乳腺癌中EGFR蛋白表达和基因扩增的检测结果比较

目的:比较免疫组织化学技术检测乳腺癌中EGFR蛋白表达和荧光原位杂交检测EGFR基因扩增的结果的符合率,为EGFR靶向治疗病例的选择提供依据。方法:随机选取2005年1月到2011年12月冷水江市人民医院和湖南省肿瘤医院病理科的147例乳腺癌档案病例,采用免疫组织化学技术检测乳腺癌组织中EGFR蛋白表达,荧光原位杂交检测EGFR的基因扩增,比较两种方法阳性结果的符合率。结果:免疫组化染色结果显示EGFR在原发性和转移性乳腺癌中的阳性表达率分别为85%(105/123)和79%1(9/24),两组比较无显著差异(P0.05)。FISH检测结果显示原发性和转移性乳腺癌中分别有12%(15/123)和8%(2/24)存在EGFR基因扩增,两组比较结果无显著差异(P0.05)。所有存在EGFR基因扩增的原发性和转移性乳腺癌的EGFR免疫组织化学结果均为阳性。在原发性和转移性乳腺癌中,免疫组化阳性和基因扩增程度间呈显著正相关(P0.05),但免疫组化结果预测基因扩增的特异性较低。结论:免疫组织化学检测EGFR只能作为EGFR靶向治疗病例选择的初步筛选,进一步进行荧光原位杂交检测EGFR基因扩增是必须的。     

乳腺癌中EGFR蛋白表达和基因扩增的检测结果比较

目的：比较免疫组织化学技术检测乳腺癌中EGFR蛋白表达和荧光原位杂交检测EGFR基因扩增的结果的符合率,为EGFR靶向治疗病例的选择提供依据。方法：随机选取2005年1月到2011年12月冷水江市人民医院和湖南省肿瘤医院病理科的147例乳腺癌档案病例,采用免疫组织化学技术检测乳腺癌组织中EGFR蛋白表达,荧光原位杂交检测EGFR的基因扩增,比较两种方法阳性结果的符合率。结果：免疫组化染色结果显示EGFR在原发性和转移性乳腺癌中的阳性表达率分别为85%（105/123）和79%1（9/24）,两组比较无显著差异（P〉0.05）。FISH检测结果显示原发性和转移性乳腺癌中分别有12%（15/123）和8%（2/24）存在EGFR基因扩增,两组比较结果无显著差异（P〉0.05）。所有存在EGFR基因扩增的原发性和转移性乳腺癌的EGFR免疫组织化学结果均为阳性。在原发性和转移性乳腺癌中,免疫组化阳性和基因扩增程度间呈显著正相关（P〈0.05）,但免疫组化结果预测基因扩增的特异性较低。结论：免疫组织化学检测EGFR只能作为EGFR靶向治疗病例选择的初步筛选,进一步进行荧光原位杂交检测EGFR基因扩增是必须的。     

Erythrovirus B19 infection in acquired immunodeficiency syndrome: screening by histopathology, immunohistochemistry, and in situ hybridization

Erythrovirus B19 infects erythrocytic progenitors, transiently interrupting erythropoiesis. In AIDS patients it causes chronic anemia amenable to treatment. We looked for evidences of B19 infection in stored bone marrow material from patients with acquired immunodeficiency syndrome. Histological sections were made from stored paraffin blocks from 33 autopsies (39 blocks) and 35 biopsies (45 blocks, 30 patients) performed from 1988 to 2002. They were examined after hematoxylin-eosin (HE) staining, immunohistochemical (IHC), and in situ hybridization. HE revealed intra-nuclear inclusion bodies ("lantern cells") suggesting B19 infection in 19 sections corresponding to 19 of 63 patients examined with this test. Seven of 78 sections subjected to immunohistochemistry were positive, corresponding to 7 of 58 patients examined with this test. Fourteen sections corresponding to 13 of the 20 HE and/or IHC positive patients were subjected to in situ hybridization, with six positives results. Among the 13 patients subjected to the three techniques, only one gave unequivocal positive results in all and was considered a true positive. The frequency of B19 infection (1/63 patients) in the material examined can be deemed low.     

