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Biomedical subjects

J H Brock

Publications and source records attributed to J H Brock.

At least 19 recordsLinked to original sources
PubMed2002

Expression of ferritin, transferrin receptor, and non-specific resistance associated macrophage proteins 1 and 2 (Nramp1 and Nramp2) in the human rheumatoid synovium.

OBJECTIVE: To gain a better understanding of how iron accumulates in human rheumatoid synovium. METHODS: The distribution of ferritin, transferrin receptor, and non-specific resistance associated macrophage proteins 1 and 2 (Nramp1 and Nramp2) in the human rheumatoid synovium was investigated by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Both heavy and light ferritin subunit types were detected in the lining layer and the subinitimal zone of rheumatoid synovium, heavy ferritin generally being more abundant than light. Both heavy and light ferritin were detected in isolated synovial macrophages and fibroblasts. Transferrin receptor expression was largely confined to fibroblasts of the synovial lining layer. Nramp2 was detected by PCR in both isolated synovial macrophages and fibroblasts, whereas Nramp1 was detected by PCR and immunocytochemistry in macrophages and neutrophils in the lining and subinitimal zone, and in inflammatory infiltrates, but was absent from fibroblasts. CONCLUSION: A complex chain of events, perhaps initiated by proinflammatory cytokines, may culminate in a toxic build up of iron in the rheumatoid joint.

Arthritis, Rheumatoid↗
PubMed2001

Growth inhibition of Bacteroides fragilis by hemopexin: proteolytic degradation of hemopexin to overcome heme limitation.

The stimulatory effect of heme on growth of Bacteroides fragilis, an anaerobic human pathogen, was strongly inhibited by hemopexin, an avid (Kd<1 pM) heme-binding plasma protein. Both rabbit and human hemopexins were bacteriostatic for a limited period of time, suggesting an adaptation by B. fragilis to heme-limited growth, and that hemopexin-bound heme can eventually be utilized by the bacteria. The inhibitory effect of hemopexin was lost when heme in the medium was replaced by protoporphyrin IX, which is bound less strongly by hemopexin (Kd approximately 1 microM). Protease activity was detected in the culture supernatant of B. fragilis grown in the presence of heme plus hemopexin but not in the presence of free heme, protoporphyrin IX or protoporphyrin IX plus hemopexin, suggesting that the enzyme(s) is induced by heme macrocycle limitation due to the scavenging effect of hemopexin. This protease activity was able to degrade rabbit hemopexin and human hemopexin, as well as human transferrin and ovalbumin, and may be a due to a serine protease since it was inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by EDTA, leupeptin, pepstatin A or aprotinin. Thus, B. fragilis may overcome hemopexin-mediated heme limitation by secreting inducible protease(s), shown here to make protein-bound heme available to the microorganism.

Animals↗
PubMed2001

Bacteriostatic activity of human lactoferrin against Staphylococcus aureus is a function of its iron-binding properties and is not influenced by antibiotic resistance.

The in vitro antistaphylococcal activity of lactoferrin and the antibiotic resistance of clinical Staphylococcus aureus isolates obtained from three different sites of infection were examined. Antibiotic, but not lactoferrin resistance correlated with selective antibiotic pressure, and nosocomial and most community isolates were antibiotic resistant, whereas only a third of each group was resistant to lactoferrin. The antimicrobial activity of lactoferrin, both in defined medium and in normal human plasma serum, was dependent upon its ferrochelating properties. Therapeutic approaches based on the use of ferrochelating agents such as lactoferrin combined with antimicrobial drugs may help to counteract the reduced efficacy of current antibiotics.

Anti-Bacterial Agents↗
PubMed2001

Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria.

Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains of S. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.

Adhesins, Bacterial↗
PubMed2001

Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases.

