PubMed HealthSearch

Biomedical subjects

W E Thomas

Publications and source records attributed to W E Thomas.

At least 19 recordsLinked to original sources

Characterisation of ramified microglial cells: detailed morphology, morphological plasticity and proliferative capability.

Several cellular properties of brain microglia in the rat were investigated using both whole tissue and cultures of dissociated cerebral cortical cells. As revealed by thiamine pyrophosphatase histochemistry, tissue microglia possessed a highly distinctive cellular morphology. Stained microglia showed similar overall features of morphology and distribution in both preparations; however, the cells in culture displayed some slight differences from those of the tissue, including larger somata and less developed processes. Through studying living ramified cells in culture, both morphological plasticity as evidenced by patterned variations in soma size and mitotic activity were directly confirmed. It was concluded that ramified microglia definitely possess proliferative capability, and this may reduce the need for blood cell recruitment in brain immune responses. In addition, cultured microglia exist in a somewhat more activated state than those in normal tissue, and in some instances undergo further activation as macrophages. This cortical tissue culture system should provide an amenable preparation for investigating the regulation of microglial function.

Animals

Evidence for motility and pinocytosis in ramified microglia in tissue culture.

Ramified microglial cells were investigated in primary cultures of dissociated cerebral cortical tissue from rats. The identification of these cells was confirmed through immunohistochemical staining with 7 monoclonal antibodies selective for microglia. While there was significant variation in staining intensity with different antibodies, all stained the identified ramified cells; the antibodies OX-42 and ED1 yielded the most intense immunoreactivity. Based on distinctive morphological features, the microglia could be identified in living cultures where they were monitored using time-lapse video recording. This technique revealed extremely dynamic features of cellular plasticity and motility. Ramified microglia exhibited constant and rapid alterations in the size and shape of their cell body with an associated extension and retraction of processes; concomitantly, the cells moved about in a circumscribed area. These features of plasticity and motility were unique to this cell type, and correlated with OX-42 immunostaining. The microglia also possessed a differentially high level of pinocytotic activity; this too was correlated with OX-42 staining. From the nature of their morphological plasticity and motility, high pinocytosis, and cellular distribution, it is hypothesized that the ramified microglia specifically function as a system of fluid cleansing in normal brain tissue.

Animals

Pinocytotic activity in ramified microglia.

Pinocytotic activity was investigated in rat cerebral cortex using the soluble tracers horseradish peroxidase and Lucifer yellow. A subpopulation of cells selectively accumulated both compounds and the labelling was mainly present in pinocytotic vesicles associated with the cell body. Labelled cell bodies were small, round to oval, and distributed in an almost regular array throughout the tissue. Based on distinctive morphological features, some of the labelled cells could be determined as ramified microglia. This identification was confirmed by immunofluorescence staining with the monoclonal antibody OX-42, which specifically recognizes microglia; OX-42 staining consistently co-localized with pinocytotic labelling. The possibility that ramified microglial cells perform a normal function of continuous fluid exchange in brain tissue is discussed.

Animals

Characterization of ramified microglia in tissue culture: pinocytosis and motility.

Functional properties of ramified microglia were investigated in primary cultures of rat cerebral cortical cells. These microglia could be readily identified in both fixed and living cultures through previously established features. Based on their destruction by 5 mM L-leucine methyl ester, a high level of intrinsic endocytotic activity was established. When cultures were incubated with fluorescent latex beads to assess phagocytosis, little or no such activity was exhibited by ramified cells. However, when cultures were incubated with dyes or other soluble tracer compounds, these cells always exhibited labeling. This labeling was selective for ramified microglia in the cultures and was demonstrated using a variety of compounds, including trypan blue, lucifer yellow, horseradish peroxidase (HRP), and India ink. Intracellular label could be observed in vesicular structures; this localization corresponded to an active cellular process. Also, cellular labeling was inhibited by the presence of colchicine. These features supported the inference that the labeling was attributable to pinocytosis, and this process appeared to account for the vast majority of endocytotic activity in the ramified microglia. Possible physiological significance of this pinocytotic activity was indicated by the accumulation of various neurotransmitters/modulators: gamma-aminobutyric acid and vasoactive intestinal polypeptide (VIP). Ramified cells in these cultures have been previously noted to exhibit a constant and rapid pattern of motility, which was consistently observed here through time-lapse video recording; pinocytosis and rapid motility were shown to concur in individual cells. Based on their high intrinsic pinocytotic activity and pattern of cellular motility, the ramified microglia specifically are suggested to serve a constitutive function of fluid cleansing within the interstitial spaces of brain tissue.

