Biomedical subjects
Publications and source records attributed to P Monaghan.
A malignant rhabdoid tumour of the bladder is reported from a girl aged 6. Detailed immunohistochemical and ultrastructural investigations provide evidence which suggests a histiocytic origin for this controversial neoplasm.
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An antibody, 21N, raised against a synthetic peptide from the predicted sequence of the c-erbB-2 protein has been used immunocytochemically in a retrospective study of formalin fixed paraffin embedded breast biopsies. Fourteen out of 103 infiltrating ductal carcinomas exhibited positive membrane staining. Fifty-four of these tumours had lymph node involvement of which nine contained stained cells. These were all cases where the primary tumour was positive. In this series there was no correlation between c-erbB-2 overexpression and lymph node status. In five of the positive cases studied there was an associated in situ component which was also positively stained. Ten out of 24 pure intraduct carcinomas showed membrane staining, but none of the 149 benign conditions studied, which included 22 radial scars and 13 cases of atypical ductal proliferation, demonstrated the pattern of staining associated with overexpression. It is concluded that the c-erbB-2 protein is overexpressed in a minority (approximately 14%) of infiltrating ductal carcinomas and only in cells that are cytologically malignant. Overexpression of c-erbB-2 is considered in relation to pathogenesis.
We have estimated the frequency of synonymous (KS) and amino acid replacement nucleotide substitutions (KA) among gag and env genes of published HIV sequences. The ratio of KS to KA can be used as an indicator of the intensity of the selective constraints on the amino acid sequence of a protein. By this approach we have shown that for both gp120 and gp41, the rate of change in amino acid sequence relative to the overall rate of change in nucleotide sequence is higher than for any other protein yet analyzed. The gag proteins p15 and p17 evolve slightly less rapidly, but p24 is relatively strongly conserved. We have compared the env gene of HIV with those of two other retroviruses for which appropriate data are available. In neither murine leukemia virus nor feline leukemia virus do the envelope glycoproteins evolve particularly rapidly. These results suggest that HIV is unusual among retroviruses in that both gp120 and gp41 are evolving under extremely weak selective constraints relative to the rest of the viral genome; accordingly, much of the observed variation may be selectively neutral.
Human embryonal carcinoma cells sometimes display the developmental potential of early embryonic stem cells. While available data do not clearly identify a counterpart of these tumor cells in normal development, previous comparisons of human embryonal carcinoma and yolk sac carcinomas indicated that these cell types are closely related, and suggested that embryonal carcinoma cells might resemble the progenitors of extraembryonic endoderm. To analyse further cell-differentiation lineage in these tumors, we produced monoclonal antibodies to cytostructurally associated antigens of human embryonal carcinoma cells. Spleen cells from mice immunized with a detergent-insoluble extract of cultured human embryonal carcinoma cells were fused to NS-1 myeloma cells, and hybridoma supernatants were screened by indirect immunofluorescence on the immunizing cell line, then on a panel of cell lines derived from human embryonal carcinomas, yolk sac carcinomas, and a range of neoplastic and normal tissues. Monoclonal antibody GCTM-1 stained the nuclei of all human cells tested and served as a positive control; this antibody immunoprecipitated proteins of 85 and 66 k Da from human embryonal carcinoma cells. GCTM-2 recognized an epitope on a 200-k Da extracellular protein present on the surface of embryonal carcinoma cells, and stained the surface of visceral yolk sac-type carcinoma and colorectal carcinoma cells as well. Enzymatic analysis of carbohydrate residues on the GCTM-2 antigen revealed that it was a keratan sulphate proteoglycan, and suggested that the epitope recognized by the antibody lies on the core protein. In immunoblots, antibody GCTM-3 bound to a 57-k Da cytoskeletal protein expressed in human embryonal carcinoma. This antibody decorated filamentous arrays in cell lines from human embryonal carcinoma, visceral yolk sac carcinoma, parietal yolk sac carcinoma (endodermal sinus tumour), and adenocarcinoma and large cell carcinoma of the lung. Antibody GCTM-4 recognized a determinant present on a 69-k Da polypeptide, associated with a component of the lysosomal compartment, which was expressed in embryonal carcinoma cells, but no other cell type tested. The results with this antibody panel thus allow distinction between human embryonal carcinoma and yolk sac carcinoma, but provide further evidence of a close relationship between these cell types.
