Human Organ Culture Research Articles

HIV binding, penetration, and primary infection in human cervicovaginal tissue

We have developed human cervicovaginal organ culture systems to examine the initiating events in HIV transmission after exposure to various sources of HIV infectivity, including semen. Newly infected cells were detected in the cervical submucosa 3-4 days after exposure to a primary HIV isolate. At earlier times, extensive and stable binding occurred when cervical surfaces were exposed to virions or seminal cells. Cervical mucus provided some protection for the endocervical surface, by physically trapping virions and seminal cells. Confocal microscopy combined with 3D surface reconstruction revealed that virions could both bind to the external surface of the cervical epithelium and actually penetrate beneath the epithelial surface. In quantitative assays, pretreatment with a blocking antibody directed against beta1 integrin reduced HIV virion binding. Collectively, these results highlight a continuum of complex interactions that occurs when natural sources of HIV infectivity are deposited onto mucosal surfaces in the female reproductive tract.

Ultrastructural analysis of the pathogenesis of Neisseria gonorrhoeae endometrial infection

We have studied gonococcal infection in human endometrium organ culture and in human primary endometrial epithelial cells using various microscopic techniques including scanning electron microscopy, transmission electron microscopy, bright field light microscopy and laser scanning confocal microscopy. Here we describe the interactions between Neisseria gonorrhoeae and human endometrial luminal epithelial cells at the ultrastructural levels. N. gonorrhoeae attached to cilia but were not observed associated with the plasma membrane of ciliated epithelial cells or internalized into ciliated epithelial cells. N. gonorrhoeae could be found in intracellular vacuoles in secretory epithelial cells. N. gonorrhoeae have diverse interactions with endometrial epithelium. These include intimate association and colocalization with asialoglycoprotein receptor (ASGP-R) and CEACAM, lamellipodia and ruffle formation and colocalization with CR3, and microvillus engagement. These studies indicate that N. gonorrhoeae utilize multiple mechanisms to associate with endometrial epithelial cells and can associate with both ciliated and secretory cells. This diversity is consistent with a role of the endometrium as a transition zone between frequently asymptomatic cervical gonorrhoea and symptomatic pelvic inflammatory disease.

Animal Compound–Free Medium and Poloxamer for Human Corneal Organ Culture and Deswelling

Eliminating fetal calf serum (FCS) from corneal organ culture (OC) media has long been a challenge. This study was an assessment of a new animal compound-free (ACF) medium for corneal storage and of its combination with poloxamer for end-of-storage corneal deswelling. A randomized controlled study with masked assessment compared the ACF medium to standard commercialized media containing 2% FCS and their combination with dextran for deswelling. Paired human corneas were randomly allocated at procurement, one to the ACF medium and the other to the FCS media, and then assessed at day (D)2 and D30 of OC storage and after 48 hours of deswelling. Comparison criteria were endothelial cell density (ECD) and morphometry by a corneal analyser, quality of endothelial visualization (using saline), EC mortality (trypan blue), corneal thickness, corneal transparency, and folding. Fifty-six corneas (28 pairs) with ECD of 2000 cells/mm(2) or more were enrolled. Data were compared using paired tests with P < 0.01 deemed significant. Parameters were similar at baseline (D2) between groups. Daily EC loss during the 30 days of storage was reduced with the ACF compared with standard (-0.31% +/- 0.30% vs. -0.88% +/- 0.38%, P < 0.001). With poloxamer 188 (Lutrol F68; BASF, Ludwigshafen, Germany), EC loss was substantially reduced (-1.43% +/- 3.60 vs. -15.41% +/- 10.13%, P < 0.001) and morphometry better preserved, despite thickness reduction, transparency improvement and folding reduction comparable to dextran. After 30 days of storage in ACF medium and deswelling in poloxamer 188, ECD was 30% higher (2466 +/- 447 cells/mm(2) vs. 1729 +/- 281 cells/mm(2), P < 0.001). ACF medium alone and combined with poloxamer 188 considerably facilitated EC visualization at D30 and after deswelling. The ACF medium combined with poloxamer 188 for deswelling showed superiority over standard FCS medium in its ability to preserve EC viability and facilitate endothelial visualization. This innovative use of poloxamer for deswelling appears far less toxic than does dextran.

