Growth acceleration and stem cell expansion in Dexter-type cultures by neutralization of TGF-beta.

Hematopoietic lineage-restricted stem cell growth has been shown to be significantly inhibited by the addition of exogenous transforming growth factor-beta (TGF-beta) to Dexter-type long-term murine bone-marrow cultures. In order to examine whether TGF-beta produced by these cells has a role in hematopoietic growth regulation, Dexter cultures have been treated with either 1D11.16, a monoclonal antibody that neutralizes the biological activity of TGF-beta types 1, 2, and 3, or with a control antibody. The composition and cellularity of the nonadherent cell populations in these cultures were assessed weekly. Treatment with anti-TGF-beta antibody resulted in a five- to 20-fold increase in nonadherent cells in the cultures when compared to either the control or untreated cultures by week 4. The majority of these cells were granulocyte/macrophage-lineage cells as assessed by histologic and flow-cytometric analysis. There was also a significant increase of megakaryocytes in cultures treated with anti-TGF-beta antibody. Stem-cell analysis, using a colony-forming unit-spleen (CFU-S) assay that combined both the adherent and nonadherent populations from either 4- or 6-week cultures, showed that there are an equivalent number of hematopoietic stem cells per 10(6) cells regardless of antibody treatment. Therefore, cultures treated with anti-TGF-beta antibody contained at least three times as many stem cells as the control cultures. Finally, kinetics studies show that the presence of anti-TGF-beta antibody is required from the onset of culture to produce these effects. These results suggest that TGF-beta is involved in normal growth regulation of bone-marrow hematopoietic cells. By addition of a neutralizing antibody, the normal TGF-beta negative growth signal is disrupted, allowing for expanded growth of several cell populations.

Transforming growth factor beta 1 systemically modulates granuloid, erythroid, lymphoid, and thrombocytic cells in mice.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to inhibit the development of most early hemopoietic progenitors in vitro. The present series of in vivo experiments show that TGF-beta 1 can simultaneously augment and suppress distinct cell lineages in peripheral and central hemopoietic compartments. Mice treated daily for 7-14 days with s.c. injections of TGF-beta 1 exhibited up to a 95% reduction in circulating platelets and a 50% reduction in red cell counts, whereas a 50%-400% increase occurred in circulating white cells with the morphology of small lymphocytes. Decreased erythrocytes were also evident in the splenic red pulp and bone marrow sinusoids. A dramatic increase in granulopoiesis occurred in the spleen and bone marrow, followed by a peripheral neutrophilia 1 week after treatments ceased. All effects were completely reversible, with normal histologic and hematologic profiles evident 2 weeks after cessation of treatments. Thus, TGF-beta 1 can differentially regulate multiple hemopoietic pathways in a systemic, reversible, and dose-dependent fashion. These actions may be mediated by the direct effects of TGF-beta 1 or through modulation of secondary cytokines and receptors.

Antigens specified by the Tla locus are expressed on the surface of murine Langerhans cells.

A monoclonal antibody against the murine thymus leukemia antigen TL, was employed to demonstrate the presence of the antigen on the surface of dendritic cells in murine epidermis of Tla-positive strains, B.10A and A.TH. Immunofluorescence and immunoperoxidase staining of EDTA-separated epidermal sheets demonstrated dendritic cells with a distribution pattern and density comparable to that noted for anti-IAk staining. Tla-negative mouse strains such as A.TL, C3H/HeJ, and C57BL/6 did not show any staining of dendritic epidermal cells. Epidermal cell suspensions similarly contained 2-4% cells with discrete surface staining with anti-TL antibody. Capping was noted in these cells. Once again positive results were noted only in appropriate Tla-positive strains. Control staining was carried out in all cases on frozen sections of thymii from mice. Thymocytes in the cortical zones and some dendritic cells at the corticomedullary junction were stained. TL antigen in mouse appears to be analogous to T-6 antigen previously detected on human Langerhans cells.

Differential modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin.

