Biomedical subjects

S Nagarajan

Publications and source records attributed to S Nagarajan.

At least 19 recordsLinked to original sources

PubMed2001

Designer cancer vaccines made easy: protein transfer of immunostimulatory molecules for use in therapeutic tumor vaccines.

Advances in the understanding of the immune response to tumors has led to the development of new strategies to design therapeutic vaccines. One of these strategies is the development of protein transfer of immunostimulatory molecules onto the surface of tumor cells, thereby directing the immune response to the tumor antigens carried by the modified tumor cells. This strategy has been developed as an alternative to gene transfer, the more classical technique of introducing immunostimulatory molecules onto tumors. In this report we briefly review current strategies for immunotherapy and then focus on several approaches to protein transfer and their historical basis. Finally, the application of these protein transfer approaches to develop cancer vaccines are reviewed and discussed.

Animals↗

PubMed2001

Development of therapeutic vaccines by direct modification of cell membranes from surgically removed human tumor tissue with immunostimulatory molecules.

The addition of immunostimulatory molecules to tumor cells has been proposed as a potentially useful strategy to induce anti-tumor immunity. In this report we have investigated the application of using isolated tumor membranes modified by transfer of a glycosyl-phosphatidylinositol (GPI)-anchored form of the costimulatory molecule, B7-1 (CD80), as a cell free cancer vaccine for clinical use. Isolated tumor cell membranes were prepared from established tumor cell lines and the optimum conditions necessary for modification and clinical application were determined. GPI-B7-1 transferred optimally onto isolated human tumor membranes at physiological temperature (37 degrees C) in a dose dependent manner. Transfer of GPI-B7-1 to isolated membranes resulted in stable expression and costimulatory function. These modified membranes could be stored for repeated immunizations while retaining expression of GPI-B7-1. Critically, isolated tumor membranes, prepared directly from surgically removed human tumor tissue, could be modified by GPI-B7-1 and costimulate T cells. Finally, membranes isolated from tumor tissue expressed MHC class II, unlike the cell line established in vitro from the same patient. This novel approach to express immunostimulatory molecules on isolated membranes derived from a patient's tumor tissue will make the preparation of autologous therapeutic cancer vaccines available to patients from which tumor cell lines can not be established.

Adjuvants, Immunologic↗

PubMed2001

Quantifying the impact of membrane microtopology on effective two-dimensional affinity.

Just as interactions of soluble proteins are affected by the solvent, membrane protein binding is influenced by the surface environment. This is particularly true for adhesion receptors because their function requires tightly apposed membranes. We sought to demonstrate, and further, to quantify the possible scale of this phenomenon by comparing the effective affinity and kinetic rates of an adhesion receptor (CD16b) placed in three distinct environments: red blood cells (RBCs), detached Chinese hamster ovary (CHO) cells, and K562 cells. Effective affinity reflects both the intrinsic receptor-ligand kinetics and the effectiveness of their presentation by the host membranes. Expression of CD16b, a low affinity Fcgamma receptor, was established by either transfection or spontaneous insertion via its glycosylphosphatidylinositol anchor. Binding to IgG-coated RBCs, measured using a micropipette method, indicated a 50-fold increase in effective affinity for receptors on RBCs over CHO and K562 cells, whereas the off rates were similar for all three. Electron microscopy confirmed that specific tight contacts were broad in RBC-RBC conjugates but sparse in CHO-RBC conjugates. We suggest that through modulation of surface roughness the cytoskeleton can greatly impact the effectiveness of adhesion molecules, even those with no cytoplasmic structures. Implications for locomotion and static adhesion are discussed.

Animals↗

PubMed2000

Disruption of the neural response to rapid acoustic stimuli in dyslexia: evidence from functional MRI.

