Hexosaminidase Assay Research Articles

Abstract 3055: Wnt inhibitory factor 1 inhibits cervical cancer cell growth in vitro

Abstract Background and Purpose: Cervical cancer is one of the most common malignancies and major causes of death in women worldwide. Aberrant activation of Wnt/β-catenin pathway is associated with various cancers including cervical cancer. Mutations within intracellular components of the Wnt pathway are rare in cervical cancer. We hypothesize that alterations in secreted Wnt antagonists might play a major role in Wnt activation. Here we characterize the status of the Wnt inhibitory factor 1 (WIF1) in cervical cancer cell lines and determined the in vitro effects of WIF1 on cervical cancer cells. Methods: Three cervical cancer cell lines (HeLa, C33A and CC1) were treated with the demethylating agent 5-aza-2′-deoxycytidine (DAC) for 4 days, total RNA was isolated and real-time RT-PCR was performed to determine WIF1 mRNA expression. HeLa cells were transfected simultaneously with either pCI blast (empty vector) or pCI blast-WIF1 using LipoD293 transfection reagent and assessed for cell proliferation and apoptosis. Cell proliferation was determined at different time intervals (24, 48, 72 and 96 h) by hexosaminidase assay. Cell cycle analysis was performed after 72 h by flow cytometry using FACSCalibur analyzer. Caspase 3/7 activity was measured using the Apo-one Homogeneous Caspase-3/7 Assay kit. Results: We demonstrated that WIF1 is down-regulated in cervical cancer cells. Treatment with DAC caused a significant increase in WIF1 mRNA expression in all the cell lines tested compared with vehicle treatment. Furthermore, transfection with a plasmid expressing WIF1 significantly inhibited cervical cancer cell proliferation at all time points studied. To probe the possible mechanisms involved in WIF1 cell growth inhibition, we performed cell cycle analysis and caspase 3/7 assay. Interestingly, cell cycle analysis demonstrated that transfection with WIF1 significantly increased the G2-M cell population and the number of apoptotic cells. In addition, WIF1 significantly induced caspase 3/7 activity suggesting that WIF1 induced apoptosis is mediated through caspase 3/7 activation. Conclusions: Our data suggests that WIF1 is silenced in cervical cancer cells and WIF1 restoration inhibits cervical cancer cell growth by inducing G2-M arrest and apoptosis. This study emphasizes the importance of WIF1 as a potential therapeutic strategy in the treatment of cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3055.

Abstract 5046: SR1264, a novel tetrafluoro analog of curcumin prevents pancreatic tumor growth in vivo by suppressing Notch signaling pathway

Abstract Dysregulated notch signaling plays an important role in the progression of pancreatic cancer. Previous studies have demonstrated that curcumin, the active ingredient in the spice turmeric can modulate notch activity. In this study, we have developed a novel curcumin analog SR1264 and have determined its effects on notch signaling in pancreatic cancer cells. Methods: Five pancreatic adenocarcinoma cell lines (AsPC-1, MiaPaCa-2, PanC-1, BxPC-3 and Pan02) were used in the study. Cell proliferation was measured by hexosaminidase and colony formation assays. Apoptosis was determined by measuring caspase 3/7 activity and western blot analysis for caspase 3. Cell cycle analysis was performed by propidium iodide staining and flow cytometry. Real Time PCR and western blot analyses were used to measure mRNA and protein levels. For in vivo studies, nude mouse xenografts were developed with mouse pancreatic cancer cells (Pan02). The resulting effects of SR1264 treatment was assessed by measuring tumor volume. Immunohistochemistry was performed for CD31 (endothelial marker, surrogate for angiogenesis) and for COX-2, VEGF and Notch-1 expression. Results: SR1264 treatment resulted in a dose and time dependent inhibition of proliferation and in colony formation in all 5 pancreatic cell lines. Conversely, SR1264 did not affect the proliferation of normal mouse embryo fibroblasts. The compound also induced apoptosis by activating caspase 3. Cell cycle analyses demonstrated that SR1264 induced G2/M cell cycle arrest, which was followed by the induction of apoptosis as evidenced by caspase-3 activation and an increased number of cells with a sub-G1 DNA fraction. Western blot analyses demonstrated that SR1264 downregulated the expression of cell cycle related proteins cyclin A, D1 and E, while upregulating p21Waf1. In addition, SR1264 significantly downregulated the expression of Notch-1, Jagged-1, Hey-1, Hes-1, COX-2, IL-8 and VEGF. To determine the effect of SR1264 on tumor growth in vivo, nude mice harboring pancreatic cancer tumor xenografts in their flanks were administered with the compound intraperitoneally every day for 21 days. SR1264 significantly inhibited tumor xenograft growth, with notably lower tumor volume and weight. Microvessel density, based on CD31 staining was also significantly lower in the tumors following SR1264 treatment when compared to tumors in controls. Real Time PCR and immunohistochemistry analyses demonstrated significant inhibition of COX-2, VEGF, and Notch-1, and cyclin D1 mRNA and protein expression in the tumors. Conclusion: Together, these data suggest that SR1264 is an effective inhibitor of pancreatic tumor growth in vivo in part through downregulating Notch-1 protein and its downstream targets. Therefore, SR1264 may be a novel therapeutic agent for pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5046.

