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Biomedical subjects

N Bhardwaj

Publications and source records attributed to N Bhardwaj.

At least 55 records · Page 3Linked to original sources
PubMed1999

Changes in frequency of HIV-1-specific cytotoxic T cell precursors and circulating effectors after combination antiretroviral therapy in children.

Combination antiretroviral therapy has had a major role in reducing human immunodeficiency virus type 1 (HIV-1) plasma viral loads in HIV-1-infected adults but a variable effect in infants, in whom complete viral suppression appears to be less readily achieved. In adults, after the reduction in plasma viremia, there is a decrease in the numbers of circulating cytotoxic T cell (CTL) effectors and precursors in the majority of patients. This longitudinal study assessed the effect of combination drug therapy on the frequency of HIV-1-specific CTL responses in 8 HIV-1-infected children. Following treatment, the frequency of HIV-1-specific CTL responses initially increased, especially in children with incomplete viral suppression but with increasing CD4+ cell counts. In children with complete viral suppression, the frequency of HIV-1-specific CTL responses decreased, suggesting that viral replication is required to maintain CTL responses in the systemic circulation.

Anti-HIV Agents↗
PubMed1999

Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.

Adolescent↗
PubMed1998

Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells.

Cells from the bone marrow can present peptides that are derived from tumors, transplants, and self-tissues. Here we describe how dendritic cells (DCs) process phagocytosed cell fragments onto major histocompatibility complex (MHC) class II products with unusual efficacy. This was monitored with the Y-Ae monoclonal antibody that is specific for complexes of I-Ab MHC class II presenting a peptide derived from I-Ealpha. When immature DCs from I-Ab mice were cultured for 5-20 h with activated I-E+ B blasts, either necrotic or apoptotic, the DCs produced the epitope recognized by the Y-Ae monoclonal antibody and stimulated T cells reactive with the same MHC-peptide complex. Antigen transfer was also observed with human cells, where human histocompatibility leukocyte antigen (HLA)-DRalpha includes the same peptide sequence as mouse I-Ealpha. Antigen transfer was preceded by uptake of B cell fragments into MHC class II-rich compartments. Quantitation of the amount of I-E protein in the B cell fragments revealed that phagocytosed I-E was 1-10 thousand times more efficient in generating MHC-peptide complexes than preprocessed I-E peptide. When we injected different I-E- bearing cells into C57BL/6 mice to look for a similar phenomenon in vivo, we found that short-lived migrating DCs could be processed by most of the recipient DCs in the lymph node. The consequence of antigen transfer from migratory DCs to lymph node DCs is not yet known, but we suggest that in the steady state, i.e., in the absence of stimuli for DC maturation, this transfer leads to peripheral tolerance of the T cell repertoire to self.

Animals↗
PubMed1998

Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes.

Dendritic cells, but not macrophages, efficiently phagocytose apoptotic cells and cross-present viral, tumor, and self-antigens to CD8(+) T cells. This in vitro pathway corresponds to the in vivo phenomena of cross-priming and cross-tolerance. Here, we demonstrate that phagocytosis of apoptotic cells is restricted to the immature stage of dendritic cell (DC) development, and that this process is accompanied by the expression of a unique profile of receptors, in particular the alphavbeta5 integrin and CD36. Upon maturation, these receptors and, in turn, the phagocytic capacity of DCs, are downmodulated. Macrophages engulf apoptotic cells more efficiently than DCs, and although they express many receptors that mediate this uptake, they lack the alphavbeta5 integrin. Furthermore, in contrast to DCs, macrophages fail to cross-present antigenic material contained within the engulfed apoptotic cells. Thus, DCs use unique pathways for the phagocytosis, processing, and presentation of antigen derived from apoptotic cells on class I major histocompatibility complex. We suggest that the alphavbeta5 integrin plays a critical role in the trafficking of exogenous antigen by immature DCs in this cross-priming pathway.

Antigen Presentation↗
PubMed1998

Utility of transesophageal echocardiography during port-access minimally invasive cardiac surgery.

