Periodontal disease in free-ranging koalas (Phascolarctos cinereus) from the Mount Lofty Ranges, South Australia, and its association with koala retrovirus infection.

BACKGROUND: In northern Australian koala populations (Queensland and New South Wales), periodontal disease (gingivitis and periodontitis) is common while koala retrovirus subtype A is endogenous, with other subtypes transmitted exogenously. Koala retrovirus has been hypothesised to cause immune suppression and may predispose koalas to diseases caused by concurrent infections. In southern Australia populations (Victoria and South Australia) periodontal disease has not been investigated, and koala retrovirus is presumably exogenously transmitted. This study described oral health in South Australian koalas and investigated if an association between periodontal disease and koala retrovirus exists. METHODS: Oral health was examined for wild-caught koalas from the Mount Lofty Ranges (n = 75). Koala retrovirus provirus was detected in whole blood using nested PCR and proviral load determined with qPCR. Periodontal disease severity was recorded and used to calculate the Final Oral Health Index (0-normal, 24-severe).Results Periodontal disease was observed in 84% (63/75) of koalas; 77% had gingivitis (58/75) and 65% (49/75) had periodontitis. The average Final Oral Health Index was 5.47 (s.d 3.13). Most cases of periodontal disease were associated with the incisors. Koala retrovirus-infected koalas were more likely to present with periodontitis (p = 0.042) and the Final Oral Health Index was negatively correlated with proviral load (ρ = -0.353, p = 0.017). CONCLUSION: South Australian koalas had a high prevalence of gingivitis and periodontitis. Periodontal disease was more prevalent in the incisors. Exogenous koala retrovirus infection may also facilitate the development of periodontitis by modulation of the immune response to concurrent oral bacterial infections.

The involvement of superoxide anions in the nitro blue tetrazolium chloride reduction mediated by NADH and phenazine methosulfate.

Oxygen inhibits competitively the reduction of nitro blue tetrazolium chloride (nitro BT) by NADH and phenazine methosulfate (PMS). The oxygen-dependent inhibition is stronger in the presence of superoxide dismutase, whereas cyanide counteracts the oxygen interference. On the other hand, the oxidation of NADH mediated by PMS and dioxygen is affected only marginally by superoxide dismutase and cyanide. Therefore, it is concluded that the involvement of superoxide anions occurs at the level of nitro BT reduction via a nitro blue tetrazolinyl radical, as has been suggested by Picker and Fridovich [1984) Arch. Biochem. Biophys. 228, 155-158) and not at the level of PMS oxidation. The inhibition of the oxygen interference in the nitro BT reduction by cyanide is dependent on the cyanide concentration, whereas in nitrogen cyanide has no effect on the reduction. It is caused by competition between cyanide and oxygen to reduce or oxidize the nitro BT radical to either formazan with concomitant cyanogen production or nitro BT, respectively. For the histochemical localization and analysis of electropherograms of NAD(P)+-dependent dehydrogenase activities, the interference of oxygen can be avoided by anaerobic incubations or by the use of 5 mM nitro BT when incubating aerobically.

Enhancement of class I HLA antigen expression by cytomegalovirus: role in amplification of virus infection.

We have investigated the effect of cytomegalovirus (CMV) infection on the expression of class I HLA antigens on fibroblasts in vitro. Scanning and integrating microdensitometry was used to quantitate the level of cytoplasmic class I antigen expression, and an antibody binding assay was used to quantitate cell surface expression of class I HLA molecules. CMV infection resulted in a significant increase in the level of cytoplasmic and cell surface class I HLA antigen expression of fibroblast monolayers. The maximal effect was seen at 72 hours postinfection and was observed with both the laboratory strain of CMV, strain AD169, and with CMV purified directly from clinical specimens. Part of the increased HLA expression was mediated by interferon released from infected cells; however, an additional direct effect of the virus itself has not been ruled out. Interferon-induced increased expression of class I HLA antigens was accompanied by increased binding of CMV to the cells, consistent with our recent demonstration that class I HLA molecules can function as a cellular receptor for CMV.

