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Biomedical subjects

K L Brown

Publications and source records attributed to K L Brown.

At least 19 recordsLinked to original sources
PubMed2006

Delayed diagnosis of congenital heart disease worsens preoperative condition and outcome of surgery in neonates.

OBJECTIVES: To assess whether the route by which neonatal congenital heart disease (CHD) is first recognised influences outcome after surgery. METHODS: Surgical neonates admitted to a tertiary cardiac unit between March 1999 and February 2002 were retrospectively reviewed with analysis of risk factors for outcome. Three routes to initial recognition of CHD were compared: antenatal diagnosis, detection on the postnatal ward, and presentation after discharge to home. Outcome measures were mortality and duration of perioperative ventilation. RESULTS: 286 neonates had cardiac surgery with a median duration of ventilation of 101 h and in-hospital mortality of 12%. Recognition of CHD was antenatal in 20%, on the postnatal ward in 55% and after discharge to home in 25%. Multiple regression analyses, including the cardiac diagnosis, associated problems and other risk factors, indicated that severe cardiovascular compromise on admission to the cardiac unit was significantly related to mortality and prolonged ventilation. Considered in isolation, the route to recognition of heart disease did not influence mortality or ventilation time. Route to initial recognition did, however, influence the patient's condition on admission to the cardiac unit. Cardiovascular compromise and end organ dysfunction were least likely when recognition was antenatal and most common when presentation followed discharge to home. CONCLUSION: The setting in which neonatal CHD is first recognised has an impact on preoperative condition, which in turn influences postoperative progress and survival after surgery. Optimal screening procedures and access to specialist care will improve outcome for neonates undergoing cardiac surgery.

Analysis of Variance↗
PubMed2004

Predicting outcome in ex-premature infants supported with extracorporeal membrane oxygenation for acute hypoxic respiratory failure.

OBJECTIVE: To identify predictors of outcome in ex-premature infants supported with extracorporeal membrane oxygenation (ECMO) for acute hypoxic respiratory failure. METHODS: Retrospective review of ex-premature infants with acquired acute hypoxic respiratory failure requiring ECMO support in the United Kingdom from 1992 to 2001. Review of follow up questionnaires completed by general practitioners and local paediatricians. RESULTS: Sixty four ex-premature infants (5-10 each year) received ECMO support, despite increased use of advanced conventional treatments over the decade. The most common infective agent was respiratory syncytial virus (85% of cases). Median birth gestation was 29 weeks and median corrected age at the time of ECMO support was 42 weeks. Median ECMO support duration was relatively long, at 229 hours. Survival to hospital discharge and to 6 months was 80%, remaining similar throughout the period of review. At follow up, 60% had long term neurodisability and 79% had chronic pulmonary problems. Of pre-ECMO factors, baseline oxygen dependence, younger age, and inpatient status were associated with non-survival (p < or = 0.05). Of ECMO related factors, patient complications were independently associated with adverse neurodevelopmental outcome and death (p < 0.01). CONCLUSIONS: Survival rates for ex-premature infants after ECMO support are favourable, but patients suffer a high burden of morbidity during intensive care and over the long term. At the time of ECMO referral, baseline oxygen dependence is the most important predictor of death, but no combination of the factors considered was associated with a mortality that would preclude ECMO support.

Acute Disease↗
PubMed2004

Cardiac ECMO for biventricular hearts after paediatric open heart surgery.

OBJECTIVE: To delineate predictors of hospital survival in a large series of children with biventricular physiology supported with extracorporeal membrane oxygenation (ECMO) after open heart surgery. RESULTS: 81 children were placed on ECMO after open heart surgery. 58% (47 of 81) were transferred directly from cardiopulmonary bypass to ECMO. Hospital survival was 49% (40 of 81) but there were seven late deaths among these survivors (18%). Factors that improved the odds of survival were initiation of ECMO in theatre (64% survival (30 of 47)) rather than the cardiac intensive care unit (29% survival (10 of 34)) and initiation of ECMO for reactive pulmonary hypertension. Important adverse factors for hospital survival were serious mechanical ECMO circuit problems, renal support, residual lesions, and duration of ECMO. CONCLUSIONS: Hospital survival of children with biventricular physiology who require cardiac ECMO is similar to that found in series that include univentricular hearts, suggesting that successful cardiac ECMO is critically dependent on the identification of hearts with reversible ventricular dysfunction. In our experience of postoperative cardiac ECMO, the higher survival of patients cannulated in the operating room than in the cardiac intensive care unit is due to early effective support preventing prolonged hypoperfusion and the avoidance of a catastrophic cardiac arrest.

