Gestational age-dependent repair kinetics of microsurgical defects in monolayers of human amniocytes.

OBJECTIVES: There is paucity of data on the capacity of fetal membranes to repair surgical defects following trauma. We aimed at developing an in vitro model using monolayers of human amnion epithelial cells to study fetal membrane healing. METHODS: Term (n = 6) and preterm (n = 3) fetal membranes were collected at caesarean section. The amnion was digested twice in a trypsin solution. Amniocytes were seeded (250,000-750,000/ml) and incubated at 37 degrees C and 5% CO(2) and 21 or 5% O(2). A microsurgical injury was made centrally in the monolayers and the cultures were incubated for 48 h. Every 6 h, slides were fixed and immunohistochemical staining was performed to quantify proliferation at the site of the defect and centrally in the monolayer. The closure rate was evaluated by measuring the defect size every 6 h. RESULTS: The closure rate of the defects was higher in preterm versus term cultures. Proliferation was significantly higher in the defect zone versus the peripheral zone, and also higher in the preterm group. CONCLUSION: We describe a new model for the study of fetal membrane healing and observed gestational age-dependent repair capacity of the amnion.

Mutation profile and clinical outcome of mixed endometrioid-serous endometrial carcinomas are different from that of pure endometrioid or serous carcinomas.

Clinical outcome of 23 patients with mixed endometrioid and serous endometrial carcinomas (mixed EEC-SC) was compared to that of pure endometrioid (EEC) and pure serous (SC) carcinomas. Hotspot mutation frequencies in KRAS, PIK3CA, PTEN, and TP53 and microsatellite instability (MSI) status were determined in mixed EEC-SC, as well as in their EEC and SC microdissected components separately, and alterations were compared to frequencies in pure EEC and SC. Relapse-free (RFS) and overall survival (OS) differed significantly between mixed EEC-SC and pure EEC and SC, revealing that outcome of mixed EEC-SCs was intermediate to that of pure EEC and pure SC. PTEN mutations were absent in pure SC, but occurred in 20 % of pure EEC, and 13 % of mixed EEC-SC. In contrast, TP53 mutations were more frequent in pure SC (17 %) and mixed EEC-SC (22 %) than in pure EEC (2 %). Mutations in mixed EEC-SC were shared by the two microdissected components in 30 %, whereas in 35 %, some mutations were component-specific. Mutation analysis confirms similarities between the EEC and SC components of mixed EEC-SC with pure EEC and pure SC, respectively. However, PTEN and KRAS mutations were more frequent in the SC component of mixed EEC-SC than in pure SC, while TP53 mutations were more frequent in the EEC component of mixed EEC-SC than in pure EEC. Presence of different clonal mutation pattern between EEC and SC components of mixed EEC-SC raises the possibility of divergent tumor heterogeneity or biclonal origin in some cases.

Once-daily dosing decreases renal accumulation of gentamicin and netilmicin.

The pathogenesis of aminoglycoside nephrotoxicity is intimately related to the extent of drug accumulated in the renal cortex. In the framework of searching for preventive measures of aminoglycoside-induced nephrotoxicity, we investigated the influence of dosage regimen on the renal cortical accumulation of gentamicin and netilmicin in humans. Patients with a tumor partly involving one kidney, with normal renal function, and scheduled for nephrectomy received one dose of either gentamicin (4.5 mg/kg) or netilmicin (5 mg/kg) as a single short-term infusion or as 24-hour continuous infusion. Treatment started 24 hours before surgery. Serum aminoglycoside pharmacokinetics were examined during treatment and renal cortical tissue was sampled at the moment of operation for drug determination. The short-term infusion schedule yielded cortical concentrations of 103.2 +/- 36.3 and 137.4 +/- 34.6 micrograms/gm for gentamicin and netilmicin, respectively. Tissue levels after continuous infusion were 158.1 +/- 52.9 and 178.5 +/- 21.8 micrograms/gm for gentamicin and netilmicin, respectively. For each aminoglycoside, a single short-term infusion resulted in significantly lower renal drug levels than did a continuous infusion of the same dose. From the nephrotoxicity point of view, these data support the administration of gentamicin and netilmicin as once-daily injections. This also supports the appropriateness of further studies to determine clinical efficacy of once-a-day dosing for aminoglycosides.

