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Aim To perform a cross-sectional study on the carrier frequency of JP2 and non-JP2 genotypes of A. actinomycetemcomitans in Moroccan school children and relate the presence of these genotypes to the periodontal status in the mixed dentition. Material and Methods A plaque sample from 513 children was analysed by PCR. JP2 genotype-positive subjects (n = 46), an equally sized group of non-JP2 genotype-positive subjects, and an A. actinomycetemcomitans-negative group were randomly chosen among the remaining subjects for clinical and radiographic examination. Results Among 513 children, 46 (9.0%) carried the JP2 genotype and 186 (36.3%) were positive for non-JP2 genotypes, whereas A. actinomycetemcomitans could not be detected in the remaining 281 subjects. Among 75 subjects with mixed dentition and selected for clinical examination, clinical attachment loss (CAL) ≥3 mm at two or more periodontal sites on primary teeth was found in the JP2 genotype-positive group only. In total, 6.7% of subjects with primary teeth present showed CAL ≥3 mm at two or more sites. Conclusions The carrier frequency of the JP2 genotype of A. actinomycetemcomitans was at a comparable level to frequencies previously found in Moroccan adolescent populations. Clinical attachment loss, manifesting already in the primary dentition, was found only in the group of Moroccan children carrying the JP2 genotype of A. actinomycetemcomitans.

We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalactiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping results obtained by latex agglutination. Among 281 human isolates 27% could not be typed by latex agglutination. All 312 isolates except 5 could be typed by the three PCR methods combined. Two of these methods were multiplex assays. Among the isolates that were typeable by both latex agglutination and capsular gene typing, 94% showed agreement between the two methods. However, each of the PCR methods showed limitations. One of the methods did not include all 10 recognized serotypes, one misidentified eight isolates of serotypes Ib and IV as serotype Ia, and one did not distinguish between serotypes VII and IX. For five isolates that showed aberrant patterns in the capsular gene typing, long-range PCR targeting the cps operon disclosed large insertions or deletions affecting the cps gene cluster. A sensitive flow cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination revealed that approximately one-half of these did express capsular polysaccharide. A procedure for convenient and reliable capsular gene typing to be included in epidemiological and surveillance studies of S. agalactiae is proposed.

Summary Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat-in-toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α-haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA-mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA-mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS-exposure is preceded by increments in the intracellular Ca2+ concentration ([Ca2+]i). Interestingly, LtxA-mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA-induced [Ca2+]i increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2-mediated auto- and paracrine signalling may prevent LtxA-mediated cell damage.

Elamin A, Albandar JM, Poulsen K, Ali RW, Bakken V. Prevalence ofAggregatibacter actinomycetemcomitansin Sudanese patients with aggressive periodontitis: a case–control study. J Periodont Res 2011; 46: 285–291.©2011 John Wiley & Sons A/S Background and Objective: Aggregatibacter actinomycetemcomitans is considered a possible etiological agent for aggressive periodontitis. The aim of this study was to determine the prevalence of the JP2 clone and non-JP2 genotypes of A. actinomycetemcomitans in the subgingival plaque of patients with aggressive periodontitis and controls among Sudanese high-school students. Material and Methods: In a previous study we examined a large representative sample of students attending high schools in Khartoum, Sudan. In this population, 17 patients with aggressive periodontitis and 17 controls (14–19 years of age) consented to participate in the present study. The subjects underwent a clinical periodontal examination, and subgingival dental plaque samples were collected using paper points. The presence of the A. actinomycetemcomitans JP2 clone and non-JP2 genotypes were assessed using loop-mediated isothermal amplification (LAMP) and the PCR. Results: The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of either the cases or the controls. Non-JP2 types of A. actinomycetemcomitans were detected in the subgingival plaque of 12 (70.6%) patients with aggressive periodontitis and from only one (5.9%) control subject, showing a significantly higher frequency of detection in cases than in controls (p = 0.0001). The odds ratio for the detection of A. actinomycetemcomitans in the subgingival plaque of the patients with aggressive periodontitis was 38.4 (95% confidence interval: 4.0–373.0; p = 0.002). The PCR and LAMP methods showed identical results pertaining to the identification of non-JP2 types of A. actinomycetemcomitans. Conclusions: The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of high school subjects in Sudan. The detection of non-JP2 types of A. actinomycetemcomitans may be a useful marker of increased risk for development of aggressive periodontitis in young subjects.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.

24 hours after nephrectomy there is an increase in plasma angiotensinogen which is 2 to 3 times increased in unilaterally nephrectomized rats and 5 to 12 times increased in bilaterally nephrectomized rats. In normal rats, DOCA + salt treated rats and renal hypertensive rats, the postnephrectomy changes are about the same, independent of the marked differences in their pre-operative plasma and renal renin. The postnephrectomy increase in angiotensinogen must therefore be due to loss of some internal factor other than renin or to loss of some external renal function. Both shamoperation and unilateral nephrectomy are followed by a marked decrease in plasma renin, the postoperative fall being more pronounced after removal of a clamped kidney than after removal of a normal.

