Effect of the cathepsin K inhibitor odanacatib administered once weekly on bone mineral density in Japanese patients with osteoporosis--a double-blind, randomized, dose-finding study.

UNLABELLED: The efficacy and safety of oral placebo or odanacatib 10, 25, or 50 mg once weekly for 52 weeks were evaluated in a double-blind, randomized, multi-center study in Japanese female and male patients with osteoporosis. INTRODUCTION: Odanacatib is a selective and reversible cathepsin K inhibitor that decreases bone resorption and increases bone mineral density (BMD). METHODS: The primary efficacy endpoint was percent change from baseline to week 52 in lumbar spine BMD. Secondary endpoints included percent change in total hip, femoral neck, and trochanter BMD and in bone biomarkers after treatment for 52 weeks. RESULTS: In this study, 286 patients [94% female, mean age (SD) 68.2 (7.1) years] were included in the analysis. The least-squares mean percent changes from baseline to week 52 in the groups receiving placebo, 10, 25 and 50 mg of odanacatib for lumbar spine (L1~L4) BMD were 0.5, 4.1, 5.7, and 5.9% and for total hip BMD were -0.4, 1.3, 1.8, and 2.7%, respectively. The changes in femoral neck and trochanter BMD were similar to those at the total hip. Bone turnover markers were reduced in a dose-dependent manner. However, the effects of odanacatib on bone formation markers were less compared with the effects on bone resorption markers. Tolerability and safety profiles were similar among all treatment groups with no dose-related trends in any adverse events. CONCLUSIONS: Weekly odanacatib treatment for 52 weeks increased BMD at the lumbar spine and at all hip sites in a dose-dependent manner and was well tolerated in Japanese patients with osteoporosis.

Late-onset alopecia areata: a retrospective study of 73 patients from Taiwan.

BACKGROUND: Alopecia areata (AA) is regarded to be mediated by autoimmune process, and manifests as patchy non-scarring hair loss with occult onset. Little is known about AA occurring later in life. OBJECTIVE: To define the characteristics of late-onset AA. METHODS: Patients with first onset of AA at age 50 years and above were retrospectively recruited from two separate institutes in southern and northern Taiwan. The onset age, patterns, severity, past history, serological findings and therapeutic responses were reviewed. RESULTS: Seventy-three AA patients were enrolled, including 49 females (67%) and 24 males (33%). The onset age ranged from 50-78 years with the median age of 57 years. Multifocal lesions (41%) constituted the most common pattern and 55% of the recruited patients had a hair loss of less than 10%. Seventeen patients (23%) had co-existent dermatological or systemic diseases while six patients (8%) had a history of malignancy. Among 27 patients (37%) with available laboratory data, positive anti-nuclear antibody, anti-microsomal antibody and anti-thyroglobulin antibody was demonstrated in 26%, 40% and 30% of them, respectively. Association with personal or family history of atopy was absent. In 15 patients of follow-up longer than 6 months, a complete hair regrowth was found in three patients with mild disease severity. CONCLUSION: Late-onset AA is characterized by marked female predominance and milder disease activity with increasing age. The link to cancer in the old age remains to be determined. The influence of aging on the pathogenesis and prognosis of AA deserves further studies.

Guidelines for management of androgenetic alopecia based on BASP classification--the Asian Consensus Committee guideline.

BACKGROUND: Androgenetic alopecia (AGA), or pattern hair loss, is a common disorder in both Asian men and women. There are several guidelines for the treatment of AGA which are suitable for Caucasian patients; however, each of these has some limitations. Furthermore, in comparison with Caucasian patients, Asian patients with AGA have different types of hair loss and family histories which may alter the treatment response. There is currently no published AGA guideline for Asian patients. OBJECTIVES: The Asian Consensus Committee for Androgenetic Alopecia aimed to develop an algorithmic guideline, based on the basic and specific (BASP) classification, for the treatment of AGA especially in Asian patients. METHODS: The committee collaborated extensively on reviewing available literature on AGA treatment in order to formulate an algorithmic guideline on AGA management. RESULTS: Previously published guidelines based on pre-existing classifications of AGA cannot easily classify the patterns of AGA that are more frequently seen in Asians. The BASP classification not only facilitates the development of a unified and simplified algorithm, but also overcomes the disadvantages of previously reported classification systems. CONCLUSIONS: The proposed treatment guideline for AGA based on the BASP classification may be useful for dermatologists in their approach to treating Asian patients with AGA in clinical practice. Ideally, clinicians should try to utilize this guideline consistently in their practice to monitor treatment response with the goal of enhancing successful outcomes. This will help boost patients' confidence and self-esteem, thus improving patient' compliance with the prescribed treatments.