Linearity in dehydrogenase reaction rate studies in tissue sections is affected by loss of endogenous substrates during the reaction.

We studied the effect of section thickness on the reaction rate of glucose-6-phosphate dehydrogenase (G6PD) activity in unfixed sections of rat liver by use of continuous monitoring by microdensitometry of the reaction product as it formed in the section during incubation. Tetranitro BT or nitro BT was used as final electron acceptor and polyvinyl alcohol as tissue stabilizer. Each test minus control reaction curve deviated from linearity during the first 2 min of incubation. This was mainly due to loss of low molecular weight endogenous dehydrogenase substrates from the surface of the section. For any given reaction, the same absolute amount of endogenous substrate was lost from each section, and hence a much greater proportion was lost from the thinner sections. Such losses lead to a deficit in (nonspecific) formazan production. There was a greater loss from, and hence a greater deficit in, formazan production in sections incubated at 30 degrees C than at 37 degrees C and when nitro BT was used instead of tetranitro BT, but the greatest loss of endogenous substrates occurred in sections incubated in control media. Therefore, greater losses seemed to occur when the reactions were slower because of failure to overcome the critical supersaturation level of the formazan. A consequence of this was a non-linear test minus control response during the first minutes of the incubation.

Effect of lack of noradrenaline on myocardial oxygen consumption in denervated dog hearts.

Surgical cardiac denervation was carried out in dogs under halothane anaesthesia. In a paired experimental design control biopsy specimens were obtained before surgical denervation. The dogs were allowed to recover and three weeks to elapse before the second biopsy specimen was taken. Both right and left ventricular specimens had higher in vitro oxygen consumption after denervation than before. Other specimens were immediately cooled in hexane at -60 degrees C and stored under liquid nitrogen until analysed. Succinate dehydrogenase and cytochrome oxidase activities were then measured histochemically in sequential 10 or 12 microns sections. There was no significant difference between the enzyme activities measured before or after cardiac denervation (succinate dehydrogenase 20.3(6.3) before, 19.4(4.02) pmol.H2.cm-2.s-1, after; cytochrome oxidase 223(73.4) before, 263(61.6) (measured as extinction coefficient) after). Thus the changes in oxygen consumption in the chronically denervated dog heart are not due to any lack of these mitochondrial enzymes.

The quantitation of HLA-DR expression on human cells using immunocytochemistry.

An immunocytological method that can be used to quantify the comparative expression of Class II HLA-DR antigens on human cells has been developed. Tissue sections or cytospins are incubated with the monoclonal antibody RFDR1 (anti-HLA-DR), conjugated to the enzyme glucose oxidase (GO). The bound McAb is identified by incubation with the substrate beta-D-glucose and capture of the reducing equivalents generated by a tetrazolium salt forming an insoluble formazan. Under standard conditions the amount of formazan precipitated is proportional to the amount of McAb bound to the cells or tissue and thus the amount of HLA-DR expressed. The reaction has been quantified both by spectrophotometry (on tissue sections) and by microdensitometry (on cell spreads). Computer analysis has been used to relate density of HLA-DR expression to cell area. Repeated 'blind' measurements by separate investigators have confirmed the reproducibility of the results. It is suggested that this method adds a new dimension to immunohistological/cytological analysis of tissues and single cells.

A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity.

A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed. For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor. The apparent Km of the reaction is 0.83 mM. Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP+ (Ki = 1.50 mM) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity. An NADP+ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition). The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal. Continuous microdensitometric monitoring of the reaction product as it is formed has shown that the specific reaction is linear with incubation up to 10 min, thus allowing end-point measurements to be used for cytophotometric analysis.

The density of HLA-DR antigen expression on alveolar macrophages is increased in pulmonary sarcoidosis.