While hypotension elicited a marked increase in plasma renin concentration in conscious normal mice, no increase was provoked in previously nephrectomized mice in spite of the high renin content of their submaxillary glands. The role of the increased release of renal renin for the homeostasis of the blood pressure was shown by the decrease in pressure which followed blockade of the renin system. Contrary to Saralasin which did not change the blood pressure in nephrectomized mice, injections of SQ 20.881 did in some mice result in a decrease in blood pressure, which was probably caused by its ability to inhibit bradykininases. Both Saralasin and SQ 20.881 elicited marked increases in plasma renin in normal, but not in nephrectomized mice, showing that, while renal renin release is controlled by the plasma angiotensin II concentration, this does not apply to submaxillary renin release.

Inhibition of the angiotensin I converting enzyme with SQ 20.881 results in a 2 to 35 fold increase in plasma renin concentration in normal rats and in spontaneously hypertensive rats. The effect is transient, lasting for 1 to 3 hours even in the presence of prolonged blockade. The relative increase is independent of the pretreatment plasma renin concentration. The blood pressure is unchanged in conscious rats in which the effect of SQ 20.881 on plasma renin is believed to be due to a blockade of the negative feedback of angiotensin II on renin release. In anaesthetized rats, SQ 20.881 has an additional hypotensive effect which augments the increase in plasma renin. Saralasin is without effect on blood pressure and plasma renin in conscious normal rats and in spontaneously hypertensive rats, while it causes a transient 3 to 27 fold increase in plasma renin concentration in anaesthetized rats. It is suggested that this increase is hardly due to an interception of the feedback, but to the concomitant fall in blood pressure, as a similar hypotension and increase in plasma renin is produced by dihydra-lazine. It is furthermore found that Saralasin blocks renin release induced by SQ 20.881. This demonstrates that Saralasin is bound to the receptors in the juxtaglomerular cells and has slight, agonistic properties there. Both in conscious rats and in anaesthetized adrenalectomized rats substituted with DOCA and salt, SQ 20.881 as well as Saralasin causes transient increases in plasma renin concentration. If such rats are only substituted with salt and not with DOCA, the effects of both blockers are in the form of severe hypotension and a permanent elevation of plasma renin.

A variety of insulins and insulin fragments were shown to be active as inhibitors of angiotensin I converting enzyme from guinea pig plasma when tested in vitro. Angiotensin I was used as substrate and the consumption rate of angiotensin I as well as the generation rate of angiotensin II were measured by means of specific radioimmunoassays for angiotensin I and II. The B-chain of insulin from different species possessed about the same inhibitory capacity as the whole insulin molecule. In an attempt to locate the active part of the sequence of the B-chain, the five peptides which resulted from tryptic digestion of the duck insulin B-chain were studied. They were, however, all active as inhibitors and on molar basis equal to or slightly more potent than the B-chain and insulin. This demonstrates the unspecific nature of converting enzyme. Its affinity for insulin and its peptide fragments were, however, two to four orders of magnitude lower than it was for the most potent inhibitor described so far, the synthetic nonapeptide SQ 20.881. This difference was confirmed in in vivo experiments.

After gentle manipulation with subsequent removal of the submaxillary glands in nephrectomized mice there is a vast increase in the concentrations of high molecular weight renin (“prorenin”?) as well as active renin in plasma. The increase in active renin is continuous even after removal of glands as well as kidneys. Using in vitro incubation and replacement transfusion experiments, the continuous rise was found not to be due to activation of the high molecular weight renin. The continuous increase could instead be shown to be due to a rapid transfer of submaxillary lymph and (or) interstitial fluid to the surrounding tissues, with a subsequent slower release from these tissues to the blood. Continued release of renin from extra-submaxillary tissue-depots is probably also the cause of the continuance of the high plasma renin concentrations for several hours after the removal of the manipulated glands, which contrasts with the much more rapid decline in the concentration after injection of extracts of submaxillary glands as well as after injection of pure submaxillary renin.

The expression of angiotensin (Ang) II receptors, active renin and prorenin in porcine and bovine ovarian follicles and corpora lutea was investigated and compared. In the wall tissue of porcine follicles, the Ang II receptor density was 47 (range 19-97; n = 13) fmol/mg membrane protein. The active renin concentration was 1.32 (0.40-3.43; n = 23) GU/kg wet tissue. These values were about 35-fold and 15-fold lower, respectively, than previously found in bovine follicles. No prorenin could be detected in the porcine follicular wall tissue. Ang II receptors of subtype 2 (AT 2 receptors) with a dissociation constant (K d ) of 1.01 (0.64-1.79; n = 8) nmol/l for [Sar 1 -Ile 5 -Ile 8 ]-Ang II were demonstrated in the bovine corpus luteum. The receptors density was 22.7 (1.9-93; n = 26) fmol/mg membrane protein, which was about 10-fold higher than in porcine corpora lutea. The active renin concentration was 20.7 (2.2-60.0; n = 26) GU/kg tissue in bovine and 0.40 (0.16-1.09; n = 17) GU/kg tissue in porcine corpora lutea. No prorenin could be detected in corpora lutea from both species. The variation between species in expression of the ovarian renin-angiotensin system indicates the existence of species differences in the physiological role.

