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Biomedical subjects

S Agrawal

Publications and source records attributed to S Agrawal.

At least 181 records · Page 10Linked to original sources
PubMed1997

Pharmacokinetics of oligonucleotides.

The effectiveness of antisense oligonucleotides as therapeutic agents depends on their pharmacokinetics, tissue disposition, stability, elimination and safety profile. Pharmacokinetic data allow one to determine the frequency of administration and any potential toxicity associated with chronic administration. Phosphorothioate oligonucleotides degrade from the 3' end, the 5' end, and both the 3' and 5' ends in a time- and tissue-dependent manner. After intravenous administration in mice, rats and monkeys, phosphorothioate oligonucleotides are detected in plasma; they distribute rapidly and are retained in the majority of tissues. The major route of elimination is the urine. The pharmacokinetic profile is similar following subcutaneous, intradermal or intraperitoneal administration, but with lower maximum plasma concentrations. Phosphorothioate oligonucleotides have a short plasma half-life in humans. End-modified, mixed-backbone oligonucleotides (MBOs) contain nuclease-resistant 2'-O-alkylribonucleotides or methylphosphonate internucleotide linkages at both the 3' and 5' ends of phosphorothioate oligonucleotides. These end-modified MBOs have pharmacokinetic profiles similar to those of the parent phosphorothioate oligonucleotides, but they are significantly more stable in vivo and they can be administered orally. Centrally modified MBOs contain modified RNA or DNA in the centre of a phosphorothioate oligonucleotide. They show controlled degradation and elimination following administration in rats. The pharmacokinetics of antisense oligonucleotides depends on the sequence, the nature of the oligonucleotide linkages and the secondary structure.

Animals↗
PubMed1997

Single-stranded DNA and RNA targeted triplex-formation: UV, CD and molecular modeling studies of foldback triplexes containing different RNA, 2'-OMe-RNA and DNA strand combinations.

We studied the influence of different 2'-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D.D:D, R*.D:D, D.D:R*, R*.D:R*, D.R:D, R*.R:D, D.R:R*, and R*.R:R*; where the first, middle, and last letters stand for the Hoogsteen Pyrimidine, Watson-Crick [WC] purine and WC pyrimidine strands, respectively, and D, R and R* stand for DNA, RNA and 2'-OMe-RNA strands, respectively) indicate more stable foldback triplex formation with a DNA purine strand than with an RNA purine strand. Of the four possible WC duplexes with RNA/DNA combinations, the duplex with a DNA purine strand and a 2'-O-Me-RNA pyrimidine strand forms the most thermally stable triplex, although its thermal stability is the lowest of all four duplexes. Irrespective of the duplex combination, a 2'-OMe-RNA Hoogsteen pyrimidine strand forms a stable foldback triplex over a DNA Hoogsteen pyrimidine strand confirming the earlier reports with conventional and circular triplexes. The CD studies suggest a B-type conformation for an all DNA homo-foldback triplex (D.D:D), while hetero-foldback triplex spectra suggest intermediate conformation to both A-type and B-type structures. A novel molecular modeling study has been carried out to understand the stereochemical feasibility of all the combinations of foldback triplexes using a geometric approach. The new approach allows use of different combinations of chain geometries depending on the nature of the chain (RNA vs. DNA).

Circular Dichroism↗
PubMed1997

Upregulation of HLA class-I gene transcription in K562 cells by analogs of splenopentin (SP-5)

We report here the capacity of three analogs of Splenopentin, to up regulate transcription of the HLA-B7 gene in the K562 cell line. These cells normally do not transcribe the HLA class I genes. The Splenopentin analogs used in this study are effective at very low molar concentrations and are non-toxic to cells at these levels. Electrophoretic Mobility Shift assays indicate that this transcriptional up regulation of HLA class I genes may be related to the appearance of novel class I promoter binding factors induced in the nuclei of treated cells.

Cell Nucleus↗
PubMed1997

In vivo metabolic profile of a phosphorothioate oligodeoxyribonucleotide.

