RESUMEN
Experiments to evaluate the effect of the level and duration of endotoxaemia on the meningeal inflammatory response were performed in order to determine if systemic inflammation alters meningitis. Rabbits received either saline or Escherichia coli O111:B4 lipopolysacharide (LPS) intravenously at various doses (1, 3 or 10 microg) and times (-8, -2 or 0 h) before an intracisternal injection of 20 ng LPS. An intracisternal LPS injection together with saline intravenously produced a peak cerebrospinal fluid (CSF) tumour necrosis factor (TNF) level (95 +/- 26 ng/ml) at 2 h and peak leucocyte level (5413 +/- 764 cells/microl) at 4 h post-injection. Blood leucocytes were slightly elevated (12 000 +/- 500/microl at 0 h; 16 900 +/- 280/microl at 8 h) but plasma TNF was always undetectable (< 0.05 ng/ml). Conversely, intravenous injection of 3 or 10 microg LPS 2 h prior to intracisternal LPS injection impaired pleocytosis (peak < 220 cells/microl) and delayed ( approximately 4 h) and reduced peak CSF TNF levels (3 microg LPS 5.0 +/- 1.2 ng/ml; 10 microg LPS 6.9 +/- 1.9; P < 0.05). Intravenous administration of 1 microg LPS was less inhibitory to CSF inflammation, but delayed onset (peak 1100 +/- 60 leucocytes/microl CSF at 8 h; 6.3 +/- 0.3 ng TNF/ml CSF at 4 h; both P < 0.05). Neutropenia nadirs were dependent on LPS dose (1 microg, 4500 +/- 1700; 3 microg, 1900 +/- 60; 10 microg, 1100 +/- 100 all at 4 h post-intravenous dose). Peak plasma TNF levels were not dose-dependent (> 8 ng/ml), but plasma TNF was always detectable (> 0.2 ng/ml at 10 h post-intravenous dose). Intravenous LPS administration at 0 h also blocked pleocytosis, but the inhibitory effect was lost when administration at -8 h. In conclusion, the degree and duration of endotoxaemia affect the meningeal inflammatory response to LPS in experimental meningitis.
Asunto(s)
Endotoxemia/inmunología , Meningitis Bacterianas/inmunología , Modelos Animales , Animales , Líquido Cefalorraquídeo/microbiología , Relación Dosis-Respuesta Inmunológica , Escherichia coli , Inyecciones/métodos , Inyecciones Intravenosas , Recuento de Leucocitos , Lipopolisacáridos/farmacología , Masculino , Conejos , Factor de Necrosis Tumoral alfa/líquido cefalorraquídeoRESUMEN
Interleukin-8 (IL-8) is elevated in the cerebrospinal fluid (CSF) of patients with meningitis and is proposed to participate in subarachnoid-space pleocytosis. However, intracisternal injection of IL-8 into rabbits failed to induce indices typical of meningitis (leukocyte, tumor necrosis factor, or protein accumulation in the CSF or histopathological changes), indicating that merely increasing the CSF level of this chemokine is insufficient to induce inflammation in this anatomical site. IL-8 treatment did not affect inflammatory responses to subsequently intracisternally administered lipopolysaccharide (LPS). IL-8 was chemotactic for rabbit neutrophils in vitro, and subcutaneous injection of IL-8 (diluted in buffer or CSF) proved the in vivo activity of this peptide and suggested the absence of an IL-8 inhibitor in normal rabbit CSF. LPS-dependent pleocytosis was only slightly diminished by intracisternally administered murine anti-rabbit IL-8 monoclonal antibody (MAb) WS-4 but was dramatically reduced by intravenously administered MAb. Therefore, elevated CSF IL-8 levels may contribute to, but cannot solely account for, neutrophil influx into the subarachnoid space during meningitis. However, inhibition of IL-8 activity of the bloodstream side of the blood-brain barrier effectively reduces pleocytosis, indicating a central role of IL-8 in neutrophil influx into CSF during bacterial meningitis. Thus, inhibition of IL-8 is a possible therapeutic target for adjunct treatment of meningitis.
