Metabotropic glutamate 7 receptor subtype modulates motor symptoms in rodent models of Parkinson's disease.

Metabotropic glutamate (mGlu) receptors modulate synaptic transmission in the central nervous system and represent promising therapeutic targets for symptomatic treatment of Parkinson's disease (PD). Among the eight mGlu receptor subtypes, mGlu7 receptor is prominently expressed in the basal ganglia, but its role in restoring motor function in animal models of PD is not known. The effects of N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), the first selective allosteric activator of mGlu7 receptors, were thus tested in different rodent models of PD. Here, we show that oral (5 mg/kg) or intrastriatal administration (0.1 and 0.5 nmol) of AMN082 reverses haloperidol-induced catalepsy in rats. AMN082 (2.5 and 5 mg/kg) reduces apomorphine-induced rotations in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats. In a more complex task commonly used to evaluate major akinetic symptoms of PD patients, 5 mg/kg AMN082 reverses the increased reaction time to respond to a cue of bilateral 6-OHDA-lesioned rats. In addition, AMN082 reduces the duration of haloperidol-induced catalepsy in a mGlu7 receptor-dependent manner in wild-type but not mGlu7 receptor knockout mice. Higher doses of AMN082 (10 and 20 mg/kg p.o.) have no effect on the same models of PD. Overall these findings suggest that mGlu7 receptor activation can reverse motor dysfunction associated with reduced dopamine activity. Selective ligands of mGlu7 receptor subtypes may thus be considered as promising compounds for the development of antiparkinsonian therapeutic strategies.

mGluR7 facilitates extinction of aversive memories and controls amygdala plasticity.

Formation and extinction of aversive memories in the mammalian brain are insufficiently understood at the cellular and molecular levels. Using the novel metabotropic glutamate receptor 7 (mGluR7) agonist AMN082, we demonstrate that mGluR7 activation facilitates the extinction of aversive memories in two different amygdala-dependent tasks. Conversely, mGluR7 knockdown using short interfering RNA attenuated the extinction of learned aversion. mGluR7 activation also blocked the acquisition of Pavlovian fear learning and its electrophysiological correlate long-term potentiation in the amygdala. The finding that mGluR7 critically regulates extinction, in addition to acquisition of aversive memories, demonstrates that this receptor may be relevant for the manifestation and treatment of anxiety disorders.

Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1)).

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

Unexpected thymic hyperplasia in transgenic mice harboring a neuronal promoter fused with simian virus 40 large T antigen.

The hypothalamic peptide growth hormone-releasing factor (GRF) regulates the secretion and production of growth hormone from the anterior pituitary (M. C. Gelato and G. R. Merriam, Annu. Rev. Physiol. 48:569-591). To study GRF gene regulation, transgenic mice were generated that harbor the human GRF promoter fused to the coding sequences from the simian virus 40 early region. These mice had normal hypothalamic functions but unexpectedly suffered from severe thymic hyperplasia. Immunohistochemical analysis revealed that large T antigen was expressed in the thymic epithelial cells. These cells have endocrine properties and are known to produce thymic hormones [corrected]. The thymic hyperplasia was the apparent consequence of inappropriate production of T-cell maturation factors by epithelial cells and could involve increased self renewal of apparently normal T stem cells in the thymus.

Mice transgenic for the human carcinoembryonic antigen gene maintain its spatiotemporal expression pattern.

The tumor marker carcinoembryonic antigen (CEA) is predominantly expressed in epithelial cells along the gastrointestinal tract and in a variety of adenocarcinomas. As a basis for investigating its in vivo regulation and for establishing an animal model for tumor immunotherapy, transgenic mice were generated with a 33-kilobase cosmid clone insert containing the complete human CEA gene and flanking sequences. CEA was found in the tongue, esophagus, stomach, small intestine, cecum, colon, and trachea and at low levels in the lung, testis, and uterus of adult mice of independent transgenic strains. CEA was first detected at day 10.5 of embryonic development (embryonic day 10.5) in primary trophoblast giant cells and was found in the developing gut, urethra, trachea, lung, and nucleus pulposus of the vertebral column from embryonic day 14.5 onwards. From embryonic day 16.5 CEA was also visible in the nasal mucosa and tongue. Because this spatiotemporal expression pattern correlates well with that known for humans, it follows that the transferred genomic region contains all of the regulatory elements required for the correct expression of CEA. Furthermore, although mice apparently lack an endogenous CEA gene, the entire repertoire of transcription factors necessary for correct expression of the CEA transgene is conserved between mice and humans. After tumor induction, these immunocompetent mice will serve as a model for optimizing various forms of immunotherapy, using CEA as a target antigen.