Research trends of Janus Kinase inhibitors: a bibliometric and visualized study from 2012 to 2023.

OBJECTIVE: Janus Kinase (JAK) inhibitors have been extensively evaluated for their potential in the management of various diseases. Despite previous research on this topic, there is a lack of bibliometric analysis that summarizes research trends on JAK inhibitors. This study aims to provide a comprehensive overview of the top 100 most frequently cited studies on JAK inhibitors over the last ten years. MATERIALS AND METHODS: The Web of Science database was used to screen and extract relevant studies on JAK inhibitors. The top 100 studies most cited within the JAK inhibitor-related research were identified and evaluated, and various data such as the year of publication, study focus and keywords, author information, and number of citations were extracted and analyzed for further examination. RESULTS: In the top 100 most cited studies of JAK inhibitors, more than 70% of studies focused on the role of JAK inhibitors in disease treatments, with 42% of these studies focused on using JAK inhibitors as treatment for autoimmune diseases and 19 of them focused on the treatment of neoplasms. Time trend analysis revealed that the keywords "tofacitinib", "atopic dermatitis", and "rheumatoid arthritis" were widely mentioned in 2016, while new trends emerged in 2018, with "ruxolitinib" and "baricitinib" being more commonly mentioned. CONCLUSIONS: The top 100 most frequently cited studies on JAK inhibitors focused primarily on the safety and efficacy of these inhibitors in the management of various diseases, particularly inflammatory diseases and neoplasms. The results can serve as a valuable reference for rheumatologists and immunologists interested in the development of JAK inhibitors and expanding future research fields.

Using brachial-ankle pulse wave velocity to associate arterial stiffness with cardiovascular risks.

BACKGROUND AND AIMS: This study aimed to elucidate the relationship between brachial-ankle pulse wave velocity (baPWV) and conventional cardiovascular risk factors. METHODS AND RESULTS: A total of 192 subjects with low to intermediate risk was enrolled in a cardiovascular evaluation program. A multiple regression model was built to find significant cardiovascular biomarkers for predicting baPWV. A logistic regression model was developed to associate baPWV and other biomarkers with the risk of cardiac diastolic dysfunction. A total of 123 men (mean age: 52.6+/-12.0) and 69 women (mean age: 51.7+/-10.4) was included. Age, blood pressure, C-reactive protein, serum homocysteine, heart rate, and blood urea nitrogen were positively predictive of increased pulse wave velocity. In turn, baPWV increased the risk (odds ratio: 1.257 for each m/s, 95% CI: 1.105 approximately 1.430, p<0.001) and high-density lipoprotein decreased the risk for cardiac diastolic dysfunction (0.962 for each mg/dl, 95% CI: 0.925 approximately 1.000, p=0.05). The correlation between baPWV and Framingham 10-year risk was moderate (men: r=0.306, p=0.002; women r=0.548, p<0.001). CONCLUSION: The results suggest that baPWV is a composite risk factor for early atherosclerotic change and a predictor for the development of diastolic dysfunction and long-term cardiovascular risk.

A randomized controlled trial of the efficacy and safety of a fixed triple combination (fluocinolone acetonide 0.01%, hydroquinone 4%, tretinoin 0.05%) compared with hydroquinone 4% cream in Asian patients with moderate to severe melasma.

