Immuno-PCR for the early serological diagnosis of acute infectious diseases: the Q fever paradigm.

To reduce the delay in diagnosis of Q fever, we have adapted the ultrasensitive immuno-PCR method for the detection of Phase II IgM anti-Coxiella burnetii. We compared its performance to ELISA, IFA and PCR using 31 acute Q fever sera and 50 control sera. The best sensitivity was obtained by iPCR (27 out of 31) followed by PCR (18 out of 31), ELISA (12 out of 31) and IFA (10 out of 31). A specificity of 92% was found by iPCR (3 false positive out of 40), 92% for ELISA (3 false positive out of 40) whereas PCR and IFA exhibited a specificity of 100%. Among the 31 Q fever sera, we compared the four methods for the detection of the early sera sampled during the two first weeks after the onset of symptoms and found a sensitivity of 90% by iPCR, 55% for PCR, 35% for ELISA and 25% for IFA. The results presented in this study suggest that iPCR is a promising, sensitive and specific method that can be used for the early diagnosis of acute Q fever and more generally for acute infections where traditional methods lack sensitivity.

The hypervirulent Coxiella burnetii Guiana strain compared in silico, in vitro and in vivo to the Nine Mile and the German strain.

OBJECTIVE: Q fever epidemic outbreaks have been reported in French Guiana and in The Netherlands. To determine whether the C. burnetii strains involved in these epidemics had a peculiar virulence pattern, we compared the pathogenicity of the Guiana and the German strain (a clone of The Netherlands strain), in silico, in vitro, and in vivo versus the Nine Mile strain. METHOD: The pan-genomes of the Guiana (Cb175), German (Z3055), and the referent Nine Mile (RSA 493) C. burnetii strains were compared. In vitro, the growth rate and the morphological presentation were compared. In vivo (SCID and Balb/c mice), weight loss, histological lesions, C. burnetii bacterial load in deep organs, and serological response were reported according to each C. burnetii strain studied. RESULTS: The Guiana strain had 77 times more missing genes and 12 times more unique genes than the German strain. The Guiana strain presented as large cell variants (LCVs) and led to the most pronounced fatality rate in SCID mice (100% at 4 weeks). The German strain presented as small cell variants (SCVs), and had an intermediate fatality rate (75% at 4 weeks). Both the Guiana and the German strains led to a significant higher serological response at 2 and 4 weeks post infection (p <0.05). CONCLUSION: The Guiana strain was the most virulent strain, followed by the German strain and the referent Nine Mile strain. Unique and missing genes could be implicated but further investigations are necessary to specify their role.

Changes in lipoxygenase activities in human erythroleukemia (HEL) cells during diosgenin-induced differentiation.

A human erythroleukemia (HEL) cell line was used as a model to study dynamic changes in human 12-, 15-, 5-lipoxygenases, five lipoxygenase activating protein (FLAP), and leukotriene A4 (LTA4) hydrolase gene expression during megakaryocytic differentiation induced by diosgenin (Beneytout, J.L., Nappez, C., Leboutet, M.J. and Malinvaud, G., Biochem. Biophys. Res. Commun., 207 (1995) 398-404). The study was performed at the transcriptional level: 12- and 5-lipoxygenase mRNAs, FLAP mRNA and LTA4 hydrolase mRNA were detected before and after diosgenin treatment in HEL cells while 15-lipoxygenase mRNA was undetected. When HEL cells were incubated with arachidonic acid, 5-, 12-, 15-hydroxyeicosatetraenoic acids (HETE) and LTC4 were synthesized. In contrast, the diosgenin treatment induced the suppression of 12-lipoxygenase activity and only 5-, 15-HETEs and LTC4 were synthesized.

trans,trans-2,4-decadienal: cytotoxicity and effect on glutathione level in human erythroleukemia (HEL) cells.

The effects of trans,trans-2,4-decadienal (DDE), an isomer of a lipid peroxidation product were investigated on the human erythroleukemia cell line (HEL TIB 180). DDE strongly inhibits cell growth and affects cell viability without any differentiating effects. DDE treatment of HEL cells leads to a marked variation of the cellular glutathione level (GSH) and is involved in the beginning of DNA fragmentation.

A plant steroid, diosgenin, a new megakaryocytic differentiation inducer of HEL cells.

We investigated the effect of plant steroids 5 alpha-spirosten-3 beta-ol (diosgenin), 5 alpha-spirostan-3 beta-ol (tigogenin) and 5 alpha-spirostan-3 beta-ol-12-one (hecogenin) on the human erythroleukemia cell line (HEL TIB 180) and found that diosgenin addition to HEL cell cultures induces morphological and biochemical changes characteristic for megakaryocyte cells. Diosgenin-treated cells exhibit, at the ultrastructural level, increases in size in cytoplasmic and nuclear complexity. At the biochemical level, we demonstrated that diosgenin-treated HEL cells increased glycoprotein Ib (GpIb) expression as previously described in the megakaryocytic differentiation of HEL cells induced by nanomolar dose phorbol myristate acetate treatment.