Sclerotium rolfsii lectin inhibits proliferation and induces apoptosis in human ovarian cancer cell line PA-1.

OBJECTIVES: Sclerotium rolfsii lectin (SRL), isolated from soil born phytopathogenic fungus Sclerotium rolfsii, exhibits exquisite binding specificity to the oncofoetal Thomsen-Friedenreich (Galß1,3GalNAcα-O-Ser/Thr, T or TF) antigen and associated glycans. In the present study, we report anti-proliferative activity of SRL and investigate underlying mechanisms of SRL-induced apoptosis, in the human ovarian cancer cell line PA-1. MATERIALS AND METHODS: SRL-induced anti-proliferative effects were determined using MTT assay and induction of apoptosis was determined by flow cytometry, confocal microscopy and western blot analysis. RESULTS: SRL inhibited population growth of PA-1 cells in a dose- and time-dependent manner with maximum inhibition (71.3 ± 1.9%) occurring at concentration of 50 µg/ml after 72 h incubation. Observed effects of SRL could be blocked by competing glycoproteins, asialomucin, mucin and fetuin. Treatment with SRL resulted in increase in hypodiploid cell population as determine by cell cycle analysis. Increase in numbers of annexin V-PI positive cells, and cleavage of PARP confirmed apoptosis-inducing activity of SRL. Involvement of caspases in SRL-mediated apoptosis was determined by cleavage of caspases-3, -8 and -9 in a time-dependent manner, thereby suggesting possible involvement of both intrinsic and extrinsic caspase-dependent pathways. CONCLUSION: The present study demonstrates anti-proliferative and apoptosis-inducing activity of SRL that can be exploited for potential application in ovarian cancer research.

Hypercalcemia induced by parathyroid hormone-related peptide after treatment of squamous cell carcinoma of oral cavity.

Squamous cell carcinoma of the head and neck is a rare cause of humoral hypercalcemia of malignancy. This paraneoplastic syndrome is usually one of the presenting symptoms of the disease. We report a case of squamous cell carcinoma of the oral cavity that presumably elaborated parathyroid hormone-related peptide (PTH-rP) and caused hypercalcemia only after radiotherapy and chemotherapy.

Influence of non-enzymatically glycated collagen on monocyte-macrophage differentiation.

Blood monocytes (mo) on transendothelial migration interact with extracellular matrix components (ECM) and differentiate into macrophages (m(phi)), which play an important role in both physiological, and pathological conditions, particularly, atherosclerosis. In order to study whether modification of ECM such as non-enzymatic glycation occurring in diabetes influences mo-m(phi) differentiation, an in vitro system using isolated human peripheral blood mononuclear cells (PBMC) maintained on non-enzymatically glycated COL I (NEG COL I) was used. M(phi) specific functions such as receptor mediated endocytosis of modified proteins, production of m(phi) specific 92 and 72 kDa matrix metalloproteinases (MMPs), expression of surface antigen and loss of myeloperoxidase (MPO) activity were assessed. Endocytosis of 125[I] acetyl BSA was significantly higher in cells maintained on NEG COL I than those on COL I. Kinetic analysis revealed that the rate of uptake of modified BSA and production of MMPs by cells maintained on NEG COL I were higher than those on COL I suggesting a faster rate of differentiation of cells maintained on modified substrata. FACS analysis of the expression of surface antigen showed that the rate of down-regulation of monocyte specific CD14 and the rate of up-regulation of m(phi) specific CD71 were high in cells maintained on NEG COL I. These results suggest that the interaction of monocyte with non-enzymatically glycated matrix protein in the vessel wall may result in faster rate of induction of mo-m(phi) differentiation leading to foam cell formation, a critical early event in the initiation and development of atherosclerosis.

Neuroblastoma cell lines--a versatile in vitro model in neurobiology.

