Incidence of neoplasia in patients with unilateral epiphora.

BACKGROUND: Nasolacrimal duct obstruction is common and is usually a result of benign stricture formation.Although neoplasia near or around the lacrimal system may produce epiphora, the incidence of neoplasia from within the lacrimal system as a cause of nasolacrimal duct obstruction is not well documented. METHODS: A retrospective study was performed on all patients undergoing dacryocystorhinostomy with a history of epiphora. The incidence of patients with operative findings of intra-lacrimal neoplasm was sought. Histopathologically confirmed cases were included. RESULTS: The study comprised 537 patients, who underwent a total of 631 endoscopic dacryocystorhinostomy procedures between January 1998 and July 2013. Non-stenotic causes of nasolacrimal duct obstruction were encountered in 3.01 per cent of dacryocystorhinostomy procedures, and included neoplastic, inflammatory and infectious pathologies. Inverted papilloma was the most common cause, encountered in 0.79 per cent of dacryocystorhinostomy operations. CONCLUSION: These findings suggest that neoplasia is an uncommon but not a rare cause of nasolacrimal duct obstruction. Surgical teams performing high numbers of dacryocystorhinostomy procedures should be aware of such pathology and patients counselled appropriately.

Vasculitis primaria del sistema nervioso central, diagnóstico diferencial de demencia subaguda: Caso clínico / Primary vasculitis of the Central Nervous System: report of one case

Primary central nervous system vaculitis is an uncommon and invalidating disease, which has a fatal course if left untreated. We report a 63 year-old woman presenting with a history of two months of cognitive impairment, dysarthria, gait instability and tremor. After four months of evolution a right hemianopsia and a flaccid paresis of upper right limb appeared. A brain biopsy was performed and the histological findings confirmed the suspicion of primary cerebral vasculitis. The patient was treated with cyclophosphamide and prednisone, observing a partial recovery of cognitive and motor function.

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.

Evaluation of a pen device for self-administration of recombinant human FSH in clomiphene citrate-resistant anovulatory women undergoing ovulation induction.

This open-label multicentre study evaluated ease of use, safety, and efficacy of a pen device for self-administration of recombinant follicle-stimulating hormone (rFSH) in 43 subjects undergoing ovulation induction. Follitropin beta was administered subcutaneously with the Follistim Pen within 3 days of onset of menses. A 75 IU starting dose could be increased by 25 or 50 IU on days 8 and 15 if no ovarian response was observed. Human chorionic gonadotrophin (HCG; 10,000 IU) was administered when one follicle > or =18 mm or two to three follicles > or =15 mm were observed. Subjects received standardized instruction for the pen device and subject comprehension was recorded as subjects practised and prepared injections. Ease of use was also evaluated by questionnaire. Forty-four subjects enrolled; 43 were treated with rFSH and 41 were treated with HCG. The comprehension questionnaire revealed that during the mock injection, 100% of subjects properly loaded the cartridge into the pen device, while 95% selected the correct dose and 100% self-injected the medication prescribed. During the second actual injection, 100% of subjects comprehended these pen-related steps. The ease-of-use questionnaire showed that 100% of the subjects rated the overall experience of self-administering with the pen as 'very good' to 'good'. Mean duration and total amount of follitropin beta were 11.4 +/- 4.2 days and 1070.3 +/- 580.3 IU respectively. Ovulation rate was 95%. Biochemical and ongoing pregnancy rates per attempt were 34.9 and 30.2% respectively. Three subjects experienced serious adverse events [asthma; ovarian hyperstimulation syndrome (OHSS) and pain; OHSS]. In conclusion, the pen device provides an easy, safe, and effective way for women to self-administer follitropin beta during ovarian stimulation.

Administration of recombinant human FSH (solution in cartridge) with a pen device in women undergoing ovarian stimulation.

