Search

The pathophysiology of takotsubo cardiomyopathy remains enigmatic. Here we attempted to define the link between the coronary arteries and the histopathological involvement of the left ventricle. We observed similarities and discrepancies between patients. All patients experienced stress prior to the event. We found a reduced coronary flow reserve in all patients and signs of hibernating myocardium on biopsy specimen. This raises a strong suspicion of stress-induced endothelial dysfunction with hibernating myocardium in the pathogenesis of this cardiomyopathy.

Myxoinflammatory fibroblastic sarcoma (MIFS) is a rare low-grade sarcoma of the distal extremities characterized by a myxohyaline stroma, a dense inflammatory infiltrate and virocyte- and lipoblast-like giant cells. Up to now, only two cases have been investigated cytogenetically, showing complex and heterogeneous karyotypes, in part with supernumerary ring chromosomes. We characterized two further cases of MIFS immunohistochemically and performed comparative genomic hybridization as well as DNA image cytometry analyses. Both tumors showed the characteristic histomorphological pattern of MIFS and were positive for Vimentin and CD68. Moreover, both cases presented aberrant karyotypes including distinct DNA copy number changes involving chromosome 7 and disclosed DNA aneuploidy.

Visceral pleural invasion (VPI) increases the T category of non-small cell lung carcinomas (NSCLCs) that otherwise only meet T1 criteria to T2. The American Joint Committee on Cancer provides no guidelines on what constitutes VPI. Penetration beyond the visceral pleural elastic layer (VPEL) has been proposed as the minimum criterion but is not internationally accepted. The purposes of this study were to elucidate current international practices regarding assessment of VPI and identify histologic features pathologists consider necessary for VPI. We examined responses to an online quiz consisting of 15 NSCLCs adjacent to or involving the visceral pleura. Of 103 participants from 22 countries, 84.5% were in academic practice; 42.7% had a subspecialty interest in pulmonary pathology. Interobserver percentage agreement about whether VPI was present, absent, or indeterminate ranged from 36.9% to 93.2% (mean, 73.0%). The K for participants for all quiz cases was 0.35. There was considerable diagnostic variability in cases with extensive pleural elastosis and when tumor cells were intermingled with the VPEL. It seems that the majority of participants consider penetration of the VPEL necessary and also sufficient to categorize VPI as present. However, the formation of internationally recognized guidelines for assessing VPI by NSCLC is likely to improve diagnostic consensus.

Presented here are three cutaneous sebaceous tumors (one carcinoma and two sebaceomas), each demonstrating a focal glandular pattern representing apocrine differentiation. The patients, two males and one female, each clinically presented with a small solitary nodule or tumor on the scalp. None of the patients had features of Muir-Torre syndrome. Surgical removal of the lesions was performed in all cases. None of the patients developed recurrence or metastasis after surgery (follow-up ranged from 18 to 24 months). The glandular areas represented a minor but significant component of the lesions and appeared as glands of various complexity, mostly as simple round or elongated tubular structures lined by a row of cuboidal to columnar cells with eosinophilic cytoplasm and round nuclei, with or without a distinct nucleolus. Decapitation secretion was evident but not prominent. In both sebaceomas, at least a portion of the glands had a peripheral small-cell layer that appeared similar to the basal/myoepithelial cells of normal eccrine and apocrine ducts. In some glands, the basal/myoepithelial cells seemed to have undergone hyperplasia, resulting in two or more rows of cells that even formed small islands, with an overall appearance reminiscent of basal cell hyperplasia in the prostate, arising in the basal layer of the prostatic glands. The descriptive terms seboapocrine carcinoma or seboapocrine sebaceoma are proposed for such lesions. These tumors may be viewed as rare histopathological variants of sebaceous carcinoma and sebaceoma, with a second type of differentiation along the lines of the folliculosebaceous-apocrine unit.

