Assessment of measurement methods of posterior inflammation in stromal choroiditis: the value of quantitative outcome measures versus the presently qualitatively based paradigm.

PURPOSE: To assess posterior inflammation using a fluorescein (FA)/indocyanine-green angiography (ICGA) scoring system, and compare them to the presently recommended outcome measure, the standardization of uveitis nomenclature vitreous haze score (SUN-VH) in stromal choroiditis. METHODS: This was a retrospective study on patients with a diagnosis of ocular sarcoidosis(OS), ocular tuberculosis(OT), Birdshot retinochoroiditis(BRC) and Vogt-Koyanagi-Harada disease(VKH) seen in the Centre for Ophthalmic Specialized Care, Lausanne, Switzerland. Angiography signs were quantified according to an established FA/ICGA scoring system. Vitritis was assessed using SUN-VH. Results were compared. RESULTS: 65 newly diagnosed patients (128 eyes) with stromal choroiditis were included. Angiographic scoring showed variable degrees of choroidal versus retinal involvement (87% for OS, 72% for OT, 62.5% for BRC and 100% for VKH). On the other hand, a mere 22 of 128 eyes (17%) showed a SUN-VH score ≥ 2 necessary for inclusion in clinical trials. Moreover, FA/ICGA values followed a normal distribution curve and presented inter-examiner variations greater than 1-SD in only 8.4% of cases. SUN-VH values' distribution was non-normal and showed inter-examiner discrepancies greater than 1-SD in 51.7% of cases. CONCLUSION: This study highlights the precise measurement of global posterior inflammation achieved by a dual FA/ICGA scoring system in stromal choroiditis. In contrast, SUN-VH scale appears imprecise and inadequate, as only a minute percentage of the studied eyes could have been included in a clinical trial based on this criterion. To evaluate posterior intraocular inflammation meaningfully in stromal choroiditis, the use of dual FA/ICGA is strongly advised and should replace the presently recommended SUN-VH system.

Evolution of choroidal thickness over time and effect of early and sustained therapy in birdshot retinochoroiditis.

PurposeTo follow choroidal thickness (ChT) over time in birdshot retinochoroiditis (BRC) using enhanced depth imaging optical coherence tomography (EDI-OCT) and study the effect of early and sustained treatment on ChT.Patients and methodsEighteen patients were included and EDI-OCT measurements of ChT were analyzed retrospectively in five groups of patients with follow-up times ranging from 1 year to ≥15 years. The OCT images were evaluated and ChT was calculated under the foveola and 1500 µm temporal, nasal, superior, and inferior to the foveola. To assess the effect of treatment, 13 patients with a disease duration ≥10 years were divided into two groups depending on their treatment status: early and sustained therapy vs insufficient, late, or no treatment. ChT was compared in these two groups along with the number of typical fundus BRC lesions.ResultsThe ChT decreased (r=-0.41, P=0.0018) over the disease duration, which ranged from <1 year to ≥15 years. In patients with a disease duration ≥10 years, a significant difference in ChT was noted between adequately and undertreated patients (288.3±76.9 µm vs 161.4±39.2 µm; P=0.004). At the last follow-up, in the group with insufficient therapy 10 of 11 eyes presented typical fundus BRC lesions vs 2 of 13 eyes in the treated group (P≤0.0006, F-test).ConclusionsChoroidal thickness decreases significantly over time in BRC. If undertreated, patients show thinner choroids compared with adequately treated individuals and present significantly more BRC lesions.

Productivity and growth of a natural population of the smallest free-living eukaryote under nitrogen deficiency and sufficiency.

The influence of dissolved inorganic nitrogen (DIN) enrichments on cell-normalized carbon uptake rate, chlorophyll a content, and apparent cell size of a picoeukaryote (<1 microm) ( Ostreococcus tauri, the smallest eukaryotic cell) from a natural summer phytoplanktonic assemblage (<200 microm) in a northern Mediterranean Lagoon (Thau Lagoon) was studied in 20-L enclosures in June 1995. The natural planktonic community was incubated in situ for 24 h with initial ammonium and nitrate enrichments and compared to a control without enrichment. O. tauri cell-normalized productivity was estimated from the combination of flow cytometric (FCM) enumeration and 2-h (radioactive) carbonate incorporation measured on post-incubation size fractions (<1microm). No difference between the effects of the two DIN sources of enrichment on the studied biological parameters was measured during this experiment. Growth of natural O. tauri was perturbed by the low DIN availability in the control with drastic changes in cell productivity, chlorophyll content, and cell cycle (from the variations in apparent cell size) as compared to the DIN sufficiency conditions. On the other hand, a very high specific growth rate for natural O. tauri, up to 8 day(-1) under DIN enrichments, has been estimated from production and abundance data obtained during this experiment. This supports values measured in culture and suggests that the yearly high contribution of picophytoplankton to the total primary production in Thau Lagoon is likely to be due to their high growth rate rather than the previously suggested lack of grazing pressure.