Antisense phosphorothioate oligodeoxyribonucleotides (PS oligonucleotides) have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. Following administration in vivo, PS oligonucleotides are rapidly cleared from the plasma and distributed to various organs. However, the manner in which administered oligonucleotides are metabolized in plasma and tissues is poorly understood. In this study, a 25-mer PS oligonucleotide (GEM91) complementary to the gag gene mRNA of the human immunodeficiency virus (HIV-1) was administered to mice through intravenous injections to investigate its metabolism. The PS oligonucleotide was extracted from plasma at 1 hour postadministration and from kidney and liver at 24 hours postadministration. After extraction, the PS oligonucleotide and its metabolites were tailed with dA and annealed to a dT-tailed plasmid. The recombinant plasmid was ligated and used to transform competent bacteria. The region of interest containing the PS oligonucleotide was then sequenced. Our results show that degradation of the PS oligonucleotide in plasma was primarily from the 3'-end. However, in kidney and liver, degradation was primarily from the 3'-end, but a large proportion of the PS oligonucleotide was degraded from the 5'-end as well. We also studied the metabolism of PS oligonucleotide in plasma after 2-hour intravenous infusion in HIV-infected patients. The degradation of the PS oligonucleotide in plasma was primarily from the 3'-end. This study is important in understanding the metabolism of antisense PS oligonucleotide in vivo in general but also provides guidance for designing second-generation antisense oligonucleotides with improved stability and safety profile.

Animals↗
PubMed1997

Pattern and kinetics of cytokine production following administration of phosphorothioate oligonucleotides in mice.

Phosphorothioate oligonucleotides with certain sequences or structure motifs can stimulate the immune system. We administered to mice a 27-mer phosphorothioate oligonucleotide (sequence 5'-TCG TCG CTG TCT CCG CTT CTT CTT GCC-3'), which has previously been shown to cause splenomegaly and hypergamma-globulinemia on in vivo administration in mice, and studied the pattern and kinetics of cytokine production at both the splenic mRNA and serum protein levels. Following i.p. administration of 50 mg/kg of oligonucleotide, significant increases in the splenic mRNA levels of IL-6, IL-12p40, IL-1 beta, and IL-1Ra and serum levels of IL-6, IL-12, MIP-1 beta, and MCP-1 were observed. In contrast, no significant differences in splenic mRNA levels of IL-2, IL-4, IL-5, IL-9, IL-13, IL-15, IFN-gamma, or MIF or serum levels of IL-2, IL-4, IL-5, IL-10, IFN-gamma, or GM-CSF were detected. The induction of IL-12 secretion was dependent on the sequence and dose of the oligonucleotides. One oligonucleotide (sequence 5'-GAG AAC GCT CGA CCT TCG AT-3') induced a high level of IL-12 secretion even at 5 mg/kg, whereas another oligonucleotide (sequence 5'-CTC TGC CAC CCA TCT CTC TCC TTC T-3') did not induce significant IL-12 secretion even at 50 mg/kg. IL-12 secretion induced by various doses of oligonucleotide has the same kinetics but differs in magnitude. These studies show a distinct pattern and kinetics of cytokine production following oligonucleotide administration and further demonstrate that cytokine induction is not a general property of phosphorothioate oligonucleotides but is dependent on the sequence and dose of the oligonucleotides.

Animals↗
PubMed1997

Toxicologic effects of an oligodeoxynucleotide phosphorothioate and its analogs following intravenous administration in rats.

The aim of the present study is to evaluate the in vivo toxicologic effects of a phosphorothioate oligodeoxynucleotide (PS oligo) and three of its analogs [PS oligo containing four methylphosphonate linkages at the 3' and 5'-ends (MBO 1), PS oligo containing four 2'-O-methylribonucleosides at both the 3'- and 5'-ends (MBO 2), and PS oligo containing an 8 bp loop region at the 3'-end (self-stabilized oligo)]. Oligodeoxynucleotides were administrated intravenously to male and female rats at doses of 3, 10, and 30 mg/kg/day for 14 days. Rats were killed on day 15, blood samples were collected for hematology and clinical chemistry determinations, and tissues, including lymph nodes, spleens, livers, and kidneys, were subjected to pathologic examinations. The toxicity profiles of the four oligodeoxynucleotides were very similar, but differed in magnitude. In terms of the severity of the abnormalities caused by the oligodeoxynucleotides, the order was MBO 2 > PS oligo > self-stabilized oligo > MBO 1. Alterations in hematology parameters included thrombocytopenia, anemia, and neutropenia. Abnormalities in clinical chemistry parameters observed with PS oligo or MBO 2 were dose-dependent elevation of liver transaminases and reduction of the levels of alkaline phosphatase, albumin, and total protein. In addition, MBO 2 caused elevation of the total bilirubin level in male rats at the 30 mg/kg dose. No major alterations in hematology or clinical chemistry were observed in rats receiving MBO 1 or self-stabilized oligo. Dose-dependent enlargements of spleen, liver, and kidney were observed, especially in rats receiving PS oligo and MBO 2. Pathologic studies showed a generalized hyperplasia of the reticuloendothelial (RE) system in the tissues examined. Alterations in the spleen were mainly RE cell hyperplasia and hematopoietic cell proliferation. In addition to RE cell hyperplasia, lymph nodes showed necrosis, hepatocytes showed cytologic alterations and necrosis, and kidneys showed renal tubule regeneration. The severity of pathologic changes observed was oligodeoxynucleotide dependent, in the order of MBO 2 > PS oligo > self-stabilized oligo > MBO 1.

