Chlamydia trachomatis Infection Is Associated with E-Cadherin Promoter Methylation, Downregulation of E-Cadherin Expression, and Increased Expression of Fibronectin and α-SMA-Implications for Epithelial-Mesenchymal Transition.

Chlamydia trachomatis (Ct) can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT) contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE) with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring.

Fibroblasts profiling in scarring trachoma identifies IL-6 as a functional component of a fibroblast-macrophage pro-fibrotic and pro-inflammatory feedback loop.

Trachoma is a conjunctiva scarring disease, which is the leading infectious cause of blindness worldwide. Yet, the molecular mechanisms underlying progressive fibrosis in trachoma are unknown. To investigate the contribution of local resident fibroblasts to disease progression, we isolated conjunctival fibroblasts from patients with scarring trachoma and matching control individuals, and compared their gene expression profiles and functional properties in vitro. We show that scarring trachoma fibroblasts substantially differ from control counterparts, displaying pro-fibrotic and pro-inflammatory features matched by an altered gene expression profile. This pro-inflammatory signature was exemplified by increased IL-6 expression and secretion, and a stronger response to macrophage-mediated stimulation of contraction. We further demonstrate that scarring trachoma fibroblasts can promote Akt phosphorylation in macrophages in an IL-6 -dependent manner. Overall this work has uncovered a distinctive molecular fingerprint for scarring trachoma fibroblasts, and identified IL-6- as a potential contributor to the chronic conjunctival fibrosis, mediating reciprocal pro-fibrotic/pro-inflammatory interactions between macrophages and fibroblasts.

Increased Epithelial Expression of CTGF and S100A7 with Elevated Subepithelial Expression of IL-1ß in Trachomatous Trichiasis.

PURPOSE: To characterize the histological appearance and expression of pro-inflammatory mediators, growth factors, matrix metalloproteinases and biomarkers of epithelial-mesenchymal transition (EMT) in healthy control and trachomatous trichiasis (TT) conjunctival tissue. METHODS: Conjunctival biopsies were taken from 20 individuals with TT and from 16 individuals with healthy conjunctiva, which served as controls. Study participants were of varying ethnicity and were living in a trachoma-endemic region of northern Tanzania. Formalin-fixed paraffin-embedded tissue sections were stained using hematoxylin and eosin or by immunohistochemistry using antibodies against: IL-1ß, IL-6, IL-17A, IL-22, CXCL5, S100A7, cleaved caspase 1 (CC1), PDGF, CTGF, TGFß2, MMP7, MMP9, E-cadherin, vimentin, and αSMA. RESULTS: Tissue from TT cases had a greater inflammatory cell infiltrate relative to controls and greater disruption of collagen structure. CTGF and S100A7 were more highly expressed in the epithelium and IL-1ß was more highly expressed in the substantia propria of TT cases relative to controls. Latent TGFß2 was slightly more abundant in the substantia propria of control tissue. No differences were detected between TT cases and controls in the degree of epithelial atrophy, the number of myofibroblasts or expression of EMT biomarkers. CONCLUSIONS: These data indicate that the innate immune system is active in the immunopathology of trachoma, even in the absence of clinical inflammation. CTGF might provide a direct link between inflammation and fibrosis and could be a suitable target for therapeutic treatment to halt the progression of trachomatous scarring.

Queratitis por Stenotrophomonas maltophilia tratada con trimetoprima-sulfametoxazol / Stenotrophomonas maltophilia keratitis treated with trimethoprim-sulfamethoxazole

CASO CLÍNICO: Mujer de 84 años con defecto epitelial persistente e infiltrado estromal denso tras trasplante corneal. El análisis microbiológico reveló una Stenotrophomonas maltophilia (S. maltophilia), resistente a todos los antibióticos salvo a trimetoprima-sulfametoxazol (TMP/SMX) Se consiguió su resolución tras 3 semanas de tratamiento con TMP/SMX tópico y oral. DISCUSIÓN: S. maltophilia es un microorganismo oportunista raramente descrito en oftalmología. Se asocia con conjuntivitis, queratitis, escleritis, dacriocistitis, celulitis y endoftalmitis, con importante morbilidad. El tratamiento es complicado por sus múltiples resistencias a antibióticos de amplio espectro. El TMP/SMX en monoterapia puede considerarse como una opción de tratamiento para estas queratitis

