Radiographic evaluation of cases referred for surgical endodontics.

AIM: The aim of this study is to test the hypothesis that more patients with failed root-canal treatment or other endodontic problems are referred for periradicular surgery rather than nonsurgical re-treatment. METHODOLOGY: Three sets of 100 periapical radiographs representing typical cases referred for surgical treatment were collected in three departments of oral and maxillo-facial surgery situated in different parts of the Netherlands. Of these, a total of 278 radiographs were evaluated to determine whether endodontic surgery was indicated or whether the primary endodontic treatment or endodontic re-treatment was a realistic option. An oral and maxillo-facial surgeon, an endodontist and a general dental practitioner viewed the radiograph independently under standard conditions. RESULTS: Overall, orthograde root-canal (re-)treatment was considered possible in 63% of the cases. The results differed between the three examiners with the oral surgeon reporting that 41% of cases were amenable to conventional treatment, for the general dental practitioner and the endodontists the figures were 67 and 80%, respectively. CONCLUSIONS: Based on these observations, it is concluded that most of the teeth referred for surgical treatment to an oral surgeon could be treated by orthograde nonsurgical root-canal treatment.

Analysis of the cellular infiltrate in the iris during experimental autoimmune encephalomyelitis.

PURPOSE: Previous studies have shown that experimental autoimmune encephalomyelitis (EAE) and anterior uveitis (AU) develop in Lewis rats immunized with myelin basic protein (MBP). The purpose of this study was to characterize the dynamics, distribution, and phenotype of infiltrating cells in the iris during EAE-associated AU. METHODS: Lewis rats were immunized with MBP emulsified in complete Freund's adjuvant (CFA) or with CFA alone. Cellular infiltration of the iris was analyzed at various time points by immunohistochemistry of wholemounts, flow cytometry, and immunoelectron microscopy, by using monoclonal antibodies specific for monocytes/macrophages (ED1), T lymphocytes (R73, W3.25, OX8), T-cell activation markers (OX39, OX40), granulocytes (HIS48), major histocompatibility complex (MHC) class II (OX6), and neurofilament (2H3). RESULTS: MBP-immunized rats showed development of characteristic monophasic EAE, followed, after resolution of paralysis, by mild self-limited AU. Initially, focal infiltrates of round MHC class II(+) and ED1(+) cells were found in the iris. During the course of AU, the midiris became massively infiltrated with ED1(+) monocytes-macrophages, R73(+) T cells, granulocytes (HIS48(+)), and MHC class II(+) cells. The influx of T cells consisted of CD4(+) and CD8(+) cells, of which only a small fraction (<14 and 11%, respectively) expressed activation markers. The infiltrating cells accumulated in proximity to myelinated and nonmyelinated nerve bundles and in the vicinity of blood vessels in the iris. No evidence was found for demyelination or nerve degradation. Neither EAE nor AU developed in CFA-treated control rats. CONCLUSIONS: These data show that EAE-associated AU is characterized by a transient mixed cellular infiltrate consisting of monocytes-macrophages, granulocytes, and CD4 and CD8 T cells. The preferential accumulation of inflammatory cells in the vicinity of nerve fibers suggests that AU in this model may result from autoreactivity to nerve antigens.

Effect of macrophage depletion on immune effector mechanisms during corneal allograft rejection in rats.

