Comparative study on Saliva and Nasopharyngeal swabs and the outcome of RT-PCR test in patients with mild symptoms of SARS-CoV-2 / Estudio comparativo de muestras de saliva y nasofaríngeas y el resultado de la prueba RT-PCR en pacientes con síntomas leves de SARS-CoV-2

Aim A simple and reliable method for diagnosing COVID 19 infections is the needed. The role of saliva in the transmission of the infection has already been established. Method Saliva and nasopharyngeal swabs from patients suspected to have COVID 19 infections were taken simultaneously, and the results of the RT-PCR were compared. Result Total 405 samples were collected, of which 250 males and 155 females. In the 391 samples included for analysis, 370 (94.63%) samples were found to have concordance results, and 21 (5.37%) samples had discordant results. Conclusion The use of saliva to diagnose COVID 19 infection is reliable, and its use can be recommended. (AU)

Objetivo Un método simple y confiable para diagnosticar infecciones por COVID 19 es necesario. Ya se ha establecido el papel de la saliva en la transmisión de la infección. Método Se tomaron simultáneamente hisopos de saliva y nasofaríngeos de pacientes con sospecha de infección por COVID 19 y se compararon los resultados de la RT-PCR. Resultado Se recogieron 405 muestras, de las cuales 250 hombres y 155 mujeres. En las 391 muestras incluidas para el análisis, se encontró que 370 (94,63%) muestras tenían resultados de concordancia y 21 (5,37%) muestras tenían resultados discordantes. Conclusión El uso de la saliva para diagnosticar la infección por COVID 19 es confiable y se puede recomendar su uso. (AU)

Phosphatase activity of the control of virulence sensor kinase CovS is critical for the pathogenesis of group A streptococcus.

The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains.

Telomerase activity impacts on Epstein-Barr virus infection of AGS cells.

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva.

Quantification of the adenylate cyclase toxin of Bordetella pertussis in vitro and during respiratory infection.

Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ≈ 10(8) CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ≈ 10(8) CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ≈ 60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

Nasopharyngeal lactate dehydrogenase concentrations predict bronchiolitis severity in a prospective multicenter emergency department study.

We reexamined the finding of an inverse relationship between values of nasopharyngeal lactate dehydrogenase, a marker of the innate immune response, and bronchiolitis severity. In a prospective, multicenter study of 258 children, we found in a multivariable model that higher nasopharyngeal lactate dehydrogenase values in young children with bronchiolitis were independently associated with a decreased risk of hospitalization.

Protein tyrosine phosphatase PTPN14 is a regulator of lymphatic function and choanal development in humans.

The lymphatic vasculature is essential for the recirculation of extracellular fluid, fat absorption, and immune function and as a route of tumor metastasis. The dissection of molecular mechanisms underlying lymphangiogenesis has been accelerated by the identification of tissue-specific lymphatic endothelial markers and the study of congenital lymphedema syndromes. We report the results of genetic analyses of a kindred inheriting a unique autosomal-recessive lymphedema-choanal atresia syndrome. These studies establish linkage of the trait to chromosome 1q32-q41 and identify a loss-of-function mutation in PTPN14, which encodes a nonreceptor tyrosine phosphatase. The causal role of PTPN14 deficiency was confirmed by the generation of a murine Ptpn14 gene trap model that manifested lymphatic hyperplasia with lymphedema. Biochemical studies revealed a potential interaction between PTPN14 and the vascular endothelial growth factor receptor 3 (VEGFR3), a receptor tyrosine kinase essential for lymphangiogenesis. These results suggest a unique and conserved role for PTPN14 in the regulation of lymphatic development in mammals and a nonconserved role in choanal development in humans.

Oral sulforaphane increases Phase II antioxidant enzymes in the human upper airway.