CASE REPORT: An 84 year-old woman with persistent epithelial defect and a dense stromal infiltrate post-corneal transplantation. According to the microbiological results, it was due to a Stenotrophomonas maltophilia (S. maltophilia) resistant to all antibiotics except trimethoprim-sulfamethoxazole (TMP/SMX). Healing was achieved after three weeks of treatment with oral and topical TMP/SMX. DISCUSSION: S. maltophilia is an opportunistic microorganism rarely described in ophthalmology. It is associated with conjunctivitis, keratitis, scleritis, dacryrocystitis, cellulitis, and endophthalmitis with significant morbidity. Treatment is complicated because of its resistances to broad-spectrum antibiotics. TMP/SMX monotherapy can be considered an option of treatment for this type of keratitis

Doxycycline prevents matrix remodeling and contraction by trichiasis-derived conjunctival fibroblasts.

PURPOSE: Trachoma is a conjunctival scarring disease, which is the leading infectious cause of blindness worldwide. Elimination of blinding trachoma is being held back by the high rate of trichiasis recurrence following surgery. There is currently no treatment available to suppress the profibrotic state and reduce recurrence. Although the mechanisms underlying trichiasis development are unknown, the profibrotic phenotype has been linked to matrix metalloproteinase (MMP) expression. Doxycycline, a well-known tetracycline antibiotic, can act as a broad MMP inhibitor and has showed some success in preventing fibrosis in various clinical contexts. The purpose of this work was to assess the antiscarring properties of doxycycline in an in vitro model of trichiasis fibroblast-mediated tissue contraction. METHODS: Primary cultures of fibroblasts were established from conjunctival samples obtained from normal donors or during surgery for trachomatous trichiasis. The effect of doxycycline on matrix contraction was investigated in our standard collagen gel contraction model. Cell morphology and matrix integrity were assessed using confocal reflection microscopy. Quantitative real time polymerase chain reaction and a FRET-based assay were used to measure MMP expression and activity, respectively. RESULTS: Doxycycline treatment successfully suppressed the contractile phenotype of trichiasis fibroblasts, matrix degradation, and significantly altered the expression of MMP1, MMP9, and MMP12 associated with the profibrotic phenotype. CONCLUSIONS: In view of the results presented here and the wider use of doxycycline in clinical settings, we propose that doxycycline might be useful as a treatment to prevent recurrence following trichiasis surgery.

Azithromycin 1.5% ophthalmic solution: in purulent bacterial or trachomatous conjunctivitis.

The second-generation macrolide azithromycin is available as a 1.5% ophthalmic solution for use in the treatment of bacterial or trachomatous conjunctivitis. This article reviews the pharmacological properties of azithromycin 1.5% ophthalmic solution and its clinical efficacy and tolerability in patients with purulent bacterial conjunctivitis or trachomatous conjunctivitis caused by Chlamydia trachomatis. Azithromycin 1.5% ophthalmic solution had good in vitro activity against Haemophilus influenzae and C. trachomatis, and achieved good concentrations in tear samples from healthy volunteers. Azithromycin 1.5% ophthalmic solution for 3 days (1 drop twice daily) was noninferior to tobramycin 0.3% ophthalmic solution for 7 days (1 drop every 2 hours) in paediatric and adult patients with purulent bacterial conjunctivitis, with regard to clinical cure and bacteriological resolution on day 9, in a randomized, investigator-masked, multicentre study. In children with trachomatous inflammation, 3-day treatment with azithromycin 1.5% ophthalmic solution was noninferior to a single dose of azithromycin oral suspension, with regard to clinical cure rate in the worst eye at 60 days, in a randomized, double-masked, multicentre study. Azithromycin 1.5% ophthalmic solution was well tolerated in patients with bacterial or trachomatous conjunctivitis. Most events were of mild to moderate severity.

