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Background and Objective: There have been few studies of gender differences in response to periodontitis. Thus, we compared gender-specific differences in systemic cytokine concentrations in rats with and without ligature-induced periodontitis., Material and Methods: Experimental periodontal disease was initiated in Sprague-Dawley rats by placing a ligature around the crowns of the second right maxillary molar tooth. Sham-operated control groups were also created. Two weeks later, the right and left maxillary quadrants of teeth, liver and serum were collected from all the rats, and uterine horns were collected from the female rats. Liver and uterine samples were ground in phosphate-buffered saline (10 mg of tissue/mL of phosphate-buffered saline + protease inhibitor) containing a protease inhibitor, and cytokine concentrations were determined by enzyme-linked immunosorbent assay. Digital radiographs were made of maxillary quadrants, and the distance from cemento-enamel junction to alveolar crest was measured using image analysis software. Data were compared by factorial analysis of variance and a post-hoc Tukey test., Results: Female rats with ligatures had greater, but not significantly different, alveolar bone loss than males with ligatures. However, they had higher serum concentrations of interleukin-6, tumor necrosis factor-alpha and C-reactive protein, and liver C-reactive protein (p < 0.05). These females also had higher interleukin-6, tumor necrosis factor-alpha and vascular endothelial growth factor concentrations within the uterine horn, compared to female controls (p < 0.05). Male animals with ligatures had lower serum concentrations of C-reactive protein and higher interleukin-6 and tumor necrosis factor-alpha concentrations within serum, compared to male controls (p < 0.05)., Conclusion: Our study suggests that females with periodontal disease have a greater risk for inflammatory-based systemic diseases than males.

Objective: The objective of this study was to determine the effects of induced periapical abscesses on pregnant rats., Design: In 1/2 of the animals (n=16), the pulps of the maxillary right molars were exposed to the oral environment, which resulted in a periapical abscess. The other 1/2 (n=16) were sham-operated. 1/2 of the animals of both groups became pregnant 2 weeks later. The pregnancy duration, and weight and number of pups were assessed at delivery. Serum, liver and uterine horn samples were taken from all animals at euthanasia and serum IL-6, endothelin-1, TNF-alpha, IL-10, cortisol and insulin were determined by ELISA. Liver concentrations of IL-6, CRP and IL-6 and uterine horn concentrations of IL-6, vascular endothelial growth factor (VEGF), TNF-alpha, IL-10 and IL-1-beta were assessed by ELISA. Blood glucose concentrations were determined using a glucometer. Outcome variables were compared by factorial ANOVA, a post hoc Tukey test, and Pearson's correlation test., Results: Pregnant rats with periapical abscesses had a significantly longer pregnancy and delivered pups with a significantly higher birthweight (p<0.05). They had significantly higher concentrations of IL-6, VEGF, IL-1-beta, and IL-10 within the uterine horn and IL-6, CRP and TNF-alpha within the liver (p<0.01). Blood glucose and serum TNF-alpha, IL-6, endothelin-1, IL-10, and insulin concentrations were significantly higher in the pregnant animals with pulpal abscesses (p<0.01)., Conclusion: The significant increase in serum TNF-alpha, taken together with significant increases in blood glucose and serum insulin concentrations, suggest that animals with induced periapical abscesses developed insulin resistance, which significantly affected their pregnancy outcomes.

Most pulpal afferent neurons have cytochemical features in common with the class of nociceptors that express neuropeptides and respond to NGF, while very few bind the plant lectin IB4, a widely used marker for the class of nociceptors that respond to the GDNF family of neurotrophic factors. The present study was undertaken to determine whether the GDNF receptor, GFRalpha-1, is expressed by pulpal afferents, and, further, to determine whether tooth injury evokes changes in expression of the GDNF and NGF receptors among pulpal afferents. The tracer, fluoro-gold (FG), was applied to shallow cavities in dentin of first and second maxillary molars. After 4 weeks, the molars of one side received a test injury consisting of a deeper cavity that exposed pulp horns. Animals were perfusion fixed 2 days later, and sections of the trigeminal ganglia were double immunostained with combinations of antibodies against GFRalpha-1, and TrkA. Under control conditions, GFRalpha-1 immunostaining was observed in 72% of neurons that projected to the molar pulp, TrkA in 78%, and immunostaining for both receptors was observed in 65% of pulpal afferents. Tooth injury evoked up-regulation of GFRalpha-1 expression (to 93%) and a slight down-regulation of TrkA expression (67%) among tooth afferents, while there was no discernable change in the proportion of pulpal afferents that expressed both TrkA and GFRalpha-1 (to 61%).

