The role of complement in immunological demyelination of the mammalian spinal cord.

STUDY DESIGN: Specificity of serum complement component to elicit immunological demyelination. OBJECTIVES: To assess the role of complement components and pathways in experimental immunological demyelination of the adult rat spinal cord. SETTING: ICORD, University of British Columbia, Vancouver, Canada. SUBJECTS: We used 32 adult male Sprague-Dawley rats, of approximately 220 g weight. METHODS: Rats received intraspinal infusions of demyelinating reagents, delivered by osmotic minipump, for a 7-day infusion at 0.5 microl/h. Reagents consisted of a polyclonal antibody to galactocerebroside and human serum complement. Complement sera deficient for a single component were used to assess the role of the alternative pathway, the classical pathway, and the membrane attack complex. Demyelination was assessed, at 7 days, ultrastructurally. RESULTS: Removal of C3 protein, common to classical and alternative complement pathways, or C4 protein, a classical pathway protein, resulted in no demyelination. However, complement deficient in Factor B, an alternative pathway protein, produced effective demyelination. Upon removal of C5 or C6, membrane attack complex proteins, demyelination was also observed. CONCLUSION: This suggests that the classical pathway is sufficient for the protocol to demyelinate the adult rat spinal cord, and that the membrane attack complex is also not required.

Adenine toxicity and transport in the moderately halophilic eubacterium Halomonas elongata.

The presence of adenine in the L-alanine defined medium substantially inhibited the growth of the moderately halophilic eubacterium Halomonas elongata. Extensive attempts to reverse the adenine toxicity for growth were made using a variety of purine and pyrimidine compounds, vitamins, and amino acids. Of the compounds tested, only cytosine was found to reverse the adenine growth inhibition. This indicates a mechanism similar to that found for some strains of Escherichia coli in which the presence of exogenous purines (e.g. adenine) was found to stop purine de novo synthesis and repress the synthesis of the pyrimidine salvage enzyme cytosine deaminase. H. elongata was found to possess an active adenine uptake system that was sodium dependent with only lithium having a considerable capacity to replace the sodium. A competition study indicated that the adenine transport system was quite specific. This paper represents the initial study of purine and pyrimidine salvage pathways and adenine uptake for the moderately halophilic eubacteria.

Immunological myelin disruption does not alter expression of regeneration-associated genes in intact or axotomized rubrospinal neurons.

The inability of axotomized neurons to regenerate within the CNS has been partially attributed to a number of inhibitory factors associated with CNS myelin that are extrinsic to the severed neurons. However, some neurons are capable of limited regeneration after injury and this ability has been shown to correlate with the expression of certain regeneration-associated genes (RAGs) intrinsic to injured neurons. It has therefore been postulated that neutralization of inhibitory factors, as well as the induction of an appropriate neuronal cell body response, would facilitate improved regrowth of injured CNS axons. In previous studies we have shown that immunological removal of myelin from the spinal cord facilitates axonal regeneration by rubrospinal neurons, as indicated by retrograde transport of a fluorescent dye placed distal to the site of injury. Here, we investigated whether the immunological focal removal of spinal cord myelin, following a thoracic spinal cord injury, concomitantly stimulated an increase in the expression of RAGs in rubrospinal neurons. In situ hybridization for Talpha-1 tubulin and GAP-43 at days 7, 14, and 21 revealed no significant increase in gene expression in rubrospinal neurons following immunological demyelination. The ability of various neuronal populations to sprout or slowly regrow without expressing the previously characterized cell body response is reviewed. We conclude that the recently demonstrated regeneration of rubrospinal tract, after immunologically directed spinal cord demyelination, is the result of either axonal sprouting or slow axonal regrowth without the increased expression of RAGs characteristic for fast axon regeneration.

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.

Nicotine and smokeless tobacco effects on gingival and peripheral blood mononuclear cells.

The pathogenesis of tobacco-related periodontal diseases is not well understood. The purpose of this study was therefore to investigate smokeless tobacco extract (ST) and nicotine effects on prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) secretion by peripheral blood mononuclear cells (PBMC, consisting of monocytes and lymphocytes) and gingival mononuclear cells (GMC). Both peripheral blood and gingival tissue adjacent to the alveolar crest were taken from non-smoking adult periodontitis patients. Gingival tissue was treated with collagenase and deoxyribonuclease and GMC and PBMC were isolated by Ficoll-Hypaque centrifugation. GMC and PBMC (100,000 cells/200 microl) were cultured for 24 hours in supplemented RPMI 1640 alone (control), or in supplemented RPMI 1640 containing 1% ST, 100 microg/ml nicotine, 1 microg/ml Porphyromonas gingivalis LPS, or 1 microg/ml P. gingivalis LPS and either 100 microg/ml nicotine or 1% ST. Enzyme immunoassays were used to quantify PGE2 and IL-1beta. Treatments were compared by repeated measures ANOVA. 100 microg/ml nicotine (7-fold, p<0.02) and 1% ST (3.5-fold, p<0.004) significantly increased secretion of PGE2 by PBMC relative to control cultures. 100 microg/ml nicotine and 1% ST, however, had no effect on IL-1beta secretion by PBMC. Enhanced PGE2 secretion also was seen when PBMC were treated with P. gingivalis LPS+ 100 microg/ml nicotine relative to P. gingivalis LPS alone (p<0.007). In contrast, 100 microg/ml nicotine significantly downregulated IL-1beta secretion by GMC relative to medium alone (p<0.008) and had no effect on PGE2 secretion by GMC. These data indicate that while nicotine and ST can stimulate PBMC to secrete PGE2, they cannot activate further mononuclear cells extracted from gingiva, possibly due to maximal previous stimulation in the periodontitis lesion.

