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PubMed1982

Endogenous androgen levels in epithelium and stroma of human benign prostatic hyperplasia and normal prostate.

5 alpha-Dihydrotestosterone (DHT) and 5 alpha-androstane-3 alpha, 18 beta-diol (3 alpha-diol) were extracted from epithelium and stroma of human benign prostatic hyperplasia (BPH) and of normal prostate and quantified by RIA. The main results were: (1) concerning the BPH, DHT is mainly located in the nuclear fraction of epithelium and stroma, whereas 3 alpha-diol was completely of extranuclear origin, (2) in the nuclei derived from BPH stroma the DHT content (7.1 +/- 0.88 pmol/mg DNA, mean +/- SEM [n = 14]) was significantly higher (P less than 0.01) than in the nuclei derived from BPH epithelium (3.8 +/- 0.38 pmol/mg DNA [n = 14]). The DHT content in the nuclear fraction derived from unseparated, i.e. whole tissue, was 5.6 +/- 0.60 pmol/mg DNA [n =14], (3) the biological significance of the overwhelming DHT accumulation in the stromal nuclei for the BPH tissue is reflected by a significant correlation between the DHT values in the nuclei from stroma and whole tissue (rs = 0.710, P less than 0.01) and (4) in four normal prostates the DHT content in the nuclear fraction of epithelium (1.3 +/- 0.37 pmol/mg DNA) and stroma (2.2 +/- 0.93 pmol/mg DNA) was significantly lower (P less than 0.01, each) compared with the respective BPH fraction. These data support earlier findings which indicate that the stroma of BPH is a preferential tissue for androgen metabolism.

Adult↗
PubMed2004

Gap junctions in hematopoietic stroma control proliferation and differentiation of blood cell precursors.

We examined gap junction communication in an in vitro model of hematopoiesis, using the murine bone marrow stroma cell line S-17, and primary cultures of murine marrow-derived blood cell precursors. S-17 cells express several connexins, the major one being connexin 43. Connexin expression and formation of functional gap junctions is modulated by stroma cell density. Transfection of S-17 cells with a vector containing connexin 43 sense or anti-sense sequences increased or decreased, respectively, connexin 43 synthesis and intercellular dye coupling. Under these conditions, modulation of gap junction-mediated communication modified the growth pattern of stroma itself, as well as the ability of the stroma to sustain hematopoiesis. Increased connexin 43 expression was associated with a delay in differentiation of blood cells, resulting in increased production of hematopoietic precursors, while decreased connexin 43 expression elicited an accelerated differentiation of myeloid blood cell precursor cells. These results suggest that connexin-mediated coupling in the stroma modulates the ratio between proliferation and differentiation of hematopoietic precursors. We therefore propose that increased gap junction communication in the stroma elicits an enhanced production of immature bone marrow cells through the delay in their terminal differentiation, inducing consequently an extended proliferation period of blood cell precursors.

Animals↗
PubMed1987

Protein profiles of benign hypertrophic prostate: stroma-abundant distribution of BPH-associated nonhistone proteins.

Mechanically isolated epithelium and stroma from benign hypertrophic prostates were highly pure on the basis of histochemical and biochemical criteria. By electrophoretic analyses, whole cellular and nuclear proteins were compared among whole tissues, epithelium, and stroma. The characteristic protein profiles of benign hypertrophic prostates were reflected in the electrophoretic patterns of the stroma. Two-dimensional gel electrophoretic patterns of the epithelium were different from those of the stroma with exception of about 18 major spots that were common to both fractions. Of the protein species separated, 35K/6.7 (molecular weight/pI) and 36K, which was composed of two species with pI of 4.4 and 4.6, were abundant in the epithelium and stroma, respectively. Nuclei prepared from whole tissues of benign hypertrophic prostates contained three kinds of nonhistone proteins (NHP) closely associated with benign prostatic hypertrophy (BPH); 42 K-NHP, 55 K-NHP and 190 K-NHP. Electrophoretic analysis of the nuclear proteins revealed that all the BPH-associated nonhistone proteins were abundantly distributed in the nuclei of the stroma.

DNA↗
PubMed1980

Preferential binding of beta C relative to beta S globin to stroma in hemoglobin SC disease.