Pulse-field Gel Electrophoresis for Epidemiological Studies of Streptococcus pneumoniae.

Electrophoresis techniques have allowed for very effective separation of nucleic acids on the basis of molecular weight. Conventional DNA electrophoresis can only separate DNA fragments smaller than 50 kb. Above this size, DNA fragments are larger than the pore size of the matrix. They can travel through a gel matrix by deforming their shape, parallel to the field in order to pass through the pores. This mode of migration is called "reptation" and all large molecules migrate at the same rate. Shwartz et al. (1) were the first to introduce the concept that molecules larger than 50 kb can be separated by using two alternating electric fields, one homogeneous and the other nonhomogeneous. In pulsed field gel electrophoresis (PFGE) the electrical field is applied alternately in two directions. The orientation of DNA molecules is perturbed and they have to reorient to move efficiently in response to the new field. The time required for this reorientation has been found to be proportional to the molecular weight. Larger DNA molecules take more time to realign after the fields are switched than do smaller ones. Thus, the reorientation introduces a size dependence that is absent during simple "reptation." The timing of the electrical field in each direction is called the pulse time: too fast or too slow and no separation occurs. However molecules of DNA, whose reorientation times are less than the period of the electric pulse, will be fractionated according.

Molecular biology of Streptococcus pneumoniae: an everlasting challenge.

Streptococcus pneumoniae is a model for elucidating: 1) recombination steps of DNA, from its discovery to polarity of integration; 2) long-patch mismatch repair, short-patch repair triggered by A/G and exclusion of deletions; 3) resistance to beta-lactam antibiotics; and 4) factors of virulence. Several of these topics remain a challenge for future investigations.

Structural organization of the Streptococcus pneumoniae chromosome and relatedness of penicillin-sensitive and -resistant strains in type 9V.

Fragmentation of Streptococcus pneumoniae genomic DNA with low-frequency-cleavage restriction endonucleases and separation of the fragments by field-inversion gel electrophoresis (FIGE) provides a DNA-fingerprint of a strain. This method enables us to construct a physical and genetic map of the R6 laboratory strain what will be presented. The origin of replication containing several Dna boxes was located in the dnaA region. It was of interest to compare the profiles of subclones. Two clones of strain R36A (R6 and C13) were cultivated separately for more than 15,000 generations in two laboratories. FIGE profiles differed by only one band. Another R36A descendant, isolated in 1958 by Ravin, strain Rx was of interest since it was deficient in Dpn restriction enzymes and methylases and in the hex B function. Its origin was questionable; its profile is identical to others R6 descendants, demonstrating that Rx is derived from R36A. FIGE analysis was carried out on several penicillin-resistant strains of type 9V because penicillin-resistance in this type increased recently. The profiles of a collection of a number of these resistant isolates were very similar, showing that they result from a clone. The profiles of penicillin sensitive isolates of the same type are very similar to the resistant isolates. This suggests that the 9V type has spread recently from a clone, and the resistance genes have mutated and were selected when penicillin was extensively used.

Electrotransformation of Streptococcus pneumoniae: evidence for restriction of the DNA on entry.

Electrotransformation is a method generally used in biotechnology to introduce recombinant DNA into a wide range of bacteria. However the mechanism of DNA entry is poorly understood. We report that in Streptococcus pneumoniae, a naturally transformable species, electrotransformation efficiently introduces a plasmid replicon. DNA is strongly restricted by the restriction-modification systems DpnI and DpnII which degrade methylated and nonmethylated DNA respectively at GATC sequences. This suggests that in electrotransformation double-strand DNA penetrates into these bacteria without a single-strand step in contrast to natural transformation. Single-strand DNA by itself is able to electrotransform very weakly and linearized double-stranded plasmid DNA yields barely detectable levels of transformants.