Evidence has accumulated for the existence of local tissue renin-angiotensin systems in the reproductive tissues. In the ovary the local renin-angiotensin system (RAS) has been shown to influence ovulation and steroidogenesis. In this review we focus on aspects of a local RAS in the uteroplacental unit. High renin concentrations have been found in all tissues of the uteroplacental unit. The presence of renin mRNA in the endometrium, choriodecidua and the fetal part of the placenta indicates local renin synthesis. Angiotensin (Ang) II, formed by the enzymatic action of tissue renin and Ang I converting enzyme, acts by interaction with its receptors. These receptors have been demonstrated in high densities in the placenta and uterus, indicating an autocrine or paracrine action of Ang II. Several probable effects of the uteroplacental RAS can be defined. It is very likely that the uteroplacental RAS plays an important role during implantation and placentation by stimulation of decidualization and angiogenesis. Furthermore, Ang II may regulate synthesis and secretion of other hormones formed locally in the uteroplacental unit. During labour the action of Ang II may be important for contraction of the uterine musculature, Ang II is also involved in the complex regulation of the uteroplacental blood flow. Species difference in the expression of the uteroplacental RAS exists, and one may speculate that the physiological and pathophysiological roles vary between species.

Multiple sclerosis (MS) is an inflammatory disease where phagocytic cells infiltrate the nerve tissue and act as terminal agents in destruction of the myelin sheath. However, the mechanism that triggers the ability of these cells to recognize myelin remains obscure. We show that myelin basic protein (MBP), a major autoantigen in MS, is a potent and specific ligand for the integrin αMβ2 (Mac-1, CD11b/CD18) expressed mainly on phagocytic cells. MBP undergoes a dramatic conformational change when liberated from the lipid-rich environment of the myelin sheath. The MS drug glatiramer acetate mimics the conformationally labile regions of MBP, interacts in the unfolded state strongly with αMβ2, and inhibits the MBP binding to αMβ2. Our study reveals a link between MBP, glatiramer acetate, and the αMβ2 integrin, and suggests a new model for MS pathogenesis based on the recognition of unfolded MBP by the αMβ2 integrin.

Bacterial IgA1 proteases share the ability to cleave human IgA1 at the hinge region. Nature has developed this trait along at least five independent evolutionary lineages. To obtain further insight into the phylogeny and function of IgA1 proteases, the nucleotide sequence of theigagene that encodes the IgA1 protease was determined from twoStreptococcus mitisstrains and oneGemella haemolysansstrain. Heterologous expression inEscherichia coliconfirmed that the genes encode human IgA1-cleaving activity. IgA1 proteases fromStreptococcusandG. haemolysansshared structural features, including a motif typical for zinc-dependent metalloproteases of clan MA(E) family M26 and an N-terminal signal sequence followed by an LPXTG cell-wall-anchor motif and two putative membrane-spanning domains. In addition, they all harboured a repeat region preceding the active site of the protease. In the streptococcal IgA1 proteases, a G5 domain, which has been suggested to bindN-acetylglucosamine, was identified. Conservation of these structures in otherwise diverse proteases suggests that they are essential to the biological function of the enzyme. The phylogenetic distribution of homologousigagenes and conservation of gene order in theigagene region in differentStreptococcusspecies, combined with the sequence homologies, strongly suggest that theigagene is more ancient inStreptococcusthan inG. haemolysans, and therefore that the IgA1 protease gene was transferred fromStreptococcustoG. haemolysans.

Udgivelsesdato: 2004-Oct The JP2 clone of Actinobacillus actinomycetemcomitans has been implicated in the etiology of periodontitis in adolescents. The aim of this two-year longitudinal study was to describe clinical attachment loss (CAL) progression and to assess its association with baseline occurrence of the JP2 and non-JP2 types of A. actinomycetemcomitans. Clinical re-examination of 121 adolescents in Morocco was performed. Progression of CAL > or = 1 mm, > or = 2 mm, > or = 3 mm, and > or = 4 mm on at least one site was found in 58%, 48%, 22%, and 6% of the subjects, respectively. Subjects who, at baseline, harbored the JP2 clone had a significantly higher progression of CAL than did subjects harboring non-JP2 types of A. actinomycetemcomitans. Subjects harboring non-JP2 types displayed a marginally higher CAL progression than did subjects who were culture-negative for A. actinomycetemcomitans.

ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes.

Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse β 2 -integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express β 2 -integrin or not. Clearly, removal of sialic acid groups significantly reduces a β 2 -integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express β 2 -integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since α-hemolysin from Escherichia coli induced cell lysis of both β 2 -integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to β 2 -integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.

The genetic structure of a population of Pseudomonas aeruginosa , isolated from patients with keratitis, endophthalmitis, and contact lens-associated red eye, contact lens storage cases, urine, ear, blood, lungs, wounds, feces, and the environment was determined by multilocus enzyme electrophoresis. The presence and characteristics of virulence factors were determined by restriction fragment length polymorphism analysis with DNA probes for lasA , lasB , aprA , exoS , exoT , exoU , and ctx and by zymography of staphylolysin, elastase, and alkaline protease. These analyses revealed an epidemic population structure of P. aeruginosa , characterized by frequent recombination in which a particular successful clone may increase, predominate for a time, and then disasappear as a result of recombination. Epidemic clones were found among isolates from patients with keratitis. They were characterized by high activity of a hitherto-unrecognized size variant of elastase, high alkaline protease activity, and possession of the exoU gene encoding the cytotoxic exoenzyme U. These virulence determinants are not exclusive traits in strains causing keratitis, as strains with other properties may cause keratitis in the presence of predisposing conditions. There were no uniform patterns of characteristics of isolates from other types of infection; however, all strains from urinary tract infections possessed the exoS gene, all strains from environment and feces and the major part of keratitis and wound isolates exhibited high elastase and alkaline protease activity, and all strains from feces showed high staphylolysin activity, indicating that these virulence factors may be important in the pathogenesis of these infectious diseases.