In vitro evaluation of human osteoblast adhesion to a thermally oxidized γ-TiAl intermetallic alloy of composition Ti–48Al–2Cr–2Nb (at.%)

Ti-48Al-2Cr-2Nb (at.%) (gamma-TiAl), a gamma titanium aluminide alloy originally designed for aerospace applications, appears to have excellent potential as implant material. Thermal treatment of gamma-TiAl renders this alloy extremely corrosion resistant in vitro, which could improve its biocompatibility. In this study, the surface oxides produced by thermal oxidation (at 500 degrees C, and at 800 degrees C for 1 h in air) on gamma-TiAl were characterized by X-ray photoelectron spectroscopy (XPS). hFOB 1.19 cell adhesion on thermally oxidized gamma-TiAl was examined in vitro by a hexosaminidase assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) after 1, 7 and 14 days. Ti-6Al-4V surfaces were used for comparison. Hexosaminidase assay data and CLSM analysis of focal contacts and cytoskeleton organization showed no differences in cell attachment on autoclaved and both heat-treated gamma-TiAl surfaces at the different time points. SEM images showed well organized multi-layers of differentiated cells adhered on thermally oxidized gamma-TiAl surfaces at day 14. Unexpectedly, thermally oxidized Ti-6Al-4V surfaces oxidized at 800 degrees C exhibited cytotoxic effects on hFOB 1.19 cells. Our results indicate that thermal oxidation of gamma-TiAl seems to be a promising method to generate highly corrosion resistant and biocompatible surfaces for implant applications.

Population‐based Tay‐Sachs screening among Ashkenazi Jewish young adults in the 21st century: Hexosaminidase a enzyme assay is essential for accurate testing

Tay-Sachs disease (TSD) carrier screening, initiated in the 1970s, has reduced the birth-rate of Ashkenazi Jews with TSD worldwide by 90%. Recently, several nationwide programs have been established that provide carrier screening for the updated panel of Jewish genetic diseases on college campuses and in Jewish community settings. The goals of this study were to determine the performance characteristics of clinical TSD testing in college- and community-based screening programs and to determine if molecular testing alone is adequate in those settings. Clinical data for TSD testing were retrospectively anonymized and subsequently analyzed for 1,036 individuals who participated in these programs. The performance characteristics of the serum and the platelet Hexosaminidase assays were compared, and also correlated with the results of targeted DNA analysis. The serum assay identified 29 carriers and the platelet assay identified 35 carriers for carrier rates of 1/36 and 1/29, respectively. One hundred sixty-nine samples (16.3%) were inconclusive by serum assay in marked contrast to four inconclusive samples (0.4%) by the platelet assay. Molecular analysis alone would have missed four of the 35 carriers detected by the platelet assay, yielding a false negative rate of 11.4% with a sensitivity of 88.6%. Based on the results of this study, platelet assay was superior to serum with a minimal inconclusive rate. Due to changing demographics of the Ashkenazi Jewish population, molecular testing alone in the setting of broad-based population screening programs is not sufficient, and biochemical analysis should be the assay of choice.