Asunto(s)
Interleucina-8/inmunología , Leucocitosis/inmunología , Lipopolisacáridos/inmunología , Meningitis Bacterianas/inmunología , Neutrófilos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Quimiotaxis de Leucocito , Humanos , Interleucina-8/administración & dosificación , Interleucina-8/sangre , Interleucina-8/líquido cefalorraquídeo , Pruebas de Neutralización , ConejosRESUMEN
Release of iron from transferrin, the iron-transporting protein of the circulation, is a concerted process involving remote amino acid residues as well as those at the two specific iron-binding sites of the protein. Previous studies of fluoresceinated transferrin have suggested Lys 569 as a kinetically active site in the C-terminal lobe of the protein. We have therefore turned to site-directed mutagenesis to investigate the role of Lys 569 in the release process at pH 5.6, the pH of the endosome where iron is transferred from transferrin to the iron-dependent cell. Mutation of positively charged Lys 569 to an uncharged Gln results in a protein in which release of iron from the mutated lobe to pyrophosphate is slowed by a factor of 15-20 and in which release kinetics switch from a complex saturation-linear to a simple saturation function. Acceleration of release by chloride is also substantially less than in native transferrin. When Lys 569 is replaced by a positively charged Arg, in contrast, observed release rates and chloride dependence are close to those of the native protein. The mechanism of release from the C-lobe site therefore appears to be sensitive to positive charge at position 569. Binding of chloride or other simple anion accelerates and is essential for release from the C-lobe; a muted response of K569Q to chloride concentration suggests that Lys 569 may function as a kinetically active anion-binding residue in the C-lobe. Despite the kinetic effects of the K569 mutation on iron release, rates of iron uptake by K562 cells from the C-lobes of native, K569Q, and K569R proteins are almost identical. In contrast to the C-lobe, iron release from the N-lobe is insensitive to charge at residue 233, the site in that lobe homologous to residue 569, with chloride retarding rather than accelerating release. K233, therefore, is not a kinetically active anion-binding site in the N-lobe. Release mechanisms differ substantially in the two lobes of transferrin despite the identity of ligands and their nearly identical arrangements in the lobes.
Asunto(s)
Lisina/química , Transferrina/química , Humanos , Hierro/química , Lisina/genética , Mutagénesis Sitio-Dirigida , Transferrina/genéticaRESUMEN
The inflammatory response associated with Staphylococcus aureus osteomyelitis results in extensive bone damage characterized by apparent increases in bone resorption and formation. These results suggest an increased local release of agents capable of modulating bone remodelling. Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine proposed to play an important role both in normal bone remodelling and in bone diseases; however, its potential role in osteomyelitis is unclear. This study evaluated changes in bone TNF levels during infection, using a rat model of acute osteomyelitis due to S. aureus. Following direct tibial infection, bacterial counts in bone were persistently high (approximately 6 log10 CFU/g of bone over 63 days) and bone weights increased. TNF activity was undetectable in uninfected bone (<0.01 ng/g of bone) but dramatically higher in infected bone (up to 5.2 +/- 3.5 ng/g of bone). Although TNF-alpha mRNA was weakly detected in uninfected bone, osteomyelitis was associated with up to 37-fold increases in expression of both the 1.6- and 2.4-kb transcripts. Both TNF activity and mRNA transcript levels remained elevated throughout the course of infection. TNF-alpha mRNA detected by in situ hybridization was present in osteoblasts as well as in populations of marrow cells and/or inflammatory infiltrate cells. Histopathology of infected bone indicated extensive bone resorption and adjacent areas of formation that were associated with cells expressing TNF-alpha mRNA. These data suggest that the elevated TNF levels induced by experimental infection may be directly related to changes in the histology of bone during osteomyelitis.