We report an investigation on a collection of multidrug-resistant Enterobacter cloacae isolates from a Hungarian neonatal intensive care unit. All the isolates (n=142) were examined by antimicrobial susceptibility testing and ERIC-PCR. The seven ESBL positive isolates (derived from six patients) made up a separated group with regard to their patterns by antimicrobial susceptibility testing and ERIC-PCR and were further tested by class-1-integron PCR and plasmid electrophoresis. The ESBL isolates were found indistinguishable in each of these laboratory tests, one genetic clone were revealed in the background of ESBL cases by PFGE. The ESBL positive isolates were proven to harbour a approximately 62 Md plasmid and two class-1 integrons (0.9 kb, 1.875 kb). With respect to their clinical relatedness and our laboratory findings there was a small outbreak caused by the ESBL clone. PCR-sequencing were performed on the outbreak strain and has revealed a blaSHV gene that encodes for an SHV-2a type ESBL enzyme. This is the first description of SHV-2a positive E. cloacae strains isolated in Hungary.

This Internet-based quiz (http://kathrin.unibas.ch/polyp/) tested the diagnostic variability of 168 pathologists in the diagnosis of 20 colorectal polyps on 3 representative images, including hyperplastic polyps (HPs), traditional serrated adenomas (TSAs), sessile serrated adenomas (SSAs), and tubulovillous adenomas (TVAs). Interobserver variability for each of the 20 lesions was significant and was most pronounced for SSAs. Correct answers were independent of the participant's experience with TVAs, HPs, and TSAs. Participants with gastrointestinal subspecialty training and those who had read a reference article on serrated polyps gave a significantly higher percentage of correct answers for SSAs. The nomenclature used for serrated polyps was generally inconsistent. Our results suggest significant shortcomings in the routine H&E diagnosis of serrated colorectal polyps. A diagnostically unifying concept for lesions of the serrated neoplasia pathway, standardization of nomenclature, training of pathologists, and possibly development of ancillary techniques are of paramount importance for accurate patient management.

The relationship between HER-2 overexpression and gene amplification is well evaluated in breast cancers but remains unclear or controversial in many other tumor entities. Therefore, we tested the HER-2 status in more than 120 different tumor entities. 5751 tumor samples were analyzed on TMAs by immunohistochemistry (Hercept-Test, DAKO) and fluorescence in situ hybridization (PathVysion, Abbott-Vysis) under highly standardized conditions. HER-2 overexpression (score 2/3+) and amplification occurred most often in breast cancers but was also seen in 18 other tumor entities including cancers of the urinary bladder (amplification in 14.3%, overexpression in 6.7%), stomach (8.3/4.9%), endometrium (6.6/6.8%), lung (2.8/3.1%) and ovary (2.3/1.2%). Remarkably, a strong association between overexpression and amplification was seen in all examined cancer entities. Trastuzumab therapy is highly efficient in HER-2 amplified breast cancer both in metastatic disease and as an adjuvant therapy. A variety of other tumor entities including frequent neoplasms and cancers with often limited therapeutic options have similar patterns of HER-2 alterations as observed in breast cancer (ie high overexpression due to high level gene amplification). Such tumor entities should be carefully evaluated for a possible utility of trastuzumab treatment.

Cell cycle and apoptosis regulatory proteins are markers of tumor progression in colorectal cancer. In a series of 1,274 mismatch repair-proficient colorectal cancers, immunohistochemical analysis of p21, p27, p53, and bcl-2 expression was performed using the tissue microarray technique. In univariate analysis, p21 expression was associated with tumor location and pN0; p27 expression with tumor location, lower tumor grade, early pT, and pN; p53 expression with tumor location; and bcl-2 with early pT and pN. Expression of p27 identified subgroups with worse prognosis in pT3 N0 and pT3 N+ patients. None of the 4 tumor markers were independent prognostic indicators of survival. The multimarker phenotypes p21/p27/p53 and p21/p27/bcl-2 were associated with survival. In mismatch repair-proficient colorectal cancer, p27 expression is associated with a better prognosis, and pT, pN, and vascular invasion are independent prognostic factors. In addition, multimarker phenotypes are useful to identify colorectal cancer subgroups with different prognoses.

