JM Cells Research Articles

Lensless imaging-based discrimination between tumour cells and blood cells towards circulating tumour cell cultivation.

Circulating tumour cells (CTCs) are recognized as important markers for cancer research. Nonetheless, the extreme rarity of CTCs in blood samples limits their availability for multiple characterization. The cultivation of CTCs is still technically challenging due to the lack of information of CTC proliferation, and it is difficult for conventional microscopy to monitor CTC cultivation owing to low throughput. In addition, for precise monitoring, CTCs need to be distinguished from the blood cells which co-exist with CTCs. Lensless imaging is an emerging technique to visualize micro-objects over a wide field of view, and has been applied for various cytometry analyses including blood tests. However, discrimination between tumour cells and blood cells was not well studied. In this study, we evaluated the potential of the lensless imaging system as a tool for monitoring CTC cultivation. Cell division of model tumour cells was examined using the lensless imaging system composed of a simple setup. Subsequently, we confirmed that tumour cells, JM cells (model lymphocytes), and erythrocytes exhibited cell line-specific patterns on the lensless images. After several discriminative parameters were extracted, discrimination between the tumour cells and other blood cells was demonstrated based on linear discriminant analysis. We also combined the highly efficient CTC recovery device, termed microcavity array, with the lensless-imaging to demonstrate recovery, monitoring and discrimination of the tumour cells spiked into whole blood samples. This study indicates that lensless imaging can be a powerful tool to investigate CTC proliferation and cultivation.

Adenosine Deaminase 1 as a Biomarker for Diagnosis and Monitoring of Patients with Acute Lymphoblastic Leukemia.

SummaryBackgroundAcute lymphoblastic leukemia (ALL) is known as the most prevalent pediatric malignancy all around the world. Identification of specific biomarker is necessary for early diagnosis and effective therapy. It is believed that Adenosine deaminase (ADA) as an enzyme involved in the purine salvage pathway increases in ALL patients. Herein, the quantity and pattern of ADA isoenzymes were surveyed among ALL patients in comparison to healthy subjects.MethodsSerum and RBC samples of three different groups of ALL patients, including newly diagnosed cases without any drugs administration, subjects with the relapsed disease, patients in the remission stage after therapy, and the healthy subjects were enrolled in the study. Then, the activity and pattern of ADA1, ADA2 and ADA1+cp were determined using ADA kit and electrophoresis on SDS-PAGE, respectively. To confirm the presence of ADA enzyme, the fresh serums, extractions from erythrocytes, JM cell line as a human T lymphocyte line and J774 A.1 as mouse monocyte line were electrophoresed on 1.2% agarose gel and stained with the specific dye.ResultsThe activities of ADA1 isoenzyme and total ADA in new cases and subjects with the relapsed disease were significantly higher than their activities in the patients in the remission stage and healthy controls (p<0.001). The unbounded ADA1 isoenzyme was found to exist in the erythrocyte, lymphocyte and monocyte. But in serum, all the ADA1 was bounded to the cp protein.ConclusionsADA1 is the key isoenzyme elevating in ALL patients, therefore this isoenzyme could be a useful biomarker to diagnose ALL patients and monitor their therapies.

Functionality and opposite roles of two interleukin 4 haplotypes in immune cells

Cytokines expression can be influenced by polymorphisms in their respective coding genes. We associated the CTI/TTD haplotype (Hap-1) and TCI/CCI haplotype (Hap-2) in the IL4 gene formed by the −590, +33 and variable number of tandem repeat polymorphisms with the severity of chronic periodontitis in humans. The functionality of these IL4 haplotypes in the response of immune cells to phorbol 12-myristate 13-acetate (PMA) with Ionomycin and IL-1β (as inflammatory stimuli) was evaluated. Gene expression (quantitative real-time PCR), profile of secreted cytokines (multiplex) and phenotypic polarization of T cells (flow cytometry) were the outcomes assessed. Green fluorescent protein reporter plasmid constructs containing specific IL4 haplotype were transiently transfected into JM cells to assess the influence of the individual haplotypes on promoter activity. In response to inflammatory stimuli the immune cells from Hap-1 haplotype had increased expression of anti-inflammatory IL4; conversely, the Hap-2 haplotype showed higher levels of pro-inflammatory cytokines. The haplotype CTI proved to be the most important for the regulation of IL4 promoter, regardless of the nature of the inflammatory stimulation; whereas the polymorphism in the promoter region had the least functional effect. In conclusion, IL4 haplotypes studied are functional and trigger opposite immune responses: anti-inflammatory (Hap-1) and pro-inflammatory (Hap-2). In addition, we identified the CTI haplotype as the main responsible for the regulation of IL4 transcriptional activity.