TcR alpha, beta, and gamma chain negative cytotoxic NK-like cells were cloned from alloantigen-stimulated PBL obtained from nai;ve channel catfish. Stimulation with allogeneic cells and growth promoting factors are required for their continued in vitro proliferation and cytotoxic activity. These granular cells kill not only the stimulating allogeneic cells, but also unrelated allogeneic targets by a perforin/granzyme-mediated apoptosis pathway. In addition, they are negative for markers that define neutrophils, monocytes/macrophages, and non-specific cytotoxic cells. Although these NK-like clones kill a number of different allogeneic targets, they display interclonal variation in cytotoxicity toward a panel of allogeneic targets, i.e. some clones have no apparent target specificity, while others display a target preference. In addition, flow cytometric analyses revealed that expression of a putative FcmuR, an LFA-1-like molecule, and a putative thymocyte/T cell antigen varies among the different clones, with no clear correlation between surface antigen expression and cytotoxic activity. Although not all clones express a putative FcmuR, it was noted that they all expressed an ITAM containing FcepsilonR gamma chain homolog. This finding suggests that the catfish FcepsilonR gamma chain may potentially be used as an accessory molecule for not only FcmuRs, but also for other unknown activation receptors. These results support the hypothesis that catfish NK-like cells are heterogeneous in terms of target specificities and cell surface phenotype.

The dental pulp is a favorable model for studies of interactions between nociceptive sensory neurons and their peripheral target tissues. In the present study, we retrogradely labeled pulpal afferent neurons with an improved method that permits monitoring of changes in neuronal phenotype in response to controlled tooth injuries. The capacity of retrograde neuronal tracers to diffuse through dentinal tubules was exploited, thereby avoiding the severe injury to the pulp associated with previous tracer application methods. The strategy was to apply the durable fluorescent tracer, Fluoro-gold (FG), to exposed dentin in the floor of shallow cavities in molars, in order to pre-label pulpal neurons in trigeminal ganglia of young adult Sprague-Dawley rats. A high percentage of pupal afferent neurons were retrogradely labeled by application of FG to exposed dentin and the FG fluorescent signal persisted in most labeled neurons for at least 8 weeks. Following tracer application to dentin, the pulp tissue appeared normal histologically, with the exception that a layer of reactive dentin was deposited at the pulp-dentin border beneath the shallow cavities. Assessment of expression of calcitonin gene-related peptide (CGRP) and brain derived neurotrophic factor (BDNF) indicated that pulpal neurons remained in a quiescent, baseline condition cytochemically following application of tracer to cavities in dentin and upregulation of these markers could be detected in neurons that projected to teeth that received a test injury subsequent to tracer application. Thus, labeling of trigeminal neurons via dentinal tubules provides the basis for a useful model for precisely assessing properties of pulpal afferents in both quiescent and activated states.

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.

