Species-specificity of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens

Prevotella intermedia and Prevotella nigrescens are not easily distinguished, making it difficult to assess their roles in disease. This study examined the specificity of three monoclonal antibodies (mAbs) for these species. Differentiation between P. intermedia (13 isolates) and P. nigrescens (24 isolates) was by the electrophoretic mobility of their malate and glutamate dehydrogenase enzymes or by DNA homology grouping. All P. intermedia reacted strongly with mAb 40BI3.2.2 whereas P. nigrescens strains did not. Monoclonal antibodies 37BI6.1 and 39BI1.1.2 recognised all strains of both species but most P. nigrescens reacted weakly with mAb 39BI1.1.2. Monoclonal antibody 40BI3.2.2 therefore recognises an antigen specific for P. intermedia but not P. nigrescens and provides an easy and reliable means of distinguishing between these species. Three vaginal isolates identified biochemically as P. intermedia had enzymes with mobilities corresponding to neither P. intermedia nor P. nigrescens. These isolates were not recognised by mAbs 39BI1.1.2 or 40BI3.2.2 and may represent an undescribed taxon within this group of organisms.     

Incidence of Prevotella intermedia and Prevotella nigrescens in periodontal health and disease

The incidence of black-pigmented rods (BPRs), especially Prevotella intermedia and Prevotella nigrescens, in periodontal health and disease were examined. Furthermore, the degradative enzyme activities of P. intermedia were compared among the strains from periodontal health and disease. Microbiological specimens were collected from subgingival crevice or periodontal pocket by paper point. The BPRs were found in 71.1% of periodontally healthy subjects (n=45), and in 47.1% of healthy sites (n=34) and 87.8% of active sites (n=41) among periodontally diseased patients. Porphyromonas gingivalis was detected only in active sites of periodontally diseased patients (17.8% of 180 strains). P. intermedia was the predominant BPR in both healthy and active sites (37.3 and 41.7%, respectively) of the patients. However, P. nigrescens was the predominant BPR (70.5% of 173 strains) in periodontally healthy subjects. The enzyme activities of esterase, esterase-lipase, acid-phosphatase and alpha-fucosidase of P. intermedia strains isolated from active sites in patients were significantly higher (P<0.05) than those of healthy subjects. The results suggest that P. intermedia might increase the activity of degradative enzymes under a certain condition and support the progression of periodontitis.     

Occurrence of Prevotella nigrescens and Prevotella intermedia in infections of endodontic origin

The occurrence of Prevotella intermedia and Prevotella nigrescens in endodontic infections was studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell protein to distinguish between the species. Previous studies have shown an association between black-pigmented bacteria (BPB) and endodontic infections and that Prevotella intermedia (previously known as Bacteroides intermedius) was the most commonly isolated BPB. Recently, however, strains identified as P. intermedia were shown to in fact be composed of two separate species, P. intermedia and P. nigrescens. Fifty-six strains of BPB isolated from endodontic infections and previously identified as P. intermedia were used in this study. Following SDS-PAGE, P. nigrescens showed a unique 18.6 kDa band that was used to differentiate P. nigrescens from P. intermedia. Of the 56 strains of BPB, 41 (73.2%) were identified as P. nigrescens and 15 (26.8%) as P. intermedia. This study confirms that P. nigrescens, and not P. intermedia, is the BPB most often isolated from infections of endodontic origin.     

Characterization of an IgM Fc-binding receptor on human T cells

The existence of an IgM receptor on human T cells has been suggested by T cell rosetting with IgM-coated erythrocytes. In this study we used immunofluorocytometry to demonstrate that 1) after short-term culture, a majority of the T cells can bind IgM at easily detectable levels, 2) human and mouse IgM preparations bind to human T cells but other Ig isotypes do not, 3) the IgM binding is saturable and inhibitable only by Ig of IgM isotype and 4) the Fc portion of IgM is involved in the binding to a protease-sensitive cell surface protein. Biochemical analysis of the T cell receptor for IgM reveals a cell surface protein of 60 kDa in comparison with the 58 kDa Fc mu receptors (Fc mu R) on B-lineage cells. Although Fc mu R expression is up-regulated after B cell activation, the reverse is true after T cell activation. In addition, the T cell Fc mu R is relatively resistant to phospholipase C treatment. These results indicate that T- and B-lineage cells express Fc mu R with different biologic characteristics.     