New derivatives of kanamycin B obtained by modifications and substitutions in position 6". 1. Synthesis and microbiological evaluation.

The clinical use of the potent, wide-spectrum aminoglycoside antibiotics is limited by oto- and nephrotoxicities. The latter is related to the binding of these polycationic drugs to negatively charged phospholipids and to the subsequent inhibition of lysosomal phospholipases. In order to explore the influence of a modification of the hydrophobic/hydrophilic balance at a specific site of an aminoglycoside, kanamycin B has been chemically modified in position 6" by substitution of the hydroxyl group with a halogen atom (or a pseudohalogen group), or an amino, an amido, a thioalkyl, or an alkoxy group, each series containing increasingly bulkier chains. Examination of the antibacterial activity of the synthesized compounds revealed a negative correlation between the size of the 6"-substituent and the antibacterial activity against kanamycin B sensitive Gram-positive and -negative organisms. Only derivatives with small substituents in position 6", namely chloro, bromo, azido, amino, methylcarbamido, acetamido, methylthio, methylsulfinyl, O-methyl, O-ethyl, and O-isopropyl, showed acceptable activity (geometric mean of minimum inhibitory concentrations for Gram-negative strains less than or equal to 2.5 mg/L; value for kanamycin B, 0.5 mg/L). In vitro toxicological evaluation of all derivatives and computer-aided conformational analysis of selected compounds inserted in a phosphatidylinositol monolayer are presented in the following paper in this issue.

New derivatives of kanamycin B obtained by combined modifications in positions 1 and 6". Synthesis, microbiological properties, and in vitro and computer-aided toxicological evaluation.

Substitution of the C-1 atom in the 2-deoxystreptamine moiety of gentamicin C2, a broad-spectrum aminoglycoside antibiotic, by an axial hydroxymethyl group has been reported to confer protection against most clinically important bacterial enzymes inactivating aminoglycosides, while simultaneously reducing the nephrotoxic potential of this drug. We report here on a similar modification of kanamycin B. Microbiological evaluation, however, revealed no useful protection, as established by the almost complete lack of activity of 1-C-(hydroxymethyl)kanamycin B against an array of organisms producing defined types of aminoglycoside-inactivating enzymes and against which 1-C-(hydroxymethyl)gentamicin C2 and amikacin (1-N-[(S)-2-hydroxy-4-aminobutyryl]kanamycin A) are active. Moreover, toxicological evaluation, based on the in vitro measurement of the drug inhibitory potential toward lysosomal phospholipases, a predictive test of the intrinsic nephrotoxic potential of aminoglycosides, showed not decreased but rather increased toxicity. Comparative conformational analysis of the interactions of the drug with a phosphatidylinositol monolayer explained the lack of protective effect, since no significant change of the mode of insertion of the derivative in this monolayer was detected compared to that of kanamycin B. Combination of a 1-C-(hydroxymethyl) substituent with a 6"-chloro, 6"-acetamido substituent resulted in a partial improvement of the toxicological behavior with no loss of activity for the 6"-chloro and the 6"-azido derivatives, but not to the extent of obtaining better derivatives than kanamycin B itself. We, therefore, suggest that the advantages of an axial hydroxymethyl substituent at C-1 are probably restricted to the gentamicin family and do not extend to kanamycins. It might be concluded that the structural differences between gentamicins and kanamycins play an important, still undescribed role both in their effective recognition by aminoglycoside-inactivating enzymes, which are responsible for most of the clinically important cases of resistance to aminoglycosides, and also in the interactions with phospholipids, which in turn cause nephrotoxicity.

Temocillin sensitivity of Enterobacteriaceae species resistant to cefotaxime.

The in vitro activity of temocillin, cefotaxime, aztreonam and ceftazidime was tested against selected cefotaxime-resistant isolates (43 Enterobacter species, 37 Serratia marcescens, 16 Morganella morganii, 8 Klebsiella species) and cefotaxime-susceptible strains of the same species. Cross-resistance with a high degree of correlation was observed between cefotaxime and aztreonam or ceftazidime, but not between cefotaxime and temocillin.

Antibacterial activity of temocillin in combination with other beta-lactam antibiotics.

