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Biomedical subjects

T D Allen

Publications and source records attributed to T D Allen.

At least 91 records · Page 5Linked to original sources
PubMed1991

Cellular interactions in erythroblastic islands in long-term bone marrow cultures, as studied by time-lapse video.

Long-term bone marrow cultures (LTBMC) are readily converted from the usual granulopoietic to erythropoietic production by the addition of anemic mouse serum (AMS). The "statics" of proliferation and maturation, previously shown by ultrastructural methods to closely mirror the in vivo situation, were studied dynamically using a time-lapse video system. Several cell pedigrees were followed, but the most complete series showed three successive divisions and subsequent enucleations in the progeny of three synchronously mitotic cells observed in the culture; this is indicative of a five division sequence in the erythron. As in erythroblastic islets observed in marrow in vivo, the striking synchrony of maturation was maintained in vitro. Furthermore, when some of the erythroid progeny became displaced to other macrophages, the synchrony, which was maintained by the original erythroid group on the original erythroblastic islet macrophage, was lost. Time-lapse video, which is inexpensive to run and can be maintained in continuous recording for many weeks, is an ideal technique for recording both erythroid cell pedigrees, and the initial events leading to the formation of an erythroblastic islet in vitro after stimulation with AMS.

Animals↗
PubMed1990

Stromal cells in haemopoiesis.

Stromal cells of the bone marrow can provide the growth-promoting and differentiation-inducing molecules which are necessary for haemopoiesis. While the nature of these stimuli is largely unknown, the development of haemopoietic cells in association with stromal cells requires intimate cell contact. Molecules of the extracellular matrix, such as heparan sulphate, are able to bind growth factors and in this way the stromal cells may form microenvironmental niches which preferentially promote development of multipotent and committed cells along discrete lineages. Cells from some patients with acute and chronic myeloid and lymphoid leukaemias are defective in their ability to interact with stromal cells and consequently cannot survive in stromal cell-mediated long-term marrow cultures. We have exploited this phenomenon to obtain normal haemopoietic cells from patients with leukaemia, and to use these cells for successful autografting in patients with acute and chronic myeloid leukaemias.

Animals↗
PubMed1990

Ultrastructural morphometric study of efferent nerve terminals on murine bone marrow stromal cells, and the recognition of a novel anatomical unit: the "neuro-reticular complex".

In order to extend our understanding of the role of nerve fibers in the structure and function of bone marrow stroma, we have examined nerve terminals, arterioles, and capillaries in femoral bone marrow tissues of 50 C57BL strain mice, using electron microscopy and morphometric methods. Within the adventitia of arterioles, a particular type of cell, termed periarterial adventitial (PAA) cell, is characterized by a thin veil-like cytoplasm which concentrically surrounds both nerves and arterioles. Nerve fibers containing both unmyelinated and myelinated axons are distributed mainly between the layers of PAA cells, but are found rarely on the sinus walls or within the hematopoietic parenchyma. Quantitatively, the efferent nerve terminals with many synaptic vesicles are distributed mainly beside arterial smooth muscle cells (Type I: 58.8%) or between the layers of PAA cells (Type III: 33.2%), and rarely in hematopoietic parenchyma (Type II: 5.3%) or on sinus walls (Type IV: 2.7%). In the case of Type II-IV nerve terminals, efferent (autonomic) nerves and bone marrow stromal cells which are connected by gap junctions (sinus adventitial reticular cells, intersinusoidal reticular cells, and PAA cells) appear to constitute a potential functional unit for signal conduction. We would like to propose a new term for this anatomical unit in marrow, the "neuro-reticular complex."

Animals↗
PubMed1990

Prostatomembranous urethral disruptions: management by suprapubic cystostomy and delayed urethroplasty.

