Biomedical subjects
Publications and source records attributed to A M Rich.
A case is reported in a 15-year-old white girl who had a swollen lower face and lips; a diagnosis of orofacial granulomatosis was made. It was suspected that her condition had an allergic basis because an increase in clinical signs and symptoms was shown to be related to the food additive monosodium glutamate. Treatment with a restricted diet resulted in resolution of the facial swelling.
It has been suggested that the size of the nuclei of epithelial basal cells can be used in predicting the likelihood of malignant transformation of epithelium. This proposition was assessed in rat palatal epithelium after the carcinogen 4-nitroquinoline-1-oxide had been applied to the epithelium for varying periods of time. No consistent alterations in basal cell nuclear size, including area, perimeter, diameter and regularity of form were found with routine light microscopy as the epithelium passed through various stages of dysplasia to carcinoma. This finding casts doubt on the value of using a variation of basal cell nuclear size as a predictor of malignant transformation.
Accurate diagnosis of oral mucosal lichen planus (OMLP) is essential if appropriate management is to be instituted. Direct immunofluorescence microscopy (DIF) has facilitated the diagnosis of some dermatologic diseases with oral manifestations, but its use in the diagnosis of OMLP is less well documented. In the present study, 165 biopsies from consecutive cases for which a provisional diagnosis of OMLP was suggested clinically or on the basis of routine histopathologic, or DIF assessment were studied. In 27 cases the results of DIF were non-contributory. In 13 cases the diagnosis could only be established by DIF examination. Nine cases clinically diagnosed as OMLP were assigned to a different condition on the basis of routine histology and DIF. It was concluded that DIF was an integral step in the diagnosis of OMLP.
Quantitative analysis of Langerhans cells stained with OKT6 in the epithelium of 42 patients with oral mucosal lichen planus and 14 patients with oral mucosal leukoplakia showed significantly greater numbers of Langerhans cells in lichen planus. This may reflect the role of Langerhans cells in the pathogenesis of this condition and their identification could help in distinguishing lichen planus from leukoplakia.
The relevance of phosphoinositide remodeling to calcium movements and to the physiological response of superoxide anion (O2-) generation was probed in neutrophils stimulated by the chemotactic peptide fMet-Leu-Phe and the lectin concanavalin A. fMet-Leu-Phe and concanavalin A triggered O2- generation but elicited different patterns of calcium mobilization and phosphoinositide remodeling. fMet-Leu-Phe (10(-7) M) triggered a rise in cytosolic calcium by mobilization of intracellular calcium (fura-2) and increased calcium permeability (45Ca uptake), while concanavalin A (100 micrograms/ml) elicited a rise in cytosolic calcium, primarily by uptake of extracellular calcium (45Ca uptake). fMet-Leu-Phe triggered rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate and phosphatidylinositol, and generation of inositol 1,4,5-trisphosphate (IP3). In contrast concanavalin A triggered breakdown of phosphatidylinositol, but not PIP2, nor was there a significant increase in IP3. However, both fMet-Leu-Phe and concanavalin A triggered a rapid biphasic increase in levels of labeled diacylglycerol (in [3H]arachidonate or [14C]glycerol prelabeled cells) and a 3-fold increase in [32P] phosphatidic acid. These results are concordant with a role for PIP2 breakdown and generation of IP3 specifically in intracellular calcium mobilization but not for other aspects of the signaling pathway for O2- generation. Calcium permeability changes were associated with elevated diacylglycerol and [32P]phosphatidic acid, although a cause and effect relationship is not apparent. Ligands such as concanavalin A enhance cytosolic calcium and trigger generation of O2- without significant PIP2 remodeling; elevated diacylglycerol and cytosolic calcium are the common events associated with ligand-induced O2- generation.
Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O2-) which are potent mediators of inflammation. Various agents elicit different responses; N-formylmethionylleucylphenylalanine (fMLP) (0.1 microM) provokes brisk generation of superoxide anion; leukotriene B4 (LTB4, 0.1 microM) is a poor stimulus. In contrast, phorbol myristate acetate (PMA, 1.6 microM) acting directly via protein kinase C is a potent stimulus for O2-. We compared the kinetics of appearance of various "second messengers" with the capacity of these ligands to elicit O2- generation. Kinetic analysis showed a two-phase response to membrane ligands; both an "early" (less than or equal to 15 s) and a "late" (greater than 15 s) increase in [3H]- and [14C]diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB4 elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Cytochalasin B increased the late phase of DG labeling elicited by all agonists. Moreover, comparison of increases in [3H]DG versus those of [14C]DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Early increments of DG were also accompanied by addition of plasma membrane (ultrastructural morphometry); the ratio of surface perimeter to area increased rapidly (10 s) and persisted (60 s) in response to fMLP. Increments were more gradual in response to PMA. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 Mr protein was phosphorylated with kinetics consistent with the production of O2- and DG in response to fMLP (early and late) and PMA (late). In contrast, LTB4 provoked only early phosphorylation of this protein. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only "triggering" (the rapid generation of a signal) but also "activation" (the maintenance of a signal) to sustain responses.
Direct immunofluorescence is an important technique in establishing the diagnosis of a variety of diseases. It has become a useful adjunct to the diagnosis of oral mucosal diseases, yet many practitioners find the field confusing. This paper summarises the published findings and presents a table for ready reference for clinicians.
Quantitative analysis of rat palatal mucosa after the carcinogen 4- nitroquinoline-1-oxide had been applied to the epithelium for varying periods of time showed that there was a significant increase in epithelial thickness, due largely to an increase in thickness of the basal compartment. This alteration was measurable before epithelial dysplasia could be recognised.
Leukotriene B4 (5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/10(7) cells); no omega-oxidized metabolites of LTB4 were found. Adding 10 microM-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 microM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its omega-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 microM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10(-6) M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (less than 0.01 ng/10(7) cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity of neutrophil 5-lipoxygenase. However, adding 50 microM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its omega-oxidized products (fMLP, 20.2 ng/10(7) cells; IgG, 17.1 ng/10(7) cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.
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Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases.
Crystalline monosodium urate (MSU) produces inflammation in vivo and kills phagocytes in vitro. A plausible hypothesis to account for crystal-induced cell death is that ingested crystals perforate the phagocytic vacuole into which lysosomes have degranulated: "lysis from within." However, it has also been contended that degranulation is not required for crystal-induced cell death. To resolve this controversy, we have prepared neutrophil-derived cytoplasts ("neutroplasts") which are devoid of most cellular organelles, including lysosomal granules. Both intact neutrophils and neutroplasts ingested MSU crystals, but inhibition of phagocytosis by cytochalasin B reduced crystal-induced death of neutrophils (release of lactate dehydrogenase) from 42% to 16% without altering lysis of neutroplasts (27% with MSU alone and 26% with MSU and cytochalasin B). Moreover, addition of serum, which prevents direct interaction of crystals with the outer plasma membrane, reduced lysis of neutrophils, reducing cell death from 42% to 25%. After 60 min incubation, serum was totally ineffective in reducing neutrophil death but continued to reduce lysis of neutroplasts from 61% to 13%. Thus, the MSU lysed neutroplasts under conditions in which it ruptured membranes of nonphagocytic structures (erythrocytes, liposomes), i.e., in the absence of serum: "lysis from without." These data suggested that death of neutrophils after internalization of MSU requires a component that is lacking in neutroplasts. Granules (lysosomes) are the best candidates for this component, supporting the general validity of the "lysis from within" hypothesis.
Activation of the neutrophil by interaction of a ligand such as f-Met-Leu-Phe with its specific receptor elicits a prompt breakdown of PIP2 and the generation of PA via diacylglycerol. There is a rapid elevation of cytosolic calcium and activation of protein kinase C. Calcium and protein kinase C act synergistically to elicit the physiological responses. Although PIP2 breakdown and PA generation are prompt responses of neutrophils to receptor occupancy by chemoattractants, these steps can be bypassed by stimuli which directly activate protein kinase C or increase cytosolic calcium. Elevation of cytosolic Ca and activation of protein kinase C did not elicit breakdown of PIP2, indicating that phosphoinositide remodeling is not caused by activation of protein kinase C or by elevation of cytosolic calcium, nor is such a breakdown or the generation of phosphatidic acid required for the subsequent responses. The evidence indicates that PIP2 breakdown is an early event in stimulus-response coupling and is correlated with receptor-initiated generation of the signal.
