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Biomedical subjects

J Verhoef

Publications and source records attributed to J Verhoef.

At least 37 records · Page 2Linked to original sources

Pharmacokinetics of ciprofloxacin after intraperitoneal administration in uninfected patients undergoing CCPD.

Ciprofloxacin is increasingly used to treat peritoneal dialysis related peritonitis. We studied the pharmacokinetics of intraperitoneally administered ciprofloxacin in six uninfected CCPD patients. In a randomized cross-over setting ciprofloxacin was added either to a long dwell exchange (lastbag) or to four short dwell exchanges (dwell time 1.5 h). Addition of ciprofloxacin (25 mg/l) during the four short dwell exchanges resulted in dialysate levels of 21.1-13.3 mg/l during these exchanges. In the subsequent last bag devoid of ciprofloxacin a dialysate Cmax,D of 1.38 mg/l was observed at 30 min. Mean +/- SD serum Cmax,S was 0.59 +/- 0.29 mg/l after 5.4 h. Instillation of 100 mg/l ciprofloxacin in the last-bag yielded Cmax,D of 99.1 mg/l, falling with a t1/2 of 3.3 h towards levels < 2 mg/l at 19.8 h. A mean +/- SD serum Cmax,S of 0.69 +/- 0.19 was reached after 4 h. During four subsequent 1.5h exchanges without ciprofloxacin dialysate levels were < 0.1 mg/l. Instillation of 25 mg/l ciprofloxacin in the last-bag yielded a Cmax,D of 21.7 mg/l, falling towards levels < 2 mg/l at 15 h with a t1/2 of 3.85 h. A mean +/- SD serum Cmax,S of 0.26 +/- 0.03 was reached after 8 h. We conclude that the rapid absorption of ciprofloxacin from the dialysate into the tissues requires ciprofloxacin to be administered to all CCPD bags to ensure bactericidal dialysate levels. When therapeutic serum levels are required higher intraperitoneal doses or an oral or i.v. loading dose is warranted.

Adult

Bacteroides and Staphylococcus glycocalyx: chemical analysis, and the effects on chemiluminescence and chemotaxis of human polymorphonuclear leucocytes.

Glycocalyx (or slime), which is an important virulence factor of many pathogenic bacteria, was isolated from Bacteroides fragilis, Bacteroides thetaiotaomicron and Staphylococcus epidermidis. Organisms were grown for 24 h in a chemically defined, dialysable liquid medium. Bacteria were centrifuged and the supernatant was concentrated and dialysed against distilled water. Total carbohydrate and protein were estimated using standard methods. Thin layer and gas-liquid chromatography of trifluoro acetic acid hydrolysed and non-hydrolysed samples provided evidence for the presence of polysaccharide, the absence of nucleic acids and lipopolysaccharide and for the identification of the individual sugar residues. Glucose, mannose and galactose (B. fragilis), glucose (B. thetaiotaomicron), and glucose and heptose (S. epidermidis) were the sugar residues detected. Uronic acid and hexosamine were detected in all species. Glycocalyx preparations (1 mg/ml) from Bacteroides and Staphylococcus significantly inhibited the chemiluminescence and chemotactic responses of viable human polymorphonuclear leucocytes (PMNL), but were not toxic for PMNL.

Bacteroides

Intranasal estradiol administration to oophorectomized women.

An open, dose-finding study into the effects of a new intranasal 17 beta-estradiol formulation using dimethyl-beta-cyclodextrin as a solubilizer was conducted in nine hysterectomized and oophorectomized patients with symptoms of estrogen deficiency. After nasal delivery of 0.34 mg estradiol in three patients, concentration-time curves of estradiol in serum and its metabolite estrone in plasma were established. High initial estradiol serum levels were found in the order of 5 nmol/l. Estrone/estradiol ratios were below unity, whereas, in contrast, estrone/estradiol ratios after oral administration of estradiol are known to exceed unity. Biological activity, as reflected by suppression of FSH and LH, was manifest. In nine patients symptoms of estrogen deficiency were assessed by a questionnaire. After 3 and 6 months of treatment, a clinically and statistically significant decrease (P less than 0.01) of the total score according to this questionnaire was found compared to the beginning of the study. Thus nasal delivery of estradiol using this cyclodextrin formulation has potential for further clinical evaluation.

