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Objectives: To investigate the incidence of levofloxacin resistance in Streptococcus pneumoniae isolates cultured by Lancashire Teaching Hospitals NHS Foundation Trust (LTHTR), and detect cases of in vivo resistance development., Methods: During the study period (September 2004-February 2007), isolates of S. pneumoniae cultured by the LTHTR microbiology laboratory were examined by Etest to determine MICs of levofloxacin. Isolates from patients in whom there was a shift towards colonization with S. pneumoniae of reduced levofloxacin susceptibility were further characterized by serotyping, multilocus sequence typing (MLST) and sequencing of parC and gyrA genes., Results: Eight hundred and sixty-five isolates were collected; however, 772 isolates from 652 patients were recoverable; 412 (53.4%) came from hospitalized patients, 12 (1.6%) were resistant to levofloxacin according to the BSAC breakpoint (>2 mg/L) and 29 (3.8%) had MICs at the breakpoint (MIC = 2 mg/L). Of six patients in whom there was a shift towards isolates with reduced levofloxacin susceptibility, five had acquired new distinct strains. One patient, who had a parC mutation (Ser79Phe) in the original susceptible isolate and an additional second-step mutation in the gyrA gene (Ser81Phe) of the later resistant one, had isolates belonging to the same pneumococcal clone., Conclusions: S. pneumoniae resistance to levofloxacin was uncommon and we managed to identify only one case of probable in vivo resistance development in the 2.5 years of the study. Strain replacement accounted for the majority of incidences where there was an apparent shift towards colonization with isolates of reduced levofloxacin susceptibility.

Objective: The aim of the study was to determine whether Nasal Provocation Tests (NPT) could help in the diagnosis of occupational rhinitis (OR)., Methods: Changes in nasal airway resistance (NAR), measured by posterior rhinomanometry during specific nasal challenge associated with per and post test clinical scores, were compared to a prior probability, based on the patient's history, determined by occupational physicians, in 41 hairdressers and 33 bakers referred for suspected OR., Results: A DeltaNAR >or= 150% defined the positivity of the NPT. DeltaNAR demonstrated 50% sensitivity and a 86% specificity in hairdressers and a 95% sensitivity with 100 % specificity in bakers. DeltaNAR presented significant positive correlations with both per (p = 0.0003, r = 0.48) and post test clinical scores (p < 0.005, r = 0.39). The addition of clinical scores increased the sensitivity to 100% in hairdressers with 81% specificity., Conclusions: The NPT constitutes a safe procedure of nasal reactivity with good levels of sensitivity and specificity in both hairdressers and bakers when nasal resistance and clinical scores are taken into account.

The clinical activity of the preimplantation genetic diagnosis (PGD) at Erasme Hospital was carried out since September 1999 for a 47,XYY patient. Up to 31 December 2007, 79 PGD cycles were carried out (45 couples) for either chromosomal structural abnormalities (robertsonian and reciprocal translocations, pericentric inversion, deletion) (n = 41), chromosomal numerical abnormalities (47,XXY, 47,XYY, 45,X/46,XX) (n = 10), aneuploidy screening for recurrent miscarriages or multiple in vitro fertilization failures (n = 10), autosomal recessive diseases (cystic fibrosis and sickle cell anaemia) (n = 12) or X-linked disorders (n = 6). A total of 475 embryos were biopsied for genetic analysis. Unaffected embryos were transferred in 58 cycles, resulting in 22 pregnancies, including fifteen clinical pregnancies. Up to now, 9 babies were born and 3 pregnancies are still ongoing. After a learning curve, our current PGD efficiency shows a total pregnancy rate per transfer of 60.0% and an implantation rate of 28.6%. Each PGD result was confirmed by prenatal or postnatal diagnosis. Our data demonstrate that PGD is a valid technique to allow couples at high risk of transmitting a genetic abnormality to increase their chances of a healthy pregnancy, but considering its complexity, patients must be counselled and selected rigorously.

