Solubilisation partial characterisation of the alpha-MSH receptor on primary rat Schwann cells.

The ACTH/MSH melanocortin core peptide sequence possesses neurotrophic properties in peripheral nerve. During functional neuroanatomical recovery after damage to peripheral nerves, Schwann cells play a significant role in facilitating regeneration. Here we employ a modified super-potent alpha-MSH analogue to solubilise alpha-MSH receptor proteins from cultured primary rat Schwann cells. [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]-alpha-MSH photoaffinity labelled proteins from Schwann cells were analyzed by SDS-PAGE followed by autoradiography. The results indicate that the alpha-MSH receptor proteins labelled have a molecular weight of 42-45 kDa. These data are the first to demonstrate solubilisation and characterisation of alpha-MSH receptors from non-melanoma cells.

Melanocortin analogue Org2766 binds to rat Schwann cells, upregulates NGF low-affinity receptor p75, and releases neurotrophic activity.

Binding of the stable melanocortin(4-9) analogue, Org2766 [Met(O2)-Glu-His-Phe-D-Lys-Phe] to cultured rat sciatic nerve Schwann cells was demonstrated using a biotinylated derivative in semiquantitative histochemical and CELISA assays. Org2766 bound to Schwann cells, but not to fibroblasts, and was displaced maximally by unlabeled Org2766, alpha-MSH and ACTH(1-24). Displacement of Org2766 from the binding sites was considerably reduced by N- and C-truncation of the peptide. Specific binding of Org2766 was also demonstrated in the immortal rat Schwann cell line SCL4.1/F7 and was more pronounced in cells displaying a differentiated morphology. Org2766 and alpha-MSH increased cyclic AMP content of Schwann cells but neither stimulated DNA synthesis when applied alone. However, in the presence of a priming (subthreshold) concentration of the mitogen, cholera toxin, Org2766 and alpha-MSH caused a delayed increase in DNA synthesis. Org2766 did not modulate the expression of several differentiation-related Schwann cell markers. However, Org2766 increased immunoreactivity for p75 low-affinity NGF receptor on Schwann cells and evoked the release of neurotrophic factor(s) that synergized with NGF in stimulating neurite outgrowth in rat DRG neurons. The results indicate that Schwann cells are a primary target for the action of Org2766 and provide evidence for an indirect mechanism by which melanocortins might stimulate neurite sprouting in regenerating peripheral nerve axons.

Diploid and hyperdiploid rat Schwann cell strains displaying negative autoregulation of growth in vitro and myelin sheath-formation in vivo.

Neonatal rat Schwann cells were cultured for several months with intermittent exposure to the mitogen, cholera toxin, and infrequent passaging to avoid premature transformation. A cell line SCL4.1/F7 was derived following the cloning of one of these long-term cultures by limiting dilution in liquid medium to select for cells capable of continuous proliferation in the absence of mitogen. F7 cells have been passaged 40 times (80-120 generations) over 14 months. Two substrains were identified at passage 20, one of which ,s diploid and the other which has trisomy 7 (t7). The cell line displays a characteristic flattened or crescent-shaped morphology, substratum adhesion which is calcium-dependent in the millimolar range, and pronounced contact-inhibition of growth. Confluent or subconfluent cultures readily cease proliferation and change to a differentiated (stellate/bipolar) morphology through the mediation of an autocrine growth-inhibitory factor. F7 cells grafted into the site of a crush injury in adult rat sciatic nerves remained viable and myelinated host axons. F7 is the first clonally derived diploid immortal Schwann cell line to have been published and should provide a suitable tool for the study of the biochemical and cellular basis of sheath cell-neuron interactions, myelin stabilization in peripheral nerve and Schwann cell growth autoregulation.

Axonal cytoskeleton changes in experimental optic neuritis.

