Differential expression of interferon responsive genes in rodent models of transmissible spongiform encephalopathy disease

Background

Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD.

Results

Adeno-associated viral (AAV) vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits.

Conclusion

The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that whilst expression of Aβ42 alone is sufficient to initiate Aβ deposition, both Aβ40 and Aβ42 may contribute to cognitive deficits.     

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

Background  

The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).     

Transvaginal fine needle aspiration biopsy.

OBJECTIVE: To assess the role of transvaginal fine needle aspiration biopsy (FNAB) in the evaluation of palpable gynecologic masses. STUDY DESIGN: Transvaginal FNABs from 1994 to 1999 were identified from the files of Barnes-Jewish Hospital. Histologic correlation was obtained using the Pathology Department's computer database. Two pathologists reviewed the pathologic samples. Pertinent clinical information was obtained by reviewing the medical records. RESULTS: Twenty-two transvaginal FNABs from 22 patients were studied. The patients' mean age was 59 years (range, 29-84). Most patients (77%) had a previous history of a gynecologic malignancy, and 73% had a previous total abdominal hysterectomy and bilateral salpingo-oophorectomy. The size of the lesion sampled was provided in 15 cases and ranged from <1 to 5.4 cm in diameter. The location of the mass was reported as follows: vaginal (10 cases), vaginal cuff (5), rectovaginal septum (2), cul-de-sac (1), fornix (1), vaginal apex (1), right side of pelvis (1), and not specified (1). The cytologic diagnoses were: negative for malignancy (10 cases), positive for malignancy (9) and unsatisfactory (3). Most cases (77%) had histologic correlation or clinical follow-up. There was one false negative and no false positive cytologic diagnosis. CONCLUSION: Cytologic interpretation of transvaginal FNAB is an effective toolfor the evaluation of palpable pelvic and vaginal masses. Its specificity and sensitivity are 100% and 88%, respectively.     

Routes to improving the reliability of low level DNA analysis using real-time PCR

Background  

Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.      

Columnar cell variant of papillary thyroid carcinoma. Report of a case with cytologic findings

BACKGROUND: The columnar and tall cell variants of papillary thyroid carcinoma (PTC) are uncommon variants and have generally been regarded as more aggressive forms in comparison to the more common classic papillary and follicular subtypes. Cytologic diagnosis of these rare variants is elusive since the characteristic nuclear features of the usual papillary thyroid carcinoma are very often absent or inconspicuous. We present a case of the columnar cell variant of PTC in a young woman that demonstrates the diagnostic challenge. CASE: A 24-year-old woman presented with a solitary, 3-cm mass in the left aspect of the thyroid. The aspirate consisted of a moderately cellular sampling of sheets, papillary clusters and microfollicles of cells with oval nuclei and uniform, finely granular chromatin. These cells were arranged in a peudostratified manner around well-defined fibrovascular cores. There were no intranuclear inclusions or well-defined nuclear grooves in the cells of the aspirate. There was also absence of colloid despite the presence of well-formed follicles. The resected thyroid revealed a columnar cell variant of PTC. CONCLUSION: The cytologic features of columnar cell-type PTC are at variance with those of classic PTC and are elusive in fine needle aspiration cytology. It is the lack of classic cytologic features of PTC that is distinctly apparent, yet it is the monomorphism of cells in the aspirate, their papillary configuration and their pseudostratification in well-formed fibrovascular cores that are the keys to the diagnosis. Immunohistochemical staining to rule out other thyroid neoplasms can be performed to aid in the diagnosis.     

Fine needle aspiration cytology of a dedifferentiated liposarcoma: report of a case with histologic and immunohistochemical follow-up

BACKGROUND: Dedifferentiation is a histologic progression of a neoplasm from low grade to high grade histology. It occurs in tumors of the retroperitoneum and in those undergoing treatment. This usually occurs in the setting of radiation or chemotherapy or as a spontaneous process over a long period. The features of dedifferentiation can be toward any mesenchymal element of the underlying neoplastic process. CASE: We report the cytologic features of a dedifferentiated liposarcoma arising in a 76-year-old man who had a history of well-differentiated liposarcoma. Papanicolaou- and Diff-Quik-stained smears from a radiologically guided fine needle aspiration biopsy showed a hypercellular sample. The smears showed a mixed population of cells. There were multinucleated, pleomorphic giant cells with abundant cytoplasm, smaller clusters of cells with a high nuclear/cytoplasmic ratio and cells with spindled and elongated nuclear features. The follow-up surgical resection specimen showed a dedifferentiated liposarcoma with strong and diffuse immunoreactivity to vimentin, desmin and CD68 in the large, pleomorphic cells; focal and weak immunoreactivity to smooth muscle actin and S-100 in these cells; and strong and focal immunoreactivity to desmin, smooth muscle actin and muscle-specific actin in the spindle cells. This supports the dedifferentiated components of this tumor to be of fibrohistiocytic and leiomyosarcomatous differentiation. CONCLUSION: Dedifferentiation of a well-differentiated liposarcoma should be entertained in the setting of a mass lesion in the retroperitoneum in patients with prior histories of well-differentiated liposarcoma. The radiologic features of a particular neoplastic process can be very helpful in determining the nature of this process.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.