Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release.

Clearance of apoptotic cells is critical to tissue homeostasis and resolution of inflammatory lesions. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well. In the lactating mammary gland following weaning, alveolar epithelial cell death is massive, yet the gland involutes rapidly, attaining its prepregnancy state in a matter of days. We found histologic evidence of apoptotic cell phagocytosis by viable mammary epithelial cells (MEC) in the involuting mouse mammary gland. Cultured MEC were able to engulf apoptotic cells in vitro, utilizing many of the same receptors used by macrophages, including the phosphatidylserine receptor (PSR), CD36, the vitronectin receptor alpha(v)beta3, and CD91. In addition, MEC, like macrophages, produced TGFbeta in response to stimulation of the PSR by apoptotic cells or the anti-PSR ab 217G8E9, and downregulated endotoxin-stimulated proinflammatory cytokine production. These data support the hypothesis that amateur phagocytes play a significant role in apoptotic cell clearance and its regulation of inflammation.

The retroviruses human immunodeficiency virus type 1 and Moloney murine leukemia virus adopt radically different strategies to regulate promoter-proximal polyadenylation.

Maximal gene expression in retroviruses requires that polyadenylation in the 5' long terminal repeat (LTR) is suppressed. In human immunodeficiency virus type 1 (HIV-1) the promoter-proximal poly(A) site is blocked by interaction of U1 snRNP with the closely positioned major splice donor site (MSD) 200 nucleotides downstream. Here we investigated whether the same mechanism applies to down-regulate 5' LTR polyadenylation in Moloney murine leukemia virus (MoMLV). Although the same molecular architecture is present in both viruses, the MoMLV poly(A) signal in the 5' LTR is active whether or not the MSD is mutated. This surprising difference between the two retroviruses is not due to their actual poly(A) signals or MSD sequences, since exchange of either element between the two viral sequences does not alter their ability to regulate 5' LTR poly(A) site use. Instead we demonstrate that sequence between the cap and AAUAAA is required for MSD-dependent poly(A) regulation in HIV-1, indicating a key role for this part of the LTR in poly(A) site suppression. We also show that the MoMLV poly(A) signal is an intrinsically weak RNA-processing signal. This suggests that in the absence of a poly(A) site suppression mechanism, MoMLV is forced to use a weak poly(A) signal.

A lipoprotein-containing particle is transferred from the serum across the mammary epithelium into the milk of lactating mice.

To investigate the role of low-density lipoprotein (LDL) in the delivery of cholesterol to the mammary gland during pregnancy and lactation, we examined the distribution of radioactivity from (125)I-tyramine cellobiose-LDL injected into the tail vein of female mice at various stages of the reproductive cycle. Changes in the proportion of isotope taken up by the mammary gland largely reflected the increased weight of the gland in pregnancy and lactation. In addition, during lactation, radioactivity was found in the milk and was associated with a protein of the molecular weight of apoB-100. Quantitatively similar results were obtained with mice homozygous for disruption of the LDL receptor gene (LDLR null). Analysis of endogenous lipoproteins showed that the milk lipoprotein particles were denser than the corresponding serum lipoproteins and largely depleted of triglyceride and cholesterol. Using fluorescence microscopy we visualize the sorting of apoB protein from the LDL lipid phase at the basal surface of the mammary epithelial cell of both wild-type and LDLR-null mice. Our findings provide evidence that the mammary epithelium of the lactating mouse is able to take up LDL from the plasma by a non-LDLR-mediated process. An apoB-containing particle from which the cholesterol has been removed is transferred into milk.

Vesicular transport of soluble substances into mouse milk.

Utilizing a novel protocol to study transport of substances into mouse milk in situ, we have shown that many "fluid-phase" markers are taken up by mammary epithelial cells and deposited in milk. Since the tight junctions are closed and impermeable even to small molecules, extra-alveolar substances (those not synthesized by the alveolar cells) must be transported into the milk by the epithelial cells themselves. The markers we have used include dextran, lucifer yellow dye, horseradish peroxidase, and albumin. Using these markers and immunostaining for endogenous proteins, we have visualized transcytotic vesicles involved in transporting these markers to milk.

The effect of serum iron concentration on iron secretion into mouse milk.