The dental pulp is richly innervated by peptidergic nociceptive neurones that are of special interest because of their central role in dental pain and because they have some features that are not typical of other somatic nociceptors. Here, (35)S-riboprobes were used to determine whether pulpal afferents of adult (2-month-old) rats express the nerve growth-factor (NGF) receptors, p75(NTR) and trkA, which are characteristic of peptidergic nociceptors, and additionally, whether these cells express receptors (trkB and trkC) for other members of the neurotrophin family. In order to begin characterizing the postnatal role of NGF in regulating these neurones, the susceptibility of pulpal afferents to antiserum-mediated early postnatal NGF depletion spanning the period of pulpal innervation development was also examined. In control animals, about 200 trigeminal ganglion cells were labelled after application of the retrograde tracer Fluoro-gold to the first maxillary molar. Among the labelled cells, 79% had positive hybridization signals for p75(NTR), 72% for trkA, 34% for trkB, 1% for trkC, and 77% for BDNF. Neonatal NGF depletion reduced the number of retrogradely labelled pulpal afferents by 33%, with numbers of smaller neurones being most strikingly subnormal. This reduction could be attributed to a partial depletion of the neurone population that expressed p75(NTR) and trkA. Consistent with reports that NGF-responsive neurones also express BDNF, NGF deprivation resulted in a reduction in the number of pulpal afferents that expressed BDNF to an extent similar to that seen for trkA. In contrast, anti-NGF exposure had little effect on the number of pulpal afferents that expressed trkB. These findings indicate that most pulpal afferents in the adult express the NGF receptors p75(NTR) and trkA, and thus have a continuing potential susceptibility to NGF-mediated regulation of functions such as neuropeptide and BDNF synthesis. However, only a subpopulation of this group of neurones requires NGF in order to develop connections to the pulp during the neonatal period. Few, if any, pulpal afferents express the high-affinity neurotrophin-3 (NT3) receptor trkC, although many have large cell bodies typical of NT3-responsive sensory neurones. A small subpopulation of pulpal afferents seems to express no neurotrophin receptors, yet it is unlikely that these cells belong to the class of small sensory cells known to bind isolectin IB4.

The rodent dentition has become an important model for investigations of interactions between dental tissues and peripheral neurons. Although experimental nerve injury has been widely used for such studies, there is uncertainty about the courses of nerve fibers supplying the mandibular teeth. In order to clarify this, we used a mixture of monoclonal antibodies against neurofilament proteins to enhance demonstration of nerve fibers so that small nerves could be readily traced in serial frozen sections of mandibles of Sprague Dawley rats ranging in age from embryonic day (E) 18 to postnatal day (P) 90. The 1st molar and anterior portion of the 2nd molar were innervated by small nerves that emerged as distinct branches of the IAN trunk at or near the mandibular foramen. In contrast, the nerve supply to the 3rd molar and posterior part of the 2nd molar was a branch of the lingual nerve that bypassed the mandibular canal altogether. The IAN trunk split into the mental nerve and a large branch to the incisor about 2 mm anterior to the mandibular foramen. Thick branches of the incisor nerve descended into the incisor socket to form a dense plexus of nerve fiber bundles extending along the length of the incisor periodontium. The sparse pulpal innervation of the incisor was provided by a few thin fascicles that emerged from the caudal portion of the periodontal plexus to enter the incisor apex. The dental branches of the IAN and lingual nerve seen in the adult were well established and readily identifiable at age E18 even though their targets were limited to the follicles of the developing teeth. These studies show that the trigeminal branches that supply the mandibular teeth can be identified at a wide range of ages as distinct nerves at a considerable distance proximal to their targets. This detailed information on the courses taken by the dental nerves can provide an anatomical basis for increased precision in characterization and perturbation of neural pathways from the molars and incisor., (Copyright 1999 Wiley-Liss, Inc.)

This study examines cytotoxic mechanisms used by channel catfish peripheral blood-derived effector cells. Transmission electron microscopy (TEM), coupled with [(3)H]thymidine DNA fragmentation (JAM) and terminal deoxynucleotidyl nick-end labeling (TUNEL) assays, provided the first evidence that catfish peripheral blood cytotoxic effectors killed allogeneic targets via an apoptotic pathway. TEM demonstrated that the effector cell population present within peripheral blood leukocytes (PBLs) was composed of agranular lymphocytes that formed conjugates with, and induced apoptosis in, allogeneic target cells. Both JAM and TUNEL assays showed that PBLs induced target cell DNA fragmentation within 1 h of coculture. In addition, fixed effectors did not induce target cell necrosis or apoptosis, and target cell lysis was completely inhibited by chelation of free Ca(2+) by EGTA. These results suggest that catfish peripheral blood-derived effector cells utilize a secretory mechanism rather than a ligand-based mechanism to trigger apoptosis., (Copyright 1999 Academic Press.)