Neuroblastoma (NB) cell lines are transformed, neural crest derived cells, capable of unlimited proliferation in vitro. These cell lines retain the ability of differentiation into neuronal cell types on treatment with various agents. This ability of NB cells to proliferate as well as to differentiate makes it an excellent in vitro system for various studies. This review article focuses on the applications and potential uses of murine and human NB cell lines. NB cells are extensively used for testing neurotoxicity of putative drugs such as antimalarial or anticancer agents. NB cell lines have wide applications in virus research to understand various aspects of virus-host cell interactions at the molecular and cellular levels. They have been used to dissect the relationships between proliferation, differentiation and apoptosis. This feature has been useful in understanding the pediatric cancer--neuroblastoma and for development of newer therapies.

Differential responses of staurosporine on protein kinase C activity and proliferation in two murine neuroblastoma cell lines.

We studied the effect of staurosporine (SSP), a broad-spectrum protein kinase inhibitor, on the levels of protein kinase C (PKC) activity and proliferation in two murine neuroblastoma cell lines, Neuro2a and its clone NB41A3. The PKC activity was examined in whole cell lysate, cytosolic and particulate fractions. A differential response to SSP treatment in the enzyme activity in whole cell lysate and particulate fractions was demonstrated in the two cell lines. The data on proliferation indicated that Neuro2a cells were more sensitive to the SSP treatment with significant inhibition in DNA synthesis in a time course study. Our findings suggest that the data on basal levels of PKC activity in tumors will be of significance in studies using PKC inhibitors as an approach for therapeutic intervention.

Antiproliferative activity of conditioned medium from lymphocytes of neuroblastoma (NB) patient and inhibition with NB serum.

Neuroblastoma (NB) is a pediatric malignancy and results in high mortality rate. Cellular immunity has been shown to play an important role in killing tumors 'in vitro'. Human lymphocytes were activated in vitro with phytohaemagglutinin (PHA) and the effect of supernatants collected at 24, 48, 72 and 96 h were tested on proliferation of human NB cell line-SK-N-MC and glioma cell line U87-MG. The SK-N-MC cells were observed to be more susceptible to the supernatants compared to U87-MG with higher inhibition of proliferation as evaluated by [3H]thymidine incorporation (P < 0.05 for 24 and 72 h and P < 0.0005 for 48 and 96 h). Conditioned medium from lymphocytes of NB patient collected at 48 and 96 h after activation inhibited proliferation (P < 0.005) of SK-N-MC cells. The presence of serum from NB patient decreased the antiproliferative activity of supernatants from normal lymphocytes and NB patient's autologous lymphocytes (P < 0.01). This preliminary data demonstrates the capability of the activation of lymphocytes from NB patient undergoing aggressive multiagent chemotherapy and controlling proliferation of tumor cells on one hand and the role of serum from NB patient in abrogating to a certain extent the effect of activated immune cells thereby protecting tumor cells, on the other hand. Both these aspects need to considered with equal importance to study mechanisms in designing strategies for immune therapies.

Polaroid scotometer: a new device to chart suppression scotomata.

Suppression, an active inhibition of vision in one eye under binocular conditions, is a sensory adaptation in concomitant strabismus. The characteristics and extent of suppression differ not only in different types of strabismus but also in the same type under different testing conditions, which may differ in their degree of dissociation. The findings in exodeviations have thus varied from regional suppression to hemiretinal trigger, and in esodeviations from two point scotoma to single large scotoma. There is a need for a simple but less dissociative test to chart the fields under binocular conditions. We have devised a new, simple instrument based on polaroid dissociation. The findings corroborate the findings of more sophisticated tests like phase-difference haploscope.

The foetal immune response to maternal tetanus toxoid immunization.

Paired maternal and cord blood samples were collected at delivery from 150 women who received varying doses of tetanus toxoid during pregnancy. Tetanus specific IgM and IgG antibodies were measured in them by standard ELISA with a sensitivity for IgM of 0.001 mg/ml, and for IgG of 0.0003 IU/ml. In 22 infants an additional estimation of tetanus antibody was made 1 month after birth. The presence of specific IgM in 78% of cord samples established an active foetal immune response. The titre did not alter significantly with the number of TT doses given to the mother. Foetal IgM rose in 60% of cases at one month of age compared to cord blood levels. At this time IgG levels were uniformly diminished in accord with a maternally derived passively transferred antibody. No switch of foetal IgM to IgG production was evident. The foetal immune response thus did not confer active protection against tetanus.