A particular clone (JP2) of Actinobacillus actinomycetemcomitans with increased leukotoxin production has been isolated from individuals with early-onset periodontitis (EOP). The aim of this study was to determine the frequency of carriers of this clone and its association with EOP in Moroccan schoolchildren. Of 217 plaque samples, 131 (60.4%) were culture-positive for A. actinomycetemcomitans. A total of 19 of these isolates had a 530-bp deletion in the leukotoxin promoter region characteristic of the JP2 clone. A strong association between the presence of A. actinomycetemcomitans with the 530-bp deletion and EOP was found (adjusted OR = 29.4; 95% CI = 8.3 - 104.4; p < 0.0005), while no association could be demonstrated between the presence of A. actinomycetemcomitans without the deletion and EOP (adjusted OR = 1.3; 95% CI = 0.5 - 2.9; p = 0.750). The study demonstrates that the endemic presence, in a human population, of the highly leukotoxic JP2 clone may result in an unusually high prevalence of EOP.

Recent evidence strongly suggests that the microbiota of the nasal cavity plays a crucial role in determining the reaction patterns of the mucosal and systemic immune system. However, little is known about the normal microbiota of the nasal cavity. The purpose of this study was to determine the microbiota in different parts of the nasal cavity and to develop and evaluate methods for this purpose. Samples were collected from 10 healthy adults by nasal washes and by swabbing of the mucosa through a sterile introduction device. Both methods gave results that were quantitatively and qualitatively reproducible, and revealed significant differences in the density of the nasal microbiota between individuals. The study revealed absence of gram-negative bacteria that are regular members of the commensal microbiota of the pharynx. Likewise, viridans type streptococci were sparsely represented. The nasal microbiota was dominated by species of the genera Corynebacterium, Aureobacterium, Rhodococcus, and Staphylococcus, including S. epidermis, S. capitis, S. hominis, S. haemolyticus, S. lugdunensis and S. warneri. These studies show that the microbiota of the nasal cavity of adults is strikingly different from that of the pharynx, and that the nasal cavity is a primary habitat for several species of diphtheroids recognized as opportunistic pathogens. Under special circumstances, single species, including IgA1 protease-producing bacteria, may become predominant in a restricted area of the nasal mucosa.

The sequence of infB, encoding the prokaryotic translation-initiation factor 2 (IF2), was determined in eight strains of Streptococcus agalactiae (group B streptococcus) and an alignment revealed limited intraspecies diversity within S. agalactiae. The amino acid sequence of IF2 from S. agalactiae and from related species were aligned and revealed an interspecies conserved central and C-terminal part, and an N-terminal part that is highly variable in length and amino acid sequence. The diversity and relationships in a collection of 58 genetically distinct strains of S. agalactiae were evaluated by comparing a partial sequence of infB. A total of six alleles were detected for the region of infB analysed. The alleles correlated with the separation of the same strains of S. agalactiae into major evolutionary lineages, as shown in previous work. The partial sequences of infB were furthermore used in phylogenetic analyses of species closely related to S. agalactiae, yielding an evolutionary tree which had a topology similar to a tree constructed using 16S rRNA sequences from the same species.

Proteinases of Staphylococcus aureus are emerging as potential virulence factors which may be involved in the pathogenecity of staphylococcal diseases. We describe here the structure of the gene encoding the metalloproteinase referred to as aureolysin. This gene occurs in two allelic forms and is strongly conserved among S. aureus strains, implying the possibility that the proteinase may have important housekeeping functions.

Summary This short review describes how competitive inhibitors were synthesized and how the solubility was increased in order to make them effective at physiological pH. Their use for in vitro and in vivo inhibition of renin is discussed and their use for purification of renin demonstrated.

In normal valvular collagen turnover in the rat, angiotensin (Ang) II and angiotensin-converting enzyme (ACE) seem to be involved. In common human and canine valvular diseases, changes in valvular collagen play a pathogenetic role and the valvular renin-angiotensin system is therefore of particular interest in these species. Healthy mitral valve leaflets and adjacent left ventricular myocardium were taken from five rats and five dogs immediately after euthanasia, and from five humans at autopsy. The valvular and myocardial Ang II receptors and ACE were detected and measured by quantitative autoradiography. In rat valves, high levels of Ang II receptors and ACE were found. In human and canine valves, insignificant levels were found. Significant myocardial levels of Ang II receptors and ACE were found only in the rat. The study demonstrated major species differences regarding the level of valvular and myocardial Ang II receptors and ACE in man, dog and rat. The lack of valvular Ang II receptors and ACE in man and dog, suggest that the renin-angiotensin system plays a minor, if any, role in the physiological valvular collagen formation in these two species. The findings in humans, however, need to be confirmed using fresh material.