The antibacterial interactions of temocillin with other beta-lactam antibiotics were tested by the checkerboard combination method in Mueller-Hinton agar against 146 strains of Enterobacteriaceae, 35 Pseudomonas aeruginosa strains, and 35 Staphylococcus aureus strains. Most combinations showed either a moderate degree of synergism or indifference. Strains showing occasional antagonism at a particular proportion of concentrations of the test combination, were found to only be indifferent when the mean index of the fractional inhibition concentration of all checkerboard combinations was calculated.

In vitro activity of temocillin against clinical isolates.

The minimum inhibitory concentrations of temocillin against more than 1000 clinical isolates were determined by an agar dilution method. Temocillin showed excellent activity against Haemophilus influenzae, pathogenic Neisseria species and Branhamella catarrhalis, including beta-lactamase producing strains, but showed very low activity or was inactive against Gram-positive cocci and Campylobacter, Bacteroides, Acinetobacter and Pseudomonas species. Good activity was obtained against 702 Enterobacteriaceae, including isolates resistant to the other penicillins and first- and second-generation cephalosporins, with 92% of all the strains inhibited at a concentration of 8 mg/L. However, the most striking property of temocillin was its high beta-lactamase stability which resulted in both a very narrow range of MICs within which all the isolates were inhibited, and a small influence of inoculum size on the MICs.

Interaction of interstitial trophoblast with placental bed capillaries and venules of normotensive and pre-eclamptic pregnancies.

While endovascular trophoblast invasion of the human placental bed spiral arteries has been studied extensively, no information is available on the interaction between interstitially invading trophoblast and uterine capillaries and venules. Placental bed biopsies of eight normotensive and 15 pre-eclamptic patients were double-immunostained for cytokeratin and the endothelial marker CD31, providing satisfactory staining results in six and 10 biopsies, respectively. Interstitial trophoblast tissue density did not differ between the two series of biopsies, implying that this pathway of invasion is not impaired in pre-eclampsia. Both groups showed a similar incidence of approach of non-arterial vascular structures by perivascular trophoblast. Differences in CD31 staining intensity were noticed in different vascular cross-sections. Lower staining intensity was related to the presence of perivascular trophoblast. Because of the identity of CD31 with the platelet-endothelial cell adhesion molecule (PECAM)-1, the trophoblast-dependent downregulation of CD31 may play a role in the control of leukocytic traffic within the placental bed. The phenomena described in this paper did not show any difference between the normotensive and pre-eclamptic patients, implying that interaction of interstitial trophoblast with venous and capillary structures is not related to the pathogenesis of pre-eclampsia.

Disappointing dipstick screening for urinary tract infection in hospital inpatients.

AIM: To compare the performance of leucocyte esterase and nitrite dipstick tests with microscopic examination and culture of first morning urines (n = 420) of hospital inpatients. RESULTS: The sensitivity, specificity, and negative predictive value of the leucocyte esterase test for the cutoff of > 10 WBC/microliter were 57%, 94%, and 68%, respectively. For > 5 WBC per high power field (HPF) these variables were 84%, 90%, and 93%. For > 10(5) colony counts/ml, the sensitivity of the nitrite test was 27%, specificity 94%, and negative predictive value 87%. When either leucocyte esterase or nitrite positivity was accepted as a marker of urinary tract infection, the sensitivity was 78%, specificity 75%, and negative predictive value 94%, and there were 22% false negative results. Semiquantitative microscopic estimation of bacteria per HPF yielded 40% false positives. CONCLUSIONS: Leucocyte esterase and nitrite dipstick tests are not suitable for screening for urinary tract infections.

Comparative antimicrobial activity of ceftibuten against multiply-resistant microorganisms from Belgium.