Development of a Novel Cell Monitoring System Based on Lens-Less Imaging Toward Cultivation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are defined as tumor cells circulating in the peripheral blood of patients with metastatic cancer. As CTCs could provide useful information regarding malignancy and metastatic property of primary tumor, as well as its response to anticancer agents, fundamental techniques for recovery and analysis of CTCs have been intensively studied in this decade. Although state-of-the-art microfluidic tools and whole genome amplification techniques are now available to investigate single CTCs, it is still extremely difficult to cultivate CTCs that, for example, enables examination of multiple anticancer agents toward identical CTCs. This could be due to the lack of knowledge of division and proliferation behavior of CTCs. The critical difficulty in investigation of CTC proliferation is that cell division events of CTCs are very rare, and thus conventional microscopy could hardly identify the dividing CTCs from the samples containing a number of blood cells. In this study, we propose to use a wide-field imaging system for investigation of CTC cultivation. The imaging system is composed of a light emitting diode (LED, peak wavelength: 465 nm) and a complementary metal oxide semiconductor (CMOS) imaging sensor by which an area of 6.55 × 4.92 mm2can be visualized in one shot. A cell culture dish is placed on the CMOS image sensor, and images are acquired under the LED illumination without using any lens systems (referred to as “lenss-less imaging”). The CMOS image sensor allows to develop a space-saving and inexpensive platform, so that whole system can be introduced in a general incubator, and real-time monitoring of cell growth is possible. Using this lens-less imaging system, we attempted to observe tumor cell proliferation and distinguish tumor cells from blood cells. HeLa cells were used as model tumor cells. A culture dish containing HeLa cells in 2 ml of the high glucose Dulbecco's modified Eagle's medium (DMEM, 5,000 cells/ml) was placed on the lens-less imaging system, and incubated for 18 hours at 37°C with 5% CO2. Images were captured every 15 min. For comparison, microscopic observation was also performed. As a result, HeLa cells were recorded as a spherical patterns with high intensity by a CMOS image sensor. When divided, HeLa cells were once illustrated as a dark patterns, and then two daughter cells gradually appeared with increasing the intensity. It is well known that adhesive cells temporarily go through a non-adhesive state before their division process. Indeed, we confirmed that the spherical patterns with high intensity represents the HeLa cells adhere on the bottom of the dishes, and the subsequent dark patterns do the cells in the non-adhesive state by microscopic observation. This data indicates that the lens-less imaging system can detect individual cell division events. Taking advantage of the wide field of view, it was possible to monitor the cell division events of several hundreds of cells simultaneously. Recovery of CTCs from blood samples is the first step for the cultivation and further analysis, however it is still hard to avoid blood cell contamination despite the development of sophisticated technology for CTC separation and enrichment nowadays. Consequently, it is essential to differentiate CTCs and the contaminated blood cells, otherwise accurate evaluation of CTC proliferation is impossible. To test whether tumor cells and blood cells were distinguishable based on lens-less imaging, we compared the lens-less images of HeLa cells, red blood cells and JM cells (model white blood cells). A number of differentiation parameters were extracted from the images, and computed for cluster analysis. As a result, it was successfully demonstrated to discriminate HeLa cells, red blood cells and JM cells without any labeling process, suggesting the development of a simple, inexpensive and rapid cell typing system. In summary, the lens-less imaging system could monitor cell division events of HeLa cells in a wide field of view, and differentiate HeLa cells and blood cells with high accuracy. We believe that this system could be a powerful tool to monitor the proliferation of CTCs recovered from patient blood samples.