1. The aim of the present study was to characterize the angiotensin II (AngII) receptor subtypes in the porcine uterus and the variation of receptor densities and renin concentrations during gestation. 2. In myometrium from non-pregnant sows, the AngII receptors were almost exclusively AT2 receptors. During gestation, the AngII receptor density was decreased and the AT1 receptor became predominant in the last part of gestation as a result of a down-regulation of the AT2 receptor. 3. In the endometrium, the AT1 receptor was predominant both in non-pregnant sows and throughout gestation. The AngII receptor density was decreased during gestation as a consequence of down-regulation of the AT1 receptor. 4. The renin concentrations in the myometrium and endometrium of pregnant sows did not differ from those in non-pregnant animals. 5. The finding of enzymatically active renin and high densities of AngII receptors in the porcine uterus is in accordance with a functional renin-angiotensin system (RAS), which may be important for an increased vascular permeability and stimulated angiogenesis in early pregnancy and for contraction of the myometrial smooth muscle cells during parturition. The predominance of AT1 receptors in the endometrium of non-pregnant sows differs from an earlier finding in non-pregnant women, where AT2 receptors were predominant in the endometrium. This is in accordance with earlier studies, indicating species differences in the expression and possibly also the physiological roles of the RAS in reproductive tissues.

Udgivelsesdato: 1996-Oct Bacterial immunoglobulin A1 (IgA1) proteases constitute a very heterogenous group of extracellular endopeptidases which specifically cleave human IgA1 in the hinge region. Here we report that the IgA1 protease gene, iga, of Streptococcus pneumoniae is homologous to that of Streptococcus sanguis. By using the S. sanguis iga gene as hybridization probe, the corresponding gene from a clinical isolate of S. pneumoniae was isolated in an Escherichia coli lambda phage library. A lysate of E. coli infected with hybridization-positive recombinant phages possessed IgA1-cleaving activity. The complete sequence of the S. pneumoniae iga gene was determined. An open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,927 amino acids with a molecular mass of 215,023 Da was preceded by a potential -10 promoter sequence and a putative Shine-Dalgarno sequence. A putative signal peptide was found in the N-terminal end of the protein. The amino acid sequence similarity to the S. sanguis IgA1 protease indicated that the pneumococcal IgA1 protease is a Zn-metalloproteinase. The primary structures of the two streptococcal IgA1 proteases were quite different in the N-terminal parts, and both proteins contained repeat structures in this region. Using a novel assay for IgA1 protease activity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we demonstrated that the secreted IgA1 protease was present in several different molecular forms ranging in size from approximately 135 to 220 kDa. In addition, interstrain differences in the sizes of the pneumococcal IgA1 proteases were detected. Southern blot analyses suggested that the S. pneumoniae iga gene is highly heterogenous within the species.

Publisher Summary This chapter elaborates the structural chemistry and the biological aspects of IgA protease of Clostridium ramosum. Bacterial IgA1 proteases are a group of endopeptidases with a remarkable specificity to cleave human IgAl molecules in the hinge region at a single Pro–Ser or Pro–Thr peptide bond. This unique microbial activity that separates the IgA1 molecule into intact Fab and Fc fragments was first identified in fluids of the human digestive tract, and subsequently isolated from Streptococcus sanguis. The enzyme was named IgA protease. C. ramosum IgA protease is highly specific. The enzyme is unable to cleave other human immunoglobulins. The amino acid sequence of the C. ramosum IgA protease deduced from the IgA gene revealed a translation product of 1234 amino acids. The first 30 residues constitute the signal peptide. In the C-terminus, a putative cell wall anchor was identified. The sequence SPQTG presumably constitutes the sortase recognition site. Other features including a small spacer, DNSN, separating the SPQTG motif from a transmembrane domain, IFLWFALLFSAAGVTGITAY, followed by a positively charged tail, NKKKKEHAE, at C-terminus. This strongly argues that the C. ramosum IgA protease is subject to sortase activity.

Summary Cloning and sequencing of the IgA1 protease gene (iga) from Neisseria meningitidis strain HF13 showed an overall structure equivalent to iga genes from Neisseria gonorrhoeae and Haemophilus influenzae, although no region corresponding to the gonococcal α-peptide was evident. An additional 18 N. meningitidis and 3 H. influenzae iga genes were amplified by the polymerase chain reaction technique and sequenced corresponding approximately to the N-terminal half of the mature enzyme. Comparative analyses of a total of 29 iga genes showed that pathogenic Neisseria have iga genes with a significantly lower degree of heterogeneity than H. influenzae iga genes. Recombinational events indicated by mosaic-like structures corresponding to those found among N. gonorrhoeae protease genes were detected among N. meningitidis iga genes. One region showed characteristic differences in sequence and length which correlated with each of the different cleavage specificities. Meningococci were extremely conserved in this region with no evidence of recombination between isolates of different cleavage specificities. Sequences further downstream showed no obvious relationship with enzyme cleavage type. This region consisted of conserved areas interspersed with highly variable areas. Amino acid sequence homologies in the variable regions of meningococci reflected the antigenic types defined by using polyclonal neutralizing antibodies.