Asunto(s)
Osteomielitis/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Enfermedad Aguda , Animales , Huesos/patología , Hibridación in Situ , Masculino , Osteomielitis/patología , ARN Mensajero/análisis , Ratas , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The purpose of the present study was to evaluate the combination of azithromycin and rifampicin on experimental chronic osteomyelitis due to Staphylococcus aureus. Alterations in bone bacterial titre, activity of tumour necrosis factor (TNF), a cytokine implicated in inflammation-induced bone pathology, and histopathological changes during infection and following antibiotic treatment were evaluated. Rats were infected with S. aureus by direct tibial inoculation and then randomized 56 days after infection to receive saline treatment or a combination of azithromycin and rifampicin (50 mg/kg po and 25 mg/kg sc respectively) once daily for 21 days. The combination of azithromycin and rifampicin was successful as determined by dramatic reduction in bone bacterial counts (approximately log 4 cfu), but regrowth of the organisms occurred suggesting that the duration of treatment was insufficient. TNF alpha mRNA and TNF activity were constantly elevated by approximately 20- and >200-fold, respectively, and remained elevated irrespective of antimicrobial treatment. Bone histology revealed extensive increase in bone turnover in both the infected and antibiotic treated bones with no difference being observed between the groups. This suggests that, in infected bone, the elevated TNF levels observed may be directly related to the bone pathology and both remain largely unchanged despite potent antibiotic therapy.
Asunto(s)
Azitromicina/farmacología , Quimioterapia Combinada/farmacología , Osteomielitis/tratamiento farmacológico , Rifampin/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibióticos Antituberculosos/farmacología , Antibióticos Antituberculosos/uso terapéutico , Azitromicina/uso terapéutico , Huesos/efectos de los fármacos , Huesos/microbiología , Huesos/patología , Enfermedad Crónica , Modelos Animales de Enfermedad , Quimioterapia Combinada/uso terapéutico , Osteomielitis/microbiología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Rifampin/uso terapéutico , Staphylococcus aureus/efectos de los fármacos , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Proton magnetic relaxation times T1 and T2 were measured at field strengths from 0.05 T to 1.5 T in solutions of ferritin with loading factors from 90 to 3600 iron atoms per molecule. 1/T2 increased linearly with field strength, as previously observed, and the slope per unit iron was approximately the same in all samples. This latter finding indicates that the field dependence of T2 may be used as a measure of ferritin-bound iron, regardless of loading factor. A possible explanation is presented, based on the presumed antiferromagnetic structure of the ferritin core and the linear dependence of 1/T2 on core magnetization. A nonzero contribution to 1/T2 in the limit of low field and a contribution to 1/T1 were also found, both of which increase linearly with loading factor for constant protein concentration; these effects represent quantum mechanical dipole-dipole relaxation of water protons either by iron atoms on the surface of the core or by the iron core itself. Finally, the extrapolated intercept at LF = 0 for both 1/T1 and 1/T2 indicates a contribution from a small number of iron ions bound to the protein shell. These results may help in the use of MRI to measure brain iron and possibly even ferritin loading factor.
Asunto(s)
Ferritinas/química , Espectroscopía de Resonancia Magnética , Animales , Apoferritinas/química , Encéfalo/metabolismo , Caballos , Hierro/química , Quelantes del Hierro/química , Modelos Lineales , Imagen por Resonancia Magnética , Peso Molecular , Proteínas/química , Bazo/química , Agua/químicaRESUMEN