Animals↗
PubMed1997

Role of preoperative donor-specific transfusion and cyclosporine in haplo-identical living related renal transplant recipients.

A prospective randomized trial of use of donor-specific transfusion and cyclosporine given 24 h before operation was performed in living related renal transplant recipients. The benefits, disadvantages and effect on graft and patient outcome was analyzed. Cyclosporine was started 72 h before operation and 48 h before donor-specific transfusion (DST). Fifteen patients received DST while another 15 age- and sex-matched living related renal allograft recipients on similar immunosuppression served as controls. Patient and donor demographics were similar in the two groups. The DST group had significantly fewer rejection episodes than the control group (0.26 vs. 1.1 rejection episode per patient, p < 0.01). There were fewer episodes of acute rejection in the first 3 months posttransplant in the DST group. Hyperresponder recipients (as tested by mixed lymphocyte cultures) also benefitted by DST which significantly reduced the number of acute rejection episodes (0.25 vs. 1 episode per hyperresponder patient, DST vs. control, p < 0.05). The need for dialysis, incidence of infections and other complications were similar in the two groups. Graft function at 3, 6, 9 and 12 months after transplant was significantly better in the DST group (p < 0.05). Graft survival at 1 year in DST group (85.5%) was not statistically different than control (74.8%). In conclusion, DST and cyclosporine given 24 h before live related renal transplantation is effective in improving graft function and reducing the number of acute rejection episodes which could have a beneficial effect on long-term graft survival.

Acute Disease↗
PubMed1997

Factors affecting agreement between alcohol abusers' and their collaterals' reports.

OBJECTIVE: Because of their low cost and ease of use, collaterals' reports are the most frequent source of independent corroboration with alcohol abusers' self-reports of drinking and related events. Although several reviews have shown that we can have confidence in the accuracy of alcohol abusers' reports of their drinking and in the use of collateral reports as an independent validity criterion, neither data source is error free. This study examined factors that influence the level of agreement between collaterals' and alcohol abusers' reports. METHOD: Using data from a study of natural recoveries from alcohol-related problems, this study examined how agreement between 120 alcohol abusers' (79.2% male) and their collaterals' reports varied as a function of collateral type and of the collaterals' ratings of their confidence in the accuracy of their reports of the subjects' drinking and related behaviors. Collaterals' awareness of nonalcohol-related levels was also examined. RESULTS: The best agreement occurred for reports from alcohol abusers' spouses who were fairly confident about the information provided. For all variables, some proportion of collaterals respond to demand characteristics of the interview by providing very specific information about subjects' behavior yet admit to being unsure of this information. CONCLUSIONS: Collaterals who are fairly sure of the information they provide are the preferred informants to corroborate alcohol abusers' reports of drinking and related behaviors. In some cases the best collaterals are spouses who are fairly sue of the information they reported. It is also recommended that treatment outcome studies should accept reports only from collaterals who are confident about the information they report.

Adult↗
PubMed1997

Molecular typing of HLA class I and class II antigens in Indian kala-azar patients.

HLA has been shown to be associated with many diseases. To find out whether host genetic factors like the HLA are involved in susceptibility to kala-azar (visceral leishmaniasis) in India, we formulated an association study with genetically related controls. All samples were typed by PCR SSOP (sequence specific oligonucleotide probes) for HLA class I (A and B) and class II (DR) antigens. The test of association we used was the transmission disequilibrium test (TDT). No significant evidence for association with any of the three HLA loci was obtained.