The distribution, genome location, and evolution of the four paralogous zinc metalloproteases, IgA1 protease, ZmpB, ZmpC, and ZmpD, in Streptococcus pneumoniae and related commensal species were studied by in silico analysis of whole genomes and by activity screening of 154 representatives of 20 species. ZmpB was ubiquitous in the Mitis and Salivarius groups of the genus Streptococcus and in the genera Gemella and Granulicatella, with the exception of a fragmented gene in Streptococcus thermophilus, the only species with a nonhuman habitat. IgA1 protease activity was observed in all members of S. pneumoniae, S. pseudopneumoniae, S. oralis, S. sanguinis, and Gemella haemolysans, was variably present in S. mitis and S. infantis, and absent in S. gordonii, S. parasanguinis, S. cristatus, S. oligofermentans, S. australis, S. peroris, and S. suis. Phylogenetic analysis of 297 zmp sequences and representative housekeeping genes provided evidence for an unprecedented selection for genetic diversification of the iga, zmpB, and zmpD genes in S. pneumoniae and evidence of very frequent intraspecies transfer of entire genes and combination of genes. Presumably due to their adaptation to a commensal lifestyle, largely unaffected by adaptive mucosal immune factors, the corresponding genes in commensal streptococci have remained conserved. The widespread distribution and significant sequence diversity indicate an ancient origin of the zinc metalloproteases predating the emergence of the humanoid species. zmpB, which appears to be the ancestral gene, subsequently duplicated and successfully diversified into distinct functions, is likely to serve an important but yet unknown housekeeping function associated with the human host., IMPORTANCE The paralogous zinc metalloproteases IgA1 protease, ZmpB, ZmpC, and ZmpD have been identified as crucial for virulence of the human pathogen Streptococcus pneumoniae. This study maps the presence of the corresponding genes and enzyme activities in S. pneumoniae and in related commensal species of the genera Streptococcus, Gemella, and Granulicatella. The distribution, genome location, and sequence diversification indicate that zmpB is the ancestral gene predating the evolution of today’s humanoid species. The ZmpB protease may play an important but yet unidentified role in the association of streptococci of the Mitis and Salivarius groups with their human host, as it is ubiquitous in these two groups, except for a fragmented gene in Streptococcus thermophilus, the only species not associated with humans. The relative sequence diversification of the IgA1 protease, ZmpB, and ZmpD is striking evidence of differences in selection for diversification of these surface-exposed proteins in the pathogen S. pneumoniae compared to the closely related commensal streptococci.

The close phylogenetic relationship of the important pathogen Streptococcus pneumoniae and several species of commensal streptococci, particularly Streptococcus mitis and Streptococcus pseudopneumoniae , and the recently demonstrated sharing of genes and phenotypic traits previously considered specific for S. pneumoniae hamper the exact identification of S. pneumoniae . Based on sequence analysis of 16S rRNA genes of a collection of 634 streptococcal strains, identified by multilocus sequence analysis, we detected a cytosine at position 203 present in all 440 strains of S. pneumoniae but replaced by an adenosine residue in all strains representing other species of mitis group streptococci. The S. pneumoniae -specific sequence signature could be demonstrated by sequence analysis or indirectly by restriction endonuclease digestion of a PCR amplicon covering the site. The S. pneumoniae -specific signature offers an inexpensive means for validation of the identity of clinical isolates and should be used as an integrated marker in the annotation procedure employed in 16S rRNA-based molecular studies of complex human microbiotas. This may avoid frequent misidentifications such as those we demonstrate to have occurred in previous reports and in reference sequence databases.

To investigate the presence of A. actinomycetemcomitans, including the highly virulent JP2 clone, in young adult patients with aggressive periodontitis, and associate the findings with the two forms of the disease.Seventy Moroccan subjects with aggressive periodontitis, aged less than 35 years, were recruited. Among these, 41 had LAgP and 29 had GAgP. Plaque samples were collected from periodontal pockets and examined using a PCR that detects the presence of A. actinomycetemcomitans and which differentiates between JP2 and non-JP2 genotypes of the bacterium.total of 58 (83%) from the 70 AgP patients were positive for A. actinomycetemcomitans, among whom 77% were positives for the JP2 clone. The JP2 clone was detected in 34 (83%) of the LAgP patients compared to 20 (69%) of the GAgP patients (p = 0.17). Fourteen (20%) of the patients harbored non-JP2 genotypes of A. actinomycetemcomitans, although most of these patients (10/14) also harbored the JP2 clone.The presence of the JP2 clone of A. actinomycetemcomitans is strongly associated with both LAgP and GAgP in young adults in Morocco. This implies that treatment of AgP in this population should include microbiological screening and aim at eradication of the bacterium when present.

SUMMARY 1. The aim was to analyse the in vivo variations with time of prorenin and active renin and their relationship to steroid hormones in ovarian follicular fluid during follicular growth in heifers. 2. Thirty one beef heifers were assigned to two groups after oestrous synchronization: an unstimulated and a follicle-stimulating hormone (FSH)-treated (superovulated) group. Within each group, animals were slaughtered at different times of the follicular phase of the oestrous cycle. Ovarian follicular fluids were aspirated and analysed for the concentrations of active renin, prorenin, oestradiol-17β (E2) and progesterone (P4). 3. Prorenin and active renin concentrations in follicular fluid remained constant until the luteinizing hormone (LH) peak, after which time they increased four- and two-fold, respectively, in superovulated heifers. 4. In follicular fluid, prorenin and active renin correlated negatively with oestradiol and E2/P4 ratio but positively with progesterone during follicular growth in superovulated heifers. Prorenin also correlated negatively with oestradiol and E2/P4 ratio in unstimulated heifers. 5. The increase of renin concentrations in ovarian follicles after the LH peak and the correlations to steroid hormones suggest an important role of the ovarian renin-angiotensin system in bovine follicular growth and maturation.