Administration, Intranasal

Analysis of Escherichia coli isolates from subjects with travellers' diarrhoea using DNA probes and serotyping.

Escherichia coli isolated from faeces of 54 healthy volunteers who visited Tunisia for eight days were examined. These volunteers participated in a randomized double-blind placebo-controlled study to establish whether ciprofloxacin could prevent travellers' diarrhoea. Escherichia coli strains isolated before travel, during episodes of travellers' diarrhoea, immediately after return and five weeks after return were serotyped and tested for the presence of virulence genes indicating diarrheogenic properties by hybridization with a set of four non-radioactively labelled DNA probes. Subjects receiving ciprofloxacin prophylactically to prevent travellers' diarrhoea were asymptomatic and no Escherichia coli could be cultured shortly after return home. Sixty-four percent of subjects (18/28) who did not receive antibiotic prophylaxis suffered from travellers' diarrhoea. Hybridization tests detected 8 enterotoxigenic Escherichia coli strains producing heat stable toxin, 13 enterotoxigenic strains producing heat labile toxin and 10 strains which produced both heat labile and stable toxin. Of the 31 probe positive strains, 29 (94%) were cultured from 11 volunteers with travellers' diarrhoea. A bacterial cause was thus determined in 61% of the volunteers who experienced travellers' diarrhoea.

Adult

Evaluation of the magnetic immuno PCR assay for rapid detection of Salmonella.

A new technique, the Magnetic Immuno PCR Assay (MIPA), has been developed for the detection of Salmonella. The assay utilizes magnetic particles coated with monoclonal antibodies against Salmonella to extract these bacteria from the sample. Trapped bacteria are lysed, and the supernatant, which contains bacterial DNA, is then subjected to the polymerase chain reaction (PCR) using primers from the Salmonella typhimurium origin of DNA replication to amplify a 163 bp region. The specificity of the primer set was tested in the PCR; amplification occurred with all 25 Salmonella strains tested but not with 19 other species of Enterobacteriaceae tested. A sensitivity of 100 cfu Salmonella typhimurium was achieved for the MIPA by visualization of the amplified products by ethidiumbromide stained agarose gel electrophoresis. A ten-fold higher sensitivity was obtained by Southern blotting of the amplified products. The presence of 10(7) cfu Escherichia coli did not interfere with these detection levels. The MIPA thus specifically detected 100 cfu of Salmonella within 5 h and may be potentially useful for rapid detection of Salmonella in clinical specimens and food.

Base Sequence

Species differences in impairment and recovery of alveolar macrophage functions following single and repeated ozone exposures.

Effects of single (0.4 ppm for 3, 6, or 12 hr) and repeated (0.4 ppm, 12 hr/day for 3 or 7 days) in vivo ozone exposures on rat and mouse alveolar macrophage functions and cell number were investigated. Single ozone exposure of rats resulted in a small (approximately 15%) decrease in Fc-receptor-mediated phagocytosis and phorbol ester-induced superoxide production by the alveolar macrophages and was followed by recovery above control levels within 12 hr of exposure. Repeated exposures of rats for up to 7 days did not alter alveolar macrophage functions, with the exception of the effects of 3 days of exposure on superoxide production (71 +/- 9% as compared with the controls). In mice, significant changes in alveolar macrophage functions were not observed until 12 hr of exposure (at that timepoint phagocytosis was 74 +/- 2%). Repeated ozone exposures of mice did not cause a further decrease in phagocytosis (at Day 7, 74 +/- 14%). Both after 3 and 7 days of repeated ozone exposure of mice, superoxide production by the alveolar macrophages was inhibited approximately 50%. In rats and mice, repeated ozone exposures led to an increase in the number of alveolar macrophages. In mice, this increase appeared at a later time point (at Day 7 vs Day 3) and was less pronounced (at Day 7, 139 +/- 9% vs 179 +/- 17%) as compared with rats. In summary, our data show that rat and mouse alveolar macrophages have different susceptibilities to both single and repeated in vivo ozone exposures.