Interaction of Enteropathogenic Escherichia coli with Human Intestinal Mucosa: Role of Effector Proteins in Brush Border Remodeling and Formation of Attaching and Effacing Lesions

Enteropathogenic Escherichia coli (EPEC) strains deliver effector proteins Tir, EspB, Map, EspF, EspH, and EspG into host cells to induce brush border remodeling and produce attaching and effacing (A/E) lesions on small intestinal enterocytes. In this study, the role of individual EPEC effectors in brush border remodeling and A/E lesion formation was investigated with an in vitro human small intestinal organ culture model of EPEC infection and specific effector mutants. tir, map, espB, and espH mutants produced "footprint" phenotypes due to close bacterial adhesion but subsequent loss of bacteria; an espB mutant and other type III secretion system mutants induced a "noneffacing footprint" associated with intact brush border microvilli, whereas a tir mutant was able to efface microvilli resulting in an "effacing footprint"; map and espH mutants produced A/E lesions, but loss of bacteria resulted in a "pedestal footprint." An espF mutant produced typical A/E lesions without associated microvillous elongation. An espG mutant was indistinguishable from the wild type. These observations indicate that Tir, Map, EspF, and EspH effectors play a role in brush border remodeling and production of mature A/E lesions.

A new generation organ culture arising from cross‐talk between multiple primary human cell types

The inability to experiment directly on humans strongly constrains biomedical research, creating a great need to develop cultures that mimic human tissues and organs as experimental systems that can be used to directly understand and manipulate biological processes. The advent of availability of primary human cells now makes possible engineering of such organ cultures. Here we report the generation of a human "skin" arguably the simplest human tissue. Beginning with three primary cell types taken from adult tissues, this organ culture develops into a mature tissue containing a stratified epithelium and an interconnected network of mature microvessels, with appropriate matrix molecules and cytokines. Surprisingly, pericytes and monocytes appear adjacent to and within "blood" vessels, respectively. These cultures respond appropriately to stimulators of specific biological processes, providing a vehicle to investigate basic biological processes, such as 1) cell-cell and cell-microenvironment interaction; 2) transdifferentiation of one cell type to another and/or differentiation from stem cells present in adult tissues; and 3) opportunities for genetic manipulation of human tissues to understand function. Moreover, this "skin" can potentially be developed into a tailored "living bandage" for patients with impaired healing and can serve as prototype for the development of other human organ cultures.

A Comparison of Six Statins on the Development of Intimal Hyperplasia in a Human Vein Culture Model

Objective Intimal hyperplasia (IH) threatens the patency of up to 35% of saphenous vein (SV) bypass grafts. In addition to reducing cholesterol levels, statins may modulate smooth muscle cell proliferation and migration. Statins inhibit matrix metalloproteinase (MMP) activity. We therefore investigated the effect of six statins on neointimal formation and MMP activity in human SV organ culture. Study design Human SV specimens were cultured for 14 days in the presence of six different statins and subsequently processed for measurement of neointimal thickness and MMP activity. The drug concentrations chosen corresponded to the manufacturers' Cmax. Results The six statins all significantly reduced IH development ( P=0.004) in association with reduced expression of proMMP-2 and 9 ( P=0.03) and reduced activity of activated MMP-2 and 9 ( P=0.03). Conclusion This study suggests that the potential benefit of statins in reducing IH is a class effect and not confined to specific statins. The reduction of IH produced by statins may in part be due to their inhibition of MMPs.