The biological basis for developmental dyslexia remains unknown. Research has suggested that a fundamental deficit in dyslexia is the inability to process sensory input that enters the nervous system rapidly and that deficits in processing rapid acoustic information are associated with impaired reading. Functional magnetic resonance imaging (fMRI) was used to identify the brain basis of rapid acoustic processing in normal readers and to discover the status of that response in dyslexic readers. Normal readers showed left prefrontal activity in response to rapidly changing, relative to slowly changing, nonlinguistic acoustic stimuli. Dyslexic readers showed no differential left frontal response. Two dyslexic readers participated in a remediation program and showed increased activity in left prefrontal cortex after training. These fMRI results identify left prefrontal regions as normally being sensitive to rapid relative to slow acoustic stimulation, insensitive to the difference between such stimuli in dyslexic readers, and plastic enough in adulthood to develop such differential sensitivity after intensive training.

Acoustic Stimulation↗

PubMed2000

The membrane anchor influences ligand binding two-dimensional kinetic rates and three-dimensional affinity of FcgammaRIII (CD16).

Kinetic rates and affinity are essential determinants for biological processes that involve receptor-ligand binding. By using a micropipette method, we measured the kinetics of human Fcgamma receptor III (CD16) interacting with IgG when the two molecules were bound to apposing cellular membranes. CD16 is one of only four eukaryotic receptors known to exist natively in both the transmembrane (TM, CD16a) and glycosylphosphatidylinositol (GPI, CD16b) isoforms. The biological significance of this anchor isoform coexistence is not clear. Here we showed that the anchor influenced kinetic rates; compared with CD16a-TM, CD16a-GPI bound faster and with higher affinities to human and rabbit IgGs but slower and with lower affinity to murine IgG2a. The same differential affinity patterns were observed using soluble IgG ligands. A monoclonal antibody bound CD16a-GPI with higher affinity than CD16a-TM, whereas another monoclonal antibody reacted strongly with CD16a-TM but weakly with CD16a-GPI. No major differential glycosylation between the two CD16a isoforms was detected by SDS-polyacrylamide gel electrophoresis analysis. We suggest a conformational difference as the mechanism underlying the observed anchor effect, as it cannot be explained by the differing diffusivity, flexibility, orientation, height, distribution, or clustering of the two molecules on the cell membrane. These data demonstrate that a covalent modification of an Ig superfamily receptor at the carboxyl terminus of the ectodomain can have an impact on ligand binding kinetics.

Amino Acid Sequence↗

PubMed2000

Cell-specific, activation-dependent regulation of neutrophil CD32A ligand-binding function.

Neutrophils express 2 low-affinity FcgammaR, FcgammaRIIIB (CD16B), and FcgammaRIIA (CD32A). CD16B is a glycosyl-phosphatidyl inositol-anchored molecule, whereas CD32A is a polypeptide-anchored molecule. These 2 receptors also differ in their signaling. The biological significance of coexpression of 2 FcgammaRs with distinct membrane anchors and signaling capacities is not clearly understood. Using neutrophils from a CD16B-deficient donor and normal neutrophils treated with anti-CD16 monoclonal antibodies, the authors demonstrated that affinity modulation of CD32A is one of the mechanisms by which neutrophils regulate their FcgammaR-dependent functions. Neutrophils isolated from a CD16B(- )donor rosetted poorly with sheep erythrocytes opsonized with rabbit IgG (EA) (12% +/- 2% versus 80% +/- 6% for control) and were unable to mediate immunophagocytosis. However, activation of CD16B(-) neutrophils with fMLP, a bacterial chemotactic peptide, increased the CD32A-dependent EA rosetting to 58%. The CD32A-dependent rosetting of fMLP-activated normal neutrophils also increased nearly 5-fold, but there was no increase in CD32A expression. The CD32A-dependent immune complex (IC) binding was also increased in activated neutrophils. This affinity regulation was not observed with CD32A expressed on Chinese hamster ovary cells. These results suggest that in resting neutrophils CD32A is in a low-affinity state and that these cells primarily engage CD16B for IC binding. However, once the neutrophils are activated, the CD32A is converted to a high-affinity state that leads to CD32A-dependent ligand binding and signaling. These results suggest that neutrophils adopt a novel strategy to engage the 2 different FcgammaR selectively during physiologic and pathologic conditions to carry out their functions efficiently.