Management of prostatomembranous urethral disruptions associated with pelvic fractures remains a major controversy in urology. A group of 64 patients who suffered a prostatomembranous urethral disruption in association with a pelvic fracture and who were managed initially by suprapubic cystostomy with delayed urethroplasty was compared to 17 patients managed initially by primary realignment. No statistically significant difference in the incidence of impotence or urinary incontinence was found between the 2 groups (p greater than 0.5) Secondary reconstructions for impassable strictures developed in 95% of the patients treated by a suprapubic tube alone compared to 53% of those treated by primary realignment. Indeed, only 1 patient in the latter group achieved urethral continuity that did not require further intervention. We conclude that while primary realignment is associated with no increase in the instance of impotence and urinary incontinence, it subjects the patient to a major operation at a critical time and provides little in the way of long-term positive gains for the effort expended. In the final analysis the outcome is more dependent upon the nature of the injury and the quality of the repair than upon the order in which the repair is effected.

Cystostomy↗
PubMed1990

Modulation of extracellular matrix biosynthesis by bovine retinal pericytes in vitro: effects of the substratum and cell density.

Bovine retinal pericytes plated on a two-dimensional substratum display a characteristic stellate morphology. In post-confluent cultures these cells aggregate spontaneously to form multicellular nodules. The same cells plated within a three-dimensional collagen matrix display an elongated sprouting morphology. Sprouting pericytes may be embedded within a gel either as individual cells or as multicellular aggregates. We have compared the nature of the matrix proteins synthesised by pericytes displaying these different phenotypes. Stellate pericytes cultured on plastic dishes synthesised predominantly type I collagen, some type III collagen and only traces of type IV collagen. The same collagen types were secreted when nodules had formed in postconfluent cultures on plastic, and by sprouting cells plated as single cells within the collagen gel. By contrast, sprouting pericytes plated as aggregates within the collagen gel secreted increased levels of type IV collagen and reduced amounts of type I collagen. Fibronectin was synthesized by pericytes under all experimental conditions examined; thrombospondin was produced in relatively large amounts by cells grown on plastic dishes, whereas only trace amounts could be detected in the medium when the cells were cultured within a collagen gel matrix. Transmission electron microscopy revealed that pericyte aggregates within a collagen gel contained cells in close apposition surrounded by a dense extracellular matrix. In contrast, cells in the centre of a nodule on plastic appeared to be separated from each other by loose extracellular material. These results suggest that the morphological and biosynthetic phenotypes of retinal pericytes are modulated by cell-matrix and/or cell-cell interactions.

Animals↗
PubMed1990

Pericytes derived from the retinal microvasculature undergo calcification in vitro.

Pericytes isolated from the bovine retinal microvasculature retain characteristic features of their in vivo counterparts, such as the presence of glycogen deposits, long filamentous processes, prominent microfilament bundles and the ability to display two distinct and reversible phenotypes. Time-lapse video-microscopy demonstrated that pericytes tend to overlap and aggregate, even in sparse cultures. After reaching confluence, they form multilayered areas that retract away from each other, resulting in the formation of multicellular nodules. These nodules increase in size and cellularity by going through repeated 5- to 6-h cycles of anchoring, spreading, cell proliferation and retraction. Alkaline phosphatase was not detected in pericytes at subconfluent or confluent densities, but this enzyme was expressed in areas of high cell density, such as multilayers and nodules. Pericytes synthesise and deposit an extracellular matrix at all stages of their in vitro development, including nodule formation. The matrix within the nodules contains cross-striated collagen fibres and matrix vesicles. Needle-like crystals of hydroxyapatite appear to be deposited within the matrix, thus leading to massive calcification of the nodule. Calcification, as assessed by electron microscopy, histochemical staining and X-ray microprobe analysis, occurred on plastic and collagen substrate in the absence of disodium-beta-glycerophosphate. The addition of this compound at 5 or 10 mM or the use of a collagen substratum (rather than plastic), brought forward the process of nodule formation and calcification by 3-6 days. Our results suggest that retinal pericytes may differentiate in vitro along the osteogenic pathway.

Animals↗
PubMed1989

Cellular interactions between 3T3 cells and interleukin-3-dependent multipotent haemopoietic cells: a model system for stromal-cell-mediated haemopoiesis.