The murine rac1 gene: cDNA cloning, tissue distribution and regulated expression of rac1 mRNA by disassembly of actin microfilaments.

A 886 bp cDNA encoding the murine rac1 protein has been isolated. The abundance of rac1 mRNA was determined in fourteen tissues from both mouse and pig. The mRNA 5' non-coding sequence is very rich in G + C, and has the potential to form several stable secondary structures. In addition, this region contains a putative open reading frame of 57 amino acids. Disruption of the actin microfilament network by cytochalasin B in LLC-PK1 cells results in down regulation of rac1 mRNA. In agreement with the proposed role of rac1 in exocytosis, these results could explain the inhibitory effect of cytochalasin B on secretory processes.

Transgenes encoding mutant simian virus 40 large T antigens unmask phenotypic and functional constraints in thymic epithelial cells.

Transgenes encoding simian virus 40 (SV40) T antigen (Tag) can cause hyperplastic or tumorigenic lesions of desired but also of unforeseen cellular origin. Unexpectedly the human growth hormone-releasing factor (GRF) gene promoter directs expression of SV40 Tag specifically in thymic epithelial (TE) cells. Expression starts in the neonate, in which GRF-Tag+ cells display strict numerical and spatial constraints. Tag supersedes mechanisms that constrain these features and GRF-Tag mice develop thymic hyperplasia. To characterize GRF-Tag+ TE cells and their putative normal counterparts we compared phenotypic and functional effects caused by transgenes encoding mutant large T antigens. This strategy is applicable to any situation in which T antigen is used to alter development. One large Tag mutant (K1 + 5080) does not cause thymic hyperplasia. GRF-Tag (K1 + 5080)+ TE cells display strict temporal and spatial constraints throughout life. TE cells expressing other mutant large T antigens that cause thymic hyperplasia do not obey these rules and reveal that phenotypically distinct GRF-Tag+ TE-cell stages exist in vivo. Analysis of conditional immortal GRF-Tag(tsA58)+ TE cells expressing a temperature-sensitive large Tag shows that large Tag blocks differentiation in these cells. Phenotype and functions in these cells are regulated by cellular differentiation and interleukin 4 (IL-4).

Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7.

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7(-/-)). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7(-/-), but not in mGluR7(+/-), mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7(-/-) mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.

Evidence of blood-brain barrier alteration and activation in HIV-1 gp120 transgenic mice.

OBJECTIVE: To verify whether HIV envelope protein gp120 changes the blood-brain barrier in vivo, as a fundamental mechanism of early central nervous system damage by HIV-1. DESIGN: Analysis of the functional integrity and immune activation of the blood-brain barrier in brains of HIV-1 gp120 transgenic mice secreting circulating gp120 at levels similar to those detected in AIDS patients. METHODS: Number of vessels/mm2 section area with perivascular albumin and percentage of vessels expressing adhesion molecules (ICAM-1 and VCAM-1) were determined by immunohistochemistry in frozen brains from autopsied transgenic and non-transgenic mice. The percentage of vessels showing substance P immunoreactivity was also calculated, as this neuropeptide is known to mediate the increase in permeability of the rat brain endothelium in vitro caused by HIV-1 gp120. RESULTS: The number of vessels with albumin extravasation was significantly higher in transgenic than non-transgenic mice brains (P = 0.0003). A greater percentage of ICAM-1- and VCAM-1-positive brain vessels in transgenic than non-transgenic mice was shown (P = 0.0017 and P = 0.0008 respectively). Significant immunoreactivity for substance P was detected in brain vessels in transgenic mice and a significant correlation was found between the percentage of substance P-positive and ICAM-1-positive brain vessels (P < 0.0001) in transgenic mice. CONCLUSIONS: These findings demonstrate that HIV-1 gp120 is capable of changing and activating in vivo the vascular component of the blood-brain barrier.