The myoepithelial-type cell line, Rama 712, derived from a normal rat mammary gland, deposits an extracellular matrix containing type-IV collagen and other basement membrane proteins round its cellular periphery. After transformation with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV) the cells fail to deposit an extracellular matrix at the permissive temperature (35 degrees C), but retain the capacity to do so at the non-permissive temperature (41 degrees C). The synthesis of type-IV collagen is not affected by the temperature shift. Rama 712 cells fail to form tumours in syngeneic rats. However, Rama 712-tsRSV cells form tumours that are locally invasive but fail to metastasize. In histological sections, the tumour cells stain with an antibody to type-IV collagen, but do not deposit any extracellular type-IV collagen. Cells isolated from the tumours (Rama 712T) remain temperature-sensitive for the extracellular deposition of type-IV collagen when grown in vitro. Rama 712, Rama 712-tsRSV and Rama 712T fail to produce any detectable type-I or type-IV collagenase at either 35 degrees C or 41 degrees C. These results show that in this system extracellular deposits of basement membrane proteins are lost from invasive tumours produced by myoepithelial-type cells by mechanisms other than those due to the production of collagenolytic enzymes.
Fragments of human breast epithelium, devoid of all stromal and basal lamina components, which maintain their in vivo topological organisation can be cultured for up to 28 days within a reconstituted rat-tail-derived collagen matrix. These organoids initially undergo a loss of structural and 3-dimensional organisation, typified by loss of lumina formed by epithelial cells, and myosin from myoepithelial cells. Their subsequent reorganisation is dependent on the presence of serum, insulin, hydrocortisone, and cholera toxin in tissue culture medium. After this preliminary phase, a reduction in the concentration of serum, insulin, hydrocortisone, and cholera toxin is necessary to allow the structural differentiation of epithelial and myoepithelial cells. The myoepithelial cells also regain their ability to produce the basal lamina component laminin. The use of bovine-dermal collagen as the matrix, rather than rat-tail-derived collagen is shown to result in more stable organisation and differentiation of the organoids. The successful use of single-cell pellets (derived by trypsinisation of the organoids) in place of organoids in such cultures illustrates that there is no requirement for pre-existing cell/cell contact or topological organisation of cells prior to embedding within the collagen matrix.
This technique for fabrication of a semirigid custom cementation aid is quick, fairly simple, and the device is easy to use. Patients have reported no discomfort when this device has been used, and accepted clinical measurement standards indicate that this device performs effectively.
Fragments of ductal and lobular epithelium ('organoids') produced by collagenase digestion of reduction-mammoplasty specimens were injected into athymic 'nude' mice. These heterospecific tissues were accepted without rejection, and the presence of xenografts was confirmed by cytology, immunocytochemistry and chromatin staining. Lactation, as confirmed by immunocytochemical and ultrastructural criteria, was observed in the grafted human epithelium during murine pregnancy at both intra- and extra-mammary sites.
