Biomedical subjects

K L Buchanan

Publications and source records attributed to K L Buchanan.

17 recordsLinked to original sources

PubMed2000

An upstream Oct-1- and Oct-2-binding silencer governs B29 (Ig beta) gene expression.

The B cell-specific B29 (Igbeta) gene is activated in the earliest B cell precursors and is expressed throughout B cell development. Tissue-specific expression of the murine B29 gene is controlled by a B cell-specific promoter whose activity is governed by a cassette of upstream transcriptional silencers. This study describes a potent new silencer that is located 5' of the previously identified B29 silencer elements, FROG and TOAD. Like these known elements, the new B29 silencer is not restricted to the B29 promoter. Nuclear proteins from all cell lines tested interacted with this A+T-rich sequence, which closely resembled a noncanonical octamer binding motif and also conformed to the consensus sequence for nuclear matrix attachment regions. Interaction of Oct-1 and Oct-2 with the B29 A+T-rich sequence was confirmed using octamer-specific Abs. Oct-1/Oct-2 binding was required for the inhibitory activity of this sequence because mutations that blocked Oct-1/Oct-2 binding also eliminated inhibition of the B29 promoter. This B29 A+T-rich sequence specifically interacted with isolated nuclear matrix proteins in vitro, suggesting that it may also function as a matrix attachment region element. Maintenance of the level of B29 gene expression through the interaction of the minimal promoter and the upstream silencer elements FROG, TOAD, and the A+T-rich Oct-1/Oct-2 binding motif may be essential for normal B cell development and/or function.

5' Untranslated Regions↗

PubMed2000

Song as an indicator of male parental effort in the sedge warbler.

Repertoire size has been found to be a sexually selected trait in a number of bird species, although the advantages of mating with a male who possesses a complex song remain unclear. We studied the potential role of song as an indicator of male parental effort in the sedge warbler Acrocephalus schoenobaenus. The male provisioning rate was used as a measure of male parental effort and was found to increase with nestling age and brood size. When controlling for chick age, brood size and other variables, we found a highly significant positive correlation between a measure of song complexity (repertoire size) and male parental effort. Both male parental effort and repertoire size were found to be positively correlated with chick weight when controlling for chick age. We found no correlation between a measure of song output (amount of song flighting) or territory size and parental effort. Repertoire size is known to be the most important cue in female choice amongst sedge warblers and we discuss the possible reasons for this. We suggest that, in choosing a male with a large repertoire, a female obtains not only indirect benefits but also direct benefits in the form of increased parental effort.

Animals↗

PubMed2000

Requirement for CD4(+) T lymphocytes in host resistance against Cryptococcus neoformans in the central nervous system of immunized mice.

The importance of cell-mediated immunity (CMI) and CD4(+) T lymphocytes in host resistance against Cryptococcus neoformans is well documented and is exemplified by the high susceptibility to progressive infection with this pathogen of AIDS patients with reduced CD4(+) T-cell numbers. Although much has been learned about the role of CMI in the clearance of C. neoformans from the lungs and other internal organs, less is known about the protective mechanisms in the brain, the organ most frequently involved with a fatal outcome of cryptococcosis. We hypothesized that host resistance mechanisms against C. neoformans in the central nervous system (CNS) were similar to those outside the CNS (i.e., gamma interferon [IFN-gamma], CD4(+) T cells, and others). To test this hypothesis, we used a murine model of cryptococcal meningitis whereby cryptococci are introduced directly into the CNS. In experiments where mice were immunized to mount an anticryptococcal CMI response, our results indicate that immunization induced protective mechanisms that could be detected in the CNS by inhibition of the growth of viable yeast cells. Flow cytometric analyses of leukocytes in brain and spinal cord homogenates revealed that T lymphocytes, macrophages, and neutrophils accumulated in C. neoformans-infected brains of immune mice. In vivo depletion of CD4(+) T cells, but not CD8(+) T cells, resulted in significantly reduced leukocyte accumulation in the brains of immune mice. Furthermore, depletion of CD4(+) T cells or neutralization of IFN-gamma exacerbated CNS infection in immune mice, suggesting a critical role for CMI mechanisms in acquired protection in the CNS.

Animals↗

PubMed1999

Differential regulation of immune responses by highly and weakly virulent Cryptococcus neoformans isolates.