A literature survey of 106 articles shows that standard electrostimulation is an effective treatment of urinary incontinence and urinary disorders with bladder instability. Bladder inhibition is obtained by applying an alternating current at a frequency of between 5 and 25Hz and with a pulse width of between 0.2 and 0.5ms. In 19 articles (including three randomized, placebo-controlled studies), good results were achieved in 60 to 90% of cases, depending on the exact method (i.e. chronic or acute stimulation). Standard electrostimulation is also efficient in stress urinary incontinence. Urethral closure is obtained by applying a 50Hz alternating current with, again, a pulse width of between 0.2 and 0.5ms. In 21 articles (including two randomized, placebo-controlled studies), good results were achieved in 47.5 to 77% of cases. Treatments combining perineal rehabilitation (behavioural education, muscle improvement and biofeed-back) and electrostimulation are reported by 10 authors, with good results in 70 to 80% of cases after 10 to 12 sessions. According to 14 studies, neuromodulation is also an efficient treatment for complex urinary disorders, urgency, pollakiuria and dysuria. The recommended stimulation parameters are a frequency of 10 to 15Hz and a pulse width of 210ms. Good results were found in 34 to 94% of cases (with between 60 and 75% in an international, multicenter study). The overall results different from one study to another because of the need to harmonize stimulation parameters, choice of the study population and treatment follow-up with self-training programs and therapeutic education.

Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumoniae.

Objective: To describe the presentation, management, and outcome of 43 cases of pneumococcal-associated hemolytic uremic syndrome (P-HUS). An increased incidence of P-HUS has been noted in the United Kingdom between January 1998 and May 2005., Study Design: Cases with microangiopathic hemolytic anemia (Hb <10 g/dL with fragmented RBCs), thrombocytopenia (platelet count < 130 x 10(9)/L), acute renal impairment with oliguria and elevated plasma creatinine for age, confirmed or suspected pneumococcal infection and/or T-activation were included., Results: The median age at presentation was 13 months (range, 5-39 months). Pneumococcus was identified in 34 of 43 cases; T-activation was identified in 36 of 37 cases. Twelve strains were serotyped: serotypes 3 (n = 2), 6A (n = 2), 12F (n = 1), 14 (n = 1), 19A (n = 6). Empyema was present in 23 of 35 pneumonia cases; 13 cases had confirmed (9) or suspected (4) pneumococcal meningitis; 36 cases required dialysis (median, 10 days; range, 2-240 days). The mortality rate was 11%, comprising 3 cases of meningitis, 1 case of sepsis and 1 case of pulmonary embolism at 8 months follow up while on dialysis. Follow-up data were available for 35 of 38 patients who survived (median follow-up period, 9 months; range, 1-63 months); of these, 10 patients had renal dysfunction, 1 patient was dialysis-dependent, 5 patients had hypertension and 8 patients had at least 1+ proteinuria on urinalysis., Conclusion: P-HUS has increased compared with historic surveys (0/288 in 1985-1988; 8/413 in 1997-2001, 43/315 in 1998-May 2005). Early mortality remains high (8-fold that of VTEC-induced HUS). Ten of 12 strains identified would not be covered by the PCV7 vaccine.

Secondary hypokalemic periodic paralysis is rare. However, when it occurs, it is usually associated with Graves' disease and it is mostly diagnosed in Asiatic male patients. In this report, we analyze the diagnostic procedure in three cases of hypokalemic periodic paralysis associated with Graves' disease, diagnosed in three different emergency care units over the last 3 years. Three Caucasian men (26, 30, and 39 years of age) came to the emergency care unit for acute tetraparesia. One of them had suffered 15 stereotypical episodes of tetraparesia during the last 2 years. Goiter was present in each case. Kalemia was 1.8, 2.1, and 3 mmol/l, respectively. Triggering events such as considerable sugar intake and physical exercise were present in two cases. In all cases, low TSH levels, high FT4 levels, and anti-TSH receptor antibodies led to the diagnosis of Graves' disease. All patients were treated with potassium supplementation and neomercazole. Outcome was good with a follow-up of 6, 9, and 24 months, respectively. Emergency care practitioners should be aware of this diagnosis, which may affect Caucasian patients presenting with transient tetraparesia in a primary care unit.