Axonal loss and degeneration in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE) have been suggested by brain imaging, pathological and axonal transport studies. Further elucidation of the processes and mechanisms of axonal degeneration in demyelinating diseases is therefore of potential importance in order to alleviate the permanent disabilities of MS patients. However, detailed studies in this area are impeded by the small number of reliable models in which the onset and location of demyelination can be well-controlled. In this study, microinjection of polyclonal rabbit anti-galactocerebroside (anti-Gal C) antibody and guinea pig complement was used to induce local demyelination in the rat optic nerve. We found that treatment with appropriate volumes of the antibody and complement could induce local demyelination with minimal pressure- or trauma-induced damage. Local changes in neurofilaments (NFs) and microtubules (MTs) were examined with both immunohistochemistry (IHC) and electron microscopy (EM). On day 1 after microinjection, we observed moderate NF and MT disassembly in the local demyelinated area, although in most cases, no apparent inflammatory cell infiltration was seen. The NF and MT changes became more apparent on days 3, 5, 7 after microinjection, along with gradually increased inflammatory cell infiltration. These results suggested that acute demyelination itself may induce local cytoskeleton changes in the demyelinated axons, and that the ensuing local inflammation may further enhance the axonal damage. When the lesions were stained with specific antibodies for T lymphocytes, macrophages, and astrocytes, we found that most of the cells were macrophages, suggesting that macrophages may play a greater role in inflammation-related axonal degeneration and axonal loss. These results were confirmed and further characterized on the ultrastructural level.

Identification and characterization of species of the family Bacteriodaceae by polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis was used to compare protein profiles of members of the family Bacteriodacceae. Cell-free extracts were prepared and the protein components separated by multistage polyacrylamide gel electrophoresis. The profiles were distinct and reproducible thus allowing identification of species, subspecies, and minor strain differences. Lyophilization of the cell-free extracts did not alter the major protein components. The results indicate that the techniques used may be useful in conjunction with conventional tests in identification at the species level.

Modulation of in vitro human lymphocyte responses by an exopolysaccharide from Capnocytophaga ochracea.

An exopolysaccharide (EP), purified from spent culture fluid previously inoculated with Capnocytophaga ochracea strain 25, suppressed in vitro human peripheral blood lymphocyte responses to the mitogen concanavalin A. EP was suppressive when added to cultures before the mitogen, but enhanced responses when added 24 or 48 hr after the mitogen.

HLA-D types and serum IgG responses to Capnocytophaga in diabetes and periodontitis.

Serum IgG responses to the cell envelope proteins (CEPs) from Capnocytophaga sputigena, Capnocytophaga ochracea, and Capnocytophaga gingivalis were examined in periodontally healthy and periodontitis subjects, both with and without type 1 diabetes (n = 60). Serum IgG responses to CEPs were determined by immunoblotting with biotin-goat anti-human IgG and an alkaline phosphatase-streptavidin system. Reactivity was analyzed by transmission densitometry, digitization, and computer manipulation. The patients with diabetes showed significantly (p < 0.01) fewer responses to 14 CEPs (from 81 to 10 kDa) from C. sputigena, 5 CEPs (from 90 to 17 kDa) from C. gingivalis, and the 27-kDa CEP from C. ochracea than in the non-diabetic group. The periodontitis patients showed significantly (p < 0.01) fewer responses to the 25- and 11-kDa CEPs from C. sputigena, the 125- and 17-kDa CEPs from C. gingivalis, and the 42-kDa CEP from C. ochracea than in the periodontally healthy group. HLA-DR4, HLA-DR53, and HLA-DQw3 were associated with periodontitis, while only HLA-DR4 was associated with diabetes (p < 0.02). Significant (p < 0.01) correlations were found between HLA-DR2 and IgG reactivity patterns associated with non-diabetics, and between HLA-DR4 and IgG reactivity patterns associated with diabetic and periodontitis subjects. These results indicate that both type 1 diabetics and periodontitis subjects have a depressed IgG antibody profile to Capnocytophaga, which may account for an increased susceptibility to periodontitis infection. Periodontitis in type 1 diabetes may be related more to the HLA-D type and altered immune function than to the diabetes itself.

Evaluation of four modalities of periodontal therapy. Mean probing depth, probing attachment level and recession changes.

Eighty-two periodontally involved patients were treated in a split mouth design such that one quadrant received coronal scaling (CS), root planing (RP), modified Widman surgery (MW), and flap with osseous resection surgery (FO). The therapy was performed in three phases: Phase I: the teeth previously designated to receive RP, MW, and FO were thoroughly root planed and the teeth designated to receive CS were scaled with no subgingival instrumentation, plaque control was initiated and reinforced for the entire mouth; Phase II: the designated teeth received MW or FO surgery; and Phase III: maintenance therapy every three months. The CS teeth received coronal scaling and polishing during maintenance appointments, while RP, MW, and FO teeth received supragingival instrumentation, subgingival instrumentation and polishing. Clinical measurements were taken initially, four weeks post-Phase I, 10 weeks post-Phase II, and after each of two years of maintenance care. All therapy modalities resulted in a decrease of mean probing depth with the FO producing the greatest decrease followed by MW, RP, and CS. The deeper the initial probing depth, the greater was the mean reduction of probing depth. FO created a loss of mean probing attachment in the 1 to 4 mm category. RP and MW produced the greatest gain of mean probing attachment in the 5 to 6 mm category. RP, MW, and FO produced similar gains in the greater than or equal to 7 mm category. FO created the most gingival recession followed by MW, RP, and CS.