Extracts of Prevotella intermedia and Actinobacillus actinomycetemcomitans inhibit alkaline phosphatase activity in osteoblastic cells in vitro

A prospective, randomized study was carried out to evaluate functional and biomicroscopic long-term results of different posterior chamber intraocular lenses (IOLs) over a period of two years after clear corneal cataract surgery. PATIENTS AND METHODS: 2 years after phacoemulsification through a temporal two-step clear corneal incision a total of 67 patients were examined. In 26 eyes (group A) a foldable plate-haptic silicone IOL (Chiron Adatomed, C10), in 25 eyes (group B) a foldable disc silicone IOL (Chiron Adatomed, 90D) had been implanted through a 3.5 (group A) or 4 mm (group B) corneal incision using a cartridge injector. In 16 eyes (group C) a one-piece PMMA-IOL (Pharmacia & Upjohn, 809C) had been implanted through a 5 mm incision. All eyes underwent functional and biomicroscopic examinations, as well as computerized videokeratographic analysis to obtain corneal topography data. RESULTS: In group A uncorrected visual acuity valued 0.64 (+/-0.29 SD), in group B 0.59 (+/-0.24) and in group C 0.56 (+/-0.27). Median of uncorrected visual acuity was 0.6 for all groups. Corrected visual acuity was 0.81 (+/-0.29) in group A, 0.8 (+/-0.25) in group B and 0.83 (+/-0.3) in group C. Intraocular pressure (mm Hg) was 13 (+/-2.5) in group A, 14.7 (+/-2) in group B and 15.1 (+/-2.5) in group C. Fibrosis of the anterior capsular rim occurred in 42% of the cases. One eye demonstrated folds in the posterior capsule (group B). Posterior capsular opacification valued 11.9% for all groups. In one eye a Nd:YAG-capsulotomy had already been performed. In group A a decentration of more than 1 mm was objected in one case, in group B in two cases and in group C in one case, but no patient complained about any functional impairment. Two years postoperatively, no signs of a re-flattening in the incision area could be detected using difference mapping tools in the videokeratographic analysis. CONCLUSION: Two years after implantation of foldable silicone IOLs and PMMA-IOLs via a temporal clear corneal tunnel incision after phacoemulsification only slight functional and morphologic differences between the three IOL-types could be observed.     

The binding and utilization of hemoglobin by Prevotella intermedia

Personalism in ethics denotes any system based upon the value of the person. Several versions of personalist morals have been developed over the past 50 years. Some have had particular interest in the field of medical ethics. Here the question is being studied about one such system, the so-called Leuven personalist morals and its usefulness in today's world of bioethics. In order to test the usefulness of this system the case of artificial insemination is examined both in the early 1970s in the context of the Leuven clinics and, subsequently, in the 1990s in a US policy document. The investigation reveals strengths and weaknesses of this personalism. Regarding AID it reveals unresolved oppositions. The conclusion seems to be that this personalism had, no doubt, a profound impact upon medical ethics within its own circle but, as regards the universal usefulness of the system, serious doubts remain.     

Antimicrobial susceptibilities of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens spp. isolated in Spain

The susceptibilities of 143 Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens isolates to 18 antimicrobial agents were tested. All P. gingivalis isolates were susceptible. In contrast, some Prevotella spp. (17%) were resistant to beta-lactams, erythromycin, clindamycin, or tetracycline and carried resistance genes, ermF or tetQ, or beta-lactamases.     

Purification and functional analysis of the DnaK homologue from Prevotella intermedia OMZ 326

Large-volume leukapheresis (LVL), defined as the processing of at least three blood volumes in a single session for peripheral blood progenitor cell (PBPC) collection, was performed in 32 small children weighing < or = 25 kg, aged 10 months to 8 years, with a variety of malignancies. Harvesting of PBPC was started after 4 days of cytokine (G-CSF, 12 micrograms/kg s.c.) alone. Procedures were performed using a continuous flow blood cell separator (COBE Spectra). The automated program of lymphocytapheresis was modified to achieve a collection rate of 0.9 ml/min. The extracorporeal line was primed with a unit of a packed red blood cells before the procedure. Acid citrate dextrose (ACD) was used as anticoagulant with an ACD inlet ratio of 1:14 and an ACD infusion rate of 1.1 ml/min/L of total blood volume. The inlet flow ranged between 6 and 35 ml/min (median 20 ml/min). A total of 37 apheresis procedures were performed (median 1, range 1-3). In 84% of patients, a single apheresis yields the minimum number of PBPC cells required for transplantation. No consistent side effects were observed, and LVL was well tolerated by children. A median of 7.7 x 10(8) kg MNC, 5.4 x 10(6)/kg CD34+, and 6.2 x 10(4)/kg CFU-GM per apheresis were harvested. Patients with neuroblastoma had a significantly lower yield than other patients. To date, 27 patients have been transplanted after myeloablative treatment, and rapid and sustained engraftment was achieved in all cases. The number of CD34+ cells infused was highly correlated with engraftment kinetics. LVL can be safely and easily performed in small children, allowing adequate PBPC collection for transplantation with rapid hematologic recovery.     