The results of analysis of blood-meal remnants in unfed nymphs, despite relatively low detection levels (49.4%, n = 322), support the conclusion from an earlier study that small rodents are relatively unimportant as reservoir hosts of B. burgdorferi s.l. in this particular area, and suggest that songbirds (Passeriformes) are the most significant hosts in this respect. Tick (Ixodes ricinus) abundance was greater in the present study, but the overall Borrelia burgdorferi s.l.-infection prevalence of nymphal ticks was the same (12.2%), and the relative proportions of the various Borrelia burgdorferi s.l. genospecies were similar. B. garinii and B. valaisiana were the most frequent, B. burgdorferi s.s the least frequent, and B. afzelii of intermediate frequency. An unusually high proportion of nymphs (39%) with multiple infections of different B. burgdorferi genospecies was detected, and Borrelia spp. related to relapsing-fever spirochetes were detected in Ireland for the first time. The results of the present study contribute to the validation of blood-meal analysis as a means of determining the host origin of certain pathogens in unfed questing ticks, and raise some questions concerning the extent of B. burgdorferi s.l. host specificity.

XHRT1 is a member of the HRT/Hey protein subfamily that are known as Notch effectors. XHRT1 is expressed in the developing floor plate and encodes a basic helix-loop-helix (bHLH) transcription repressor. Here, we show that XHRT1 misexpression in the neural plate inhibits differentiation of neural precursor cells and thus may be important for floor plate cells to prevent them from adopting a neuronal fate. Deletion analysis indicated that inhibition of differentiation by XHRT1 requires the DNA-binding bHLH motif and either the Orange domain or the C-terminal region. XHRT1 could efficiently homodimerize and heterodimerize with hairy proteins. Among those hairy genes, Xhairy2b shows extensive overlap of expression with XHRT1 in floor plate precursors and may be a biologically relevant XHRT1 partner. Dimerization is mediated through both the bHLH and downstream sequences, the Orange domain being particularly important for the efficiency of the interaction. Using chimeric constructs between XHRT1 and the ESR9 bHLH-O protein that does not interact with Xhairy1 and Xhairy2b, we found that both the bHLH domain and downstream sequences of XHRT1 were required for heterodimerization with Xhairy2b, while only the XHRT1 sequences downstream of the Orange domain are required for the interaction with Xhairy1. Together, these results suggest that XHRT1 plays a role in floor plate cell development and highlight the importance of the Orange and downstream sequences in dimerization and in the selection of the bHLH partners.

Hairy-related transcription factors (HRTs) constitute a recently identified subfamily of basic-helix-loop-helix transcription factors containing an Orange domain (bHLH-O factors). As compared to the related HES proteins, HRTs exhibit distinct DNA-binding activities in vitro and the molecular mechanisms underlying their transcriptional activity remain poorly understood. We have identified here the sequence "ggCACGTGcc" as predominant binding site for Xenopus HRT1 (XHRT1). In transiently transfected 3T3 cells, XHRT1 represses the expression of a luciferase reporter gene under the control of multimerized XHRT1 binding sites. Deletion analysis indicated that repression by XHRT1 requires the presence of the DNA-binding bHLH motif and the Orange domain. However, the presence of the sequence motif YRPWGTEIGAF located at the very C-terminus of XHRT1 is dispensable. Accordingly, the groucho co-repressor, which is known to mediate transcriptional repression by HES factors through binding their C-terminal WRPW sequence, does not recognize the related YRPW motif present in the C-terminal part of XHRT1 significantly in vitro. As the C-terminus of HRTs is well conserved, our observation indicates that this part of HRTs, unlike the corresponding part of HES proteins, does not recruit the groucho co-repressor efficiently.