Improved formulation of liposome-encapsulated hemoglobin with an anionic non-phospholipid

We are developing liposome-encapsulated hemoglobin (LEH) as an artificial oxygen carrier for resuscitation in indications, such as acute blood loss and surgery. Earlier attempts to formulate a viable LEH met with constraints of scale up and limited hemoglobin content. In this work, we report an LEH formulation containing novel anionic non-phospholipid (CHHDA) that enhances the encapsulation efficiency of hemoglobin inside the liposome bilayer. CHHDA was synthesized from inexpensive ingredients in high yields. The formulation was evaluated in vitro to investigate the cytotoxic effects on RAW 264.7 macrophages and HUVEC endothelial cells in culture by LDH, MTT and hexosaminidase assays. Under optimal conditions of manufacturing, the presence of 28 mol% of CHHDA enhanced the hemoglobin content to over 4 g/dl. The LEH containing CHHDA shows some cytotoxicity in HUVEC and RAW cells in vitro, especially by LDH assay. MTT assay was negative for cytotoxicity in both cells lines. By hexosaminidase assay, the proliferation of RAW cells, but not HUVEC cells, was inhibited. When CHHDA-LEH was incubated with isolated human platelets in vitro, no platelet activation was observed. The LEH formulation with novel anionic lipid and high hemoglobin content reported in this article is an improvement from the past preparations.

Serum IgA1 from patients with IgA nephropathy up-regulates integrin-linked kinase synthesis and inhibits adhesive capacity in podocytes through indirect pathways

To investigate the influence of IgA1 isolated from IgA nephropathy (IgAN) patients on integrin-linked kinase (ILK) synthesis and adhesive capacity of podocytes through indirect pathways. IgA1 was isolated from healthy control or IgAN patients' sera using jacalin affinity chromatography and S-200 chromatography. Podocytes were treated with medium from mesangial cells incubated with aggregated IgA1 (aIgA1, 100 microg/ml), in the presence or absence of valsartan (10(-5)M) or neutralizing antibodies of tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml). Adhesive capacity of podocytes was assessed by cell counting manually and hexosaminidase assay. Real-time PCR and western blotting were used to detect the expression of ILK. Medium from mesangial cells incubated with aIgA1 from IgAN patients reduced podocyte adhesion to collagen compared with medium from mesangial cells incubated with control medium(RPMI-1640 with 0.5% FBS) (35.0+/-4.8% vs. 60.0+/-2.0%; P < 0.05). While medium from mesangial cells incubated with aIgA1 from IgAN patients upregulated ILK expression in podocytes at mRNA and protein levels compared with medium from mesangial cells without aIgA1 incubated (1.6-fold and 1.38-fold higher than control, respectively, P < 0.05). Defects in podocyte adhesion and up-regulation of ILK synthesis induced by medium from mesangial cells incubated with aIgA1 from IgAN patients can be partially reversed by the pre-treatment for 1 hour with valsartan(P < 0.05), while pre-treatment with neutralizing antibodies of TNF-alpha produced no protective effect on podocytes (P > 0.05). Serum IgA1 from IgAN patients may inhibit adhesive capacity and up-regulate ILK synthesis in podocytes through indirect pathways.

The regulatory role of nerve growth factor and its receptor system in fibroblast‐like synovial cells