BACKGROUND: Cellular oxidative stress is an important factor in asthma and is thought to be the principle mechanism by which oxidant pollutants such as ozone and particulates mediate their pro-inflammatory effects. Endogenous Phase II enzymes abrogate oxidative stress through the scavenging of reactive oxygen species and metabolism of reactive chemicals. OBJECTIVE: We conducted a placebo-controlled dose escalation trial to investigate the in vivo effects of sulforaphane, a naturally occurring potent inducer of Phase II enzymes, on the expression of glutathione-s-transferase M1 (GSTM1), glutathione-s-transferase P1 (GSTP1), NADPH quinone oxidoreductase (NQO1), and hemoxygenase-1 (HO-1) in the upper airway of human subjects. METHODS: Study subjects consumed oral sulforaphane doses contained in a standardized broccoli sprout homogenate (BSH). RNA expression for selected Phase II enzymes was measured in nasal lavage cells by RT-PCR before and after sulforaphane dosing. RESULTS: All subjects tolerated oral sulforaphane dosing without significant adverse events. Increased Phase II enzyme expression in nasal lavage cells occurred in a dose-dependent manner with maximal enzyme induction observed at the highest dose of 200 g broccoli sprouts prepared as BSH. Significant increases were seen in all sentinel Phase II enzymes RNA expression compared to baseline. Phase II enzyme induction was not seen with ingestion of non-sulforaphane containing alfalfa sprouts. CONCLUSION: Oral sulforaphane safely and effectively induces mucosal Phase II enzyme expression in the upper airway of human subjects. This study demonstrates the potential of antioxidant Phase II enzymes induction in the human airway as a strategy to reduce the inflammatory effects of oxidative stress. CLINICAL IMPLICATIONS: This study demonstrates the potential of enhancement of Phase II enzyme expression as a novel therapeutic strategy for oxidant induced airway disease. CAPSULE SUMMARY: A placebo-controlled dose escalation trial demonstrated that naturally occurring sulforaphane from broccoli sprouts can induce a potent increase in antioxidant Phase II enzymes in airway cells.

Resistance to mucosal lysozyme compensates for the fitness deficit of peptidoglycan modifications by Streptococcus pneumoniae.

The abundance of lysozyme on mucosal surfaces suggests that successful colonizers must be able to evade its antimicrobial effects. Lysozyme has a muramidase activity that hydrolyzes bacterial peptidoglycan and a non-muramidase activity attributable to its function as a cationic antimicrobial peptide. Two enzymes (PgdA, a N-acetylglucosamine deacetylase, and Adr, an O-acetyl transferase) that modify different sites on the peptidoglycan of Streptococcus pneumoniae have been implicated in its resistance to lysozyme in vitro. Here we show that the antimicrobial effect of human lysozyme is due to its muramidase activity and that both peptidoglycan modifications are required for full resistance by pneumococci. To examine the contribution of lysozyme and peptidoglycan modifications during colonization of the upper respiratory tract, competition experiments were performed with wild-type and pgdAadr mutant pneumococci in lysozyme M-sufficient (LysM(+/+)) and -deficient (LysM(-/-)) mice. The wild-type strain out-competed the double mutant in LysM(+/+), but not LysM(-/-) mice, indicating the importance of resistance to the muramidase activity of lysozyme during mucosal colonization. In contrast, strains containing single mutations in either pgdA or adr prevailed over the wild-type strain in both LysM(+/+) and LysM(-/-) mice. Our findings demonstrate that individual peptidoglycan modifications diminish fitness during colonization. The competitive advantage of wild-type pneumococci in LysM(+/+) but not LysM(-/-) mice suggests that the combination of peptidoglycan modifications reduces overall fitness, but that this is outweighed by the benefits of resistance to the peptidoglycan degrading activity of lysozyme.

Identificating cathepsin D as a biomarker for differentiation and prognosis of nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis.

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.

Expression of CYP2E1 in human nasopharynx and its metabolic effect in vitro.