Active trachoma is associated with increased conjunctival expression of IL17A and profibrotic cytokines.

The immunological basis of scarring trachoma is not well understood. It is unclear whether it is driven primarily through cell-mediated adaptive or epithelial-cell-derived innate responses. The purpose of this study was to investigate the expression of the inflammatory and fibrogenic mediators which may be involved. We conducted a cross-sectional survey of children living in an untreated trachoma-endemic community in Tanzania. The children were examined for signs of trachoma, and swabs were collected for bacteriological culture and RNA and DNA isolation. Chlamydia trachomatis was detected by the Amplicor PCR test. The expression of the following genes was measured by quantitative reverse transcription-PCR (RT-PCR): S100A7, IL1B, IL17A, IL23A, CXCL5, CCL18, TLR2, NLRP3, KLRD1, CTGF, and MMP9. Four hundred seventy children under the age of 10 years were included. Follicular trachoma (TF) was detected in 65 children (14%), C. trachomatis was detected in 25 (5%), and bacterial pathogens were cultured in 161 (34%). TF was associated with significantly increased expression of S100A7, IL17A, CCL18, CXCL5, and CTGF. Expression was increased further in the presence of papillary inflammation. Nonchlamydial bacterial infection was associated with increased expression of IL17A, CXCL5, CCL18, and KLRD1. IL17A expression was associated with increased expression of S100A7, CXCL5, CCL18, KLRD1, and CTGF. These data are consistent with a role for IL-17A in orchestrating the proinflammatory response in trachoma. Its activity may be promoted either as part of the cell-mediated response or through innate pathways. It may drive a range of proinflammatory factors leading to excessive tissue damage and repair involving fibrosis.

Identification of novel single nucleotide polymorphisms in inflammatory genes as risk factors associated with trachomatous trichiasis.

BACKGROUND: Trachoma is the leading preventable cause of global blindness. A balanced Th1/Th2/Th3 immune response is critical for resolving Chlamydia trachomatis infection, the primary cause of trachoma. Despite control programs that include mass antibiotic treatment, reinfection and recurrence of trachoma are common after treatment cessation. Furthermore, a subset of infected individuals develop inflammation and are at greater risk for developing the severe sequela of trachoma known as trachomatous trichiasis (TT). While there are a number of environmental and behavioral risk factors for trachoma, genetic factors that influence inflammation and TT risk remain ill defined. METHODOLOGY/FINDINGS: We identified single nucleotide polymorphisms (SNP) in 36 candidate inflammatory genes and interactions among these SNPs that likely play a role in the overall risk for TT. We conducted a case control study of 538 individuals of Tharu ethnicity residing in an endemic region of Nepal. Trachoma was graded according to World Health Organization guidelines. A linear array was used to genotype 51 biallelic SNPs in the 36 genes. Analyses were performed using logic regression modeling, which controls for multiple comparisons. We present, to our knowledge, the first significant association of TNFA (-308GA), LTA (252A), VCAM1 (-1594TC), and IL9 (T113M) polymorphisms, synergistic SNPs and risk of TT. TT risk decreased 5 times [odds ratio = 0.2 (95% confidence interval 0.11.-0.33), p = 0.001] with the combination of TNFA (-308A), LTA (252A), VCAM1 (-1594C), SCYA 11 (23T) minor allele, and the combination of TNFA (-308A), IL9 (113M), IL1B (5'UTR-T), and VCAM1 (-1594C). However, TT risk increased 13.5 times [odds ratio = 13.5 (95% confidence interval 3.3-22), p = 0.001] with the combination of TNFA (-308G), VDR (intron G), IL4R (50V), and ICAM1 (56M) minor allele. CONCLUSIONS: Evaluating genetic risk factors for trachoma will advance our understanding of disease pathogenesis, and should be considered in the context of designing global control programs.