Tissue responses to injury are regulated by neurotrophins and neurotrophin receptor levels and can involve both retrograde and paracrine/autocrine trophic signaling. To determine how neurotrophins may contribute to the injury response, the timing and the extent of the up-regulation of neurotrophins and their receptors was examined in a model system which is particularly well suited for the analysis of trophic signaling pathways in response to injury. Injury to the occlusal surfaces of rat molar cusps induces a localized increase in nerve growth factor (NGF) expression in the dental pulp within 4-6 h. Radiolabeled NGF was transported in a receptor-mediated fashion from the teeth to a subset of neurons in the trigeminal ganglion within 15 h, indicating that these neurons possess NGF receptors (trk A and/or p75NTR). To test for NGF responses in the tooth sensory afferent neurons, levels of expression of neurotrophins and their receptors were examined by in situ hybridization in the trigeminal ganglion at 0, 4, 12, 20, 28 and 52 h post-injury. Within the maxillary division of the trigeminal ganglion, trk A expression was elevated at 4 h post-injury, with a maximum increase (2-fold) after 52 h. p75NTR was increased by 28 h post-injury and was increased 1.35-fold by 52 h. BDNF mRNA was increased 12 h after injury (1.8-fold), and 2.5-3-fold at 52 h post-injury. The trk B expression was increased only late after injury (28 and 52 h). To determine the receptor/neurotrophin phenotype of trigeminal neurons with projections to the molar teeth, these neurons were double-labeled with the retrograde tracer fluoro-gold and probes for either BDNF or trk B. The results show that tooth-innervating trigeminal neurons express BDNF, but not trk B. The timing of mRNA expression after injury and the phenotype of identified trigeminal neurons suggests a complex signaling cascade in which NGF at the injury site regulates NGF receptor expression at the levels of the cell body as well as increases in BDNF expression. Upregulated BDNF may act in a paracrine fashion on neighboring trigeminal cells expressing trk B. This signaling cascade may be a common feature of the response to mild peripheral inflammatory injuries within nociceptive pathways., (Copyright 1998 Elsevier Science B.V.)

The first aim of the present study was to determine whether depletion of endogenous nerve growth factor (NGF) during early postnatal development results in a long-term deficit in the number of trigeminal ganglion cells and axons projecting to the molar pulp. The second aim was to identify selectivity of the effects of NGF deprivation for any specific size group among pulp neurones. Newborn Sprague-Dawley rats were given subcutaneous injections of either rabbit anti-mouse-NGF serum or non-immune (control) rabbit serum for a period of 1 month. At age 4 months, Fluoro-gold (FG) was applied to the pulp chamber of the right maxillary first molar. One week later the animals were perfusion-fixed, and the trigeminal ganglia were removed and serially sectioned with a cryostat. Labelled neurones were seen only in the trigeminal ganglia ipsilateral to the injected teeth. The area of every labelled cell profile was measured, and from these data, estimates of the true number and size distribution of FG-labelled cells were obtained by recursive translation. Ganglia of control animals had a mean of 197 labelled neurones, all in the maxillary division, and most of the somas were of medium or large diameter. NGF-deprived animals had significantly fewer (mean = 145) FG-labelled cells in the trigeminal ganglion ipsilateral to the injected tooth. Neurones with somas of less than 30 microns dia were most strikingly subnormal in anti-NGF treated animals (64% of controls). In accordance with the greater susceptibility of small neurones to anti-NGF exposure, deficits in apical nerve fibres of the mandibular first molar were greater in degree and duration for unmyelinated axons than for myelinated axons. It is concluded that NGF is an important mediator in regulation of postnatal development of the sensory innervation of the dental pulp. The results also indicate that postnatal development of at least one class of larger pulpal afferent neurones is regulated by factors other than NGF.

