Serum neurofilament light for detecting disease activity in individual patients in multiple sclerosis: A 48-week prospective single-center study.

BACKGROUND: Serum neurofilament light (sNfL) reflects neuroaxonal damage and is now used as an outcome in treatment trials of relapsing-remitting multiple sclerosis (RRMS). However, the diagnostic properties of sNfL for monitoring disease activity in individual patients warrant further investigations. METHOD: Patients with suspected relapse and/or contrast-enhancing lesions (CELs) were consecutively included and performed magnetic resonance imaging (MRI) of the brain at baseline and weeks 28 and 48. Serum was obtained at baseline and 2, 4, 8, 16, 24, and 48 weeks. Neurofilament light concentration was measured using Single molecule array technology. RESULTS: We included 44 patients, 40 with RRMS and 4 with clinically isolated syndrome. The median sNfL level peaked at 2 weeks post-baseline (14.6 ng/L, interquartile range (IQR); 9.3-31.6) and reached nadir at 48 weeks (9.1 ng/L, IQR; 5.5-15.0), equivalent to the median sNfL of controls (9.1 ng/L, IQR; 7.4-12). A baseline Z-score of more than 1.1 (area under the curve; 0.78, p < 0.0001) had a sensitivity of 81% and specificity of 70% to detect disease activity. CONCLUSION: One out of five patients with relapse and/or CELs did not change significantly in post-baseline sNfL levels. The utility of repeated sNfL measurements to monitor disease activity is complementary rather than a substitute for clinical and MRI measures.

Immunohistochemical identification of collagenase in carrageenin granuloma.

The collagenase present in experimental carrageenin granuloma in the guinea pig has been purified to homogeneity in acrylamide gel electrophoresis by a combination of ammonium sulfate salting out and affinity chromatography on Sepharose 4B--collagen-packed columns. The single protein band thus obtained was used as an antigen to obtain a monospecific antibody in heterologous conditions. Several immunodiffusion, immunoaffinity chromatography, and immunoinhibition tests of the antibody against the specific antigen and various possible serum and tissue contaminants suggested that the antibody was specifically directed against the enzyme protein collagenase. Indirect immunohistochemical staining of carrageenin granulomas, samples at different developmental phases with this specific anti-collagenase antibody, revealed that the specific antigenic protein (the enzyme collagenase) is universally present on the extracellular structures at both the collagen-deposition and the collagen-resorption stages. A hypothesis is proposed to account for these findings, namely, that the enzyme collagenase is bound to its substrate (collagen) under both normal and pathological conditions, and that the critical point of control of collagen degradation must be the activation of the collagen-bound enzyme.

Recurrent bacteriuria and primary biliary cirrhosis: ABO blood group, P1 blood group, and secretor status.

Patients with primary biliary cirrhosis have an abnormally high incidence of urinary tract infection (35%). Susceptibility to urinary infection and other infectious diseases has been linked with certain blood group antigens and secretor status. We have therefore studied these characteristics in patients with primary biliary cirrhosis. We were unable to show any abnormal distribution in blood groups or secretor status in patients with primary biliary cirrhosis (compared with a normal population) which might reflect their predisposition to urinary infection. The distribution of blood groups and secretor status in patients with primary biliary cirrhosis with a history of urinary infections was not significantly different from patients without such a history. Escherichia coli strains isolated from patients with primary biliary cirrhosis did not bind in any greater numbers to the uroepithelial cells of primary biliary cirrhosis patients than to the cells of a normal healthy control. We therefore conclude that blood group distribution, abnormal secretor status, and epithelial cell type are not important factors in the predisposition of primary biliary cirrhosis patients to urinary infections.

Anaerobic periurethral flora of healthy women and women susceptible to urinary-tract infection.

The anaerobic periurethral microbial flora of 25 healthy women was compared with that of 29 women attending the urinary-tract-infection clinic at the Royal Free Hospital. The latter group consisted of 19 patients receiving long-term prophylactic antimicrobial therapy and 10 with proven recurrent urinary-tract infection not receiving prophylactic treatment. The numbers and species of anaerobes isolated from each group were similar. Lactobacillus spp. were the most frequently isolated organisms in each group and the most numerous. Bacteroides spp. were the next most frequently isolated. In any one subject, the anaerobic flora varied considerably during the study period of approximately 6 months. Thus, the anaerobic flora is not affected by recurrent urinary-tract infection in the past nor by the use of prophylactic chemotherapy. It does not appear to exert a protective role against the initiation of urinary-tract infection.

Relationship between adhesion of Escherichia coli to uro-epithelial cells and the pathogenesis of urinary infection: problems in methodology and analysis.

