Hisayuki Ueno

Absence of Chlamydia psittaci in ocular adnexal lymphoma from Japanese patients

Low-grade malignant lymphomas arising from mucosa-associated lymphoid tissue (MALT) represent a distinct clinicopathological entity of B-cell non-Hodgkin lymphoma (Isaacson & Wright, 1984). MALT lymphoma tends to remain localised and the extranodal sites of involvement include the gastrointestinal tract, salivary gland, thyroid and ocular region. MALT lymphoma often occurs in relation to autoimmune disorders or chronic antigen stimulation, but the pathogenetic mechanisms of this type of lymphoma have not been well defined. Recently, Ferreri et al (2004) reported that 32 (80%) of 40 Italian patients with ocular adnexal lymphoma harboured Chlamydia psittaci DNA. The findings suggested that C. psittaci infection might contribute to the development of these lymphomas. Soon after this, two other groups independently investigated ocular adnexal lymphoma for the presence of C. psittaci DNA in 57 patients from south Florida (Rosado et al, 2005) and 11 patients from the north-eastern USA (Vargas et al, 2005) and found that none of them carried C. psittaci DNA. One explanation for this discrepancy concerns geographic differences in the incidence of C. psittaci infection. However, all of these reports are based on findings from patients in the USA and Europe, and therefore additional surveys evaluating the association between C. psittaci and ocular adnexal lymphomas including MALT lymphoma in other geographical regions of the world would be warranted. There has been no report from Asia thus far. In this study, we looked for C. psittaci DNA in tumour specimens from Japanese patients with primary ocular adnexal lymphoma. Tissues obtained by an incisional biopsy from 24 Japanese patients (12 males and 12 females, mean age 56 years; range 34–73 years) with localised lymphoproliferative conditions of the ocular adnexa, including 18 MALT lymphomas, three nonMALT lymphomas and three reactive ocular lymphoid hyperplasias, were studied. The localities of the tumours were the orbit in 14 cases, lacrimal glands in four cases, and conjunctiva in six cases. DNA was obtained from frozen or paraffinembedded tissues using the phenol/chroroform extraction technique or DNA extraction system according to the manufacturer’s instructions (Takara, Tokyo, Japan). All samples were successfully amplified by polymerase chain reaction (PCR) for human b-globin sequences, yielding a 123-bp amplicon, which indicated that amplifiable DNAs were present. Three independent PCRs were used to detect C. psittaci DNA in this study. First, we used the same method employed in the previous studies (Ferreri et al, 2004; Rosado et al, 2005; Vargas et al, 2005). The touchdown enzyme time release PCR with CPS100 and CPS101 primers produces a 111-bp amplicon (Madico et al, 2000). The primers amplify a highly conserved region encoding the 16S ribosomal RNA gene. The second method was a nested PCR to detect the same DNA region with the first-step primers (5¢-ACGGAATAATGACTTCGG-3¢ and 5¢-TACCTGGTACGCTCAATT-3¢) and the second-step primers (5¢-ATAATGACTTCGGTTGTTATT-3¢ and 5¢-TGTTTTA GATGCCTAAACAT-3¢), generating a 127-bp amplicon. The PCR primers for the third C. psittaci PCR were derived from another DNA region encoding the ompA gene, and the primer sequences were 5¢-GCCTTAAACATCTGGGATCG-3¢ and 5¢-GCACAACCACATTCCCATAAAG-3¢, giving a 248-bp fragment. All three PCRs failed to detect C. psittaci DNA in any of our samples, while positive control reactions using DNA prepared from C. psittaci strain Cal-10 yielded amplicons of expected size. In addition, our samples were negative for Chlamydia pneumoniae and Chlamydia trachomatis DNA sequences. Our results showed no correlation between C. psittaci infection and ocular adnexal lymphoma in Japan, supporting the observation from the USA (Rosado et al, 2005; Vargas et al, 2005). All these studies were based on testing C. psittaci DNA sequences. Given the serological evidence of an association between chronic chlamydia infections (C. pneumoniae and C. trachomatis) and lymphomas (Anttila et al, 1998), serological surveys for C. psittaci may also help to better understand the relationship between C. psittaci and ocular adnexal lymphoma. Although the ‘hit-and-run’ mechanism cannot be fully excluded, the present findings indicate that C. psittaci is unlikely to have played a pathogenetic role in ocular adnexal lymphoma in the Japanese population.