Immunohistochemical localization of metabotropic glutamate receptors, mGluR7a and mGluR7b, in the central nervous system of the adult rat and mouse: a light and electron microscopic study.

The distributions of two alternative splicing variants of metabotropic glutamate receptor mGluR7, mGluR7a and mGluR7b, were examined immunohistochemically in the rat and mouse by using variant-specific antibodies raised against C-terminal portions of rat mGluR7a and human mGluR7b. Many regions throughout the central nervous system (CNS) showed mGluR7-like immunoreactivities (LI). The distribution patterns of mGluR7-LI in the rat were substantially the same as those in the mouse, although some species differences were observed in a few regions. Intense mGluR7a-LI was seen in the main and accessory olfactory bulbs, anterior olfactory nucleus, islands of Calleja, superficial layers of the olfactory tubercle, piriform cortex and entorhinal cortex, periamygdaloid cortex, amygdalohippocampal area, hippocampus, layer I of the neocortical regions, globus pallidus, superficial layers of the superior colliculus, locus coeruleus, and superficial layers of the medullary and spinal dorsal horns. The distribution of mGluR7b was more restricted. It was intense in the islands of Calleja, substantia innominata, hippocampus, ventral pallidum, and globus pallidus. The medial habenular nucleus also showed intense mGluR7a-LI in the rat but not in the mouse. For both mGluR7a- and mGluR7b-LI, localization in the active zones of presynaptic axon terminals was confirmed electron microscopically at synapses of both the asymmetrical and symmetrical types. It is noteworthy that mGluR7a-LI is seen preferentially in relay nuclei of the sensory pathways and that both mGluR7a- and mGluR7b-LI are observed not only in presumed glutamatergic axon terminals, but also in non-glutamatergic axon terminals including presumed inhibitory ones. Thus, mGluR7 may play roles not only as an autoreceptor in glutamatergic axon terminals, but also as a presynaptic heteroreceptor in non-glutamatergic axon terminals in various CNS regions.

Immunocytochemical localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of the rat.

This study evaluates the localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of rats using preembedding immunocytochemical methods. At the light microscopic level, punctate labeling was evident in layers Ia and Ib of the piriform cortex, and immunolabeled fibers were present in layers II and III. Following bilateral destruction of the olfactory bulb, the density of labeled puncta in layer Ia decreased. These results suggest that the receptor is present on the terminals of the lateral olfactory tract (LOT). Electron microscopic evaluation of layers Ia and Ib revealed that mGluR4a was localized in synaptic terminals in layers Ia and Ib. The terminals had clear, round synaptic vesicles and terminated on asymmetric synapses on dendritic spines and shafts. There was also immunolabeling of some dendritic profiles in layers Ia and Ib that were postsynaptic to unlabeled presynaptic terminals. These observations suggest that mGluR4a is present on presynaptic terminals in the layers of the piriform cortex that receive LOT and associational synapses. This is the same area in which previous studies have revealed the presence of mGluR7 and mGluR8, suggesting that all three receptors may be colocalized.

Developmental expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body of the rat.

A preembedding immunocytochemical method for light microscopy was used to study the postnatal development of expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body (MNTB) of the rat. Immunoreactivity for mGluR4a was localized in axonal endings wrapping the principal globular neurons in MNTB, known as calyces of Held. The percentage of calyces of Held immunoreactive for mGluR4a increased progressively from postnatal day 3 (PND3), showing the highest density of labeled calyces by PND9. From this postnatal age on, a gradual reduction in the number of mGluR4a-immunopositive calyces of Held was observed, reaching the lowest level of labeled profiles in adult tissue. The developmental expression of mGluR4a in calyces of Held correlates well with previous studies in young animals showing a modulation of synaptic neurotransmission by group III mGluRs in these giant excitatory synapses made on MNTB principal neurons. All these observations together suggest that the expression of mGluR4a mainly between PND7 and PND12 might be relevant to the maturation and modulation of synaptic transmission at the calyces of Held.