Immunohistochemical staining of skin sections with two polyclonal antibodies (anti-CC 1-30 and anti-LC 1-30), specific for transforming growth factor-beta 1, revealed increased extracellular and decreased intracellular expression of transforming growth factor-beta 1 in retinoic acid-treated, compared to vehicle-treated, skin. Transforming growth factor-beta 1 staining, with both antibodies, was most marked in the upper layers of the epidermis, although dermal staining was also evident. The modulation of transforming growth factor-beta 1 expression by retinoic acid occurred in the absence of any change in its mRNA level. Transforming growth factor-beta 1 protein, as detected by rabbit polyclonal antibody (anti-LC 50-75) and mRNA, were only minimally detected in either retinoic acid- or vehicle-treated skin. Similar changes in TGF-beta 1 and TGF-beta 2 immunoreactivity and mRNA levels, as observed in retinoic acid-treated skin, were observed in skin following topical application of the irritant sodium lauryl sulfate, indicating that the alterations induced by retinoic acid were not specific. In contrast, mucin deposition, which is induced by transforming growth factor-beta, was elevated in retinoic acid-treated but not sodium lauryl sulfate-treated skin. Cultured adult human keratinocytes also expressed predominantly transforming growth factor-beta 1 protein, as measured by ELISA, and mRNA. Treatment of keratinocytes with retinoic acid resulted in a 50% induction of transforming growth factor-beta 1 protein, without any detectable change in transforming growth factor-beta 2. These data demonstrate disassociation of modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin in vivo. Whereas alterations in transforming growth factor-beta 1 expression were observed in both retinoic acid- and sodium lauryl sulfate-treated skin, accumulation of mucin was specific to retinoic acid-treated skin.

Morphometric analysis of thymic medullary non-lymphoid cell changes during postnatal development.

Thymic non-lymphoid cells have been shown to influence the differentiation of T-lymphocytes. Large numbers of non-lymphoid cells are concentrated in the thymic medullary zone. A morphometric analysis of these cells in the medulla identified little change in their absolute number and volume density during thymic development in the rat. Expansion of thymic medullary volume with age was documented by planimetry. Several subtypes of medullary non-lymphoid cell were identified by electron microscopy, including both squamous and cystic epithelial cells, interdigitating cells and macrophages. Changes in their relative proportion during development were determined with the percentage of squamous epithelial cells declining, interdigitating cells increasing and the other cell types exhibiting little change. Subcellular evidence of secretory activity was sought by analysis of changes in the volume density of organelles in each of the non-lymphoid classes. Cystic enlargement in epithelial cells and a decrease in the volume density of macrophage inclusion bodies were noted but no clear morphometric trend could be correlated with increased secretory activity expected in the developing endocrine thymus.

Glial fibrillary acidic protein and S-100 protein in pineal supportive cells: an electron microscopic study.

Pineal supportive cells in a teleost were shown to contain the glial fibrillary acidic (GFA) and S-100 proteins by electron microscopic immunocytochemistry. Both proteins are known to exhibit a high degree of evolutionary conservation. The pineal photoreceptor cells did not stain for these marker proteins. A relationship between supportive cells and macroglial elements is therefore implied.

Thymic interdigitating reticulum cells demonstrated by immunocytochemistry.

The lymphoid interdigitating cell (IDC) was investigated in human and rat thymus. A glial protein, S-100, was demonstrated in IDCs found in the human and rat thymic medulla by light microscopic immunocytochemistry. This marker served to distinguish IDCs from conventional macrophages of the thymic cortex. IDCs were S-100+, lysozyme-. Cortical macrophages were S-100-, lysozyme+. Thymic epithelial cells and lymphocytes possessed none of these markers. Examination of human fetal thymic tissue revealed that the IDC is present within the thymus very early in embryogenesis. Ultrastructural analysis of a developmental series of the rat thymus identified IDCs and macrophages. Medullary IDCs possessed many of the morphologic features of the epidermal Langerhans cell including the Birbeck granule. Cortical macrophages contained many inclusion bodies and lysosomes indicative of active phagocytosis. Some changes in IDC shape and structure were noted during maturation in the rat that may reflect the migration of this cell into the thymic parenchyma.

Langerhans cells and extra-epidermal dendritic cells. An investigation in laboratory animals and man with immunomorphological methods.

S-100 protein was demonstrated in the cytoplasm of dendritic cells (DCs) in normal and pathologic lymphoid tissues and epidermis in man and several other species. The presence of S-100 protein served to distinguish these cells from other mononuclear cells, most importantly from those of macrophage/histiocyte lineage. Fractionation procedures to isolate and enrich suspensions of DCs were coupled with immunocytochemical techniques to identify S-100-positive cells. Langerhans cells in the epidermis and in aural cholesteatomata and nodal, splenic, and thymic interdigitating cells were S-100-positive. Lymph node and splenic follicular dendritic cells (except in rats) were negative, indicating that this DC may be a separate cell type.

Extravasation of macromolecules and possible trapping of transforming growth factor-beta in venous ulceration.