BACKGROUND: Melasma is an acquired, chronic hypermelanosis for which therapy remains a challenge. OBJECTIVES: To compare the efficacy and safety of a triple combination [TC: fluocinolone acetonide 0.01%, hydroquinone (HQ) 4%, tretinoin 0.05%] vs. HQ 4% after 8 weeks of treatment of moderate to severe facial melasma in Asian patients. METHODS: This was a multicentre, randomized, controlled, investigator-blinded, parallel comparison study. East and South-East Asian patients aged 18 years or older, with a clinical diagnosis of moderate to severe melasma, were enrolled in this study. Patients were enrolled at baseline and treated daily for 8 weeks with TC cream (one application at bedtime) or HQ cream (twice daily). There were four study visits: at baseline and weeks 2, 4 and 8. The primary efficacy variable was the melasma global severity score (GSS). Other outcome measures included Melasma Area and Severity Index, global improvement and patient satisfaction. Safety was assessed through the reporting of adverse events. RESULTS: TC had superior efficacy to HQ for the primary variable: 77/120 patients (64.2%) on TC had GSS 'none' or 'mild' at week 8 vs. 48/122 patients (39.4%) on HQ (P < 0.001). The secondary efficacy variables confirmed these results. Patient satisfaction was in favour of TC (90/127, 70.8%, vs. 64/129, 49.6%; P = 0.005). More patients had related adverse events on TC (63/129, 48.8%) than on HQ (18/131, 13.7%) but most were mild and none was severe. CONCLUSIONS: Efficacy in Asians and patient satisfaction were superior with the fixed TC than with HQ 4%.

Cys shotgun labeling of macrophages adhering to and engulfing Ig-opsonized cells.

A method is being developed to study cytoskeletal reorganization in cell adhesion processes. The initial model process is adhesion and phagocytosis of beads or red blood cells by macrophages. Live cell labeling with Cys reactive fluorophores is performed before and during phagocytosis with different color labeling dyes. Since Cys is a relatively hydrophobic amino acid, its differential exposure and labeling in principle reflects changes in tertiary or quaternary structure of specific proteins. Similar studies conducted on red blood cells under fluid shear conditions showed that specific domains in spectrin undergo extensible unfolding within sheared cells. The initial work here with macrophages also suggests some structural changes in phagocytosis although the proteins and specific sites have yet to be identified.

Identification of DNA recognition sequences and protein interaction domains of the multiple-Zn-finger protein Roaz.

Roaz, a rat C2H2 zinc finger protein, plays a role in the regulation of olfactory neuronal differentiation through its interaction with the Olf-1/EBF transcription factor family. An additional role for the Roaz/Olf-1/EBF heterodimeric protein is suggested by its ability to regulate gene activation at a distinct promoter lacking Olf-1/EBF-binding sites. Using an in vitro binding-site selection assay (Selex), we demonstrate that Roaz protein binds to novel inverted perfect or imperfect repeats of GCACCC separated by 2 bp. We show that Roaz is capable of binding to a canonical consensus recognition sequence with high affinity (Kd = 3 nM). Analysis of the structural requirement for protein dimerization and DNA binding by Roaz reveals the role of specific zinc finger motifs in the Roaz protein for homodimerization and heterodimerization with the Olf-1/EBF transcription factor. The DNA-binding domain of Roaz is mapped to the N-terminal 277 amino acids, containing the first seven zinc finger motifs, which confers weak monomeric binding to a single half site and a stronger dimeric binding to the inverted repeat in a binding-site-dependent manner. Full-length protein can form dimers on both the inverted repeat and direct repeat but not on a single half site. These findings support the role of the TFIIIA-type Zn fingers in both protein-protein interaction and protein-DNA interaction and suggest distinct functions for specific motifs in proteins with a large number of zinc finger structures.

Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A.