This study evaluated the first multiple-use pen device for the self-administration of recombinant FSH. The pen device is used for the subcutaneous injection of a pre-mixed ready-to-use solution of follitropin beta from a multiple-dose cartridge, and has flexible dosing capabilities. In the ease-of-use questionnaire, 90% of subjects rated the overall experience of self-injecting follitropin beta using the pen device as 'very good' (on day 6). The comprehension questionnaire revealed that prior to the first injection and during the second injection, the follitropin beta cartridge was properly loaded into the pen device by 96.7 and 100% of the subjects respectively. The questionnaire also showed that the correct dose was selected and self-administered by 98.3 and 100% of the subjects respectively. Biochemical and ongoing pregnancy rates per attempt were 56.7 and 45.0% respectively. The pen device is safe, effective, and easy to use for self-administering recombinant FSH during ovarian stimulation.

Motility-related proteins as markers for head and neck squamous cell cancer.

HYPOTHESIS: Increased cell motility is a hallmark of cancer cells. Proteins involved in cell motility may be used as molecular markers to characterize the malignant potential of tumors. METHODS: Molecular biology and immunohistochemistry techniques were used to investigate the expression of a selected panel of motility-related proteins (Rho A, Rac 2, Cdc42, PI3K, 2E4, and Arp2) in normal, premalignant, and squamous cell cancer cell lines of human head and neck origin. To assess the clinical potential of these proteins as molecular markers for cancer, immunohistochemistry was performed on paraffin-fixed head and neck cancer specimens (n = 15). RESULTS: All six motility-associated proteins were overexpressed in the premalignant and squamous cell cancer cell lines relative to normal keratinocytes. Immunohistochemistry with Rho A and Rac 2 showed increased staining in areas of cancer but not in normal tissue. CONCLUSION: Proteins involved in cell motility can be used as markers for head and neck squamous cell carcinoma. The head and neck cell lines used in this study may be used as a model to further investigate cell motility. Molecular markers of motility could have a significant impact on the diagnosis and staging of cancers originating from differentiated non-motile cells.

Spectral karyotyping analysis of head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: The genetic content of head and neck squamous cell carcinomas is ill defined. Spectral karyotyping (SKY) is a new technique that allows the simultaneous detection of all chromosomal translocations by labeling each individual chromosome with different fluorescent agents. In the current study we used SKY to analyze cell lines and a primary tumor derived from head and neck squamous cell carcinomas (HNSCC) to delineate recurrent translocations and breakpoints. STUDY DESIGN: Spectral karyotyping analysis of head and neck cancer. METHODS: Two cell lines (MDA886 and MSK922) and one primary tumor in short-term culture were subjected to metaphase growth arrest with colcemide in their exponential growth phase and fixed onto glass slides. Painting probes for each of the autosomes and the sex chromosomes were generated from flow-sorted human chromosomes using sequence-independent DNA amplification. The probes were labeled using a polymerase chain reaction-based reaction and hybridized to metaphase preparations for 2 days at 37 degrees C. Biotinylated probes were detected using avidin Cy5 and digoxigenin-labeled probes with an anti-mouse digoxigenin antibody followed by goat anti-mouse antibody conjugated to Cy5.5. Chromosomes were counterstained with 4,6-diamino-2-phenyliodole (DAPI), and a minimum of five metaphases were captured and analyzed for each case. Breakpoints on the SKY-painted chromosomes were determined by comparison of corresponding DAPI banding. RESULTS: Spectral karyotyping analysis revealed a complex pattern of chromosomal abnormalities. A total of 66 translocations were identified in the three cases, with one new recurrent translocation at (der(4)t(4;20)(q35;?)). Nine complex translocations, involving three or more chromosomes, were identified in these cases. Overall, 96 breakpoints were assigned to metaphase chromosomes and another 74 breakpoints could not be assigned. Breakpoints most commonly involved chromosomes in genetic rearrangements were 1, 3, 5, 8, 13, 16, and 17. CONCLUSIONS: Spectral karyotyping analysis reveals the true complexity of chromosomal aberrations in cell lines derived from head and neck squamous cell carcinomas. The use of SKY, in combination with other techniques, may allow for a more complete assessment of the genetic abnormalities of head and neck cancers and serve as a starting point for gene identification.

Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification.