We have studied Microciona prolifera cells as a model for inflammation and secretion. Dissociated in Ca-, Mg-free seawater with 2.5 mM EDTA, the cells aggregate when exposed to Ca (greater than 5 mM) and Ca ionophores. Extracellular Ca is not required over the course of aggregation; brief pulses of Ca suffice. Aggregation was induced by A23187 in excess EDTA after cells were prepared by pulse Ca. It appeared that Ca ionophore stimulated the secretion of Microciona aggregation factor (MAF) to a locus or in a form inaccessible to external EDTA. Pulse-induced aggregation depended on MAF because it was inhibited by MAF fragments, which are ligands for MAF-binding sites. Sponge cells were preloaded with three fluorescent dyes that monitor aspects of stimulus-secretion coupling: 1) 3,3'-dipropylthiadicarbocyanine iodide (dis-C3-(5)), a carbocyanine dye presumed to report changes in membrane potential; 2) 9-aminoacridine (9AA), which presumably reports secretion from acid vesicles; and 3) chlortetracycline (CTC), presumed to report mobilization of membrane-associated Ca. Exposure of cells either to constant Ca or to pulse Ca stimuli caused prompt decreases in the fluorescence of cells with diS-C3-(5) and increases in fluorescence of cells with 9AA. In contrast, although constant Ca provoked decreases in fluorescence of cells with CTC, a pulse Ca was without effect. Moreover, inhibitors of stimulus-response coupling (e.g., aspirin, sodium salicylate, 5 mM; diclofenac, 100 microM) inhibited sponge aggregation induced by either constant or pulse stimuli. In contrast, like the endogenous mediator of inflammation, leukotriene B4, trienoic alkyl catechols (urushiol) from poison ivy provoked aggregation. These studies suggest the utility of this marine model for analysis of stimulus-response coupling in cells of higher species that also respond to secretagogues in the absence of external Ca.
Squamous cell carcinomas of the tongue can be produced reliably in rats by repeated application of the carcinogen 4-nitroquinoline-1-oxide. The histopathological changes in the lingual epithelium during carcinogenesis were progressive, passing through mild to moderate to severe epithelial atypia before the development of squamous cell carcinoma. Initiation, with its putative irreversible cellular changes, occurred within 4 weeks.
Dissociated cells of the marine sponge, Microciona prolifera, aggregate in response to a species-specific aggregation factor (MAF) and Ca ionophores. We now report that leukotriene B4, a 5-lipoxygenase product, also causes aggregation of sponge cells. No other lipoxygenase products provoked aggregation. However, nordihydroguaiaretic acid (NDGA), colchicine, indomethacin, piroxicam and ibuprofen inhibited MAF-induced aggregation; the latter three agents inhibited Ca movements. Inhibition of prostaglandin release cannot be the mechanism whereby these agents inhibit aggregation of sponge cells, since the cells do not respond to exogenous cyclooxygenase products.
Activated neutrophils aggregate, generate superoxide (O-2), and degranulate. The role of Ca as "second messenger" in neutrophil activation was examined using as agonist the chemotactic peptide fMet-Leu-Phe and its antagonist t-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe to systematically vary the time of receptor occupancy. Release of enzymes from specific and azurophil granules showed a finite requirement for receptor occupancy; the cells were committed to full degranulation after 10 s of receptor-agonist interaction. In contrast, continuous occupation of the receptor by agonist was required to initiate and maintain O-2 generation and aggregation. Cytosolic Ca ( Quin2 fluorescence) increased immediately in response to fMet-Leu-Phe, requiring less than 2 s of agonist-receptor interaction to initiate an optimal response. Mobilization of membrane-associated Ca (chlorotetracycline fluorescence) also demonstrated a finite time requirement; the cells were fully committed after 10 s of agonist-receptor interaction. Increased Ca permeability (45Ca uptake) was fully launched after 15 s of agonist-receptor interaction. The data indicate that Ca movements ( quin2 , chlorotetracycline fluorescence, 45Ca uptake) are both necessary and sufficient to account for degranulation by neutrophils activated by fMet-Leu-Phe. However, neutrophil aggregation and the generation and release of O-2 in response to the same stimulus require a further unknown factor(s) associated with receptor occupancy to maintain these responses.
Three cases of oral lichenoid reaction linked with the administration of allopurinol are presented. Withdrawal of the drug resulted in considerable clinical improvement in one case and complete resolution of the ulcerative lesions in the other two cases. The consideration of drug-induced reactions in the differential diagnosis of oral mucosal diseases has been emphasized.