Background: Equivocal atypia in respiratory cytology can be a diagnostic challenge. In such cases fluorescence in situ hybridization (FISH) may be used for the analysis of chromosomal aberrations and often allows a reliable distinction of benign and malignant cells., Methods: An online picture gallery of 30 respiratory cytologic preparations comprising 23 specimens with equivocal cytology as well as 5 positive and 2 negative controls was prepared (www.unibas.ch/patho/lungenzyto/loesung). The final diagnoses were confirmed by clinical follow-up or biopsy or both. Each of the illustrated cell groups was analyzed by multitarget FISH after PAP image capturing and automatic relocalization., Results: The online questionnaire was completed by 137 cytomorphologists from all continents. The control cases were assessed accurately to a significantly higher percentage than the equivocal cases. In equivocal cases participants more often made false-positive than false-negative diagnoses. In 2 patients with benign conditions (tuberculosis and pulmonary capillaritis) the rate of false-positive answers was remarkably high (31.4% and 62.8% respectively). The result of the 20 best-performing participants for the 5 cases with the highest percentage of inaccurate answers was not better than if they had chosen their answer by chance., Conclusions: These data illustrate that single cells or cell clusters of a subgroup of equivocal lung cytology are a diagnostic challenge even for highly experienced morphologists. Internet-based tests are able to reveal limitations of cytomorphology.

Urinary cytology is limited by high interobserver variability in the evaluation of cells with little atypia. We set up an online quiz on urinary cytology and tested the performance of 246 international participants. The quiz consisted of still images of 42 urinary specimens with equivocal morphologic features and 10 control cases with an unequivocal cytologic diagnosis. The nature of the cells on the 292 quiz images had been verified by multitarget fluorescence in situ hybridization in addition to the information obtained by cystoscopy, clinical follow up, and/or histologic examination. The original quiz cases and the percentage of answers given by the participants can be viewed at: http://kathrin.unibas.ch/urinzyto/. High-grade cancers were diagnosed correctly in 76.0% and low-grade cancers in only 33.9%. Remarkably, 54.5% of all participants misclassified decoy cells as malignant. This study shows that large-scale international online quizzes may be used to find educational deficits in cytopathology.

The category of equivocal respiratory cytology is a common diagnostic dilemma to both cytopathologists and clinicians. Chromosomal alterations are a hallmark of cancer but are rare or absent in benign conditions. The goal of this study was to test the ability of multitarget fluorescence in situ hybridization (FISH) for dissecting equivocal respiratory cytology into reactive and malignant categories. A consecutive series of 54 Papanicolaou-stained cytologic specimens of the lung was analyzed. The Papanicolaou-stained atypical cell groups were photographed, and the exact locations on the specimens were saved using automated stage and relocation software. The specimens were hybridized with a multitarget FISH probe that contains a mixture of fluorescent probes to the centromeric region of chromosome 6 and to the 5p15, 8q24 (site of the MYC gene) and 7p12 (site of the EGFR gene) loci. The hybridized atypical cells were selectively scored after relocation. A final diagnosis was available in 45 patients, revealing lung carcinoma in 55.5% (n = 25), no evidence of malignancy in 37.8% (n = 17), and pulmonary metastasis of another primary carcinoma in 6.7% (n = 3). FISH results were negative in all 17 patients with benign pulmonary disease and positive in 20 of the 25 patients (80%) with lung carcinoma (p < 0.0001). The sensitivity, specificity, and positive and negative predictive values for detection of malignancy were 79%, 100%, 100%, and 74%, respectively. These data suggest that multitarget FISH in conjunction with automated relocation is a powerful approach for the elucidation of equivocal lung cytology.