With the aid of a multipotent stem cell line (FDCP-mix cells) co-cultured with either normal or irradiated Swiss 3T3, cellular interactions between stromal cells and haemopoietic stem cells were studied by electron microscopy and time-lapse video microscopy. When cultured in the presence of interleukin 3 (IL-3) but in the absence of stromal cells, the FDCP-mix cells have a characteristic blast morphology. In the absence of IL-3, the cells die unless they are co-cultured with marrow stromal cells or 3T3 cells. In the latter case, they attach, proliferate, and differentiate on both normal and irradiated Swiss 3T3 cell layers without the addition of extrinsic growth factor (IL-3). At the initial attachment sites of these two cell lines, cellular recognition seemed to be mediated by the formation of microvillus cytoplasmic projections and extracellular matrix. These areas may well be the sites of plasma-membrane-bound signalling/adhesional molecules between the interacting cells.

Animals↗
PubMed1989

Ureteropelvic junction obstruction associated with ureteral hypoplasia.

We present 3 cases in which a neonate had unilateral hydronephrosis associated with a ureter hypoplastic throughout its length. In all 3 infants the affected kidney failed to visualize on excretory urography and function of the kidney proved to be disappointing despite its relatively normal appearance on external examination. In each case significant renal dysplasia was present microscopically and ultimately the kidney had to be removed. A retrograde pyelogram should be considered whenever a hydronephrotic kidney in an infant fails to visualize, and if this shows a long hypoplastic ureter that cannot be bypassed easily associated renal dysplasia should be suspected and consideration given to nephrectomy rather than pyeloplasty as the primary treatment modality.

Female↗
PubMed1989

Fine-structural aspects of bromodeoxyuridine incorporation in sister chromatid differentiation and replication banding.

The structure of harlequin-stained chromosomes following substitution with low levels of 5-bromodeoxyuridine (BrdUrd) over two cell cycles and high levels over the last part of one cycle (replication banding) was studied in Chinese hamster ovary (CHO) cells. By using correlative light (LM) and scanning electron microscopy (SEM), it was shown that the effects of both the ultraviolet light (u.v.) and hot SSC treatment steps of the harlequin staining procedure were necessary to obtain sister-chromatid differentiation (SCD) or replication banding. u.v. treatment alone resulted in dark Giemsa staining of both chromatids with SEM morphology of short compact protuberances and an overall flattened smooth appearance in both the unsubstituted and BrdUrd-substituted chromatids, a morphology essentially similar to that of untreated chromosomes. SSC alone on the other hand resulted in dark-staining chromatids with an SEM morphology of raised, loosely packed loops of fibres in both types of chromatids. u.v. and SSC treatment together resulted in differentiation, with dark-staining unifilarly (TB) chromatids in the LM corresponding to raised loosely packed loops in the SEM and pale bifilarly (BB) chromatids corresponding to the smooth compact flattened SEM appearance. Where the BrdUrd-substituted strand became the template (BT), or when the nascent strand TB contained high levels of BrdUrd substitution in replication banding, the chromatid stained pale and showed the compact smooth appearance in the SEM. The Giemsa staining ability and ultrastructural morphology of harlequin staining is discussed with respect to putative DNA loss and also in terms of preferential protein-protein, protein-DNA cross-linkage in BrdUrd-containing DNA. These changes are also compared with the ultrastructural morphology observed after other banding methods, where deterioration of protein and DNA-protein interaction resulting in aggregation of chromatin fibres appears to be the major mechanism.

Animals↗
PubMed1989

The translaminal fibrils of the human amnion basement membrane.

