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N Arora

Publications and source records attributed to N Arora.

At least 37 records · Page 2Linked to original sources
PubMed2000

Allergens of Epicoccum nigrum grown in different media for quality source material.

BACKGROUND: The Epicoccum nigrum (EN) extract used in allergy disorders exhibits batch-to-batch variations in protein composition and allergenic potency. In this study, the allergens of EN grown in different media were investigated. METHODS: EN was grown in five different nutrient media as stationary cultures at 25 degrees C for 5-23 days. The growth pattern was characterized by measuring dry weight, protein and carbohydrate content. The antigenic and allergenic content of EN extract was evaluated with EN-positive patients' sera and antibodies raised in rabbit. RESULTS: The growth of EN in Czapeck Dox medium yielded insufficient material, while Sabouraud's broth with yeast extract (SBY) gave maximum spore-mycelial mass and protein content. Potato dextrose broth (PDB) and potato dextrose agar (PDA) showed higher dry weight and protein in 7-9-day cultures. SDS-PAGE resolved 26, 22, and 21 protein bands in EN extracts from cultures of day-13 SBY, day-7 PDB, and day-9 PDA, respectively. IgE/IgG immunoblots showed more allergenic (25)/antigenic (25) bands in EN cultured in SBY than in the others. Specific IgE ELISA and intradermal tests showed EN extract from day-13 culture in SBY to be the most potent. CONCLUSIONS: The day-13 culture of EN in SBY was the most potent and may be selected for preparing EN extracts for diagnosis of allergy and future studies.

Adolescent↗
PubMed2000

Anthrax toxin-mediated delivery of cholera toxin-A subunit into the cytosol of mammalian cells.

The protective antigen (PA) component of anthrax toxin mediates delivery of either lethal factor (LF) or oedema factor into the cytosol of mammalian cells. The N-terminal domain of LF(1-254) (amino acids 1-254 of LF) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. In the present study, we fused the catalytic subunit of cholera toxin (CT-A) with LF(1-254) and showed that the fusion protein LF(1-254)-CT-A retains ADP-ribosylation activity in solution and increased intracellular cAMP levels in J774A.1 macrophage cells when added together with PA. A mutant fusion protein, in which arginine-7 of CT-A was replaced with lysine, did not show ADP-ribosylation activity in solution and failed to increase cAMP levels in macrophage cells. The data show that LF(1-254)-CT-A retains its catalytic activity in solution as well as when translocated into the cytosol of eukaryotic cells via an alternative pathway to the GM(1) receptor used by CT.

Adenosine Diphosphate↗
PubMed2000

Purification and partial characterization ofa 67-kD cross-react ive allergen from Imperata cylindrica pollen extract.

BACKGROUND: Grass pollens are known to induce type I allergic reactions in a large number of genetically predisposed individuals. Earlier studies have recognized Imperata cylindrica (Ic) pollen as an important source of aeroallergen which contained 7 IgE binding proteins in the MW range of 85-16 kD. OBJECTIVES: To isolate, purify and characterize a cross-reactive allergenic protein from Ic pollen extract for diagnosis and therapy of grass pollen allergy. METHODOLOGY: Ic pollen extract was fractionated using DEAE Sephadex A-50, Sephadex G-200 and Mono Q column. Allergenic activity of the fractions was checked by ELISA, skin tests, ELISA inhibition and immunoblot using sera of Ic-sensitive patients. A 67-kD protein was purified to homogeneity from Ic-VIII. The allergenic determinants of this protein were identified by SDS-PAGE and immunoblot after CNBr treatment. RESULTS: Among Ic fractions, Ic-VIII was highly potent by ELISA, skin tests and showed cross-reactivity with 4 other tropical grasses by immunoblot and ELISA inhibition. The subfraction Ic-VIIIe1 of Ic-VIII showed a band at 67 kD on SDS-PAGE. On CNBr treatment, it gave 7 peptides, 3 of which were found to be allergenic. CONCLUSION: A 67-kD protein (Ic-VIIIe1) was isolated, purified to homogeneity and partially characterized. It showed cross-reactivity with tropical grasses tested and contained at least three allergenic determinants.