SUMMARY 1. The ovarian follicular renin—angiotensin system was investigated by using an in vitro incubation method, based on tissue incubation of individual bovine follicles. 2. Very high and varying concentrations of active renin (median 18.0 GU/kg, range 2.1–107 GU/kg; n= 101) and prorenin (11.7 GU/kg, range

1. The purpose of this study was to investigate angiotensin II (AII) receptors in isolated bovine ovarian follicles and the relationship of their density to follicular concentrations of prorenin, active renin, oestradiol and progesterone. 2. Displacement of [125I]-[Sar1-Ile5-Ile8]-AII binding by the AII receptor antagonists PD 123319 and Losartan (DuP 753) confirmed that follicular AII receptors are of subtype 2 (AT2 receptor). 3. The dissociation constant (Kd) for [Ile5]-AII (human AII) was 0.84 (range 0.51-1.47) nmol/L. The receptor density varied between 90 and 5990 (mean 1640) fmol/mg membrane protein. 4. The follicular AII receptor density correlated positively with follicular diameter (Spearman's rho = 0.518; P < 0.003) and tissue weight (Spearman's rho = 0.636; P < 0.0001), and negatively with the active renin concentration in the follicular wall (Spearman's rho = -0.399; P < 0.02). The AII receptor density did not correlate with the follicular fluid concentrations of prorenin, active renin, oestradiol (E2), progesterone (P4) or the E2/P4 ratio. The follicular fluid concentrations of prorenin correlated negatively with the E2/P4 ratio (Spearman's rho = -0.716; P < 0.0001). 5. The inverse relationship between AII receptor density and the high active renin concentrations in the follicular wall suggests an active regulated tissue renin-angiotensin system. A high AII receptor density is a general feature of large bovine ovarian follicles.

To examine the global diversity of Streptococcus agalactiae (group B streptococci [GBS]) and to elucidate the evolutionary processes that determine its population genetics structure and the reported changes in host tropism and infection epidemiology, we examined a collection of 238 bovine and human isolates from nine countries on five continents. Phylogenetic analysis based on the sequences of 15 housekeeping genes combined with patterns of virulence-associated traits identified a genetically heterogeneous core population from which virulent lineages occasionally emerge as a result of recombination affecting major segments of the genome. Such lineages, like clonal complex 17 (CC17) and two distinct clusters of CC23, are exclusively adapted to either humans or cattle and successfully spread globally. The recent emergence and expansion of the human-associated and highly virulent sequence type 17 (ST17) could conceivably account, in part, for the increased prevalence of neonatal GBS infections after 1960. The composite structure of the S. agalactiae genome invalidates phylogenetic inferences exclusively based on multilocus sequence typing (MLST) data and thereby the previously reported conclusion that the human-associated CC17 emerged from the bovine-associated CC67., IMPORTANCE Group B streptococci (GBS) (Streptococcus agalactiae) have long been recognized as important causes of mastitis in cattle. After 1960, GBS also became the most prevalent cause of invasive and often fatal infections in newborns. At the same time, GBS are carried by a substantial proportion of healthy individuals. The aims of this study were to elucidate the genetic mechanisms that lead to diversification of the GBS population and to examine the relationship between virulence and host preference of evolutionary lineages of GBS. Genetic analysis of GBS isolates from worldwide sources demonstrated epidemic clones adapted specifically to either the human or bovine host. Such clones seem to emerge from a genetically heterogeneous core population as a result of recombination affecting major segments of the genome. Emergence and global spread of certain clones explain, in part, the change in epidemiology of GBS disease and may have implications for prevention.

Neisseria meningitidis is one of several important bacterial pathogens that secrete a specific protease capable of cleaving human immunoglobulin A1 (IgA1) in the hinge region. To obtain further information on this putative virulence factor, we examined the IgA1 protease and iga gene region of 133 isolates of N. meningitidis assigned to 88 multilocus enzyme genotypes and representing major epidemics and carrier strains from 19 countries. Of the two IgA1 cleavage specificities previously observed, isolates associated with epidemics of meningococcal disease showed exclusively type 1 IgA1 protease activity. Considerable heterogeneity of the N. meningitidis IgA1 protease was demonstrated at both the protein and gene levels. Thus, five different forms of IgA1 protease were detected with enzyme-neutralizing antibodies raised in rabbits. An antiserum raised against a single type 2 IgA1 protease inhibited the enzyme activity of all strains examined, a finding of potential significance for the possible application of IgA1 protease in a vaccine against meningococcal disease. Examination of the iga gene region with restriction endonucleases revealed a high degree of polymorphism among strains belonging to some multilocus enzyme genotypes. The different iga gene types did not correlate with cleavage type or inhibition of the IgA1 protease. Our findings indicate that horizontal genetic exchange occurs in vivo with considerably different frequency in different clones of meningococci.

Udgivelsesdato: 1992-May-11 Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.

Figure S3 contains errors. Please view the corrected file here: Click here for additional data file.(12M, tif)

Udgivelsesdato: 2009-Sep The JP2 clone of Aggregatibacter actinomycetemcomitans is strongly associated with aggressive periodontitis. To obtain information about colonization dynamics of the JP2 clone, we used PCR to examine its presence in 365 Moroccan juveniles from whom periodontal plaque samples were collected at baseline and after one and two years. Periodontal attachment loss was measured at baseline and at the two-year follow-up. At baseline, 43 (12%) carriers of the JP2 clone were found. Nearly half (44 %) of these were persistently colonized with the clone. The relative risk for the development of aggressive periodontitis, adjusted for the concomitant presence of other genotypes of A. actinomycetemcomitans, was highest for individuals continuously infected by the JP2 clone (RR = 13.9; 95% CI, 9.0 to 21.4), indicating a relationship between infectious dose and disease, which further substantiates the evidence for the JP2 clone as a causal factor in aggressive periodontitis.