Early inflammatory responses, delayed-type hypersensitivity (DTH) responses, and cytokine profiles were studied in mice infected by the pulmonary route with either a highly virulent isolate (NU-2) or a weakly virulent isolate (184A) of Cryptococcus neoformans. After infection, NU-2 remained in the lungs and the capsule became more pronounced during the first 24 h, whereas 184A induced an immediate inflammatory reaction and was rapidly cleared from the lungs. Cryptococcal antigen (GXM) appeared in sera early after infection with NU-2 and increased over the entire observation period. There was no detectable GXM in sera from 184A-infected mice. Both C. neoformans isolates induced anticryptococcal cell-mediated immune responses, but the responses had different profiles. DTH in NU-2-infected mice appeared at day 15 after infection and waned by day 21, whereas DTH in 184A-infected mice was present by day 5 and continued to increase. T helper 1 (Th1) cytokines (interleukin 2 [IL-2] and gamma interferon) were made by spleen cells early after infection with either isolate. NU-2-infected mice lost their ability to produce these cytokines, but 184A-infected mice retained it. IL-4, a Th2 cytokine, was not detected in infected mice. The regulatory cytokine IL-10 was made by spleen cells early but not later after infection with the highly virulent isolate and was not produced by spleen cells from 184A-infected mice. IL-10-deficient mice survived an NU-2 infection significantly longer than wild-type mice, suggesting that IL-10 is important in down-regulating the protective immune response. The induction of anergy appears to be responsible for the inability of NU-2-infected mice to control a C. neoformans infection.

Acute-Phase Reaction↗

PubMed1998

Expression of bright at two distinct stages of B lymphocyte development.

The B cell regulator of Ig heavy chain transcription (Bright) is a DNA-binding protein that was originally discovered in a mature Ag-specific B cell line after stimulation with IL-5 and Ag. It binds to the intronic heavy chain enhancer and 5' of the V1 S107 family V(H) promoter. Several studies suggested that Bright may increase transcription of the heavy chain locus, and expression in cell lines was limited to those representing mature B cells. We have now analyzed normal hemopoietic tissues for the expression of Bright during B lymphocyte differentiation. We expected to find Bright expression in a subset of mature spleen cells, but also observed Bright in a subset of normal B lymphocytic progenitors in both adult bone marrow (BM) and in fetal liver as early as day 12 of gestation. Bright was also expressed in the small percentage of CD4(low) cells in the thymus that are newly arrived from the BM and are not yet committed to the T lymphocyte lineage, but was not observed at later stages of T cell differentiation in either the spleen or thymus. Bright mRNA was not detected in the immature B lymphocytes that initially populate the spleen after migration from the BM. In addition, new splice variants of Bright were observed in fetal tissues. Thus, Bright expression is highly regulated in normal murine lymphocytes and occurs both early and late during B cell differentiation. These findings may have important implications for the function of Bright in regulating Ig transcription.

Animals↗

PubMed1997

Family-specific differences in transcription efficiency of Ig heavy chain promoters.

Murine Ig variable region heavy chain genes (V(H)) are grouped into families based on coding sequence homology. We observed that the accompanying promoter sequences were also conserved in a family-specific manner. Remarkably, no one has directly compared the transcription efficiencies of V(H) genes from different families. Using an in vitro transcription system, we found that transcription efficiencies of different V(H) promoters differed by as much as 70-fold. These differences could be attributed to variation in the octamer-heptamer and TATA sequences, as well as to the presence or absence of initiator elements. The J558 family promoter exhibited the highest level of transcription and specifically interacted with an Oct-1 dimer not bound by other V(H) promoters. These data suggest that differential transcription and regulation of V(H) promoters could occur in vivo. The increased transcription efficiency of the J558 promoter relative to other V(H) promoters also presents a possible explanation for the abundance of J558 sterile transcripts observed before V(H)DJ(H) rearrangement.

Animals↗

PubMed1997

Reassessment of germline heavy chain transcripts from two murine VH families.

While expression of functional heavy chain immunoglobulin mRNA requires rearrangement of variable (VH), diversity (D) and (JH) gene segments, these individual gene segments can be transcribed prior to their rearrangement. It has been proposed that the resulting germline, or sterile, transcripts play an important role in the rearrangement process because strong correlations between rearrangement frequency and sterile transcript levels have been observed in some studies. Murine VH genes have been grouped into families on the basis of coding sequence homology. VH families rearrange in a developmentally regulated manner, so that rearrangements of genes from several VH families are detected earlier than rearrangements of J558 family genes. Paradoxically, the only VH family for which sterile transcripts have been documented is the J558 family. We used RT-PCR analyses to ask whether sterile transcripts from other VH families could be detected in fetal liver samples prior to their rearrangement. While J558 family germline transcripts were easily detected, no sterile transcripts were observed from the S107 family. Our studies also revealed the ability of small quantities of degraded genomic DNA to nonspecifically prime cDNA synthesis, emphasizing the need for caution in interpreting RT-PCR data in which family-specific oligos are used for cDNA production. These results cast doubt on the idea that sterile transcripts are required for V(H)DJ(H) rearrangement.