Objectives: Investigating the role of nerve growth factor (NGF) and its receptors (NGF‐R) in inflammatory diseases is an active field of research. Inflammatory diseases of the joint are the commonest cause of human morbidity but very little is known about the effect of NGF on synovial tissue biology. Here we have studied NGF/NGF‐R and their functional significance on cultured fibroblast‐like synovial cells (FLS) collected from the synovial tissue of five healthy subjects.Methods: NGF/NGF‐R expression was determined in the basal condition and after stimulation with tumour necrosis factor (TNF)α and interleukin (IL)‐1β by enzyme‐linked immunosorbent assay (ELISA) and fluorescence‐activated cell sorting (FACS). Proliferation studies were performed by cell count, hexosaminidase assay, and the MTT assay. The synovial fluid (SF) NGF level was studied by ELISA in 12 psoriatic arthritis (PsA), 14 rheumatoid arthritis (RA), and 10 osteoarthritis (OA) patients.Results: FACS studies showed that unstimulated FLS expressed low levels of NGF and the high‐affinity NGF‐tyrosine kinase receptor TrkA, and TNFα and IL‐1β increased NGF and TrkA expression in FLS. NGF (100 ng/mL) increased FLS proliferation by 400% compared to the control (medium only). The NGF level was significantly higher in the PsA group (365.5±85.2 pg/mL) than in the RA (120±35 pg/mL) and OA groups (30±6 pg/mL).Conclusions: Upregulation of NGF/TrkA in proinflammatory cytokine‐activated FLS, the mitogenic effect of NGF on FLS, and the increased NGF level in SF of inflammatory arthritis suggest that there is cross‐talk between NGF/NGF‐R and FLS. These results also suggest that dysregulated production of NGF may lead to synovial cell proliferation and thus could influence the inflammatory and proliferative cascades of inflammatory arthritis.

Hexosaminidase assays

beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

Diphenyl Difluoroketone: A Curcumin Derivative with Potent In vivo Anticancer Activity

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was reported to inhibit proliferation of a variety of cancer cells in vitro. However, the efficacy and in vivo mechanism of action of EF24 in gastrointestinal cancer cells have not been investigated. Here, we assessed the in vivo therapeutic effects of EF24 on colon cancer cells. Using hexosaminidase assay, we determined that EF24 inhibits proliferation of HCT-116 and HT-29 colon and AGS gastric adenocarcinoma cells but not of mouse embryo fibroblasts. Furthermore, the cancer cells showed increased levels of activated caspase-3 and increased Bax to Bcl-2 and Bax to Bcl-xL ratios, suggesting that the cells were undergoing apoptosis. At the same time, cell cycle analysis showed that there was an increased number of cells in the G(2)-M phase. To determine the effects of EF24 in vivo, HCT-116 colon cancer xenografts were established in nude mice and EF24 was given i.p. EF24 significantly suppressed the growth of colon cancer tumor xenografts. Immunostaining for CD31 showed that there was a lower number of microvessels in the EF24-treated animals coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA and protein expression. Western blot analyses also showed decreased AKT and extracellular signal-regulated kinase activation in the tumors. Taken together, these data suggest that the novel curcumin-related compound EF24 is a potent antitumor agent that induces caspase-mediated apoptosis during mitosis and has significant therapeutic potential for gastrointestinal cancers.

Bacterial contamination positively interferes with β-glucosidase and hexosaminidase assays in white blood cell lysates

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The effect of PLGA sphere diameter on rabbit mesenchymal stem cells in adipose tissue engineering

To investigate the effect of injectable PLGA sphere's diameter on adipose tissue engineering, rabbit mesenchymal stem cells were attached to various diameters of injectable PLGA spheres (<75; 75-100; 100-150; 150-200; and 200-250 microm). These five groups were cultured in adipogenic media for 2 weeks in vitro and injected into necks of nude mice. Prior to in vivo study, cell proliferation and adipogenic differentiation were determined by hexosaminidase assay and Oil red O staining after 2 weeks. Group C (100-150 microm) showed the highest adipogenic differentiation and the proliferation capacity of Group B (75-100 microm) was significantly higher than that of any other group. We harvested newly formed tissues from necks of nude mice after 1 and 4 weeks. Although PLGA spheres have not been degraded and there was no significant histological difference among various sizes of spheres after 1 week, well-organized fat pads (PLGA spheres were completely degraded) could be observed, and the histology of the 100-150 microm groups resembled that of native tissue after 4 weeks. Based on these experiments, we could conclude that the optimal size of PLGA spheres for adipogenesis was 100-150 microm.