Cardiopulmonary Bypass↗
PubMed2003

Near-fatal grape aspiration with complicating acute lung injury successfully treated with extracorporeal membrane oxygenation.

OBJECTIVE: In this report of a near-fatal case of grape aspiration successfully treated with extracorporeal membrane oxygenation (ECMO), we highlight the danger of feeding seedless grapes to young children and demonstrate that ECMO can provide cardiopulmonary support for cases of acquired large-airway disruption and can facilitate therapeutic intervention. DESIGN: Case report. SETTING: A tertiary pediatric intensive care unit and ECMO center. PATIENT: A healthy 14-month-old boy aspirated a seedless grape while playing at home and suffered a cardiopulmonary arrest of 15 mins in duration. He responded to advanced life support with return of cardiac output but developed intractable cardiopulmonary failure secondary to aspirated grape particles and postobstructive pulmonary edema. INTERVENTIONS: The patient was emergently transferred to the regional ECMO center and placed on venoarterial ECMO. Bronchoscopies were performed in the stable environment provided by ECMO, aspirated particles were removed from the large airways, and lung recovery was facilitated. MEASUREMENTS AND MAIN RESULTS: End-organ perfusion was restored via ECMO during a period of severe intractable cardiopulmonary failure. Pulmonary recovery occurred during a 6-day ECMO run and was facilitated by therapeutic bronchoscopy. The patient was reviewed 1 yr later and has made a full neurodevelopmental recovery, despite a 15-min out-of-hospital cardiac arrest. CONCLUSIONS: Aspiration of a seedless grape is a life-threatening event in a small child. This danger is not fully appreciated by parents in the UK. ECMO may be life saving in cases of acquired large-airway disruption resulting in severe cardiopulmonary failure, including foreign body aspiration, as long as end-organ perfusion is maintained.

Cardiac Output, Low↗
PubMed2001

Enzymatic activity of coenzyme B(12) derivatives with altered axial nucleotides: probing the mechanochemical triggering hypothesis in ribonucleotide reductase.

Theoretical studies (J. Inorg. Biochem. 2001, 83, 121) of the involvement of the bulky 5,6-dimethylbenzimidazole (Dmbz) ligand of coenzyme B(12) (5'-deoxyadenosylcobalamin, AdoCbl) in the mechanism of activation of the carbon-cobalt bond of the coenzyme for homolytic cleavage by AdoCbl-dependent enzymes (the "mechanochemical triggering" mechanisms) have shown that a purely steric, ground-state mechanism can supply only a few kilocalories per mole (of the observed 13-16 kcal mol(-1)) of activation, but that an electronic mechanism, operating to stabilize the transition state, can explain all of the observed catalytic effect. To address these mechanisms experimentally, analogues of AdoCbl in which the Dmbz ligand is replaced by benzimidazole (Ado(Bzim)Cbl) or by imidazole (Ado(Im)Cbl) have been prepared and characterized. Both of these analogues support turnover in the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii at 100% of the activity of AdoCbl itself, but the Ado(Im)Cbl analogue has a significantly higher K(m). 5'-Deoxyadenosylcobinamide, the analogue in which the axial nucleotide has been chemically removed, in contrast, is inactive in the spectrophotometric assay, which indicates that it has at most 1% of the activity of AdoCbl. Stopped-flow spectrophotometric measurements of the formation of cob(II)alamin at the enzyme active site show that RTPR binds Ado(Bzim)Cbl slightly more weakly than it does AdoCbl, but binds Ado(Im)Cbl 8-fold more weakly. While the equilibrium constant for cob(II)alamin formation is nearly the same for Ado(Bzim)Cbl and AdoCbl, it is 5-fold smaller for Ado(Im)Cbl. Finally, the forward rate constant for enzyme-induced Co-C bond homolysis was about the same for Ado(Bzim)Cbl and for AdoCbl but was 17-fold smaller for Ado(Im)Cbl. These results are consistent with a small contribution from ground-state mechanochemical triggering, but they do not in themselves rule out transition-state mechanical triggering.