Immunohistochemical detection of interferon-gamma: fake or fact?

Immunohistochemistry is a widely accepted tool to investigate the presence and immunolocalization of cytokines in tissue sections at the protein level. We have tested the specificity and reproducibility of IFNgamma immunohistochemistry on tissue sections with a large panel of anti-IFNgamma antibodies. Thirteen different commercially available anti-IFNgamma antibodies, including seven advertised and/or regularly applied for immunohistochemistry/-cytochemistry, were tested using a three-step streptavidin-biotin-peroxidase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzyme double staining was used to identify the IFNgamma-positive cells. Serial cryostat sections were used of human reactive hyperplastic tonsils, rheumatoid synovium, and inflammatory abdominal aortic aneurysms, known to possess a prominent Th1-type immune response. In vitro phorbol myristate acetate/ionomycin-stimulated T-cells served as positive control; unstimulated cells served as negative control. Cultured T-cells were used adhered to glass slides (immunocytochemistry), in suspension (FACS), or snap-frozen and sectioned (immunohistochemistry). Immunocytochemistry and FACS analysis on stimulated cultured T-cells showed positive staining results with 12 of 13 anti-IFNgamma antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFNgamma immunohistochemical single- and double staining analysis of the tissue sections showed huge variations in staining patterns, including positivity for smooth muscle cells (n = 8), endothelial cells (n = 4), extracellular matrix (n = 4), and CD138+ plasma cells (n = 12). Specific staining of T-cells, as the sole positive staining, was not achieved with any of the 13 antibodies. IFNgamma-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFNgamma antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFNgamma-immunohistochemistry must be interpreted with great caution.     

Study of infection by human papillomavirus in severe dysplasias and carcinomas in situ of the uterine cervix using immunohistochemistry and in situ hybridization.

Diagnosis of human papillomavirus (HPV) infection in uterine cervical lesions is usually based on histopathological criteria and, in some cases, is confirmed by immunohistochemistry. The recent development of in situ hybridization techniques has facilitated the detection of HPV in these lesions. Consequently, we carried out a study on 18 uterine cervical biopsy specimens histopathologically diagnosed as severe dysplasias and carcinomas in situ, using an immunohistochemical method with a rabbit polyclonal antibody against the HPV common structural antigen and in situ hybridization techniques with three biotinylated DNA probes for HPV types 6/11, 16/18, and 31/35/51. By immunohistochemistry only one case (5.5%) proved to be positive, whereas by in situ hybridization 12 HPV-positive cases were obtained (66.6%), of which 7 were positive for HPV types 16/18 (38.8%) and 6 for HPV types 31/35/51 (33.3%). One case was positive with positive with both DNA probes. From our results it can be inferred that in situ hybridization is a more sensitive technique than immunohistochemistry for confirming the presence of HPV in severe dysplasias and carcinomas in situ of the uterine cervix. Furthermore, in situ hybridization provides much more information than immunohistochemistry since it permits the identification of the HPV types causing the lesion.     

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed,Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.     

Noise and incongruence: interpreting results of the incongruence length difference test

Incongruence between data sets is an important concept in molecular phylogenetics and is commonly measured by the incongruence length difference (ILD) test (J. S. Farris et al., Cladistics 10, 315-319). The ILD test has been used to infer specific evolutionary events and to determine whether to combine data sets for phylogenetic analysis. However, the interpretation in the literature of the test's results varies because authors have conflicting expectations of the effect that noise will have. Using simulations we demonstrate that noise can by itself generate highly significant results in the ILD test and demonstrate why this is the case. To clarify the interpretation of test results, we suggest an additional procedure in which the result is compared against a frequency distribution generated from completely shuffled data. As examples, we apply this approach to two previous studies that have reported incongruence.     