Tay-Sachs and Sandhoff diseases: enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards

Background: Tay-Sachs disease (TSD), Sandhoff disease (SD) and variants are caused by deficient activity of the lysosomal enzymes hexosaminidase A (HA) and total hexosaminidase (TH) (hexosaminidase A plus B), respectively. For diagnosis, these enzymes are usually measured in plasma or extracts of leukocytes. We describe methods for the assay of hexosaminidase A and total hexosaminidase activities in dried blood spots (DBSs) on filter paper. Materials and Methods: We studied 163 healthy controls, 9 Tay-Sachs patients, 4 Sandhoff patients, 18 obligate carriers and the newborn-screening cards from two patients with Tay-Sachs and one patient with Sandhoff disease. To tubes containing a 3-mm-diameter blood spot, we added elution liquid and substrate solution. After incubation at 37 °C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Results and conclusions: The described methodology is useful to distinguish patients with Tay-Sachs disease or Sandhoff disease from carriers and controls using samples that are sufficiently stable to be transported to the testing laboratory by mail. The diagnosis of both diseases from a newborn-screening card (NSC) was clearly demonstrated, even after storage for up to 38 months at room temperature. The newborn-screening card has been added to the biological materials that allow the identification of patients with Tay-Sachs disease and Sandhoff disease.

Effect of nerve growth factor on endothelial cell biology: proliferation and adherence molecule expression on human dermal microvascular endothelial cells.

In addition to its effect on the central nervous system, nerve growth factor (NGF) appears to play a key role in the initiation and maintenance of inflammation in many organs. NGF degranulates mast cells, recruits inflammatory cellular infiltrates and activates T cells. Extravascular migration of leukocytes is initially controlled by the interaction of cell surface adhesion molecules of leukocytes and endothelial cells. A marked upregulation of NGF in keratinocytes is also observed in conditions characterized by angiogenesis such as psoriasis and wound healing. In this study we investigated the role of NGF in inflammation by studying its effects on endothelial cell proliferation and intracellular adhesion molecule expression by endothelial cells. The effect of NGF on human dermal microvascular endothelial cell (HDMEC) proliferation was measured using the hexosaminidase assay. ICAM-1 expression on HDMEC was measured by ELISA. The function of ICAM-1 was assessed by adherence of peripheral blood mononuclear cells (PBMC) to HDMEC using 51Cr-labeled PBMC. There was a significant increase in proliferation of HDMEC stimulated with NGF as compared to unstimulated HDMEC (P < 0.001). NGF-neutralizing antibody decreased the mitogenic effect of NGF significantly (P < 0.05). NGF also increased ICAM expression on HDMEC as compared to unstimulated HDMEC (P < 0.05). NGF-neutralizing antibody decreased ICAM expression on NGF-stimulated HDMEC (P < 0.05). The percentage of PBMC adherence was higher in NGF-stimulated HDMEC (P < 0.001). Anti-ICAM antibody decreased PBMC adherence. In the study reported here, the role of NGF in two important aspects of inflammation, i.e. angiogenesis and inflammatory cell recruitment at the site of inflammation, was investigated.

Assessment of acute cyto- and genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes.

Whilst a patient is undergoing orthodontic treatment, dental appliances based on non-precious metals or titanium remain in the oral cavity for up to several years. Throughout this period the appliance is in either direct or indirect contact with the oral mucosa. To investigate the possibility of cell damage occurring as a result of appliance corrosion, monolayer cultures of immortalized human gingival keratinocytes were assessed for acute cyto- and genotoxicity using the hexosaminidase assay and the Comet assay respectively. The materials tested included 1. a nickel-free wire, 2. a UK-1 bond, 3. nickel-free as well as nickel-containing brackets with and without color signature and 4. a titanium expansion screw. Each of the test materials was corroded in a solution consisting of equal amounts of lactic acid and sodium chloride (0.1 M) for 1, 3, 7 and 14 days. The cell cultures were then exposed to eluates exhibiting the highest ion concentrations. None of the eluates was found to exhibit acute cytotoxicity, regardless of the type of test system used. Qualitative assessment using neutral red dye for live cells and either trypan blue or propidium iodide to disclose dead cells failed to reveal any significant increase in cell damage when exposed cells were compared to control cultures. Unrestricted cell vitality was confirmed by quantifying viable cells through measurement of hexosaminidase enzyme activity. Furthermore, assessment of genotoxicity revealed no apparent DNA damage to immortalized gingival keratinocytes following exposure to the test eluates. Because the materials tested in this study were corroded using the exacting methods normally applied to precious metals or gold-containing alloys, the lack of either acute cyto- or genotoxic effects following exposure to the test eluates indicates that the materials tested exert no adverse effects on cells similar to those of the target tissue exposed to the materials in situ.