Elevated levels of IgG specific antimyosin antibodies in acute rheumatic fever (ARF): differential profiles of antibodies to myosin and soluble myocardial antigens in ARF, acute glomerulonephritis and group A streptococcal pharyngitis.

Group A streptococcus is the common etiologic agent associated with group A streptococcal pharyngitis (SAP) and its sequelae: acute rheumatic fever (ARF) and acute glomerulonephritis (AGN). However, hyperresponsiveness to cardiac antigens stemming from the shared antigenic determinants with streptococcal antigens is believed to play a role only in the pathogenesis of ARF. A Profile of IgM and IgG immune responses to soluble myocardial antigen (SMA) and myosin was evaluated in ARF, AGN and SAP. A modified ELISA measuring the area under curve (AUC) for quantitation of antibodies to SMA and rabbit muscle myosin was employed. Proteins in the SMA were resolved by SDS-PAGE. Immune responses to a major protein band of congruent to 205 kD, corresponding to the molecular weight of the heavy chain of cardiac myosin was also evaluated. In the ARF group while a significant elevation of both IgM and IgG anti-SMA antibody levels was observed, only antibodies of IgG isotype were elevated against rabbit muscle myosin and 205 kD protein of human cardiac tissue in comparison with normal controls, AGN and SAP groups. There was a significant positive correlation of antibodies against skeletal muscle myosin with antibodies against 205 kD protein of human cardiac tissue for both IgM and IgG specificities in ARF alone. The incidence of positive sera (values greater than mean + 2SD of control values) for IgM and IgG anti-SMA and antimyosin antibodies was higher in ARF than in AGN and SAP. None of the AGN and SAP sera had elevated levels of antibodies against SMA whereas low incidence of positive sera for antimyosin antibodies was observed in these groups. Although group A streptococcus etiology is associated with ARF, AGN and SAP, differential profiles of immune responses to cardiac antigens is observed in these diseases. Elevated IgG specific response to myosin and 205 kD cardiac protein was demonstrated in ARF and not in other groups with a similar etiology. It may be worthwhile, therefore to explore the possibility of using this as an additional parameter in diagnosis of ARF.

Antibodies to N-acetylglucosamine and heparin in acute and remission phases of rheumatic fever.

The etiology of acute rheumatic fever (ARF) is believed to involve an immunological response to group A streptococcal antigens. Antibodies to group A carbohydrate (A-CHO) have been reported in ARF and rheumatic heart disease patients. As N-acetylglucosamine (GlcNAc) units form the major immunodominant regions in A-CHO antigens, we investigated levels of antibodies to GlcNAc and heparin (with repeated sequences of GlcNAC) in rheumatic fever (RF) patients. Serum samples from 26 acute cases (ARF), 18 remission cases and 17 normal healthy subjects were analyzed for IgG and IgM levels of antibodies to GlcNAc and heparin. High titres of IgG antibodies to heparin and GlcNAc were observed in the ARF group in comparison with controls (p less than 0.0025 and 0.0125 respectively). There was no difference in the levels of IgM antibodies. Remission group demonstrated low titres of IgG to heparin and GlcNAc (p less than 0.01 and 0.0125 respectively) in comparison with ARF group. Heparin antibodies of IgM class was comparatively lower in remission group (p less than 0.005). While the role of these antibodies in different phases of RF needs to be investigated, we conclude that GlcNAc antibodies do not play any role in the pathogenesis of RF or rheumatic heart disease.

Persistence of heart reactive antibodies (HRA) in acute rheumatic fever (ARF) and rheumatic heart disease (RHD) patients.