Conjunctival chlamydial 16S ribosomal RNA expression in trachoma: is chlamydial metabolic activity required for disease to develop?

BACKGROUND: Nucleic acid amplification testing frequently detects conjunctival Chlamydia trachomatis infection in subjects without clinical signs of trachoma. It is unclear whether such subjects are actually infected. We measured chlamydial 16S ribosomal RNA (rRNA) expression, a marker of chlamydial metabolic activity, in comparison with the quantitation of a chlamydial DNA target in subjects exposed to trachoma. METHODS: Subjects from 2 Gambian villages where trachoma was endemic were examined. Conjunctival samples were tested for the presence of C. trachomatis DNA using a quantitative real-time polymerase chain reaction (PCR) assay for the omp1 gene and for the expression of C. trachomatis 16S rRNA using a 1-step, real-time reverse-transcriptase PCR assay. RESULTS: A total of 248 people were examined. The prevalence of clinically active trachoma was 16.9%. C. trachomatis was detected in 19.8% and 6.8% of subjects by the omp1 and 16S rRNA assays, respectively. For subjects with positive results for both tests, the number of copies of 16S rRNA was approximately 100-fold greater than the number of copies of the omp1 gene. In samples from subjects in whom the omp1 gene was detected in the absence of 16S rRNA, typically only a few copies of omp1 were present. The expression of 16S rRNA was strongly associated with the presence of clinical signs of active trachoma. CONCLUSIONS: The use of 16S rRNA expression for the detection of chlamydial metabolic activity appears to usefully discriminate established infections from the inoculation of the conjunctiva with dead or subviable organisms, which probably occurs frequently in settings in which trachoma is endemic. The data support conclusions from primate challenge studies that live Chlamydiae species or antigens derived from them are needed to provoke the clinical signs of disease.

Expression of growth factors in the conjunctiva from patients with active trachoma.

PURPOSE: The blinding complications of trachoma are associated with progressive conjunctival fibrosis due to excessive accumulation of extracellular matrix (ECM) components. We studied the processes involved in the regulation of fibrosis in trachoma by investigating the expression of the fibrogenic and angiogenic connective tissue growth factor (CTGF) and basic fibroblast growth factor (bFGF), the angiogenic vascular endothelial growth factor (VEGF), the angiogenesis-associated endothelial cell marker CD105 (endoglin), and the ECM protein tenascin in the conjunctiva. METHODS: Conjunctival biopsy specimens from six patients with active trachoma, and six control subjects were studied by immunohistochemical techniques using monoclonal and polyclonal antibodies directed against CTGF, bFGF, VEGF, CD105, and tenascin. RESULTS: In the normal conjunctiva, weak immunoreactivity for VEGF was observed in epithelial cells. There was no immunoreactivity for the other antibodies. In all trachoma specimens, immunoreactivity for CTGF and bFGF was localized in monocytes/macrophages, positive for the CD68 marker. Strong immunoreactivity for VEGF was observed in epithelial cells and on vascular endothelial cells. CD105 immunoreactivity was observed on vascular endothelial cells. Immunoreactivity for tenascin was noted in the upper substantia propria. CONCLUSIONS: These findings suggest that macrophages play an active role in conjunctival scarring, upregulated local production of CTGF, bFGF, and VEGF contributes to both fibrous tissue growth and angiogenesis, vascular endothelial cells are activated and are undergoing active angiogenesis, and deposition of tenascin reflect remodelling of the conjunctiva in trachomatous conjunctivitis.

Relationship between Tear TNF-alpha, TGF-beta1, and EGF levels and severity of conjunctival cicatrization in patients with inactive trachoma.