Mitotic figure (MF) counting is important in the evaluation of many tumor types. Inadequate fixation, crush artefacts, the presence of many apoptoses, or the rarity of MFs in a given lesion can make the determination of the mitotic index a very time-consuming or even impossible task, especially for novices. We evaluated the potential of the two commercially available mitotic markers MPM-2 and Phospho-Histone H3 Ser28 (PHH3) for improving mitotic counting. Formalin-fixed tissue of 1 lymphoma, 19 epithelial, 25 mesenchymal, and 10 melanocytic tumors was immunohistochemically stained with both antibodies. Mitotic counts of each tumor sample were determined by a pathologist and three residents in the hematoxylin and eosin and in both immunohistochemical stainings. Because of the higher sensitivity of the immunohistochemical stainings for MFs, average mitotic counts per 10 HPF were higher with MPM-2 (11.0) and PHH3 (10.1) than with hematoxylin and eosin (5.9) staining. The precise distinction of MFs from apoptoses and the visualization of the distribution of MFs uncovering mitotic hotspots, even at low magnification, turned out to be major advantages of both mitotic markers. In addition, the average time needed to establish the mitotic count was reduced by 40.3% with MPM-2 and by 50.4% with PHH3. MPM-2 and PHH3 were subjectively rated by all pathologists involved in this study to be very helpful in mitotic counting, especially in melanocytic and mesenchymal lesions but less so in epithelial tumors. Both markers have hence been successfully introduced in our laboratory for the routine assessment of MFs in melanocytic and mesenchymal tumors.

Pneumocephalus is commonly seen after head and facial trauma, tumors of the skull base, after neurosurgery or otorhinolaryngology, and rarely spontaneously. Venous air embolism can result from right-to-left shunting or pneumocephalus. We report two cases of pneumocephalus, one after surgery of the paranasal sinus and the other after transabdominal prostatectomy. Clinical signs of the pneumocephalus were headache followed by epileptic seizures and, respectively, severely impaired consciousness after the operation. Treatment was either revision of the skull base with craniotomy or conservative therapy of the brain edema. Based on these case reports and the medical literature on pneumocephalus, we review the causes and treatment of this rare condition.

Purpose: We set out to evaluate alterations of the therapeutic target genes KIT (CD 117), EGFR, and HER-2 in human retinoblastoma., Methods: Ninety-five formalin-fixed, paraffin-embedded retinoblastomas were brought into a tissue microarray (TMA) format. Immunohistochemistry was performed to analyze the expression of CD117, EGFR, and HER-2. Fluorescence in situ hybridization (FISH) was utilized for detection of EGFR amplifications. Three tumors with strong CD117 positivity were sequenced for KIT exon 11 mutations., Results: Detectable CD117 expression was seen in 19% of all interpretable cases. Sequence analysis of the three tumors with the strongest CD117 expression revealed no mutations. EGFR was positive in 14% of all cases. No EGFR amplification was observed by FISH, however. All tumors were negative for HER-2 expression., Conclusions: Our data suggest that selected cases of retinoblastoma may be candidates for anti-EGFR and imatinib mesylate (STI571) therapy.

Purpose: KIT (CD117) is a transmembrane tyrosine kinase representing a target for STI571 (Glivec) therapy. Some KIT-overexpressing solid tumors have responded favorably to STI571, potentially because of the presence of KIT-activating mutations., Experimental Design: To investigate the epidemiology of KIT overexpression and mutations, we investigated a series of 1654 breast cancers. All tumors were analyzed by immunohistochemistry in a tissue microarray format., Results: KIT expression was always present in normal breast epithelium. However, cancer analysis revealed the only 43 of 1654 (2.6%) tumors were KIT-positive. KIT expression was more frequent in medullary cancer (9 of 47 positive; 19.1%) than in any other histological tumor subtype (P < 0.001). KIT expression was significantly associated with high tumor grade (P < 0.0001) but unrelated to pT and pN categories or patient survival. Mutation analysis of exons 2, 8, 9, 11, 13, and 17 was negative in 10 KIT-positive tumors., Conclusions: Overall, our data show that a high level of KIT expression occurs infrequently in breast cancer. KIT-positive breast cancers may not reflect "KIT up-regulation" because KIT is also expressed in normal breast epithelium. The lack of KIT mutations also argues against the therapeutic efficacy of STI571 in breast cancer.