Engagement of 4-1BB Inhibits the Development of Experimental Allergic Conjunctivitis in Mice

The 4-1BB receptor acts as a costimulator in CD8+ T cell activation. Agonistic stimulation through this molecule by treatment with anti-4-1BB Abs has been demonstrated to inhibit various experimentally induced diseases in animals. However, the effect of anti-4-1BB Abs on experimental allergic diseases has not been reported. We investigated the effect of anti-4-1BB Abs on the development and progression of experimental allergic conjunctivitis in mice. To examine the effects of Abs during the induction or effector phase, actively immunized mice or passively immunized mice by splenocyte transfer were treated with agonistic anti-4-1BB Abs, blocking anti-4-1BB ligand Abs, or normal rat IgG. Eosinophil infiltration into the conjunctiva was significantly reduced in wild-type mice by the anti-4-1BB Ab treatment during either induction or effector phase. Th2 cytokine production by splenocytes and total serum IgE were significantly reduced by the anti-4-1BB Ab treatment, while IFN-γ production was increased. The anti-4-1BB Ab treatment induced a relative increase of CD8-positive cell numbers in the spleens. Moreover, inhibition of eosinophil infiltration by the treatment with anti-4-1BB Abs was also noted in actively immunized IFN-γ knockout mice. Taken altogether, in vivo treatment with agonistic anti-4-1BB Abs in either induction or effector phase inhibits the development of experimental allergic conjunctivitis, and this inhibition is likely to be mediated by suppression of Th2 immune responses rather than up-regulation of IFN-γ.

Search for accumulation of p53 protein and detection of human papillomavirus genomes in sebaceous gland carcinoma of the eyelid

Twenty-one Japanese patients with sebaceous carcinoma of the eyelid were investigated for tumour incorporation of human papillomavirus (HPV) types-6, 11, 16, 18, 31, and/or 33 DNA by in situ hybridization with fluorescein isothiocyanate-labelled DNA probes, and for p53 protein accumulation by immunohistochemical analysis with an antibody to p53 protein. Thirteen tumours (61.9%), including 9 cases of multiple infections, were positive for HPV DNA. Positive signal in the nucleus was observed not only in the cancer cells, but also in the cells of surrounding normal sebaceous glands and epidermis. Positive nuclear staining of cancer cells with the antibody to p53 protein was detected in 12 cases (57.1%). p53 protein accumulation was more frequently observed in the clinically advanced cases, occasionally in association with recurrence and/or metastasis. Among the 12 p53-positive cases, 7 were also positive for the presence of HPV DNA. HPV infections exist in a high percentage of sebaceous carcinomas of the eyelid in Japan; the overexpression of p53 protein may be important in both carcinogenesis and progression.

Dissection of antigen-specific humoral and cellular immune responses for the development of experimental immune-mediated blepharoconjunctivitis in C57BL/6 mice

Purpose: Allergic conjunctivitis is characterized by allergen-specific IgE in the serum and infiltration of eosinophils into the conjunctiva. However, it remains unclear whether early-phase reaction (EPR) mediated by Ag-specific IgE links to late-phase reaction (LPR) in the conjunctiva. We aimed to investigate whether LPR is mediated by either cellular or humoral immune responses. Methods: Experimental immune-mediated blepharoconjunctivitis (EC) was induced in C57BL/6 mice by either active immunization or passive immunization by transfer of ragweed (RW)-primed lymphocytes and RW-specific IgE, followed by RW challenge onto the conjunctiva. Transferring RW-primed lymphocytes were prepared from RW-primed splenocytes which were stimulated in vitro with RW for 3 days. Fifteen minutes after RW challenge, clinical findings were evaluated and 24 hr after challenge, the conjunctivas and sera were harvested for histologic analysis and measurement of IgE, respectively. Results: EPR was most prominent when EC was induced by transfer of RW-specific IgE. EPR was hardly detectable if EC was induced by transfer of RW-primed lymphocytes. Mild EPR was noted when EC was induced by active immunization. LPR, evaluated by infiltration of eosinophils into the conjunctiva, was most severe when EC was induced by transfer of RW-primed lymphocytes. Minimal, but definite LPR was induced when EC was induced by transfer of RW-specific IgE. Intermediate severity of LPR was induced when EC was induced by active immunization. Conclusions: LPR in the conjunctiva is dominantly mediated by cellular immune responses, whereas EPR in the conjunctiva is putatively mediated by humoral immune responses. Importantly, LPR in the conjunctiva is inducible by Ag-specific IgE alone, although minute.