Administration, Inhalation

The influence of the adjuvant Quil A on the epitope specificity of meningococcal lipopolysaccharide anti-carbohydrate antibodies.

Rabbits were immunized with immunotype L3,7,9 phosphoethanolamine (PEA) group containing oligosaccharide-tetanus toxoid conjugates both with and without the addition of the adjuvant Quil A. The epitope specificity of the antibodies present in these antisera was analysed in an immunotype L2 and L3,7,9 specific inhibition ELISA using the homologous and heterologous lipopolysaccharide, oligosaccharide and partial dephosphorylated oligosaccharide as inhibitors. Two groups of antisera could be identified. In one group of antisera, at least two antibody populations are present, namely directed against the PEA group containing determinants on immunotype L3,7,9 lipopolysaccharide and against immunotype L2 specific epitopes in which no PEA group is present. In the second group of antisera, one but probably more antibody populations are detected with a similar specificity towards the conserved epitopes of both immunotypes. In general, immunization with the conjugates only resulted in the induction of antibodies against the PEA group containing epitopes on the L3,7,9 lipopolysaccharide (80%). Antibodies directed against the conserved epitopes of both immunotypes are mainly evoked with the conjugates in combination with the adjuvant Quil A (80%). Although these results suggest that the epitope specificity of the antibodies induced depends on the use of Quil A, the influence of genetic factors cannot be excluded. At the moment it is not known whether the differences in epitope specificities are reflected in biological function of these antibodies. However, the induction of antibodies with clearly different epitope specificities after immunization of different rabbits with the same antigen stresses the importance of this kind of analysis when developing a vaccine based on oligosaccharide-protein conjugates.

Animals

Effects of ozone on cellular ATP levels in rat and mouse alveolar macrophages.

Hydrogen peroxide (H2O2) is thought to be a major intermediate in the toxicity of ozone. In a previous study we demonstrated that ATP depletion may play an important role in the H2O2-induced inhibition of the phagocytic functions of alveolar macrophages. Ozone exposure can adversely affect the phagocytic capacities of alveolar macrophages. In the present study we investigated whether a decrease in the cellular ATP concentration may underly the effects of ozone on alveolar macrophages. Neither following single (6 and 12 h) exposure nor repeated (12 h/day for 3 and 7 days) exposures of mice or rats to 0.4 ppm ozone, were decreased levels of ATP found in the alveolar macrophages. In contrast, repeated exposures of mice for 7 days to ozone led to a significant increase (1.5-fold) in the ATP content of the alveolar macrophages. In vitro ozone exposure of rat and mouse alveolar macrophages also did not lead to a decrease in the cellular ATP concentration. These results showed that ATP depletion does not play a role in the toxicity mechanism of ozone for alveolar macrophages.

Adenosine Triphosphate

Effect of sub-minimal inhibitory concentrations of ciprofloxacin and fleroxacin on the bacterial capsular antigen and opsonophagocytosis by human polymorphonuclear leukocytes.