Primary microcephaly (microcephalia vera) is a developmental abnormality resulting in a small brain, with mental retardation. It is usually transmitted as an autosomal recessive trait, and six loci have been reported to date. We analyzed a translocation breakpoint previously reported in a patient with apparently sporadic primary microcephaly, at 1q31, where locus MCPH5 maps. The patient was lost to follow-up, and we sampled a maternal aunt who carried the familial translocation. FISH analyses showed that the insert of BAC clone RP11-32D17 spanned the breakpoint. The breakpoint was further located within a fragment of this insert corresponding to intron 17 of the ASPM gene, resulting in a predicted transcript truncated of more than half of its coding sequence. It is very likely that the proband carried a second ASPM mutation in trans, but he was not available for sampling and hence we could not confirm this hypothesis. Our observation adds to the mutation spectrum of ASPM in primary microcephaly, and is to our knowledge the second example of a constitutional, reciprocal translocation responsible for a bona fide autosomal recessive phenotype.

Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity., (Copyright 2003 Wiley-Liss, Inc.)

In this study, we have developed molecular methods for the identification of reservoir hosts of sylvatic tick-borne zoonoses. The methods are based on the analysis of the blood meal remnant in the tick gut and include detection of pathogens and identification of the host origin of the blood meal. For host identification, a universal primer pair was used to amplify part of the vertebrate 18S rRNA gene followed by reverse line blot hybridization using subgroup-specific probes. Analyses of DNA from whole blood of vertebrates identified the correct subgroup of a broad range of vertebrate species (e.g., Ruminantia, Leporidea, Canidae, Murinae, Arvicolinae, Insectivora, Galliformes, Passeriformes) using probes based on the 18S rDNA sequences. Host DNA in the remnants of larval blood meals was detected in the gut of Ixodes ricinus nymphs maintained under natural conditions up to 9 mo after molting. For pathogen identification, a multiplex polymerase chain reaction was used that targeted parts of the 18S rRNA gene of piroplasm protozoa, the 16S rRNA gene of bacteria, and the intergenic spacer of the Borrelia burgdorferi genospecies complex. The utility of both methods was demonstrated under laboratory conditions by detecting Babesia microti (Franca) and gerbil DNA in 3-mo-old I. ricinus nymphs that had fed on B. microti-infected gerbils as larvae, and under field conditions by analyzing unfed ticks that were collected in a forest. The field study showed that the majority of ticks had fed on ruminants or birds and few on rodents, which is in accord with our knowledge of the fauna in this forest. Few pathogens were detected but the discovery of Borrelia valaisiana and B. burgdorferi s.s. in ticks that had fed on deer and Borrelia afzelii in a tick that had fed on a bird raises questions about the mode of transmission of these spirochetes and possibly about their host specificity.

We have isolated a Xenopus homologue of the mammalian hairy and Enhancer of split related gene HRT1. XHRT1 expression in late gastrula and early neurula embryos is restricted to two stripes of cells in the medial neural plate and in dorsal endodermal cells. At later stages, XHRT1 is expressed in the floor plate, in hypochord cells and in the somitogenic and anterior presomitic mesoderm. By tailbud stage, XHRT1 is also highly expressed in the dorsal hindbrain, telencephalon and eye vesicles, olfactory placodes, pronephros, branchial arches and tail fin. We also show that XHRT1 expression in medial neural cells is induced by Notch signaling and that there are differences in the way XHRT1 and other H/E(spl) genes are regulated.