In this study, we sought to determine the use of transesophageal echocardiography (TEE) as the primary imaging technique to assist in the placement of endovascular catheters during minimally invasive, port-access cardiac surgery. The recent development of endovascular catheters that are placed via the femoral artery and vein has enabled patients to be placed on cardiopulmonary bypass without the need for direct visualization of the heart or great vessels via sternotomy. This has allowed cardiac surgery to be performed through smaller thoracotomy incisions. Placement of these catheters has previously been performed with fluoroscopic guidance, which has major imaging limitations. Thirty-six patients underwent port-access cardiac surgery at our institution during the study period. All patients underwent intraoperative TEE. We used TEE to visualize the coronary sinus os, right atrium and superior vena cava, and thoracic aorta to assist with placement of the coronary sinus catheter, venous cannula, and endoaortic clamp. Twenty patients underwent mitral valve surgery, 14 patients coronary artery bypass grafting, 1 patient aortic valve replacement, and 1 patient repair of an atrial septal defect by the port-access approach. TEE was able to adequately visualize the cardiac structures and assist in the placement of the endovascular catheters in all patients. Fluoroscopy was only helpful as an aid to TEE for placement of the coronary sinus catheter. TEE is an excellent imaging modality for the proper placement of these new endovascular catheters, obviating the need for fluoroscopy, except to be on standby and for placement of the coronary sinus catheter.

Adult↗
PubMed1998

Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs.

CD8+ cytotoxic T lymphocytes (CTLs) mediate resistance to infectious agents and tumours. Classically, CTLs recognize antigens that are localized in the cytoplasm of target cells, processed and presented as peptide complexes with class I molecules of the major histocompatibility complex (MHC). However, there is evidence for an exogenous pathway whereby antigens that are not expected to gain access to the cytoplasm are presented on MHC class I molecules. The most dramatic example is the in vivo phenomenon of cross-priming: antigens from donor cells are acquired by bone-marrow-derived host antigen-presenting cells (APCs) and presented on MHC class I molecules. Two unanswered questions concern the identity of this bone-marrow-derived cell and how such antigens are acquired. Here we show that human dendritic cells, but not macrophages, efficiently present antigen derived from apoptotic cells, stimulating class I-restricted CD8+ CTLs. Our findings suggest a mechanism by which potent APCs acquire antigens from tumours, transplants, infected cells, or even self-tissue, for stimulation or tolerization of CTLs.

Antigen Presentation↗
PubMed1998

The distinctive features of influenza virus infection of dendritic cells.

CD8+ cytolytic T lymphocytes (CTLs) are considered to be critical mediators for resistance to influenza virus infection. We have previously demonstrated that dendritic cells are potent antigen presenting cells in the development of anti-influenza CTLs. Here we identify distinctive features of the interaction of influenza virus with dendritic cells. Exposure of dendritic cells to influenza virus at MOIs of 2-4:1 leads to > 90% infection, as manifested by the expression of the viral proteins HA and NS1. The infection is non-toxic as viral protein expression is sustained for > 2 days with retention of viability, but little infectious virus is produced. Substantial induction of the anti-viral cytokine IFN-alpha also occurs. Influenza infection of macrophages also results in viral protein expression in a majority of cells, and synthesis of IFN-alpha. In contrast to dendritic cells, macrophages display evidence of apoptosis within 10-12 hours, and the majority of cells die within 24-36 hours. During this interval macrophages synthesize > 10-fold higher levels of virus than dendritic cells. Infected dendritic cells but not macrophages, can induce substantial CTL responses from purified blood CD8+ T cells in the absence of exogenous cytokines such as IL-2. Low levels of infection (MOIs of 0.02) are sufficient to generate potent CTL responses. Influenza virus expressing non-cleaved HA does not elicit CTLs indicating that virus must access the cytoplasm of dendritic cells to utilize traditional class I processing pathways. These observations indicate that DCs are distinct in their handling of influenza virus and for the induction of anti-viral immunity.

Animals↗
PubMed1998

Tumor-specific killer cells in paraneoplastic cerebellar degeneration.

Models for immune-mediated tumor regression in mice have defined an essential role for cytotoxic T lymphocytes (CTLs); however, naturally occurring tumor immunity in humans is poorly understood. Patients with paraneoplastic cerebellar degeneration (PCD) provide an opportunity to explore the mechanisms underlying tumor immunity to breast and ovarian cancer. Although tumor immunity and autoimmune neuronal degeneration in PCD correlates with a specific antibody response to the tumor and brain antigen cdr2, this humoral response has not been shown to be pathogenic. Here we present evidence for a specific cellular immune response in PCD patients. We have detected expanded populations of MHC class I-restricted cdr2-specific CTLs in the blood of 3/3 HLA-A2.1+ PCD patients, providing the first description, to our knowledge, of tumor-specific CTLs using primary human cells in a simple recall assay. Cross-presentation of apoptotic cells by dendritic cells also led to a potent CTL response. These results indicate a model whereby immature dendritic cells that engulf apoptotic tumor cells can mature and migrate to draining lymph organs where they could induce a CTL response to tissue-restricted antigens. In PCD, peripheral activation of cdr2-specific CTLs is likely to contribute to the subsequent development of the autoimmune neuronal degeneration.