To study the activity of ceftibuten, we obtained multiply-resistant isolates from approximately 20 hospitals in Belgium. Against Enterobacteriaceae, all of the tested comparative compounds were more active than cefaclor, and ceftibuten and tigemonam were the most active of the agents tested. Ceftibuten MIC50s were less than or equal to 1 microgram/ml for most enteric bacilli species and 85% of strains were susceptible (less than or equal to 8 micrograms/ml). This level of activity compared favorably to that recorded for cefaclor (less than or equal to 8 micrograms/ml), cefetamet (less than or equal to 4 micrograms/ml), and cefteram (less than or equal to 1 microgram/ml), that is, 37%, 69%, and 59%, respectively. Ceftibuten, cefetamet, cefteram, and tigemonam were highly active against isolates of Haemophilus influenzae and Neisseria gonorrhoeae. None of the comparative agents were as active as cefaclor against staphylococcal isolates. Against streptococci, cefteram was the most active, and tigemonam the least active of the agents. The MIC90s of ceftibuten for strains of Streptococcus pneumoniae and Streptococcus pyogenes were 2 micrograms/ml and 0.5 microgram/ml, respectively. Strains of Streptococcus agalactiae were resistant to both ceftibuten and tigemonam; cefaclor and cefteram inhibited 100% of isolates of this species. Strains of Enterococcus faecalis and Pseudomonas aeruginosa were consistently resistant to all of the compounds. Overall, ceftibuten exhibited potent activity against many multiply-resistant clinical isolates.

Evaluation of Bactec high blood volume resin media.

BACTEC PLUS high-blood-volume resin media (aerobic BP 26 vial and anaerobic BP 27 vial) were compared with standard BACTEC media (aerobic NR 6A and anaerobic NR 7A vial). A total of 2253 blood culture sets, each consisting of the four vials, were collected. Positive cultures were obtained from 403 sets and grew 428 organisms; 271 organisms were considered as significant. The BACTEC PLUS high blood volume resin (BP-HBV) media grew significantly more Staphylococcus aureus, coagulase-negative staphylococci, Candida albicans, Enterococcus faecalis, and Pseudomonas aeruginosa. After taking into account the difference of blood volume between the two systems, only S. aureus was significantly more detected by the aerobic BP 26 vial. An enhanced recovery rate with the anaerobic BP 27 vial could not be established. BP-HBV media had an enhanced recovery rate over the standard BACTEC media for S. aureus and C. albicans in patients receiving antibiotics.

Prospective study of catalase-positive coryneform organisms in clinical specimens: identification, clinical relevance, and antibiotic susceptibility.

During a 6-month period, all clinical isolates of catalase-positive coryneform organisms, which were isolated during the routine processing of clinical specimens, were characterized in the laboratory of the 1800-bed University Hospital of Leuven. The distribution of the species in the corynebacteria was: Corynebacterium amycolatum 70 (53%), Corynebacterium jeikeium 16 (12%), Corynebacterium striatum 11 (8%), Corynebacterium afermentans 10 (7%), Corynebacterium minutissimum 9 (6%), CDC coryneform group G 4 (3%), Corynebacterium urealyticum 4 (3%), Corynebacterium glucuronolyticum 1 (0.7%), and Corynebacterium xerosis 1 (0.7%). Of the 150 isolates, 37 (25%) were considered to be infection related and the remaining 113 (75%) were of questionable clinical significance. Susceptibility of the corynebacteria to 12 antibiotics active against Gram-positive organisms was evaluated. C. amycolatum, C. jeikeium, and C. urealyticum were multiresistant, but all isolates were susceptible to teicoplanin and vancomycin. Most of the C. amycolatum strains, and all strains of C. jeikeium and C. striatum, were susceptible to the vibrocidal compound O/129.

Evolution of Streptococcus pneumoniae serotypes and antibiotic resistance in Belgium-update (1994-98).

OBJECTIVE: To follow the evolution of capsular types and resistance of Streptococcus pneumoniae, isolated from deep sites. METHODS: More than 100 Belgian laboratories permanently collect S. pneumoniae strains isolated from puncture specimens (blood, cerebrospinal fluid, middle ear fluid, etc.) and forward them to the reference center in Leuven, in order to determine the capsular serogroups and types (SGTs) and their resistance. RESULTS: From 1994 to 1998, the 5486 S. pneumoniae strains examined belonged to 39 of the 46 currently identified SGTs. The 10 most frequent SGTs accounted for 78.9% of the isolates, and 97% of all isolates belonged to SGTs included in the 23-valent vaccine. Overall mortality of patients with pneumococcal bacteremia or meningitis was 9.7%, and 23.8% in patients over 80 years. From 1994 to 1998, resistance to penicillin (P) increased from 7.6% to 14.2%, to tetracycline (T) from 14.9% to 28.0%, and to erythromycin (E) from 22.9% to 31%. Triple resistance (PTE) increased from 0.9% in 1994 to 6.6% in 1998. Five SGTs (6, 9, 14, 19 and 23) accounted for 50% of the isolates, but for > 90% of the penicillin-resistant or erythromycin-resistant isolates. CONCLUSIONS: Resistance of S. pneumoniae to penicillin, erythromycin and tetracycline is steadily increasing and is concentrated in five serotypes included in the 23-valent pneumococcal vaccine. Increasing resistance and high mortality of invasive infections are an incentive to vaccinate vulnerable groups.