Escherichia coli strains isolated from patients with urinary infections were tested for their ability to adhere to human uro-epithelial cells. In any single experiment, the numbers of bacteria adhering to individual uro-epithelial cells showed great variations; some cells had hundreds of bacteria adhering to them whereas other cells had few or none. This non-Normal distribution of bacterial attachment must be taken into account when carrying out statistical analyses of the results. The wide discrepancies reported in the literature regarding bacterial adhesion to uro-epithelial cells must, in part, be related to the type of statistical analysis used. In many cases, a Normal rather than a non-Normal distribution has been assumed. We found that even when all variables were kept constant, the experiment was still not reproducible. Therefore the technique shows a high degree of both inter- and intra-experimental error. Adhesion depended on such factors as the type of fimbriae produced by the bacteria, differing viability of uro-epithelial cells and varying pH of the medium used for a particular experiment. It is concluded that the results of in-vitro experiments demonstrating adhesion of E. coli to uro-epithelial cells are difficult to relate to bacterial adhesion in vivo but better results could be obtained if more attention were paid to standardisation of methods and their analysis.

Bacterial vaginosis in pregnancy: distribution of bacterial species in different gram-stain categories of the vaginal flora.

Vaginal swabs for microbiological culture were taken from 174 pregnant women whose vaginal flora had been evaluated by Gram's stain; 50 had grade III flora (bacterial vaginosis, BV), 50 grade II (intermediate), 41 had vaginal flora graded as abnormal which then reverted to grade I (revertants) and 33 had normal flora (controls). The aim was to determine whether bacterial species isolated from women with grade II flora differed from those with grade III flora. Isolation of Lactobacillus spp. decreased from grade I to grade III and that of other aerobic and anaerobic bacterial species increased. There was little difference in the species isolated from women with grade II and grade III flora, but there was a distinct order in which organisms in different species increased in numbers. The vaginal flora of revertants was intermediate between that of healthy controls and those with grade II flora. Coagulase-negative Staphylococcus spp. were isolated from a greater number of revertants than grade I controls but the incidence did not increase in grade II or grade III. Bifidobacterium spp. were isolated from a greater number of revertants than grade I controls and increased further in grade II and grade III. However, Gardnerella vaginalis and Mycoplasma hominis were isolated from a much larger number of women with grade III flora than the other groups. The conclusion is that grade II is a transitional phase between grade I and grade III and that some organisms such as G. vaginalis and M. hominis only reach large numbers in the late stage. The sequence of appearance of the various bacterial species may be a result of the pathophysiological alteration of the vaginal ecosystem associated with BV.

The use of lipid-linked oligosaccharides (neoglycolipids) in the identification of carbohydrate receptors for microbial pathogens.

Specific oligosaccharide chains on the host cell surface act as receptors for many microbial pathogens. Identification of receptor structures is an important step in the understanding of the pathogenesis of infection. Glycolipid receptors have been identified by direct binding assays. However, technical difficulties have prevented demonstration of bacterial binding to the oligosaccharides of glycoproteins; these have been identified mainly by inhibition assays. By a novel technique developed in our laboratory, oligosaccharides released from glycoproteins are linked to lipids to form neoglycolipids. These can be used in bacterial binding assays. The feasibility of this approach has been demonstrated using type 1 fimbriated Escherichia coli binding specifically to neoglycolipids rich in mannose residues. The application of the method has resulted in a demonstration of a new type of adhesive specificity for E. coli and differences in the binding specificities of E coli and Pseudomonas aeruginosa. Further application of this technique by generating oligosaccharides purified from mucus glycoproteins from patients with cystic fibrosis to use in binding assays with P aeruginosa is currently being undertaken. The basic knowledge gained by this approach may in time see the development of novel therapy in the form of receptor blocking agents.

Relation between Gram-stain and clinical criteria for diagnosing bacterial vaginosis with special reference to Gram grade II evaluation.

The aim of this study was to analyse how the results of Gram-staining vaginal smears correlated with the clinical criteria for determining the existence of bacterial vaginosis (BV) and, in particular, how the category defined as 'intermediate' or Gram grade II did so and its significance. Women attending an antenatal clinic with an abnormal vaginal flora, that is those who had Gram-stains of grades II or III, the latter considered to equate with BV, were given clindamycin or a placebo intravaginally and examined again on up to three occasions. Gram-stain readings of grade III correlated with the clinical criteria for BV on 356 (91.7%) of 388 occasions. Grade II readings covered the spectrum of clinical criteria and correlated with those for BV on 35 (37.2%) of 94 occasions. Grade I, recorded 231 times and seen usually after clindamycin treatment, was associated with BV only once. The sensitivity, specificity, positive predictive value and negative predictive value of the Gram stain for the diagnosis of BV, based on a combination of grades II and III, were 99.7%, 71.6%, 81% and 99.6%, respectively; based on grade III only, the values were 99.7%, 87.7%, 91.6% and 99.6%, respectively. Women reported a malodorous vaginal discharge on 49.2% of the occasions a grade III flora was seen and 13.3% of the times grade II was recorded. It was not associated with grade I and would seem a useful adjunct to the accepted clinical criteria for diagnosing BV. Each of the clinical criteria was found in about equal proportions (87%-91%) for women whose Gram grade was III. For grade II, an increased discharge was noted most often (76.5%) and 'clue' cells least often (24.5%). A positive amine test was the most specific, being associated with <1% of grade I smears. Of women with grade III status, 91% reverted to grade I after treatment with clindamycin for three days. In contrast, of women with grade II status, 53% reverted to grade I, as did 47% of those who were given a placebo. The 'intermediate' (grade II) category is a Gram-stain diagnosis and not one that can be made clinically. It is important to recognize as a distinct entity not only because amalgamation with grade III diminishes the specificity and positive predictive value of the Gram-stain for diagnosing BV, but also because women of grade II status usually fail to respond to clindamycin treatment, whereas those of grade III do not.