In Vitro Evaluation of Antiproliferative Potential of Calcium Channel Blockers in Human Tenon's Fibroblasts

The calcium cation has been known to play a central and diverse role in the proliferation of numerous types of cells including fibroblasts. Therefore, it follows that calcium channel blockers (CCBs) may potentially inhibit fibroblast attachment and proliferation. The time and dose-related effects of five commonly used CCBs—verapamil, diltiazem, nicardipine, trifluoperazine, and dantrolene—on human Tenon's fibroblast attachment and proliferation were studied. Fibroblasts were incubated with different concentrations of each drug. To evaluate the effect of each drug on fibroblast attachment, cell density was quantified by Coulter counter and hexosaminidase assays after 24 hours of incubation. To evaluate the effect of each drug on fibroblast proliferation, cell density was quantified by Coulter counter, hexosaminidase, and3H-thymidine uptake assays on days 1, 3, and 7. Dantrolene had minimal effect, failing to cause even 20% inhibition at 10−5M, the highest concentration tested. Verapamil, diltiazem, nicardipine, and trifluoperazine all inhibited fibroblast attachment and proliferation. Trifluoperazine was the most potent inhibitor of attachment and proliferation withID50s in the 10−5Mrange. Verapamil, diltiazem, and nicardipine had similar potency in inhibiting attachment and proliferation withID50s in the 10−4Mrange. Because CCBs seem to inhibit fibroblast attachment and proliferation, future clinical studies may show that these agents reduce collagen production, scar formation, and bleb failure following glaucoma filtration surgery.

Comparative efficacy of antiviral drugs on human ocular fibroblasts

The effects of several antiviral drugs on fibroblast attachment and proliferation from human Tenon's capsule were investigated. These drugs included purine nucleoside analogs, vidarabine and acyclovir (ACV): pyrimidine nucleoside analog, AZT; and a synthetic cyclic primary amine, amantadine. Fibroblast attachment and proliferation inhibition were determined by Coulter counter, a colorimetric assay of the enzyme hexosaminidase, and a 3H-thymidine uptake assay. Amantadine and AZT inhibited fibroblast attachment at concentrations higher than 6·61 × 10 −4 m and 3·73 × 10 −4 m, respectively. Amantadine and AZT had inhibitory effects on fibroblast proliferation as early as day 1, whereas vidarabine and ACV manifested their inhibitory effects after day three by Coulter counter and hexosaminidase assays. For amantadine, AZT, ACV and vidarabine, the 50% inhibitory dose (ID 50) were 4·94 × 10 −5 m, 1·26 × 10 −5 m, 4·6 × 10 −4 m, and 1·52 × 10 −5 m at day 9, respectively, as measured by 3H-thymidine uptake assay. All four antiviral agents tested had inhibitory effects on human ocular fibroblast proliferation and their inhibitory potential decreased in the order of amantadine ⩾ vidarabine > AZT ⩾ ACV.

Influence of various cytokines on the interleukin-2-dependent lysis of melanoma cells in vitro.