Regeneration of brainstem-spinal axons after lesion and immunological disruption of myelin in adult rat.

We previously observed that the transient developmental suppression of myelination or disruption of mature myelin, by local intraspinal infusion of serum complement proteins along with a complement-fixing, myelin-specific antibody (e.g., anti-Galactocerebroside), facilitated avian brainstem-spinal axonal regeneration after spinal transection. We now report the effects of similar immunological protocols on axonal regeneration in the injured adult rat spinal cord. After a lateral hemisection injury of the T10 spinal cord, infusion of the above reagents, over 14 days at T11, facilitated the regeneration of some brainstem-spinal axons. The hemisection lesion enabled comparisons between the retrograde labeling within an injured brainstem-spinal nucleus and the uninjured contralateral homologue. The brainstem-spinal nucleus examined in detail was the red nucleus (RN), chosen for its relatively compact descending pathway within the dorsolateral cord. Comparing the number of labeled neurons within each RN, of an experimentally myelin suppressed animal, indicated that approximately 32% of injured rubrospinal projections had regenerated into the caudal lumbar cord. In contrast, control-treated animals (e.g., PBS vehicle alone, GalC antibody alone, or serum complement alone) showed little or no axonal regeneration. We also examined the ultrastructural appearance of the treated cords. We noted demyelination over 1-2 segments surrounding the infusion site (T11) and a further two segments of myelin disruption (delamination) on either side of the demyelinated zone. The demyelination is an active process (< 3 days) with microglia and/or macrophages engulfing myelin. Thus, the facilitation of axonal regeneration through the transient suppression of CNS myelin may be fundamental to all higher vertebrates.

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.

Experimental gingivitis in periodontitis-susceptible subjects.

The purpose of this study was to evaluate clinical, microbiological, and gingival crevicular fluid (GCF) profiles in periodontitis-resistant and periodontitis-susceptible subjects during 4 weeks of experimental gingivitis. Experimental groups of similar ages were defined as gingivitis controls (GC; n = 10) and history of rapidly progressive periodontitis (RPP; n = 10), respectively. Prior to baseline, all subjects achieved good plaque control (plaque index (P1I) approximately 0) and gingival health (gingival index (GI) = 0), and had probing depths < or = 4 mm on experimental teeth. For 4 weeks after baseline, oral hygiene around maxillary 2nd premolar and 1st molar teeth was inhibited by a plaque guard. The plaque guard was removed weekly for GCF sampling to determine interleukin (IL)-1 beta and prostaglandin (PG)E2 amounts by ELISAs. In addition, P1I, GI, probing depth, and gingival recession measurements were made. Subgingival plaque darkfield microscopy and DNA probe analysis also were performed. Results indicated that clinical signs of inflammation, microbiological patterns and GCF profiles progressed similarly in both groups. However, plaque accumulated more rapidly in the susceptible subjects. P1I in RPP at 4 weeks was 2.1 +/- 0.1 compared to 1.5 +/- 0.2 in GC, with an incidence of P1I > of 100% versus 50%, respectively (logistic regression; p < 0.0001). Hence, the clinical, microbiological and host factors selected for this study were unrelated to previous susceptibility to periodontitis when evaluated in the experimental gingivitis model. However, the increased rate of plaque accumulation, following thorough plaque removal, in RPP patients suggests a potential factor in disease recurrence in these susceptible subjects.

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.

Nicotine effects on PGE2 and IL-1 beta release by LPS-treated human monocytes.