Bacteriological and crystallographical analysis of urinary calculi: aid to patient management.

In an analysis, by both crystallographic and microbiological methods, of 50 urinary calculi recently removed by surgical operation, 33 proved to be of metabolic origin (mostly calcium oxalate and some uric acid or urate) and 17 of 'infective' origin (struvite, apatite or a mixture of the two). Metabolic stones were usually bacteriologically sterile or contained only small numbers (less than 10(3)/g of stone) of bacteria which did not produce urease, while infective stones always contained urease-producing organisms, usually Proteus mirabilis, in large numbers (greater than 10(5)/g). The combined approach of stone analysis by crystallography and microbiological culture yields more information than conventional techniques on which to base the treatment of urinary calculi and the prevention of their recurrence.

Urinary calculi: microbiological and crystallographic studies.

Although referred to as "urinary calculus disease", the formation of stone in the urinary tract is not caused by a single etiological agent. As such, diverse clinical investigations to diagnose the cause of stone formation must be carried out and the course of management after diagnosis must inevitably be different in each case. This review will cover all aspects of calculus formation, but will give particular attention to calculi caused by infection of the urinary tract with urease-producing bacteria. This is a recurrent, potentially life-threatening disease which has led clinicians to refer to the condition as "stone cancer". Because the etiology of infection stones is so different from stones caused by metabolic disorders, the two disease patterns should be considered separately, a fact often overlooked in epidemiological studies of stone formation. The importance of analysis of calculi as an aid to management is thus emphasized; identification of stone type will help to indicate appropriate therapy. A review of methods of analysis will be covered, particularly crystallographic analysis. Inhibition of bacterial urease as a means of management of infection stones will be discussed together with problems encountered and brighter hopes for the future.

The effect of acetohydroxamic acid on the induction of bacterial ureases.

The ureases of Proteus mirabilis, Proteus vulgaris, and Proteus rettgeri are inducible by urea. Induction is increased when both urea and acetohydroxamic acid, an inhibitor of urease, are present during bacterial growth. Acetohydroxamic acid alone does not cause induction, but, by preventing the hydrolysis of urea, it minimizes pH increases and allows induction to occur much more effectively. The ureases of Proteus morganii and other bacterial genera are not inducible by this method. The relevance of our findings to the formation and management of infection stones is discussed.

Role of urease in the formation of infection stones: comparison of ureases from different sources.

Bacterial and vegetable ureases were found to differ in certain important respects. For maximal clinical relevance, in vitro studies on the pathogenic role of urease should use whole bacterial cells of Proteus spp., and urease inhibitors should be assessed without preincubation of enzyme with inhibitor. Urease from Proteus morganii was very different from ureases of other species of Proteus; this factor should be taken into account when infections with P. morganii are being treated.

Differences in the binding specificities of Pseudomonas aeruginosa M35 and Escherichia coli C600 for lipid-linked oligosaccharides with lactose-related core regions.

Membrane glycolipids contain the lactose sequence (galactose linked to glucose), and the oligosaccharide is variously extended such that there is a cell-type-specific repertoire. In this study, binding of Pseudomonas aeruginosa M35 to lipid-linked lactose (Gal beta 1-4Glc [structure 1]), lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc [structure 2]), lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc [structure 3]), and asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc [structure 4]) was evaluated and compared with binding of Escherichia coli C600 to these compounds. Oligosaccharides were linked to the lipid phosphatidylethanolamine dipalmitoate, and the resulting neoglycolipids were resolved on thin-layer chromatograms or coated onto plastic microtiter wells. Lipid-linked structures 1 to 4 were bound by P. aeruginosa and E. coli in the chromatogram assay, but only structure 4 was bound in the microtiter well assay. As shown previously for E. coli binding to lipid-linked structures 1 to 3, binding to lipid-linked structure 4 was not inhibited with oligosaccharide, indicating a requirement for lipid and oligosaccharide. With few exceptions, sialylation and fucosylation of structures 1 to 4 resulted in impaired or abolished binding. Comparisons of binding intensities in the chromatogram assay indicated that recognition by P. aeruginosa and recognition by E. coli are not identical. Presence of the additional disaccharide unit, as in structure 2, resulted in enhanced binding of P. aeruginosa but diminished binding of E. coli relative to lactose binding; fucosylation at galactose of lactose resulted in markedly diminished binding of P. aeruginosa only. In the microtiter well assay, binding of E. coli to asialo GM1 was much weaker than P. aeruginosa binding. The saccharide-plus-lipid-dependent adhesion may be an important factor in increased susceptibility to infection of epithelia already damaged by microbial and chemical agents; the differing strengths of adhesion to the structural variants may relate to tissue tropism.