The aims were to demonstrate sympathetic ganglion neurones projecting to the rat molar pulp and to determine whether deprivation of nerve growth factor (NGF) in neonatal rats eliminates this source of pulpal innervation. Newborn Sprague-Dawley rats were given subcutaneous injections of rabbit anti-mouse-NGF serum for 1 month. Control animals included litter mates treated with preimmune serum and untreated, age-matched rats. AT 4 months of age, Fluoro-gold (FG) was applied to the pulp chamber of the right first maxillary molar. One week later, the animals were perfusion fixed, and the superior cervical ganglia (SCG) were removed, embedded in paraffin, and serially sectioned at 10 microns. FG-labelled cells were detected by epifluorescence microscopy with a u.v. filter set. Control animals had 5-10 FG-labelled neurones widely distributed throughout the SCG ipsilateral to the injection site and no labelled cells in the contralateral SCG. NGF-deprived animals had either no FG-labelled cells or a single labelled cell in the ipsilateral SCG. These results indicate that, in rats, (1) the number of SCG neurones projecting to the molar pulp is rather low, (2) SCG neurones that innervate the dental pulp of the maxillary molar pulp are dispersed throughout the ganglion, (3) the projection from SCG to the molar is exclusively ipsilateral, and (4) neonatal NGF deprivation induces a permanent, almost total, loss of sympathetic neurones projecting to the dental pulp.

The present study was undertaken to test the hypothesis that development of the pulpal innervation is dependent on nerve growth factor (NGF). Newborn rats were given subcutaneous injections of a rabbit anti-mouse NGF serum on alternate days for the first 24 days postnatally. Control animals were untreated and normal rabbit serum-treated litter mates. The animals were deeply anesthetized on postnatal day 26, perfused with fixative and the first mandibular molars were processed for transmission electron microscopy to obtain a complete census of axons entering the four roots. The composition of the mental nerve was also examined. Compared to control animals, the apical innervation of molars from anti-NGF-treated rats had only 62% as many myelinated fibers and 41% as many unmyelinated axons. Those myelinated fibers present in antiserum-treated animals were slightly, but significantly, smaller in average diameter than controls. In teeth of control animals, about 20% of all unmyelinated axons were located in fibers coursing outside of nerve fascicles; these isolated fibers were disproportionately rare after antiserum exposure. The average number of unmyelinated axons per Schwann cell unit was also significantly lower. Postnatal exposure to anti-NGF had milder effects on mental nerve composition compared to the tooth innervation. Numbers of myelinated fibers were 83% of controls, unmyelinated axons were 74% of controls and there was no change in the average number of unmyelinated axons per Schwann cell unit. We conclude that development of dental innervation is highly susceptible to postnatal NGF deprivation. This may be a consequence of the mostly nociceptive composition of dental nerves and their late development.

The rat molar has become an important model for studies of interactions between nerves and the pulp-dentin complex, yet there is only limited quantitative information on the number and size distribution of axons entering the roots of this tooth. This study was undertaken to provide such a detailed characterization of the apical innervation of the rat molar. An additional objective was to compare the apical nerve composition of young, recently erupted rat molars with that of mature teeth in order to determine whether there is ongoing maturation of the innervation after the teeth have attained functional occlusion. A complete census was made of the nerve fibers entering the roots of both mature and recently erupted juvenile mandibular first molars in Sprague-Dawley rats. Each of the four roots of the first molars was processed for electron microscopy of thin sections near the apex. The majority of intradental nerve fibers entered the molar via the two larger (mesial and distal) roots. Within the apical root pulp, most, but not all, axons occurred within well-defined fascicles associated with blood vessels. Molars from adult animals (age 4 months) had a mean total of 232 (S.D. = 49, N = 7 teeth) myelinated fibers and 806 (S.D. = 143) unmyelinated axons entering the four roots. Fibers exceeding the A delta size range (circumference > or = 19 microns) accounted for only 4% of the myelinated axons at the apex. Molars from juvenile animals (age 4 weeks) had fewer myelinated fibers (mean 176, S.D. 18, N = 8), but more unmyelinated axons (mean 1,174, S.D. 160) than adults. The mean ratio of unmyelinated axons to myelinated axons was 6.6:1 for juveniles compared to 3.5:1 for adults. Juvenile teeth contained no myelinated fibers that exceeded 19 microns in circumference. These results indicate that the innervation of the rat molar resembles that of teeth of non-rodent mammals in that (1) innervation density is high, (2) there is a high ratio of unmyelinated axons, and (3) most of the myelinated fibers are of thin caliber. Furthermore, it appears that after the molar erupts, maturation of the nerve fiber composition continues with processes that include both a marked decrease in the number of unmyelinated axons and an increase in the number and size heterogeneity of myelinated fibers.