Rapamycin attenuates vascular wall inflammation and progenitor cell promoters after angioplasty

Rapamycin combines antiproliferative and antiinflammatory properties and reduces neointima formation after angioplasty in patients. Its effect on transcriptional programs governing neointima formation has not yet been investigated. Here, we systematically analyzed the effect of rapamycin on gene expression during neointima formation in a human organ culture model. After angioplasty, renal artery segments were cultured for 21 or 56 days in absence or presence of 100 ng/ml rapamycin. Gene expression analysis of 2312 genes revealed 264 regulated genes with a peak alteration after 21 days. Many of those were associated with recruitment of blood cells and inflammatory reactions of the vessel wall. Likewise, chemokines and cytokines such as M-CSF, IL-1beta, IL-8, beta-thromboglobulin, and EMAP-II were found up-regulated in response to vessel injury. Markers indicative for a facilitated recruitment and stimulation of hematopoetic progenitor cells (HPC), including BST-1 and SDF-1, were also induced. In this setting, rapamycin suppressed the coordinated proadhesive and proinflammatory gene expression pattern next to down-regulation of genes related to metabolism, proliferation, and apoptosis. Our study shows that mechanical injury leads to induction of a proinflammatory, proadhesive gene expression pattern in the vessel wall even in absence of leukocytes. These molecular events could provide a basis for the recruitment of leukocytes and HPC. By inhibiting the expression of such genes, rapamycin may lead to a reduced recruitment of leukocytes and HPC after vascular injury, an effect that may play a decisive role for its effectiveness in reducing restenosis.

Adenovirus type 7 induces interleukin-8 in a lung slice model and requires activation of Erk.

Adenovirus (Ad), particularly Ad type 7 (Ad7), causes severe lung infection and pneumonia. Initially, Ad causes neutrophilic inflammation of the distal airways and alveoli. Interleukin-8 (IL-8) is the major lung neutrophil chemotaxin, and we have shown that Ad7 induces IL-8 release from the A549 alveolar epithelial cell line. We sought to determine whether ex vivo human and bovine lung tissue containing primary pneumocytes could be used as a more accurate and relevant model to study Ad acute inflammation. We found that cultured lung tissue preserved normal lung architecture for more than 10 days. IL-8 was generated upon exposure of the lung organ culture to Ad7. IL-8 production required activation of the Ras/Erk pathway, since a pharmacological inhibitor blocked the appearance of IL-8 in the medium. Both human and bovine lung explants supported replication of Ad7, and immunohistochemistry experiments demonstrated the presence of the Ad hexon antigen within alveolar epithelial cells. These findings show that our novel human lung organ culture accurately reproduces the in vivo infectious disease process. Thus, this organ culture model represents a valuable tool for studying the acute innate immune response to respiratory infections.

HIV-1 replication and pathogenesis in the human thymus.

How HIV replicates and causes destruction of the thymus, and how to restore thymic function, are among the most important questions of HIV-1 pathogenesis and therapy in adult as well as pediatric patients. The thymus appears to function, albeit at reduced levels, throughout the life of adults, to respond to T cell depletion induced by HIV and to be suppressed by HIV. In this review, we summarize recent findings concerning HIV replication and pathogenesis in the human thymus, focusing on mechanistic insights gleaned from studies in the SCID-hu Thy/Liv mouse and human fetal-thymus organ culture (HF-TOC) models. First, we discuss HIV viral determinants and host factors involved in the replication of HIV in the thymus. Second, we consider evidence that both viral factors and host factors contribute to HIV-induced thymocyte depletion. We thus propose that multiple mechanisms, including depletion and suppression of progenitor cells, paracrine and direct lytic depletion of thymocytes, and altered thymocyte selection are involved in HIV-induced pathology in the thymus. With the SCID-hu Thy/Liv mouse and HF-TOC models, it will be important in the coming years to further clarify the virological, cell biological, and immunological mechanisms of HIV replication and pathogenesis in human thymus, and to correlate their significance in HIV disease progression.