We aimed to immunohistochemically examine the expression of KIT (CD 117) in human posterior uveal melanoma and to analyze KIT-positive tumors for gene mutations. Brought into a tissue microarray (TMA) format were 101 formalin-fixed, paraffin-embedded posterior uveal melanomas. Immunohistochemistry was performed using the polyclonal anti-CD117 antibody from Dako (A4502). In ten selected KIT-positive tumors, exons 2, 8, 9, 11, 13 and 17 were sequenced. Of the 101 cases, 89 (88%) could be evaluated on the TMAs. Immunohistochemistry for CD 117 was weakly positive in 5 cases (6%), moderately positive in 10 cases (12%) and strongly positive in 57 cases (69%). No KIT mutations were detected in the analyzed exons. In conclusion, human posterior uveal melanoma frequently expresses CD117 at high levels. Although KIT mutations could not be found, it appears justified to investigate the utility of imatinib mesylate in the treatment of these patients.

Hepatoid adenocarcinoma (HAC) is a special type of extrahepatic adenocarcinoma, which has a striking morphologic similarity to hepatocellular carcinoma. Seven HACs arising in the stomach and one in the lung, all with liver metastasis, were studied. They shared clinical features, such as old age, high serum alpha-fetoprotein level, aggressive behavior, and hepatic tumor in absence of risk factors for hepatocellular carcinoma (HCC). Morphologically, tumors were characterized by an admixture of tubulo-and/or papillary adenocarcinoma with hepatoid foci. In six cases, liver metastases showed an exclusive hepatoid differentiation, virtually indistinguishable from HCC with solid growth pattern. As HAC and HCC cannot be differentiated on the basis of morphology alone, differences in immunohistochemical reaction patterns would be of considerable diagnostic help. Immunostaining for CK7, CK8, CK18, CK19, CK20, alpha-fetoprotein, p-CEA, and HepPar1 revealed that hepatoid areas of both primary and metastatic HAC have a specific immunoprofile, distinctive of this entity. On the one hand, positivity of virtually all HACs for alpha-fetoprotein, CK8, CK18, and the membranous, canalicular staining for polyclonal carcinoembryonic antigen underline its hepatoid nature. On the other hand, positive staining for CK19 and CK20 and frequent negativity for HepPar1 in both primary tumors and their metastases were distinctive features of HAC. Furthermore, HAC differs from combined hepatocellular cholangiocarcinoma, being negative for CK7. In addition, for comparison of immunohistochemical results, we stained with the same antibody panel a tissue microarray of 121 HCCs. Comparative genomic hybridization study of three HAC supports their hepatoid differentiation as aberrations found in HAC are common in HCC (4q-, 8p-), and hepatoblastoma (Xq+), respectively.

Multiple sclerosis (MS) is an inflammatory and possibly autoimmune mediated demyelinating disease of the CNS. Autoimmunity within the CNS may be triggered by dysfunction of peripheral immune tolerance mechanisms via changes in the homeostatic composition of peripheral T cells. We have assessed the release of naive T lymphocytes from the thymus in patients with relapsing remitting MS (RRMS) to identify alterations in the equilibrium of the peripheral T cell compartment. Thymic T cell production was estimated by measuring TCR excision circles (TRECs) as a traceable molecular marker in recent thymic emigrants. A total of 46 treatment-naive patients with active RRMS and 49 gender- and age-matched healthy persons were included in the study. The levels of TREC-expressing CD4(+) and CD8(+) T lymphocytes were significantly decreased in MS patients, and TREC quantities overall matched those of 30 years older healthy individuals. The average concentrations of TRECs/10(6) CD4(+) and CD8(+) T lymphocytes derived from MS patients and healthy donors were 26 x 10(3)/10(6) and 28 x 10(3)/10(6) vs 217 x 10(3)/10(6) and 169 x 10(3)/10(6), respectively. To account for any influence of T cell proliferation on TREC levels, we assayed T lymphocytes from additional patients with MS and normal individuals for telomere length (n = 20) and telomerase activity (8 MS patients, 16 controls), respectively. There were no significant differences between CD4(+) and CD8(+) T cells from MS patients and controls. Altogether, our findings suggest that an impaired thymic export function and, as a consequence, altered ability to maintain T cell homeostasis and immune tolerance may play an important pathogenic role in RRMS.