The density of HLA-DR antigen expression on alveolar macrophages obtained by bronchoalveolar lavage from sarcoidosis patients and normal control subjects was quantified using scanning and integrating microdensitometry in conjunction with a monoclonal mouse anti-human HLA-DR antibody directly conjugated to fungal glucose oxidase. The density of HLA-DR antigen expression on alveolar macrophages was significantly increased in the pulmonary sarcoidosis patients compared with normal control subjects examined. The reproducibility, specificity and sensitivity establish the reliability and potential importance of this method of direct quantification of cell surface antigen expression. The demonstration of an increased density of Class II Major Histocompatibility Complex (MHC) antigen expression on alveolar macrophages in pulmonary sarcoidosis suggests that these cells may play a role in the induction of the immune responses at sites of disease activity in this poorly understood condition.

Reaction rate studies of glucose-6-phosphate dehydrogenase activity in sections of rat liver using four tetrazolium salts.

The reaction rate of glucose-6-phosphate dehydrogenase activity in liver sections from fed and starved rats has been monitored by the continuous measurement at 37 degrees C of the reaction product as it is formed using scanning and integrating microdensitometry. Control media lacked either substrate or both substrate and coenzyme. All reactions were nonlinear; however, subtraction of either of the controls from the test response produced linearity. Differing responses in sections of livers from fed and fasted rats indicate that the appropriate control medium for use in the assay of this dehydrogenase is one lacking both substrate and coenzyme rather than a medium containing coenzyme. The reaction rate was the same with each of the final acceptors. Problems with the diffusion of the formazan of BPST and with the failure to precipitate the formazan of Neotetrazolium make Tetranitro BT and Nitro BT the tetrazolium salts of choice in quantitative dehydrogenase assays.

Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts.

The properties of the four most commonly used tetrazolium salts, neotetrazolium, nitro blue tetrazolium (nitro-BT), tetranitro-BT, and 2-(2-benzothiazolyl-3-(4-phthalhydrazidyl)-5-styryl-te trazolium (BPST), have been compared for their effects on the localization of nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases under optimal incubation conditions in cryostat sections of rat liver. Glucose-6-phosphate dehydrogenase has been selected as an example of these dehydrogenases. It was found that the use of nitro-BT and tetranitro-BT, unlike neotetrazolium and BPST, in combination with an exogeneous electron carrier and azide, resulted in localization patterns in agreement with the sites of activity as determined by microchemical techniques. In the absence of an intermediate carrier the localization was very similar to that of NADPH cytochrome c (P450) reductase as demonstrated immunocytochemically. BPST did not properly localize dehydrogenase activity, most probably because of the redistribution of formazan, due to its lack of firm substantivity. Neotetrazolium reduction in nitrogen gave the localization pattern, both in the presence and absence of carrier, of the reductase, suggesting that the transfer of reducing equivalents from the exogenous electron carrier to neotetrazolium proceeds via cellular electron transport systems. The reduction of nitro-BT and tetranitro-BT via intermediate carriers was oxygen sensitive in parenchymal cells, but not in the non-parenchymal liver cells. This oxygen sensitivity could be blocked by azide. With neotetrazolium, oxygen inhibited both carrier-mediated and carrier-independent reactions, effects that were not reversed with azide. Possible mechanisms of action between oxygen, reduced carriers, and tetrazolium salts are discussed.

Diffusion during dehydrogenase reactions: the effects of intermediate electron acceptors.

A hamster cheek pouch model has been used to study the diffusion of reactants from the epithelium into adjacent muscle and connective tissue during the histochemical demonstration of glucose-6-phosphate dehydrogenase activity. The effects of the addition of intermediate electron acceptors to the incubation medium varied considerably from one acceptor to another, but were independent of the grade of polyvinyl alcohol incorporated into the medium. Menadione was the least effective intermediate both in transferring reducing equivalents from the primary dehydrogenase to Neotetrazolium chloride and in preventing diffusion. Phenazine methosulphate, Methylene Blue and Thionin were more efficient intermediates. Nevertheless, considerable diffusion occurred in the presence of Phenazine methosulphate, although very little diffusion was detectable with either of the thiazine dyes. It is suggested that these differences are related to different modes and sites of action of the carriers.