PURPOSE: In rats, corneal allograft rejection is delayed for at least 100 days by clodronate liposomes. These liposomes selectively deplete macrophages. To investigate the immunologic basis for absence of graft rejection in treated rats, the effect of these liposomes on the generation of cytotoxic T lymphocytes (CTLs) and antibody production after orthotopic corneal allotransplantation was determined. METHODS: Transplantations of corneal buttons from PVG rats were performed in AO rats. After surgery, one group received clodronate liposomes subconjunctivally at five time points, and the other group remained untreated. On postoperative day (POD) 3, 7, 12, or 17, rats were killed, the presence of CTLs was investigated at three different anatomic locations, and antibodies against donor antigens were tested. RESULTS: No significant differences were found between the groups tested 3 and 7 days after surgery. But on POD 12 (the time of onset of rejection in the untreated group) and on POD 17, the CTL activities detected in the submandibular lymph nodes (P < or = 0.008) and the spleen (P < or = 0.009) were significantly less in the treated groups compared with the untreated groups. In the untreated groups complement-independent antibodies were present only on POD 17, whereas no antibodies were found in the treated rats. CONCLUSIONS: Local treatment with clodronate liposomes was shown to downregulate local and systemic CTL responses and to prevent the generation of antibodies. Local depletion of macrophages in the initiation phase of the immune response appears to lead to a less vigorous attack on the grafted tissue and therefore to promote graft survival.

Cytokines in aqueous humour and serum before and after corneal transplantation and during rejection.

Cytokine profiles in aqueous humour were studied in relation to corneal disease and subsequent corneal graft survival or rejection. Cytokine levels in samples obtained from eyes with clear grafts (n = 59) were all within the normal range. At the time of penetrating keratoplasty (n = 146), intraocular levels of IL-6 were increased in 38% (50/131), most markedly in eyes with previous allograft failure or herpetic stromal keratitis. The level of IL-10 was increased in 1 eye (n = 144) and of IL-4 and IFN-gamma in none. During rejection (n = 10), the levels of IL-6 in aqueous humour were increased in 75% (3/4), of IL-10 in 50% (3/6), of IL-4 in none (0/4) and of IFN-gamma in 40% (2/5). In conclusion, the levels of total protein and IL-6 were increased prior to penetrating keratoplasty in eyes with previous inflammation. These results could however not predict the final outcome of the graft. Increased intraocular levels of IL-6, IL-10 and IFN-gamma were observed during rejection.

Apoptosis of infiltrating cells in experimental autoimmune uveoretinitis.

OBJECTIVE: To investigate the cellular phenotype and apoptosis of infiltrating cells involved in experimental autoimmune uveoretinitis (EAU). METHODS: Immunohistochemical staining and in situ apoptosis staining were performed using monoclonal antibodies to monocytes and macrophages (ED1), major histocompatibility complex (MHC) class-II antigen (OX6), T lymphocytes (R73) and TACS 1 Klenow kit on both ocular sections and wholemounts from Lewis rats after immunization with interphotoreceptor retinoid-binding protein (IRBP). RESULTS: EAU was induced in 12 of 16 Lewis rats with a mean clinical inflammation score of 1.29 +/- 0.7. Influx of monocytes, lymphocytes and MHC class II-positive cells into the uvea and retina was noted after immunization with IRBP. Apoptosis of infiltrating cells was observed in the uvea and retina, and more apoptotic cells were present in the iris and ciliary body compared with those in the choroid and retina. CONCLUSION: Apoptosis of infiltrating cells occurs at the early stage of EAU, which may greatly contribute to the rapid regression of the inflammation induced by IRBP.

Cytotoxic T lymphocytes and antibodies after orthotropic penetrating keratoplasty in rats treated with dichloromethylene diphosphonate encapsulated liposomes.

PURPOSE: To investigate the immunological basis for the prolonged corneal allograft survival after subconjunctival injections of liposomes filled with dichloromethylene diphosphonate (Cl2MDP-LIP). METHODS: F344 rats received orthotropic DA corneal grafts. One group of rats was treated with subconjunctival injections of Cl2MDP-LIP on days 0, 2, 4, 6 and 8 postoperatively, the control groups received no treatment. Nineteen or 42 days postoperatively cytotoxic T lymphocyte (CTL) activity was measured in the lymph nodes draining the grafted and the contralateral eyes, in the spleen and the mesenteric lymph nodes. Sera taken at the same time points were tested for presence of lytic alloantibodies. RESULTS: On day 19 CTL activity in submandibular lymph nodes draining the grafted eyes was similar in the 2 groups. In the mesenteric lymph nodes high CTL activity was found in the untreated rats and low in the Cl2MDP-LIP rats. The spleen showed high CTL activity in the untreated group but no activity in the liposome group. Forty two days postoperatively a decline in CTL activity was seen in both groups. Complement dependent anti-donor antibodies were absent in the Cl2MDP-LIP group at both time points whereas antibodies were present on days 19 and 42 in the untreated group. CONCLUSIONS: Repeated subconjunctival injection of Cl2MDP-LIP restricts the induction of cellular cytotoxicity against donor antigens to the regional lymph nodes and suppresses cytotoxic antibody formation.