Tear tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta 1 (TGF-beta1), and epidermal growth factor (EGF) levels were determined in patients with inactive trachoma, and a possible relation between these cytokines and conjunctival cicatrization severity was investigated. Forty-four patients with inactive trachoma who were admitted to the Department of Ophthalmology at the Harran University, Sanliurfa, Turkey, were included in this study. The control group consisted of 20 age- and sex-matched healthy subjects. The levels of cytokines in tears were measured by ELISA. Tear samples were collected from the conjunctival cul-de-sac by means of blunted-tip glass capillary tubes. Eyes with inactive trachoma were classified into three subgroups with respect to conjunctiva cicatrization: mild, moderate, and severe. In 44 patients with inactive trachoma, conjunctival cicatrization was found, including mild (n = 15), moderate (n = 16), and severe (n = 13) cases. In patients with inactive trachoma, decreases in tear EGF (p = 0.000) concentrations and increases in tear TGF-beta1 (p = 0.006) and TNF-alpha (p = 0.046) levels with respect to the control group were found to be concordant with conjunctival cicatrization severity. Statistically significant correlations in tear TNF-alpha (p = 0.018), TGF-beta1 (p = 0.007), and EGF (p = 0.043) levels were found between mild and severe cicatrization groups. TNF-alpha and TGF-beta1 have been implicated in the fibrogenic process. Elevated tear levels of inflammatory/fibrogenic cytokines may play an important role in scar formation in trachoma. It is possible that decreased tear levels of EGF, which may be important for the maintenance of corneal epithelial integrity, are related to fibrosis in the lacrimal gland ductules.

Corneal thickness in trachomatous dry eye.

PURPOSE: To investigate the effect of severe desiccation on corneal thickness in scarring trachoma by comparing the thickness of normal and trachomatous dry eye corneas. METHODS: Ultrasonic pachymetry was used to measure the corneal thickness at nine points in the central and peripheral cornea (superior, superonasal, nasal, inferonasal, inferior, inferotemporal, temporal, superotemporal) in 45 eyes of 27 patients with trachomatous dry eye and 54 eyes of 31 normal subjects. RESULTS: The average thickness of the nine sites in the central and midperipheral cornea was significantly less in trachomatous dry eyes than normal eyes. The superior cornea was the thickest area in both groups, measuring 574.03+/-31.62 microm in trachomatous dry eyes and 611.33+/-34.99 microm in normal eyes (p<0.001). The centre of the cornea was the thinnest, measuring 510.43+/-32.12 microm in trachomatous dry eyes and 546.27+/-36.20 microm in normal eyes (p<0.001). CONCLUSIONS: The thickness of the central and midperipheral cornea was significantly reduced in patients with trachomatous dry eye. The chronic state of severe desiccation, tear film instability and increased immune activation in trachomatous dry eye may contribute to this thinning.

Immunology of trachomatous conjunctivitis.

Trachoma, a chronic follicular conjunctivitis caused by infection with Chlamydia trachomatis, is a leading cause of preventable blindness. The blinding complications are associated with progressive conjunctival scarring. Our immunohistochemical studies of conjunctival biopsies from children with active trachoma demonstrated the presence of both humoral and cell-mediated immune responses. Antichlamydial antibodies can neutralize Chlamydiae, block attachment and internalization of the organism, and can produce partial immunity. Our observations suggest a role for T-lymphocytes and cell mediated immunity in the genesis of conjunctival scarring. Conjunctival epithelial cells expressed major histocompatibility complex (MHC) class II antigens which might allow conjunctival epithelial cells to present Chlamydial antigens to T-cells enhancing the immune response. The epithelial cells expressing MHC class II antigens might present autoantigens to T-cells leading to induction of an autoimmune reaction. We have demonstrated that the conjunctival epithelial cells from patients with trachoma expressed interleukin (IL)-1 alpha and IL-1 beta. In addition, we have detected cytoplasmic expression of IL-1 alpha, IL-1 beta, tumor necrosis factor alpha and platelet-derived growth factor by macrophages. These cytokines have the potential to influence the remodeling and fibrosis observed in trachoma. Alterations of extracellular matrix components and collagen metabolism occur in the conjunctival tissue from patients with trachoma. New collagen type V formation was noted in active trachoma and scarred trachoma. The conjunctival tissue from patients with active trachoma contained increased amounts of collagen types I, III and IV. Scarred trachoma is characterized by marked increase in basement membrane--collagen IV and marked decrease in collagen types I and III. In addition, we demonstrated increased activity of gelatinase B and numbers of inflammatory cells containing gelatinase B in trachoma patients suggesting that this enzyme might be involved in matrix degradation and promotion of conjunctival scarring in trachoma.