Animals↗

PubMed1997

Kinetics of cellular infiltration and cytokine production during the efferent phase of a delayed-type hypersensitivity reaction.

Cell-mediated immunity is a primary host resistance mechanism against many infectious organisms and is responsible for leucocyte recruitment to the infection site. Delayed-type hypersensitivity (DTH) reactions are in vivo correlates of cell-mediated immunity and have long been used to assess the level of cell-mediated immune (CMI) responsiveness to specific antigens. It has been difficult to study the kinetics of cellular influx and cytokine composition at the site of an on-going CMI reaction. Consequently, knowledge of the sequential events occurring during the efferent phase of a CMI response is incomplete. Here we report on the use of a gelatin sponge model for evaluating the progression of events during the effector phase of a DTH reaction to antigens of the mycotic organism Cryptococcus neoformans. Previously, we have shown that 24 hr after antigen injection into sponges in infected or immune mice, the leucocyte types infiltrating the sponges are consistent with a classical murine DTH reaction. Through kinetic studies, we show here that neutrophils are the first leucocytes to appear in DTH-reactive sponges, followed by increases in lymphocyte and then monocyte numbers. Tumour necrosis factor (TNF), interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-5 were elevated in DTH-reactive sponges compared with control sponges, and each cytokine had a relatively unique temporal profile. IL-4 was not detectable in the sponges. Together our data indicate that the expression of a CMI response comprises a well-regulated sequential influx of leucocytes that contribute to the lymphokine composition of the reaction.

Animals↗

PubMed1996

Prehospital education: effectiveness with total hip replacement surgery patients.

In-hospital education can reduce anxiety, improve coping and shorten hospital stays of surgical patients. However, hospitals are containing costs by shortening pre- and postoperative stays and reducing the time available for in-hospital teaching. This study evaluated prehospital education for total hip replacement (THR) surgery. Half of the patients waiting for admission for THR surgery were randomly selected to receive a THR education booklet in the mail 4-6 weeks before their scheduled THR surgery. Compared to the No-Booklet patients, patients who had received the booklet were less anxious at the time of hospital admission and at discharge, were more likely to have practised physiotherapy exercises prior to hospitalization, and required significantly less occupational therapy and physiotherapy while in hospital. There were no group differences for length of hospital stay.

Adolescent↗

PubMed1995

Differential transcription efficiency of two Ig VH promoters in vitro.

Each Ig variable region gene segment is transcribed from its own unique promoter. While all of these promoters share common consensus elements that contribute to the B cell-specific expression of these genes, the DNA sequence of each promoter is distinct. In this study, we have directly compared the transcription efficiencies of two murine heavy chain (VH) promoters in a murine B cell in vitro transcription system. We found that the promoters differed in both transcription efficiency and the ability to bind specific protein complexes. While some of the transcription differences may be attributed to differences in basal promoter elements, the spacing between the octamer and the heptamer consensus elements was found to be important. Others have reported a direct correlation between transcription efficiency and the probability that individual variable region gene segments will rearrange. Our studies may be of direct importance to those interested in identifying B cell-specific transcription factors and may ultimately help to explain differences in the expression of some VH gene segments.

Animals↗

PubMed1994

Regulation of cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response.

Effects of both positive and negative regulatory T cells on cellular infiltration and cytokine production during the expression phase of the anticryptococcal immune response were examined. Tamp cells, which are induced by cryptococcal antigen, significantly amplify the anticryptococcal delayed-type hypersensitivity response, whereas a cascade of T suppressor (Ts) cells inhibits the response and decreases the clearance of Cryptococcus neoformans during an infection. By using the gelatin sponge implantation model, we found that Tamp cells do not stimulate a significant increase in cellular infiltration into the sponges in response to cryptococcal antigen compared with that into delayed-type hypersensitivity-reactive sponges in immune control mice. However, Tamp cells do stimulate significant increases in the production of gamma interferon and interleukin-2 (IL-2) in the antigen-injected sponges over the level of the representative cytokine in antigen-injected sponges from the immune control mice. Likewise, Ts1 cells, induced with cryptococcal antigen, do not significantly affect antigen-induced cellular infiltration into sponges in immune mice. In contrast, decreased levels of gamma interferon and IL-2 are observed in antigen-injected sponges from Ts1-cell-recipient, immunized mice compared with those of the positive immune controls. The presence of either Tamp or Ts1 cells in immunized mice stimulates increased production of IL-5 but not IL-4 over that of the positive immune controls.