Blocking of cell-free and cell-associated HIV-1 transmission through human cervix organ culture with UC781

To determine the efficacy of UC781 in preventing HIV-1 transmission through cervical tissue. Use of human cervical tissue organ culture, in which the cervix is in the upper chamber of a transwell and transmission of infective virus is quantified in the lower chamber. Five-millimeter pieces of cervical tissues are exposed to UC781. After thorough removal of the drug, the tissues are exposed to high doses of cell-free or cell-associated HIV-1. Transmission of HIV-1 through the cervix is measured by determining infection of target cells in the lower chamber. Exposure of cervix to 0.5 microM UC781 for 30 min, followed by extensive washing away of the residual drug, resulted in 95% reduction of subsequent viral transmission. Exposure of the cervix to 1 microM UC781 for 20 min, or 10 microM UC781 for 2 min, resulted in neutralization of T- and M-tropic HIV-1 isolates of various clades, and prevention of cell-associated HIV-1 transmission. Moreover, a 20 min incubation with 10 microM UC781 abolished HIV-1 transmission through the cervix for 48 h after drug pretreatment. Importantly, UC781 was not toxic, even when the cervical tissues were exposed to 20 microM UC781 for 24 h. UC781 was effective against transmission of both cell-free and cell-associated HIV-1 also when formulated into a non-spermicidal (Replen) or spermicidal (BufferGel) gel. Exposure of the cervix to UC781 results in blocking of subsequent HIV-1 transmission with no toxicity. Therefore, UC781 is an excellent candidate microbiocide.

Expression of optineurin, a glaucoma-linked gene, is influenced by elevated intraocular pressure

Optineurin (optic neuropathy inducing protein; OPTN) was recently linked to 16.7% of families with primary open-angle glaucoma. The function of OPTN in the eye is not known, but is present in the trabecular meshwork, which is responsible for maintenance of intraocular pressure (IOP). To gain insight into the role of OPTN in the development of glaucoma we studied its expression in response to factors known to be associated with the disease: elevated IOP, tumor necrosis factor-α (TNFα), and dexamethasone (DEX). We performed the treatments in human organ cultures under conditions mimicking physiological pressure. We find OPTN significantly upregulated after 2, 4, and 7 days of sustained elevated IOP. OPTN expression is also induced 2.3-fold by TNFα and 2.6-fold by prolonged DEX treatment. These results demonstrate that OPTN is part of the transcriptome responding to glaucomatous insults and support the protective role of this protein in the trabecular meshwork.

Human internal mammary artery organ culture model of coronary stenting: a novel investigation of smooth muscle cell response to drug-eluting stents.

Local drug delivery by coronary stents is of current research interest. Organ culture of human vascular tissue is a model of intimal hyperplasia. We report an ex vivo organ culture model of stented vessels. This allows stent-artery interactions to be studied in living tissue. The recognized anti-restenosis agent paclitaxel was chosen to test the organ culture model. Mammary artery specimens were cultured 'closed' (i.e. without opening them flat) for 72 h. Phosphocholine-coated stents, half of them loaded with the anti-restenosis drug paclitaxel, were implanted. The absorption and elution characteristics of paclitaxel were established. Artery tissue remained viable at 72 h when cultured closed, despite stent implantation. Specimens developed smooth muscle cell proliferation. The stents absorbed up to 127+/-29 microg of paclitaxel, with a biphasic elution curve. A mean of 13% of the absorbed paclitaxel remained after a 24 h perfusion. In mammary artery, these paclitaxel stents reduced or abolished smooth muscle cell proliferation compared with controls. This model allows the effects of stenting on human arterial tissue to be studied for at least 72 h, long enough to demonstrate effects on smooth muscle cell proliferation. Phosphocholine-coated stents absorb adequate doses of paclitaxel, which is eluted gradually, inhibiting muscle cell proliferation. Such an organ culture model of stented mammary artery will provide useful data in addition to that from animal or cell culture models of drug-eluting stents.