The cloned cuboidal epithelial cell line Rat Mammary (Rama) 25 converts at low frequency in culture to elongated cells that possess some of the properties of myoepithelial cells; one such clonal cell line is termed Rama 29. Three morphologically intermediate clonal cell lines have been isolated from Rama 25 which form a morphological series in the order: Rama 25 cuboidal cells, Rama 25-Intermediate 2(I2), Rama 25-I1, Rama 25-I4, and Rama 29 elongated cells. This same order is largely maintained for increasing percentages of elongated cells, decreasing percentages of cuboidal cells, decreasing tubular structures on collagen gels, and increasing times of appearance of tumors in nude mice. The fully elongated cells fail to revert to cuboidal cells and to form tumors. Binding of antisera to epithelial-specific milk fat globule membranes and human keratin declines whereas binding of antisera to myoepithelial-associated laminin, vimentin, and Thy-1 increases in the cell lines in the same order. Similarly 7 polypeptides characteristic of elongated cells increase and 4 polypeptides characteristic of cuboidal cells decrease in the cell lines in the same way. Anti-actin serum binds equally to all cell lines grown on plastic, except for Rama 25-I4, where its binding is increased. Rama 25-I1 and Rama 25-I4 cells also give rise to anti-actin, anti-myoglobin, and phosphotungstic acid hematoxylin-staining giant, striated cells on collagen gels and in tumors that also have ultrastructural characteristics of skeletal muscle. Fresh elongated converts of Rama 25 bind appreciably more anti-actin serum than many of the clonal elongated cell lines such as Rama 29. Ultrastructural analysis confirms the gradual loss of epithelial characteristics and the acquisition of immature myoepithelial characteristics in the same sequence of cell lines. It is suggested that such a linear sequence of intermediate morphological states occurs between the Rama 25 cuboidal cells and the elongated myoepithelial-like cells in vitro, and that a similar morphological sequence may exist in terminal ductal structures in vivo.
The pontine nuclei form the key relay nuclei in the cerebropontocerebellar pathway. Although a great deal of information is available regarding the anatomy of this region, the identity of the neurotransmitter(s) contained in the neurons of the pontine gray are not known. The aim of the present investigation is to utilize immunohistochemical techniques to determine whether glutamate, a putative excitatory transmitter, and the enzymes responsible for its metabolism, are found in pontine neurons. Both glutaminase, an enzyme which converts glutamine to glutamate, and aspartate aminotransferase, an enzyme which is involved in the interconversion between glutamate and aspartate, have been proposed to be markers of neurons which use excitatory amino acids as neurotransmitters. The present study utilizes a monoclonal antibody against carbodiimide-fixed glutamate and polyclonal antisera against glutaminase and aspartate aminotransferase in conjunction with the indirect peroxidase technique or the peroxidase-labeled biotin-avidin procedure to localize glutamatergic neurons in the pontine nuclei of the rat. Numerous neurons in all subdivisions of the pontine nuclei were found to contain carbodiimide-fixed glutamate-like immunoreactivity, glutaminase-like immunoreactivity or aspartate aminotransferase-like immunoreactivity. Horseradish peroxidase was injected into the cerebellum of four rats for use with a combined retrograde transport-immunohistochemical procedure. Double-labeled neurons were observed in all subdivisions of the pontine nuclei, indicating that pontine neurons which contain glutamate-like immunoreactivity project to the cerebellum. Based on the hypothesis that increased levels of glutamate, glutaminase and aspartate aminotransferase reflect a transmitter role for glutamate, the present data raise the possibility that glutamate may be a major neurotransmitter of pontocerebellar fibers.
A myoepithelial-like cell line (Rama 401), isolated from rat mammary gland, has been transformed with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV). Rama 401-tsRSV cells adopt a spindle morphology and fail to deposit basement membrane proteins when grown at the permissive temperature (35 degrees C). When switched to the non-permissive temperature (41 degrees C), the cells flatten (with a 5-fold increase in area), and deposit an extracellular matrix containing basement membrane proteins. When the cells are switched from 35 degrees C to 41 degrees C in the presence of monensin (an ionophore that inhibits protein secretion), basement membrane proteins are no longer deposited extracellularly although the cells flatten, their area increasing by ninefold. Cells switched from 35 degrees C to 41 degrees C in the presence of cycloheximide still flatten and deposit basement membrane proteins, whereas the morphological change on switching from 41 degrees C to 35 degrees C is inhibited by cycloheximide. These experiments indicate that the ability of Rama 401-tsRSV cells to spread on a plastic substratum is not dependent on the de novo synthesis and deposition of basement membrane proteins.