Adenine Nucleotides↗
PubMed2001

Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14(+) peripheral blood monocytes.

Activation of T cells by Ag or stimulation of monocytes with inflammatory cytokines induces CD44 to bind to hyaluronan (HA), an adhesion event implicated in leukocyte-leukocyte, leukocyte-endothelial cell, and leukocyte-stromal cell interactions. We have previously shown that TNF-alpha induces CD44 sulfation in a leukemic cell line, which correlated with the induction of HA binding and CD44-mediated adhesion. In this study, we establish that TNF-alpha and IFN-gamma induce HA binding and the sulfation of CD44 in CD14(+) PBMC, whereas no induced HA binding or CD44 sulfation was observed in CD14(-) PBMC stimulated with TNF-alpha. Treatment of cells with NaClO(3), an inhibitor of sulfation, prevented HA binding in a significant percentage of CD14(+) PBMC induced by TNF-alpha, LPS, IL-1beta, or IFN-gamma. Furthermore, stimulation with TNF-alpha or IFN-gamma in the presence of NaClO(3) reduced the ability of isolated CD44H to bind HA, demonstrating a direct effect of CD44H sulfation on HA binding. In contrast, the transient induction of HA binding in T cells by PHA was not affected by NaClO(3), suggesting that activated T cells do not use sulfation as a mechanism to regulate HA binding. Overall, these results demonstrate that inducible sulfation of CD44H is one mechanism used by CD14(+) peripheral blood monocytes to induce HA binding in response to inflammatory agents such as TNF-alpha and IFN-gamma.

Humans↗
PubMed2001

Equilibrium and kinetic studies on the reactions of alkylcobalamins with cyanide.

Ligand substitution equilibria of different alkylcobalamins (RCbl, R = Me, CH(2)Br, CH(2)CF(3), CHF(2), CF(3)) with cyanide have been studied. It was found that CN(-) first substitutes the 5,6-dimethylbenzimidazole (Bzm) moiety in the alpha-position, followed by substitution of the alkyl group in the beta-position trans to Bzm. The formation constants K(CN) for the 1:1 cyanide adducts (R(CN)Cbl) were found to be 0.38 +/- 0.03, 0.43 +/- 0.03, and 123 +/- 9 M(-1) for R = Me, CH(2)Br, and CF(3), respectively. In the case of R = CH(2)CF(3), the 1:1 adduct decomposes in the dark with CN(-) to give (CN)(2)Cbl. The unfavorable formation constants for R = Me and CH(2)Br indicate the requirement of very high cyanide concentrations to produce the 1:1 complex, which cause the kinetics of the displacement of Bzm to be too fast to follow kinetically. The kinetics of the displacement of Bzm by CN(-) could be followed for R = CH(2)CF(3) and CF(3) to form CF(3)CH(2)(CN)Cbl and CF(3)(CN)Cbl, respectively, in the rate-determining step. Both reactions show saturation kinetics at high cyanide concentration, and the limiting rate constants are characterized by the activation parameters: R = CH(2)CF(3), DeltaH = 71 +/- 1 kJ mol(-1), DeltaS = -25 +/- 4 J K(-1) mol(-1), and DeltaV = +8.9 +/- 1.0 cm(3) mol(-1); R = CF(3), DeltaH = 77 +/- 3 kJ mol(-1), DeltaS = +44 +/- 11 J K(-1) mol(-1), and DeltaV = +14.8 +/- 0.8 cm(3) mol(-1), respectively. These parameters are interpreted in terms of an I(d) and D mechanism for R = CH(2)CF(3) and CF(3), respectively. The results of the study enable the formulation of a general mechanism that can account for the substitution behavior of all investigated alkylcobalamins including coenzyme B(12).

Cyanides↗
PubMed2001

Human papillomavirus type 16 E6 and E7 cooperate to increase epidermal growth factor receptor (EGFR) mRNA levels, overcoming mechanisms by which excessive EGFR signaling shortens the life span of normal human keratinocytes.