We applied a combination of molecular cytogenetic methods, including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization, to characterize the genetic aberrations in a panel of 11 cell lines derived from head and neck squamous cell carcinoma and 1 cell line derived from premalignant oral epithelium. CGH identified recurrent chromosomal losses at 1p, 3p, 4, 8p, 10p, and 18q; gains at 3q, 5p, 8q, 9q, and 14q; and high-level amplification at 3q13, 3q25-q26, 5q22-q23, 7q21, 8q24, 11q13-q14, 12p13, 14q24, and 20q13.1. Several recurrent translocations including t(1;13)(q10;q10), t(13;13)(q10;q10), t(14;14)(q10;q10), i(8)(q10), and i(9)(q10) and breakpoint clusters at 1p11, 1q21, 3p11, 5q11, 5q13, 6q23, 8p11, 8q11, 9p13, 9q13, 10q11, 11q13, 13q10, 14q10, and 15q10 were identified by SKY. There was a good correlation between the number of aberrations identified by CGH and SKY (r = 0.69), and the analyses were both confirmatory and complementary in their assessment of genetic aberrations. Amplification at 3q26-q27 was identified in 42% of cases. Although SKY defined the derivation of 3q gain, the precise breakpoint remained unassigned. Positional cloning efforts directed at the amplified region at 3q26-q27 identified three highly overlapping nonchimeric yeast artificial chromosome clones containing the apex of amplification. The use of these yeast artificial chromosome clones as a probe for fluorescence in situ hybridization analysis allowed a detailed characterization and quantification of the 3q amplification and refinement of unassigned SKY breakpoints.

Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation.

We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.

Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck.

The purpose of this study was to determine whether cyclooxygenase-2 (COX-2) was overexpressed in squamous cell carcinoma of the head and neck (HNSCC). Quantitative reverse transcription-PCR, immunoblotting, and immunohistochemistry were used to assess the expression of COX-2 in head and neck tissue. Mean levels of COX-2 mRNA were increased by nearly 150-fold in HNSCC (n = 24) compared with normal oral mucosa from healthy volunteers (n = 17). Additionally, there was about a 50-fold increase in amounts of COX-2 mRNA in normal-appearing epithelium adjacent to HNSCC (n = 10) compared with normal oral mucosa from healthy volunteers. Immunoblotting demonstrated that COX-2 protein was present in six of six cases of HNSCC but was undetectable in normal oral mucosa from healthy subjects. Immunohistochemical analysis showed that COX-2 was expressed in both HNSCC and adjacent normal-appearing epithelium. Taken together, these results suggest that COX-2 may be a target for the prevention or treatment of HNSCC.

Inhibition of cyclooxygenase-2 expression. An approach to preventing head and neck cancer.

Cyclooxygenase (COX) catalyzes the formation of prostaglandins (PG) from arachidonic acid. A large body of evidence has accumulated to suggest that COX-2, the inducible form of COX, is important in carcinogenesis. In this study, we determined whether (1) COX-2 was overexpressed in squamous cell carcinoma of the head and neck (HNSCC) and whether (2) retinoids, a class of chemopreventive agents, blocked epidermal growth factor (EGF)-mediated activation of COX-2 expression. Levels of COX-2 mRNA were determined in 15 cases of HNSCC and 10 cases of normal oral mucosa. Nearly a 100-fold increase in amounts of COX-2 mRNA was detected in HNSCC. By immunoblot analysis, COX-2 protein was detected in 6 of 6 cases of HNSCC but was undetectable in normal mucosa. Because retinoids protect against oral cavity cancer, we investigated whether retinoids could suppress EGF-mediated induction of COX-2 in cultured oral squamous carcinoma cells. Treatment with EGF led to increased levels of COX-2 mRNA, COX-2 protein, and synthesis of PG. These effects were suppressed by a variety of retinoids. Based on the results of this study, it will be important to establish whether newly developed selective COX-2 inhibitors are useful in preventing or treating HNSCC. Moreover, the anticancer properties of retinoids may be due, in part, to inhibition of COX-2 expression. Combining a retinoid with a selective COX-2 inhibitor may be more effective than either agent alone in preventing cancer of the upper aerodigestive tract.