Allergens↗
PubMed2000

Hepatitis C virus in the hemodialysis setting: detecting viral RNA from blood port caps by reverse transcription-polymerase chain reaction.

BACKGROUND: A widely observed increased risk of hepatitis C virus (HCV) infection in chronic hemodialysis patients has been previously attributed to violations of "Universal Precautions" for the control of blood-borne pathogens, as well as in part, to other risk factors, such as a history of blood transfusion or injection drug use. However, specific factors responsible for transmission have not been identified and the possibility that flaws in dialysis procedures, including sterilization, could increase the risk of transmission, has not been excluded. METHODS: We investigated reuse procedures for hemodialysis equipment and tested dialyzer blood port caps for detection of hepatitis C virus RNA (HCV RNA) by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Following artificial contamination of the blood port caps with blood or fluids from human HCV-positive patients, and overnight soaking in 1% Renalin, HCV RNA was detected on 4 of 20 caps contaminated with blood, 1 of 10 contaminated with serum and 8 of 24 contaminated with dialyzer blood compartment residue. HCV RNA was also detected on 1 of 111 pairs of blood port caps collected post dialysis from HCV positive patients, after soaking the caps overnight in 1% Renalin. CONCLUSION: The results suggest that HCV RNA might be detectable on reused dialysis equipment post sterilization procedures, if residual blood or serum is not entirely or almost entirely removed prior to sterilization. This may warrant evaluation of sterilization procedures to ensure that procedures are adequate and that protocols are rigorously followed. Further studies of sterilization procedures by sensitive techniques such as RT-PCR may be indicated.

Equipment Reuse↗
PubMed2000

Pulse oral versus pulse intraperitoneal calcitriol: a comparison of efficacy in the treatment of hyperparathyroidism and renal osteodystrophy in peritoneal dialysis patients.

Controversy exists among various studies in regard to the efficacy of oral (p.o.) versus parenteral calcitriol. Some studies suggest that intravenous (i.v.) calcitriol is superior to p.o. calcitriol for treating renal osteodystrophy in hemodialysis patients; others suggest that these routes of administration are equivalent. To our knowledge, no large, prospective, randomized study compares intraperitoneal (i.p.) to p.o. calcitriol in adult peritoneal dialysis patients. We conducted a prospective randomized study in 76 patients (38 on i.p. calcitriol and 38 on p.o. calcitriol), whom we followed for 48 months. Of the 76 patients, 34 (18 in the i.p. group and 16 in the p.o. group) completed the 48-month study period. Calcitriol dosing was similar in both groups (3-6 micrograms per week in three divided doses). Dose adjustments were made depending on levels of parathyroid hormone (PTH), serum calcium, phosphorus, and calcitriol. No significant difference was seen between the groups in regard to age, sex, race, body mass index, dialysis duration, or cause of ESRD. Neither was any difference in the incidence of peritonitis seen between the groups. In the first 3-6 months, PTH decreased equivalently in both groups. The PTH level remained suppressed in the i.p. group throughout the remainder of the study, but, in the p.o. group, PTH returned to its pretreatment level after 3-6 months. Mean serum calcium was not different in the two groups. In the p.o. group, a considerably higher mean follow-up phosphorus level (6.8 +/- 2.3 mg/dL versus 4.7 +/- 1.4 mg/dL, p = 0.008), PTH level (384 +/- 146 pg/mL versus 162 +/- 64 pg/mL; p = 0.005), and alkaline phosphatase level (178 +/- 37 IU/L versus 72 +/- 21 IU/L, p = 0.02) were seen as compared to the i.p. group. In the i.p. group, resolution of osteodystrophy occurred in all patients at the end of the study; in the p.o. group, 5 patients maintained or developed osteodystrophy by the end of the study (p = 0.016). We conclude that i.p. calcitriol is more effective than pulse p.o. calcitriol in lowering PTH and alkaline phosphatase levels and in resolving renal osteodystrophy, and that i.p. calcitriol is associated with a lower incidence of hyperphosphatemia and elevated Ca x PO4 byproduct.