Long-term evaluation of periodontal therapy: II. Incidence of sites breaking down.

Eighty-two patients were treated in a split mouth design with coronal scaling (CS), root planing (RP), modified Widman surgery (MW), and flap with osseous surgery (FO) which were randomly assigned to the various quadrants in the dentition. Following phase I and phase II therapy, the patients received supportive periodontal treatment (SPT) at 3-month intervals for up to 7 years. Clinical attachment level (CAL) was determined initially, post-phase I, post-phase II and prior to each SPT appointment. If a site lost > or = 3 mm of CAL from its baseline, it was classified as a breakdown site. Baselines were the initial exam for sites treated by CS and 10 weeks post-phase II for sites treated by RP, MW, and FO. Data were grouped by probing depth (PD) severity at the initial exam and at post-phase II. The breakdown for CS sites was assessed separately from RP, MW, and FO sites because of different baselines and retreatment protocols. Sites treated by CS had a higher incidence of breakdown than the other therapies through year 1 of SPT. The breakdown incidences/year for RP and MW sites were similar and greater than for FO sites in 1 to 4 mm and 5 to 6 mm PD categories. Breakdown incidence of RP sites was greater than MW sites which was greater than FO sites initially > or = 7 mm. Differences in incidence of breakdown between therapies after recategorizing data by post-phase II PD were the same as above, except no difference was present between RP and MW sites > or = 7 mm. Breakdown incidences were greater in increasing PD severities regardless of when they were categorized. There was no further loss of CAL one year after retreatment in 88% of sites. Patients with higher breakdown incidences tended to be smokers at the initial exam.

Long-term evaluation of periodontal therapy: I. Response to 4 therapeutic modalities.

Eighty-two periodontal patients were treated in a split mouth design with coronal scaling (CS), root planing (RP), modified Widman surgery (MW), and flap with osseous resection surgery (FO) which were randomly assigned to various quadrants in the dentition. Therapy was performed in 3 phases: non-surgical, surgical, and supportive periodontal treatment (SPT) < or = 7 years. Clinical data consisted of probing depth (PD), clinical attachment level (CAL), gingival recession (REC), bleeding on probing (BOP), suppuration (SUP), and supragingival plaque (PL). Because of the necessity to exit many CS treated sites due to breakdown, data for CS were reported only up to 2 years. All therapies produced mean PD reduction with FO > MW > RP > CS following the surgical phase for all probing depth severities. By the end of year 2 there were no differences between the therapies in the 1 to 4 mm sites. There were no differences in PD reduction between MW and RP treated sites by the end of year 3 in the 5 to 6 mm sites and by the end of year 5 in the > or = 7 mm sites. FO produced greater PD reduction in > or = 5 mm sites through year 7 of SPT. Following the surgical phase, FO produced a mean CAL loss and CS and RP produced a slight gain in 1-4 mm sites. RP and MW produced a greater gain of CAL than CS and FO following the surgical phase in 5 to 6 mm sites, but the magnitude of difference decreased during SPT. Similar CAL gains were produced by RP, MW, and FO in sites > or = 7 mm. These gains were greater than that produced by CS and were sustained during SPT. Recession was produced with FO > MW > RP > CS. This relationship was maintained throughout SPT. The prevalences of BOP, SUP, and PL were greatly reduced throughout the study and were comparable between sites treated by RP, MW, and FO while the CS sites had more BOP and SUP.

HLA-D and T lymphocyte reactivity to specific periodontal pathogens in type 1 diabetic periodontitis.