Globin synthesis was measured in blood samples of four patients with hemoglobin SC Disease. In the stroma-free supernatant, globin synthesis was balanced with alpha/(beta S + beta C) = 1. In the exhaustively washed stroma, there was a preferential binding of beta chains to stroma, similar to that reported for Sickle Cell Anemia. However, while the beta S/beta C ratio was balanced in the supernatant (0.8-0.9), the beta S/beta C ratio of the stroma varied from 0.2 to 0.4. Therefore, beta C is bound to stroma in preference to beta S in hemoglobin SC disease. These findings are consistent with the concept that binding of globin to stroma is related to the electrophoretic charge of the hemoglobin.

Anemia, Sickle Cell↗
PubMed1975

[Stroma formation in epithelial malignant tumors].

The mechanism of stroma-formation and rearrangement of the stroma was studied on 221 cases of cancer of the lung, stomach and mammary gland using histological, histochemical and electron-microscopy methods of investigation. It was established that multiplying epithelial elements of teh tumour induced proliferation of histiogenic and vascular fibroblasts, activating them. Active fibroblasts play a double role: they either produce fibres and interstitial matter of the tumorous stroma, or participate in desintegration of preceding and newly formed collagenous structures. The processes of stroma-formation comply with the conventional schemes of normal fibrillogenesis and rearrangement of the interstitial tissue. It was shown that fibroblasts of the tumour stroma was capable of phagocytosis of a mature collagen. It is supposed that in the course of the tumorous growth correlation between the parenchyma and stroma is maintained, the leading role being played by the epithelium.

Acid Phosphatase↗
PubMed1988

Fibrin-fibronectin compounds in human ovarian tumor ascites and their possible relation to the tumor stroma.

Covalently linked heterogeneous fibrin-fibronectin compounds were detected in ascitic fluid of 31 patients with advanced ovarian cystadenocarcinoma by means of enzyme-linked immunosorbent assay techniques, immunoaffinity chromatography, and Western blot analysis. Deposition of fibrin and fibronectin could also be demonstrated immunohistochemically in Carnoy-fixed tissue sections. Fibrin and fibronectin were found in the tumor stroma within tumor nests and more prominently in stroma surrounding the tumor nests. The association of fibrin and fibronectin was especially pronounced in the stroma surrounding the tumor islands. Fibronectin was also found to be associated with stroma cells. Areas within the tumor stroma showed superimposed staining for both fibrin and fibronectin supporting the assumption that the covalently linked fibrin-fibronectin conjugates found in ascitic fluid may stem from the provisional tumor stroma by proteolytic release.

Ascitic Fluid↗
PubMed1983

Morphogenesis of rabbit corneal stroma.

Corneas of fetal and young albino rabbits were examined by light and transmission electron microscopy. In addition, DNA and hydroxyproline content were measured in developing stroma. The results were compared with similar data from healing corneas in adult rabbits and from developing corneas of other animal species. In the fetal rabbit, the prospective corneal stroma region contains an unorganized, sparse extracellular matrix until about the 13th day of gestation, when mesenchymal cells and capillaries from the hyaloid vessels move in to form the vascular pupillary membrane, endothelium, and stroma. Stromal growth is due to alteration in the density and morphology of the cell population early in development, along with a sequential thickening and thinning of the whole stroma. These events are similar to those reported in primates, but differ markedly from those reported in avian species. Normal developing cornea and healing adult cornea both involve migration of stomal fibroblasts and deposition of extracellular matrix. Stromal fibroblasts in the rabbit fetus are oriented with their long axis parallel to the corneal surface early in development compared with randomly oriented fibroblasts in the early healing wound of adult rabbit corneas. Although collagen and cell number progressively increase throughout the developmental periods studied, the ratio of cells to collagen is high initially but decreases with development. In contrast, the proportion of cells to collagen in the young scar tissue of adult cornea is low initially, indicating a marked deposition of collagen in comparison to that in the early normal developing stroma. The results suggest that the healing tissue differs from the normal fetal stroma in its coordination of cell population growth with collagen deposition and cellular organization.

Animals↗
PubMed1983

A comparison of three-dimensional growth characteristics of cultured primitive marrow stroma derived from genetically anemic and normal mice.