We have studied the effect of sub-minimal inhibitory concentrations (sub-MIC) of ciprofloxacin and fleroxacin on capsulated (K+) and non-capsulated (K-) Gram-negative bacilli (Escherichia coli O1:K1, O7:K1, O1:K-, O7:K-, and Klebsiella oxytoca) as well as on Staphylococcus aureus and we investigated the interaction of antibiotic pretreated bacteria with human serum and polymorphonuclear leukocytes (PMN). Following overnight growth in the presence of 1/2 MIC of the antibiotics, bacteria were opsonized in human serum and incubated with PMN. Opsonophagocytosis was quantified as the ratio of uptake by PMN of radioactively labeled bacteria. Ciprofloxacin and fleroxacin enhanced the phagocytosis rate of E. coli K+ strains (control 5-10%; 1/2 MIC of ciprofloxacin and fleroxacin 70-80%) of K. oxytoca (control O-2%; 1/2 MIC of ciprofloxacin and fleroxacin 35-40%) as well as of Staph. aureus (control 5-10%; 1/2 MIC of the antibiotics 35-40%). Opsonophagocytosis of K- strains was not altered. The enhancement of opsonophagocytosis was a complement dependent process. Exposure of capsulated E. coli to ciprofloxacin as well as to fleroxacin resulted in decreased amounts of the capsular antigen.

Agglutination Tests

Immunization of mice with antibiotic-treated Escherichia coli results in enhanced protection against challenge with homologous and heterologous bacteria.

The murine immune response to Escherichia coli exposed to subminimal inhibitory concentrations of four antibiotics was investigated. Groups of mice were injected for 8 weeks with formalin-killed bacteria and subsequently challenged with 10 x LD50 of viable E. coli. Mice receiving saline only (controls) died within 24 h. The mortality of mice immunized with ciprofloxacin-treated E. coli was significantly lower than that of mice immunized with E. coli untreated or treated with other antibiotics. Sera from mice immunized with ciprofloxacin-treated bacteria showed better bacteriostatic capacity and enhanced production of antibodies that bound to homologous and heterologous lipopolysaccharide isolated from several smooth and rough gram-negative strains. The better protection observed in mice immunized with ciprofloxacin-treated E. coli was probably due to an enhanced production of antibodies to epitopes on lipopolysaccharide that became better exposed and so more accessible after treatment with ciprofloxacin.

Animals

Influence of polymorphonuclear granulocytes on the zona-free hamster oocyte assay.

To gain more insight into a possible detrimental influence of polymorphonuclear granulocytes (PMN) on fertility, PMN were added to spermatozoa and oocytes in the hamster oocyte assay. Addition of 2.5 x 10(6)/ml PMN to the sperm suspension at the start of the first incubation period (capacitation period) resulted in a significant decrease (P less than 0.03) in the number of decondensed sperm-heads per hamster oocyte (the decondensation index). When a concentration of 0.5 x 10(6)/ml PMN was used, no decrease was found. Stimulation of PMN by phorbol myristate acetate (PMA) did not enhance the inhibitory action of PMN. After incubation of oocytes with PMN for 1 h, an almost total inhibition of decondensation was observed. A significant inhibition could also be obtained when unstimulated PMN (2.5 x 10(6] were added at the start often 3 h coincubation period of spermatozoa and oocytes (P less than 0.004). The inhibition by PMN decreased to a nonsignificant level when the hydroxyl radical scavenger thiourea was present. The possible mechanisms by which PMN affect the results of the hamster oocyte assay and the possible implications of the presence of PMN in the genital tract are discussed.

Adult

Characterisation and functional aspects of monoclonal antibodies specific for surface proteins of coagulase-negative staphylococci.

Mouse monoclonal antibodies (MAbs) raised against whole cells of Staphylococcus epidermidis strain 354 were characterised morphologically and functionally. Nine MAbs showed strong reactivity with coagulase-negative staphylococci (CNS). Only two MAbs were specific for CNS; both belonged to the IgG1 subclass, and one, MAb 36.4, reacted only with the strain used for immunisation. In immunoblotting, both CNS-specific MAbs 36.3 and 36.4 reacted strongly with cell-wall protein bands of 220 Kda of S.epidermidis strain 354 and weak reactivity was observed with a 110-Kda band. MAb 36.3 reacted also with 220-230 Kda bands of two other S.epidermidis strains (291 and ATCC 35984) and a 160-180 Kda band of S.epidermidis strain 354. Only MAb 36.4 promoted phagocytosis of strain 354 by polymorphonuclear leucocytes (PMNL) and monocytes, whereas MAb 36.3 and the other MAbs lacked this activity. Opsonisation of S. epidermidis with MAb 36.4 in the presence of complement enhanced uptake by PMNL, but not by monocytes. Furthermore, S.epidermidis strain 354 opsonised with MAb 36.4 induced chemiluminescence of PMNL. Immuno-gold electronmicroscopy with both MAbs 36.3 and 36.4 demonstrated a homogeneous distribution of gold particles on the surface as well as close to the surface of S.epidermidis.