The organisation of extracellular matrix beneath the human amniotic epithelium was investigated in order that the co-ordinate synthesis of basal lamina and stroma by these cells could be better understood. Transmission electron microscopy of intact tissue confirmed that stromal matrix fibrils are located between the cell surface and the basal lamina, and also penetrate the lamina. The distribution of the supralaminal fibrils and their association with the lamina was further investigated by scanning electron microscopy (SEM) after removal of the overlying epithelium. Five complementary procedures were used to remove the cells from the underlying lamina. Trypsin-EDTA treatment caused the epithelial cells to retract or detach from the lamina. SDS or ammonium hydroxide was used to extract the epithelium, which was then removed by physical shearing. Transmission electron microscopy (TEM) confirmed that the lamina densa and supralaminal fibres were present after extraction by these agents. Incubation in CHAPS, a zwiterionic detergent, did not remove the epithelium but permitted exposure of the basal lamina by mechanical scoring. Extraction with boric acid followed by osmium tetroxide produced epithelial disruption and revealed the lamina and stroma in different areas. Although the extraction pattern was different in each case, all of the five methods confirmed that individual fibrils and fibril bundles are present on the apical surface of, and enter, the lamina densa. Examination of the stromal surface of the basal lamina after fracture revealed fibrils passing from the stroma into the lamina densa. We therefore suggest that, in this tissue, newly synthesised stromal matrix components appear in an assembled fibrillar form between the basal cell surface and the basal lamina before becoming associated with the sublaminal stroma.

Amnion↗
PubMed1989

The organization and substructure of chromatin fibres in the interphase nucleus as studied by scanning electron microscopy.

The high packaging ratio of DNA in both interphase nuclei and metaphase chromosomes presents great difficulties to our understanding of the three dimensional organisation of processes such as DNA replication and transcription in the nucleus. Although the higher order structure of DNA, in terms of the way it is organised into the unit fibre of chromatin has received much attention over the last decade, the highest levels of packaging of chromatin in both nuclei and chromosomes have hardly begun to be elucidated. Much of the difficulty in investigating fibre organisation with conventional methods is the inherent two dimensional nature of sectioned or spread material in the transmission microscope. Three dimensional imaging from the SEM has, until recently, been limited by the available resolution. Our own previous studies of chromosome structure have shown that a combination of 'in lens' imaging combined with the high signal generation imparted by osmium impregnation have been adequate to routinely visualise chromatin fibre organisation in metaphase chromosomes, and the changes that occur as a result of a variety of banding techniques. More recent experiments using the same techniques on interphase nuclei extracted from a variety of tissue culture cells have indicated that Scanning electron microscopy of nuclei is a potentially useful technique for studying chromatin organisation, which may be made more accessible by a variety of biochemical extraction methods.

Animals↗
PubMed1989

Long-term bone marrow cultures: their use in autologous marrow transplantation.

When cultured together, hemopoietic cells and marrow stromal cells can support hemopoiesis in vitro in the absence of added growth factors. The continued production of stem cells, progenitor cells, and mature cells within these "long-term bone marrow cultures" (LTBMC) requires their physical association with the stromal cells. For reasons that are not well understood, leukemic cells often fail to survive in such cultures. We and others have undertaken studies to examine whether LTBMC can be exploited as a way of purging the bone marrow of leukemia patients prior to autologous marrow transplantation.

Bone Marrow Cells↗
PubMed1989

The use of scanning electron microscopy for investigations into the three dimensional organisation of the interphase nucleus.

In an attempt to elucidate three dimensional information on the organisation of the nucleus, nuclei have been extracted from ascites tumour cells or tissue culture cells by a variety of biochemical techniques, and prepared for high resolution scanning electron microscopy using an osmium-thiocarbo-hydrazide infiltration procedure which has previously proved successful for analysis of chromosome structure. Nuclei were prefixed with either Methanol-Acetic acid, glutaraldehyde or formaldehyde and then extracted by a variety of detergents with the aim of a 'biochemical dissection' of their outer elements to allow surface visualisation of the nuclear lamina. Continued extraction removed all aspects of the nuclear periphery and allowed direct visualisation of the in situ organisation of the chromatin, apparent as at least two levels of supercoiling.

Animals↗
PubMed1989

A comparative morphometric study on the ultrastructure of adherent cells in long-term bone marrow culture from normal and congenitally anemic mice.