1P1-V10 流体力・磁力を利用する連続的マイクロ流体細胞セパレータの開発(ナノ・マイクロ流体システム)

Selection of specific cells based on multiple factors is an essential process for clinical diagnosis/treatment and biological research. In this study, we propose a simple microfluidic cell separator based on two factors (size and surface marker), by combining HDF and magnetophoresis. Cells conjugated with immunomagnetic particles are sorted according to size due to the principle of HDF. Continuously, cells are subjected to the magnetic field, and cells conjugated with a larger number of immunomagnetic particles move downward greater than those labeled with less particles. Consequently, cells are continuously sorted based on the two factors. In the experiment, a mixture of JM cells having CD4, and HeLa cells without CD4 were prepared, and anti-CD4 magnetic immunoparticle were added. As a result, we confirmed that the enrichment/depletion of specific cells was possible, and relatively rapid cell sorting was achieved.

Interkinetic nuclear migration: cell cycle on the move

Interkinetic nuclear migration (INM) is a common feature of developing neuroepithelia, consisting of the periodic movement of the cell nucleus in phase with cell-cycle progression. In this issue of The EMBO Journal, Kosodo et al provide a first molecular mechanism to couple nuclear migration and cell cycle: the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process during the S-G2 transition, modulating microtubule organization to promote apical nuclear migration.

Single-cell detection using a thin film transistor photosensor with micro-partitions

A thin film transistor (TFT) photosensor was applied to single-cell detection by identifying cell surface molecules based on chemiluminescence. Micro-partitions were directly fabricated on the TFT photosensor surface by photolithography. The surface of each pixel was surrounded by 25 μm-height partitions, forming areas of approximately 30 μm × 30 μm for cell entrapment and photosensing. Visualization of individual JM cells, stained with mouse anti-human CD8 IgG1 primary antibody and Horseradish peroxidase (HRP)-labeled anti-mouse IgG1 secondary antibody, as bright-pixels was successfully achieved using the micro-partitioned TFT photosensor integrated into a microfluidic chamber. Furthermore, real-time monitoring of HRP-labeled JM cells was also accomplished. The fabrication of micro-partitions on the surface of the TFT photosensor allows highly efficient single-cell entrapment and chemiluminescence-based detection of JM cells. This is the first report of single-cell entrapment and subsequent signal detection on the photosensing area of individual pixels of TFT photosensor. This system will allow high-throughput and real-time analysis of more than 10(4) cells with minimum optical system requirements.

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC 50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml −1) and by HeLa, HT29 and JM cells with IC 50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml −1, all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.

Ricin toxicity to microglial and monocytic cells

Microglial cells, like macrophages, are very sensitive to ricin, a galactose-specific toxic lectin belonging to the family of ribosome-inactivating proteins. This toxin can be taken up by most cells through the binding of its B chain to galactose-containing molecules on the cell membrane. In macrophagic cell types it can be internalised also by mannose receptors which are present on the surface of these cells. Endocytosis of the toxin by either pathway was evaluated by ricin toxicity to primary cultures of rat microglial cells and to a microglial N11 cell line in the presence or absence of lactose and mannan, which compete for the endocytosis via the ricin lectin chain or cellular mannose receptors, respectively. Results were compared with those obtained in cultures of mouse macrophages, human monocytes, and a monocytic JM cell line. All cultures were protected from ricin toxicity more by lactose than by mannan, indicating that ricin endocytosis via its lectin B chain is prevalent over that mediated by cellular mannose receptors. However, a partial protection by mannan was observed in all cases but not-stimulated N11 cells, either in the form of direct protection or of significant additional protection over that afforded by lactose. Mannose receptor expression by N11 cells was negative before, and positive after, treatment with endotoxin, as assessed by the specific binding of 125I-mannose-bovine serum albumin. Moreover, a partial protection from ricin toxicity by mannan was induced in the N11 microglial line after stimulation, consistently with an inducible expression of the mannose receptor by activated cells switched towards a microglial phenotype.

Different sublines of Jurkat cells respond with varying susceptibility of internucleosomal DNA degradation to different mediators of apoptosis.