It was evident that nitrosamines can act directly on target tissue and result in carcinogenesis. As has been shown, the carcinogenic activity of nitrosamines relied on its bioactivation by Cytochrome P450 2E1 (CYP2E1). In this study, we investigated the expression of CYP2E1 in Nasopharyngeal carcinoma (NPC) cells, embryonic nasopharyngeal epithelial tissue (ENET) specimens, and NPC biopsies by RT-PCR analysis. CYP2E1 was expressed in all NPC cell lines (6/6, including 7429) and ENET (6/6), and 80% of NPC biopsie (8/10). The fact that Human nasopharynx expresses CYP2E1 suggests that CYP2E1 may play an important role in the course of NPC by indirect carcinogens nitrosamines. To further evaluate the function of CYP2E1, the CYP2E1 was stably expressed in the cell line NIH 3T3/rtTA under a tetracycline-controlled transactivator. The expression of CYP2E1 was tightly regulated in a dose-dependent manner by Doxycycline (Dox) When the catalytic activity of CYP2E1 was assayed, the result showed that the generation of 6-hydroxychlorzoxazone (6-OH-CZ) from chlorzoxazone (CZ) was dose- and time-dependent on Dox addition to the medium. In the presence of 1 microg/ml Dox, the CZ 6-hydroxylase activity of the cell line was found to be 0.986 +/- 0.034 nmol/10(6) cells/h. The metabolic activation of Tet/3T3/2E1-6 cells was also assayed by N,N'-dinitrosopiperazine (DNP) cytotoxicity, and the viability of Tet/3T3/2E1-6 cells treated with Dox was lower than that of untreated cells with a significant difference between them in 80 and 160 microg/ml DNP (P ( 0.05, t test. This cell line will be useful not only to assess the metabolic characteristics of CYP2E1, but also will be useful to investigate the role of CYP2E1 in metabolic activation of carcinogenic nitrosamines in vitro.

EBV latent membrane protein 1 up-regulates hypoxia-inducible factor 1alpha through Siah1-mediated down-regulation of prolyl hydroxylases 1 and 3 in nasopharyngeal epithelial cells.

Hypoxia-inducible factor 1 (HIF1) is up-regulated in most malignant tumors usually via interruption of ubiquitination and proteasomal degradation of its subunit alpha. Recently, we have shown that the principal EBV oncoprotein, latent membrane protein 1 (LMP1), activates HIF1alpha and subsequently expression of HIF1-responsive genes in epithelial cells. Here, we explore the mechanism for HIF1alpha activation by LMP1 in nasopharyngeal epithelial cells: LMP1 up-regulates the level of Siah1 E3 ubiquitin ligase by enhancing its stability, which subsequently induces proteasomal degradation of prolyl HIF-hydroxylases 1 and 3 that normally mark HIF1alpha for degradation. As a result, LMP1 prevents formation of von Hippel-Lindau/HIF1alpha complex, as shown by coimmunoprecipitation analyses. Thus, Siah1 is implicated in the regulation of HIF1alpha and is involved in a recently appreciated aspect of EBV-mediated tumorigenesis, namely, the angiogenesis process triggered by LMP1.

Expression of 5-lipoxygenase in specialized epithelial cells of nasopharyngeal-associated lymphoid tissue.

Leukotrienes are lipid mediators that are produced primarily by certain types of leukocytes. The synthesis of the leukotriene LTB(4) is initiated by the enzyme 5-lipoxygenase and completed by LTA(4) hydrolase. Epithelial cells constitutively express LTA(4) hydrolase but normally lack 5-lipoxygenase. In this study, we report that the stratified squamous epithelial cells from inflamed or hyperplastic tissues of palatine and pharyngeal tonsils (nasopharyngeal-associated lymphoid tissue) express 5-lipoxygenase protein. The localization of 5-lipoxygenase was indicated by immunohistochemical staining and presence confirmed by immunoblot. Positive staining for 5-lipoxygenase in infiltrating leukocytes in inflamed tissues served as internal positive controls for immunohistochemical staining. Staining for 5-lipoxygenase in appendix tissue was negative for epithelial cells while positive for polymorphonuclear leukocytes, indicating that 5-lipoxygenase expression is not a general feature of epithelial cells in mucosa-associated lymphoid tissue. In tonsils, 5-lipoxygenase staining was pronounced in broad regions but reduced or absent in others, suggesting regional regulation of expression. Epithelial cells of tonsils were also positive for 5-lipoxygenase activating protein and leukotriene A(4) hydrolase, indicating a capacity to produce LTB(4). Taken together, these results suggest that the specialized epithelial cells of the mucosa-associated lymphoid tissue of human tonsils can synthesize LTB(4). This lipid mediator may serve to modulate the function of cells within the lymphoid tissue as well as promote an inflammatory response.

[Expression of cytochrome P450 enzymes in human nasopharyngeal carcinoma and non-cancerous nasopharynx tissue].