To obtain information about useful combinations of various cytokines in melanoma therapy, we studied the influence of interleukin-2 (IL-2) in combination with interferon-alpha (IFN-alpha), IFN-gamma and tumour necrosis factor-alpha (TNF-alpha) on the lytic activity of IL-2-stimulated cells in vitro. Peripheral mononuclear cells (PMC) were incubated for 4 days with various combinations of cytokines and used as effector cells. Two different melanoma cell lines (M19 and M26) were used as target cells. The lytic activity of stimulated PMC was determined using a modified hexosaminidase assay. IL-2 was mainly responsible for the lytic activity of the effector cells in a dose-dependent manner. IFN-alpha, IFN-gamma and TNF-alpha did not enhance lytic activity with an optimal IL-2 dose (50 IU/ml IL-2). Using a suboptimal IL-2 dose (5 IU/ml), they increased cytotoxicity. The specific lysis of M19 cells was significantly increased by pretreatment of the cells with 5 IU/ml IFN-alpha together with 50 IU/ml TNF-alpha (t-test, P < or = 0.001), while the specific lysis of M26 cells was increased by pretreatment with 5 IU/ml IFN-gamma. We conclude that the lysis of melanoma cells by cytotoxic cells in vitro can be enhanced by various cytokines. The optimal cytokine combination differed for the two melanoma cell lines tested.

In Vitro Evaluation of Antiproliferative Potential of Topical Cyclo-oxygenase Inhibitors in Human Tenon's Fibroblasts

Pharmacologic agents which inhibit fibroblast proliferation and scar formation may improve the success of glaucoma filtration surgery. To evaluate the potential use of nonsteroidal anti-inflammatory drugs as an antifibrotic treatment following glaucoma filtration surgery, the time and dose-related effects of four commonly used topical cyclo-oxygenase inhibitors—diclofenac, flurbiprofen, piroxicam, and suprofen—on human Tenon's fibroblast attachment and proliferation were studied. Fibroblasts were incubated with different concentrations of a drug and analysed for dose response 1, 3, and 8 days following drug addition. The cell density was quantified by Coulter counting, hexosaminidase assay, and [3H]thymidine uptake into the DNA. All four drugs inhibited fibroblast attachment at the highest concentrations. Diclofenac was the most effective agent in inhibiting fibroblast proliferation, with inhibition occurring at a range of concentrations above 10-3-10-4 mmol I-1. Flurbiprofen, piroxicam, and suprofen were comparable to each other in their antiproliferative activities, with inhibition occurring at a range of concentrations above 10-1-10-2 mmol I-1. These results suggest a potential role for the commonly used topical nonsteroidal anti-inflammatory drugs in preventing excessive fibroblast proliferation following GFS, but further clinical studies are indicated to assess their activity as anti-inflammatory and antiproliferative agents in preventing bleb failure.

Neuropeptides Modulate Leukotriene B4 Mitogenicity Toward Cultured Human Keratinocytes

Neuropeptides released in skin from nerve fibers may interact with endogenous growth factors (or other mitogenic agents) to induce psoriasis lesions characterized by proliferating epidermal keratinocytes. The mitogenic effects of two neuropeptides, substance P (SP) and vasoactive intestinal peptide (VIP), on human adult and newborn keratinocytes were observed in the presence or absence of leukotriene B4 (LTB4) and leukotriene C4 (LTC4). In the presence of SP or VIP, LTB4 (but not LTC4) demonstrated substantial increase in thymidine incorporation into DNA, which was confirmed by cell-growth observations using the hexosaminidase assay. In the absence of either neuropeptide, LTB4 had only marginal effects, especially with adult (but not newborn) keratinocytes. With adult keratinocytes, LTB4 (but not LTC4) demonstrated synergy with both SP and VIP. VIP was mitogenic to keratinocytes at concentrations as low as 10(-12)M and exhibited a different dose-response curve depending on whether adult or newborn keratinocytes were used. The mitogenic effects of SP were abrogated by the SP antagonist spantide and those of VIP by the VIP antagonist [Ac-Tyr1, D-Phe2] growth-hormone-releasing factor (1-29) amide. This study suggests that the mitogenic effects of LTB4, which are elevated in psoriatic lesions, may be enhanced by the presence of neuropeptides, especially VIP. These effects can be reversed by antagonists that may have potential as drugs for the disease.

Tay-Sachs disease heterozygote detection: use of a centrifugal analyser for automation of hexosaminidase assays with two different artificial substrates.

An assay for measuring hexosaminidase A in serum and leucocytes is described in which a centrifugal analyser is used for automation of the enzyme assays after manual heat inactivation. The...