Green tea regulates cell cycle progression in oral leukoplakia.

BACKGROUND: A complete in vitro multi-stage carcinogenesis model for oral cancer was developed to examine chemopreventive strategies. In the present study, the effects of EGCG [(-)-epigallocatechin-3-gallate], the major constituent of green tea, is being examined to understand mechanisms of action. METHODS: Effects of EGCG on the cell populations were examined with growth assays, cell cycle analysis, and western blots for retinoblastoma protein (pRB). RESULTS: In each cell type, EGCG inhibited growth, with a decrease in efficacy as cells progressed from normal to cancer. A G1 block was induced with an increase in the underphosphorylated form of pRB; EGCG-induced inhibition was not permanent, cells recovered, and no resistance developed. CONCLUSIONS: Our multistage carcinogenesis model for chemoprevention was effective in defining the chemopreventive value of EGCG. The observation that cancerous oral epithelium was less responsive than normal or dysplastic tissues has implication in the use of this agent, and the mechanisms responsible for this result remain to be defined.

Quantitation of chemopreventive synergism between (-)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells.

An in vitro model for oral cancer was used to examine the growth inhibitory effects of chemopreventive agents when used singly and in combination. The model consists of primary cultures of normal oral epithelial cells, newly established cell lines derived from dysplastic leukoplakia and squamous cell carcinoma. Two naturally occurring substances, (-)-epigallocatechin-3-gallate (EGCG) from green tea and curcumin from the spice turmeric were tested. Cells were treated singly and in combination and effects on growth determined in 5-day growth assays and by cell cycle analysis. Effective dose 50s and the combination index were calculated with the computerized Chou-Talalay method which is based on the median-effect principle. Agents were shown to differ in their inhibitory potency. EGCG was less effective with cell progression; the cancer cells were more resistant than normal or dysplastic cells. In contrast, curcumin was equally effective regardless of the cell type tested. Cell cycle analysis indicated that EGCG blocked cells in G1, whereas curcumin blocked cells in S/G2M. The combination of both agents showed synergistic interactions in growth inhibition and increased sigmoidicity (steepness) of the dose-effect curves, a response that was dose and cell type dependent. Combinations allowed for a dose reduction of 4.4-8.5-fold for EGCG and 2.2-2.8-fold for curcumin at ED50s as indicated by the dose reduction index (DRI). Even greater DRI values were observed above ED50 levels. Our results demonstrate that this model which includes normal, premalignant and malignant oral cells can be used to analyse the relative potential of various chemopreventive agents. Two such naturally-occurring agents, EGCG and curcumin, were noted to inhibit growth by different mechanisms, a factor which may account for their demonstrable interactive synergistic effect.

Synergistic effects of 13-cis retinoic acid and arachidonic acid cascade inhibitors on growth of head and neck squamous cell carcinoma in vitro.

Products of arachidonic acid metabolism can influence normal and malignant cell growth. In vivo, inhibitors of arachidonic acid metabolism have been associated with inhibition of tumor growth, including head and neck squamous cell carcinoma (HNSCC). This has not been evaluated extensively in vitro in an HNSCC model. Therefore we investigated the effects of several arachidonic acid cascade inhibitors (AACIs) (indomethacin, curcumin, phenidone, nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, and 13-cisretinoic acid) on the growth of two HNSCC cell lines (MDA 886Ln and 1483). We found that AACIs caused dose-dependent growth inhibition of both cell lines. In an effort to inhibit HNSCC cell growth at lower concentrations of these drugs, we evaluated the effects of a variety of AACIs in combination with 13-cis retinoic acid. We observed synergistic growth inhibition when the drugs were used in all combinations, with the exception of indomethacin. These results suggest that AACIs may have some utility in the direct treatment of HNSCC, and a strategy combining 13-cis retinoic acid with other AACIs may prove to be even more effective.

Modulation of galectin-1 content in human head and neck squamous carcinoma cells by sodium butyrate.