Liposomes prepared from naturally occurring biodegradable and nontoxic lipids are good candidates for local delivery of therapeutic agents. Treatment of arthritis by intra-articular administration of anti-inflammatory drugs encapsulated in liposomes prolongs the residence time of the drug in the joint. We have previously shown that intra-articular injection of human lactoferrin (hLf), a glycoprotein that possesses anti-inflammatory and antimicrobial activities, into mice with collagen-induced arthritis reduces inflammation. We have now investigated the possibility of using liposome-entrapped hLf as a delivery system to prolong hLf retention at sites of local inflammation such as the rheumatoid joint. Entrapment of hLf in negatively charged liposomes enhanced its accumulation in cultured human synovial fibroblasts from rheumatoid arthritis (RA) patients, compared with positively charged formulations or free protein. However, in the presence of synovial fluid, positively charged liposomes with entrapped hLf were more stable than the negatively charged formulations. In vivo experiments in mice with collagen-induced arthritis showed that the positive liposomes were more efficient in prolonging the residence time of hLf in the inflamed joint as compared with other liposomes. Thus, the amount of hLf retained in the joint after 2 hr was 60% of the injected dose in the case of positive liposomes and only 16% for negative pH-sensitive liposomes. The results suggest that entrapment of hLf in positively charged liposomes may modify its pharmacodynamic profile and be of therapeutic benefit in the treatment of RA and other local inflammatory conditions.

Animals↗
PubMed2000

A novel endoproteolytic processing activity in mitochondria of erythroid cells and the role in heme synthesis.

The erythroid isoform of aminolevulinate synthase (eALAS) protein is a major control point in erythroid heme synthesis and hemoglobin formation. Erythroid cells were extracted from mouse blood and bone marrow and metabolically labeled with (35)S-methionine. This was followed by immunoprecipitation of eALAS protein products. The results show that the N-terminus of the expected full-length 59-kd form of the eALAS protein is truncated in bone marrow erythroid cells by approximately 7 kd. More differentiated erythroid cells in the peripheral blood exhibit very little of this protein truncation. Erythroid cells from the bone marrow were isolated using monoclonal antibody TER-119 and were shown to contain a unique endoprotease activity that could cleave the eALAS protein to the shorter form in vitro. With or without the mitochondrial signal sequence, the eALAS protein could serve as a substrate for the cleavage. This cleavage renders a functional eALAS protein and only removes a domain of unclear function, which has previously been reported to vary in size as a result of alternative RNA splicing. The protease activity was enriched from the membranes of mitochondria from bone marrow cells and was shown to be different from mitochondrial processing peptidase, medullasin, and other known proteases. Apart from the mitochondrial processing peptidase that cleaves the import signal sequence, this is the first description of a mitochondrially located site-specific processing protease activity. (Blood. 2000;96:740-746)

5-Aminolevulinate Synthetase↗
PubMed2000

Inhibition of binding of lactoferrin to the human promonocyte cell line THP-1 by heparin: the role of cell surface sulphated molecules.

Lactoferrin, an iron-binding protein of the transferrin family, is a highly basic protein which interacts with many acidic molecules, including heparin proteoglycans. Such interactions may modify some of the biological properties of lactoferrin. In the present work we found that heparin caused a dose-dependent inhibition of specific binding of both human and bovine lactoferrin to human monocytic THP-1 cells. Low-affinity binding sites (Kd 500 nM) were more susceptible to inhibition by heparin than the high-affinity sites (Kd 100 nM). The effect was mediated by interaction between lactoferrin and heparin rather than by competition between heparin and lactoferrin for common binding sites on the cells. Pretreatment of cells with NaClO3 to prevent sulphation of surface glycosaminoglycans reduced lactoferrin binding, and de-N-sulphated heparin did not inhibit binding of lactoferrin to THP-1 cells. These results suggest that heparin binding and monocyte/macrophage binding by lactoferrin both involve interactions between basic regions in the N1 domain of lactoferrin and sulphate groups. The N-terminal Arg2-Arg5 sequence of human lactoferrin may be involved, but it does not seem to be the key element in these interactions.

Animals↗
PubMed2000

The effects of local administration of lactoferrin on inflammation in murine autoimmune and infectious arthritis.