Age-specific differences in treatment and survival of patients with cervical cancer in the southeast of The Netherlands, 1986-1996.

Age at diagnosis has been proven to be an important determinant of the choice of initial treatment for several sites of cancer. Elderly patients are more likely to receive no treatment or less intensive treatment modalities. This study analysed the influence of age on treatment choice and survival in patients diagnosed with cervical cancer. This population-based study used data on 1176 new cases of invasive cervical cancer diagnosed in the period of 1986-1996 from three regional cancer registries in the Netherlands. All available information on treatment and survival (on 1 January 1998) was recorded. Relative survival rates were calculated according to the Hakulinen method. Relative risks (RR) for excess mortality due to the diagnosis of cervical cancer were calculated with a regression model for relative survival rates. Only 5% of the patients aged 70 years and older (n=224) were diagnosed with stage IA disease, compared with 11 and 30% of the patients aged 50-69 years and 49 years and younger, respectively. Almost 50% of the 70+ patients with stage IB-IIA were treated with radiotherapy as a single treatment modality, whereas 64% of the patients aged < or =49 years were treated with surgery alone. In all age groups, treatment for advanced stage disease (stage > or =IIB) was radiotherapy alone. No treatment was given to 10% of the patients aged 70 years and older, 5% of those aged 50-69 years and 1% of those aged 49 years and younger. Five-year relative survival was 69% (95% Confidence Interval (CI): 66-72%) and differed significantly (P=0.001) with age (70+ years: 49%; 50-69 years 58%; < or =49 years: 81%). Multivariate analyses on a subset of patients showed that age was not an independent prognostic factor, whereas stage and treatment modality were very important prognostic factors. Although elderly cancer patients were sometimes treated differently from younger patients, this was in accordance with the guidelines. Relative survival rates differed significantly by age. The multivariate analyses on the subset of patients also revealed that excess mortality increased with age. However, when adjustment was made for stage and treatment, this difference disappeared. The influence of treatment on survival is likely to be due to the selection of patients based on other characteristics, such as tumour volume, comorbidity and performance status.

Generation of chimeric monoclonal antibodies from mice that carry human immunoglobulin Cgamma1 heavy of Ckappa light chain gene segments.

Gene targeting in mouse embryonic stem (ES) cells was used to replace (i) the mouse immunoglobulin heavy chain (IgH) Cgamma2a gene segment (mCgamma2a) with the human Cgamma1 gene segment (hCgamma1), and (ii) the mouse immunoglobulin light chain (IgL) Ckappa gene segment (mC kappa) with its human counterpart (hC kappa). ES cells carrying these gene conversions were used to generate chimeric mice that transmitted the human alleles through the germ line. Mice homozygous for both gene alterations were generated by breeding. Serum from homozygous mutant mice contained comparable amounts of antibodies with chimeric kappa or mouse lambda light chains but only small fractions of basal serum IgG or antibodies elicited against immunizing agents contained chimeric heavy chains. A relative increase in immunogen-specific hCgamma1 antibodies was seen following immunization in combination with the saponin adjuvant QS-21. The effect of this was to shift the IgG1-dominated response to an IgG subclass profile that included significant amounts of IgG2a, IgG2b and IgG3 and chimeric IgG. The amounts of antibody secreted by hybridomas derived from mutant and wild-type mice were similar. Sequencing confirmed correct splicing of hCgamma1 and hCkappa gene segments to mouse J gene segments in hybridoma Ig gene transcripts. In conclusion, IgHhCgamma1/IgLhCkappa double mutant mice provide a useful animal model for deriving humanized antibodies with potential applications in immunotherapy and diagnostics in vivo as well as for investigating hCgamma1 associated functions.

A novel splice variant of a metabotropic glutamate receptor, human mGluR7b.