Comparative histochemical study of Bowen's disease and actinic keratosis: preserved normal basal cells in Bowen's disease

The degree of DNA-instability as revealed by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) has been recently used as a marker of malignancy. This technique was applied to examine 17 skin tissue samples of Bowen's disease, 47 of actinic keratosis, 15 of squamous cell carcinoma, 5 of seborrheic keratosis, and 10 of normal skin. All benign neoplastic cells of seborrheic keratosis and normal epidermal cells were negative. On the other hand, all cancer cells were positive with the DNA-instability test, indicating their malignancy, but all basal cells in Bowen's disease were completely negative. Compatible with this result, the basal cells in Bowen's disease were characteristically normal as evident in other histochemical examinations. Thus, they were negative with p53 immunohistochemistry, with normal signals of chromosome 17 in situ hybridisation and argyrophilic nucleolar organiser region, and showed slightly enhanced proliferative activity as revealed by proliferating cell nuclear antigen immunohistochemistry. Immunohistochemical staining with 34 beta E12 (monoclonal antibody against cytokeratins 1, 5, 10, and 14), which stains all normal epidermal keratinocytes including basal cells, showed that only the basal cells of Bowen's disease stained strongly and homogeneously, while all cancer cells in the upper layers of Bowen's disease and all layers of actinic keratosis were only sporadically or weakly stained. Staining with 34 beta B4 (monoclonal antibody against cytokeratin 1), which recognises the whole epidermis except for the basal layer in the normal epidermis, showed that the basal cells in the Bowen's disease were completely negative, and lower layer cells in the actinic keratosis and upper layer cells in Bowen's disease were only sporadically stained positive, although the superficial layer cells in actinic keratosis stained strongly and homogeneously. Our findings clearly indicate that the basal cells in Bowen's disease are normal. In support of this conclusion, the same cells showed normal morphology on electron microscopy with preserved basement membrane, although the latter was often damaged in actinic keratosis.     

Realities of diagnosing Helicobacter pylori infection in clinical practice: a case for non-invasive indirect methodologies.

BACKGROUND: The current, arbitrarily defined gold standard for the diagnosis of H. pylori infection requires histologic examination of two specially stained antral biopsy specimens. However, routine histology is potentially limited in general clinical practice by both sampling and observer error. The current study was designed to examine the diagnostic performance of invasive and non-invasive H. pylori detection methods that would likely be available in general clinical practice. METHODS: The diagnostic performance of rotating clinical pathology faculty using thiazine staining was compared with that of an expert gastrointestinal pathologist in 38 patients. In situ hybridization stains of adjacent biopsy cuts were also examined by the expert pathologist for further comparison. Receiver operator characteristic (ROC) analysis was performed to evaluate whether the diagnostic performance of the expert pathologist differed depending upon the histologic method employed. A similar analysis was made to evaluate the diagnostic performance of pathology trainees relative to the expert. In the absence of an established invasive gold standard, non-invasive testing methods (rapid serum antibodies, formal Elisa antibodies and carbon-14 urea breath testing) were evaluated in 74 patients by comparison with a gold standard defined using a combination of diagnostic tests. RESULTS: Using either rapid urease testing of biopsy specimens or urea breath testing as the gold standard for comparison, the diagnostic performance of the rotating clinical pathology faculty was inferior to that of the expert gastrointestinal pathologist especially with regard to specificity (e.g., 69 percent for the former versus 88 percent, with the latter relative to rapid urease testing). Although interpretation of in situ hybridization staining by the expert appeared to have an even higher specificity, ROC analysis failed to show a difference. The mean ROC areas for thiazine and in situ hybridization staining for trainee pathologists relative to the expert were 0.88 and 0.94, respectively. In untreated patients, urea breath testing had a sensitivity and specificity of 100 percent as compared with thiazine staining with a sensitivity of 83 percent and a specificity of 97 percent. Post-therapy, breath testing had a sensitivity of 100 percent but a specificity of only 86 percent as compared with invasive testing with a sensitivity and specificity of 100 percent. Rapid serum antibody testing and formal Elisa antibody testing agreed in 93 percent of cases (Kappa 0.78) with the rapid test being correct in three of the four disagreements. CONCLUSIONS: The current study illustrates a number of realities regarding H. pylori diagnosis. There is no diagnostic gold standard in general clinical practice. Accurate interpretation of specially stained slides is a learned activity with a tendency towards overdiagnosis early on. Urea breath testing is likely to be the diagnostic method of choice for untreated patients in general clinical practice although antibody testing is almost as accurate. Rapid antibody tests are at least as accurate as formal Elisa antibody tests. Urea breath testing is useful for confirming cure after therapy, but false-positive results may occur in some patients.     