Influx of immunoglobulins from the vascular compartment into a grafted cornea.

PURPOSE: To determine the effect of a fresh corneal wound or a healed corneal scar on the immunodiffusion of immunoglobulins into the cornea. METHODS: F344 rats were immunized with human serum albumin (HSA) 1 week before an autologous rotational keratoplasty of the right cornea or 1 year after an autograft was performed. One group of rats also was treated with gentamicin-dexamethasone ointment in the grafted eye for 1 week after transplantation to reduce the postsurgical inflammatory signs. A serum sample was drawn every week and booster injections with HSA were given after 2 and 3 weeks. At various times after immunization, groups of rats were killed, blood and aqueous humor samples were taken, and the corneas of both eyes were removed. The corneas were divided into the graft or a 3-mm central button and the peripheral rim and weighed. The anti-HSA titer was determined in serum, aqueous humor, and both parts of the corneas. RESULTS: Up to 5 weeks after transplantation, the grafted cornea contained more anti-HSA immunoglobulins than did the control eye. One year postgrafting, no difference was seen. In the first weeks after keratoplasty, influx of anti-HSA from the peripheral into the central cornea was, however, neither obstructed nor enhanced. CONCLUSIONS: Surgical trauma in itself causes increased influx of anti-HSA immunoglobulins into the cornea. Within the cornea, a wound or a scar does not appear to be a barrier for centripetal immunoglobulin diffusion.

Prevention of corneal allograft rejection in rats treated with subconjunctival injections of liposomes containing dichloromethylene diphosphonate.

PURPOSE: The drug dichloromethylene diphosphonate (CL2MDP) encapsulated in liposomes depletes macrophages but not other immunocompetent cells. The authors investigated whether subconjunctival injection of CL2MDP containing liposomes (CL2MDP-LIP) could prolong survival of corneal allografts in rats. METHODS: Male Fisher rats received orthotopic corneal grafts of Dark Agouti origin. Rats were treated postoperatively with subconjunctival injections of 0.1 ml CL2MDP-LIP at the time of transplantation and on days 2, 4, 6, and 8 after transplantation. Control groups received either liposomes containing phosphate-buffered saline subconjunctivally at the same time points or no additional treatment. Corneal grafts were evaluated every other day and were scored for neovascularization, opacity, and edema. Immunohistologic evaluation was performed 12 and 19 days after surgery. RESULTS: Corneal grafts in both control groups were rejected within 17 days. In the Cl2MDP-LIP treated rats, grafts were not rejected during the maximum follow-up of 100 days. Cellular infiltration in these grafts was clearly reduced. There was also a strong reduction in neovascularization of the cornea. CONCLUSIONS: Rejection of orthotopic allogeneic corneal grafts could be prevented by repeated subconjunctival injection of Cl2MDP-LIP.

Genetic and clinical determinants for the T cell mediated immune response against the cornea specific protein BCP 54.

