration of the corneal cells. The significance of IFN-γ in the control of microsporidial infections has been previously demonstrated (Didier et al. 1994, Achbarou et al. 1996). Macrophages activated by IFN-γ kill microsporidia by the nitric oxide-dependent mechanism (Didier 1995). This study also demonstrated the production of IL-6 and IL-8 in our study which has been linked to the recruitment of neutrophils and lymphocytes, suggesting that corneal epithelial cells play a role in inducing neutrophil chemotaxis to the parasite infection site. Like IL-6, TNF-α is a potent proinflammatory cytokine and is produced mainly by activated macrophages and T cells and has been reported to be released by the epithelial cells of the mouse central cornea in response to lipopolysaccharide (Sekine-Okano et al. 1996). However, this study showed the absence of TNF-α expression, the reason for this could not be estabilished. Previous studies have demonstrated the essential role of IL-6 as a potent stimulator of VEGF production (Nakahara et al. 2003, Wei et al. 2003) from corneal epithelial cells after HSV infection. In addition, the intrastromal injection of IL-6 resulted in corneal Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 146 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas neovascularisation in a VEGF-dependent process (Banerjee et al. 2004). This data substantiates the close relationship between proinflammatory cytokines and VEGFinduced corneal neovascularisation. In addition, this study also showed minimal EGF expression that has been reported to stimulate epithelial cell proliferation (Klenkler and Sheardown 2004). Nevertheless, the ability of the epithelium to produce multiple proinflammatory cytokines in response to microsporidial infection indicates that the epithelium is an important part of the innate response system for the cornea and plays a role in eliciting the infiltration of inflammatory cells into the tissue. Studies of parasite infections have played a major role in establishing the veracity of the Th subset paradigm. The balance between Th1 and Th2 helper cells and their associated cytokine patterns can in an immune response influence the phenotype and progression of several clinical diseases (Alexander and Bryson 2005). Th1 cells produce type 1 cytokines (IL-2, TNF-α, IFN-γ), while Th2 cells produce type 2 cytokines (IL-4, IL-6, IL-10, IL13). In this study, IL-6 and IFN-γ are coexpressed, the expression of IL-2 and IL-4 were down-regulated. In addition, TNF-α, IL-1 and IL-10 are suppressed, suggesting a skewed cytokine production and a mixed pattern of Th response. This is the first study in the literature to investigate the balance of Th1/Th2 in microsporidiosis in corneal cells. Further investigations should be focused on the polarization of the immune response and the role of various subpopulations of lymphocytes in the elimination of microsporidia from the host cornea. Selective receptor blockage of highly expressed chemokines or proinflammatory cytokines in inflamed eye tissue could then be explored as a reasonable strategy to inhibit the recruitment of leukocytes. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 147 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas 7.3 INVESTIGATION OF THE POTENTIAL INFECTIVITY OF DONOR CORNEAS LEADING TO MICROSPORIDIAL KERATITIS FOLLOWING KERATOPLASTY 7.3.1 Introduction The infection of the corneal graft is one of the most serious complications following keratoplasty (Morris and Bates 1989, Varley and Meisler 1991). In many instances, it can be treated successfully with intensive topical and sub-conjunctival antibiotics. However, when this therapy is ineffective, a surgical approach is considered. Two cases of microsporidial epithelial keratoconjunctivitis occurring in the corneal graft of individuals who were locally immunocompromised have been already described in Chapter 3. These patients had no history of owning pets, river swimming, or wearing contact lenses. The only possible associated risk in both these cases was the use of topical steroids, leading to a localized immunosuppressed state, resulting in secondary infection by microsporidia. The use of immunosuppression as a therapeutic goal following keratoplasty, therefore, may increase the risk for acquiring microsporidia infections in these patients. It is tempting to speculate the possible presence of these organisms in the donor corneas and subsequent infection in these patients. To test this hypothesis, the presence of microsporidia in donor corneas from the eye bank was evaluated by the most sensitive nucleotide amplification method currently available in the laboratory. The study objective was to determine the potential infectivity of donor corneas leading to microsporidial keratitis following keratoplasty. 