Animals

Comparative in vitro antibacterial activity of sparfloxacin (AT-4140; RP 64206), a new quinolone.

The in vitro activity of sparfloxacin (AT-4140; RP 64206), a new fluoroquinolone, was compared with those of 10 other agents against 1,222 clinical isolates. Sparfloxacin and ciprofloxacin were the most active quinolones against members of the family Enterobacteriaceae and nonfermenting gram-negative bacilli; sparfloxacin had superior activity against gram-positive cocci in comparison with the activities of ciprofloxacin and the other quinolones tested (norfloxacin, lomefloxacin, and pefloxacin). Among the inhibited strains, several were resistant to the tested beta-lactam antibiotics or to aminoglycosides. The activity of sparfloxacin was not influenced by the medium that was used; lowering of the pH to 5 had a marked effect on the MICs for two strains each of Enterobacter cloacae and Pseudomonas aeruginosa and one strain each of Escherichia coli and Staphylococcus aureus; the MBC of sparfloxacin was within 1 to 2 dilution steps of the MIC for the strains that were tested.

4-Quinolones

Minimal oligosaccharide structures required for induction of immune responses against meningococcal immunotype L1, L2, and L3,7,9 lipopolysaccharides determined by using synthetic oligosaccharide-protein conjugates.

The 12 types of meningococcal lipopolysaccharide (LPS) (immunotypes) contain immunotype-specific and cross-reactive epitopes situated on the oligosaccharide part of the LPS molecules. To identify useful cross-reactive epitopes and to determine minimal oligosaccharide structures required for the induction of an immune response against the most prevalent immunotypes, L1, L2, and L3,7,9, synthetic as well as native LPS-derived oligosaccharides were conjugated with tetanus toxoid. L3,7,9 phosphoethanolamine (PEA) group-containing oligosaccharide-tetanus toxoid conjugates evoked high immunoglobulin G (IgG) antibody levels in rabbits which were detected by an L2-, L3,7,9-, and, depending on the antiserum, L1-specific enzyme-linked immunosorbent assay (ELISA). Inhibition studies revealed that an identical antibody population was detected by L1 and L3,7,9 ELISA, indicating a similar tertiary structure of the inner core oligosaccharide of these two immunotypes. These antibodies recognize PEA group-containing epitopes present on the L1 and L3,7,9 LPS. An L2 PEA group-containing oligosaccharide-tetanus toxoid conjugate elicited L2- and L3,7,9-specific IgG antibodies, but in contrast with the L3,7,9 conjugates, no L1-specific IgG antibodies were evoked. These results indicate that L1 and L2 LPS do not contain cross-reactive epitopes, whereas both L2 and L3,7,9 LPS and L1 and L3,7,9 LPS possess common determinants. Three linear oligosaccharides and one branched oligosaccharide, representing partial structures of the inner core oligosacchardes of meningococcal LPS, were synthesized. Only the branched synthetic oligosaccharide-containing conjugate was able to induce and L1- and L3,7,9-specific immune response, whereas the linear oligosaccharide-protein conjugates evoked L2-specific immune responses. The branched oligosaccharide (beta-D-Glcp(1----4)-[L-alpha-D-Hepp(1----3)]-L-alpha-D-Hepp ) is therefore considered a minimal structure required for the induction of an immune response against L1 and L3,7,9 LPS and part of a cross-reactive epitope between these two immunotypes. For L2-specific immune responses, oligosaccharide structures terminating in beta-D-Glcp(1----4), alpha-D-GlcNAcp(1----2), or L-alpha-D-Hepp(1----5) are needed. The results suggest that it is possible to prepare an oligosaccharide structure with the ability to evoke an immune response against L1, L2, and L3,7,9 LPS. A feasible structure for such a "hybrid" oligosaccharide is discussed.