The ability of two different Jurkat sublines, termed standard and JM, to form DNA ladders was investigated after various apoptotic stimuli. Exposure to a broad spectrum of drugs interfering with signal transduction or cellular metabolism revealed distinct differences between both Jurkat sublines with regard to the pattern of DNA degradation. In standard Jurkat cells, internucleosomal DNA cleavage occurred only after treatment with the protein kinase inhibitor staurosporine. In contrast, the JM subline responded with internucleosomal DNA fragmentation to exposure to gemcitabine, cycloheximide or staurosporine. All drugs induced the formation of DNA fragments of about 50 kb in both sublines, as revealed by pulse field electrophoresis, except H2O2, which caused unspecific DNA degradation. The staurosporine-induced DNA ladder formation was accompanied by an increase in caspase-3 activity in both lines which, however, was considerably lower in Jurkat JM cells after gemcitabine or cycloheximide exposure. When the analysis of internucleosomal DNA degradation was carried out after mycoplasma infection, both Jurkat lines responded with DNA ladder formation after exposure to all drugs used (here only shown for the standard subline). Employing the zymogram technique, nuclease activities of 47 kDa and 54 kDa were detected in culture supernatants, cell homogenates and nuclear extracts only when mycoplasma-infected, whereas the samples obtained from mycoplasma-free sublines were nuclease-negative using this technique, indicating that these endonucleases were of mycoplasmal origin. After drug exposure, the mycoplasmal nucleases must have gained access to the cytoplasm and nuclei of their host cells by an unknown mechanism.

Basis of Relative Insensitivity of HIV-Infected JM Cells to AZT

The basis of azidothymidine (AZT) insensitivity in the human JM T-cell line has been investigated. It was shown that uptake of radiolabelled thymidine or AZT into cellular acid-soluble pools of JM cells was about 10-fold lower than that seen in AZT-sensitive HeLa cells. Thymidylate kinase, however, was apparently not inhibited by AZTMP in JM cells to the extent observed in most cells and, as a result, AZTTP formation proceeded at a greater rate than in HeLa cells, which exhibited accumulation primarily of AZTMP. Thus the deficit in phosphorylation in JM cells cannot solely account for the decreased AZT sensitivity. Instead, it was shown that JM cells excreted AZTMP into the culture medium and that, whereas HeLa cells continued to accumulate AZT nucleotides over time, JM cells did not. This excretion of AZTMP in JM cells also led to a failure to sustain lowered competing TTP pools. It is concluded that it may not be appropriate to use the JM cell line for testing of novel anti-HIV nucleotides designed to circumvent requirements for phosphorylation.

Resistance to anti-IgM-induced apoptosis in a WEHI-231 subline is due to insufficient production of ceramide.

We describe the properties of a physiological cell death (PCD)-resistant subline of WEHI-231 generated from the PCD-susceptible WEHI-231.7 JM cell line maintained in our laboratory. The PCD-resistant WEHI-231.7 JMRE subline was uniquely resistant to anti-immunoglobulin (Ig)M-induced PCD but not to irradiation and etoposide. In these sublines, we compared the expression of genes implicated in regulating PCD. Northern analysis of c-myc, c-fos, egr-1, Fas, p53 and retinoblastoma revealed similar basal levels of expression in all sublines tested and comparable responses to anti-IgM treatment. Similarly, the expression of bcl-2, bcl-x, bax and IL-1 beta converting enzyme did not correlate with susceptibility to anti-IgM-induced PCD. Next, we systematically studied signal transduction events including: tyrosine phosphorylation, Ca++ flux, and ceramide production in the Jm and JMRE sublines. The tyrosine phosphorylation patterns and the Ca++ influx generated following sIgM engagement were very similar in the JM and JMRE sublines. In contrast, the generation of ceramide differed in the PCD-resistant and PCD-susceptible sublines. Ceramide is produced following cross-linking sIgM on WEHI-231.7 JM cells and causes PCD. Ceramide levels in anti-IgM-treated WEHI-231.7 JMRE cells are low and appear to be insufficient to induce PCD.

Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase.

Intercellular adhesion molecule (ICAM)-3, a recently described counter-receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1-mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.

Synthesis and anti-HIV activity of some novel diaryl phosphate derivatives of AZT

Novel diaryl phosphate triester derivatives of the anti-HIV nucleoside analogue AZT have been prepared by phosphorochloridate chemistry. These materials were designed to act as membrane-soluble pro-drugs of the bio-active free nucleotides. In particular, novel para-substituted diaryl phosphate derivatives were prepared. In vitro evaluation revealed the compounds to have a pronounced and selective antiviral effect, the magnitude of which varied considerably with the nature of the aryl substituent. In particular, strongly electron-withdrawing aryl substituents correlate with high anti-HIV potency in C8166 cells. Along with AZT, the compounds are poorly effective in JM cells, which appear to lack thymidine kinase, indicating the phosphates to act as pro-drugs of the nucleoside rather than of the free phosphate.