Relatedness of penicillin-resistant Streptococcus pneumoniae serogroup 9 strains from France and Spain.

Pulsed-field gel electrophoresis of the genomic DNA of penicillin-resistant strains of Streptococcus pneumoniae was carried out. Eleven clinical strains of serogroup 9 from different French towns and Paris hospitals were tested. The restriction enzymes Apal and Smal were used to digest intact chromosomes, and the fragments were resolved by field-inversion gel electrophoresis (FIGE). Five strains were similar using Apal and Smal. Four others were closely related when using Apal, and five others were closely related when using Smal. These results suggest that 10 of these strains are genetically related and have a clonal origin. The profile of the eleventh strain was completely different. Thus, in a given serotype the spreading of penicillin resistance can result from both clonal and independent events. Five strains had similar FIGE profiles to strains first isolated in Spain, suggesting that a resistant strain had spread from Spain to France.

Pulsed field gel electrophoresis for molecular epidemiology of penicillin resistant Streptococcus pneumoniae strains.

The emergence of strains of Streptococcus pneumoniae resistant to penicillin and other antibiotics has become a major concern for antimicrobial therapy of pneumococcal infections. The spread of that resistance over the world increases the need for their epidemiological surveillance: specific epidemiological markers are required. In this study, pulsed field gel electrophoresis of genomic DNA was carried out on sixteen resistant isolates of S. pneumoniae from different parts of the world and fifteen resistant isolates from Toulouse. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. The polymorphism obtained with FIGE was greater than that obtained with serotyping which appeared to be not a good criterion for genetic relatedness. Three common clones could be recognized among the penicillin-resistant isolates. Two clones were found in Spain and in Toulouse and were associated with serotypes 6B and 9V, respectively. The third clone was isolated in South Africa and in Spain and contained serotype 23F isolates and one serotype 19F strain. The FIGE profiles observed in this study also demonstrated that serogroup 23 multiresistant strains isolated in Toulouse are genetically closely related and might have originated from the same Spanish 23F clone. These results underline the importance of the geographic spread of resistant clones in the increase in the incidence of penicillin-resistant pneumococci. They indicate that pulsed field gel electrophoresis should be an effective tool for the typing of resistant S. pneumoniae strains capable of tracing their origin.

A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae.

Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for beta-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this beta-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, ciaR and ciaH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase CiaH into two putative domains: an N-terminal extracellular sensor part, and an intracellular C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of CiaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated ciaH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.

Genetic studies of cefotaxime resistance in Streptococcus pneumoniae: relationship to transformation deficiency.

A laboratory pneumococcal strain resistant to cefotaxime was studied by DNA-induced transformation in order to characterize its genetic structure. At least three independent genes were required to confer the highest level of resistance to this beta-lactam antibiotic. The accumulation of mutations in these three genes accounted for three levels of resistance. Mutation of the gene encoding penicillin-binding protein 2x was very likely responsible for the first step of resistance, which was a prerequisite for sequential increase in resistance. Additionally, strains highly resistant to cefotaxime were defective for natural transformation. Revertants of these strains were frequently observed. Such strains had recovered full transformability, suggesting a correlation between the inability to be transformed and a high level of resistance to cefotaxime. The possibility of electrotransforming these highly resistant strains suggests that natural transformation is probably blocked at the DNA-uptake level.

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae.

A physical map of the Streptococcus (Diplococcus) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.

Conversion of deletions during recombination in pneumococcal transformation.

Genetic analysis of 16 deletions obtained in the amiA locus of pneumococcus is described. When present on donor DNA, all deletions increased drastically the frequency of wild-type recombinants in two-point crosses. This effect was maximal for deletions longer than 200 bases. It was reduced for heterologies shorter than 76 bases and did not exist for very short deletions. In three-point crosses in which the deletion was localized between two point mutations, we demonstrated that this excess of wild-type recombinants was the result of a genetic conversion. This conversion extended over several scores of bases outside the deletion. Conversion takes place during the heteroduplex stage of recombination. Therefore, in pneumococcal transformation, long heterologies participated in this heteroduplex configuration. As this conversion did not require an active DNA polymerase A gene it is proposed that the mechanism of conversion is not a DNA repair synthesis but involves breakage and ligation between DNA molecules. Conversion of deletions did not require the Hex system of correction of mismatched bases. It differs also from localized conversion. It appears that it is a process that evolved to correct errors of replication which lead to long heterologies and which are not eliminated by other systems.