Background: Smooth muscle cells (SMCs) play a key role in intimal thickening in atherosclerosis and restenosis. The precise signaling pathways by which the proliferation of SMCs is regulated are largely unknown. The TR3 orphan receptor, the mitogen-induced nuclear orphan receptor (MINOR), and the nuclear receptor of T cells (NOT) are a subfamily of transcription factors belonging to the nuclear receptor superfamily and are induced in activated SMCs. In this study, we investigated the role of these transcription factors in SMC proliferation in atherogenesis., Methods and Results: Multiple human vascular specimens at distinct stages of atherosclerosis (lesion types II to V by American Heart Association classification) derived from 14 different individuals were studied for expression of these transcription factors. We observed expression of TR3, MINOR, and NOT in neointimal SMCs, whereas no expression was detected in medial SMCs. Adenovirus-mediated expression of a dominant-negative variant of TR3, which suppresses the transcriptional activity of each subfamily member, increases DNA synthesis and decreases p27(Kip1) protein expression in cultured SMCs. We generated transgenic mice that express this dominant-negative variant or full-length TR3 under control of a vascular SMC-specific promoter. Carotid artery ligation of transgenic mice that express the dominant-negative variant of TR3 in arterial SMCs, compared with lesions formed in wild-type mice, results in a 3-fold increase in neointimal formation, whereas neointimal formation is inhibited 5-fold in transgenic mice expressing full-length TR3., Conclusions: Our results reveal that TR3 and possibly other members of this transcription factor subfamily inhibit vascular lesion formation. These transcription factors could serve as novel targets in the treatment of vascular disease.

We describe here a selection strategy allowing the cloning of sequences that contain a functional nuclear targeting signal. Our method relies on the use of green fluorescent protein fusion proteins to identify nuclear targeting sequences. Transfected cells expressing nuclear protein fusions were isolated on the basis of their nuclear fluorescence using flow cytometry and the transfected DNAs were recovered after bacterial transformation with total DNA from pools of sorted cells. Starting from a cDNA expression library, in which only 1% of the expressed proteins were nuclear, we obtained a 70-fold enrichment in nuclear protein-encoding clones after a single round of selection. Among the 63 clones that have been partially sequenced to date, 25 (40%) corresponded to known nuclear proteins and 13 (20%) to previously uncharacterized sequences. Despite their ability to target the green fluorescent protein marker to the cell nucleus, about half of the cloned sequences did not encode canonical basic or bipartite nuclear localization signals. The method can thus be applied to the large-scale cloning of functional nuclear targeting sequences, which opens the way to a wide investigation of nuclear import mechanisms and to the identification of previously unknown nuclear proteins., (Copyright 2000 Academic Press.)

To assess the Lyme borreliosis vector population density we set up a methodology for sampling the Ixodes ricinus L. population host questing on the vegetation. We focused on the collection of the nymphal stage, which is the principal stage of disease transmission to humans. This study was carried out in Rambouillet forest (Yvelines, France) where seven study areas were demarcated. These areas are maximally homogeneous for plant species using a finer scale than the phytosociological classification as defined by the method of landscape diagnostics. Out of 23 collections performed from March 1997 to May 1998, 2,906 I. ricinus nymphs were collected. The sampling technique chosen was the cloth lure technique. The technical parameters were studied and fixed (cloth type, cloth size, sample size, researcher position). It appeared that toweling was the best cloth type to optimize the number of ticks collected; the position of the researcher had no effect on tick samples. To satisfy the criteria for correct sampling, we studied representativity, randomness, and nonselectivity of our methodology. The spatial distribution of nymphs in a homogeneous area was close to random and thus very few subsamples were needed to obtain a relative density which was representative. No significant differences were found between random samples and following transect samples; and nonselectivity was totally satisfied because we only worked on questing nymphs. We grouped the samples that presented no significant differences to attribute a density index, which varied from 0 to 5. This methodology, applied with the same parameters, offers potential for producing comparable results from studies in different geographical areas and at different times of the years.