This study examines the ultrastructure of mouse defective marrow stroma when cultured as a three-dimensional organization of cells. Gelfoam sponges impregnated with agar medium allowed the three-dimensional organization of newly formed stromal cells derived from the crevices of marrow-depleted bones of Steel mutant mice (Sl/Sld) with defective stroma and also from mice with normal stroma (Sl +/+, W/Wv, and W +/+. Ultrastructural comparisons of 5- to 14-day cultures revealed that the mutant defective stromal cells developed normally, viz. i) proliferated and formed a three-dimensional organization of stroma, ii) stimulated residual hemopoietic precursors to form myeloid cells, and iii) formed a variety of stromal cell types characterized by variable quantities of Golgi bodies and ER, glycogen, filaments, and round cytoplasmic granules. The Steel-Dickie strain, however, included bacilliform electron-dense granules in both normal and mutant stroma. The only ultrastructural deficiency in Sl/Sld stroma was the absence of a category of "activated" cells that occurred within normal cell populations.

Anemia↗
PubMed1995

Identification of a growth factor for primary murine stroma as macrophage colony-stimulating factor.

Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. We took advantage of an assay that permits analysis of primary stroma-initiating cells (SICs) on the clonal level, and further characterized SICs and the factors that regulate SICs. Stroma formation in this assay is dependent on a high-molecular-weight factor secreted by the stromal cell line AC3.U. Here we show that this factor is identical to macrophage colony-stimulating factor (M-CSF), and that purified M-CSF is sufficient for induction of stroma formation. M-CSF, isolated from the line AC3.U, as well as from L929 cells and COS cells transfected with an expression vector encoding M-CSF, migrated in two peaks as 160- and 650-kD species after gel filtration. These molecular-weight species encompassed all stroma-inducing activity, and both stimulated macrophage colony formation. Affinity chromatography and blocking studies with antibodies specific for M-CSF and c-fms confirmed M-CSF as the sole factor in the supernatant of the stromal cell line AC3.U that promotes stroma formation. Culture of marrow, for as little as 1 week, depleted M-CSF-dependent SIC while increasing the incidence of replatable, factor-independent SIC. This suggests that culture changes the properties of SICs, perhaps by inducing differentiation into mature stromal cells. Thus, our results show a novel function of M-CSF as an important modulator of stroma formation.

Animals↗
PubMed1994

Clonal analysis of primary marrow stroma: functional homogeneity in support of lymphoid and myeloid cell lines and identification of positive and negative regulators.

Stromal cells play an important role in regulating early hematopoiesis. How stromal cells exert their different functions and the factors that regulate stromal cells themselves remain to be elucidated definitively, however. We describe here a limiting dilution assay for primary stroma colonies from murine marrow. This system permits a critical analysis of stromal cell function and regulation on the clonal level. We report that stroma formation was dependent on an activity secreted by the long-term cultured stromal line AC-3.U. Differential ultrafiltration of AC-3.U supernatant (SN) suggests that this potentially novel activity is represented by molecules with apparent molecular weights (m.w.) of > 100 < 300 kD and > 300 kD. In contrast to the AC-3.U activity, hydrocortisone (HC) acts as a negative regulator of stroma colony formation. We used the stroma colony assay to analyze potential stromal cell heterogeneity. We found that most, if not all, primary stromal colonies supported expansion of both myeloid and lymphoid cell lines. In contrast, long-term cultured stromal cell lines differed not only among lines, but also on the level of sublines, in their ability to sustain myeloid and lymphoid cells. This intraclonal variation suggests that the heterogeneity of cell lines can be a reflection of ongoing culture adaptation. The functional homogeneity of primary stromal colonies, together with their susceptibility to regulators, indicates that the performance of primary stroma is subject to external control. The establishment of a clonal assay system has paved the way to analyze the molecules that regulate primary stroma and thereby hematopoietic cells.

Animals↗
PubMed1993

Distribution and histochemical characteristics of mast cells in stroma of the cervix squamous cell carcinoma.