An immunohistological study in the human breast and the rodent breast (from inbred Ludwig/Wistar/Olac rats) was conducted with the use of a murine monoclonal antibody, which reacts with the common acute lymphoblastic antigen, a glycosylated polypeptide of a molecular weight of 100,000. The epitope, as recognized by this antibody, is expressed on myoepithelial cells of the normal human and rat breasts and was studied in the developing rodent mammary gland. Ultrastructural studies in the normal human breast clearly demonstrated the presence of the antigen on the lateral membrane of the myoepithelial cells with no staining of luminal cells, blood vessels, or stromal elements. The antigen survived prolonged enzymatic digestion of human breast tissue and could be demonstrated on myoepithelial cells in single-cell suspensions of human breast where it stained approximately 3-14% of the total cell population. The presence of this antigen on myoepithelial cells is discussed in the context of myoepithelial differentiation in the breast and the potential utility of the antibodies for cell separation.
Cellular proliferation during different stages of pregnancy and lactation in the rat mammary gland was studied by use of autoradiography at the ultrastructural level or in combination with immunocytochemistry. The study consisted of two groups of animals, young 45-day-old rats undergoing their first pregnancy and old multiparous rats undergoing their third pregnancy. Both groups of animals were injected with [3H]thymidine, 2 hours before sacrifice. The timing of proliferative activity through pregnancy and lactation was similar in the two groups. A peak level of proliferation was observed on the 5th day of gestation and the rate of proliferation was at its lowest at the end of gestation. A second small peak was observed on the 3rd day of lactation. In virgin animals, both epithelial and myoepithelial cells showed a high level of proliferative activity. The epithelial cells continued to divide during all stages of their differentiation, throughout pregnancy and lactation. Myoepithelial cells showed active division in early pregnancy but their rate of proliferation declined as the cells acquired abundant cytoplasmic filaments for their contractile function. Intra-alveolar dendritic cells, which have been recently identified in the lactating rat breast, showed a high proliferative rate at the end of gestation.
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A comparison has been made of the effects of different fixation protocols upon the ultrastructure of normal human breast. Pale (B) and dark (A) epithelial cells as described by previous authors are present in tissue fixed with hyperosmotic Karnovsky-type fixatives, but not where the primary fixative is isosmotic glutaraldehyde. The observed subpopulations of pale and dark epithelial cells have been directly compared with sub-groups of epithelial cells recognized by the monoclonal antibody LICR-LON-M8. The pale and dark cells bear no relation to the subpopulations of epithelial cells depicted by LICR-LON-M8.
Normal and neoplastic human breast tissues have been stained with antibodies recognizing neuroendrocrine differentiation. Fifteen out of 44 (34 per cent), breast carcinomas stained positively with monoclonal antibody LICRLON-E36, and 11 out of 44 (25 per cent) of these tumours stained with an antibody raised against neuron-specific enolase (NSE). Eight tumours stained positively with both antibodies. No correlation was observed between staining with these antibodies and the tumour histology, nor with the degree of cellular differentiation as indicated by staining with several cell surface directed monoclonal antibodies. Ultrastructural analysis of a series of breast tumours showed the presence of membrane-bound dense-core vesicles in almost all tumours, including E36 and NSE positive and negative tumours. The presence of these structures appears to be of little value in predicting neuroendocrine differentiation.
Dendritic cells which express Ia antigen have been demonstrated for the first time in the lactating rat mammary gland. Ultrastructurally, the dendritic cells appear as electron-lucent pale cells interspersed among the epithelial cells of the alveoli, forming a cell population distinct from classical macrophages. They show morphological resemblance to the dendritic cells of lymphoid organs as well as the Langerhans cells of skin. The Ia antigen has been localised by electron microscopic immunocytochemistry on the cell membrane and endocytotic vesicles and tubules. Ia positive cells are also seen in the stroma of the mammary gland. It is proposed that the dendritic cells of the mammary gland belong to the lineage of epidermal Langerhans cells and lymphoid dendritic cells, subserving an immunological role in the lactating breast.