Animals↗

PubMed1993

Developmental study of GnRH neuronal projections to the medial basal hypothalamus of the male Djungarian hamster.

The present study in the male Djungarian hamster determined the neuroanatomical distribution and morphology of gonadotropin-releasing hormone (GnRH) neurons which innervate the medial basal hypothalamus during sexual maturation. Prepubertal, peripubertal, and postpubertal males were perfused, brains were removed, and crystals of the fluorescent tract tracer, DiI, were implanted directly into the median eminence of the brain. Eight weeks later, brains were sectioned and processed for GnRH immunofluorescence. At all ages, GnRH cell bodies were bipolar or unipolar; both subtypes were labeled with DiI in proportion to their respective numbers in each brain region. GnRH perikarya were distributed in a diffuse ventromedial continuum from the septum through the anterior hypothalamus. In prepubertal males, DiI was present in the majority of GnRH neurons (54% of total) that were located in brain regions rostral to and including the medial preoptic area. In lateral and caudal brain areas, fewer GnRH perikarya contained DiI (28% of total or less). With sexual maturation, fewer GnRH somata were labeled with DiI in areas rostral to the hypothalamus. The data suggest that bipolar and unipolar GnRH neurons in the forebrain, rostral to the preoptic area, are major contributors to the GnRH innervation of the median eminence in the male Djungarian hamster. With the onset of puberty, the finding that decreasing numbers of GnRH perikarya directly project to the medial basal hypothalamus suggests that fewer GnRH neurons constitute the final common pathway that controls gonadotropin secretion.

Aging↗

PubMed1993

Retinal input to the suprachiasmatic nucleus before and after puberty in Djungarian hamsters.

Retinal projections to the suprachiasmatic nucleus (SCN) mediate the effect of photoperiod to entrain circadian rhythms and to control reproductive maturation in the Djungarian hamster. To determine whether the retinal innervation of the SCN had fully developed by the onset of puberty in this hamster species, prepubertal and postpubertal hamsters received an intraocular unilateral injection of horseradish peroxidase (HRP), and after 24 h, the anterograde transport of HRP to the SCN was studied. In prepubertal hamsters, the retinohypothalamic tract (RHT) was found to project to the medial and caudal SCN, principally the ventrolateral regions and, to an extent, the dorsomedial portion of the nucleus. RHT innervation was asymmetric; the SCN contralateral to the monocular injection received the dominant projection. A similar pattern of retinal projections was found postpubertally; however, the ipsilateral SCN was less extensively labelled with HRP and smaller as determined by Nissl counterstain compared to that in prepubertal hamsters. These findings indicate that modifications in the retinal innervation of the SCN occur as late as puberty, and may be part of a developmental change in the mechanism which processes photoperiodic information during sexual maturation.

Animals↗

PubMed1993

Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response.

Cryptococcosis, an increasingly important opportunistic infection caused by the encapsulated yeast-like organism Cryptococcus neoformans, is limited by an anticryptococcal cell-mediated immune (CMI) response. Gaining a thorough understanding of the complex anticryptococcal CMI response is essential for developing means of controlling infections with C. neoformans. The murine cryptococcosis model utilizing footpad swelling to cryptococcal antigen (delayed-type hypersensitivity [DTH]) has proven to be a valuable tool for studying the induction and regulation of the anticryptococcal CMI response, but this technique has limitations with regard to evaluating the role of the final effector cells recruited by an ongoing CMI response. The purpose of this study was to assess the types of cells and cytokines induced into the site of cryptococcal antigen deposition in C. neoformans-infected and -immunized mice compared with those for control mice. We used a gelatin sponge implant model to examine the cells and cytokines present at the site of an anticryptococcal DTH response. Sponges implanted in infected mice and injected with cryptococcal culture filtrate antigen (CneF) 24 h before assessment had significantly increased numbers of infiltrating leukocytes compared with saline-injected sponges in the same animals. Exaggerated influxes of neutrophils and mononuclear cells were the major contributors to the increase in total numbers of cells in the DTH-reactive sponges. The numbers of CD4+ and LFA-1+ cells were found to be significantly increased in the CneF-injected sponges of infected and immunized mice over the numbers in control sponges. The numbers of large granular lymphocytes were also increased in DTH-reactive sponges compared with control sponges. Gamma interferon, interleukin 2 (IL-2), and IL-5 are clearly relevant cytokines in the anticryptococcal CMI response, since they were produced in greater amounts in the CneF-injected sponges from C. neoformans-infected and -immunized mice than in control sponges. IL-4 was not associated with the expression of DTH to cryptococcal antigen. The gelatin sponge model is an excellent tool for studying cells and cytokines involved in specific CMI responses.