Epidermal growth factor receptor (EGFR) levels are dramatically increased in human keratinocytes (HKc) immortalized with full-length human papillomavirus type 16 (HPV16) DNA (HKc/HPV16), but increases in EGFR levels actually precede immortalization. In some normal HKc strains, acute expression of HPV16 E6 (but not HPV16 E5, HPV16 E7, or HPV6 E6) from LXSN retroviral vectors produced an increase in EGFR mRNA levels detectable at 24 h and stable for up to 10 days after infection. However, about one-half of the individual normal HKc strains we analyzed proved unresponsive to E6 induction of EGFR mRNA despite the robust expression of E6 and degradation of p53. E6 responsiveness of normal HKc strains correlated inversely with initial EGFR levels: although HKc strains expressing relatively low basal EGFR levels grew poorly and tolerated the infection protocol with difficulty, they responded to E6 with an increase in EGFR mRNA and protein and with robust proliferation. However, those HKc strains expressing high basal EGFR levels grew well, but did not respond to E6 with increased EGFR levels or with proliferation. Immunostaining of paraffin-embedded foreskin tissue for the EGFR confirmed that there is an intrinsic interindividual variability of EGFR expression in HKC: These results prompted us to investigate the effects of overexpression of the EGFR in normal HKC: Infection of normal HKc with a LXSN retrovirus expressing the full-length human EGFR cDNA resulted in a dramatic reduction in growth rate and a shorter life span. Although acute expression (1-10 days after infection) of HPV16 E7 alone did not induce the EGFR, acute expression of E6 and E7 together increased EGFR levels in normal HKc unresponsive to E6 alone. Also, HKc infected with E7 alone expressed increased EGFR levels at early stages of extended life span (at passage 9 after infection), and HKc immortalized by HPV16 E7 alone expressed EGFR levels comparable with those of E6/E7-immortalized cells. These results support a key role of the EGFR in HPV16-mediated transformation of HKC: In addition, these data show that normal HKc do not tolerate excessive EGFR levels/signaling, and such intolerance must be overcome in order for HKc to become immortalized by HPV16. We conclude that both E6 and E7 contribute to increasing EGFR levels, but with different mechanisms: although E6 can increase EGFR levels, it cannot overcome the resistance of normal HKc to excessive EGFR signaling. On the other hand E7, which alone does not acutely increase EGFR mRNA or protein, allows for EGFR overexpression in normal HKC:

Cell Survival↗
PubMed2001

Fetal cell grafts provide long-term protection against scrapie induced neuronal loss.

We have transplanted fetal neurons to prolong hippocampal pyramidal cell survival in a mouse scrapie model in which 50% of CA1 pyramidal cells have died by day 180 of the 250-day incubation period. Cells prepared from embryonic PrP deficient mice were intracerebrally injected into infected mice on day 150 and groups killed on day 171 and with terminal disease. Neuron counts and CA1 depth measurements were made on semi-serial sections using an image analysis system. Both grafted groups retained more CA1 neurons than controls injected with medium alone, and showed greater depth of CA1 than controls. This new approach may have potential as a late-stage therapy for TSEs for which there are currently no available treatments.

Animals↗
PubMed2001

Molecular modeling of the mechanochemical triggering mechanism for catalysis of carbon-cobalt bond homolysis in coenzyme B12.