Animals↗

PubMed2000

Concurrent and independent binding of Fcgamma receptors IIa and IIIb to surface-bound IgG.

Fc receptor-antibody interactions are key mechanisms through which antibody effector functions are mediated. Neutrophils coexpress two low-affinity Fcgamma receptors, CD16b (FcgammaRIIIb) and CD32a (FcgammaRIIa), possessing overlapping ligand binding specificities but distinct membrane anchor and signaling capacities. Using K562 cell transfectants as a model, the kinetics of both separate and concurrent binding of CD16b and CD32a to surface-bound IgG ligands were studied. CD16b bound human IgG with 2-3 times higher affinity than did CD32a (A(c)K(a) = 4.1 and 1.6 x 10(-7) microm(4), respectively) and both FcgammaRs had similar reverse kinetic rates (k(r) = 0.5 and 0.4 s(-1), respectively). Because CD16b is expressed on neutrophils at a 4-5 times higher density than CD32a, our results suggest that CD16b plays the dominant role in binding of neutrophils to immobilized IgG. The question of possible cross-regulation of binding affinity between CD16b and CD32a was investigated using our multispecies concurrent binding model (Zhu and Williams, Biophys. J. 79:1850-1857, 2000). Because the model assumes independent binding (no cooperation among different species), the excellent agreement between the model predictions and the experimental data suggests that, when coexpressed on K562 cells, these two FcgammaRs do not interact in a manner that alters the kinetic rates of either molecule.

Antibodies, Blocking↗

PubMed2000

Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria.

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients' CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Antigens, CD34↗

PubMed2000

In vivo qualitative changes of 31P NMR in stressed maize roots vis-à-vis carbon substrate determining the degree of stress.

High resolution 31P nuclear magnetic resonance spectroscopy was used to investigate the changes in phosphate metabolism and intracellular pH in intact maize (Zea mays L) root segments to hyper osmotic shock. The results were compared with the happenings under field conditions, when the stress was given gradually. Effect of sugar substrate on adaptation of tissue to both kinds of situations was also studied. The hyper osmotic shock resulted in large vacuolar alkalinization and a decrease in pH across tonoplast membrane. There was gradual build up of phosphocholine and decrease in glucose 6P and UPDG levels. In gradual stress, the root segments were able to adapt to the stress and maintained pH gradient across tonoplast, with marginal alkalinization of vacuoles. The presence of sugar substrate reduced the impact of stress significantly, commensurate with the increased activity of plasmalemma H(+)-ATPase. The latter providing the driving force for uptake of organic molecules and ions required for osmoregulation.

Carbon↗

PubMed1999

Cortical auditory signal processing in poor readers.

Magnetoencephalographic responses recorded from auditory cortex evoked by brief and rapidly successive stimuli differed between adults with poor vs. good reading abilities in four important ways. First, the response amplitude evoked by short-duration acoustic stimuli was stronger in the post-stimulus time range of 150-200 ms in poor readers than in normal readers. Second, response amplitude to rapidly successive and brief stimuli that were identical or that differed significantly in frequency were substantially weaker in poor readers compared with controls, for interstimulus intervals of 100 or 200 ms, but not for an interstimulus interval of 500 ms. Third, this neurological deficit closely paralleled subjects' ability to distinguish between and to reconstruct the order of presentation of those stimulus sequences. Fourth, the average distributed response coherence evoked by rapidly successive stimuli was significantly weaker in the beta- and gamma-band frequency ranges (20-60 Hz) in poor readers, compared with controls. These results provide direct electrophysiological evidence supporting the hypothesis that reading disabilities are correlated with the abnormal neural representation of brief and rapidly successive sensory inputs, manifested in this study at the entry level of the cortical auditory/aural speech representational system(s).