Anti-CD4 activity of normal human immunoglobulin G for therapeutic use. (Intravenous immunoglobulin, IVIg)

The effects of intravenously administered normal immunoglobulin G (IVIg) in autoimmune diseases are dependent on the ability of IVIg to interact with surface molecules of lymphocytes. In the present study, we demonstrate the presence of anti-CD4 activity in IVIg by showing the ability of IVIg to bind to CD4 and to inhibit CD4-dependent cellular functions. Binding of IVIg to recombinant soluble human CD4 was assessed by ELISA, immunoblotting and real time analysis of complex formation. Anti-CD4 antibodies isolated from IVIg by affinity-chromatography bound to human CD4+ T cells. These anti-CD4 antibodies inhibited proliferative responses in MLR and infection of CD4+ human T cells with HIV. These results indicate that IVIg contains antibodies reactive with human CD4 and that these anti-CD4 antibodies exhibit biological functions. The presence of anti-CD4 antibodies in IVIg may be relevant to the immunoregulatory effects of normal polyspecific immunoglobulin G.     

Laser Raman investigation of the conformation of human immunoglobulin G

Laser Raman spectra of human immunoglobulin G in neutral solution, as well as in the lyophilized and alkaline-denatured states are presented. In the spectrum of the native protein, the amide III band appears at 1240 cm-1 and is assigned to the presence of beta-sheet structure. From its intensity, using a procedure described in this paper, we evaluate the beta-structure content to 37 +/- 4%. This result is supported by the strong amide I' band at 1667 cm-1 and by the presence in the spectra of two bands at 991 and 1078 cm-1, respectively assigned to the C-C and C-N skeletal stretching modes. The differences between the spectrum of the lyophilized powder and that of the solution show that the lyophilization process induces conformational changes that perturb the local environment of some of the tryptophan residues and alter the secondary structure of immunoglobulin G. The beta-structure appears to be more uniform and more abundant in solution. When the protein is denatured at pH 11, the amide III and amide I'bands, which become weaker and broader, shift in frequency from 1240 to 1248 cm-1 and from 1667 to 1656 cm-1 respectively. These changes indicate a decrease in the amount of beta-structure and a transition toward a much more disordered conformation. During the denaturation, the intensities of many bands of the aromatic chromophores change, notably the tryptophan peaks at 879, 1359 and 1573 cm-1.     

Characterization of fertilization-blocking monoclonal antibody 1G12 with human sperm-immobilizing activity

A mouse hybridoma (1G12) producing sperm-immobilizing MoAb to human sperm was established and characterized in order to study the antigens relevant to sperm immobilization by antibodies. MoAb 1G12 had strong sperm-immobilizing and agglutinating activities and also showed a fertilization-blocking activity on in vitro fertilization tests. The antibody absorption experiments showed that MoAb 1G12 reacted not only to ejaculated sperm but also human seminal plasma, suggesting that the corresponding antigen might be a sperm coating antigen. The MoAb also reacted with peripheral blood lymphocytes. In histochemical studies, the epithelia of corpus epididymis were most strongly stained. Ejaculated sperm were stained with a granular pattern for their entire surface by immunofluorescence. MoAb 1G12 recognized polymorphic glycoproteins of 15-25 kD in the ejaculated sperm extract in Western blot analysis. After deglycosilation of the sperm extract, only a single staining band of under 15 kD was detected by MoAb 1G12. This suggests that the antigen epitope recognized by MoAb 1G12 might be a peptide of the core portion of the glycoprotein. MoAb 1G12 might be a useful tool for studying the mechanism of egg-sperm interaction, and also be applied to identifying the corresponding antigen by using gene technology.     

Anti-idiotypic activity against anti-myeloperoxidase antibodies in pooled human immunoglobulin