Matrix metalloproteinases-2 and -9 and their endogenous tissue inhibitors in tissue remodeling after sealing of the fetal membranes in a sheep model of fetoscopic surgery.

OBJECTIVES: We studied collagen plugging of the fetoscopic access site in an in vivo fetal lamb model for fetoscopic surgery and possible role for matrix metalloproteinase (MMP)-2 and -9 and tissue inhibitors (TIMPs). METHODS: Eight ewes had fetoscopic balloon occlusion of the trachea as an experimental treatment for congenital diaphragmatic hernia between days 88 and 99 of gestation (term 145 days) with sampling of amniotic, allantoic, and tracheal fluid. Nonoperated cotwins were used as controls. The fetoscopy port was closed using a collagen plug. Ten days (range 9-12) later, fluids were sampled and plug sites collected for histologic analysis. Activity of MMP-2 (72 kDa, gelatinase A) and MMP-9 (92 kDa, gelatinase B) was determined in the fluids by zymography and secretion of TIMPs (27-30 kDa; TIMP-1, glycosylated TIMP-3 and TIMP-4, 24 kDa; unglycosylated TIMP-3, 21 kDa; TIMP-2) by reverse zymography and quantified by densitometric analysis. RESULTS: No pregnancy was complicated by amniorhexis or preterm labor. At cesarean, normal volumes of amniotic and allantoic fluid were present in all cases. Histology of the plug sites revealed good integration of the collagen plug without complete restoration of membrane integrity. MMP-2, MMP-9, and TIMPs were detected in all fluids. In the operated animals, significantly (P <.05) higher activity of MMP-9 was found in amniotic fluid, with lower concentrations of TIMPs in allantoic fluid (P <.01). Tracheal occlusion was associated with a significant (P <.02) increase in both MMP-2 and -9 in tracheal fluid. CONCLUSION: Collagen plugging of the fetoscopic access port sites in sheep resulted in functionally effective sealing of the fetal membranes. Changes in MMP-2, MMP-9, and TIMPs suggest an active remodeling of both the fetal lung and the fetal membranes.

Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae from Rwanda, 1984-1990.

A total of 383 clinical isolates of Streptococcus pneumoniae, obtained from an equal number of patients in Kigali, Rwanda, was tested for resistance to penicillin G with a 1 microgram oxacillin disc. Of these isolates, 99 (25.8%) showed reduced zones of inhibition. By means of an agar dilution method, 21% all isolates were confirmed as relatively resistant (MIC > or = 0.12- < or = 1.0 mg/l) strains of Streptococcus pneumoniae (RRSP). A high degree of resistance to penicillin G (MIC > or = 2 mg/l) was not observed. Resistance to chloramphenicol (MIC > or = 8 mg/l) was found in 31% RRSP and in 6% penicillin susceptible strains (PSSP). Doxycycline resistance was common in both RRSP and PSSP strains. All isolates remained fully susceptible to erythromycin. Children more often harboured a strain giving a reduced inhibition zone than did adults (74/230 versus 25/153; P = 0.0005). A total of 32 serotypes or serogroups were identified, seven of them relating to 64.8% all isolates typed. Of all the isolates 84% belonged to a serotype represented in the 23-valent vaccine or to a cross-reacting serotype. Serotype 25, not included in the vaccine, accounted for 10.7% typed isolates from adults but only for 2.0% typed isolates from children. Results of susceptibility testing and clinical experience suggest that penicillin G, ampicillin and chloramphenicol should not be used alone as empirical treatment for pneumococcal meningitis in patients in Rwanda.