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. Potential use of BoNT as a biothreat agent has made development of sensitive assays for toxin detection and potent antitoxin for treatment of intoxication a high priority. To improve detection and treatment of botulism, molecular evolution and yeast display were used to increase the affinity of two neutralizing single chain Fv (scFv) antibodies binding BoNT serotype A (BoNT/A). Selection of yeast displayed scFv libraries was performed using methods to select for both increased association rate constant (k(on)) and decreased dissociation rate constants (k(off)). A single cycle of error prone mutagenesis increased the affinity of the 3D12 scFv 45-fold from a K(D) of 9.43x10(-10)M to a K(D) of 2.1x10(-11)M. Affinity of the HuC25 scFv was increased 37-fold from 8.44x10(-10)M to 2.26x10(-11)M using libraries constructed by both random and site directed mutagenesis. scFv variable region genes were used to construct IgG for use in detection assays and in vivo neutralization studies. While IgG had the same relative increases in affinity as scFv, (35-fold and 81-fold, respectively, for 3D12 and HuC25) higher solution equilibrium binding constants were observed for the IgG, with the 3D12 K(D) increasing from 6.07x10(-11)M to 1.71x10(-12)M and the HuC25 K(D) increasing from 4.51x10(-11)M to 5.54x10(-13)M. Affinity increased due to both an increase in k(on), as well as slowing of k(off). Higher affinity antibodies had increased sensitivity, allowing detection of BoNT/A at concentrations as low as 1x10(-13)M. The antibodies will also allow testing of the role of affinity in in vivo toxin neutralization and could lead to the generation of more potent antitoxin.

The 'metabolon,' CD47, and the 'phagocytic synapse': molecular co-localization and species divergence.

CD47 is a widely expressed integral membrane protein, found also on red blood cells where it reportedly has a key role in inhibiting phagocytic clearance of RBC by signaling within a multi-molecular 'phagocytic synapse'. Calreticulin is postulated to be on the RBC surface and stimulate phagocytosis, whereupon CD47 on the RBC binds SIRPalpha on the phagocyte and signals a block against phagocytosis. While studies of mouse suggest such an inhibitory role for CD47, CD47 seems to have distinct interactions in human RBC--particularly within a 'metabolon' complex of CD47, Rh proteins, and several other proteins. We have assessed the relative density, co-clustering, and mobility of some of the implicated proteins on human RBC versus murine RBC (hu-RBC and mu-RBC, respectively), and we find a few major differences. While RBC from both species express similar densities of CD47 and SIRPalpha interactions are measurably modest, the interactions prove species-specific. While RBC from both species also have detectable calreticulin, fresh hu-RBC are found to have 10-100-fold more calreticulin binding sites on their surface. Imaging of clusters of SIRPalpha-CD47 on both species of RBC show that RhD does co-localize with CD47 on hu-RBC, but neither calreticulin nor Glycophorin-A appear enriched in the metabolon complexes. Furthermore, mouse-cells alone tend to aggregate due to cross-bridging by SIRPalpha complexes, showing accumulation of CD47 in the adhesion zone, which is consistent with a high mobility of CD47 unique to mu-RBC.

Cell contact regulates fate choice by cortical stem cells.

Cell fate is determined by intrinsic programs and external cues, such as soluble signals and cell-cell contact. Previous studies have demonstrated the roles of soluble factors in the proliferation and differentiation of cortical stem cells and cell-cell contact in maintaining stem cells in a proliferative state. In the present study, we focused on the effect of cell-cell interaction on cell-fate determination. We found that density could exert a strong influence on the cell-type composition when cortical stem cells differentiate. Multipotent stem cells, which normally gave rise to neurons, astrocytes, and oligodendrocytes under high-density culture condition, differentiated almost exclusively into smooth muscle at low density. Clonal analysis indicated that smooth muscle and astrocytes were derived from a common precursor and that the density effect on cell types used an instructive mechanism on the choice of fate rather than an effect of selective survival and/or proliferation. This instructive mechanism depended on the local and not the average density of the cells. This local signal could be mimicked by membrane extract. These findings demonstrate the importance of membrane-bound signals in specifying lineage and provide the first evidence for a short-range regulatory mechanism in cortical stem cell differentiation.

Nitric oxide synthesis inhibition retards surgical reversal of one-kidney Goldblatt hypertension in rats.