Genomic variation of human immunodeficiency virus type 1 (HIV-1): molecular analyses of HIV-1 in sequential blood samples and various organs obtained at autopsy.

Length polymorphisms and partial nucleotide sequences were determined for the hypervariable regions, V1 to V5, of the human immunodeficiency virus type 1 (HIV-1) env gene obtained from proviral DNA of sequential peripheral blood samples, from viral RNA in plasma, and from proviral DNA obtained from different organs of individuals at autopsy. The lengths of several env regions of HIV-1 proviral DNA differed markedly when obtained from different organs of an individual. Nucleotide sequences of the hypervariable V3 region of HIV-1 obtained from different organs of one patient demonstrated distinct viral variants. Most proviral DNA sequences found in organs were also present in viral RNA obtained from plasma. The majority of HIV-1 V3 variants present in the lymph tissue could be found in the plasma viral population obtained at autopsy and in the sequential blood samples obtained before death, but were absent from the cardiac blood provirus population obtained at autopsy. However, sequence variants found in the brain proviral DNA were not detected in either plasma or the sequential blood samples. Sequence differences were observed at the apex of the V3 loop between HIV-1 variants present in sequential blood samples and in blood lymphocytes and nervous tissue, lymph tissue and plasma obtained post-mortem. The potential effect of lymph tissue on the long-term persistence of different viral variants is discussed. Virus obtained from the two sequential blood samples produced syncytia in primary cultures and was easily transmitted to the continuous JM cell line. Consensus (majority) V3 loop sequences determined for the adapted viruses demonstrated that some, but not all, sequences were represented within the in vivo viral population.

Deoxyribonucleoside triphosphate pools and thymidine chemosensitization in human T-cell leukemia

Thymidine kills cells by depleting dCTP stores. The present experiments tested whether deoxycytidine, by replenishing dCTP pools, could prevent thymidine cytotoxicity and thymidine's enhancement of carboplatin killing in two human T-cell acute leukemia cell lines. MOLT3 and JM cells were exposed to combinations of thymidine, deoxycytidine, and carboplatin and then assessed for survival, the magnitude of thymidine-carboplatin chemosensitization, and changes in deoxyribonucleoside triphosphate pools. For both cell lines, deoxycytidine (up to 144.5 μg/ml × 24 h) completely restored dCTP pools but only partially protected against thymidine cytotoxicity (100–1000μg/ml × 24 h) and thymidine-carboplatin sensitization (up to 60 μg carboplatin/ml during the last hour of thymidine). This contrasts with complete protection in prior studies using other cell types. Thymidine alone markedly increased dTTP and dGTP pools and decreased dCTP; dATP pools underwent a sharp decline which has not been observed before in any cell line. In subsequent studies 0.0336–137.3 μg deoxyadenosine/ml partially prevented cytotoxicity and carboplatin sensitization by 300 μg thymidine/ml. Together, deoxycytidine and deoxyadenosine completely prevented thymidine-carboplatin sensitization even though dATP and dCTP pools were not entirely returned to normal. These findings are discussed in regard to the unusual sensitivity of T-cell malignancies to thymidine toxicity, mechanisms of cytotoxicity and chemosensitization by thymidine, and the possibility of thymidine selectively sensitizing T-cell malignancies to killing by alkylating agents.

The assembly of HIV within the Golgi apparatus and Golgi-derived vesicles of JM cell syncytia

Chronic infection of the T-lymphocyte cell line JM with HIV-1 isolate GB8 results in the formation of multinucleate cells (syncytia). Transmission electron microscopy of these syncytia showed the presence of HIV particles both at the cell surface and within cytoplasmic vesicles. HIV particles were observed in dilated Golgi cisternae and Golgi-derived vesicles and in large vacuoles near the periphery of the syncytia. Immunolabelling was performed using an affinity-purified antiserum to the Golgi enzyme galactosyltransferase. This enzyme was consistently localized within both the Golgi apparatus and within virus-containing vesicles of JM syncytia, indicating that these vesicles originated from the Golgi apparatus.