Animals↗

PubMed1991

Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response.

Cell-mediated immunity is an important host resistance mechanism against Cryptococcus neoformans, the etiological agent of cryptococcosis. Previous studies from our laboratory have shown that the anticryptococcal cell-mediated immune response as measured by delayed-type hypersensitivity (DTH) is down-regulated by a cascade of antigen-specific T suppressor (Ts) cells. Recently, we have identified a population of CD4 T cells that up-regulate the anticryptococcal DTH response (Tamp cells). The Tamp cells are found in the spleens of donor mice at 6 days after immunization with cryptococcal antigen, and they amplify the anticryptococcal DTH response when transferred to syngeneic recipients at the time of immunization of the recipients. In this study, we determined the effects of C. neoformans-specific Ts cells on the induction of the Tamp cells in the Tamp cell-donor mice and on the induction and expression of the amplified anticryptococcal DTH response in the Tamp cell-recipient mice. When cryptococcal-specific Ts1 cells were given at the time of immunization of the Tamp cell-donor mice, induction of Tamp cells was inhibited. In contrast, when Ts1 cells were given at the time of adoptive transfer of Tamp cells, the recipients displayed amplified DTH responses, indicating that Ts1 cells do not affect the Tamp cells' function once the Tamp cells have been produced. C. neoformans-specific Ts2 cells given at the time of either immunization or footpad challenge of the Tamp cell-recipient mice did not alter, to any measurable extent, the amplified DTH response. These results indicate that in addition to amplifying the anticryptococcal DTH response, Tamp cells may protect the anticryptococcal TDH cells from suppression by C. neoformans-specific Ts cells, much like contrasuppressor cells do in other systems. However, further characterization of the Tamp cells revealed that they are not adherent to Viscia villosa lectin, indicating that the anticryptococcal Tamp cells do not have this characteristic in common with contrasuppressor cells of other antigen systems.

Animals↗

PubMed1991

Delayed puberty in the male Djungarian hamster: effect of short photoperiod or melatonin treatment on the GnRH neuronal system.

The effect of short days or timed melatonin treatments on the number and neuroanatomical location of gonadotropin-releasing hormone (GnRH) neurons was studied in the brain of the pubertal male Djungarian hamster. At the beginning of the rapid phase of testicular growth and onset of peak gonadotropin secretion (15 days of age), males were treated for 10 days with either short days (10 L:14 D; n = 6), or remained in long days (16 L:8 D; n = 5) and injected each afternoon with melatonin. These treatments arrested testicular growth compared to the gonadal development that occurred in long-day controls (n = 9). Every brain section (60 microns) from the olfactory bulb to the anterior hypothalamus was processed for GnRH immunocytochemistry and viewed under brightfield light microscopy. GnRH cell bodies had smooth contours and were morphologically bipolar or unipolar. The number of bipolar neurons was similar regardless of treatment (about 170/brain). However, fewer unipolar GnRH cell bodies (p less than 0.05) were found in males in short days (73 +/- 11) or in males administered melatonin (72 +/- 14) compared to the unipolar number in hamsters in long days (132 +/- 14). With respect to neuroanatomical distribution, significantly fewer unipolar GnRH neurons were found in the medial preoptic area of males treated with short days or melatonin (55-70% decrease) compared to cell numbers in long-day controls. The melatonin-treated hamsters also had reduced numbers of unipolar GnRH neurons in the diagonal band of Broca relative to the number of unipolar neurons in long-day controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

What makes Cryptococcus neoformans a pathogen?

Life-threatening infections caused by the encapsulated fungal pathogen Cryptococcus neoformans have been increasing steadily over the past 10 years because of the onset of AIDS and the expanded use of immunosuppressive drugs. Intricate host-organism interactions make the full understanding of pathogenicity and virulence of C. neoformans difficult. We discuss the current knowledge of the characteristics C. neoformans must possess to enter the host and establish progressive disease: basic growth requirements and virulence factors, such as the polysaccharide capsule; shed products of the organism; melanin production; mannitol secretion; superoxide dismutase; proteases; and phospholipases.

Animals↗