Two splice variants of the human metabotropic glutamate receptor 7, named hmGluR7a and hmGluR7b, were isolated from a human brain cDNA library. The isoforms differ by an out-of-frame insertion of 92 nucleotides close to the C-terminus of the hmGluR7 coding region, hmGluR7a has a length of 915 amino acids and represents the human homolog of the recently cloned rat mGluR7. hmGluR7b is seven amino acids longer and exhibits a novel C-terminus of 23 amino acids in length. RT-PCR analysis demonstrated the existence of mGluR7b transcripts in wild-type mouse brain and its absence in mGluR7 knockout mice. Northern blot analysis indicate that mGluR7 expression is developmentally regulated. It is expressed at high levels in human fetal brain and at a lower level in many regions of adult human brain. Stimulation of hmGluR7b with L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP) or L-glutamate in stably transfected Chinese hamster ovary (CHO) cells depressed forskolin-induced cAMP accumulation, whereas (1S,3R)-1-aminocyclopentane-1,3,-dicarboxylic acid ((1S,3R)-ACPD) and quisqualate (both at 1mM) had no significant effects. As described for rat mGluR7, the rank order of agonist potencies is: L-SOP, L-AP4 > L-glutamate > (1S,3R)-ACPD, quisqualate.

Characterization of uptake and compartmentalization of 3,5,3'-tri-iodothyronine in cultured neonatal rat cardiomyocytes.

The uptake of tri-iodothyronine (T(3)) in cultured neonatal rat cardiomyocytes was investigated and compared with the uptake of reverse T(3 )(rT(3)) and thyroxine (T(4)). Cellular compartmentalization of T(3) was studied by distinguishing T(3) activity associated with the plasma membrane from that in the cytosol or incorporated in the cell nucleus. T(3) and T(4) uptake displayed similar temperature dependencies which, in magnitude, differed from that of rT(3) uptake. T(3) uptake was Na(+ )independent, and sensitive to oligomycin and monodansylcadaverine (42-49% and 25% inhibition of 15-min cellular uptake respectively). Furthermore, T(3) uptake could be inhibited by tryptophan (20%) and tyrosine (12%), while 2-aminobicyclo[2,2,1]heptane-carboxylic acid had no effect. Co-incubation with tryptophan and oligomycin resulted in an additive inhibition of T(3) uptake (77%). We therefore conclude that (i) T(3) uptake is energy dependent, (ii) receptor-mediated endocytosis may be involved and (iii) the aromatic amino acid transport system T may play a role, while system L is not involved in T(3) transport in cardiomyocytes. Co-incubation with unlabeled iodothyronines showed that 3,3'-di-iodothyronine and T(3) itself were the most effective inhibitors of T(3) uptake (30% and 36% inhibition of 15-min cellular uptake respectively). At 15-min incubation time, 38% of the total cell-associated T(3) was present in the cytosol and nucleus, and 62% remained associated to the plasma membrane. Unidirectional uptake rates did not saturate over a free T(3) concentration range up to 3.9 microM. We have concluded that T(3) uptake in neonatal rat cardiomyocytes occurs by an energy- and temperature-dependent mechanism that may include endocytosis and amino acid transport system T, and is not sensitive to the Na(+) gradient. Elucidation of the molecular basis for the T(3) transporter is the subject of current investigation.

Uptake of tri-iodothyronine and thyroxine in myoblasts and myotubes of the embryonic heart cell line H9c2(2-1).