Stilbene disulfonic acids. CD4 antagonists that block human immunodeficiency virus type-1 growth at multiple stages of the virus life cycle

The stilbene disulfonic acids 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid and, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid bound the variable-1 immunoglobulin-like domain of CD4 on JM cells. The interaction blocked the binding of the anti-CD4 monoclonal antibody OKT4A and the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1). DIDS inhibited the acute infection of CD4+ cells by HIV-1 with a potency (IC50 approximately 30 microM) similar to that which blocked gp120 binding (IC50 approximately 20 microM) to the cellular antigen. Pretreating uninfected CD4+ C8166 cells with DIDS blocked their fusion with chronically infected gp120+ cells. DIDS covalently and selectively modified lysine 90 of soluble CD4 and abolished the gp120-binding and antiviral properties of the recombinant protein. When added to cells productively infected with HIV-1, DIDS blocked virus growth and cleared cultures of syncytia without inhibiting cellular proliferation. The stilbene disulfonic acids are a novel class of site-specific CD4 antagonists that block multiple CD4-dependent events associated with acute and established HIV-1 infections.

6-0-butanoylcastanospermine (MDL 28,574) inhibits glycoprotein processing and the growth of HIVs.

The antiviral activity of 6-0-butanoylcastanospermine (MDL 28,574) [50% inhibitory concentration (IC50: 1.1 microM)] in JM cells infected with a recent isolate of HIV-1 (GB8), was compared with other inhibitors of glycoprotein-processing enzymes. N-butyldeoxynojirimycin (BuDNJ), deoxynojirimycin (DNJ), castanospermine (CAST) or the reverse transcriptase inhibitor 2'3'-dideoxycytidine (ddC) had activities of 56, 560, 29 and 0.1 microM, respectively. MDL 28,574 was at least 50 times more active than BuDNJ and less active but better tolerated in cell culture than ddC, two compounds currently undergoing clinical trials. The CAST derivative showed good protection in H9 cells infected with HIV-1 (RF; IIIB; U455), and HIV-2 (ROD), although the potency was less than that seen in the JM/GB8 system. HIV-1 glycoproteins, gp160 and gp120, synthesized in H9 cells chronically infected with HIV-1 (RF) and treated with MDL 28,574, were characterized by an increase in relative molecular weight of approximately 7-8000 kD. The ratio of gp120 to gp160 was markedly reduced in treated cells and provided further evidence that cleavage of the gp160 precursor molecule is a major consequence of the inhibition of glycoprotein processing. The intracellular target for MDL 28,574 was verified as alpha-glucosidase-I of the processing enzymes by the analysis of high-glucose glycopeptides recovered from treated mouse cells. This activity correlated with the antiviral effect observed against the growth of a mouse retrovirus, Moloney murine leukemia virus (MOLV), in mouse cells.

Inhibition of Glycoprotein Processing and HIV Replication by Castanospermine Analogues

Inhibitors of glycoprotein processing enzymes have been shown to have activity against HIV. Several analogues of the known glucosidase I inhibitor, castanospermine (CAST), were synthesized and evaluated for their inhibitory effect on glucosidases and for antiviral activity against Moloney murine leukemia virus (MOLV) and HIV-1. The most effective analogue was 6-O-butanoyl CAST (B-CAST, MDL 28,574) with an IC50 of 0.05 micrograms/mL against MOLV. A correlation between inhibition of glucosidase I and MOLV replication was observed. This analogue was further evaluated against HIV-induced syncytial formation in HeLa T4+ cells and against productive infection in JM cells infected with HIV 1 (GB8 strain). B-CAST showed an IC50 of 0.3 micrograms/mL in the HeLa T4+ assay, compared to CAST at 11 micrograms/mL. The compound also was more potent (IC50:0.15 micrograms/mL) than CAST (4-6 micrograms/mL) in JM cells. The antiretroviral activity of B-CAST was further confirmed in Friend leukemia virus (FLV) infection in mice. B-CAST showed equivalent activity to AZT and was more potent than CAST in inhibiting FLV-induced splenomegaly in mice. The data presented herein suggest the potential of these novel glucosidase inhibitors as anti-HIV agents.