Acoustic Stimulation↗

PubMed1999

Protein transfer of glycosyl-phosphatidylinositol-B7-1 into tumor cell membranes: a novel approach to tumor immunotherapy.

Modification of tumor cells with one or more costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy. Expression of B7-1 (CD80) in tumors by gene transfer creates an immunogenic tumor cell that induces antitumor immunity and protects mice from further challenge with wild-type tumor cells. In this report, we demonstrate that protein transfer of glycosyl-phosphatidylinositol (GPI)-anchored costimulatory molecules into tumor cell membranes could be used as an alternative to gene transfer for tumor immunotherapy. Incubation of isolated tumor membranes with purified GPI-anchored B7-1 results in stable incorporation of B7-1 on tumor cell membranes within a few hours. Immunization of C57BL/6 mice with EG7 tumor membranes modified to express GPI-B7-1 by protein transfer induces tumor-specific T-cell proliferation and CTLs. In addition, immunization with these EG7 membranes protects mice from parental tumor challenge. The protein transfer approach used here does not require foreign vectors or live tumor cells and is completed within a matter of hours. Irradiated cells or membrane preparations from fresh or frozen tumor tissue can be used. Therefore, protein transfer of glycolipid-anchored molecules provides an efficient and novel approach to modify tumor membranes for human immunotherapy. This approach is not limited to costimulatory molecules because any cell surface protein can be converted to a GPI-anchored form by recombinant techniques.

Animals↗

PubMed1999

Inhaled insulin.

Inhalation of regular insulin for meal time glucose control has been found to be safe, efficacious and reliable in Type I and Type II diabetics. The administration of regular insulin through the human lungs by inhalation has been conducted in at least 14 short studies in both normal and diabetic subjects beginning as early as 1925. In all studies, significant insulin absorption and lowering of blood glucose was observed in the absence of penetration enhancers. Although a concern of variable dosing was raised in early studies, the development of new reproducible delivery systems has ensured that the variability of aerosol insulin can be as good, if not better, than subcutaneous (SC) injection. In the longest controlled studies in humans to date, both Type I and Type II insulin-dependent diabetics used a novel inhaled dry powder insulin delivery system for 3 months for meal time glucose control. The study results indicate that inhaled insulin provides equivalent glucose control, measured by hemoglobin A1c, when directly compared to SC injection. Interim results from an additional study with Type II diabetics who were failing oral hypoglycemic agents suggest that adjunctive therapy with inhaled insulin markedly improved glycemic control with a low risk of hypoglycemia. In all the 3 month studies the system was efficacious, well tolerated, well liked, and resulted in reproducible results. A potential advantage of aerosol insulin is that it is more rapidly absorbed (serum peak at 5-60 min) and cleared than SC injection (peak at 60-150 min), which provides a more relevant and convenient therapy for meal time glucose control. The relative efficiency of insulin delivery by aerosol, compared to SC injection, has been estimated from the dose measured at the exit point of the aerosol device, and found to range between 8 and 25% of SC, depending on the study.

Journal Article↗

PubMed1999

Expression and characterization of glycolipid-anchored B7-1 (CD80) from baculovirus-infected insect cells: protein transfer onto tumor cells.