The hepatic uptake of transferrin-bound iron by a nontransferrin receptor (NTR)-mediated process was investigated using the human hepatoma cell line HuH7. Because HuH7 cells also acquire iron from transferrin by a receptor (TR)-mediated process, TR expression was inhibited by transfecting the cells with a plasmid containing human TR complementary DNA in antisense orientation relative to a human cytomegalovirus promoter/enhancer element. Cell clones were obtained that expressed a 50% to 60% reduction in cell surface TR, leading to a corresponding decrease in transferrin and iron uptake compared with wild-type cells. Uptake of transferrin by a second process was nonsaturable and not inhibited by a 100-fold excess of unlabeled transferrin. The amounts of transferrin taken up by the wild-type and antisense cells by this process were similar, showing that it did not involve TR. The proteolytic enzyme Pronase reduced the uptake of transferrin, suggesting that the NTR-mediated process entailed the nonsaturable binding of transferrin to plasma membrane proteins. This process, like the TR-mediated one, involved the internalization and recycling of transferrin, leading to accumulation of iron with time. Iron uptake mediated by NTR process was saturable and displaced by 100-fold excess unlabeled transferrin and reduced by weak bases and metabolic inhibitors. Therefore, the NTR-mediated process entailed transferrin adsorption to membrane-bound proteins, internalization, and release of iron from transferrin by a pH-dependent step followed by the intracellular transport of iron into ferritin and heme by a saturable carrier-mediated mechanism.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores de Transferrina/metabolismo , Transferrina/metabolismo , Transporte Biológico Activo , Carcinoma Hepatocelular/genética , ADN sin Sentido/genética , ADN sin Sentido/farmacología , Expresión Génica , Humanos , Hierro/metabolismo , Cinética , Neoplasias Hepáticas/genética , Receptores de Transferrina/antagonistas & inhibidores , Receptores de Transferrina/genética , Células Tumorales CultivadasRESUMEN
Mutations of kinetically active residues in the recombinant N-lobe of human transferrin may accelerate or retard release of iron from the protein to pyrophosphate, thereby providing means for exploring the individual roles of such residues in the concerted mechanisms of release. Using an established spectrofluorometric method and pyrophosphate as the required iron-sequestering agent, we have compared release from unaltered native transferrin and recombinant N-lobe half-transferrin to release from six N-lobe mutants, R124S, R124K, K206R, H207E, H249Y, and Y95H. Mutation of R124, which serves as a principal anchor for the synergistic carbonate anion ordinarily required for iron binding by transferrin, accelerates release. This effect is most marked at endosomal pH, 5.6, and is also evident at extracellular pH, 7.4, pointing to a critical and perhaps initiating role of carbonate in the release process. Mutation of K206 to arginine, or of H207 to glutamine, each lying in the interdomain cleft of the N-lobe, gives products mimicking the arrangements in lactoferrin. Release of iron from these two mutants, as from lactoferrin, is substantially slower than from unaltered recombinant N-lobe. Interdomain residues not directly involved in iron or anion binding may therefore participate in the control of iron release within the endosome. The H249Y mutant releases iron much more rapidly than its wild-type parent or any other mutant, possibly because of steric effects of the additional phenolic ring in the binding site. No simple explanation is available to account for a stabilizing effect of the Y95H mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hierro/metabolismo , Proteínas Recombinantes/metabolismo , Transferrina/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transferrina/química , Transferrina/genéticaRESUMEN
The aim of the present study was to determine if human N-terminal half-transferrin (N- fragment), prepared by thermolysin cleavage of diferric transferrin, would bind to the rat hepatocyte transferrin receptor and donate iron to the cell. Competition experiments between 125I-labelled N-fragment and diferric transferrin revealed no receptor binding of the half-transferrin. Still, the N-fragment delivered iron to the cells in amounts approximately 30-fold above what could be accounted for by uptake of the fragment itself. The rate of cellular iron uptake from the fragment was comparable to what is seen with the intact transferrin. The uptake of 125I-labelled N-fragment was not inhibited by excess non-radioactive diferric transferrin. By comparison, the uptake of 59Fe from the N-fragment was inhibited 70% by excess nonradioactive diferric transferrin. This suggests that iron derived from diferric transferrin competes with the iron derived from the N-fragment for a common transport pathway. Although some cellular degradation of the N-fragment occurred, the extent of degradation was too low to explain the amount of iron accumulated by the cells. The results show that the hepatocyte has an effective transferrin-receptor-independent mechanism for accumulation of iron from transferrin.