Galectin-1 and galectin-3 are beta-galactoside-binding proteins thought to be important for cellular interactions, growth regulation and differentiation. Alterations in cellular content of galectins have been associated with differentiation, transformation and malignant progression. We examined the modulation of galectin-1 and galectin-3 expression in head and neck squamous cell carcinoma (HNSCC) cell lines by treatment with sodium butyrate, a known differentiation-modulating agent, and identified potential mechanisms of butyrate regulation of galectin-1 levels in one of the cell lines. Sodium butyrate effected an increase in galectin-1 protein concentration in 5 of 8 cell lines. One cell line, MDA-886LN, showed a marked time- and dose-dependent increase from barely detectable amounts with butyrate treatment. Concurrently with increased galectin-1 expression, butyrate treatment promoted morphologic changes, induced growth inhibition and inhibited soft agar colony formation in MDA-886LN cells. Butyrate-treated MDA-886LN cells demonstrated increased galectin-1 mRNA content, suggesting a role for butyrate in transcriptional regulation of galectin-1 expression. Treatment with other inhibitors of histone deacetylase also induced an increase in galectin-1 expression. Together, our results indicate that butyrate treatment can modulate galectin-1 content in MDA-886LN HNSCC cells as well as induce morphologic changes and growth inhibition. This action may involve a combination of transcriptional regulation and inhibition of histone deacetylation.

Native fluorescence spectroscopy of normal and malignant epithelial cells.

Native fluorescence spectroscopy of normal human oral and malignant epithelial cells was studied under uv excitation. Differences were observed in the excitation spectra between normal and malignant epithelial cells for 340 nm emission. The observed differences may be utilized for both discrimination and changes associated with the amino acid residues in the cellular proteins.

Efficient gene transfer to human squamous cell carcinomas by the herpes simplex virus type 1 amplicon vector.

BACKGROUND: This study evaluates the efficiency of herpes simplex virus (HSV) mediated gene transfer in human squamous cell carcinoma (SCC) cell lines in vitro and in vivo when delivered by selective intra-arterial perfusion. METHODS: Human head and neck SCC were exposed to HSV-LacZ and HSV-interleukin-2 (IL-2) and gene transfer and expression assessed by X-gal staining and enzyme-linked immunosorbent assay, respectively. Hamster cheek pouch tumors were perfused with HSV-LacZ or HSV-IL-2, by microcannulating the external carotid artery, and gene transfer determined. RESULTS: A ratio of 5 viral particles per tumor cell achieved gene transfer rates exceeding 50%. Interleukin-2 levels of 287 +/- 17 to 424 +/- 8.4 ng per million cells were achieved at a ratio of 2 viral particles per tumor cell. Selective intra-arterial perfusion of the HSV-IL-2 vector yielded IL-2 levels of 45.8 +/- 17.0 pg per g tumor. CONCLUSIONS: HSV amplicon vectors are efficient vehicles for gene transfer in vitro in human head and neck SCC cell lines and in vivo when introduced by selective intra-arterial perfusion.

Retinoids suppress epidermal growth factor-induced transcription of cyclooxygenase-2 in human oral squamous carcinoma cells.

Cyclooxygenase-2 (Cox-2), the inducible form of cyclooxygenase, is up-regulated in tumors and transformed cells. Because this enzyme catalyzes the formation of prostaglandins from arachidonic acid, chemopreventive strategies that suppress its expression could be useful for preventing cancer. We investigated whether retinoids suppressed basal expression of Cox-2 or EGF-mediated induction of Cox-2 in human oral squamous carcinoma cells. Treatment with retinoids [all-trans-retinoic acid (all-trans-RA), 9-cis-RA, 13-cis-RA, and retinyl acetate] suppressed both basal levels of Cox-2 and EGF-mediated induction of Cox-2 protein and synthesis of prostaglandin E2. Retinoids also suppressed the induction of Cox-2 mRNA by EGF. Transient transfection experiments showed that EGF caused about a 100% increase in Cox-2 promoter activity, an effect that was suppressed by retinoids. Levels of epidermal growth factor receptor were unaffected by retinoids. Epidermal growth factor caused a nearly 10-fold increase in mitogen-activated protein kinase activity; this effect was not blocked by retinoids.