BACKGROUND & OBJECTIVE: The etiology of nasopharyngeal carcinoma (NPC) is associated with environmental and hereditary factors. Xenobiotics from environment must be activated to derive carcinogens. Several cytochrome P450 (CYP450) metabolic enzymes participate to the activation of pre-carcinogens, and the genetic polymorphism of those genes is associated with metabolic polymorphism and susceptibility to cancer. We performed a preliminary investigation on the xenobiotics metabolism of human nasopharynx by determining the expression of CYP450 genes in NPC and non-cancerous nasopharynx tissues. METHODS: The following two methods were used: (1)A cDNA library from the mixed RNA sample of seven non-cancerous nasopharynx tissues was generated, followed by clone sequencing and bioinformatics analysis. (2)RNA of 14 NPC and 8 non-cancerous nasopharynx tissues were reversely transcribed, and the expression of CYP450 genes in those samples was determined by PCR amplification. RESULTS: Eight ESTs of CYP450 genes including CYP1B1, CYP2F1, CYP2J2, CYP4B1, CYP4F12, CYP5A(TBXAS1), CYP20A1, and CYP51A1 were detected in non-cancerous nasopharynx cDNA library, among these CYP4B1 exhibited the highest expression level with 16 copies of ESTs. Positive expression of fourteen CYP450 genes including CYP1A1, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP3A5, CYP3A7, and CYP4B1 were detected by RT-PCR, among these, CYP1B1, CYP2B6, and CYP4B1 had also been detected in the cDNA library. A total of 19 CYP450 genes expression were detected in NPC and non-cancerous nasopharynx tissues, and the expression levels of CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP3A5, and CYP4B1 were higher than those of the other genes. CONCLUSION: The expression of a great number of CYP450 genes was detected in human nasopharynx, some of which might participate to the activation of pre-carcinogen in human nasopharynx. Contribution of these genes to the risk of NPC needs further investigation.

[Epstein-Barr virus latent membrane protein 1 induces the telomerase activity in human nasopharyngeal epithelial cells].

Telomerase activation has been linked to cell immortalization in vitro and tumorigenicity in vivo. In this study, for the first, we reported that Epstein-Barr virus activated the telomerase activity of human nasopharyngeal epithelial cells in the early stage of immortalization as tested by the PCR-ELISA. The telomerase activity in nasopharyngeal epithelial cells was only observed in presenescent cells. It was implicated that Epstein-Barr virus induced the escape of nasopharyngeal epithelial cells from senescence via the activation of telomerase. We further showed that telomerase activation in infected cells was dependent on the protein level of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus using a Tetracycline regulatory cell line expressing LMP1, pTet-on-LMP1-HNE2. The activity of telomerase in nasopharyngeal cells was decreased when the protein level of LMP1 was blocked by antisense LMP1 plasmid DNA. And the activity of telmerase was also related to the carboxyl terminus of LMP1. It was implicated that the ability of Epstein-Barr virus to suppress senescence is associated with telomerase activation by LMP1.

Diagnostic usefulness of telomerase activity in nasopharyngeal carcinoma.

Telomeres are specialized structures at the ends of eukaryotic chromosomes which are composed of simple repetitive G-rich hexameric sequences. Activation of telomerase, a ribonucleoprotein that synthesizes telomeric DNA, is found in most malignant tumors. However, little data is available concerning the correlation between telomerase activity and NPC (nasopharyngeal carcinoma). In this study, telomerase activation was determined using the TRAP (telomerase repeat amplification protocol) assay in 62 nasopharyngeal biopsies (25 NPC, 25 non-malignant nasopharyngeal lymphoid tissues, 12 post-irradiated nasopharyngeal tissues). The results showed that strong telomerase activity was present in both NPC and non-malignant nasopharyngeal biopsies. Post-irradiated nasopharyngeal samples had a significantly lower telomerase activity than NPC and non-malignant nasopharyngeal lymphoid tissues. It is well known that nasopharyngeal tissue is infiltrated by numerous lymphocytes, which might retain telomerase activity. Therefore, the finding that the telomerase activation was lowest in post-irradiated nasopharyngeal tissues is reasonable because of the destruction of activated lymphocytes and NPC by radiation. NPC biopsies with positive lymph node involvement exhibited higher levels of telomerase compared to those without lymph node involvement. Our data indicate a positive association between telomerase activity and tumor potential for lymphatic spreading in limited local tumors. In addition, telomerase activity may be useful as a diagnostic marker in the detection of tumor cells in recurrent NPC, but not in primary NPC.