Administration, Oral↗
PubMed1999

Antineoplastic urinary protein inhibits Kaposi's sarcoma and angiogenesis in vitro and in vivo.

Kaposi's sarcoma (KS) is the most common tumor in human immunodeficiency virus infection and acquired immune deficiency syndrome. Recent clinical trials with human chorionic gonadotropin (hCG) prepared from early pregnancy urine have shown encouraging results in the resolution of KS lesions. A urinary protein with antitumor activity, ANUP (antineoplastic urinary protein), a dimer of 32 kD, has previously been shown to inhibit the growth of various tumor cell lines in vivo. It was thus studied for its activity in KS cell lines in vitro and in vivo to determine whether it could be a source of the anti-KS activity observed in hCG preparations. ANUP is a strong growth inhibitor for KS cell lines, but has little or no effect on fibroblast, aortic smooth muscle, T- and B-lymphocyte, and monocyte cell lines. ANUP also inhibited the proliferation of endothelial cell lines, suggesting that the in vitro effects were endothelial cell lineage-specific. However, ANUP antibodies did not block the inhibitory effect of certain commercial preparations of hCG, previously shown to be active in KS. Thus, the active protein in these commercial preparations of hCG may be distinct from ANUP. The antitumor activity of ANUP was further confirmed in a chicken allantoic membrane (CAM) assay in which vascular endothelial growth factor (VEGF) and beta fibroblast growth factor (bFGF)-induced angiogenesis was inhibited by ANUP in a dose-dependent manner. In vivo activity of ANUP was demonstrated in the murine model of KS, where ANUP inhibited tumor growth. ANUP is thus a potential candidate for development in the treatment of KS and other diseases in which angiogenesis plays an important role.

Adult↗
PubMed1999

Vascular endothelial growth factor chimeric toxin is highly active against endothelial cells.

Angiogenesis is a critical step in a benign tumor's evolution toward malignancy and metastasis. Tumor cells acquire such a phenotype by their ability to secrete angiogenic factors such as vascular endothelial growth factor (VEGF). VEGF receptors (VEGFRs) flt-1/VEGFR-1 and Flk-1/ KDR/VEGFR-2 are restricted to activated endothelial cells, with the highest expression being in the tumor vasculature. The present study was undertaken to target the VEGFRs. Targeted toxins were developed by recombinant methods by fusing VEGF165 or VEGF121 to the diphtheria toxin (DT) translocation and enzymatic domain (DT390-VEGF165 or DT390-VEGF121). Both fusion proteins were found to be highly toxic to proliferating endothelial cells but not to vascular smooth muscle cells. The fusion protein is also active in Kaposi's sarcoma, a tumor type that expresses high levels of VEGFRs. These fusion proteins completely inhibit the basic fibroblast growth factor-induced growth of new blood vessels in the chick chorioallantoic membrane assay. Furthermore, the fusion toxin substantially retards the growth of Kaposi's sarcoma tumors in mice. Because nearly all tumors induce local angiogenesis with high VEGFR expression, VEGF-derived toxins may have wide application in cancer therapy.

Animals↗
PubMed1999

Allergens of Curvularia lunata during cultivation in different media.

BACKGROUND: Despite enormous efforts toward the standardization of fungal extracts, only a few extracts have been characterized that are relevant for the diagnosis and immunotherapy of allergy-associated disorders. OBJECTIVE: The goal of this study was to determine the optimum growth condition of Curvularia lunata, an important fungal allergen for quality raw material, and to analyze the C lunata extract for IgE- and IgG-binding proteins. METHODS: C lunata was grown in synthetic (Czapeck Dox medium), semisynthetic (Sabouraud's broth [SB]), and natural media (potato dextrose [PD]) for different periods of time. The extracts were probed for allergenic and antigenic activity, with pooled patient sera and polyclonal antibodies raised against C lunata by means of ELISAs, immunoblots (in vitro), and intradermal tests (in vivo). RESULTS: The growth of C lunata was better in semisynthetic media (ie, SB) compared with other types of media. Dry weight and protein content was maximum in the 7-day culture of SB. ELISA with pooled sera from C lunata-sensitive patients exhibited that cultures grown in SB for 11 to 13 days and PD plus 1.0% agar for 5 days were the most potent. Intradermal tests with 11- to 13-day SB culture extract showed maximum skin reactivity in allergy patients. Immunoblots with patient sera showed 10 to 14 IgE-binding proteins in 5- to 15-day SB cultures. Analysis of 5- to 15-day SB extract with rabbit sera showed 10 to 16 IgG-binding proteins. CONCLUSIONS: The extract from 11- to 13-day SB cultures were most biologically potent (intradermal tests) and showed high antigenic and allergenic reactivity (ELISA and immunoblot). The addition of yeast extract did not affect the dry weight and protein content of the C lunata extract. Furthermore, addition of agar in PD medium increases the dry weight and protein content of the fungal mat. Synthetic media was not suitable for mass cultivation of C lunata.