Uptake of tri-iodothyronine (T(3)) was compared with that of thyroxine (T(4)) in the embryonic heart cell line H9c2 (2-1). These cells propagate as myoblasts and form differentiated myotubes upon reduction of the serum concentration, as indicated by a 31-fold increase in creatine kinase activity. Protein and DNA content per well were around 2-fold higher in myotubes than in myoblasts. When expressed per well, T(3) and T(4) uptake were, compared with myoblasts, 1.9- to 2-fold and 3.1- to 4-fold higher in myotubes respectively. On the other hand, the characteristics of T(3) and T(4) uptake were similar in myoblasts and myotubes. At any time-point, T(4) uptake was 2-fold higher than that of T(3), and both uptakes were energy but not Na(+) dependent. T(3) and T(4) uptake exhibited mutual inhibition in myoblasts and myotubes: 10 microM unlabeled T(3) reduced T(4) uptake by 51-60% (P<0.001), while 10 microM T(4) inhibited T(3) uptake by 48-51% (P<0.001). Furthermore, T(3) and T(4) uptake in myoblasts was dose-dependently inhibited by tryptophan (maximum inhibition around 70%; P<0.001). Exposure of the cells to T(3) or T(4) during differentiation significantly increased the fusion index (35 and 40%; P < 0.01). Finally, both myoblasts and myotubes showed a small deiodinase type I activity, while deiodinase type II activity was undetectable. In conclusion, T(3) and T(4) share a common energy-dependent transport system in H9c2(2-1) cells, that may be important for the availability of thyroid hormone during differentiation.

Uptake of triiodothyronine and triiodothyroacetic acid in neonatal rat cardiomyocytes: effects of metabolites and analogs.

Cellular and nuclear uptake of [125I]tri-iodothyronine (T3) and [125I]triiodothyroacetic acid (Triac) were compared in cardiomyocytes of 2-3 day old rats, and the effect of thyroid hormone analogs on cellular T(3) uptake was measured. Cells (5-10 x 10(5) per well) were cultured in DMEM-M199 with 5% horse serum and 5% FCS. Incubations were performed for from 15 min to 24 h at 37 degrees C in the same medium, 0.5% BSA and [125I]T3 (100 pM), or [125I]Triac (240 pM). Expressed as % dose, T(3) uptake was five times Triac uptake, but expressed as fmol/pM free hormone, Triac uptake was at least 30% (P<0.001) greater than T3 uptake, whereas the relative nuclear binding of the two tracers was comparable. The 15 min uptake of [125I]T3 was competitively inhibited by 10 microM unlabeled T3 (45-52%; P<0.001) or 3,3'- diiodothyronine (T2) (52%; P<0.001), and to a smaller extent by thyroxine (T(4)) (27%; 0.05

Chemotherapy resistant ovarian cancer in carriers of an hMSH2 mutation?

Hereditary Non-Polyposis Colorectal Cancer (HNPCC, Lynch syndrome) is an autosomal dominant condition of cancer susceptibility with high penetrance, characterised by early onset of colon tumours as well as a variety of extracolonic tumours including ovarian cancer and, in particular, cancer of the endometrium. Germline mutations in one of five DNA-mismatch repair (MMR) genes (hMLH1, hMSH2, hMSH6, PMS1, PMS2) are known to cause HNPCC. To date, mutations in two of these genes (hMSH2 and hMLH1) are found in the majority of mutation positive families. Recent literature suggests that especially hMSH2 mutations are associated with extracolonic tumours. We describe two women from an HNPCC family carrying an hMSH2 mutation (deletion of exon 6 of this gene) who developed ovarian cancer. In these patients (full cousins) the ovarian cancers were noted for their aggressive development and rapid recurrence after surgical debulking and during regular multichemotherapy including Cisplatin. This report strengthens recent in vitro studies suggesting an involvement of MMR-gene mutations in ovarian cancer cell biology with decreased susceptibility to Cisplatin therapy. The possible implications for the therapy of ovarian cancer, the screening and genetic counselling of family members are discussed.

Composition of cervical smears in patients with and without a cervical ectropion.

AIMS: To test the hypothesis that absence of squamous cells in cervical smears obtained by an endocervical sampling technique is more prominent in patients with a cervical ectropion. METHODS: Prospective study exploring the relation between the composition of cervical smears obtained using an endocervical cotton swab in patients with (n = 188) and without (n = 341) a cervical ectropion. Subjects were 529 consecutive patients from whom a cervical smear was prepared at a university gynaecological clinic. RESULTS: In 7% of the endocervical samples no squamous cells were found. There was no correlation, however, between the presence or the size of an ectropion and the absence of squamous cells in those samples. CONCLUSIONS: It was confirmed that endocervical sampling alone is insufficient to obtain good quality cervical smears. The presence of an ectropion proved to be an unreliable predictor of the absence of squamous cells.