1. The concentration of iron in mouse milk is approximately 3 times that of the serum. Although there is clear evidence for the presence of the transferrin receptor in the rodent mammary gland, the precise mechanisms of iron transfer into milk are not known. 2. Milk iron was linearly related to the serum iron:transferrin ratio in lactating mice whose serum iron ranged from 8 to 66 microM. 3. Increasing the iron binding capacity of the milk by 340 microM by targeting the lactoferrin transgene to the mammary gland did not alter the relation between milk iron and the serum iron:transferrin ratio. 4. The steady-state distribution ratio of 125I-transferrin between plasma and milk was about 0.2, indicating that transcytosed transferrin contributed a maximum of 6% of the milk iron. 5. Fluorescently labelled transferrin incubated with the in situ gland localized mainly near the basal surface of the mammary alveolar cells. 6. These experiments provide evidence that the initial and rate-limiting step in the transfer of iron into milk is binding to a basal transferrin receptor. 7. A theoretical model of the relation between milk and serum iron suggests that the affinity of apotransferrin for the basal recycling system may be higher than observed in many other cell types.

The mammary fat pad.

The mammary fat pad is essential for development of the mammary epithelium, providing signals that mediate ductal morphogenesis and, probably, alveolar differentiation. The "cleared" fat pad is often used as a transplantation site. Considering the crucial role of the fat pad, its properties have received relatively little attention from researchers in the field. Some of the questions whose investigation is pertinent to understanding both normal mammary development and carcinogenesis are outlined in this commentary in the spirit of stimulating enquiry into this important subject. It is clear from a brief perusal of the available literature that until studies are specifically designed to clearly differentiate between functional effects of the fibrous and the adipose stroma, more substantive information about their differential effects on mammary development and tumorigenesis will not be forthcoming.

Intergenic transcription and transinduction of the human beta-globin locus.

We have identified novel nuclear transcripts in the human beta-globin locus using nuclear run-on analysis in erythroid cell lines and in situ hybridization analysis of erythroid tissue. These transcripts extend across the LCR and intergenic regions but are undetectable in nonerythroid cells. Surprisingly, transient transfection of a beta-globin gene (epsilon, gamma, or beta) induces transcription of the LCR and intergenic regions from the chromosomal beta-globin locus in nonerythroid cell lines. The beta-globin genes themselves, however, remain transcriptionally silent. Induction is dependent on transcription of the globin gene in the transfected plasmid but does not require protein expression. Using in situ hybridization analysis, we show that the plasmid colocalizes with the endogenous beta-globin locus providing insight into the mechanism of transinduction.

Life stories and sickness experience: a performance perspective.

This paper concerns how a 'performance' perspective may aid our understanding of life story data in the investigation of sickness experience. The argument is illustrated through structural and thematic analysis of the life story of a young English woman with multiple sclerosis (MS). The life story genre allowed the author creatively to explore contrasting perspectives on the shaping of social relations by bodily disorder. This was in the context of her more general concerns about definition and change in social relationships and the physical body. Performance relates the form and content of the story to generative social experience as well as to multiple readings and reformulations. It thus gives insight into the constitutive processes through which bodily knowledge is produced and shared in relation to notions of person and self.

Upstream sequence elements enhance poly(A) site efficiency of the C2 complement gene and are phylogenetically conserved.

Poly(A) signals of mammalian pre-mRNA have been defined as an AAUAAA sequence 10-30 nt upstream of the cleavage/poly(A) site followed by a GU/U-rich element immediately downstream. However, a number of viral poly(A) signals have been shown to possess additional signals upstream of AAUAAA that increase poly(A) site efficiency. We describe the first non-viral example of such an upstream sequence element (USE) for the poly(A) site of the human C2 complement gene. As this gene is very closely spaced to the related Factor B gene [the C2 poly(A) site is only 421 bp from the transcription start site of Factor B] we have isolated this same intergenic sequence from four other mammals (mouse, cat, rabbit and cow). We show that the USE of the C2 poly(A) site is highly conserved between these five different mammals. Furthermore, extensive mutagenesis of the human USE indicates that most of the 53 nt sequence is required for full activity. The human C2 poly(A) site does not possess any obvious downstream GU/U-rich sequences, although sequences immediately 3' to AAUAAA as well as 13 nt of sequence following the cleavage site are both required for full activity. Interestingly the other mammalian C2 poly(A) sites do possess significant downstream GU/U-rich sequences. Finally we show that all five mammalian C2 poly(A) signals are immediately followed by conserved signals for transcriptional termination, consistent with the close proximity of the downstream Factor B gene.

Working together for the care of elderly people.

Meeting the community care needs of elderly people at home requires close co-ordination of health and social services input. Social worker Jonathan Monks and health visitor Lou Illesley report on how they work together to plan and deliver effective packages of care to elderly people registered with the GP practice to which they are both attached.

Multiple SP1 binding sites confer enhancer-independent, replication-activated transcription of HIV-1 and globin gene promoters.