Animals

Dynamic features of cells expressing macrophage properties in tissue cultures of dissociated cerebral cortex from the rat.

Two previously identified forms of macrophage were investigated in primary cultures of cerebral cortical cells. Dynamic features were revealed through time-lapse video recording and aspects of macrophage function were assessed. The two cell forms were shown to be different pre-mitotic stages of a single cell type. The cell cycle for these cells involved an initial large, flat, quiescent cell which retracted to yield a slightly rounded form with numerous processes. This latter form lost processes and developed profuse filopodia as it became very rounded just prior to division; both resulting daughter cells then regained the initial large flat appearance. These cells possessed several properties of macrophages, including phagocytosis, nucleoside diphosphatase enzyme, and CR3 receptors. These properties were transient, expressed just before and after mitosis, but subsequently down-regulated in the flat daughter cells. Because of this feature, it was difficult to determine the exact size of this cell population; however, the observed rate of proliferation suggests it may be substantial. It is suggested that these cells correspond to non-microglial macrophages of brain tissue and, because of their significant down-regulation, they may be difficult to detect. This may be important in studies of brain accessory immune cells in tissue culture.

Animals

Factors influencing the establishment of tumour-infiltrating lymphocyte cultures from human breast carcinoma and colon carcinoma tissue.

Tumour-infiltrating lymphocytes (TIL) were obtained from breast and colon tumour tissue and cultured in vitro in the presence of recombinant human interleukin-2. Seven of 35 breast tumours and five of 41 colon TIL cultures were established in vitro: proliferation rates of greater than 10(3) were achieved. The cytotoxic capacity of these cells was determined against the cell lines K562 and SW742, and percentage cytotoxicity levels of greater than 97% and 79%, respectively, were seen. An inverse relationship between the ability of TIL to kill and their proliferative capacity was observed in all cultures. The prominent phenotype was CD3 positive, with greater than 55% of TIL expressing this antigen; there was no expression of CD16. The expression of CD56 and CD25 varied, being maximumly expressed on 64% and 38% of TIL, respectively. When greater than 90% of TIL expressed CD3, the ability of the culture to kill the target cell lines was low; only when there was an increase in the proportion of cells expressing CD56 and a decrease in the expression of CD3 was there high cytotoxicity. This study indicates that the TIL which proliferate in vitro in the presence of interleukin-2 are not necessarily the mediators of cytotoxicity.

Adult

Loss of monomorphic and polymorphic HLA antigens in metastatic breast and colon carcinoma.

MHC class I antigens are intimately involved in intercellular communication, and recognition by cytotoxic T cells. Thus tumour cells that fail to express them may be at a growth or metastatic advantage. A series of ten colorectal and ten breast carcinomas, and their respective lymph node metastases, were examined immunohistologically using monoclonal antibodies (mAb) against both monomorphic and A2 polymorphic determinants, and beta-2-microglobulin (beta 2m). Four colon polypoid adenomas stained positively throughout, but 6/10 primary tumours had partial or complete loss of expression of monomorphic determinants using mAb W6/32: two node and the liver metastasis showed less, four more expression. Similar results were seen for beta 2m. HLA-A2 expression was absent or reduced in 4/4 colon tumours and all their metastases. Among the breast tumours, W6/32 staining was absent or reduced in 2/10, and node deposits showed two with less reactivity than their primary. Beta 2m staining was reduced or absent in 8/10 primaries and all the node metastases; in every case in which beta 2m was detected in the primary tumour their corresponding lymph node metastasis showed a decreased expression. HLA-A2 expression was absent or reduced in 3/4 primary breast carcinomas, and all their metastases. These results show that individual human colon and breast carcinomas often have a reduced HLA class I antigen expression, which apparently confers a metastatic advantage.

Adenoma

Partial splenectomy for massive splenomegaly secondary to Gaucher's disease.