OBJECTIVE: To determine whether lactoferrin can modify articular inflammation in murine models of autoimmune and septic arthritis. METHODS: Collagen arthritis was induced in DBA/1 mice and Staphylococcus aureus septic arthritis in Swiss mice. Joints with established inflammation were injected periarticularly with 0.5 mg or 1 mg of human lactoferrin, and arthritis was monitored for 3 days. RESULTS: DBA/1 mice injected with lactoferrin showed significantly suppressed local inflammation for up to 3 days, achieving up to 71% of the effect of corticosteroid. Periarticular injection of 125I-lactoferrin confirmed that 25% of lactoferrin was retained in paws after 6 hours. Serum levels of interleukin-6, however, were not significantly reduced, suggesting a predominantly local antiinflammatory effect. Similarly, local, periarticular administration of lactoferrin into S aureus-infected Swiss mice significantly suppressed paw inflammation and did not enhance bacterial survival. CONCLUSION: Lactoferrin may have clinical utility in reducing articular inflammation, particularly in septic arthritis, in which antiinflammatory effects may be achieved without promoting bacterial survival.

Administration, Topical↗
PubMed2000

Cellular and molecular aspects of iron and immune function.

Fe plays a critical role in the immune system and defence against infection. However, many aspects of the way in which Fe influences these processes at the molecular and cellular level are unclear. The present review summarizes the role of Fe in lymphocyte activation and proliferation, and discusses how Fe is handled by macrophages.

Humans↗
PubMed1999

Regulation of iron metabolism in murine J774 macrophages: role of nitric oxide-dependent and -independent pathways following activation with gamma interferon and lipopolysaccharide.

To elucidate the pathways by which nitric oxide (NO) influences macrophage iron metabolism, the uptake, release, and intracellular distribution of iron in the murine macrophage cell line J774 has been investigated, together with transferrin receptor (TfR) expression and iron-regulatory protein (IRP1 and IRP2) activity. Stimulation of macrophages with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS) decreased Fe uptake from transferrin (Tf), and there was a concomitant downregulation of TfR expression. These effects were mediated by NO-dependent and NO-independent mechanisms. Addition of the NO synthase (NOS) inhibitor N-monomethyl arginine (NMMA) partially restored Fe uptake but either had no effect on or downregulated TfR expression, which suggests that NO by itself is able to affect iron availability. Analysis of the intracellular distribution of incorporated iron revealed that in IFN-gamma/LPS-activated macrophages there was a decreased amount and proportion of ferritin-bound iron and a compensatory increase in insoluble iron, which probably consists mainly of iron bound to intracellular organelles. Finally, although NO released by IFN-gamma/LPS-activated macrophages increased the iron-responsive element (IRE)-binding activity of both IRP1 and IRP2, IFN-gamma treatment decreased IRP2 activity in an NO-independent manner. This study demonstrates that the effect of IFN-gamma and/or LPS on macrophage iron metabolism is complex, and is not entirely due to either NO-or to IRP-mediated mechanisms. The overall effect is to decrease iron uptake, but not its utilization.

Animals↗
PubMed1999

Benefits and dangers of iron during infection.

Iron is essential for both human and microbial metabolism, and it is therefore important that iron status is maintained at a level that permits optimal immune responses by the host but does not facilitate availability of iron to microorganisms. This paper reviews the role of iron in resistance to infection, with particular reference to malaria and hepatitis C, and discusses the desirability of iron supplementation of populations at risk of infection.

Dietary Supplements↗
PubMed1998

Iron, lactoferrin and iron regulatory protein activity in the synovium; relative importance of iron loading and the inflammatory response.

OBJECTIVES: To determine the ability of lactoferrin in rheumatoid arthritis (RA) synovial fluid to bind "free" iron, and to study the regulatory mechanisms therein that control iron homeostasis. METHODS: "Free" iron was determined by the bleomycin assay and lactoferrin concentrations by enzyme linked immunosorbent assay. The activities of iron regulatory protein (IRP) and NF-kappa B in synovial fluid cells were assayed by mobility shift assay. RESULTS: 30% of synovial fluids contained "free" iron and in these, lactoferrin concentrations were significantly lower than in those with no "free" iron (p < 0.01). Addition of exogenous lactoferrin consistently reduced the amount of "free" iron in positive synovial fluids. IRP activity in synovial cells did not correlate with synovial fluid iron concentrations but did correlate with NF-kappa B activation and with serum C reactive protein. CONCLUSION: Lactoferrin may prevent iron mediated tissue damage in RA by reducing "free" synovial iron concentration when inflammatory stimuli have disregulated IRP mediated iron homeostasis.

Adult↗