Asunto(s)
Hierro/metabolismo , Hígado/metabolismo , Transferrina/metabolismo , Animales , Células Cultivadas , Humanos , Transporte Iónico , Masculino , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores de Transferrina/metabolismoRESUMEN
The objective of adjunct anti-inflammatory therapy of bacterial meningitis is the containment of heightened inflammation caused by lysis of bacteria by antibiotics. This can be modelled by giving two consecutive intra-cisternal injections of lipopolysaccharide (LPS) to rabbits, the first at 0 h to induce inflammation to mimic that occurring during the proliferation of bacteria in the cerebrospinal fluid (CSF), and the second at 6 h to mimic inflammation subsequent to antibiotic-induced bacterial lysis. Injection of 2.5 ng of LPS induced pleocytosis at 4 h which was preceded by a peak of tumour necrosis factor (TNF) activity at 2 h. A subsequent injection of 25 ng of LPS at 6 h induced second peaks of pleocytosis and CSF TNF. Dexamethasone (1.5 mg/kg, i.v.) administered 15 min or 1 h before the second injection of LPS tended only to reduce CSF TNF, but effectively prohibited further pleocytosis. Brain TNF alpha mRNA levels were unchanged at 6 and 7 h after LPS injection, and were unaffected by dexamethasone. These results indicate that the subarachnoid space is distinct from the general circulation in that the TNF-producing cells present do not display a hypo-responsive state towards LPS as occurs when LPS is injected systemically. Furthermore, dexamethasone is able to attenuate the secondary inflammatory response resulting from a second LPS injection without eliminating a second peak of TNF activity.
Asunto(s)
Dexametasona/uso terapéutico , Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli , Lipopolisacáridos/toxicidad , Meningitis Bacterianas/tratamiento farmacológico , Animales , Secuencia de Bases , Química Encefálica , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/citología , Proteínas del Líquido Cefalorraquídeo/análisis , Cartilla de ADN/química , Modelos Animales de Enfermedad , Glucosa/análisis , Lactatos/análisis , Recuento de Leucocitos , Datos de Secuencia Molecular , ARN Mensajero/análisis , Conejos , Organismos Libres de Patógenos Específicos , Espacio Subaracnoideo/patología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The usefulness of scid mice bearing endogenous Pneumocystis carinii infection as a model for experimental chemotherapy was examined using standard compounds known to be effective against P. carinii. Trimethoprim/sulphamethoxazole was able to reduce pulmonary P. carinii cysts in a dose-dependent manner within the dose range studied (10/50 to 100/500 TMP/SMX mg/kg/d, bd, po, 5 days per week for 30 treatments). However, alterations in associated symptoms of infection (reduced body weight, increased lung weight, increased blood leucocytes and erythrocytes), was apparently not linearly dose-dependent. Blood and lung lavage fluid levels of sulphamethoxazole one hour post administration of trimethoprim/sulphamethoxazole was dose-dependent, but not linear with dose, and was apparently correlated to cyst reduction; trimethoprim was below the limit of detection at this time. Treatment of mice with 100/500 mg/kg/day trimethoprim/sulphamethoxazole required 2 weeks (bd for 10 days of treatment) before changes in indices of infection became significant. Pentamidine (20 mg/kg, sc, three times per week for 3 weeks) was nearly as effective as high-dose trimethoprim/sulphamethoxazole in reducing cysts, whereas lower doses were ineffective. Despite being unable to reduce pulmonary P. carinii infection, even low doses of pentamidine (6 or 2 mg/kg, sc, three times per week for 3 weeks) were able to reduce lung weights and blood leucocyte levels. This model of pulmonary P. carinii infections is amenable to chemotherapeutic intervention in an apparently dose-dependent fashion, and can be used to evaluate the capacity of compounds to eradicate P. carinii and resolve signs of infection.