A 16 year old girl with Type 1 Gaucher's disease presented with massive splenomegaly, hypersplenism and abdominal discomfort. Traditionally hypersplenism has been treated with splenectomy, but this results in a high incidence of overwhelming sepsis and accelerated sphingolipid deposition in both liver and bone. A 90% partial splenectomy was therefore performed leaving a fully vascularized inferior segment of the spleen and resecting 5.8 kg of splenic tissue. The patient made an uneventful recovery with a marked improvement in her haematological parameters and general condition.

Adolescent

Pinocytosis as a select marker of ramified microglia in vivo and in vitro.

Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.

Animals

Method for the identification of brain macrophages/phagocytic cells in vitro.

The use of labelled latex beads, with acute incubation conditions, for the identification of active macrophages in mixed cell cultures based on their phagocytic activity is described. Fluorescent beads provided the best results and selectively labelled active macrophage cells in cultures of blood monocytes. When this technique was applied to primary cultures of rat cerebral cortex, a specific cell type was significantly labelled. This was a small round cell, previously uncharacterized, which in addition to phagocytic activity indicated by the ingestion of beads also possessed macrophage biochemical markers. Thus, the procedure appears useful for phagocytic macrophage identification in vitro, and should be generally applicable to any tissue culture system. Additionally, this procedure can be rendered compatible with cell viability and, therefore, be utilized for long-term monitoring of macrophages.

Animals

Characterization of the dynamic nature of microglial cells.

Dynamic properties of ramified microglia were investigated in tissue cultures of rat brain cells. Microglia were identified in viable cultures based on previously determined aspects of their morphology and location, and this identification was confirmed through immunohistochemical staining for type-three complement receptors. When living microglial cells were monitored using time-lapse video recording, an extremely dynamic nature was revealed. These cells exhibited morphological alteration, surface and cytoplasmic activity, and migration. All of these properties were both constant and rapid, with individual cells being capable of assuming a completely different overall morphology within a period as short as 5-10 min. These dynamic properties were consistently observed in microglia and appeared specific to this cell type. Thus, ramified microglia appear to possess a unique and highly dynamic nature, and this feature is proposed as a potential marker for these cells. Additionally, the significance of microglial cellular dynamics in the functional role of this cell population in vivo is considered.

Animals

Morphological diversity of ependymal cells in tissue culture.

Ependymal cells were visualized in primary cultures of cerebral cortex from rat using an immunohistochemical staining technique. Five different morphological subtypes of cuboidal ependyma were recognized: 1) round, 2) triangular, 3) columnar, 4) cone- and spindle-shaped, and 5) large pleomorphic cells. These cells varied in size and almost all possessed cilia. Two distinct forms of tanycyte ependyma were detected based on the presence of cilia. These features reflect a significant level of development of the ependymal cells in culture and may correspond to functional diversity within this group.

Animals

Squamous cell carcinoma of the stomach following gastric surgery.

The development of adenocarcinoma in the gastric remnant following surgery for peptic ulceration is well recognised. However, squamous cell carcinoma of the stomach is rare, with only 80 cases being reported in the literature and none reported following gastric surgery. We report a case in which a squamous cell carcinoma with only minute areas of adenocarcinoma developed 22 years after surgery for a duodenal ulcer.

Adenocarcinoma

Gastrointestinal malignancies as a complication of chronic myeloid leukaemia.

A 70-year-old patient is presented who, after chemotherapy for chronic myeloid leukaemia, developed two separate gastrointestinal malignancies. His family history indicated a strong family tendency towards neoplasia, and it is speculated that this predisposition may have been potentiated by the chemotherapy or the leukaemic process itself. It is stressed that, in such cases of multiple malignancy, each condition should be treated on its own merits.

Adenocarcinoma

Further studies on the identification of microglia in mixed brain cell cultures.

The investigation of brain microglia in primary cultures of dissociated cerebral cortical cells has been continued here utilizing a histochemical procedure for thiamine pyrophosphatase, which selectively stains microglia in whole tissue. This procedure also selectively stained a subpopulation of cells in the cortical cultures. The stained cells were small and exhibited quite variable morphology; from these features, a corresponding cell population could be identified in viable cultures. The stained cells, which were distinct from previously identified macrophage forms, were also distinguished from similar appearing cell types--small neurons and oligodendrocytes. Based on their staining properties, morphology, and distinction from other cell types, the identified cells are proposed as equivalents of ramified microglia.

Animals