Simvastatin inhibits human saphenous vein neointima formation via inhibition of smooth muscle cell proliferation and migration

Objective: Migration and proliferation of vascular smooth muscle cells (SMCs) contributes to intimal hyperplasia in saphenous vein (SV) bypass grafts, which leads to patency-threatening stenosis. Evidence for the involvement of basement membrane-degrading matrix metalloproteinases (MMPs) and growth factors in mediating SMC migration and proliferation has been presented in a number of in vitro and in vivo models. 3-Hydroxy-3 methylglutaryl CoA reductase inhibitors (statins) are widely used in patients with atherosclerosis and are claimed to have additional effects beyond cholesterol reduction. We therefore examined the effects of simvastatin, a commonly prescribed statin, on the proliferation and migration of cultured human SV SMC and on neointima formation and MMP activity in human SV organ cultures. To clarify its mode of action, we studied in parallel the effects of a specific MMP inhibitor, marimastat. Study Design: Human SV specimens were obtained from patients who underwent coronary artery bypass grafting, and were cultured for 14 days in the presence of three concentrations of simvastatin and subsequently processed for measurement of MMP activity and neointimal thickness measurements. Cultured SV SMCs were used to construct growth curves in the presence of 10% fetal calf serum or 10% fetal calf serum supplemented with simvastatin or marimastat. Migration through a Matrigel basement-membrane matrix (invasion) was quantified with modified Boyden chambers. Results: Simvastatin dose dependently reduced neointima formation ( P = .004) in association with reduced MMP-9 activity ( P = .03). SMC proliferation and invasion also were inhibited with simvastatin ( P < .007 and P < .009, respectively). Marimastat dose dependently inhibited SMC invasion ( P < .001) but importantly had no effect on SMC proliferation ( P > .36). Conclusion: For effective control of neointimal development in vivo, a pharmacologic strategy should inhibit both SMC migration and proliferation. The ancillary properties of 3-Hydroxy-3 methylglutaryl CoA reductase inhibitors typified by simvastatin may be important in this regard. (J Vasc Surg 2002;36:150-7.)

The Polymorphism at Codon 54 of the FABP2 Gene Increases Fat Absorption in Human Intestinal Explants

Based on titration microcalorimetry and Caco-2 cell line transfection studies, it has been suggested that the A54T of the FABP2 gene plays a significant role in the assimilation of dietary fatty acids. However, reports were divergent with regard to the in vivo interaction between this polymorphism and postprandial lipemia. We therefore determined the influence of this intestinal fatty acid-binding protein polymorphism on intestinal fat transport using the human jejunal organ culture model, thus avoiding the interference of various circulating factors capable of metabolizing in vivo postprandial lipids. Analysis of DNA samples from 32 fetal intestines revealed 22 homozygotes for the wild-type Ala-54/Ala-54 genotype (0.83) and 10 heterozygotes for the polymorphic Thr-54/Ala-54 genotype (0.17). The Thr-encoding allele was associated with increased secretion of newly esterified triglycerides, augmented de novo apolipoprotein B synthesis, and elevated chylomicron output. On the other hand, no alterations were found in very low density lipoprotein and high density lipoprotein production, apolipoprotein A-I biogenesis, or microsomal triglyceride transfer protein mass and activity. Similarly, the alanine to threonine substitution at residue 54 did not result in changes in brush border hydrolytic activities (sucrase, glucoamylase, lactase, and alkaline phosphatase) or in glucose uptake or oxidation. Our data clearly document that the A54T polymorphism of FABP2 specifically influences small intestinal lipid absorption without modifying glucose uptake or metabolism. It is proposed that, in the absence of confounding factors such as environmental and genetic variables, the FABP2 polymorphism has an important effect on postprandial lipids in vivo, potentially influencing plasma levels of lipids and atherogenesis.

Human odontoblast culture method: the expression of collagen and matrix metalloproteinases (MMPs).