Congenital cystic eye: report of two cases and review of the literature.

A 13-month-old boy and a 2-week-old girl, who were considered to be anophthalmic and who later each developed a cystic lesion in the left orbit with protrusion of the lower eyelid, were studied. The fellow eye in case 1 was subsequently found to be microphthalmic with cyst and was normal in case 2. Histopathologic study of each case revealed a cyst lined externally by dense fibrous connective tissue to which skeletal muscle and adipose tissue were attached. The inner aspect of the cyst was lined by neuroglial tissue, possible immature retinal tissue, and cuboidal epithelium. No fully developed ocular structures or microphthalmos were identified. Fourteen cases of congenital cystic eye, including our cases, have been published in the English-language literature since 1964. We discuss and illustrate the findings in our cases and 10 others in which histopathologic findings were reported. Congenital cystic eye, microphthalmos with cyst, and microphthalmos with cystic teratoma should be suspected in patients with a small or unrecognizable eye and an orbital cystic mass that is detected by palpation or visualization.

Exertion of the suppressive effects of IFN-γ on experimental immune mediated blepharoconjunctivitis in Brown Norway rats during the induction phase but not the effector phase

Background/aims: Interferon gamma (IFN-γ) knockout mice exhibit severe allergic conjunctivitis (AC), indicating that IFN-γ regulates the development of AC. The authors examined whether this inhibitory effect of IFN-γ is exerted during the induction or effector phase of experimental AC. Methods: Experimental immune mediated blepharoconjunctivitis (EC) was induced in Brown Norway (BN) rats, using ovalbumin (OVA) as the antigen. To investigate the role of IFN-γ in the induction phase, EC was induced by active immunisation and IFN-γ (10 μg/time, total 70 μg), or phosphate buffered saline (PBS) as a control, was injected intraperitoneally every other day from the day of immunisation. The rats were challenged with OVA eye drops 13 days after immunisation, and 24 hours later, the eyes were harvested for histology. To examine the effects of IFN-γ in the effector phase, OVA specific T cells were transferred into syngeneic rats and IFN-γ (10 μg/time, total 50 μg) or PBS was injected each day after the transfer until induction of EC 4 days later with an OVA challenge. To investigate the role of endogenous IFN-γ during the effector phase, an anti-IFN-γ monoclonal antibody (3 mg/time) was injected on days 3 and 4. Results: Injection of IFN-γ into actively immunised rats suppressed eosinophilic infiltration but not infiltration of mononuclear cells. In contrast, neither IFN-γ nor anti-IFN-γ affected EC in passively immunised rats. Conclusion: IFN-γ is a suppressive cytokine for the development of EC and exerts this suppressive effect during the induction phase.

Involvement of programmed death-ligand 2 (PD-L2) in the development of experimental allergic conjunctivitis in mice

Background/aim: Involvement of programmed death-1 (PD-1) and its ligands has been demonstrated in experimental allergic airway disease. Here, the authors aimed to examine whether PD-1 and its ligands are involved in the development of experimental allergic conjunctivitis (EC) in mice. Methods: EC was induced in Balb/c mice by active immunisation with short ragweed pollen (RW) in alum. 10 days later (day 10), the mice were challenged with eye drops containing RW. 24 hours after the challenge, conjunctivas, spleens, and sera were harvested for histological analysis, cytokine assays, and measurement of RW specific Ig levels. The actively immunised mice were treated with anti-PD-1, anti-PD-L1, anti-PD-L2 antibodies (Abs), or normal rat immunoglobulin G (nrIgG) during either the induction (day 0, 2, 4, 6, and 8) or the effector (2 hours before RW challenge on day 10) phase. Results: Ab treatment during the induction phase did not affect eosinophil infiltration although immune responses were modulated. In contrast, treatment with anti-PD-L2 Ab, but not anti-PD-1 or anti-PD-L1 Ab, during the effector phase significantly increased eosinophil infiltration into the conjunctiva without affecting systemic immune responses. Conclusions: Similar to allergic airway inflammation, PD-L2 is involved in the development of EC during the effector phase but not the induction phase.