CD95 ligand (CD95L) immunohistochemistry: a critical study on 12 antibodies

In recent years, some studies on the expression of CD95(Fas/APO-1) ligand (CD95L) in tissues or cells raised concerns about the specificity of the antibodies used. We therefore tested 12 CD95L antibodies for their reliability in immunocyto/histochemistry by (i) staining CD95L-transfected and control CV-1/EBNA cells and (ii) comparing staining patterns in immunohistochemically labeled tissue sections with the localization of CD95L+ cells in in situ hybridization. While G247-4, NOK-1, NOK-2, 4H9, and MIKE-1 stained CD95L-transfected cells and did not significantly bind to controls, G247-4 was the only antibody giving satisfying signals in tissue sections perfectly matching the distribution of CD95L+ cells by in situ hybridization. MAb 33, C-20, and N-20 comparably stained both transfected and control cells and showed considerable background or falsely positive staining in sections. MIKE-2, 8B8, A11, and 4A5 did not or only very faintly bind to either cells and, thus, were not tested on sections. We conclude that G247-4 is the only tested antibody that is recommendable for immunohistochemistry.     

Galectin-3 is not useful in thyroid FNA

INTRODUCTION: Previous studies have suggested that galectin-3 immunohistochemistry may be useful in the fine needle aspiration (FNA) diagnosis of thyroid carcinoma as it has been reported to selectively stain carcinomas and not adenomas or goitres. METHODS: Fifty-one patients were included in a prospective study of galectin-3 in thyroid FNA; 88.2% were female and 11.8% male, mean age 53 years, range 25-87 years. Cell blocks were prepared and stained for galectin-3 if any cells were present in needle washings from the respective FNAs. RESULTS: Twelve of 51 (23.5%) of cell blocks contained epithelial cells. One benign and one inadequate FNA were negative for galectin-3 staining. One of five non-diagnostic FNA cases, a papillary carcinoma on final histology showed positive staining. Four follicular neoplasm/suspicious of carcinoma cases showed negative staining. One malignant FNA case, a papillary carcinoma showed positive staining with galectin-3 but three further carcinomas, two papillary and one follicular were galectin-3 negative. CONCLUSION: Galectin-3 immunohistochemistry does not appear to be a useful adjunct to diagnosis in thyroid FNA as it does not reliably distinguish malignant and benign lesions. Many thyroid aspirates are of low cellularity and are not suitable for cell block immunohistochemistry.     

Peripheral iron dextran induced degeneration of dopaminergic neurons in rat substantia nigra

Iron accumulation is considered to be involved in the pathogenesis of Parkinson's disease. To demonstrate the relationship between peripheral iron overload and dopaminergic neuron loss in rat substantia nigra (SN), in the present study we used fast cyclic voltammetry, tyrosine hydroxylase (TH) immunohistochemistry, Perls' iron staining, and high performance liquid chromatography-electrochemical detection to study the degeneration of dopaminergic neurons and increased iron content in the SN of iron dextran overloaded animals. The findings showed that peripheral iron dextran overload increased the iron staining positive cells and reduced the number of TH-immunoreactive neurons in the SN. As a result, dopamine release and content, as well as its metabolites contents were decreased in caudate putamen. Even more dramatic changes were found in chronic overload group. These results suggest that peripheral iron dextran can increase the iron level in the SN, where excessive iron causes the degeneration of dopaminergic neurons. The chronic iron overload may be more destructive to dopaminergic neurons than the acute iron overload.     