Allergens↗
PubMed1999

Anthrax toxin fusion proteins for intracellular delivery of macromolecules

The dominant role played by the anthrax toxin in Bacillus anthracis pathogenesis shows that the toxin has evolved to be an efficient system for delivering its two catalytic protein components, oedema factor and lethal factor (LF), into the cytosol of host cells. This system involves binding of the protective antigen (PA) toxin component to a ubiquitous (and still unidentified) receptor, proteolytic activation at the cell surface, internalization by endocytosis and translocation through an early endosome membrane to the cytosol (Leppla 1995). We and colleagues showed that the system can be exploited to deliver heterologous polypeptides to the cytosol (Arora et al. 1992; Milne et al. 1995). This work used the catalytic domains of other toxins which are normally translocated across membranes (Arora & Leppla 1994). Immunity to intracellular pathogens depends on the cytosolic processing of antigens to produce peptides that are presented on the cell surface bound to MHC Class I molecules. The anthrax toxin delivery system provides a way to mimic this process. We made a fusion protein containing the (non-catalytic) amino terminal domain of LF and the gp120 envelope glycoprotein of HIV-1. Administration of this recombinant protein along with PA to antigen-presenting cells sensitized them to cytolysis by cytotoxic T-cells specific to gp120 peptides (Goletz et al. 1997). Further exploitation of the anthrax toxin system as a cell-targeting reagent would be facilitated by achieving cell type specificity. The recent determination of the PA structure (Petosa et al. 1997) allows rational engineering to modify or replace the receptor-binding domain with specific ligand structures. A model system was produced by fusing a c-Myc peptide to the carboxyl terminus of PA so as to target hybridoma cells expressing cell surface antibodies to this peptide. Killing of the hybridoma cells was shown to be specific by competition with the peptide and with non-toxic mutants of PA (Varughese et al. 1998).

Journal Article↗
PubMed1998

Emerging treatments for epidemic (AIDS-related) Kaposi's sarcoma.

Kaposi's sarcoma (KS) is an opportunistic tumor that develops with increased frequency (100,000-fold) after HIV infection. KS causes significant morbidity from mucocutaneous involvement and mortality from complications of visceral sites of disease such as the lungs, gastrointestinal tract, and the liver. Progressive unraveling of the KS pathogenesis has lead to the development of novel therapeutic approaches. Newest therapies are first evaluated in patients with limited tumor burden. These include: 1) inhibitors of angiogenesis such as vascular endothelial growth factor signaling inhibitor (SU 5416), and several other inhibitors of angiogenesis such as the dipeptide IM 862, TNP-470, Col-3, and thalidomide; 2) topical and systemic retinoids; 3) antiviral agents specific for Kaposi's sarcoma herpesvirus and human herpesvirus-8, or HIV; and 4) pregnancy-related factors. Patients with advanced disease such as widespread mucocutaneous disease, lymphedema, and visceral disease are treated most effectively with cytotoxic agents. The most active agents include liposomal anthracyclines, paclitaxel, vinca alkaloids, and bleomycin. The combination of liposomal anthracyclines and paclitaxel, with and without the most promising biologicals, should now be studied to further reduce the toxicity, and enhance the antitumor effects. Furthermore, identification of risk factors for KS should serve to explore prophylactic therapies.

AIDS-Related Opportunistic Infections↗
PubMed1997

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein.