The distribution and histochemical characteristics of mast cells in the stroma of cervical squamous cell carcinoma were investigated using different staining methods of alcian blue-safranin, berberine sulphate and toluidine blue. The findings suggest that mast cells in the stroma of the squamous cell carcinoma of the cervix might contain a glycosaminoglycan different from that of typical connective tissue mast cell (CTMC) and mucosal mast cell (MCC). They thus, might be a specific subset of mast cells though sharing some of the staining properties of both CTMC and MMC. In the cervical carcinoma in situ, mast cells were distributed closely around the tumor or the involved glands with great density and the number was significantly higher than that of the invasive carcinoma (P < 0.01). In invasive carcinoma, mast cells were mainly distributed in the deep stroma and the stroma around the tumor, only a few in the stroma within the tumor (P < 0.01). The significance of mast cell reaction in the stroma of squamous cell carcinoma of the cervix is discussed.

Carcinoma, Squamous Cell↗
PubMed1996

Long-term survival and proliferation of precursor-B acute lymphoblastic leukemia cells on human bone marrow stroma.

Leukemic cells frequently persist in the bone marrow of patients treated for acute lymphoblastic leukemia (ALL), and may regrow to produce relapse. We have used a long-term co-culture system to analyze the interaction of ALL blasts with components of the marrow microenvironment. Blast cells from 10 cases of precursor-B ALL were cultured on allogeneic human bone marrow stromal layers at 37 degrees C in microtiter wells, and replated when stroma showed evidence of deterioration. Leukemic cells from seven of 10 cases showed evidence of survival and proliferation beyond 30 days in culture, while in three cases there was a progressive decline in the number of viable leukemic cells by 7-21 days. Two cases continued to proliferate for 149 to 332+ days, while five underwent senescence after 34-52 days. In the two cases with long-term proliferation, the leukemic identity of the cells was confirmed by immunophenotyping, cytogenetics, and demonstration of clonal immunoglobulin gene rearrangements. Evidence of selection of leukemic subclones was seen in two cases, with immumophenotypic evidence of loss of CD10 and CD34 antigens, and acquisition of CD20 and surface mu chain. The leukemic cells in these cases grew either in clumps attached to the surface of the stroma, or as'cobblestone areas' beneath the stromal cells. Survival and growth of two evaluable cases was dependent on the continuing presence of stromal cells in the culture system. In one case, direct contact with stroma was shown to be necessary to main- tain viability, while blast cells from the other case survived equally well when separated from the stroma by a 0.4-micron pore size microporous membrane. These results indicate that leu- kemic cells from the majority of cases of precursor-B ALL are able to persist and undergo proliferation in vitro in the presence of normal marrow stroma. This process appears dependent on either direct cell-cell contact, or on diffusible factors derived from the stroma. The availability of ALL cells capable of indefinite proliferation under these conditions will allow further analysis of the mechanisms mediating leukemic cell proliferation.

Adolescent↗
PubMed1998

Addition of a bone marrow "facilitating cell" population increases stem cell-derived cobblestone area formation in impaired long-term bone marrow culture stroma.

Treatment of mouse bone marrow (BM) with rabbit anti-mouse brain serum (RAMBS) plus complement (C') depletes several cell types, including T cells and facilitating cells (FCs), that is, cells that facilitate engraftment of sorted allogeneic stem cells (SCs) in vivo. In the present study, treatment of BM with RAMBS+C' resulted in the depletion of approximately half of the late cobblestone area (CA)-forming stem cells as assayed on irradiated long-term bone marrow culture (LTBMC) stroma. In addition, LTBMC of RAMBS+C'-treated BM produced functionally impaired stroma with reduced ability to support CA formation by nontreated exogenous SCs. This stromal impairment was not due to depletion of TCRalphabeta T cells in the BM, because BM cultures from TCR alpha-chain knockout mice supported normal numbers of exogenous CAs. Because CD8+/TCR- cells are enriched for FCs, we tested the effect of adding these cells back to the treated BM prior to culture. The sorted FCs alone did not produce CAs, but did improve the ability of the impaired stroma to support late CA formation by sorted SCs. These studies provide a new model for dissecting the roles of different cellular components of BM in producing functional stroma that supports CA formation by SCs, and show that the number of CAs formed depends on the "quality" of the stroma as well as the number of SCs seeded. These findings further suggest that CD8+/TCR- BM cells may be important for the establishment of functional stroma.

Animals↗
PubMed2006

Bone marrow-derived cells in normal human corneal stroma.