We investigated the ability of six different pooled human immunoglobulin (PHIG) preparations to inhibit the binding of anti-myeloperoxidase (MPO) antibodies to MPO. All six PHIG preparations inhibited the binding of anti-MPO antibodies from six sera to MPO in a concentration-dependent manner in the concentration range 0.016-10 mg/ml. There was considerable variation in the ability of each PHIG preparation to inhibit the binding of anti-MPO antibody in a given serum. Further differences were seen in the ability of a given PHIG to inhibit anti-MPO binding in different sera. F(ab')2 fragments from two PHIG preparations also inhibited in a concentration-dependent manner anti-MPO binding to MPO in all six sera in the concentration range 0.002-2.65 mg/ml, with a maximum inhibition of 42%. Little inhibition was seen with F(ab')2 of normal human IgG from individual donors (1.8-12.2% at the maximum concentration of 2 mg/ml). F(ab')2 fragments from three anti-MPO containing sera and two affinity-purified anti-MPO antibodies were eluted by affinity chromatography from Sepharose-bound PHIG F(ab')2 and showed anti-MPO antibody activity. We have shown that PHIG and F(ab')2 fragments of PHIG inhibit anti-MPO binding to MPO, and further that F(ab')2 fragments of PHIG bind to F(ab')2 fragments of anti-MPO antibodies. These observations indicate binding between the variable regions of PHIG and the antigen binding site of anti-MPO antibodies, and are consistent with an anti-idiotypic reaction. The variability seen in the inhibitory effect of the different PHIG preparations in anti-MPO-positive sera implies differences in their anti-idiotype content, while the variability of the inhibitory effect of a particular PHIG preparation between different sera suggests heterogeneity in the idiotypic repertoire of anti-MPO antibodies. Such variations in the inhibitory effect of different PHIG preparations on antibody binding may be an important determinant of their therapeutic effect.     

Human thyroid adenyl cyclase-stimulating activity in immunoglobulin G of patients with Graves' disease

We have studied the characteristics of the stimulation of adenyl cyclase (AC) activity in human thyroid plasma membranes by thyroid-stimulating hormone (TSH) and by immunoglobulin G (IgG) from the sera of patients with Graves' disease. AC activity was measured as adenosine 3',5'-cyclic monophosphate (cAMP) generated by membranes in a 10 minute incubation. IgG from two patients with Graves' disease possessed particularly potent human thyroid AC-stimulating activity; the dose-response curves with these IgGs were essentially parallel to those obtained with TSH. As little as 30 mug of the IgG of one patient with Graves' disease or 8 muU of TSH caused significant AC stimulation. A Lineweaver-Burk plot of the data suggested similarity in the site of action of both TSH and human thyroid adenyl cyclase stimulator (HTACS) in Graves' IgG. Submaximal doses of HTACS and TSH had additive effects on AC stimulation, but a large dose of a Graves' IgG with potent AC stimulating activity did not enhance AC stimulation by a maximal dose of TSH. The effect of HTACS on AC was slower in onset and longer in duration than an equipotent dose of TSH. HTACS was detectable in IgGs of 9 of 15 untreated hyperthyroid Graves' disease patients; its concentration, however, did not correlate significantly with tests of thyroid function, nor with long-acting thyroid stimulator (LATS) activity. In another 11 treated patients with Graves' disease, selected for the presence of LATS, HTACS and LATS were significantly correlated. We observed no inhibition of LATS activity in a Graves' IgG chosen for such testing because of its high titer of HTACS and no detectable LATS. However, an inhibitor of HTACS was detected in 2 of 4 IgGs; one of these two IgGs also inhibited AC stimulation by TSH. Conclusions: 1) Some Graves' disease IgGs contain a human thyroid AC stimulator (HTACS), probably different from LATS. 2) HTACS may act via a common pathway with TSH; it differs from TSH, however, in having a slower onset and a greater effect during more prolonged incubation with plasma membranes. 3) There is also an inhibitor of HTACS activity in some Graves' disease IgGs.     

Characterization of heparinase from an oral bacterium Prevotella heparinolytica

The idea that amphotericin B (AmB) may not require sterols to form ion selective channels has recently been criticized on the grounds that egg phospholipids commonly used in experiments may contain small amounts of sterol which associate with AmB to form AmB/sterol pore channel structures. It was recently shown in this laboratory that modest osmotic stress can enhance the formation of AmB channels in sterol-free egg phosphatidylcholine (eggPC) membranes. We have tested AmB's ability to form ion channels/defects in synthetic palmitoyl oleoyl (POPC), dieicosenyl (DEPC) and natural eggPC osmotically stressed large unilamellar vesicles (LUV) using pyranine fluorescence detected ion/H+ exchange. These sterol-free POPC LUV exhibit greatly increased sensitivity to cation selective AmB channel formation when osmotically stressed; even more than eggPC. Under these stressed conditions, AmB activity was observed at [AmB]/POPC ratios as low as 3.5x10(-4), corresponding to about 34 AmB molecules/vesicle. DEPC vesicles were almost completely unresponsive, demonstrating a strong bilayer thickness dependence. These results prove conclusively that AmB can form sterol-free channels and do so within therapeutic concentration ranges (>0.5-10x10(-6) M) in a stress-dependent manner. This phenomenon may allow us to use osmotic stress changes in simple model systems to spectroscopically isolate and characterize the thus-far elusive AmB channel forming aggregate. In addition, this stress dependence may be responsible for the potentiation of renal toxicity of AmB in the ascending branch of the loop of Henle which is under greatest osmotic stress.     