EMMPRIN Is an Independent Negative Prognostic Factor for Patients with Astrocytic Glioma

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as CD147, is a member of the immunoglobulin superfamily that is present on the surface of tumor cells and stimulates adjacent fibroblasts to produce matrix metalloproteinases (MMPs). It has been proved to be associated with tumor invasion and metastasis in various human malignancies. In our study, the protein expression level of EMMPRIN in 306 cases of astrocytic glioma is investigated by immunohistochemistry assay. Statistical analysis was utilized to evaluate the association of EMMPRIN with clinicopathological characteristics and prognosis of patients. It was proved that EMMPRIN protein expression was increased in glioma compared with that in normal brain tissue. Moreover, EMMPRIN immunohistochemical staining was correlated with WHO grade and Karnofsky performance score for strong positive EMMPRIN staining is more frequently detected in glioma of advanced grade or low KPS score. It is also demonstrated that EMMPRIN could be an independent negative prognostic factor in glioma for patients with glioma of strong EMMPRIN staining tend to have high risk of death. These results proved that EMMPRIN is associated with prognosis of glioma, which may also suggest the potential role of EMMPRIN in glioma management.     

宫颈特殊染色技术对子宫颈癌及宫颈上皮内瘤变初筛的临床研究

目的:探讨宫颈特殊染色技术筛查方法对宫颈癌及癌前病变的筛查意义。方法:本研究通过对1963例就诊我院妇科门诊的患者进行宫颈特殊染色检查(FRD),以组织病理学检查结果为标准,分析FRD宫颈特殊染色的临床意义。结果:1963例患者行宫颈特殊染色检查及对初筛阳性患者行阴道镜下活检,根据活检病理结果进行分析,CINI阳性率80.77%℅,CINII81.25%,CINIII100%℅,侵润癌100%,总阳性率90.50%。结论:利用亚甲蓝显色和醋酸白化反应双重定位及指示,不仅可提高宫颈癌及癌前病变的检出率,而且操作简便,判读容易,结果快速,成本低廉。     

Comparison of NCL-hTERT antibody reactivity and telomere repeat amplification protocol in situ in effusions

OBJECTIVE: To compare the performances of 2 methods, telomerase repeat amplification protocol (TRAP) in situ and antibodies to the hTERT protein, in assessing telomerase activity. STUDY DESIGN: TRAP in situ and immunohistochemistry with a commercial antibody (NCL-hTERT) was performed on 54 body cavity effusions. The results were compared and correlated to diagnosis. RESULTS: Thirty-four effusions from patients with verified malignant disease contained cytologically malignant cells. Both methods were positive in 33 of the cases, whereas only hTERT was positive in 1 case. Twenty effusions, all containing mesothelial cells, came from patients with benign conditions. In 2 fluids atypical, hyperplastic mesothelial cells were both TRAP in situ and hTERT positive. All remaining 18 fluids were TRAP in situ negative, whereas 12 of 18 were hTERT positive. Thus the results of TRAP in situ and hTERT immunohistochemistry disagreed in 1 of 34 (3%) malignant and 12 of 20 (60%) benign cases. CONCLUSION: The sensitivities for malignancy were similar for TRAP in situ and hTERT immunohistochemistry. The specificity of the applied hTERT antibody was significantly lower, due to hTERT reactivity in mesothelial cells.     

The Evaluation and Interpretation of Cervical Cytology: Application of the Likelihood Ratio Concept

The Papanicolaou smear (Pap test), used for the detection and prevention of neoplastic lesions of the cervix, is known to have both false negative and false positive results. Proper handling of the diagnostic uncertainty resulting from these errors demands quantification of flaws. Traditionally, sensitivity, specificity and predictive values are used for that aim. In this study another approach is advocated, namely the use of the likelihood ratio. For cervical cytology this ratio is the quotient of the probability of a Pap class within the diseased population to the probability of that same Pap class within the non-diseased group. This approach enables the characterization of each Pap class separately, and is therefore much better for clinical interpretation of the result. It is also a superior approach for quality assessment.     