The possible contributions of the mechanochemical triggering effect to the enzymatic activation of the carbon-cobalt bond of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl) for homolytic cleavage have been studied by molecular modeling and semiempirical molecular orbital calculations. Classically, this effect has envisioned enzymatic compression of the axial Co-N bond in the ground state to cause upward folding of the corrin ring and subsequent sterically induced distortion of the Co-C bond leading to its destabilization. The models of this process show that in both methylcobalamin (CH3Cbl) and AdoCbl, compression of the axial Co-N bond does engender upward folding of the corrin ring, and that the extent of such upward folding is smaller in an analog in which the normal 5,6-dimethylbenzimidazole axial ligand is replaced by the sterically smaller ligand, imidazole (CH3(lm)Cbl and Ado(lm)Cbl). Furthermore, in AdoCbl, this upward folding of the corrin is accompanied by increases in the carbon-cobalt bond length and in the Co-C-C bond angle (which are also less pronounced in Ado(Im)Cbl), and which indicate that the Co-C bond is indeed destabilized by this mechanism. However, these effects on the Co-C bond are small, and destabilization of this bond by this mechanism is unlikely to contribute more than ca. 3 kcal mol(-1) towards the enzymatic catalysis of Co-C bond homolysis, far short of the observed ca. 14 kcal mol(-1). A second version of mechanochemical triggering, in which compression of the axial Co-N bond in the transition state for Co-C bond homolysis stabilizes the transition state by increased Co-N orbital overlap, has also been investigated. Stretching the Co-C bond to simulate the approach to the transition state was found to result in an upward folding of the corrin ring, a slight decrease in the axial Co-N bond length, a slight displacement of the metal atom from the plane of the equatorial nitrogens towards the "lower" axial ligand, and a decrease in strain energy amounting to about 8 kcal mol(-1) for both AdoCbl and Ado(Im)Cbl. In such modeled transition states, compression of the axial Co-N bond to just below 2.0 A (the distance subsequently found to provide maximal stabilization of the transition state by increased orbital overlap) required about 4 kcal mol(-1) for AdoCbl, and about 2.5 kcal mol(-1) for Ado(Im)Cbl. ZINDO/1 calculations on slightly simplified structures showed that maximal electronic stabilization of the transition state by about 10 kcal mol(-1) occurred at an axial Co-N bond distance of 1.96 A for both AdoCbl and Ado(Im)Cbl. The net result is that this type of transition state mechanochemical triggering can provide 14 kcal mol(-1) of transition state stabilization for AdoCbl, and about 15.5 kcal mol(-1) for the Ado(Im)Cbl, enough to completely explain the observed enzymatic catalysis. These results are discussed in the light of current knowledge about class I AdoCbl-dependent enzymes, in which the coenzyme is bound in its "base-off" conformation, with the lower axial ligand position occupied by the imidazole moiety of an active site histidine residue, and the class II enzymes, in which AdoCbl binds to the enzyme in its "base-on" conformation, and the pendent 5,6-dimethylbenzimidazole base remains coordinated to the metal during Co-C bond activation.

Carbon↗
PubMed2001

Resection, rotationplasty, and femoropelvic arthrodesis in severe congenital femoral deficiency. A report of the surgical technique and three cases.

BACKGROUND: The major problems associated with severe congenital deficiency of the femur are an unstable hip joint and a femur that is more than 50% shorter than the contralateral, normal femur. The usual treatment of these extreme cases of congenital femoral deficiency is a Syme or Boyd amputation when the child begins to walk. A knee fusion is done when the child is older, and the patient functions as an above-the-knee amputee. Rotationplasty has been described as an alternative treatment that allows the patient to function as a below-the-knee amputee. None of the currently described types of rotationplasty address the problem of the unstable hip. METHODS: Three patients with severe congenital femoral deficiency underwent a unique single-stage reconstruction. In this procedure, the limb is completely detached except for the sciatic nerve and the femoral vessels. The proximal part of the dysplastic femur and some muscles are resected. The residual limb is externally rotated 180 degrees and the rotated distal part of the femur is fused to the pelvis. All of the muscles distal to the knee remain undisturbed. RESULTS: The anatomical knee in its rotated position functioned as a hip with flexion and extension, and the femoropelvic arthrodesis provided a more stable support. The rotated ankle acted as a knee, and the patients functioned as below-the-knee amputees. The duration of follow-up of these three patients was eight, six, and four years. Active hip flexion was 10 degrees to 70 degrees , 10 degrees to 90 degrees , and 0 degrees to 80 degrees , and active knee flexion was 900 and 95 . The patients' gait continued to improve as they matured. CONCLUSIONS: The femoropelvic arthrodesis provides a stable hip. Since the muscles distal to the knee are not disturbed, the problem of derotation of the limb, which is often seen following the Van Nes rotationplasty, is not seen after this operation.

Arthrodesis↗
PubMed2000

Translation/secretion coupling by type III secretion systems.

Type III secretion systems mediate export of virulence proteins and flagellar assembly subunits in Gram-negative bacteria. Chaperones specific to each class of secreted protein are believed to prevent degradation of the secreted substrates. We show that an additional role of chaperones may be to regulate translation of secreted proteins. We show that the chaperone FIgN is required for translation of the flgM gene transcribed from one mRNA transcript (a flagellar class 3 transcript), but not from another (a flagellar class 2 transcript). FIgM translated from the class 3 transcript is primarily secreted whereas FIgM translated from the class 2 transcript is primarily retained in the cytoplasm. These results suggest FIgM and other type III secretion substrates possess both mRNA and amino acid secretion signals, and supports a new role for type III chaperones in translation/secretion coupling.