Studies on mature human odontoblasts have suffered for the lack of in vitro models. We recently introduced a human odontoblast and pulp tissue organ culture method, in which the odontoblasts are cultured in the pulp chamber after removal of the pulp tissue, and the pulp tissue can be cultured separately (Tjäderhane et al., 1998a). With this method, we have studied the effects of growth factors on the expression of collagen and extracellular matrix (ECM)-degrading enzymes, matrix metalloproteinases (MMPs), in mature human odontoblasts. TGF-beta 1 was selected because of its ability to regulate the response of the dentin-pulp complex to external irritation. The effect of TGF-beta 1 (10 ng/mL) on pro alpha 1(I) collagen mRNA was analyzed by quantitative PCR, and type I procollagen propeptide (PINP) was analyzed from conditioned culture media with RIA. Odontoblast media were also assayed for respective type III procollagen propeptide (PIIINP). TGF-beta had a negligible effect on collagen mRNA expression or protein synthesis, indicating that TGF-beta alone does not markedly induce dentin matrix formation per se in the human dentin-pulp complex (Palosaari et al., 2001). However, TGF-beta 1 seems to regulate MMP expression in mature human odontoblasts differentially. A strong down-regulation of MMP-8 (Palosaari et al., 2000), a modest down-regulation of MMP-20 (Tjäderhane et al., 2000), and considerable up-regulation of MMP-9, with no apparent effect on MMP-2 expression (Tjäderhane et al., 1998b), indicate that growth factors may affect the matrix synthesis by controlling the expression and activity of MMPs instead of collagen synthesis. The altered expression of MMPs may result in altered ECM formation, which in turn may contribute to the formation of atubular reparative dentin.

Apoptosis and autoimmune thyroid disease: following a TRAIL to thyroid destruction?

In the past decade, it became apparent that immune mediated cell death in a number of autoimmune endocrine diseases was due to the induction of apoptosis in target organ cells. This was conclusively demonstrated for thyroid follicular cells in Hashimoto’s (destructive autoimmune) thyroiditis, but the mechanisms underlying this cell death were not clear. Several hypotheses were put forth involving the role of deathsignalling molecules expressed on thyroid cells. While many of these hypotheses did not hold up under close scrutiny, this stimulated work on the molecular mechanisms of thyroid destruction. Several apoptosis signalling pathways, initiated by molecules such as Fas ligand (FASL) and tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been shown to be active in thyroid cells and may be involved in destructive thyroiditis. In this review we will attempt to sort out the inconsistencies in published data on the mechanisms of death-receptor mediated thyroid destruction. We will also review recently proposed models of these mechanisms, and outline directions for research that we feel might lead to discoveries of benefit to the clinician in the treatment and prevention of destructive autoimmune thyroiditis.

Impaired replication of protease inhibitor-resistant HIV-1 in human thymus.

Many HIV-1-infected patients treated with protease inhibitors (PI) develop PI-resistant HIV-1 variants and rebounds in viremia, but their CD4+ T-cell counts often do not fall. We hypothesized that in these patients, T-cell counts remain elevated because PI-resistant virus spares intrathymic T-cell production. To test this, we studied recombinant HIV-1 clones containing wild-type or PI-resistant protease domains, as well as uncloned isolates from patients, in activated peripheral blood mononuclear cells, human thymic organ cultures and human thymus implants in SCID-hu Thy/Liv mice. In most cases, wild-type and PI-resistant HIV-1 isolates replicated to similar degrees in peripheral blood mononuclear cells. However, the replication of PI-resistant but not wild-type HIV-1 isolates was highly impaired in thymocytes. In addition, patients who had PI-resistant HIV-1 had abundant thymus tissue as assessed by computed tomography. We propose that the inability of PI-resistant HIV-1 to replicate efficiently in thymus contributes to the preservation of CD4+ T-cell counts in patients showing virologic rebound on PI therapy.

Detection of superficial zone protein in human and animal body fluids by cross-species monoclonal antibodies specific to superficial zone protein.

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.