Asunto(s)
Antiinfecciosos/uso terapéutico , Ratones SCID/microbiología , Neumonía por Pneumocystis/tratamiento farmacológico , Animales , Antifúngicos/uso terapéutico , Recuento de Células Sanguíneas , Líquidos Corporales/metabolismo , Relación Dosis-Respuesta a Droga , Pulmón/microbiología , Ratones , Tamaño de los Órganos/efectos de los fármacos , Pentamidina/uso terapéutico , Neumonía por Pneumocystis/sangre , Neumonía por Pneumocystis/microbiología , Combinación Trimetoprim y Sulfametoxazol/farmacocinética , Combinación Trimetoprim y Sulfametoxazol/uso terapéuticoRESUMEN
The postantibiotic effect (PAE) is the persistent inhibition of bacterial growth after a brief exposure to an antibiotic. Most beta-lactams do not induce a PAE for Gram-negative bacteria, but PAEs have been reported for carbapenems and penems. This study investigated the effect of sequential doses of imipenem on the PAE for Pseudomonas aeruginosa and Escherichia coli cultures in a chemostat. The PAE for the bacterial population did not change even after six successive exposures to imipenem. Nevertheless, screening of colonies isolated after repeated drug exposure identified a single P. aeruginosa mutant whose imipenem PAE was shortened, although the MIC was unchanged. The PAEs for the parent and mutant were studied in vitro in batch culture by monitoring: (i) viable counts; (ii) electrical impedance of the culture medium; (iii) incorporation of radiolabelled N-acetyl-D-glucosamine and (iv) cell volume changes. PAEs for the parent and mutant were found to be significantly different by all in-vitro methods used. Moreover, the median cell volume in antibiotic-exposed cultures remained much smaller and less heterogeneous than in the control cultures, even though both cultures were growing at the same rate. The mutant was found to have a reduced expression of a 52 kDa outer membrane protein. These observations suggest that factors in addition to suppression of bacterial growth should be considered when studying the PAE. The PAEs of imipenem for the parent and mutant were studied in a thigh infection model in leucopenic mice. Similar PAEs were observed in vivo for both parent and mutant in one experiment and no PAEs for either organism were found in a second experiment. This study showed that although the PAE is a stable in-vitro phenomenon, the lack of correlation between the in-vitro and in-vivo results warrants caution in attributing clinical significance to the PAE of imipenem.
Asunto(s)
Imipenem/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Acetilglucosamina/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Conductividad Eléctrica , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Femenino , Imipenem/farmacocinética , Leucopenia/metabolismo , Leucopenia/microbiología , Ratones , Ratones Endogámicos , Pruebas de Sensibilidad Microbiana , Mutación , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismoRESUMEN
The role of the transferrin receptor in capturing and conveying transferrin through the cell during the iron-donating cycle of receptor-mediated endocytosis has been studied extensively. Nevertheless, almost nothing is known of how human transferrin binds to its receptor. In an initial approach toward delineating the receptor-recognition site(s) of human transferrin, we have studied the interactions of proteolytically-cleaved, single-sited fragments of transferrin, representing the N- and C-lobes of the molecule respectively, with cells expressing the transferrin receptor on their plasma membranes. Only the C-fragment was found capable of donating iron to hepatoma-derived HuH-7 cells or of binding to surface receptors of HuH-7 and leukemic K562 cells. Although no association of N- and C-fragments could be demonstrated by gel chromatography, the presence of excess N-fragment strengthened the binding of C-fragment by an order of magnitude. An explanation of these observations is that the primary receptor recognition site of human transferrin is on the C-lobe of the protein, but that prior binding of this lobe to receptor enables the N-lobe to respond to receptor as well, either directly or by interaction with the bound C-lobe.
Asunto(s)
Receptores de Transferrina/metabolismo , Transferrina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Hidrólisis , Hierro/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Termolisina , Transferrina/química , Células Tumorales CultivadasRESUMEN
For clinical trials of anti-infective drugs for the treatment of vascular access device-related bloodstream infections, patients should be identified and enrolled on the basis of current standards for the clinical diagnosis of such infections. To ensure comparability of patients, only those infected with staphylococci and Candida species should be included. A prospective, randomized, double-blind design is recommended. Future protocols may include abbreviated courses of therapy, treatment with combinations of drugs, or a progression from parenteral to oral therapy. Clinical response is judged as cure, failure, or indeterminate response; there is no "improved" category. Microbiological response is categorized as eradication, persistence, or relapse and is of paramount importance. Several months of follow-up may be necessary for the detection of late relapses or metastatic infections. This guideline does not apply to studies of bacteremia or fungemia secondary to non-device-related, organ-based primary infections (e.g., pneumonia, urinary tract infection), which should be assessed in relation to the primary disorder.