Cigarette smoking is a major risk factor in the development and further progression of periodontitis. However, little is known regarding the pathogenesis of smoking-related periodontal diseases. The purpose of this study was to examine the effects of nicotine, alone and in combination with lipopolysaccharide (LPS), on monocyte secretion of bone-resorbing factors, PGE2 and IL-1 beta. Peripheral blood monocytes (PBM) were isolated by counterflow centrifugal elutriation from 15 healthy, non-smoking donors. PBM were incubated for 24 h in RPMI 1640 containing nicotine (0, 50 ng/ml, 1 microgram/ml, 10 micrograms/ml and 100 micrograms/ml) with or without 10 micrograms/ml Porphyromonas gingivalis LPS or Escherichia coli LPS. Culture supernatants were assayed for PGE2 and IL-1 beta by ELISA. None of the nicotine preparations resulted in significant PBM secretion of PGE2 and IL-1 beta above that of unstimulated cultures. However, PGE2 release was potentiated 1.7-fold by the combination of P. gingivalis LPS and 10 micrograms/ml nicotine relative to P. gingivalis LPS alone (p < 0.05, one-way ANOVA). Prostaglandin E2 release also was potentiated 3.5-fold by P. gingivalis LPS and 100 micrograms/ml nicotine relative to P. gingivalis LPS alone (p < 0.00001, one-way ANOVA) and 3.1-fold by E. coli LPS and 100 micrograms/ml nicotine relative to E. coli LPS alone (p < 0.00001, one-way ANOVA). IL-1 beta secretion was lower for either LPS plus 100 micrograms/ml nicotine relative to LPS alone, although not significantly. These data demonstrate upregulation of LPS-mediated monocyte secretion of PGE2 by nicotine and suggest a potential role for nicotine in periodontal disease pathogenesis.

Response to intracrevicular controlled delivery of 25% tetracycline from poly(lactide/glycolide) film strips in SPT patients.

Controlled local delivery of antibiotics has been shown to reduce periodontopathic micro-organisms with minimal side-effects. Clinical studies in our laboratory have shown that 25% tetracycline HCl delivered from poly(D,L-lactide/glycolide) film strips (25 TTC-PLGA) released therapeutic concentrations of tetracycline for 10 days. The present pilot study compared the intracrevicular delivery of 25% tetracycline HCl incorporated in these biodegradable film strips to scaling and root planing (SRP) in 10 adult periodontitis patients, who in spite of therapy and regular supportive periodontal treatment (SPT), continued to possess 5 bleeding periodontal pockets at least 5 mm deep. Sites were randomly selected to receive the following treatments: (1) 25 TTC-PLGA, (2) control strips without TTC (PLGA), (3) SRP, and (4) untreated control. Film-strip retention was augmented with a suture/cement technique, followed by strip removal after 2 weeks. Clinical parameters and subgingival bacterial morphotypes (darkfield analysis) were evaluated over time (0, 2.4, 8, 12, 26 weeks). Results indicated that, compared to baseline, 25 TTC-PLGA film strips caused significant (p < or = 0.01): (1) probing depth reduction for 26 weeks, (2) a clinical attachment level gain for 12 weeks, (3) lower %s of spirochetes for 4 weeks and motile rods for 8 weeks (p < or = 0.05), and (4) an accompanying increase in cocci for 4 weeks. In the scaled and root planed sites, probing depth was the only finding that demonstrated a significant change from baseline (p < or = 0.01). Controls and PLGA showed isolated reductions in probing depth and % of motile organisms. From these findings, applications of intracrevicular 25 TTC-PLGA, when compared to scaling and root planing, appears to have an enhanced antibacterial effect and a similar clinical effect in SPT patients. The results of this study indicate further investigation of 25 TTC-PLGA film strips should be undertaken using more subjects and sophisticated microbiological and clinical measurements.

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.

Axonal regeneration and physiological activity following transection and immunological disruption of myelin within the hatchling chick spinal cord.

Transections of the chicken spinal cord after the developmental onset of myelination at embryonic day (E) 13 results in little or no functional regeneration. However, intraspinal injection of serum complement proteins with complement-binding GalC or 04 antibodies between E9-E12 results in a delay of the onset of myelination until E17. A subsequent transection of the spinal cord as late as E15 (i.e., during the normal restrictive period for repair) results in neuroanatomical regeneration and functional recovery. Utilizing a similar immunological protocol, we evoked a transient alteration of myelin structure in the posthatching (P) chicken spinal cord, characterized by widespread "unravelling" of myelin sheaths and a loss of MBP immunoreactivity (myelin disruption). Myelin repair began within 7 d of cessation of the myelin disruption protocol. Long term disruption of thoracic spinal cord myelin was initiated after a P2-P10 thoracic transection and maintained for > 14 d by intra-spinal infusion of serum complement proteins plus complement-binding GalC or 04 antibodies. Fourteen to 28 d later, retrograde tract tracing experiments, including double-labeling protocols, indicated that approximately 6-19% of the brainstem-spinal projections had regenerated across the transection site to lumbar levels. Even though voluntary locomotion was not observed after recovery, focal electrical stimulation of identified brainstem locomotor regions evoked peripheral nerve activity in paralyzed preparations, as well as leg muscle activity patterns typical of stepping in unparalyzed animals. This indicated that a transient alteration of myelin structure in the injured adult avian spinal cord facilitated brainstem-spinal axonal regrowth resulting in functional synaptogenesis with target neurons.

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)

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.

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)

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.