A challenge for subunit vaccines whose goal is to elicit CD8(+) cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.

AIDS Vaccines↗
PubMed1997

Site directed mutagenesis of histidine residues in anthrax toxin lethal factor binding domain reduces toxicity.

Anthrax lethal toxin is a mixture of protective antigen (PA, 735 AA) and lethal factor (LF, 776 AA). Earlier studies have shown that 254 residues of lethal factor are sufficient for PA binding to cause internalization (Arora N and Leppla SH, J Biol Chem 268: 3334-3342, 1993). The present study was undertaken to determine residues which are important for binding of LF to PA. LF modification with diethyl pyrocarbonate (DEPC, modifies histidine residue primarily) results in the loss of binding and toxicity in mammalian cells. There are nine histidine residues in the binding domain. To locate the important residue(s), site-directed mutagenesis of these histidines were performed by recombinant methods. Replacement of His42 with Gly42 destablizes the protein and hence it could not be purified. His35 when mutagenized to Gly35 (mLF-DTA) diminishes the toxicity by 20 fold. Time dependent studies show that binding of mLF-DTA was reduced at shorter incubations and longer incubations taper off this difference. Gel shift assay suggested 8-10% less binding of mLF-DTA as compared to LF-DTA. In conclusion His35 is important for binding and His42 is critical and confers proper conformation for LF binding to PA.

Animals↗
PubMed1997

Ribonuclease activity dependent cytotoxicity of Asp fl, a major allergen of A. fumigatus.

A major allergen/antigen, Asp fl, secreted by Aspergillus fumigatus exhibits cytotoxicity towards eukaryotic cell lines. Asp fl inhibited protein synthesis in RAW cells with an IC50 of 4.5 nM and also degraded ribosomal RNA of RAW cells at a similar concentration. Ribosomal inactivation by Asp fl may be the probable mechanism for protein synthesis inhibition. Specific ribonuclease activity of Asp fl was observed to be 100,000 U/mg. Presence of strong RNase activity in Asp fl was further confirmed by agar gels containing yeast RNA. Electrophoretic run on agarose gels showed that Asp fl degrades all species of naked RNA. Modification of histidine residues of Asp fl with diethyl pyrocarbonate and alkylation of cysteines with iodoacetamide resulted in loss of ribonuclease activity and cytotoxicity of Asp fl. The current study establishes the ribonuclease activity of a purified major allergen of A. fumigatus that inhibits protein synthesis and kills the eukaryotic cells.

Allergens↗
PubMed1997

Identification and evaluation of a major cytotoxin of A. fumigatus.

Aspergillus fumigatus is a highly pathogenic fungus causing a wide spectrum of diseases in immunocompromised as well as immunocompetent hosts. The present work was undertaken to evaluate the cytotoxic nature of fractionated antigens of A. fumigatus against the mammalian cell lines (J774, RAW, CHO and L929). An enriched protein antigenic fraction of A. fumigatus was subjected to con A Sepharose and phenyl Sepharose chromatography. Antigenic fractions, ConAub (conA unbound) and PSC III (fraction III of phenyl Sepharose column) containing low mw antigens showed higher cytotoxicity as compared to other antigenic fractions. PSC III was further purified on HPLC resulting in an 18 kDa homogeneous protein. The purified protein showed high ELISA absorbance values for specific IgG and IgE antibodies in sera of ABPA patients. Monoclonal antibody raised against Asp fl, a major allergen/antigen of A. fumigatus recognised the purified 18 kDa by ELISA and western blot. The 18 kDa allergen/antigen or Asp fl showed similar toxicity towards all the four cell lines (macrophage and fibroblast) with an IC50 of 75 ng/ml or 4.16 nM. Reduction in toxicity of 18 kDa at low temperatures and potentiation in presence of ammonium chloride and monensin indicates mechanism of internalisation of 18 kDa in eukaryotic cells is similar to alpha-sarcin. The present work shows that the 18 kDa allergen/antigen (Asp fl) is a major cytotoxin secreted by A. fumigatus which may play multiple roles in the pathogenesis of Aspergillosis through allergenicity, antigenicity and cytotoxicity.

Animals↗