Pronounced forward flexion of the trunk, often termed camptocormia, is a typical symptom of patients with Parkinson's disease. In 4 parkinsonian patients with camptocormia, paraspinal muscles were studied by electromyography (EMG) and axial computerized tomography (CT) or magnetic resonance imaging (MRI) scans and muscle biopsy. EMG of the lumbar and thoracic paravertebral muscles showed abundant fibrillations, positive sharp waves, and bizarre high-frequency discharges. Spinal CT and MRI scans revealed variable degrees of atrophy and fatty replacement of the thoracolumbar paraspinal muscles on both sides. No other signs of neuromuscular disease were found. Biopsy of the paraspinal muscles revealed end-stage myopathy with autophagic vacuoles, chronic inflammatory myopathy, unspecific myopathic changes, or mitochondrial myopathy. In parkinsonian patients with pronounced forward flexion of the trunk, myopathy confined to the erector spinae muscles must be considered., (Copyright 2003 Movement Disorder Society)

This paper describes a method for the direct measurement of human sRANKL (receptor activator of NF-kappaB ligand), a cytokine of the tumor necrosis factor superfamily, which is a key player in bone metabolism. Its role in the regulation of osteogenic disorders such as osteoporosis, Paget's disease and rheumatoid arthritis is being extensively discussed in the literature at present. We developed a highly specific, simple and reliable ELISA which allows the direct measurement of uncomplexed sRANKL in human serum. Assay characteristics such as analytical precision, sensitivity, interfering factors, sample stability and dilution linearity are shown. Reference values from healthy volunteers (n=57) were found to be between 0 and 2.7 pmol/l (10th-90th percentile) with a mean serum value of 1.3 pmol/l (median 0.9 pmol/l).

An account is given using typing methods and detection of virulence genes of different serotypes of Escherichia coli isolated in Hungary. By hybridization using SLT-I and SLT-II probes and PCR method using stx1-2, eae and ehx primers we could differentiate O157 strains of different serotypes into eight (stx, eae, ehxA positive; stx, eae positive; stx, ehxA positive; stx positive; eae, ehxA positive; eae positive; ehxA positive; stx, eae, ehxA negative) types. The discriminatory power of phage typing proves to be much higher than that of the plasmid profile. RAPD typing with different primers could confirm or exclude the subtypes identity of the isolated E. coli O157 serotypes. Escherichia coli O157:HNM isolates could be sorted in six different phage types and six different RAPD types with ERIC-1, in five RAPD types with ERIC-2 and in seven types with M13 primers. Escherichia coli O157:H7 showed six different phage types and three RAPD types with ERIC-1 and ERIC-2 and five types with M13 primers. According to our results the standard PFGE protocol [32] gives the opportunity to differentiate epidemiologically independent but evolutionary related or unrelated isolates, but the practical value of PFGE method for epidemiological purposes must be confirmed by other or more restriction enzymes or using an other protocol. Summarizing our results we suggest the use of phage and RAPD typing and in doubtful cases the PFGE method.

Adjuvant chemotherapy reduces the incidence of distant metastasis and increases survival of patients with colorectal cancer. However, predictive markers are needed to define subsets of patients with stage II and III disease that may benefit from adjuvant treatment. A secreted member of the TNF receptor superfamily, the decoy receptor 3 (DcR3), was reported to be amplified in colorectal cancer as a negative regulator of Fas-mediated apoptosis. We analyzed DcR3 gene copy number and protein expression in a large series of tumors from a randomized multicenter trial of 5-fluorouracil/mitomycin C (FU/MMC) adjuvant chemotherapy of the Swiss Group for Clinical Cancer Research (SAKK 40/81), using real-time quantitative PCR and immunohistochemistry on tumor microarrays. Results of gene status and protein expression of DcR3 were correlated with disease-free and overall survival of patients. We observed amplification of the DcR3 gene in 185/294 (63%) and overexpression of the DcR3 protein in 163/223 (73%) of colorectal tumors. Multivariate analysis showed no prognostic effect of DcR3 gene amplification and protein overexpression. However, adjuvant chemotherapy was significantly more beneficial in patients with normal DcR3 gene copy number than in patients with amplification (DFS: HR 2.84, 95% CI 1.16-6.98, p = 0.02; OS: HR 3.15, 95% CI 1.19-8.32, p = 0.02), whereas DcR3 protein overexpression did not influence the effect of adjuvant chemotherapy (DFS: HR 1.02, 95% CI 0.65-1.60, p = 0.95; OS: HR 0.95, 95% CI 0.61-1.49, p = 0.83). We conclude that amplification of the 20q13 locus is a predictive marker for adjuvant chemotherapy in colorectal cancer., (Copyright 2002 Wiley-Liss, Inc.)