Tumor cells can be modified to express immunostimulatory molecules such as B7-1 by protein transfer using purified glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1). In this study recombinant baculovirus encoding GPI-B7-1 (vBacB7-1(GPI)) was established to obtain large quantities of purified GPI-B7-1 to modify tumor cells by protein transfer. vBacB7-1(GPI)-infected insect cells showed high-level cell surface expression of GPI-B7-1 that was susceptible to PIPLC treatment. GPI-B7-1 expressed in insect cells (Bac-GPI-B7-1) mediated T cell proliferation, indicating that the GPI-B7-1 retains costimulatory activity. Moreover, Bac-GPI-B7-1 was completely solubilized in Triton X-100 at 4 degrees C compared to 22% solubilization of GPI-B7-1 expressed in CHOK1 cells, suggesting that GPI-anchored proteins expressed in insect cells may not be clustered into the detergent-insoluble fraction. SDS-PAGE analysis of Bac-GPI-B7-1 showed faster mobility (45 kDa) compared to GPI-B7-1 from CHOK1 (68 kDa) and this difference may be due to a difference in glycosylation. Cell binding assays showed that immunoaffinity-purified Bac-GPI-B7-1 retained its functional ability to bind CD28(+) cells. Moreover, when human tumor cells were incubated with this functionally active purified GPI-B7-1, an efficient transfer of B7-1 onto tumor cells was observed. These results demonstrate that GPI-B7-1 can be expressed in insect cells in a functionally active form and can be used to modify tumor cells for immunotherapeutic applications.

Animals↗

PubMed1999

Time-frequency MEG-MUSIC algorithm.

We propose a method that incorporates the time-frequency characteristics of neural sources into magnetoencephalographic (MEG) source estimation. The method is based on the multiple-signal-classification (MUSIC) algorithm and it calculates a time--frequency matrix in which diagonal and off-diagonal terms are the auto and crosstime--frequency distributions of multichannel MEG recordings, respectively. The method averages this time-frequency matrix over the time--frequency region of interest. The locations of neural sources are then estimated by checking the orthogonality between the noise subspace of this averaged matrix and the sensor lead field. Accordingly, the method allows us to estimate the locations of neural sources from each time--frequency component. A computer simulation was performed to test the validity of the proposed method, and the results demonstrate its effectiveness.

Algorithms↗

PubMed1998

Stabilization of Aldehydes as Propylene Glycol Acetals.

Propylene glycol (PG) acetals (substituted 1,3-dioxalans) of a few representative aldehydes are prepared according to standardized methods in yields of 80-90%. A comparative study of the regeneration of citral from the corresponding dimethyl and PG acetals under mild acidic conditions showed that, although the former readily decomposed to an extent of 85%, the latter formed a near 1:1 equilibrium mixture with the generated aldehyde.

Journal Article↗

PubMed1998

Novel devices for the predictable delivery of nitric oxide to aqueous solutions.

Nitric oxide (NO), a recently discovered biological molecule synthesized by many cells, has many physiological roles including blood pressure regulation, neurotransmission, and inhibition of platelet adhesion. However, NO and reactive species formed in the presence of oxygen and superoxide can also be cytotoxic, mutagenic, or carcinogenic. Two novel devices were developed to deliver controlled and predictable amounts of NO to aqueous solutions for studying the effects of NO on biological systems. The devices contained either a slit or a tube composed of a gas-permeable membrane. Aqueous solution flowed on one side of the membrane while the other side of the membrane was exposed to NO gas. Mathematical models were used to predict the bulk or mixing-cup NO concentration in the aqueous solution at the exit of each delivery device following exposure to NO gas. Model predictions were in good agreement with experimental values at both 25 and 37 degreesC. One of the delivery devices was also connected to a well-stirred and oxygenated chamber. Model predictions of the chamber NO concentration, which included the reaction of NO with O2, were in excellent agreement with experiments.

Algorithms↗

PubMed1998

Thyroid gland papillary carcinoma with fibromatosis-like stroma: case report.

Papillary carcinoma of the thyroid may show a variable degree of fibrosis of the stroma, but proliferation of the stromal fibroblasts mimicking fibromatosis is rare. There appears to be a new variant of papillary carcinoma of the thyroid associated with exuberant proliferation of the fibroblasts resembling fibromatosis. We present one such case in a 50 year old woman and succinctly reviewed the relevant literature of this rare variant. The necessity of a diligent search for a papillary carcinoma in thyroid gland which shows a proliferative fibrous lesion is stressed.

Carcinoma, Papillary↗

PubMed1997

The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase.

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5' extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans approximately 25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

Acyltransferases↗