Bacterial Proteins↗
PubMed2000

Detection of PrP in extraneural tissues.

Transmissible spongiform encephalopathies (TSEs) or "prion diseases" are a group of unconventional fatal diseases. TSEs are characterised by the accumulation of a modified form of the normal host glycoprotein, PrP (PrP(c)). In the course of infection PrP(c) is converted to an abnormally protease resistant form, PrP(Sc). The exact nature of the infectious agent responsible for these diseases remains controversial. While there is compelling evidence that TSE agents contain an informational molecule, possibly a nucleic acid, some believe that the infectious agent or "prion" is solely composed of PrP(Sc). Nevertheless, PrP is required for TSE pathogenesis, as mice devoid of the PrP gene (PrP(-/-)) remain healthy when challenged with TSE isolates and are unable to replicate infectivity within the central nervous system (CNS) or in other tissues. In recent years immunocytochemistry has been used to pinpoint which cells are associated with abnormal accumulations of PrP, providing important information on the cellular targeting of TSE infection. In uninfected and scrapie-infected mice, PrP protein is found in the CNS and in extraneural tissues such as spleen and lymph nodes. In the peripheral lymphoid system, PrP is associated with follicular dendritic cells that are known to be important for replication of infectivity for at least one TSE strain. This review will focus on current methods for the immunocytochemical detection of PrP in murine extraneural tissues, mainly lymphoid tissues, and will discuss recent findings on the role of the peripheral lymphoid system in TSE pathogenesis.

Animals↗
PubMed2000

A role for the cell adhesion molecule CD44 and sulfation in leukocyte-endothelial cell adhesion during an inflammatory response?

CD44 is a widely expressed cell adhesion molecule that has been implicated in a variety of biological processes including lymphopoiesis, angiogenesis, wound healing, leukocyte extravasation at inflammatory sites, and tumor metastasis. The adhesive function of CD44, like other molecules involved in inducible adhesion, is tightly regulated. Post-translational modifications, isoform expression, aggregation state, and protein associations all can affect the ligand binding properties of CD44, and these can vary depending on the cell type and the activation state of the cell. The most extensively characterized ligand for CD44 is hyaluronan, a component of the extracellular matrix. Interactions between CD44 and hyaluronan can mediate both cell-cell and cell-extracellular matrix adhesion. In the immune system, both the selectin molecules and CD44 have been implicated in the initial binding of leukocytes to endothelial cells at an inflammatory site. Sulfation is required for selectin-mediated leukocyte-endothelial cell interactions, and, recently, inducible sulfation also was shown to regulate CD44-mediated leukocyte adhesion to endothelial cells. Sulfation, therefore, may be important in the regulation of cell adhesion at inflammatory sites. In this commentary we have reviewed the molecular aspects of CD44 and the mechanisms that regulate its binding to hyaluronan. In addition, we have summarized the role of CD44 and hyaluronan in mediating leukocyte-endothelial cell interactions and have discussed how this interaction may be regulated. Finally, we examined the potential role of sulfation as an inducible means to regulate CD44-mediated leukocyte adhesion and as a more general mechanism to regulate leukocyte-endothelial cell interactions.

Animals↗
PubMed2000

Sites of prion protein accumulation in scrapie-infected mouse spleen revealed by immuno-electron microscopy.