OBJECTIVE: To examine the normal human corneal stroma for the presence of bone marrow-derived cells. METHODS: Thirty-four corneas from donors aged 56 to 71 years were used. The stroma of the donor corneas was examined immunohistochemically by fluorescent microscopy. CD45-positive and -negative cells were separated from collagenase-digested stroma by magnetic beads, and the expression of toll-like receptor 4 was analyzed. RESULTS: CD45-positive cells were mainly found in the anterior stroma of the central and paracentral cornea as well as all stromal layers of the peripheral cornea (n = 5). These cells uniformly expressed CD11b, CD11c, CD14, and HLA-DR antigen but not CD3, CD19, CD56, or CD66, indicative of bone marrow-derived monocyte lineage cells, which can include monocytes, macrophages, or dendritic cells. CD45-positive cells isolated with magnetic beads accounted for 6.0% of total stromal cells (n = 20). Stromal CD45-positive cells, but not CD45-negative cells, expressed toll-like receptor 4 by flow cytometry and reverse-transcriptase polymerase chain reaction. CONCLUSION: Our findings demonstrate that DR antigen-positive bone marrow-derived monocyte lineage cells exist in the anterior and peripheral posterior stroma of normal human cornea. Clinical Relevance These cells may play a role in the innate and adaptive immune responses in the human cornea.

Aged↗
PubMed1994

Keratocyte loss and repopulation of anterior corneal stroma after de-epithelialization.

OBJECTIVE: To quantitate corneal keratocyte loss and repopulation of anterior stroma after de-epithelialization. METHODS: Fourteen white New Zealand rabbits, each weighing 2.3 to 3.2 kg, were divided into seven groups. Each rabbit underwent a bilateral 6-mm mechanical deepithelialization procedure. The rabbits were killed two at a time after 1, 3, 6, 8, 10, 14, and 28 days. The corneas were labeled for mitosis using 5-bromo-2-deoxyuridine and stained for keratocyte quantification. Three untreated rabbits were added as controls. RESULTS: Corneas that were not operated on showed a higher density of keratocyte nuclei within the anterior corneal stroma than in the posterior stroma. Following de-epithelialization, there was a decrease in the ratio of anterior-posterior keratocytes, with maximal decrease on the third postoperative day. Keratocyte repopulation was completed by day 14. Mitotic activity was seen on days 1, 3, and 6 in the anterior half of the de-epithelialized stroma. CONCLUSIONS: Anterior stromal keratocytes are lost after epithelial removal, but repopulation is complete within 2 weeks. These findings demonstrate a loss of keratocytes and their recovery after corneal epithelial removal, as well as an interaction between corneal epithelium and stroma in rabbits.

Animals↗
PubMedDate not supplied

Regulation of fluid flow through corneal stroma in the bullfrog.

Regulation of fluid flow through corneal stroma was investigated in the bullfrog. Corneal specimens were mounted by clamping their limbal sclera between the two chambers of a Ussing-type chamber. The epithelial surface was covered with Ringer's solution, while the endothelial surface was superfused with Ringer's solution at various pressures ranging from 0-60 mm Hg. At 0 mm Hg, the cornea swelled, while at 10 mm Hg the corneal thickness remained unchanged. Further elevation of the hydrostatic pressure of the endothelial superfusion solution caused a decrease in corneal thickness, suggesting that the hydrostatic pressure in the in vivo frog corneal stroma is about 10 mm Hg. At 10 mm Hg of endothelial superfusion pressure, piercing the epithelial cell layer with a 30-gauge needle caused only slight corneal swelling. Removing glucose from the epithelial perfusion solution induced a slowly progressing increase in corneal thickness. Iodoacetate did not interfere with the swelling of the pierced cornea after the removal of glucose from the epithelial perfusion solution. To examine the possibility that the interstitial fluid flows across the stroma-scleral boundary, corneal specimens having unclamped sclera were incubated in Ringer's solution containing 3 mmol/L dextran of various molecular weights ranging from 8800-162,000, and the volume of the preparation was monitored by sequential measurement of the weight. In the presence of dextran with a molecular weight higher than 70,000, the corneal volume decreased at the beginning of incubation, and after reaching the minimal volume it slowly increased, indicating that the stroma-scleral boundary is permeable to dextran of even a molecular weight of 162,000, although dextran molecules diffuse much more slowly than water, and the concentration of unfilterable solutes in the stroma is lower than 3 mmol/L. In experiments using the Ussing-type chamber at 10 mm Hg of endothelial superfusion pressure, a decrease of NaCl in the superfusing solution to 1/2 caused rapid corneal swelling followed by slow recovery. Adding NaCl to the 1/2 NaCl Ringer's solution caused a further corneal thinning in a concentration-dependent manner. The same extent of decrease in corneal thickness as induced by adding NaCl was achieved by the same concentration of glucose as of NaCl, implying that the value of the reflection coefficient of the endothelial cell layer to either Na+ or Cl- is about half that of glucose. Our results show that even a small difference in the concentration of low molecular weight solutes (e.g., Na+ and Cl-) exerts a force that draws water from the cornea.