Intraocular pressure elevation after intravitreal or posterior sub-Tenon triamcinolone acetonide injection.

BACKGROUND Despite the benefits of intraocular steroids for the treatment of inflammatory, neovascular, proliferative, and edematous diseases, one of the side effects is raised intraocular pressure (IOP). In this study, we attempted to identify when IOP elevates, peaks, and returns to the preinjection baseline IOP after intravitreal or posterior sub-Tenon administration of triamcinolone acetonide, as well as the factors that might affect IOP. METHODS Retrospective case review was undertaken of 69 patients (82 eyes), who received either a 4 mg intravitreal (16 eyes) or a 20 mg posterior sub-Tenon (66 eyes) triamcinolone acetonide injection. IOP assessment for each eye was completed at the preinjection baseline and at the first, third, and sixth month of follow-up. RESULTS The mean IOP of all eyes increased significantly at each follow-up. The mean maximum elevation ratio from the baseline was 4.0 (SD 5.2) mm Hg. An elevation of 5 mm Hg or greater occurred in 28 eyes (34.1%). The maximum elevation correlated significantly with age (p < 0.01). The incidence of an elevation of 5 mm Hg or greater was significantly higher among patients younger than 60 years (p < 0.01) and relatively higher among female patients (p = 0.051). The mean IOP increased significantly at the first month after intravitreal injection but at all follow-up periods after posterior sub-Tenon injection. There was no significant difference in IOP elevation according to disease type, although eyes with diabetic retinopathy tended to be at higher risk of IOP elevation. Two eyes of two female patients, who had received posterior sub-Tenon injections for the treatment of diabetic retinopathy, required glaucoma surgery. INTERPRETATION The IOP elevation of 5 mm Hg or greater observed in 34.1% of the eyes was consistent with past reports. IOP elevation was associated with patients of less than 60 years of age and with female sex, and it lasted longer after posterior sub-Tenon injection than after intravitreal injection. Careful assessment of IOP during a follow-up period of at least 6 months is paramount, especially in younger female patients after posterior sub-Tenon injection.

Properties of the voltage-gated calcium channels mediating dopamine and acetylcholine release from the isolated rat retina

We examined the properties of voltage-gated calcium channels mediating endogenous dopamine (DA) and acetylcholine (ACh) release in the isolated rat retina. Application of 30 mM KCl elicited the release of DA and ACh, and these releases were abolished in Ca(2+)-free medium. The high K(+)-evoked DA release was largely blocked by both of omega-agatoxin IVA and omega-conotoxin MVIIC, P- and Q-type calcium channel antagonists, and partly blocked by isradipine, and L-type calcium channel antagonist, and omega-conotoxin GVIA, an N-type calcium channel antagonist. omega-Agatoxin IVA at a small dose, sufficient to block P-type channels alone, was however without effect. On the other hand, the high K(+)-evoked ACh release was partly blocked by omega-agatoxin IVA and omega-conotoxin MVIIC, but was resistant to isradipine and omega-conotoxin GVIA. Flunarizine, a non-selective T-type calcium channel antagonist, did not inhibit the release of DA and ACh. Cd2+ markedly blocked the release of both DA and ACh, Co2+ and Ni2+ slightly blocked the release of DA, and the release of ACh was not blocked by these two divalent cations. These results suggest that the high K(+)-evoked release of retinal DA is largely mediated by omega-agatoxin IVA and omega-conotoxin MVIIC sensitive calcium channels (probably Q-type channels), while the release of retinal ACh is largely mediated by as yet uncharacterized Cd2+ sensitive calcium channels. The properties of voltage-gated calcium channels involved in the release of ACh in the rat retina differ from those of DA.

Cyclosporin A inhibits eosinophilic infiltration into the conjunctiva mediated by type IV allergic reactions.

Background:  Eosinophils are important effector cells in severe allergic conjunctivitis such as vernal keratoconjunctivitis. Infiltration of eosinophils into the conjunctiva is mediated by type I and type IV allergic reactions. Cyclosporin A (CsA) eye drops are administered therapeutically for severe allergic conjunctivitis, but the mechanism by which CsA acts, that is, by inhibiting type I, type IV or both types of allergic reactions, is not known. We investigated whether CsA eye drops inhibit type I, type IV or both types of allergic reactions in the conjunctiva.