Animals

Characterization of a proteinaceous adhesin of Staphylococcus epidermidis which mediates attachment to polystyrene.

Coagulase-negative staphylococci (CoNS) have evolved into important agents of foreign body-related infections. Adhesion of causative bacteria to biomaterials is considered to be an essential step in these infections. We and others have shown that adhesion of CoNS to biomaterials may be mediated by protease-sensitive surface constituents. In the present study we expanded on these investigations by characterizing a biomaterial adhesin of Staphylococcus epidermidis 354 by using a strain-specific monoclonal antibody (MAb 36.4). MAb 36.4 was strongly and exclusively reactive with strain 354 in an enzyme-linked immunosorbent assay in which whole bacteria were used as antigens. Immunoblotting of cell wall polypeptides of strain 354 revealed strong reactivity with a 200- to 220-kDa band and a weaker reaction in the 100- to 110-kDa range. Preincubation of strain 354 with MAb 36.4 resulted in a 54 to 91% (mean +/- standard deviation, 74% +/- 14%; n = 10) inhibition of adhesion to polystyrene spheres. Fab fragments prepared from MAb 36.4 also inhibited adhesion effectively, indicating specific blocking of an adhesion antigen rather than aspecific inhibition. Immunogold electron microscopy with MAb 36.4 revealed deposition of gold particles on the cell surface and possibly also on fimbrialike surface projections. It is concluded that a surface-located protein antigen of S. epidermidis 354 recognized by MAb 36.4 acts as an adhesin mediating attachment to uncoated foreign material. It is speculated that this type of adhesion to biomaterials may play an important role in the pathogenesis of foreign body-related infections caused by CoNS.

Antibodies, Monoclonal

Preparation, characterization, and immunogenicity of meningococcal immunotype L2 and L3,7,9 phosphoethanolamine group-containing oligosaccharide-protein conjugates.

A method was developed for the well-defined coupling of phosphoethanolamine group (PEA)- and carboxylic acid group-containing polysaccharides and oligosaccharides to proteins without the need for extensive modification of the carbohydrate antigens. The carboxylic acid group of the terminal 2-keto-3-deoxyoctulosonic acid moiety was utilized to introduce a thiol function in meningococcal immunotype L2 and L3,7,9 lipopolysaccharide-derived oligosaccharides. The thiol group-containing oligosaccharides were subsequently coupled to bromoacetylated proteins. Immunotype L2 and L3,7,9 PEA group-containing oligosaccharide-tetanus toxoid conjugates were prepared, and their immunogenicities were studied in rabbits. Both the immunotype L2 and immunotype L3,7,9 conjugates evoked high immunoglobulin G (IgG) antibody titers after the first booster injection. These conjugates also displayed an ability to induce long-lasting IgG antibody levels which could be detected until 9 months after one booster injection at week 3. The adjuvant Quil A enhanced the immune response to all the conjugates to a minor extent, which is in contrast with reported adjuvant effects of Quil A on these types of antigens in mice. A conjugate prepared from the dephosphorylated L3,7,9 oligosaccharides evoked a significantly lower IgG response than a similar PEA-containing conjugate, and enzyme-linked immunosorbent assay inhibition studies indicated a different epitope specificity. Furthermore, antisera elicited with the complete bacteria contained antibodies directed against PEA-containing epitopes, which stresses the importance of the presence of unmodified PEA groups in meningococcal lipopolysaccharide-derived oligosaccharide-protein conjugates. The procedure developed offers an elegant solution for the specific coupling of meningococcal PEA-containing oligosaccharides to proteins and may therefore be a very useful tool in the development of a vaccine against group B meningococci.

Animals