Latanoprost and matrix metalloproteinase-1 in human choroid organ cultures

Purpose. Latanoprost, a prostaglandin F 2a analogue and ocular hypotensive agent, alters extracellular matrix and matrix metalloproteinases (MMPs), including MMP-1, within tissues of the uveoscleral outflow pathway. In addition to these tissues, the anterior choroid also is exposed to fluid within the uveoscleral outflow pathway. The present study was undertaken to compare MMP-1 expression in the choroid with other uveoscleral pathway tissues and to determine the effect of latanoprost on MMP-1 expression in human choroid organ cultures. Methods. Choroid tissue explant cultures were generated and incubated with PhXA85, the biologically active form of latanoprost, or vehicle for 12 or 18 hours. Explant cultures from iris and ciliary body also were generated and exposed to PhXA85. Protein extracts of theses cultures, as well as from fresh tissues, were then probed for MMP-1 by Western blotting. All samples were normalized for protein content and reletive expression was evaluated by densitometry. Also, total RNA was harvested from fresh tissues or from cultures treated with PhXA85. The amount of MMP-1 mRNA in these samples was measured using real time polymerase chain reaction. These results were normalized according to simultaneous measurements of glyceraldehyde-3-phosphate dehydrogenase mRNA within the same samples. Results. Compared to the ciliary body, in which specific MMP-1 concentration (/total mg protein) was greatest, the specific MMP-1 concentrations within iris, anterior choroid, and posterior choroid were less by 24.7 ± 0.69%, 40.7 ± 1.04%, and 64.5 ± 0.52%, respectively (mean ± SD). The order of MMP-1 mRNA expression among these tissues from fresh eyes was ciliary body > anterior choroid > posterior choroid > iris. Treatment of ciliary body explant cultures with 200 nM PhXA85 for 12 hours increased MMP-1 protein content by 154 ± 34% (P = 0.004, students t test, N = 3). In contrast, similar treatment of anterior choroid, posterior choroid, or iris explant cultures minimally changed MMP-1 protein content (23 ± 22±, P = 0.124; 14 ± 8%, P = 0.462; 27 ± 16%, P = 0.037, respectively). Increased MMP-1 was observed in choroid cultures incubated for 18 hrs with 200 nM or 500 nM PhXA85. MMP-1 mRNA in 12 hour PhXA85-treated choroid cultures was the same or less than vehicle-treated cultures from 7 of 9 donors. In contrast, MMP-1 mRNA was increased in PhXA85-treated ciliary body cultures from 2 of 2 donors. Conclusions. These results suggest that the capacity for latanoprost-mediated induction of MMP-1 within the choroid is less than within the ciliary muscle. Hence, it is unlikely that induction of MMP-1 in choroid plays as important a role in uveoscleral outflow modulation as induction of MMP-1 in the ciliary muscle.

Thymocyte differentiation from lentivirus-marked CD34 + cells in infant and adult human thymus

Changes in thymic function and immune system homeostasis associated with HIV infection or chemotherapy have significant effects on the ability of patients to maintain a complete T cell receptor repertoire. Therefore, the development of in vitro systems to evaluate thymic function in children and adults may aid in the understanding of thymopoiesis and the development of new therapies to improve thymic output. Here we use a lentivirus-based gene transfer system to mark CD34 + cells with EGFP and follow their differentiation into CD4 + and CD8 + single positive thymocytes in human thymic organ cultures. Lentivirus-marked cells entered the thymus and were detected in both the cortex and medulla. Pretreatment of the thymus with 2-deoxyguanosine depleted resident thymocytes and significantly increased the percentage of EGFP + thymocytes. High frequency gene transfer into CD34 + cells and maintained expression throughout differentiation allows for the in vitro assessment of thymic function. In thymuses ranging in age from fetal to adult we observed EGFP + thymocytes at all stages of development suggesting that thymuses of all ages are capable of accepting new T cell progenitors and contributing to the maintenance of T cell homeostasis.