In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.     

Diagnostic Tools for Integrated In Situ Air Sparging Pilot Tests

In situ air sparging (IAS) pilot test procedures have been developed that provide rapid, on-site information about IAS performance. The standard pilot test consists of six activities conducted to look for indicators of infeasibility and to characterize the air distribution to the extent necessary to make design decisions about IAS well placement. In addition, safety hazards that need to be addressed prior to full-scale design are identified. Two additional pilot test activities are described in those cases where air distribution must be more precisely defined. The test activities include both chemical tests (tracking contaminant concentrations, dissolved oxygen and tracers) and physical tests (air flow rate and injection pressure, groundwater pressure response). Pilot test data from Eielson Air Force Base, Alaska illustrates implementation of the pilot test and interpretation of the data.     

Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences

Conditions for combination of DNA in situ hybridization, using biotinylated DNA probes, with immunohistochemistry were investigated on cryostat sections, cytological preparations, and paraffin sections. We found that cryostat sections and cytological preparations are suitable for in situ hybridization of target DNA after fixation in acetone, methanol, ethanol, or Carnoy without further proteinase pretreatment. Acetone is also very suitable for immunostaining of cell surface or cytoskeleton antigens. We therefore performed combined immunoenzyme and in situ hybridization staining using this fixative. The best results were obtained when immunoperoxidase staining with diaminobenzidine/H2O2 was followed directly by in situ hybridization. In addition to immunoperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) staining with naphthol ASBI phosphate and New Fuchsin as a substrate could be used. In most instances, detection of the biotinylated hybrid with a streptavidin-biotinylated polyalkaline phosphatase method using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as the substrate was preferable. The double stainings were studied on the following test models: (a) frozen tonsil sections: cell surface antigens (pan T) and ribosomal DNA; (b) frozen genital condyloma sections; cytokeratins and human papillomavirus type 6 + 11 (HPV-6/11) DNA; (c) CaSKi cells: cytokeratins and HPV-16 DNA; (d) infected fetal lung fibroblasts: vimentin and cytomegalovirus (CMV) DNA. An adapted procedure was followed on routinely formaldehye-fixed and paraffin-embedded condyloma tissue. Immunoperoxidase staining for papilloma virus capsid antigen could be combined with DNA in situ hybridization with HPV-6/11 DNA. In this model, however, the accessibility of the target DNA had to be improved by enzyme treatment after the immunostaining and before starting the in situ hybridization.     

Odorant receptor expression in the mouse cerebral cortex

Mammalian odorant receptors have been known to be involved not only in odorant detection but also in neuronal development of olfactory sensory neurons. We have examined a possibility of odorant receptor expression in nonolfactory neurons in the mouse. Mouse odorant receptors (M71, C6, and OR3), two of which were already shown to be functionally activated by odorants in heterologous systems, were detected by polymerase chain reactions (PCRs) from the cerebral cortex but not from other brain tissues. Degenerate PCR further suggested that other odorant receptors were also expressed in the mouse cerebral cortex. One of these receptors showed high sequence-match with a putative chick odorant receptor OR7 transiently expressed in the notochord during development. In situ hybridization detected signals for M71 and C6 receptors in the layer II cortical pyramidal neurons located in the occipital pole. In the M71-IRES-tauLacZ mouse, in which M71 expression was genetically marked with tauLacZ, X-gal staining signals were mostly localized in the layer II neurons in the occipital pole, being consistent with the in situ hybridization result. Fluorescent immunohistochemistry using anti-beta-galactosidase antibody further detected the tauLacZ signals in the same cells. X-gal staining began at P3, peaked at P8, and continued to adults, although signals gradually decreased. These data showed that at least a few odorant receptors are expressed not only in olfactory sensory neurons but also in pyramidal neurons in the cerebral cortex, possibly playing an important role either in chemical detection of exogenous or endogenous ligands or in a developmental process such as axon guidance and target recognition.