We demonstrate that multiple SP1 protein:DNA binding sites confer enhancer-independent activation on the HIV-1 and globin gene promoters. This activation process can be achieved either by DNA replication of the promoter-containing plasmid or by high concentrations of input plasmid DNA used in the transfections. In the case of HIV-1, the three SP1 sites adjacent to the promoters TATA box are essential for this activation process. Furthermore, the human beta globin gene, which is normally dependent on a linked enhancer for transcriptional activity, can be made enhancer independent by insertion of SP1 binding sites adjacent to its TATA box. We speculate that (SP1)n-TATA type RNA polymerase II promoters may be generally permissive when present on actively replicating DNA templates and that this property of the HIV-1 promoter may be of importance to the activation of the DNA provirus in latently infected T cells.

Occlusion of the HIV poly(A) site.

To investigate the selective use of poly(A) sites in the 3' long terminal repeat (LTR) but not the 5' LTR of retroviruses, we have studied the poly(A) site of the human immunodeficiency virus (HIV-1). Using hybrid HIV/alpha-globin gene constructs, we demonstrate that the HIV poly(A) site is inactive or occluded when adjacent to an active promoter, either the homologous HIV promoter or the alpha-globin gene promoter. Furthermore, this occlusion of the HIV poly(A) site occurs over a considerable distance of up to at least 500 bp. In contrast, two nonretroviral poly(A) sites [alpha-globin and a synthetic poly(A) site] are active when close to a promoter. We also show that a short fragment of approximately 60 nucleotides containing the HIV poly(A) site is fully active when placed at the 3' end of the human alpha-globin gene or within the rabbit beta-globin gene. This result rules out the requirement of more distant upstream elements for the activity of the HIV poly(A) site, as has been suggested for other viral poly(A) sites. Finally, we show that the GT-rich downstream region of the HIV poly(A) site confers poly(A) site occlusion properties on a synthetic poly(A) site. This result focuses attention on this more variable part of a poly(A) site in retroviruses as a possible general signal for poly(A) site occlusion.

Patients' decisions about continuing with therapy in chronic illness: a study of hyperbaric oxygen therapy in multiple sclerosis.

The criteria which may be employed by chronically ill people in their decision about whether to continue with therapy were investigated in the context of a clinical trial of hyperbaric oxygen therapy for multiple sclerosis. An open-ended question about reasons for continuing or discontinuing treatment obtained data from 48 trial participants on completion of the trial and six months later. Physical factors were the most commonly cited reasons for continuing treatment; stabilization of the condition was mentioned as well as improvement. Lack of effectiveness was more commonly given as a reason to stop therapy six months after the trial. Social and practical reasons were also cited as reasons for discontinuing therapy and there was implicit evidence of a high level of social support enabling patients to continue therapy. Most problems appeared to have surfaced by the end of the trial. Epistemological reasons provided rationales for the decisions taken and referred particularly to the time required to make an informed judgement about the effectiveness of treatment. Personal experience and knowledge of the effects of hyperbaric oxygen on others were more significant to the decision than anticipation of the trial's formal results. The participants in the trial were sophisticated and responsible decision-makers even though their judgements would not always have coincided with medical opinion.

The characteristics of a national register of people with multiple sclerosis (MS): a comparison between the ARMS (Action for Research into Multiple Sclerosis) register and 10 British MS populations.

Problems with case ascertainment in epidemiological research on multiple sclerosis (MS) make it necessary to use indirect sources. However, there is a lack of information about the characteristics of cases drawn from different sources and thus little basis on which sampling frames for large scale surveys may be constructed. The characteristics of a population drawn from the membership of Action for Research into Multiple Sclerosis (ARMS) were compared with those of 10 other British MS populations reported between 1980 and 1987. Demographic variables examined were geographical location, nationality, ethnicity, sex ratio and age. Diagnostic status, age at diagnosis and duration since diagnosis were considered together with data on the comparative populations based on date of onset. On all the variables investigated the ARMS population fell close to or within the range shown by the comparative populations. The relative youth of the ARMS population and its bias towards a high proportion of females were differences in keeping with existing knowledge about the membership of mutual support organisations. A population drawn from such an organisation may usefully complement other MS research populations provided that the likely biases in each are understood.

Experiencing symptoms in chronic illness: fatigue in multiple sclerosis.

This paper examines the various ways in which symptoms of chronic illness may be experienced. With particular reference to fatigue in multiple sclerosis, it discusses how the conceptualization of symptoms as discrete entities requiring targeted action (common in much current writing on chronic illness) is only one form in which physical disorder may be recognized by those directly affected, and one which probably applies little in the course of everyday life. The paper draws attention to the significance of personal and social context in symptom definition and to the implications for the measurement of symptoms and for advice to patients on management.