Animals↗
PubMed1998

Intraepidermal lymphocytes in psoriatic lesions are activated GMP-17(TIA-1)+CD8+CD3+ CTLs as determined by phenotypic analysis.

The onset and persistence of psoriatic lesions are linked to the presence of an inflammatory infiltrate of CD3+ lymphocytes that includes CD4+ and CD8+ subsets. Since a primary susceptibility factor for psoriasis is the Class I HLA-Cw6 molecule, we set out to learn more about the features of the epidermal CD8+ lymphocytes. The markers tested were GMP-17, a cytotoxic granule protein found in activated cytotoxic lymphocytes (CTLs), and the alpha chain of the IL-2 receptor (CD25), a plasma membrane molecule found on activated T cells. Lymphocytes in lesional skin expressed the GMP-17 protein, whereas lymphocytes in non-lesional skin, resolving lesional skin and normal skin had little or no GMP-17. By flow cytometry analysis, lesional epidermal GMP-17+ cells were CD8+CD3+, with a subpopulation expressing the activation marker CD25+. Due to the abundance of activated GMP-17+CD8+CD3+ lymphocytes (the phenotype of activated cytotoxic cells) in psoriatic lesions compared to non-lesional and normal skin, we hypothesize that they are contributing directly to the psoriatic phenotype.

CD3 Complex↗
PubMed1998

Neutralizing monoclonal antibodies block human immunodeficiency virus type 1 infection of dendritic cells and transmission to T cells.

Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14(+) blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3(+) T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1.

Antibodies, Monoclonal↗
PubMed1998

Oculocardiac reflex during retinal surgery using peribulbar block and nitrous narcotic anesthesia.

BACKGROUND AND OBJECTIVES: This study was designed to examine the effects of local and general anesthesia on the oculocardiac reflex (OCR) in adults during retinal detachment surgery, with an aim of determining the safest type of anesthesia and on which extraocular muscle traction causes a higher incidence of OCR. PATIENTS AND METHODS: The study was performed on 30 American Society of Anesthesiologists-I patients (age range 40-60 years) who were undergoing retinal detachment surgery. Fifteen patients underwent surgery with general anesthesia and 15 were given a local peribulbar block. A fixed traction of 400 g was applied to all the extraocular muscles, and the heart rate, rhythm, and electrocardiogram of each patient was monitored. RESULTS: The overall incidence of OCR was higher with general anesthesia (63.3%) than with local anesthesia (14.4%). There was a significant decrease in heart rate for the rectus muscles (P < .0005) as well as the oblique muscles (P < .005) during traction with general anesthesia. All four rectus muscles were equally sensitive in eliciting the reflex. The incidence of dysrhythmias was 20% with general anesthesia and 6.67% with local anesthesia. CONCLUSION: This study showed that local anesthesia produces less bradycardia and ectopic arrhythmias and, therefore, may be safer and better than general anesthesia for surgeries in which traction of extraocular muscles is required.

Adult↗
PubMed1998

Comparison of psychomotor functions and sedation following premedication with oral diazepam and clonidine in children.

In a prospective, double-blind, controlled, randomized study, the psychomotor functions and sedation were assessed after premedication with diazepam and clonidine in children. Forty children in the age-group of 5-8 years, undergoing elective surgery under general anesthesia were studied. Twenty children (group 1) received oral clonidine 4 microg/kg, and 20 children (group 2) received oral diazepam 0.2 mg/kg, 120 minutes before induction of anesthesia. Sedation and psychomotor functions were assessed in both groups, before and after 60-90 minutes of administration of premedication. The results of the study showed that mean sedation score in group 1 was 8 +/- 1.07, and 9 +/- 0.64 in group 2. On intergroup comparison the sedation was found to be comparatively better in group 2 than group 1 (p < 0.05). The performance of psychomotor functions decreased after premedication in both the groups as compared to that before premedication (p < 0.05). The psychomotor functions were depressed more in diazepam group than in the clonidine group (p < 0.05). Thus, it is concluded that clonidine produces good sedation and causes less effect on psychomotor functions and therefore can be used for premedication in children.