Udgivelsesdato: July 16 Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.

Periodontitis is a loss of supporting connective tissue and alveolar bone around teeth, and if it occurs in an aggressive form it can lead to tooth loss before the age of 20 years. Although the cause of periodontitis in general remains elusive, a particular clone (JP2) of the gram-negative rod Aggregatibacter (Actinobacillus) actinomycetemcomitans is considered a possible aetiological agent of the aggressive form in adolescents living in or originating from north and west Africa, where the disease is highly prevalent. We did a population-based longitudinal study of adolescents to assess the role of the JP2 clone in the initiation of aggressive periodontitis.A total of 700 adolescents from public schools in Rabat, Morocco, were enrolled in the study. We used PCR to detect A actinomycetemcomitans in plaque samples (taken from molar and incisor sites) and to differentiate between the JP2 clone and other non-JP2 genotypes of the bacterium. 18 individuals were found to already have periodontitis and were excluded. The 682 periodontally healthy adolescents (mean age 12.5 years; SD 1.0) were classified according to their A actinomycetemcomitans carrier status at baseline. After 2 years, 428 (62.8%) individuals returned for re-examination, which included recording of periodontal attachment loss measured from the cemento-enamel junction to the bottom of the periodontal pockets of all teeth present.Individuals who carried the JP2 clone of A actinomycetemcomitans alone (relative risk 18.0; 95% CI 7.8-41.2, p0.0001) or together with non-JP2 clones of A actinomycetemcomitans (12.4; 5.2-29.9, p0.0001) had a significantly increased risk of periodontal attachment loss. A much less pronounced disease risk was found in those carrying non-JP2 clones only (3.0; 1.3-7.1, p=0.012).The JP2 clone of A actinomycetemcomitans is likely to be an important aetiological agent in initiation of periodontal attachment loss in children and adolescents. Co-occurrence of non-JP2 clones of A actinomycetemcomitans reduces the risk of development of periodontitis, suggesting competition for the ecological niche between the JP2 and non-JP2 clones of this species.

The plasma concentration of inactive renin was higher (p less than 0.002) in male (0.48 Goldblatt units (GU)/l, range 0.28-0.67 GU/l; n = 20) than in female rats (0.19 GU/l, range 0.09-0.30 GU/l; n = 20). This new finding indicates a sexual dimorphism of inactive plasma renin similar to that recently reported in mice and humans. Castration of male rats (n = 10) decreased (p less than 0.01) inactive plasma renin from 0.45 GU/l (range 0.22-0.65 GU/l) to 0.26 GU/l (range 0.15-0.37 GU/l) over a period of seven weeks. Similarly, inactive renin decreased (p less than 0.01) from 0.50 GU/l (range 0.28-0.67 GU/l) to 0.30 GU/l (range 0.19-0.47 GU/l) during treatment with the anti-androgen flutamide (n = 10). In castrated male rats (n = 10), inactive plasma renin was unaffected of substitution with testosterone. In intact female rats (n = 10), inactive renin increased (p less than 0.05) from 0.18 GU/l (range 0.12-0.30 GU/l) to 0.27 GU/l (range 0.15-0.40 GU/L) during testosterone treatment. Our findings indicate that androgens may be involved in the regulation of the plasma concentration of inactive renin in rats, but also suggest that other factors may be of importance.

We developed a loop-mediated isothermal amplification method that detects the JP2 clone of Aggregatibacter actinomycetemcomitans , which induces aggressive periodontitis in adolescents of North and West African descents. Being independent of special equipment, this specific and sensitive method offers significant advantages for screening of patients on a population basis and in clinical settings.

Recent studies have shown that the pulp of human teeth contains a population of cells with stem cell properties and it has been suggested that these cells originate from pericytes. Molecules of the Notch signaling pathway regulate stem cell fate specification, while Rgs5 represents an excellent marker for pericytes. Pathological conditions such as dental trauma and carious lesion stimulate pulp stem cells to elaborate reparative dentin. Previous studies have shown that genes involved in the Notch pathway are activated in response to pulp injury in rodent and humans. To demonstrate the importance of pericytes as a source of stem cells during dental repair, we have studied Rgs5 and Notch3 mRNA expression by in situ hybridization in developing, adult intact and injured rodent teeth. Furthermore, we have examined the distribution of Notch3 protein in carious and injured human teeth using immunohistochemistry. Overlapping expression patterns of Rgs5 and Notch3 were observed during rodent tooth development as well as immediately after injury. Both genes were expressed in vascular structures during development and in perivascular and single capillary cells of injured teeth. However, the expression patterns of Rgs5 and Notch3 were different during tooth repair, with relatively extensive Rgs5 expression along the pericyte-vascular smooth muscle cell axis in central pulp arterioles. These results show co-expression of Rgs5 and Notch3 in pericytes of developing and injured teeth and furthermore indicate the importance of vascular-derived stem cells during pulp healing.