T cell mediated immune responses against the cornea specific protein BCP 54 have been observed in patients with uveitis, Fuchs' heterochromic cyclitis, and corneal disease. The pathophysiological role of this anti-BCP 54 response in corneal disease is not known. In order to ascertain whether the presence of such an immune response is related to the corneal disease itself or related to genetic influences, the anti-BCP 54 response was determined in 104 patients with severe corneal disease, using a monocyte migration inhibition assay. The results were compared with the presence of a variety of ocular parameters as well as with the distribution of HLA antigens in these patients. While only 7% of healthy controls responded to BCP 54, 37% of the patients showed a positive response (p = 0.002); in particular, patients with previous graft rejection, non-herpetic keratitis, and bullous keratopathy reacted against BCP 54. No relation with known risk factors for corneal transplantation, such as corneal neovascularisation, was observed. No significant association with the presence of any of the HLA antigens was observed. It was concluded that the main inducer of an anti-BCP 54 response is corneal disease itself, and that the presence of corneal disease is able to break the immunological privilege typical of normal corneas.

Circulating cornea-specific antibodies in corneal disease and cornea transplantation.

In order to establish the significance of circulating cornea-specific antibodies, we determined the presence of anti-corneal antibodies in the serum of 100 patients with corneal disease and in 50 healthy controls, and subsequently followed the pattern of antibody reactivity in 46 patients who underwent corneal transplantation. An indirect immunofluorescence test on cryostat sections of rabbit corneas was used for screening. The reactivity against two known bovine corneal epithelial proteins was also tested: a 54-kD protein (BCP 54) and an 11-kD protein (BCP 11/24). No significant difference in the presence and specificity of anti-corneal antibodies was observed between the group of patients with corneal disease, taken as a whole, and the healthy controls. Patients with keratoconus or non-immunological graft failure, however, were significantly more often positive for anti-corneal antibodies. Neither the presence of antibodies prior to corneal transplantation nor their appearance post-transplantation had a predictive value for corneal graft survival.

Does autoimmunity to S-antigen play a role in Fuchs' heterochromic cyclitis?

Autoimmunity directed against retinal or choroidal antigens has been suggested to play a role in the chorioretinal lesions observed in patients with Fuchs' heterochromic cyclitis. This hypothesis was addressed and patients with Fuchs' heterochromic cyclitis were tested for cellular immunity (migration inhibitory factor assay) against human retinal S-antigen. A significantly higher percentage of patients with Fuchs' heterochromic cyclitis had a positive cellular autoimmune response to S-antigen than healthy controls and other patients with anterior uveitis. This finding is remarkable since Fuchs' heterochromic cyclitis is generally classified as an anterior uveitis and patients with Fuchs' heterochromic cyclitis without chorioretinal lesions also had a positive test. In view of these results and a sensitisation against a corneal antigen reported earlier in Fuchs' heterochromic cyclitis, it is suggested that a chronic low grade grade anterior uveitis or chorioretinitis of unknown origin may cause the release of potent autoantigens in these patients.

Changes in the immune response against BCP 54 following corneal transplantation in man.

Bovine corneal protein (BCP) 54 is the major soluble protein of the cornea. Immune responses against this protein can be observed in patients with corneal disease or inflammatory disease of the anterior chamber of the eye. We wanted to determine whether an immune response against this corneal protein plays a role in corneal transplantation. A cell-mediated immune response against BCP 54 was therefore determined in 46 patients prior to and on several occasions during the first year following corneal transplantation. The presence of an anti-BCP 54 response before transplantation was not associated with any clinical parameters or with rejection following transplantation. The highest frequency of positive responses was observed three months after transplantation. Transplantation may have stimulated a temporary immune response against a previously sequestered antigen, but this tissue-specific immune response did not necessarily lead to rejection. Therefore, no prognostic value can be attributed to the anti-BCP 54 response.

Distribution of the nuclear thyroid-hormone receptor in extraocular and skeletal muscles.