Administration, Oral↗
PubMed1997

A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells.

Mature human dendritic cells can be generated in substantial numbers from nonproliferating progenitors in human blood using a two-step protocol. T cell-depleted mononuclear cells are first cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) and then exposed to monocyte conditioned medium (MCM). The dendritic cells generated using this approach are rendered terminally mature and are the most potent antigen presenting cells identified to date in humans. We sought to characterize factors in MCM that induce the terminal differentiation of dendritic cells. MCM contained substantial, although varying, quantities of several factors including tumor necrosis factor-alpha, IL-1beta, IL-6, and interferon-alpha. However, none of the four factors, individually or in various combinations, could fully substitute for the MCM to generate irreversibly differentiated dendritic cells. The yields, percentage of cells expressing the mature phase marker CD83, and mixed leukocyte reaction-stimulatory function were lower when defined cytokines were used in the place of MCM. Therefore, the full maturation of dendritic cells, because it entails changes in many known cell and molecular properties, requires a number of different cytokines that are released in tandem from appropriately stimulated monocytes. We propose that MCM-matured dendritic cells will be the most effective adjuvants for immunotherapy in vivo.

Cell Differentiation↗
PubMed1997

Generation of monocyte-derived dendritic cells from precursors in rhesus macaque blood.

While the dendritic cells (DCs) of mouse and man have been extensively studied, until recently those of non-human primates remained poorly characterized. We present a method for generating large numbers of DCs from precursors in rhesus macaque blood, based on techniques developed for human blood. For 7 days, a T cell-depleted population of mononuclear cells was cultured in 1% human plasma with GM-CSF and IL-4, both to initiate DC differentiation and to inhibit macrophage development. On day 7, 50% of the culture medium was replaced with a monocyte-conditioned medium (MCM), which is required for the final maturation of the DCs into potent stimulators of the allogeneic MLR. Between 0.5 and 1.0 x 10(6) DCs can be generated from 20 ml of rhesus macaque blood. We compared these cytokine-generated DCs to the adherent macrophages present in the same cultures. Cytokine-generated DCs were considerably more potent at stimulating allogeneic T cells than adherent macrophages. Furthermore, the DCs had a distinct morphology and phenotype, with long processes, high levels of p55, and a characteristic perinuclear collection of intracellular CD68. In contrast, adherent macrophages expressed very low levels of p55, and high diffuse levels of CD68. Macaque DCs generated by this method may be useful in vaccine development and for studies of SIV pathogenesis.

Animals↗
PubMed1997

Dendritic cells as immunogens for human CTL responses.

The cellular requirements for generating potent human CD8+ CTLs to influenza A virus in vitro have been defined. Furthermore, we have developed improved methods for generating large numbers of DCs from non-proliferating progenitors. These developments have enabled the design of new strategies to elicit CTLs in vivo. For example, together with IL-12, antigen-pulsed DCs may be a useful approach for boosting CTL responses against infectious agents and malignancies. Our results also reopen the potential use of inactivated virus preparations as immunogens for CTL responses.

Blood Cells↗
PubMed1997

Comparative study of oral clonidine and diazepam as premedicants in children.

In a prospective, double-blind, controlled study the efficacy of clonidine was assessed in children, with respect to sedation, intubation response, and recovery. Fifty children, aged 4-12 years, undergoing general anesthesia were studied. Twenty-five children (group I) received oral diazepam) 0.2 mg/kg and another 25 children (group II) received oral clonidine 3 micrograms/kg, 90-120 minutes before induction of anesthesia. The level of sedation, hemodynamic changes to laryngoscopy and intubation, the recovery from anesthesia were noted and compared between the groups. Clonidine 3 micrograms/kg produced sedation comparable to diazepam 0.2 mg/kg (p > 0.1). There was significant (p > 0.01) attenuation of hemodynamic intubation response with clonidine. The recovery with clonidine was not delayed (p < 0.01). Clinically significant hypotension and bradycardia were not observed in any of the patients. We conclude that clonidine 3 micrograms/kg produces sedation comparable to diazepam 0.2 mg/kg and also attenuates the intubation response without increasing the incidence of complications.

Adrenergic alpha-Agonists↗