Asunto(s)
Antiinfecciosos/uso terapéutico , Bacteriemia/tratamiento farmacológico , Ensayos Clínicos como Asunto , Fungemia/tratamiento farmacológico , Bacteriemia/etiología , Candidiasis/tratamiento farmacológico , Candidiasis/etiología , Catéteres de Permanencia/efectos adversos , Ensayos Clínicos como Asunto/métodos , Ensayos Clínicos como Asunto/normas , Fungemia/etiología , Humanos , Proyectos de Investigación , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/etiologíaRESUMEN
Rates of iron release from both sites of free transferrin at pH 7.4 are critically dependent upon ionic strength, because release appears to require binding of a simple nonchelating anion such as chloride to a kinetically active site of the protein. This site is distinct from the synergistic anion-binding site, occupancy of which is required for binding of iron to occur at all. Complexing of transferrin to its receptor also modulates release of iron, but in a more complex fashion. At extracellular pH, 7.4, receptor retards release, but at the pH of the endosome in which release occurs within the cell, 5.6, receptor accelerates release. The present study was undertaken to determine whether the kinetically active anion requirement is maintained at pH 5.6 and whether the effects of anion binding and receptor binding are independent of each other. A spectrofluorometric method was developed to monitor release of iron from C-terminal monoferric human transferrin and its complex with the transferrin receptor. At pH 5.6, as at pH 7.4, profiles of iron release to pyrophosphate from free and from receptor-complexed monoferric transferrin show curvilinear dependence on pyrophosphate concentration, consistent with a previously described kinetic scheme and suggestive of a similar release mechanism in all cases. Furthermore, at pH 5.6 release rates depend upon anion (chloride) concentration in free and in receptor-complexed transferrin as in free transferrin at pH 7.4, extrapolating nearly to zero as chloride concentration approaches zero. The enhancing effect of receptor on release is displayed at all concentrations of chloride tested,indicating that the release-promoting effects of receptor and chloride are independent of each other.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cloruros/farmacología , Hierro/metabolismo , Receptores de Transferrina/metabolismo , Transferrina/metabolismo , Aniones , Sitios de Unión , Cloruros/metabolismo , Femenino , Humanos , Concentración de Iones de Hidrógeno , Cinética , Fosfatos/metabolismo , Placenta/química , Conformación Proteica , Espectrometría de Fluorescencia , Transferrina/químicaRESUMEN
The effect of the duration of treatment on clinical and bacteriological efficacy is generally poorly evaluated for antimicrobial agents. Infection model studies have to a large extent concentrated on endocarditis. In clinical trials systematic and well controlled studies are abundant in the field of urinary tract infections and streptococcal tonsillopharyngitis, but almost non-existent in other types of infections. Paradoxically, most of the infections studied by experimental models are uncommon in man and evaluation of optimal treatment periods for these by clinical trial is therefore difficult. In the evaluation of new antimicrobial agents, closer collaboration of experimental model and clinical trial investigators is required to design models of infections and clinical trials to evaluate optimal duration of treatment.
Asunto(s)
Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Animales , Antibacterianos/administración & dosificación , Modelos Animales de Enfermedad , Endocarditis Bacteriana/tratamiento farmacológico , Humanos , Osteomielitis/tratamiento farmacológico , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones Estreptocócicas/tratamiento farmacológico , Infecciones Urinarias/tratamiento farmacológicoRESUMEN
A recombinant plasmid carrying human transferrin receptor cDNA in reverse orientation downstream from the human cytomegalovirus immediate early promoter/enhancer element was introduced into the HuH-7 human hepatoma cell line by lipofection. Cell surface transferrin binding and iron uptake from transferrin each decreased by about 50% in stable transfectants bearing integrated antisense DNA expression vector. Northern blot analysis indicated that the abundance of target transferrin receptor message was not altered by antisense RNA. These results suggest that the antisense transcript interferes with expression of the endogenous transferrin receptor gene at the level of translation.