Molecular dynamics simulation of glycoprotein-glycans of immunoglobulin G and immunoglobulin M

In this paper, the authors introduce a workflow model. The development of computer network technology enables us to share the distributed data in real time. It is a considerable significance in the practical application of network capabilities not only to office work but also to the medical environment. In order to construct a well-connected, managed post (environment, scene), a model is needed to design the workflow. Here we propose a workflow model to cope with the scene of unforeseen events that we usually encounter in daily clinical activities. We give careful consideration to the ability of this model to manage dynamic changes within the workflow and describe its application to a medical scene (triage) and then carry out simulations based on this model. The authors are able to demonstrate the validity of this model through this simulation.     

Binding and utilization of human transferrin by Prevotella nigrescens

To survive and multiply within their hosts, pathogens must possess efficient iron-scavenging mechanisms. In the present study, we investigate the capacity of Prevotella nigrescens and Prevotella intermedia to use various sources of iron for growth and characterize the transferrin-binding activity of P. nigrescens. Iron-saturated human transferrin and lactoferrin, but not ferric chloride and the iron-free form of transferrin, could be used as sources of iron by P. nigrescens and P. intermedia. Neither siderophore activity nor ferric reductase activity could be detected in P. nigrescens and P. intermedia. However, both species showed transferrin-binding activity as well as the capacity to proteolytically cleave transferrin. To various extents, all strains of P. nigrescens and P. intermedia tested demonstrated transferrin-binding activity. The activity was heat and protease sensitive. The capacity of P. nigrescens to bind transferrin was decreased when cells were grown in the presence of hemin. Preincubation of bacterial cells with hemin, hemoglobin, lactoferrin, fibrinogen, immunoglobulin G, or laminin did not affect transferrin-binding activity. The transferrin-binding protein could be extracted from the cell surface of P. nigrescens by treatment with a zwitterionic detergent. Subjecting the cell surface extract to affinity chromatography on an agarose-transferrin column revealed that it contained a protein having an estimated molecular mass of 37 kDa and possessing transferrin-binding activity. The transferrin-binding activity of P. nigrescens and P. intermedia may permit the bacteria to obtain iron for survival and growth in periodontal pockets.     

Conformational analysis and hemolytic activity of immunoglobulin G fragment 285-292 and its analogs

Theoretical conformational analysis was carried out for the 285-292 fragment of human immunoglobulin G (His-Asn-Ala-Lys-Thr-Lys-Pro-Arg) and its analogues containing Arg, Glu, Gly, Lys, or Trp residue instead of the His residue in position 1. Spectropolarimetic investigation of these peptides showed the analogues to have different activities in the C1q-mediated erythrocytes hemolysis assay. Comparison of the low-energy structures sets of the compounds tested allowed to suggest a model of the "biological active" conformation for the peptide molecule in the course of the C1q complement component binding.     

Studies on pharmacological activation of human immunoglobulin G by chemical modification and active subfragments. X. Effect of carboxamidemethylated Fc fragment (CM-Fc) from human immunoglobulin G on delayed type hypersensitivity

The anti-allergic activity of the carboxyamidemethylated Fc fragment (CM-Fc) from human serum immunoglobulin G (IgG) was studied using sheep red blood cell-induced delayed type hypersensitivity in mice (SRBC-DTH). CM-Fc suppressed the DTH response when administered 30 min before, or 4 h after the SRBC challenge, but not when administered 8 h or more after the challenge. The Fc fragment showed no activity. CM-Fc administration 30 min before the challenge was unable to suppress the DTH response in the cyclophosphamide (CY)-treated mice. However, adoptive transfer of splenocytes from mice treated with CM-Fc to CY-pretreated mice caused suppression of the SRBC-DTH response. These results suggest that CM-Fc suppressed the DTH response by mediating the function of CY-susceptible cells.     

Low immunoglobulin G3 levels in wheezy children

This study aimed to investigate the prevalence of IgG subclass deficiency in wheezy children aged <3 y. Serum levels of IgA, IgE and IgG subclasses were measured in 310 children with recurrent wheezing and in 100 healthy controls. IgG3 levels were below the normal lower limit in 123 (39.6%) patients. This finding may reflect delayed maturation of the immune system, predisposing young children <3 y of age to wheezing.