The effects of bafilomycin A1 and of the reduced level of endosomal epsilon-COP (coatomer protein) on the infectivity of human adenovirus type 5 were investigated in Coxsackie adenovirus receptor- (CAR-) transfected Chinese hamster ovary (CHO) cells. The endosomal proton pump inhibitor bafilomycin A1 was able to cause only partial inhibition. Using Id1F cells (an epsilon-COP thermosensitive mutant CHO cell line) the reduction of epsilon-COP level also had partial inhibitory effect. Based on these results and comparing them to existing models of the adenovirus entry, we propose a refined model in which there are two pathways of adenoviral entry: the first one involves the epsilon-COP as the downstream effector of the acidification and can be blocked by bafilomycin A1 and the second one is a pH-independent pathway.

We have isolated and characterized the 5'-flanking regulatory region of the murine serotonin 5-HT transporter (5-HTT) gene. A TATA-like motif and several potential binding sites for transcription factors, including two AP1, several AP2 and AP4 binding sites, CCAAT and GC boxes (SP1 binding sites), a nuclear factor-kappaB, and a cyclic AMP response element-like motif, are present in the 5'-flanking region. A approximately 2.2-kb fragment (-2,143 to +51 with respect to the transcription start site), which had been fused to the luciferase reporter gene and transiently expressed in a 5-HTT-expressing cell line and in serotonergic raphe neurons derived from embryonic rat brainstem, displayed both constitutive and inducible promoter activity. Functional promoter mapping revealed two clusters of activating elements from bp -82 to -527 and bp -1,001 to -1,937. A cell/neuron-selective silencer element(s) is contained between bp -294 and -527. Our findings suggest that (1) the murine 5-HTT gene promoter is active in serotonergic raphe neurons but significantly repressed in neuronal cells from frontal cortex that do not express 5-HTT, (2) the information contained within approximately 0.5 kb of the 5'-flanking sequence is sufficient to confer its cell-selective expression, (3) the promoter responds to cyclic AMP- and protein kinase C-dependent induction, and (4) the expression of the 5-HTT is regulated by a combination of positive and negative cis-acting elements operating through a basal promoter unit defined by a TATA-like motif. Fusion of the 5-HTT gene promoter unit to a gene of choice may aid its cell-selective expression in transgenic strategies.

By modulating the magnitude and duration of postsynaptic responses, carrier-facilitated serotonin (5-HT) transport into and release from the presynaptic neuron is central to the fine tuning of serotonergic neurotransmission. The 5-HT transporter (5-HTT) is the prime target for widely used antidepressants, psychostimulants, drugs of abuse and neurotoxins. We have isolated the gene encoding the murine 5-HTT and determined the sequence of all exons including adjacent intronic regions and approximately 3.6 kb of the 5'-flanking regulatory region. The murine 5-HTT gene is composed of 14 exons spanning approximately 34 kb. The single gene transcript after splicing is 2744 bp in length and it contains 186 bp of 5' untranslated region (5'-UTR) and 668 bp of 3'-UTR. A TATA-like motif and several potential binding sites for transcription factors including AP1, AP2, AP4, SP1 as well as CRE- and GRE-like motifs are present in the GC-rich 5'-flanking region. The characterization of murine 5-HTT cDNA and genomic organization will facilitate studies of 5-HT uptake function with molecular pharmacologic and transgenic strategies as well as investigations of its role in quantitative traits and psychiatric disorders.