Surgical correction of renal artery stenosis in Goldblatt hypertension rapidly normalizes blood pressure and increases renal function. This study was conducted in 1-kidney, 1 clip (1K1C) Goldblatt hypertensive rats to examine whether the unclipping-induced reversal of blood pressure and renal function is mediated by nitric oxide (NO). The 1K1C rats were prepared and given tap water with or without supplementation of NG-nitro-L-arginine methyl ester (L-NAME). Systolic blood pressure (SBP) before and after renal artery clipping was measured with the tail-cuff method. Four weeks later, surgical unclipping was performed while blood pressure and renal function responses were determined. The results show that clipping the renal artery for 4 weeks increased SBP from 140+/-5 to 183+/-6 mm Hg (P<0.05). Concurrent L-NAME treatment accelerated and aggravated the clipping-induced increases in SBP from 138+/-6 to 219+/-8 mm Hg (P<0.05). Surgical unclipping reduced blood pressure to normotensive levels within 2 hours in all hypertensive rats with and without chronic or acute L-NAME treatment. However, the magnitude of reductions in blood pressure in the initial 1 hour after unclipping was significantly less in L-NAME-treated rats than in nontreated rats (9+/-2% versus 16+/-1%, P<0.05). Despite reducing blood pressure, unclipping significantly increased glomerular filtration rate, urine flow, and sodium and potassium excretions, but the extent of the increases in these renal functions was significantly attenuated in L-NAME-treated rats. These data suggest that NO production partly contributes to the hypotensive and renal responses to unclipping but does not mediate the reversal of renovascular hypertension of this model.

Dietary model for production of experimental zinc deficiency in man.

A semipurified diet based on soy protein was developed to induce mild zinc deficiency in five male volunteers. Each of seven daily menus provided (mean +/- SD) 2248 +/- 128 kcal, 56.6 +/- 5.7 protein, 261 +/- 30 g carbohydrate, 110 +/- 21 g fat, 8.5 +/- 1.4 g fiber, and 4.8 +/- 1.3 mg zinc. The analytical value for phytate:zinc molar ratio was 21 +/- 9. One subject, who received five of the menus for 28 wk, lost approximately 200 mg body zinc and 7% weight; zinc concentration declined 25% in plasma, 30% in lymphocytes, and 55% in neutrophils. This dietary model allowed simple formulation of new menus for subjects in diverse states of health. It caused no ill effects after prolonged consumption, and all deficiency symptoms were reversed by zinc supplementation of 30 mg/d for 20 wk. With simple manipulation, this dietary model may be used safely for gradual induction of zinc and/or other micronutrient deficiencies in humans.

Nitric oxide modulates the development and surgical reversal of renovascular hypertension in rats.

OBJECTIVE: To evaluate the role of nitric oxide (NO) in the development and unclipping-induced reversal of blood pressure and bilateral renal function in two-kidney, one clip (2K1C) Goldblatt hypertensive rats. METHODS: Goldblatt hypertensive rats were prepared by clipping the left renal artery 4 weeks before unclipping experiments. NG-nitro-L-arginine methyl ester (L-NAME) was administered after clipping and during unclipping to inhibit nitric oxide (NO) synthesis. Blood pressure and bilateral renal responses were measured. RESULTS: Chronic L-NAME treatment accelerated and aggravated blood pressure elevations and increased plasma nitrite and nitrate levels in 2K1C rats. Surgical removal of the renal artery clip induced profound reductions in blood pressure in rats with and without L-NAME treatment. However, the magnitude of the unclipping-induced depressor response at the first post-unclipping hour was significantly smaller in L-NAME-treated rats compared to those without L-NAME administration (15 +/- 1 versus 22 +/- 1%, P < 0.05). Two hours after unclipping, blood pressure of both groups fell to a comparable, normal level. Acute intravenous infusion of L-NAME in established 2K1C hypertensive rats further increased blood pressure. Subsequent unclipping caused a depressor response similar to that observed in hypertensive rats treated chronically with L-NAME. Despite the marked decreases in blood pressure, unclipping induced striking increases in glomerular filtration rate (GFR), urine flow and sodium and potassium excretion rates in the ipsilateral kidney. However, the magnitudes of increases in GFR and the diuretic and natriuretic responses in rats without L-NAME treatment were significantly greater than in rats with L-NAME administration. In contrast, unclipping reduced these function indices in the contralateral kidney to a similar level in rats with and without L-NAME treatment. CONCLUSIONS: NO exerts vasodilator action and thereby lessens renal artery clipping-induced blood pressure elevation. Furthermore, unclipping-induced release of NO partially contributes to the early reduction in blood pressure and changes in bilateral renal function but does not directly mediate the normalization of blood pressure after unclipping in this hypertension model.