Asunto(s)
Carcinoma Hepatocelular/genética , Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas/genética , Oligonucleótidos Antisentido/farmacología , Receptores de Transferrina/genética , Secuencia de Bases , Northern Blotting , Southern Blotting , Carcinoma Hepatocelular/patología , Vectores Genéticos , Humanos , Neoplasias Hepáticas/patología , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , Factores de Tiempo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
We examined the effect of azithromycin (CP-62,993), a new oral macrolide-like antibiotic, alone and in combination with rifampin, as treatment for experimental staphylococcal osteomyelitis. Clindamycin was used as a comparison drug. Rats (n = 10 to 15 per group) were infected by direct instillation of Staphylococcus aureus into the tibial medullary cavity. After 10 days, 21-day treatments with azithromycin (50 mg/kg of body weight, once daily, by the oral route), rifampin (20 mg/kg, once daily, subcutaneously), or clindamycin (90 mg/kg, three times daily, by the oral route) were started. The drugs were used singly or in combination (azithromycin plus rifampin or clindamycin plus rifampin). Peak azithromycin concentrations in bone were > 30 times higher than levels in serum, but the drug had little effect on final bacterial titers (5.13 +/- 0.46 log10 CFU/g of bone; for controls, 6.54 +/- 0.28 log10 CFU/g). Clindamycin was more active than azithromycin (3.26 +/- 2.14 log10 CFU/g of bone; 20% of sterilized bones), but rifampin was the most active single drug (1.5 +/- 1.92 log10 CFU/g; 53% of sterilized bones). Therapy with rifampin or clindamycin alone was associated with the emergence of resistance. Rifampin plus azithromycin (0.51 +/- 1.08 log10 CFU/g of bone; 80% of sterilized bones) and rifampin plus clindamycin (0.87 +/- 1.34 log10 CFU/g of bone; 66% of sterilized bones) were the most active regimens. Thus, azithromycin is ineffective as a single drug for the treatment of experimental staphylococcal osteomyelitis, despite high levels in bone that markedly exceeded the MIC, but it may be an attractive partner drug for rifampin.
Asunto(s)
Huesos/metabolismo , Clindamicina/uso terapéutico , Eritromicina/análogos & derivados , Osteomielitis/tratamiento farmacológico , Rifampin/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Animales , Azitromicina , Huesos/microbiología , Clindamicina/farmacocinética , Farmacorresistencia Microbiana , Quimioterapia Combinada/farmacocinética , Quimioterapia Combinada/uso terapéutico , Eritromicina/farmacocinética , Eritromicina/uso terapéutico , Masculino , Osteomielitis/metabolismo , Osteomielitis/microbiología , Ratas , Rifampin/farmacocinética , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiologíaRESUMEN
Nuclear magnetic relaxation times T1 and T2 were measured in ferritin solutions at field strengths from 0.04 to 1.5 T. T1 was relatively constant, but 1/T2 increased linearly with field strength, in agreement with earlier MRI observations in the monkey brain. This finding supports the theory that ferritin is responsible for T2 shortening in brain nuclei containing iron. The linear dependence of 1/T2 on magnetic field is unique and not explained by present theories of the magnetic properties of ferritin.
Asunto(s)
Ferritinas/química , Imagen por Resonancia Magnética , Animales , Apoferritinas/análisis , Apoferritinas/química , Cobre/química , Sulfato de Cobre , Difusión , Espectroscopía de Resonancia por Spin del Electrón , Ferritinas/análisis , Aumento de la Imagen , Hierro/análisis , Imagen por Resonancia Magnética/métodosRESUMEN
Elevation of the environmental temperature appeared to counteract the temperature-depressing effects of urethane anesthetic and allowed rabbits intracisternally infected with Haemophilus influenzae type b to mimic the development of a fever following infection. Elevated core body temperature (greater than 39 degrees C) was associated with an inhibition of the growth of H. influenzae in cerebrospinal fluid (CSF) during the first 12 h postinfection, whereas bacterial growth was essentially unrestricted in rabbits with reduced (approximately 37 degrees C) body temperature. Bacterial densities 24 h postinfection were different, hyperthermic animals having log 6.0 +/- 0.4 CFU/ml of CSF and hypothermic rabbits having log 8.2 +/- 0.8 CFU/ml of CSF (P less than 0.05, Wilcoxon rank sum test). However, the growth of this bacterium in vitro, in either pooled rabbit CSF or brain heart infusion broth, was not inhibited at 39 degrees C. These results suggest that elevated body temperature associated with the development of fever during meningitis may be associated with restriction of the growth of H. influenzae in vivo but that this effect is apparently not due to an innate inability of the bacterium to grow at elevated temperatures.