The relationship between structure and function of bone marrow stromal tissue in adherent layers of long-term bone marrow cultures (LTBMCs) from normal and congenital anemic mice (C57BL, Sl/Sld, Sl+/Sl+, W/Wv, and W+/W+) was investigated. Many previously reported features were confirmed. However, in LTBMC from all strains of mice examined, isolated cilia with the axonemal structure of a 9 + O pattern with obvious dynein arms were observed in the blanket cells. The frequency of cilia was approximately 2%-5% of total number of profiles of blanket cells examined. Crystalloid inclusions (CI) were observed in cultured macrophages similar to those reported in vivo in all strains of murine LTBMC. The CI could be classified into four types according to their structure in the same way as in vivo (type A to type D), with a predominance of type A in the cultures. Viral particles were also apparent in adherent cells of all strains (except W/Wv and W+/W+), which were compatible with a type C retrovirus. Gap junctions occurred regularly between the adherent cells of LTBMC, particularly between blanket cells and preadipocytes. The most frequent appearance of gap junctions was found in Sl/Sld cultures. The phenomena of normal and abnormal hematopoiesis appear to be accurately reproduced in culture, thus retaining the same relationship between function and structure as occurs in vivo. The surface of isolated cilia of blanket cells, CI of macrophages, viral particles among adherent cells, and gap junctions between blanket cells and preadipocytes is discussed.

Anemia↗
PubMed1988

Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis.

The proliferation and development of haemopoietic stem cells takes place in close association with marrow stromal cells. This intimate cell contact presumably enables the stem cells and their progeny to respond to stimuli present on the stromal cell surface. While the nature of these stimuli has not been determined, it is likely that growth factors play some role. Recently, it was demonstrated that the natural and the recombinant haemopoietic growth factor, granulocyte/macrophage colony stimulating factor (GM-CSF), could be adsorbed out of solution by an extract of human marrow stromal extracellular matrix (ECM) with retention of biological activity. However, the precise ECM molecules involved were not identified. Here, we clearly demonstrate that the major sulphated glycosaminoglycan of mouse marrow stroma, heparan sulphate, possesses the ability to adsorb both GM-CSF and the multilineage haemopoietic growth factor, Interleukin 3 (IL-3). Furthermore, these growth factors, once bound, can be presented in the biologically active form to haemopoietic cells.

Adsorption↗
PubMed1988

Haemopoietic growth factors: their relevance in osteoclast formation and function.

The major recent advance in our knowledge of the haemopoietic system has been the purification and characterization of a family of haemopoietic growth factors, and their availability in recombinant form. In the bone marrow the sequences of differentiation and proliferation leading to the production of mature cells that these factors regulate may be determined by the relative availability of the factors in microenvironmental domains. The observation that growth factor-producing cells and haemopoietic progenitor cells are not evenly distributed in the bone marrow leads us to expect that the overall effect of growth factors (and other regulatory molecules) on the production and function of macrophages and osteoclasts may differ when in vivo or in vitro assays are used as end-points and, in the latter case, when whole marrow or purified cell populations are tested. The availability of an in vitro assay in which osteoclast-like cells are generated will allow these concepts to be tested.

Animals↗
PubMed1988

Expression of a secretory product by microvillous and ciliated cells of the human endometrial epithelium in vivo and in vitro.

A monoclonal antibody which identifies a component of post-ovulatory endometrial secretions is now shown to be expressed within the cytoplasm and on the cell surface of both microvillous and ciliated epithelial cells. A glandular explantation model was developed in order to study the 'carry over' of this secretion to the regenerative phase endometrium. A loss of cytoplasmic antigen was observed in vitro. However, it was retained on the cell surface in a fashion consistent with its expression at the time of explantation. Mosaicism of expression of this secretory component occurs throughout the secretory-phase and is particularly pronounced at the time of transition from proliferative to secretory phase. It is concluded that both ciliated and microvillous epithelial cells produce a post-ovulatory secretory component which may be retained on the cell surface in the absence of hormonal stimulation.

Cells, Cultured↗