A north-south comparison of blood pressure and factors related to blood pressure in the People's Republic of China: a report from the PRC-USA Collaborative Study of Cardiovascular Epidemiology.

OBJECTIVES: To compare blood pressures in northern (Beijing) and southern (Guangzhou) Chinese population samples aged 35-54 years, males and females, urban and rural, and to assess the role of blood pressure-related traits in explaining north-south differences. DESIGN: Cross-sectional surveys were conducted in 1983-1984 of northern and southern populations employed in industry (urban) or farming (rural). METHODS: In the north samples were selected from the Capital Iron and Steel Complex (urban) and Shijingshan district (rural); in the south samples from the Guangzhou Shipyard (urban) and Panyu County (rural) were used. RESULTS: The number of subjects surveyed in north and south were 4706 and 4179, respectively: 1500 and 1052 urban males, and 717 and 914 rural males; and 1300 and 1061 urban females, and 1189 and 1152 rural females, respectively. Average systolic (SBP) and diastolic (DBP) blood pressures, were consistently higher in the north than in the south. SBP and DBP were significantly and independently related to age, body mass index, heart rate, use of antihypertensive drugs, serum triglycerides level, alcohol use (males only) and inversely to cigarette smoking. Northerners were older, taller, heavier and had higher body mass index and triglycerides level than southerners. With adjustment of SBP and DBP for blood pressure-related traits, north-south blood pressure differences decreased, but remained significant for urban males, rural males and rural females, with sizeable differences for rural samples in particular. CONCLUSIONS: North-south differences in blood pressure in these samples are accounted for only partly by north-south differences in the cited blood pressure-related traits. The role of other traits requires assessment.

Effects of solvation on the ionization and conformation of raclopride and other antidopaminergic 6-methoxysalicylamides: insight into the pharmacophore.

Previous work has shown that raclopride in water at neutral pH exists in a zwitterionic form, suggesting a stereoelectronic structure largely different from that of other benzamides. In the present study, the acid-base behavior of other 6-methoxysalicylamides is shown to be comparable to that of raclopride. An extensive investigation by high-temperature molecular dynamics gave insight into the conformational behavior of neutral and zwitterionic raclopride in vacuum and in water. Partitioning of raclopride and a more rigid analogue with characterization (by first-derivative UV spectroscopy) of the predominant forms in the organic phase indicated that only neutral, internally H-bonded forms partition into the organic solvent. Thus, the predominant forms of 6-methoxysalicylamides will be very different in the aqueous and organic phases. In the latter phase, and hence presumably also in the receptor phase, the drugs exist with a neutral, internally H-bonded phenolic group and are therefore stereoelectronically similar to other substituted benzamides.

Substrate modulation of cultured rabbit conjunctival epithelial cell differentiation and morphology.