Collagen content and types in trachomatous conjunctivitis.

PURPOSE: To study alterations in conjunctival collagen in the conjunctiva of patients with active trachoma. METHODS: We studied conjunctival biopsy specimens obtained from nine subjects with active trachoma and from four control subjects. We used immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies directed against types I, III, IV and V collagen. RESULTS: In normal conjunctiva, the staining for types I and III collagen was localised to the substantia propria. Type IV collagen was located in the epithelial and capillary endothelial basement membranes. The staining for type V collagen was absent. In trachoma biopsy specimens, staining for types I and III collagen showed collagen fibrils among epithelial cells, patchy increase in staining intensity in the upper stroma, and thicker and irregularly arranged collagen fibrils in the substantia propria. Staining for type IV collagen showed irregularly thickened epithelial basement membrane. Staining for type V collagen showed patchy staining in the upper substantia propria; it was also noted in the cytoplasm of fibroblasts, in the walls of blood vessels in the substantia propria, and in the walls of accessory lacrimal glands. CONCLUSIONS: Our data indicate new type V collagen formation, and increased types I, III and IV collagen content, in the conjunctiva from patients with active trachoma.

Pharmacokinetics of azithromycin in trachoma patients: serum and tear levels.

OBJECTIVE: To determine the effectiveness of single-dose oral azithromycin in the treatment of Chlamydia trachomatis through monitoring of tear and serum levels. DESIGN: Nonrandomized, clinical trial. PARTICIPANTS: Fourteen school-age children with active trachoma (one failed to complete the study). INTERVENTION: A single dose of azithromycin (20 mg/kg) was administered orally to 14 patients, and tear and serum levels were determined with high-performance liquid chromatography at 12, 24, 48, 72, 96, 120, and 144 hours after administration. MAIN OUTCOME MEASURES: Azithromycin levels in tears and serum. RESULTS: Peak levels of 1.53 microg/ml (standard deviation [SD] +/- 0.94) and 0.15 microg/ml (SD +/- 0.04) were obtained at 12 hours in both tears and serum, gradually decreasing over 144 hours. All patients were disease-free by 6 months. CONCLUSIONS: Levels of azithromycin in patients with trachoma were found to be within minimum inhibitory concentration range for Chlamydia trachomatis (0.03-0.25 microg/ml) throughout the monitored period of 6 days.

An immunohistochemical study of collagens in trachoma and vernal keratoconjunctivitis.

PURPOSE: To analyse the distribution and types of collagen in the conjunctiva of patients with trachoma and vernal keratoconjunctivitis (VKC). METHODS: Conjunctival biopsy specimens were collected from 9 patients with active trachoma, 9 patients with scarred trachoma, 6 patients with active VKC and 9 control subjects. The presence and distribution of collagen was assessed microscopically with immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies directed against types I, III, IV and V collagen. RESULTS: In normal conjunctiva, the staining for types I and III collagen was localised to the substantia propria. Type IV collagen was located in the epithelial, vascular endothelial and accessory lacrimal gland basement membranes. Staining for type V collagen was absent. New type V collagen deposition close to basement membranes was noted in active trachoma, scarred trachoma and VKC. The extent of deposition of type V collagen was markedly increased in scarred trachoma when compared with active trachoma. Staining for type IV collagen showed irregularly thickened epithelial basement membrane in active trachoma, and a marked increase in basement membrane type IV collagen was noted in scarred trachoma. Immunoreactivity of types I and III collagen increased in active trachoma and decreased in scarred trachoma. VKC conjunctiva contained increased amounts of types I, III and IV collagen due to marked increase in the thickness of vascular endothelial basement membrane and very prominent deposition of types I and III collagen around stromal vessels. CONCLUSIONS: Our data indicate new type V collagen formation in the conjunctiva from patients with active trachoma, scarred trachoma and VKC. Increased deposition of types I, III and IV collagen is noted in VKC and active trachoma. Our findings suggest that increased deposition of type IV collagen and new type V collagen formation contributes to the development of conjunctival fibrosis in scarred trachoma.