7.3.2 Materials and Methods Postmortem donor corneas were harvested with the consent of families from 53 subjects Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 148 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas by in situ excision by the Ramayamma International Eye Bank at the L.V.Prasad Eye Institute, Hyderabad, India. These tissues were considered unsuitable for use as donor tissue by the eye bank as they were of poor quality or were from patients who had died of causes contraindicated as suitable donors (septicemia, multiple myeloma) by eye bank association of India guidelines. The corneal tissue was minced and washed in PBS buffer for DNA isolation using the ‘UNSET procedure’ (described in Chapter 6). Polymerase chain reaction (PCR) was performed using pan-microsporidia primers which is capable of amplifying a conserved region of the small-subunit rRNA of V. corneae, E. cuniculi, E. hellem, and E. intestinalis (Conners et al. 2004). The PCR conditions included 1 minute denaturation at 94°C, 2 minute annealing at 55°C and 3 minute extension at 72°C for 35 cycles. The DNA from all corneal tissues were additionally spiked with 1µl of E. hellem (ATCC 50504) to rule out the presence of PCR inhibitors in the samples. 7.3.3 Results Thirty seven out of 53 donors were male and sixteen were female. The mean age of the donors was 65.5 ± 18.9 (range 25-104) years. The lower limit of detection for the primers was determined to be 10pg when using genomic DNA as a template. The primers when tested for analytical specificity, amplified DNA from all microsporidia species tested (V. corneae, E. cuniculi, E. hellem, and E. intestinalis ) but did not amplify any of the selected mammalian, bacterial, or viral DNA. Microsporidial DNA was not detected in any of the samples tested. On the other hand none of the samples in this study showed presence of any inhibitors and an expected amplification corresponding to ~270 bp was observed in all cases after spiking them. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 149 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas 7.3.4 Discussion Corneas have transmitted rabies, hepatitis B, cytomegalovirus, herpes simplex virus, bacteria, and fungi (Eastlund 1995). Over the past several years, improvements in donor screening criteria, such as excluding potential donors with infection for HIV-1 and hepatitis B and syphilis have greatly reduced the risk of such infections. There have been few reports of additional danger of transplanting active HCV and HSV if critical assessment of the graft prior to surgery is not carried out (Sengler et al. 2001). Therefore it was tempting to know if donor screening for microspordia would help in elimination of graft associated microsporidial keratitis. Traditional methods for the diagnosis of microsporidia in clinical laboratories are still reliant on histochemical staining and light microscopy (Franzen and Muller 1999). PCR-based diagnostic methods have become increasingly popular for pathogen identification and are particularly suitable for those microbial organisms that are hard to culture in vitro, morphologically similar, and present in low infectious doses. The detection limit and specificity of the pan-microsporidia primers used in our PCR assay is satisfactory and we consider the assay advantageous, because it makes it possible to rapidly screen samples collected from corneal donors for potential microsporidial contamination. Consistent with the low prevalence, there was no case of microsporidial infection in any of the donor corneas that were screened in this study. In conjunction with the established quality control for corneal storage in eye banks, there is potential for these PCR assays to aid in rapid screening of clinical specimens for microsporidial contamination and in the diagnosis of ocular infections. Possible causes of primary graft failure could include poor Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 150 Chapter 7: Characterization of Corneal Microsporidiosis by Electron Microscopic Studies, Cytokine Responses and Assessment of Infectivity of Donor Corneas surgical technique, poor donor material and poor eye banking technique, inadequate storage in organ culture, or infection of the donor material. However, the surgery in these two cases of microsporidial infection after penetrating keratoplasty were straightforward and routine. A qualitative and quantitative assessment of the donor endothelium in those cases indicated good quality and microbiology of the medium during culture and at issue was sterile. However, it is not sure if microsporidia can establish latent infection in the cornea and therefore be transmissible by corneal transplantation as they are known to be commonly associated with immunocompromised patients (Franzen and Muller 1999). Given that immunosuppression probably is the major predisposing factor for prolonged microsporidiosis in patients after organ transplantation, patients with systemic immunosuppression and AIDS may be at higher risk of acquiring unusual infections, especially in situations with hygiene deficiencies. Conclusions Considering the fact that the prevalence of microsporidial keratitis in L. V. Prasad Eye Institute is around 0.4% (Joseph et al. 2006b), the number of donor corneas examined in this study is too small for any conclusive results. However, the absence of microsporidial DNA in all the donor corneal tissues tested rules out the risk for transmission of microsporidia by corneal transplantation. At this point of time it is concluded that screening of donor corneas for presence of microsporidia is not warranted. Investigation of Microsporidial Keratitis In India: A Clinical, Microbiological, Molecular and Proteome Approach 151