The pathogenesis of venous ulceration is thought to involve formation of pericapillary fibrin cuffs as a result of venous hypertension, and a recent hypothesis suggests that extravasated plasma proteins may bind or trap growth factors. We have compared the tissue distribution of fibrin cuffs, plasma proteins, procollagen, and transforming growth factors (TGF-beta 1 and TGF-beta 2) within venous ulcers and normally healing graft donor sites. In venous ulcers, the papillary dermis and the ulcer bed contained convoluted capillaries with phosphotungstic acid haematoxylin-positive pericapillary fibrin cuffs. By immunohistochemical staining, the cuffs were positive for actin, and contained massively redundant lamellae of basement membrane material which stained positive for type IV collagen. Extravasated factor XIIIa and alpha 2-macroglobulin were present within the fibrin cuffs. Increased numbers of type I procollagen positive fibroblasts, and increased TGF-beta 1 immunoreactivity were present within the fibrin cuffs, but not in the provisional matrix in the ulcer bed around the cuffs. In contrast, in normally healing graft donor sites, tortuous capillaries and fibrin cuffs were absent, factor XIIIa and alpha 1-macroglobulin were restricted to the lumina of vessels, and procollagen and TGF-beta immunoreactivity were present within the granulation tissue and adjacent dermal matrix at the wound margin. These observations suggest that growth factors critical in wound healing, such as TGF-beta, are present within venous ulcers, but are abnormally distributed. Their distribution within fibrin cuffs and co-localization with extravasated plasma proteins, particularly alpha 2-macroglobulin, which is a recognized scavenger molecule for TGF-beta and other growth factors, provides evidence for a possible 'trapping' of growth factors in venous ulcers.

Regulatory activity of endogenous and exogenous transforming growth factor beta in experimental intestinal immunopathology.

Acute graft-versus-host disease (GvHD) is an inflammatory disorder associated with generalised damage to epithelial tissues, including the gastrointestinal tract. There is increasing evidence that this pathology is due to the effects of cytokines on epithelial cell proliferation and differentiation. However, it is unclear whether factors derived from immune cells act directly on epithelial cells or via other mediators whose principal role is to regulate cell growth under normal or diseased conditions. We show here that the increased crypt cell turnover and lymphocytic infiltration which occurs in the jejunum of mice with graft-versus-host reaction (GvHR) is accompanied by decreased enterocyte expression of transforming growth factor beta 2. Administration of exogenous TGF beta inhibits the crypt hyperplasia of GvHR and reduces systemic manifestations of GvHR such as increased splenic natural killer (NK) cell activity. In parallel, neutralisation of endogenous TGF beta by monoclonal antibody exacerbates both the proliferative and inflammatory components of intestinal and systemic GvHR. Thus, the immune system may induce epithelial pathology at least in part by altering the production of endogenous TGF beta. This cytokine may therefore prove a useful focus for therapeutic intervention in immunopathologies such as GvHD.

Immunocytochemical localization and serologic detection of transforming growth factor beta 1. Association with type I procollagen and inflammatory cell markers in diffuse and limited systemic sclerosis, morphea, and Raynaud's phenomenon.

OBJECTIVE: To determine the presence of transforming growth factor beta 1 (TGF beta 1) and inflammatory cell markers (HLA-DR and Factor XIIIa) and to compare these with the presence of type I procollagen, in clinically uninvolved and involved skin from patients with different subsets of systemic sclerosis (SSc), and to analyze circulating levels of TGF beta 1 in SSc patients. METHODS: TGF beta 1, HLA-DR, Factor XIIIa, and type I procollagen were detected in skin biopsy sections using a biotin-streptavidin-peroxidase system. Levels of circulating TGF beta 1 were measured using a capture enzyme-linked immunosorbent assay technique. RESULTS: Patients with active diffuse cutaneous SSc (dcSSc) showed minimal TGF beta 1 but significant type I procollagen staining in involved skin, while the clinically uninvolved skin of these patients showed moderate extracellular and intra-epidermal TGF beta 1 immunoreactivity. Patients with limited cutaneous SSc (lcSSc) showed elevated TGF beta 1 staining in both involved and uninvolved skin, as well as procollagen staining. Significant TGF beta 1 reactivity, HLA-DR and Factor XIIIa immunoreactivity, numerous inflammatory cells, and procollagen staining were seen in specimens from patients with morphea. Sequential biopsies suggested the presence of cytokine activity at the earliest stages of disease, which was not maintained with progression of sclerosis. Among the disease groups studied, elevated levels of circulating TGF beta 1 were seen only in patients with morphea. CONCLUSION: The pattern of TGF beta 1 staining in dermal sections from patients with dcSSc, lcSSc, and morphea suggests that this cytokine is important in the pathogenesis of scleroderma. Furthermore, the presence of TGF beta 1 prior to the onset of fibrosis indicates an early involvement of this growth factor, possibly in the inflammatory stage of the disease.

Developmental expression of adenosine deaminase in placental tissues of the early postimplantation mouse embryo and uterine stroma.