Heart Reactive Antibodies (HRA) of IgG and IgM classes were investigated in 79 patients in four clinically classified groups. Group I comprised of 16 children with Acute Rheumatic Fever (ARF) and Group II consisted of 15 cases with active Rheumatic Heart Disease (RHD). Twenty seven cases of ARF and RHD in remission and 21 adults with quiescent RHD were included in Groups III and IV respectively. Control groups comprised of 14 normal, healthy, donors and eight Post Streptococcal Acute Glomerular Nephritis (PSAGN) cases. Low levels of HRA of IgG and IgM classes were detected in control groups. The overall incidence of HRA of IgG and IgM classes was 82.28 and 39.24% respectively in the patient group. An increased frequency of HRA-IgM antibodies was observed in Groups I, III and IV (p less than 0.01, 0.001 and 0.025 respectively). All the four groups demonstrated a highly significant increase in incidence of HRA--IgG class (p less than 0.0005). Persistence of high titres of HRA-IgG class in ARF and RHD is reported in the paper.

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types.

Efficient generation in vitro, from human peripheral blood cells, of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization.

This paper describes a simple protocol for the efficient generation of large numbers of human monoclonal antibody-producing cells. This system is based on initial limiting-dilution culture after Epstein-Barr virus exposure of highly enriched precursors selected from peripheral blood mononuclear cells. Precursors can be enriched by using rosetting or panning approaches. Antibodies to erythrocytes, a mouse mammary carcinoma, DNA, and sperm antigens, produced without any deliberate immunization, are described. Large-scale human monoclonal antibody production may be facilitated by a combination of this protocol with a human cellular fusion system. For efficient precursor analysis and short-term (2 months or more) monoclonal antibody production, however, the system described here may be sufficient.

Phytohaemagglutinin as a molecular probe to study the membrane constituents of human X- and Y-bearing spermatozoa.

Phytohaemagglutinin (PHA) was employed to study the constituents of the cell membrane in X- and Y-bearing human spermatozoa. The presence of certain receptors for PHA was revealed in the equatorial region of about 50% of the spermatozoa. To determine the association of the equatorial staining by PHA with one of the subpopulations of spermatozoa a double staining technique was developed. In this method, the membrane was first stained with PHA employing the indirect immunofluorescence technique and then followed by the conventional quinacrine staining method to demonstrate the F-body. It was shown that spermatozoa which had receptors in the equatorial region for PHA were the ones which exhibited in the F-body. Staining of the membrane with PHA is a better method for the identification of human Y spermatozoa since it does not have the drawbacks of quinacrine staining.

Use of dithiothreitol for improved visibility of the F body in human Y-bearing spermatozoa.

Dithiothreitol was used to decondense the nuclear chromatin before staining human spermatozoa with quinacrine for the F body present in Y-bearing spermatozoa. In unseparated fractions and those enriched in X- or Y-bearing spermatozoa, the number of spermatozoa with F bodies increased by 2-9% after dithiothreitol treatment.

A simple and reproducible method for separating Y-bearing spermatozoa from human semen.

PIP: A simple and reproducible method for separating Y-bearing spermatozoa from human semen is reported. Semen samples with 60-80 million count and 50% motility were obtained from healthy human donors. Ficoll-400 and sodium metrizoate density gradient were used for separation. Quinacrine hydrochloride was used for staining, and Olympus fluorescent microscopy was used for the detection of F body present in the spermatozoa bearing Y-chromosomes. The data indicate that the percentage of Y-bearing spermatozoa in normal human semen is about 5-6% less than what is expected. The observations agree with those of Barlow and Vosa that with the optical attenuation coefficient being sufficiently great, the chromosomes lying deep in the head may go undetected. A mean value of 45.22 was obtained for Y-bearing spermatozoa and the sediment contained about 62.9 Y-bearing spermatozoa. The results thus suggest that it is possible to separate Y-bearing spermatozoa from semen using Ficoll-socium metrizoate density gradient.^ieng