Repair of single- and multiple-substitution mismatches during recombination in Streptococcus pneumoniae.

The use as genetic markers, during transformation of Streptococcus pneumoniae, of 19 sequences differing from wild type, located throughout the amiA locus, enabled us to examine the fate of 24 single- and 11 multiple-mismatches during recombination. Tentative mismatch ranking as a function of decreasing repair efficiency by the Hex mismatch repair system is G/T = A/C = G/G (maximum repair: 90-95%) greater than C/T (mostly 75 to 90% repair) greater than A/A (from 50 to 90% repair) greater than T/T (50-65% repair) greater than A/G (from 0 to 20% repair) greater than C/C. No indication of correction of the latter has been obtained. Over the limited number of samples examined, we observed no influence of the base composition of the surrounding sequence on correction efficiency for both transition mismatches and for G/G and C/C. Variations in the surrounding sequence affect repair of A/G and C/T, and, even more strongly, of A/A and T/T. No simple correlation to the G:C content of the surrounding sequence is apparent from our results, in contrast to the conclusion drawn for the Mut mismatch repair system of Escherichia coli. Examination of the fate of multiple mismatches suggests that C/C may sometimes impede recognition of otherwise corrected mismatches.

Inhibition of DNA repair by neighbouring mismatched bases in Streptococcus pneumoniae.

A set of pneumococcal strains containing immediately adjacent or nearby double mutations at the amiA locus, conferring resistance to amethopterin, has been isolated by oligonucleotide site-specific mutagenesis. Repair of these double mutations has been measured by transformation of wild-type strains with DNA extracted from these strains. In several transformations we have observed an inhibition of repair by neighbouring mismatches. This inhibition ranges from mild to severe depending upon the interfering mismatch. Unrepaired mismatches can strongly inhibit repair of an adjacent repairable mutation. This suggests that the repair-complex proteins attach not only to repairable mismatches but also to some mismatches known to escape the repair system.

DNA sequences required to induce localized conversion in Streptococcus pneumoniae transformation.

In pneumococcal transformation a particular point mutation belonging to the amiA locus is able markedly to enhance recombination frequency when crossed with any other markers of this gene. This results from a polarized conversion of the mutation towards the wild-type sequence. In this report, by site-directed oligonucleotide mutagenesis, we have generated a series of mutants showing various degrees of conversion. We have found that the substitution 5'-ATTCAT----5'-ATTAAT is a sufficient signal for localized conversion. Changing individual bases within this sequence results in decreased conversion frequencies to levels that depend on the mutation, suggesting that there is a family to related sequences which may act as a substrate for a conversion system. Moreover, the length over which this conversion occurs has been estimated to be 12 base pairs on the average.

Mismatch repair during pneumococcal transformation of small deletions produced by site-directed mutagenesis.

The genetic behaviour of short non-homologous regions has been studied during transformation of Streptococcus pneumoniae. Amethopterin-resistant mutants belonging to the amiA locus were used for these investigations. Five mutants deleted for 1-5 bp were obtained by oligonucleotide-directed mutagenesis. Their efficiency of transformation was measured using recipient strains either able to excise and repair mismatched bases (Hex+) or Hex- derivatives. Deletions or insertions of 1 and 2 bp are fully recognized by the Hex system, and are efficiently repaired whereas 3-bp deletions or insertions are only partially excised and repaired. The efficiency of repair is inversely related to the size of the non-homology. Markers with 5-bp deletions or insertions are poorly repaired and thus transform at very high frequency: similar results are obtained in reciprocal crosses. It is proposed that 1- or 2-bp deletions or insertions are included in the heteroduplex structure as transition mutations. The Hex system would detect only small deviations from the normal DNA structure.

Frame-shift mutants induced by quinacrine are recognized by the mismatch repair system in Streptococcus pneumoniae.

We describe the isolation of amethopterin-resistant mutants induced by quinacrine treatment of exponentially growing cultures of Streptococcus pneumoniae. Only mutants located by recombination analysis in a few hundred base pairs were further studied. They were cloned and their DNA sequences show that most of them are +/-1-base frame-shift mutants. They are excised and repaired to a degree similar to transition mutants (low efficiency class), suggesting that the mismatches resulting from a transition or a +/-1-base mutation are similar substrates for the Hex mismatch repair system.