This study demonstrated that growth and differentiation of rabbit conjunctival epithelial cells could be promoted by such substrata as collagen gel, matrigel or various mixtures of collagen and matrigel in a defined culture system. On conventional plastic or glass culture, the conjunctival epithelial cells adopted a monolayer of small epithelioid cells in primary cultures. They became enlarged, squamoid and exhibited notable senescence upon subcultures. On collagen gel, cells formed an organized monolayer sheet with cuboid shape and cell polarity. On matrigel, cells formed globules with stratified appearance including the basal layer of the outer part of globule and the squamoid cells of the central part of globule. The epithelial origin of these cultures was verified by the positive immunostaining of anti-keratin monoclonal antibodies. Expression of mucin antigen was lost on plastic or glass culture, but promoted on collagen gel or matrigel, as demonstrated by staining with periodic acid Schiff's reagent and anti-mucin monoclonal antibody stainings. These results indicate that both collagen gel and matrigel can provide a permissive substrate environment for goblet cell differentiation. Furthermore, this unique phenotypic expression may be possessed only by a selective cell subpopulation. This culture system will allow us to further explore the mechanism by which the goblet cell differentiation is controlled.

The effects of fibroblasts on the growth and differentiation of human bulbar conjunctival epithelial cells in an in vitro conjunctival equivalent.

PURPOSE: To study the effects of collagen matrix and fibroblasts on the growth and development of human bulbar conjunctival epithelial cells. METHOD: Human bulbar conjunctival epithelial cells were cultured on three-dimensional collagen gels containing either normal human conjunctival fibroblasts (HCF), Swiss 3T3 cells, or no cells. After 1 week of culturing, half of the cultures were raised to the air-liquid interface and the rest of the cultures remained submerged. On day 14, cultures were fixed and sectioned for light and electron microscopic studies. RESULTS: Conjunctival epithelial cells cultured on fibroblast-contracted collagen lattice developed into a multicell-layer epithelium with characteristic epithelial structural features including microvilli, desmosomes, early hemidesmosomes, and basement membrane-like structures. Formation of all or some of the above features appeared to be influenced by the type of fibroblasts in the collagen lattices. Structures such as hemidesmosomes and basement membrane were only observed in epithelium developed on 3T3- but not on conjunctival fibroblast-condensed collagen lattices. In contrast, goblet cell differentiation was only observed in epithelia developed on normal HCF-supported collagen matrix. Epithelial cells cultured on acellular collagen gels did not develop into multicell-layer epithelium, and no differentiated characteristics were observed. CONCLUSIONS: These results indicate that the type of fibroblasts dispersed in the collagen matrix plays an important role in the development and differentiation of conjunctival epithelial cells. Normal HCF-dispersed collagen matrix was less growth stimulating to epithelial cells and allowed them to undergo goblet cell differentiation. In contrast, 3T3-dispersed collagen matrix was more growth stimulating, resulting in thicker epithelium with a higher degree of stratification.

Inhibition of vascular endothelial cell morphogenesis in cultures by limbal epithelial cells.

PURPOSE: To study the in vitro angiogenic activity of human conjunctival and limbal epithelial cells and conjunctival, limbal, and corneal fibroblasts in a three-cell-type coculture model. METHODS: Human umbilical vein endothelial cells (EC) were cocultured with epithelial cells, fibroblasts, or epithelial cells and fibroblasts to test their effect on EC morphogenesis. Neutralizing antibodies to some known angiogenic factors were added to the culture to see whether the EC morphogenesis may be blocked by a particular antibody. RESULTS: Conjunctival and limbal epithelial cells exhibited very little or no stimulatory effect on EC tube formation when examined in an EC- epithelial cell coculture system. In contrast, conjunctival, limbal, and corneal fibroblasts all promoted EC morphogenesis when examined under the same culture conditions. Fibroblast-induced EC morphogenesis was inhibited by addition of anti-vascular endothelial growth factor (VEGF) and/or anti-basic fibroblast growth factor (bFGF) antibodies to the culture medium. In the three-cell-type coculture system consisting of ECs, fibroblasts, and epithelial cells, limbal epithelial cells (but not conjunctival epithelial cells) exhibited a strong inhibitory effect on fibroblast-induced EC tube formation. CONCLUSIONS: The proangiogenic activity of ocular surface fibroblasts is probably mediated through a paracrine mechanism by VEGF and bFGF. Limbal epithelial cells, but not conjunctival epithelial cells, inhibit fibroblast-stimulated angiogenesis.