Bacterial antigen fragments complexed with class II major histocompatibility molecules (HLA-D) on antigen presenting cells (APCs) stimulate CD4+ T lymphocyte proliferation, presumably to protect the host. This study examined these responses to antigens of two periodontal pathogens in four groups (n = 15) of age- (young adult) and sex-matched Caucasian subjects with or without type 1 diabetes and moderate to severe periodontitis: Group DP = diabetics with periodontitis; Group DnP = diabetics without periodontitis; Group nDP = nondiabetics with periodontitis; and Group nDnP = nondiabetics without periodontitis. HLA-D phenotypes for each subject were determined by lymphocytotoxicity assays. T lymphocytes purified from peripheral blood were stimulated in cell culture with APC pulsed with various concentrations of tetanus toxoid, Porphyromonas gingivalis, and Capnocytophaga sputigena antigens. T lymphocyte reactivity (3H thymidine incorporation) was numerically lower in cultures from diabetics stimulated with unpulsed APC (not significant), and antigen-pulsed cultures showed low proliferation and no significant differences among groups. Stimulation indices in cultures from diabetic patients stimulated with P. gingivalis or C. sputigena, however, were significantly elevated at all antigen concentrations compared to nondiabetic cultures. The occurrence of HLA-DR4 was moderately associated with diabetes (P < 0.05) and highly associated with periodontitis (P < 0.001, log-linear model for categorical variables); and HLA-DR53 and HLA-DQ3 were significantly associated with periodontitis (P < or = 0.02). HLA-DR was crucial to lymphocyte stimulation (anti-HLA-DR blocking experiments), but the low peripheral blood T cell reactivity to antigens of periodontal pathogens could not be linked with HLA-D type or periodontitis susceptibility.(ABSTRACT TRUNCATED AT 250 WORDS)

Smokeless tobacco effects on monocyte secretion of PGE2 and IL-1 beta.

The use of smokeless tobacco (ST) products is associated with mucosal lesions, gingival recession, and attachment loss at the site of tobacco placement. Monocytes/macrophages are primary producers of PGE2 and IL-1 beta, inflammatory mediators which are thought to play a role in the destruction of the periodontium. The purpose of this study was to determine the effect of ST alone and in combination with a major stimulator of inflammation, bacterial lipopolysaccharide (LPS), on monocyte secretion of these mediators. Peripheral blood monocytes (PBM) were isolated by counterflow centrifugal elutriation from 15 healthy donors who were non-ST users. PBM were incubated for 24 hours in RPMI 1640 containing various concentrations of ST (0%, 0.005%, 0.01%, 1%) with or without 10 micrograms/ml LPS (Porphyromonas gingivalis LPS or Escherichia coli LPS). Of the ST preparations, only 1% ST resulted in PBM mediator secretion (7.7 +/- 2.0 ng/ml for PGE2 and 1.3 +/- 0.2 ng/ml for IL-1 beta) above that of control (unstimulated) cultures. Furthermore, the combination of 1% ST and LPS resulted in a potentiation of PGE2 release (5-fold for E. coli LPS + 1% ST and 10-fold for P. gingivalis LPS + 1% ST; P < 0.0001, one-way ANOVA) relative to the LPS preparations alone. In contrast, PBM IL-1 beta release decreased more than 2-fold upon E. coli LPS and 1% ST exposure, relative to treatment with E. coli LPS alone (P < 0.0001, one-way ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)

Gingival cell IL-2 and IL-4 in early-onset periodontitis.

The purpose of this study was to compare, using cell blot analysis, the association of gingival tissue mononuclear cells (GTMC) isolated from lesions displaying histories of early-onset periodontitis (EOP; typically B-lymphocyte dominated) and gingivitis (typically T-lymphocyte dominated) with the B-cell stimulating cytokine, interleukin (IL)-4, and the T-cell stimulating cytokine, IL-2. Eleven EOP patients and 11 age- and gender-similar gingivitis control (GC) subjects participated. Gingival tissue adjacent to the alveolar crest normally removed during surgery was digested in collagenase-containing media and GTMC were isolated by density gradient centrifugation. Cells were separated into four aliquots. One was left unstimulated; the remainder were stimulated for 2 hours with Porphyromonas gingivalis outer membrane protein, mitogen Concanavalin A, or common antigen tetanus toxoid. Cells then were centrifuged onto transfer membranes and incubated in RPMI 1640 media for 6 hours to allow absorption of secreted cytokine. Membranes were treated with monoclonal anti-IL-2 or anti-IL-4, followed by a biotin-conjugated second layer, streptavidin-alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-indolyl-phosphate (NBT/BCIP) color development. A higher percentage of GTMC from EOP patients were IL-2+ when stimulated with P. gingivalis compared with GTMC from GC patients (20 +/- 2% vs. 12 +/- 2%, P < 0.003). A higher percentage of non-stimulated GTMC from EOP patients produced IL-4 than from GC (22 +/- 4% vs. 6 +/- 3%, P < 0.00007), as well as when stimulated with P. gingivalis (22 +/- 3% vs. 13 +/- 2%, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

IgG antibody subclass response to Porphyromonas gingivalis outer membrane antigens in gingivitis and adult periodontitis.