The correlation between the occurrence of Graves' ophthalmopathy and Graves' hyperthyroidism may indicate a role for tri-iodothyronine (T3) hormone in the pathogenesis of Graves' ophthalmopathy. In Graves' ophthalmopathy the recti eye muscles are greatly enlarged whereas skeletal muscles seem unaffected. The distribution of the nuclear T3 receptor was studied in normal human and rat eye and skeletal muscles with immunohistochemistry using mouse (monoclonal) antibodies, and by in-situ hybridization for the detection of mRNA encoding the T3-receptor protein. Nuclear staining with T3-receptor antibodies was found in all types of tissues studied. Cytoplasmic staining occurred predominantly in the muscle fibres of the orbital layer of the eye muscles and was generally absent or very low in skeletal muscle fibres and hepatocytes. Immunostaining could be inhibited by preabsorbing the antibodies with bacterially expressed T3-receptor protein, implying specificity. The presence of nuclear and cytoplasmic hormone-free T3 receptor sites was indicated after preincubation of sections with T3 hormone; T3-receptor immunostaining decreased and T3-hormone staining increased. In-situ hybridization clearly revealed the presence of alpha-1 and beta-1 forms of the T3-receptor mRNA in liver, skeletal muscles, and orbital and intermediate layers of the eye muscles. The data demonstrate the presence of T3 hormone-receptor molecules in the extraocular and skeletal muscles. The different susceptibilities of these muscles to Graves' hyperthyroidism may relate to the quantitative differences in T3 hormone-receptor distribution.

Analysis of corneal inflammation following the injection of heterologous serum into the rat cornea.

The immunopathologic response following the injection of various antigens into the rat cornea was evaluated. This reaction, known as Wessely's phenomenon, was believed to be primarily triggered by antibodies and complement activation. The keratitis model was originally described in rabbits, using heterologous serum or purified proteins. In rats only, heterologous serum induced corneal inflammation with the characteristics of Wessely's phenomenon, (ie, a quiescent period of several days between antigen injection and onset of clinical signs and corneal opacification). Using rats allowed us to characterize the cellular infiltrate with immunohistochemical methods. Marked infiltration of the cornea by macrophages was observed, as was infiltration by polymorphonuclear cells, although to a lesser extent. Furthermore, T lymphocytes of the helper phenotype were demonstrated. Antibodies to complement activation product C3c showed faint staining, whereas B lymphocytes and plasma cells were absent. In addition, inflammatory cells and ocular tissues, particularly the limbal and peripheral corneal epithelium, were found to express major histocompatibility complex class II antigens during the inflammatory response. After the inflammation had subsided, macrophages and T lymphocytes remained in the corneal stroma (at least until day 30). These findings suggest that antigen-induced keratitis in rats might be mediated, at least partially, by T helper lymphocytes.

Cellular and humoral anticorneal immune response in corneal transplantation.

Immunologic responses play a role in the rejection process of corneal transplants. Both histocompatibility antigens and tissue-specific antigens may be potential targets for such an immune response. To identify relevant responses, the humoral immune response against corneal tissue and the cellular immune response against one specific corneal protein were determined in patients before and after corneal transplantation. The results were compared with known risk factors for corneal transplantation, but no correlations were observed. A conversion from negative to positive in cellular immune response against the cornea-specific protein was seen in patients who had experienced an inflammatory episode during the time interval between measurements. The anticorneal protein response may therefore be the result of an intraocular inflammatory response, but not a prognostic factor to help predict the patients at risk for rejection.

Indomethacin increases the sensitivity of the monocyte migration inhibition assay.

In ophthalmo-immunological investigations only small samples of ocular tissues and fluid are available and assays which are feasible with very small volumes or cell numbers are mandatory. Indomethacin, which is known to augment the immune response both in vivo and in vitro was therefore tested for its effect on the monocyte migration inhibition (MIF) assay using low cell or antigen doses. The sensitivity of the MIF assay may be greatly increased by adding indomethaci during the first step of the assay. Titration of either the antigen dose, the mononuclear cells number or both per assay, resulted in a 10-50-fold increase in sensitivity of the assay, with a broad inter-individual variability. Increasing the sensitivity of the MIF assay with indomethacin has clear advantages with regard to the number of cells required but also confronts us with a new problem: activation of specific cells that circulate at very low frequencies in non-immunized individuals. The enhanced response could be reversed to some extent by adding prostaglandin E2 together with indomethacin to the first step of the assay. Moreover, adding leukotriene B4 to the first step of the assay had an enhancing effect over a limited concentration range. We conclude that in the presence of indomethacin, the MIF assay provides a highly sensitive technique for the demonstration of cellular immune responses in small samples of biological fluids containing very small numbers of antigen-specific lymphocytes.