Respiratory syncytial virus infection of human respiratory epithelial cells enhances inducible nitric oxide synthase gene expression.

The induction kinetics of the mRNA of interferon regulatory factor 1 (IRF-1), inducible nitric oxide synthase (iNOS), and proinflammatory cytokines in respiratory syncytial virus (RSV)-infected human type 2 alveolar epithelial cells (A549 cells) were analyzed semiquantitatively by RT-PCR. RSV enhanced IRF-1 and iNOS mRNA expression as early as 4 h after RSV infection and this enhancement lasted several hours. No IFN-gamma gene expression was observed during the whole course of the infection. Expression of IFN-beta, IL-1beta, and TNF-alpha genes was observed slightly at 4 h and became marked 7 h after infection. Addition of neutralizing antibodies to these cytokines to the culture had no effect on the induction of iNOS mRNA. The iNOS transcriptional activity in RSV-infected cells was significantly enhanced by an exogenous cytokine mixture (IL-1beta, TNF-alpha, and IFN-gamma). An apparent nitric oxide (NO) production was identified only when cytokines were added together with RSV infection. A significant increase of iNOS gene expression was observed in nasopharyngeal exudate cells obtained from infants during the acute phase of RSV bronchiolitis. These observations suggest that RSV infection of human respiratory epithelial cells induces the iNOS gene both in vitro and in vivo; this induction may occur rather promptly and involves transcriptional activator IRF-1 induced by the RSV infection itself. The iNOS gene, which is initially induced by RSV infection, may be further enhanced in a paracrine fashion by proinflammatory cytokines released by infection-activated inflammatory cells.

Bordetella pertussis adenylate cyclase activity in nasopharyngeal aspirates for rapid diagnosis of whooping cough in relation to culture and serology.

Adenylate cyclase activity, measured in 201 nasopharyngeal aspirates from patients presenting with own or parental suspicion of whooping cough, was compared to diagnosis made by culture and by serology in the culture negative cases. The median amount of cyclic AMP in samples from culture negative patients (n = 145) was 0.60 pmoles which differed significantly (p less than 0.001) from the median value 3.28 in samples from culture positive patients (n = 56). The median value 0.70 pmoles of cyclic AMP in samples from culture negative patients who were positive by serology (n = 54) did not differ significantly from the value of 0.57 pmoles in samples from serology negative patients (n = 91). With a limit for positive cyclic AMP set at 2 pmoles, 45 samples were positive. The sensitivity of the assay was 66% (37/56) in culture positive patients while the specificity was 93% (85/91) in the serology negative patients. The positive predictive value for the c-AMP test was 82% (37/45) in relation to culture and 87% (39/45) in relation to culture and/or serology. The results confirmed that measurement of adenylate cyclase activity in nasopharyngeal aspirates by an 1-h incubation method can serve as an early and rapid diagnostic method of pertussis infection. The low sensitivity of the c-AMP assay in samples from serology positive but culture negative patients indicates however, that this assay will have to be supplemented by serology for a high diagnostic sensitivity in all cases of pertussis.

Variation in inducibility of cytochrome P-450c and aryl hydrocarbon hydroxylase in rat liver, lung, kidney, pancreas and nasopharynx.

The effect of 3-methylcholanthrene (MC) treatment on the cytochrome P-450c content of various rat tissues was examined by measuring the level of immunodetectable P-450c in conjunction with its aryl hydrocarbon hydroxylase (AHH) activity. Immunoblots revealed that P-450d was induced in the nasopharynx and pancreas in addition to its previously reported induction in the liver, lung and kidney. In contrast to P-450c, induction of the immunorelated P-450d was observed only in the liver. The specific content of immunodetected P-450c in the tissue homogenates decreased in the order: liver, nasopharynx, pancreas, lung, kidney. The corresponding AHH activities decreased in the order: liver, kidney, lung, nasopharynx, pancreas. The ratio of AHH activity to P-450c content varied widely among tissues: ratios of 37.2:1.7:0.47:0.04:0.02 were obtained for the kidney, liver, lung, nasopharynx and pancreas, respectively. The absence of a direct relationship between the levels of AHH and P-450c indicates that the extrahepatic activity may partially derive from P-450 forms other than P-450c and/or the specific activity of P-450c varies among different tissues.