In this study, we have investigated the distribution of adenosine deaminase (ADA) in embryonic, extra-embryonic, and decidual tissues of the developing mouse embryo. ADA catalyzes a key step in purine metabolism converting adenosine to inosine. ADA specific activity (nmol/min/micrograms protein) was present at low levels in the embryo-decidual unit during the first 2 days of postimplantation development but then increased starting late on Day 6 of gestation (Day 0 plug). By Day 9, ADA specific activity was 80-fold higher than on Day 6. A histochemical staining method for ADA activity was applied to cryostat sections of the implantation site. The developmental increase localized primarily to the trophoblast/antimesometrial decidua interface between Days 7 and 9 of gestation, and decidua basalis and the metrial gland by Day 11. Immunofluorescent staining with sheep anti-mouse ADA antiserum confirmed the presence of ADA antigenicity in tissues forming the maternal/fetal interface. ADA specific activity was 19-fold higher in homogenates of the Day 11 decidua/parietal yolk sac than in the thymus, a tissue generally thought of as ADA-rich. High levels of ADA activity and immunoreactivity were also detected in the embryonal plasma during organogenesis, but the embryo proper showed only low levels. These results indicate that ADA is tightly regulated within tissues forming the maternal/fetal interface during early postimplantation stages of development.

Abscess formation and local necrosis after treatment with Zyderm or Zyplast collagen implant.

The incidence, clinical presentation, pathophysiology, and possible treatment of two rare but clinically meaningful complications of tissue augmentation with Zyderm and Zyplast Collagen Implant are described. Abscesses as a manifestation of hypersensitivity to bovine collagen occur rarely (4 in 10,000 cases) and may persist for days to weeks. Periods of remission and exacerbation may occur from 1 month to more than 24 months. Localized tissue necrosis also occurs rarely (9 in 10,000 cases) after implantation and is probably the result of local vascular interruption and not hypersensitivity. The incidence varies greatly between the anatomic sites of implantation; more than half the reported cases involve the glabella. Evidence strongly suggests that the increased vulnerability of the glabellar region is due to its unique vascular distribution.

Developmental expression of adenosine deaminase in the upper alimentary tract of mice.

The distribution and localization of adenosine deaminase (ADA) was studied during postnatal development of the alimentary tract in mice. There was detectable enzyme activity in all organs examined, but a range of more than 10,000 fold in the relative levels of specific activity was observed among adult tissues. A comprehensive survey of multiple adult tissues revealed that the highest levels of ADA occur in the upper alimentary tract (tongue, esophagus, forestomach, proximal small intestine). Immunohistochemical analysis revealed that ADA was predominantly localized to the epithelial lining of the alimentary mucosa: the keratinized squamous epithelium that lines the forestomach, esophagus, and surface of the tongue; and the simple columnar epithelium of the proximal small intestine (duodenum, proximal jejunum). Biochemical analysis revealed that ADA was one of the most abundant proteins of these mucosal tissue layers, accounting for 5%-20% of the total soluble protein. Tissue-specific differences in ADA activity correlated both with levels of immunoreactive protein and RNA abundance. The level of ADA activity in the upper alimentary tissues was subject to pronounced developmental control, being low at birth and achieving very high levels within the first few weeks of postnatal life. The appearance in development of ADA-immunoreactivity coincided with maturation of the mucosal epithelium. These results suggest that ADA is subject to strong cell-specific developmental regulation during functional differentiation of certain foregut derivatives in mice.

Agents, biomarkers, and cohorts for chemopreventive agent development in prostate cancer.

Chemoprevention is the use of agents to slow progression of, reverse, or inhibit carcinogenesis thereby lowering the risk of developing invasive or clinically significant disease. With its long latency, high incidence and significant morbidity and mortality, prostate cancer is a relevant target for chemoprevention. Developing rational chemopreventive strategies for prostate cancer requires well-characterized agents, suitable cohorts, and reliable intermediate biomarkers of cancer. Chemopreventive agent requirements are experimental or epidemiologic data showing efficacy, safety on chronic administration, and a mechanistic rationale for activity. Current promising agents include antiandrogens and antiestrogens; steroid aromatase inhibitors; retinoids and their modulators; 5alpha-reductase inhibitors; vitamins D, E, and analogs; selenium compounds; carotenoids; soy isoflavones; dehydroepiandrostenedione and analogs; 2-difluoromethylornithine; lipoxygenase inhibitors; apoptosis inducers; and nonsteroidal anti-inflammatory drugs. Identifying biomarkers and validating them as surrogate endpoints for cancer incidence are critical for prostate chemoprevention trials. Potentially useful biomarkers for prostate chemoprevention are associated with histologic, proliferative, differentiation-related, biochemical, and genetic/regulatory features of prostatic disease. In that the prostate is not easily visualized, critical issues also include adequacy and consistency of tissue sampling. Various drugs for the chemoprevention of prostate cancer are now under evaluation in phase 1, 2, and 3 clinical trials. Cohort selection should be based on various patient characteristics (stage of the disease, previous cancers or premalignant lesions, or high risk factors) and should be conducted within the context of standard treatment.