Chlamydia trachomatis glycosaminoglycan-dependent and independent attachment to eukaryotic cells.

Chlamydia trachomatis consists of two biovars, lymphogranuloma venereum (LGV) and trachoma, that differ in their infectivity in vivo and in vitro. Although addition of exogenous heparin or heparan sulfate in vitro effectively inhibits infectivity of both biovars and inhibits LGV biovar attachment to host cells, trachoma biovar attachment was only modestly inhibited (approximately 30%) by exogenous heparin. To dissect the relationship of heparin inhibition of attachment and infectivity, a heparan sulfate lyase (heparitinase) was used to treat organisms and evaluated for changes in attachment and infectivity. In contrast to heparitinase-treated LGV biovar organisms that lose their ability to attach and infect, treatment of trachoma biovar organisms with a concentration of heparitinase sufficient to reduce trachoma biovar infectivity by > 90%, only inhibited attachment to host cells by approximately 40%. Significantly, attachment could be fully restored for heparitinase-treated organisms of both biovars with exogenous heparan sulfate; however, the coating of the trachoma biovar organisms with heparan sulfate rendered the trachoma biovar similar to the phenotype of the LGV biovar by > 90% sensitivity to heparin inhibition of attachment. These data suggest that the LGV biovar used predominantly a heparin-inhibitable mechanism for attaching to host cells, whereas the trachoma biovar used a heparin-independent means in addition to a heparin-dependent mechanism to adhere to host cells. Once attached, the trachoma biovar, nevertheless, relied on the heparin-dependent pathway to enter host cells.

Minocycline levels in tears of patients with active trachoma.

We determined minocycline levels in human tears and plasma samples using high-performance liquid chromatography. In the first study, we determined the trough tear and plasma levels of minocycline in patients with active trachoma 24 hours after an oral dose. After a single dose of minocycline, the mean concentration of the drug in tear samples was 189 +/- 58 ng/mL and corresponding plasma levels were 578 +/- 290 ng/mL. In a second study, we monitored the pharmacokinetics of the drug in tear and plasma samples of healthy nonfasting adults. Tear and plasma samples were collected at 1 through 6, 12, 24, 48, 72, and 96 hours. The pharmacokinetics study revealed that 48 hours following a single 200-mg dose of minocycline, the mean tear level of minocycline was 68 ng/mL, which is above the in vitro minimal inhibitory concentration required for Chlamydia trachomatis and other susceptible organisms.

The effect of cyclosporine in chlamydial eye infection.

The effect of cyclosporine (CsA) treatment in both primary and secondary Chlamydia trachomatis ocular infection in cynomolgus monkeys was studied. Following primary infection, CsA-treated animals developed severe clinical symptoms which persisted for up to 12 weeks compared to control animals which developed less severe inflammation lasting only 4 to 6 weeks. CsA treatment had no effect on the clinical course of a second ocular infection. Antigen-specific antibodies developed normally in the serum and tears of all animals, but the titer of IgM in serum and secreted IgA in tears of CsA-treated animals was significantly lower than control animals. Chlamydia-specific cell-mediated immunity, as determined by in vitro lymphoproliferation assays of peripheral blood, did not develop in either group of animals.