Prion protein (PrP) from the brains of animals with transmissible spongiform encephalopathies is partially protease resistant (PrP(res)) compared with fully sensitive PrP (PrP(sen)) from uninfected brains. In most experimental models, PrP(res) is a reliable indicator of infectivity. Light microscopic studies have suggested that both PrP(sen) and disease-specific accumulations of PrP are associated with follicular dendritic cells (FDCs). Using immunogold electron microscopy, this study has demonstrated disease-specific accumulation of PrP in the spleens of C57 BL mice, 70 days after intracerebral infection with the ME7 strain of scrapie and at the terminal stage of disease at 170 days. At both stages, tingible body macrophages contained PrP within lysosomes and PrP was also detected at the plasmalemma of FDCs. In the light zone of follicles of terminally diseased mice, all FDC dendrites were arranged in the form of highly reactive or hyperplastic labyrinthine glomerular complexes, within which PrP was consistently seen between FDC processes in association with abundant electron dense material, interpreted as antigen-antibody complexes. Within some glomeruli, fibrillar forms of PrP consistent with amyloid were seen. At 70 days after challenge, large or hyperplastic labyrinthine complexes were rare and invariably labelled for PrP. However, sparse PrP labelling was also seen on simple FDC processes at this stage. The ubiquitous accumulation of extracellular PrP in complex glomerular dendrites of FDCs in spleens from terminally affected mice, contrasted with simple FDC profiles, sparse PrP and limited electron dense deposits in all but a few FDCs of 70-day post-infected mice. This suggests that FDCs continually release PrP from the cell surface, where it is associated with trapped antigen-antibody complexes and dendritic extension. It is likely that tingible body macrophages acquire PrP following phagocytosis of PrP within iccosomes or from the extracellular space around FDC dendrites. These studies would not support an intracellular phase of PrP accumulation in FDCs but show that PrP is produced in excess by scrapie-infected cells from where it is released into the extracellular space. We suggest that PrP(sen) is involved in dendritic extension or in the process of antibody-antigen trapping, perhaps as part of the binding mechanism for antigen-antibody complexes. Reproduced with the permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd.

Animals↗
PubMed2000

Follicular dendritic cells in scrapie pathogenesis.

Scrapie pathogenesis was studied in chimaeric mice that carried the prion protein (PrP) gene only in particular cells of the immune system. These mice were produced by grafting bone marrow from PrP expressing donors into PrP deficient recipients and vice versa. As follicular dendritic cells are not replaced significantly from the bone marrow in adult mice, this procedure resulted in a mismatch in PrP genotype between these cells and bone marrow derived cells such as lymphocytes. Using these models we obtained strong evidence that follicular dendritic cells produced high levels of the normal form of PrP in uninfected mice. Furthermore, the replication of a mouse-passaged scrapie strain in the spleen depended only on the presence of PrP expressing follicular dendritic cells. PrP expression by lymphocytes or other bone marrow derived cells had no influence on replication in spleen or on neuroinvasion in these models. These results indicate that the follicular dendritic cell is a potential target for prophylactic or therapeutic intervention in transmissible spongiform encephalopathies.

Animals↗
PubMed2000

Cellular and sub-cellular localisation of PrP in the lymphoreticular system of mice and sheep.

Using immunocytochemistry or immunogold electron microscopy, abnormal PrP accumulation was found in lymphoreticular tissues of Suffolk sheep naturally exposed to scrapie and in the spleens of ME7 infected C57 BL mice at 70 days after infection and at the terminal stage of disease at 170 days. Clinically diseased scrapie affected sheep show widespread PrP accumulation within tingible body macrophages (TBMs) and follicular dendritic cells (FDCs) of secondary lymphoid follicles. Serial tonsillar biopsies taken from 171 ARQ/ARQ sheep at 4 months of age did not contain abnormal PrP accumulations but 80% of biopsies were positive by 14 months. In contrast, whole body necropsies of sheep not previously biopsied failed to detect PrP in the tonsil of sheep at 4, 8, 12 or 16 months of age. These findings suggest that the biopsy procedure of susceptible sheep but not resistant sheep may induce tonsillar infection. In spleen of mice both at 70 and 170 dpi, accumulations of PrP were found within lysosomes of TBMs and also at the plasma-lemma of FDCs. In the light zone of follicles of terminally diseased mice, all FDC dendrites were arranged in the form of highly reactive or hyperplastic labrynthine glomerular complexes. PrP was consistently seen between FDC dendrites in association with abundant electron dense antigen-antibody complexes. At 70 days after challenge, labrynthine complexes were rare and invariably labelled for PrP. However, sparse PrP labelling was also seen on simple FDC dendrites at this stage. These observations suggests that scrapie infected FDCs continually release PrP from the cell surface where it accumulates in excess in association with trapped immune complexes and dendritic extension. It is likely that TBMs acquire lysosomal PrP following phagocytosis of effete FDC processes or from the extracellular space. We suggest that the normal function of PrP may involve cell process extension or immune complex trapping.

Animals↗