To determine whether deer may play a role in the cycle of the Lyme disease spirochete Borrelia burgdorferi, we sought evidence for the presence of the pathogen in skin of deer and roe deer. Biopsies of 2 mm3 were taken at four different levels from nail to tarsus. A total of 50 animals (200 biopsies) were shot in the Lyme disease foci of Rambouillet during the hunting season 1995-1996 and 1996-1997, from the beginning of November to the end of February. Borrelia burgdorferi s.l. DNA was detected by PCR in 18 biopsies from 14 animals (28%). Borrelia burgdorferi s.str. was predominant (50%), followed by B. garinii (30%) and B. afzelii (10%). Multiple infections were detected in four animals: same species at different levels or two different species from the same biopsy or from different biopsies from the same foot. A total of 125 biopsies were cultivated on BSKH medium. Cultures at 160 days revealed immobile spiralled forms in 10 cultures. One, from a deer killed at the end of December, was confirmed by PCR as B. burgdorferi s.str. These results, frequency of detection of spirochetes by PCR in the skin, multiple infections and alive spirochetes in biopsies taken out side the season of activity of ticks strongly suggest an affinity of Lyme disease spirochetes for skin of cervids.

The N-terminal end of thyroid transcription factor-1 (TTF-1) homeodomain is composed of a stretch of five basic amino-acids that is conserved in both POU- and NK2-class homeodomains and constitutes a functional nuclear localization signal. By analyzing the cellular distribution of fusion proteins, composed of a jellyfish green fluorescent variant and different parts of TTF-1, we show here that the presence of this basic sequence is not sufficient by itself to confer complete nuclear accumulation. By mutagenesis, we identified a second region located in the center of the DNA recognition helix of the homeodomain that is also able to specify a predominantly nuclear localization of the chimeric proteins, independently of the presence of the basic NLS. The destruction, by mutagenesis, of both the basic stretch and the motif in the DNA recognition helix led to the total loss of nuclear accumulation, indicating that complete nuclear accumulation of TTF-1 results from the concerted action of these two proteic signals. Both of the regions of the homeodomain that are involved in nuclear targeting also encompass critical amino-acids responsible for DNA binding site recognition, as evidenced by the loss of DNA binding activity in vitro upon mutagenesis. Specifically, residues in the central part of the DNA recognition helix are involved in contacting bases in the major groove of DNA and are the most conserved in homeodomain proteins, suggesting that this part of the homeodomain could play a general role in the nuclear localization of members of this family of proteins.

Based on the predictive analysis of the cellular protein content from the complete genome sequence of Helicobacter pylori, discrepant results were previously reported concerning the occurrence of a protein kinase in this bacterium. To solve this ambiguity, we have directly assayed cellular extracts for their capacity of phosphorylating endogenous proteins. At least eight different proteins, ranging from 24 to 200 kDa, were found to be phosphorylated to a varying extent. Individual measurement of their phosphoamino acid composition showed that they all were modified at serine residues. These data indicate that H. pylori does contain a protein-serine kinase activity.

The Rambouillet Forest, a Lyme disease-endemic area near Paris, France, was surveyed from September 1994 to October 1995 to determine the risk periods and zones for humans. Firstly, during the period of Ixodes ricinus activity, abundance of nymphs is greater in spring than in autumn. Secondly, we observed significant variation in nymphal abundance between zones according to the density of cervids. The polymerase chain reaction (PCR) was used to detect DNA of Borrelia burgdorferi sensu lato in 461 unfed nymphs. DNA was detected in 38 nymphs (8.2%). By genospecific PCR based on the OspA gene, we detected the three pathogenic spirochetes with occurrences of 10.3, 31.1 and 58.6 for B. burgdorferi s.s., Borrelia garinii and Borrelia afzelii, respectively, indicating that B. afzelii is probably the main Borrelia species in the Rambouillet Forest. Finally, 11.5% of positive nymphs exhibited a double infection. Infection rates of I. ricinus nymphs by B. burgdorferi s.l. were not significantly different throughout the year for a given area, indicating that the risk periods of acquiring Lyme disease are mainly linked to nymph activity and correspond to spring and autumn. Likewise infection rates of nymphs were not significantly different between zones with a high density of deer (more than 100 animals per 100 ha) and zones with lower deer density (less than 20 animals per 100 ha). In addition to the role of deer as an amplifier of tick populations, these data indicate that zones with a high density of cervids should be considered as higher risk areas.