Reaction rate studies of glucose-6-phosphate dehydrogenase in rat tracheal epithelium; the effect of section thickness.

The reaction velocity of glucose-6-phosphate dehydrogenase has been quantified by continuous monitoring on a Vickers microdensitometer of the reaction product as it formed in sections of different thickness of rat tracheal epithelium. Reaction velocity was directly proportional to section thickness when either tetranitro BT or neotetrazolium was used as the final acceptor; the rate was the same with each tetrazolium salt. However, the amount of formazan deposited in a given time was not proportional to section thickness. When tetranitro BT was employed the reaction became non-linear in the thicker sections due to the inability of the instrument to record beyond a certain absorbance value. Using neotetrazolium a lag phase, due to the failure to overcome the critical supersaturation level of the formazan, preceded the linear response. The duration of this phase decreased as section thickness increased. The implications of these findings on studies using conventional "end point" methods of measurement are discussed.

A quantitative histochemical study of glucose-6-phosphate dehydrogenase activity in premalignant and malignant lesions of human oral mucosa.

The levels of glucose-6-phosphate dehydrogenase activity have been assayed by quantitative histochemistry in normal, benign, potentially malignant and carcinomatous lesions of human oral mucosa. Dysplastic epithelium showed a higher activity of this enzyme than did morphologically normal, benign hyperplastic and neoplastic lesions and this may be an aid to early diagnosis. The increases in glucose-6-phosphate dehydrogenase activity in dysplastic human oral epithelium are sufficient to warrant a longitudinal study to evaluate further the significance of these changes in the process of malignant transformation.

Dehydrogenase activity and loss of formazan from tissue sections.

Formazans can be lost from tissue sections when the incubation medium is removed at the end of a dehydrogenase reaction. The loss is not uniform and will clearly affect the interpretation of results. This suggests that both the qualitative evaluation of the reaction and the measurement of enzyme activity should be made at the time of incubation.

Inhibition of glycolysis in the denervated dog heart.

We measured glucose metabolism in five dogs before and 3 weeks after cardiac denervation; after this time myocardial norepinephrine is depleted. The discharge by the myocardium, of 14CO2 from infused 14C-D-glucose (U), decreased following denervation (P = 0.05). The ratio of 14CO2 to total CO2 production, which measured the proportion of glucose to total substrate oxidized, also decreased following denervation (P = 0.05). The inhibition of glucose oxidation by denervation was not due to an increase in arterial lactate concentration. There was an associated increase in myocardial content of fructose-6-phosphate in an additional seven dogs (P < 0.01). We postulate that myocardial tissue norepinephrine is one of the controllers of the activity of phosphofructokinase.

A quantitative cytochemical study of three oxidative enzymes during experimental oral carcinogenesis in the hamster.

The activities of three oxidative enzymes have been assayed by quantitative cytochemical methods during chemically induced carcinogenesis in the hamster cheek pouch. Changes in the levels of succinate and glucose-6-phosphate dehydrogenases were apparent in the early stages of carcinogenesis prior to the development of lesions recognisable histologically as squamous cell carcinomas. In contrast the activity of lactate dehydrogenase remained constant until malignant lesions were well established. These changes were not observed in benign hyperplastic lesions, suggesting that such enzyme assays may be of value in the diagnosis of premalignant lesions of the oral mucosa.

Studies on the Gomori acid phosphatase reaction: the preparation of the incubation medium.

The pH of the buffer, the initial concentrations and order of mixing of the reactants, and the length of the pre-incubation at 37 degrees C have all been found to be important in determining the final composition of the Gomori acid phosphatase medium. In a conventionally prepared medium, as much as 70% of the lead salt can be precipitated as lead glycerophosphate during a 24 h pre-incubation at 37 degrees C. The use of acetate buffer at pH 4.7, mixed with the other reactants after maximal dilution, has made possible the preparation of an incubation medium which contains those concentrations of substrate, lead salt and buffer originally proposed by Gomori, and yet does not precipitate.