Porphyromonas gingivalis is an important oral pathogen with a strong association with adult periodontitis. Significant titers of specific IgG antibodies to P. gingivalis can be found in the sera of both gingivitis and periodontitis patients. Since IgG subclasses have different biological characteristics, the present study dealt with the serum IgG subclass response to outer membrane antigens of P. gingivalis. Western blot analysis of P. gingivalis outer membrane was carried out using 20 adult periodontitis and 20 age- and sex-matched gingivitis patients. Antibodies in sera of both adult periodontitis and gingivitis patients recognized 38 antigen bands, ranging in molecular mass from 11.1 to 161 kDa. IgG2 was the predominant antibody subclass response in both patient groups in terms of the numbers of outer membrane antigens recognized, followed by IgG3, IgG1, and IgG4. More antigens in all IgG subclasses except IgG4 were recognized in adult periodontitis cases. Of the 23 antigens identified by IgG2 antibodies, 9 were recognized predominantly in adult periodontitis and 3 in the gingivitis group. In the IgG1 subclass, 4 antigens were recognized predominantly in the adult periodontitis group while only 1 antigen was recognized significantly more in the gingivitis group. The IgG3 response identified 14 antigens ranging in molecular mass from 11.1 to 61.2 kDa in both groups. Ten antigens were recognized significantly by the adult periodontitis group. The lowest response was seen by IgG4 antibodies, with only 3 antigens of molecular mass 61.2, 52.3, and 38.8 kDa recognized, the latter two significantly in the adult periodontitis group.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum antibody responses in human periodontitis to cellular components of Capnocytophaga.

Serum levels of IgM, IgG and IgG-antibody subclasses directed against cell envelopes, lipopolysaccharides and cytoplasmic fractions from Capnocytophaga sputigena, C. gingivalis and C. ochracea were examined in age-, race- and sex-matched periodontally healthy (n = 25) subjects and subjects with adult periodontitis (n = 25). The envelopes and cytoplasmic fractions were obtained by ballistic disintegration of the cells and ultracentrifugation. Cell envelopes were treated with DNase, RNase and lysozyme. Lipopolysaccharides were obtained by hot phenol-water extraction and treated with DNase and RNase. The relative levels of the antibodies in response to the cell fractions were measured by the streptavidinbiotin micro enzyme-linked immunosorbent assay. Both groups showed IgM and IgG antibodies to each fraction of the three Capnocytophaga species, but the frequency of positive IgG subclass responses varied. The IgG4 responses were lower than the other subclasses. There were no significant differences between the IgM antibody levels of the two groups. However, the adult periodontitis group had significantly lower IgG antibody titres to the cell envelopes and cytoplasmic fractions of C. gingivalis and C. ochracea, and lipopolysaccharide of C. gingivalis. These results were reflected in the depressed levels of IgG1 and/or IgG2 to these cellular fractions from the same bacterial species. The adult periodontitis group also showed a lower level of IgG1 to the cytoplasmic fractions of C. sputigena without any depression in the total IgG antibody level. There were no significant differences between the groups in IgG3 and IgG4 antibody levels to any of the cellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of Porphyromonas gingivalis outer membrane antigens.

Porphyromonas gingivalis is strongly associated with periodontal disease. Significant titres of specific IgG antibodies to P. gingivalis can be found in healthy individuals and those with periodontitis. In this study, 22 outer membrane antigens ranging from 15.5 to 107.6 kDa were recognized by sera from persons with periodontitis and controls. Serum from individuals with periodontitis showed a significantly higher IgG response to a 31.4-kDa antigen (p < 0.05); serum from those with gingivitis demonstrated a significantly higher response to a 15.5-kDa antigen (p < 0.05). The response to the 15.5-kDa antigen might represent a protective immune response while that to the 31.4-kDa could serve as a marker for disease susceptibility. These two antigens were purified to homogeneity and their N-terminal amino acid sequences determined. The sequences did not correspond to any previously described P. gingivalis antigens. The role of these two antigens in the pathogenesis of periodontal disease remains to be determined.