Nerve growth factor-induced gene-B (NGFI-B) is an immediate early gene first found as a part of the PC12 cell response to NGF (Milbrandt, J., Science 238 (1987) 797-799). We have previously reported that NGFI-B mRNA is strongly upregulated by thyroid-stimulating hormone (TSH) in dog thyrocytes in culture (Pichon et al., Endocrinology 137 (1996) 4691-4698). In this study, we have analyzed the regulation of NGFI-B mRNA expression by a variety of agents acting on thyrocytes proliferation and/or differentiation. We show that: (1) the induction of NGFI-B mRNA is stronger after stimulation of the cAMP cascade, but it is not restricted to this signaling pathway; (2) the powerful mitogens for thyroid cells EGF and HGF have little or no effect on NGFI-B mRNA induction; (3) NGFI-B mRNA is induced by anisomycin at a subinhibitory concentration for protein synthesis, and is superinduced by the combination of TSH and anisomycin; this treatment decreases the TSH-induced proliferation levels, but does not inhibit the induction of some differentiation markers; and (4) both in dog and in pig thyrocytes, NGFI-B mRNA induction is observed after a variety of treatments stimulating differentiation, but without proliferative effects. Our results therefore suggest that NGFI-B mRNA induction might not be related to TSH-induced thyrocyte proliferation, but could participate in the differentiation program triggered by TSH.

TTF-2 is a thyroid-specific winged-helix transcription factor which has been proposed to play a key role in the hormonal control of thyroglobulin and thyroperoxidase genes transcription in FRTL-5 cells. We have analyzed TTF-2 DNA-binding activity in primary cultures of dog thyrocytes maintained in control condition or in the presence of the cAMP agonist forskolin. Binding of 35S-labelled nuclear proteins to the TTF-2 recognition sequence identified the presence of two molecular species of 41.5 and 42.5 kDa. TTF-2 DNA-binding activity was clearly detectable in nuclear extracts from unstimulated cells and appeared increased in forskolin-treated cells. Thus, the presence of TTF-2 DNA-binding activity does not correlate with the cAMP-dependent activity of thyroglobulin and thyroperoxidase genes in this cell system. In addition, the mutation of the TTF-2 binding site in the thyroglobulin promoter resulted in a very reduced but still clearly cAMP-dependent promoter activity when assayed by transient expression in the same cells. These results do not support a dominant role for TTF-2 in the cAMP-dependent control of thyroglobulin gene transcription in primary cultured thyrocytes.

After a short historical presentation of the discovery of the pathogen and its vector, the authors present the current data on bacterial and acarologic taxonomy. Then they describe their results to assess the mechanisms of circulation of the bacteria in the forests of Ile-de-France, particularly in the forest of Rambouillet. The combined study of abundance and infection frequency of the vectors, small mammals and cervids leads to the characterization of periods and areas of higher risk. The risk periods correlate with high density of I. ricinus nymphs. The risk areas correspond to those of high density of cervids. The role of reservoir of small mammals is confirmed, to the one of large mammals, so debated, is clearly demonstrated.

Thyroid transcription factor I (TTF-1) plays a critical role in thyroid organogenesis and in the control of expression of several thyroid-specific genes, like those coding for thyroglobulin and thyroperoxidase. We have expressed the isolated DNA-binding homeodomain of TTF-1 in cultured thyroid cells by transient transfection. A specific reduction in the activity of co-transfected thyroglobulin and thyroperoxidase promoters was observed in the presence of the isolated TTF-1 homeodomain, as compared to their activity measured in the presence of a mutated homeodomain unable to bind DNA. The activity of the SV40 early promoter, used as a control, was only marginally affected in these experiments. The transactivation-deficient form of TTF-1 described here may thus be used for investigating other cellular processes that are dependent on TTF-1 transcriptional activity.