Immunity to a corneal antigen in Fuchs' heterochromic cyclitis patients.

Immunity to a major corneal antigen was studied in 28 Fuchs' heterochromic cyclitis patients and compared with the response of 44 patients with other types of uveitis and 19 healthy controls. The highest incidence of immune response was found in patients with anterior segment involvement only (anterior uveitis and Fuchs') whereas the frequency of anti-corneal immune response in patients with posterior segment involvement only was low and not significantly different from that of healthy controls. Cellular immunity to corneal antigens was found in the majority of Fuchs' heterochromic cyclitis patients, and in one-third of the anterior uveitis patients. No correlation could be established in these patients between a positive cellular response and the chronicity of the disease or the presence of keratic precipitates. Humoral immunity to the corneal antigen was also the highest in patients with anterior segment involvement, but there was no difference in response between Fuchs' and non-Fuchs' anterior uveitis patients. This study suggests that anti-corneal immunity may be triggered in inflammatory diseases of the anterior segment, especially in Fuchs' heterochromic cyclitis.

Circulating monocyte migration inhibitory factor in serum of Graves' ophthalmopathy patients: a parameter for disease activity?

A factor which inhibits the random migration of monocytes in vitro was found in the serum of nearly half of Graves' ophthalmopathy patients. This factor was found in serum of only 4% of the healthy controls. When the Graves' ophthalmopathy patients were subdivided, on clinical criteria (pain, redness, swelling and impaired function), into patients with active disease and patients with inactive disease, the serum factor was found in 61% of the patients with active inflammatory disease and only in 14% of the patients with inactive disease. Furthermore this factor was also found in the serum of patients with an acute episode of uveitis, suggesting that it may be a useful marker for assessment of a localized inflammatory process.

High incidence of corneal epithelium antibodies in Fuch's heterochromic cyclitis.

Sera obtained from 26 patients with Fuchs' heterochromic cyclitis were examined for the presence of autoantibodies directed against the anterior segment of the eye by means of immunofluorescence techniques. Antibodies against human iris tissue could not be detected, whereas autoantibodies against corneal epithelium were found in almost 90% of cases. This is the first report describing the presence of autoantibodies in patients with Fuchs' heterochromic cyclitis and it provides a further clue that immunological mechanisms might play an important role in the aetiology of Fuchs' heterochromic cyclitis.

In vivo and in vitro deposition of immunoglobulin aggregates in the mouse eye.

The possible role of specific mechanisms involved in the adherence process of immune aggregates to tissue components of the mouse eye was investigated in an experimental animal model. Passive intravenous administration of immunoglobulin aggregates to mice, resulted in the localisation of these aggregates in various organs, including the eye. In the eye a strong localisation was seen in the episcleral capillary plexus, whereas only a weak deposition was seen in the iris, ciliary body and choroid. No deposits were seen in the retina. To investigate the role of specific receptors for immune complexes in the eye, in vitro experiments were performed, whereby immunoglobulin aggregates were layered on cryostat sections of the mouse eye. These in vitro studies also showed an adherence of immune aggregates to the episcleral capillary region, but furthermore a deposition on mast cells scattered throughout the extra-ocular muscles. The in vitro binding of immunoglobulin aggregates to the episcleral capillary plexus could be inhibited by high concentrations of Fc fragments and monomeric IgG, but not with Fab fragments or 0.5 M NaCl. The in vitro adherence of aggregates to mast cells could not be blocked by the inhibitors employed in our study and could therefore be distinguished from the interaction of aggregates with the episcleral capillary plexus. These results indicate that the ocular deposition of immunoglobulin aggregates in the episcleral capillary plexus of the mouse eye is (immune) specific and mediated by Fc receptors.