The establishment of channel catfish long term cloned B cell lines, the first such cell lines from ectothermic vertebrates, is described. These diploid cell lines were developed by in vitro LPS stimulation of B cells from normal channel catfish peripheral blood in the absence of overt attempts to transform or immortalize the cells. The resultant cell lines were cloned and maintained continuously in vitro for more than 12 mo without restimulation, feeder cells, or exogenous factors. Southern blot analyses of the parental cell lines revealed multiple mu-chain gene rearrangements, suggesting a polyclonal origin for the cell lines. Additional evidence for polyclonal development was provided by the demonstration that the parental cell lines transcribed mRNA for all of the six known channel catfish VH gene families. The characterization of several clonal cell lines revealed mRNA expression for both the secreted and membrane forms of the catfish mu-chain; however, the cloned cell lines each expressed only a single VH gene and analysis of the Ig H chain locus was consistent with allelic exclusion having occurred in these cells. Flow cytometry demonstrated that the cloned and uncloned cell lines produced both cytoplasmic and cell surface IgM. This IgM contained only one of the two L chain isotypes of the channel catfish, suggesting preferential L chain usage. Although these cells did not appear morphologically to be plasma cells, they secreted moderate levels of IgM in culture. These cell lines have considerable potential for addressing questions concerning the evolution of B cell function.

Nerve growth factor (NGF) is known to have an essential role in prenatal development of spinal and trigeminal primary sensory neurons serving nociceptive functions. Investigation of a possible function of NGF in development of intradental innervation has recently begun with the demonstration by others of NGF mRNA and NGF receptor in the pulp. The pulp is of special interest in this respect because of its late development and unusual properties of its innervation. In the present study, use of a sensitive ELISA for NGF has made it possible to detect and measure NGF antigen in pulps of developing rat molars. Pulps contained relatively high concentrations of NGF before and during the time of innervation development.

Most of the nerve fibres supplying the mandibular canine on one side were interrupted by sectioning the inferior alveolar nerve (IAN) and, after 1 week, the trophic activity in each mandibular canine pulp was assessed in an in vitro assay using sympathetic neurones from 11-day chick embryos as test cells. In eight of nine animals tested, neuronotrophic activity in the denervated pulp was markedly lower than in the contralateral control pulp. Antiserum to mouse nerve growth factor had no effect on the trophic activity in either control or denervated pulps. Thus, the pulp differs from other peripheral tissues, which undergo increases in neuronotrophic activity after denervation. The basis of this difference may be the high innervation density of the pulp. The IAN distal to the site of nerve transection also had reduced survival-promoting activity.

Extracts of these dental pulps from adult cats contained a non-dialysable agent or agents which could support neurone survival and neurite development for at least three days in neurone-enriched cultures of sympathetic ganglion cells from 11-day chick embryos. The neurone survival-promoting activity differed from nerve growth factor (NGF) in that: (1) anti-mouse NGF serum did not inhibit it; (2) nearly all ganglionic neurones survived in optimum concentrations of pulp extract, whereas only about 35 per cent were supported by NGF; and (3) cell bodies of NGF-supported neurones were markedly larger than in neurones supported by pulp extracts. The neuronotrophic activity in individual dental pulps was highly variable among different cats, but similar between mandibular canines from the same animal. Smaller pulps had higher concentrations of trophic activity than larger ones. Gingival tissue and the anterior belly of the disgastric muscle contained little neuronotrophic activity.