New type of adhesive specificity revealed by oligosaccharide probes in Escherichia coli from patients with urinary tract infection.

A series of oligosaccharides derived from glycoproteins or from human milk were coupled to lipid and used as probes of the binding specificities of Escherichia coli isolated from patients with urinary tract infections. Selective binding to the glycoprotein oligosaccharide probes rich in mannose residues (high-mannose type) was demonstrated with fimbriated E coli that give mannose-inhibitable haemagglutination. This observation is in accordance with predictions from inhibition studies. Binding studies with the human milk oligosaccharide probes, which resemble structures found on host-cell membranes, revealed adhesive specificity unrelated to the presence of fimbriae. This new type of host oligosaccharide receptor is affected by the presence of the blood group genetic markers. It involves the disaccharide sequence linked to the membrane-associated lipid moiety of host-cell glycolipids, and may have a role in initiation of infection on damaged epithelial cell membranes.

Bacteriuria and primary biliary cirrhosis.

Significant bacteriuria was found in 19% of 87 women with primary biliary cirrhosis, whereas in 89 women with other types of chronic liver disease bacteriuria was present in only 7%. In 74 women with rheumatoid arthritis 8% were bacteriuric. Midstream urine specimens obtained from 144 consecutive women with primary biliary cirrhosis attending hospital over a two year period showed that 50 (35%) developed bacteriuria during 12 months of follow up. Bacteriuria was unrelated to age, raised serum bilirubin, drug therapy or urinary pH but was more common in patients with late stage (fibrotic) disease as judged by histological criteria. Fifty seven per cent of bacteriuric primary biliary cirrhosis patients suffered more than one urinary infection. Fifty nine per cent of the 156 bacteriuric episodes were asymptomatic. The types of organism isolated, the antibiotic sensitivity patterns and cure rate were similar to those reported in bacteriuric women without other underlying disease. The reinfection rate (34%), however, was double that reported for bacteriuric episodes in 'problem' women with recurrent bacteriuria, indicating a special susceptibility to urinary infection. The most common isolates were E coli (70%), which did not show abnormal adhesiveness to uroepithelial or buccal cells of normal women, or to those of primary biliary cirrhosis patients. Patients with primary biliary cirrhosis have not been reported to be more susceptible to infection in general. Bacteriuria, however, was common throughout all clinical stages of primary biliary cirrhosis. Thus there may be a unique association between bacteriuria and primary biliary cirrhosis.

Relationship between hydrogen peroxide-producing strains of lactobacilli and vaginosis-associated bacterial species in pregnant women.

This study was conducted to determine the relationship between lactobacilli and bacterial species associated with bacterial vaginosis in pregnancy and the prevalence of H2O2-producing and non-producing strains of lactobacilli in pregnant women whose vaginal flora had already been analysed. Information was available for 174 pregnant women whose vaginal flora had been evaluated previously by examining gram-stained vaginal smears: 50 had grade III flora (bacterial vaginosis). 50 grade II flora, 41 flora graded as abnormal which then reverted to grade I (revertants) and 33 normal flora (controls). Lactobacilli were isolated from 19 of 50 women whose vaginal flora was grossly abnormal culturally and categorised as grade III by Gram staining. In 6 of these 50 women lactobacilli were isolated in large numbers, i.e. 10(5)-10(6) cfu/ml. H2O2-producing strains of lactobacilli were isolated from 11 of 12 women with grade III flora who were randomly selected from this group. Thus, in those 11 women it appears that H2O2-producing lactobacilli had not protected them from developing bacterial vaginosis. Bacterial species associated with vaginosis were isolated in high numbers from a large proportion of women in the revertant and grade II groups in association with high counts of lactobacilli. Thus, in some women it is possible that a change to an abnormal flora could occur before the complete disappearance of lactobacilli. It is concluded that bacterial vaginosis may develop in some women despite the presence of H2O2-producing strains of lactobacilli and that other factors, as yet unidentified, might be conducive to the appearance of abnormal bacterial flora with progression to vaginosis.