Identification of novel erythromycin derivatives in mother liquor concentrates of Streptomyces erythraeus.

The identification of five novel compounds, pseudo-erythromycin A-6,9-hemiketal, 8,9-anhydro-pseudo-erythromycin A-6,9-hemiketal, 8,9-anhydro-pseudo-N-demethylerythromycin A-6,9-hemiketal, 5-O-beta-D-desosaminylerythronolide A and 15-nor-erythromycin C, in mother liquor concentrates of Streptomyces erythraeus is described. The pseudo-erythromycin derivatives are characterized by a 12-membered macrocyclic ring as a result of C13----C11 trans-lactonization. The five compounds have very little antimicrobial activity.

The role of radiology in Campylobacter enterocolitis.

A series of 18 patients with diarrhoea and positive stool cultures for Campylobacter jejuni is presented. The most important radiological features were thickening of ileal mucosal folds, of interhaustral indentations and of the ileocaecal valve, lymphoid hyperplasia and microulcerations. Radiology, as well as endoscopy, are both nonspecific in Campylobacter jejuni enterocolitis. The importance of radiology is to exclude more typical features of other causes of inflammatory bowel diseases. Moreover, before the result of the stool culture is available, the radiological features should suggest the suspicion of an acute infectious enterocolitis by Campylobacter jejuni as possible diagnosis.

Listeria monocytogenes meningitis.

The history and findings of all patients with Listeria meningitis admitted to the University Hospital of Leuven from 1967 to 1987 were reviewed. Listeriosis during pregnancy or the perinatal natal period was not considered. Predisposing conditions in these 23 patients included renal transplants (9), immunosuppressive therapy (2), diseases of the lympthoreticular system (3) and chronic alcoholism (1). One man had an inversed T4/T8 ratio. In 7 patients no underlying disorder was detected. Disease onset may be acute or subacute. There are no clinical features distinguishing Listeria meningitis from other acute bacterial meningitides. The number of leukocytes in the CSF varied from 3 to 3700, most often with a predominance of mononuclear cells. A decrease of the glucose level in the CSF was not always present. The initial gram stain was often unrevealing and it took up to 4 days for CSF cultures to become positive. Blood cultures were often important for the identification of the organism.

Renin-like immunoreactivity in uterus and placenta from normotensive and hypertensive pregnancies.

OBJECTIVES: (1) To identify the distribution of renin-like immunoreactivity in placental bed, placenta-free uterine wall, placenta, fetal membranes, and intertwin membranes obtained from normal pregnancies and (2) to compare the findings in normal pregnancies with those in pregnancies complicated by various hypertensive disorders. STUDY DESIGN: Biopsies were taken from 31 normotensive pregnant women, eight of whom had twin pregnancies, and from 28 women with various hypertensive disorders of pregnancy. The anti-human renal renin monoclonal antibody, F37.1A1, was used for immunostaining. Histological structures were identified with standard H&E and PAS techniques, supplemented with immunostaining using the specific cell markers CD68 and cytokeratin. RESULTS: Renin-like immunoreactivity was found in cytokeratin immunolabelled placental syncytiotrophoblast, amnionic and glandular epithelium, but most consistently in CD68 immunolabelled maternal and fetal macrophages. The distribution of renin-like immunoreactivity throughout the pregnant uterus roughly parallelled reported renin concentrations in the various tissues, while its localization conforms also with that of cathepsin D. There were no obvious differences in renin-like immunolabelling between normotensive or hypertensive women. Renin-like immunoreactivity was particularly common in the atherotic lesions that are observed more often in pregnancies complicated with hypertensive disorders of pregnancy and/or intra-uterine growth restriction. CONCLUSIONS: The data complement earlier findings showing that only two of four anti-renal renin monoclonal antibodies, both of which cross-react with cathepsin D, give a positive immunostaining in placental tissue. They question whether classical concepts on renin localisation in uteroplacental tissues all relate to one and the same enzyme. The demonstration of renin-like enzymes in different cell types, including macrophages, may explain the diversity of functions that has been attributed to uterine renin. There were no differences between tissues obtained from normotensive and hypertensive pregnancies, except for the consistent presence of renin-like immunoreactivity in atherotic lesions.