Animals↗
PubMed2003

Transparency, swelling and scarring in the corneal stroma.

PURPOSE: This paper briefly reviews current explanations for corneal transparency and uses a well-developed model to try to explain the increased light scattering either accompanying corneal swelling or following phototherapeutic keratectomy (PTK). METHODS: The direct summation of fields (DSF) method was used to compute light transmission as a function of wavelength. The method requires input of a number of structural parameters. Some of these were obtained from electron micrographs and others were calculated from X-ray diffraction data. RESULTS: By swelling sections of stroma cut from different depths in the tissue, we have shown that fluid entering the cornea causes more swelling in the posterior lamellae than in the anterior lamellae. Furthermore, posterior lamellae can reach a higher final hydration than anterior lamellae. Collagen-free regions ('lakes') exist in corneas swollen in vitro and in Fuch's dystrophy corneas, many of which may be caused by the death of cells. The DSF method shows that local fibril disordering, increased refractive index mismatch, and increased corneal thickness together can account for a 20% increase in light scattering in a Fuch's dystrophy cornea at H=5.8 compared to the normal cornea. Additional scattering is probably caused by 'lakes'. The DSF method applied to PTK rabbit stroma with high levels of haze suggests that the newly deposited collagen is not the cause of the increased light scattering. CONCLUSIONS: Fluid is not uniformly distributed within the corneal stroma when the cornea swells. Increased hydration of posterior lamellae may be because of known differences in the glycosaminoglycans between the anterior and posterior stroma. Lamellar interweave in the anterior stroma probably limits the extent to which the constituent lamellae can swell. The DSF method can be used to account for increased light scattering in oedematous corneas but cannot account for haze following PTK.

Corneal Edema↗
PubMedDate not supplied

Tissue engineering of nearly transparent corneal stroma.

We determined whether a polyglycolic acid (PGA) scaffold bearing an adherent corneal stromal cell insert could be integrated into the ultrastructure of rabbit corneal stroma without compromising tissue transparency. Stromal cells were isolated from 10 newborn rabbits and expanded by tissue culture. After reaching confluence, the cells were harvested and mixed with nonwoven PGA fibers to form cell-scaffold constructs. After 1 week of culturing, they were implanted into the corneal stroma of female rabbit recipients. Green fluorescent protein (GFP) expression in transplanted corneal stromal cells was monitored at the protein level to determine cellular origin in the reconstructed stroma. Eight weeks after implantation, transmission electron microscopy and histological evaluation were performed on stromal tissue. Insertion of PGA scaffold alone served as a sham control. After stromal implantation, implants gradually became transparent over an 8-week period. During this time stromal histology was gradually restored, as collagen fibril organization approached that of their normal counterpart. GFP-labeled corneal stromal cells were preponderant in the regions bearing inserted scaffolds, suggesting that they were derived from the implants rather than from neighboring corneal stromal cells. No reconstructed stroma developed in regions where naked PGA was implanted instead. We conclude that intrastromal implantation of PGA fiber scaffold implants bearing corneal stromal cells is a useful procedure for corneal stromal tissue reconstruction because, over an 8-week period, the implants become progressively more transparent. Marked losses of translucence during this period combined with restoration of ultrastructure indicate that the implants provide the functions needed for deturgescing initially swollen stroma.

Animals↗