Disease Susceptibility↗
PubMed1996

Hoogsteen DNA duplexes of 3'-3'- and 5'-5'-linked oligonucleotides and trip formation with RNA and DNA pyrimidine single strands: experimental and molecular modeling studies.

DNA oligonucleotide sequences containing two parallel complementary strands attached through 3'-3' and 5'-5' linkages were synthesized. These oligonucleotides from Hoogsteen base-paired parallel-stranded (PS) hairpin duplexes under appropriate conditions [Kandimalla, E. R., Agrawal, S., Venkataraman, G., & Sasisekharan, V. (1995a) J. Am. Chem. Soc. 117, 6416-6417]. UV melting experiments show that these Hoogsteen hairpin duplexes have a lower thermal stability than that of the Watson-Crick (WC) hairpin duplex (antiparallel) of the same sequence. The circular dichroism (CD) spectrum of the Hoogsteen duplex is different from the canonical B-DNA WC duplex spectrum. The formation of the Hoogsteen duplex is pH-dependent since protonation of cytosine requires lower pH conditions. Studies with oligonucleotides of different loop sizes revel that three- and two-base loops are optimum for the formation of stable Hoogsteen duplexes with 3'-3' and 5'-5' linkages, respectively. The guanine residues in the loop stabilize the duplex as a result of G-G interactions as confirmed by molecular modeling studies. The new PS Hoogsteen duplexes form stable triplexes with complementary (antiparallel to the purine domain) single-stranded RNA and DNA pyrimidine sequences in Py.Pu:Py (pyrimidine third strand-purine WC strand:pyrimidine WC strand) motif. The thermal stability of the resulting triplexes is much higher than that of the conventional triplex (binding of a Hoogsteen pyrimidine third strand to a WC duplex) of the same sequence. The CD spectra of the new triplexes are similar to those of conventional triplexes, suggesting that no conformational change occurs as a result of 3'-3' or 5'-5' linkage. A molecular modeling study was carried out to examine the stereochemical feasibility of the Hoogsteen duplexes and formation of triplexes with single-stranded pyrimidine complementary strands.

Circular Dichroism↗
PubMed1996

Modulation of oligonucleotide-induced immune stimulation by cyclodextrin analogs.

Some phosphorothioate oligonucleotides have been shown previously to stimulate cell proliferation and immunoglobulin production. In the current study, we examined the effects of cyclodextrin analogs as immunomodulatory agents for oligonucleotide-induced immune stimulation, both in vitro and in vivo. Incubation of splenocytes with a 27-mer phosphorothioate oligonucleotide that induces immune stimulation increased cell proliferation as measured by [3H]thymidine incorporation, whereas treatment of splenocytes with the phosphorothioate oligonucleotide complexed to cyclodextrin analogs markedly reduced oligonucleotide-induced cell proliferation. Similarly, administration of the 27-mer phosphorothioate oligonucleotide into mice resulted in splenomegaly and an increase in IgM production 48 hr post-administration. Administration of the oligonucleotide along with cyclodextrin analogs resulted in a significant suppression of splenomegaly and IgM response. Such suppression was dependent on the concentration of cyclodextrin analogs and was observed with various other immune stimulatory phosphorothioate oligonucleotide sequences. Administration of cyclodextrin analogs alone had no effect on splenomegaly or immune stimulation.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1996

Repair of thalassemic human beta-globin mRNA in mammalian cells by antisense oligonucleotides.

In one form of beta-thalassemia, a genetic blood disorder, a mutation in intron 2 of the beta-globin gene (IVS2-654) causes aberrant splicing of beta-globin pre-mRNA and, consequently, beta-globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human beta-globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human beta-globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

3T3 Cells↗
PubMed1996

Effect of different chemically modified oligodeoxynucleotides on immune stimulation.

Based on previous studies that certain oligonucleotides can stimulate cell proliferation and immunoglobulin production, this study was carried out to establish the relationship between the stimulatory effect and the chemical modification of the oligonucleotide. First, the effects of oligonucleotide and analogs on immune stimulation were studied in vitro using murine splenic lymphocytes. Our results show that cell proliferation and immunoglobulin production (IgG and IgM) depend on the sequence and the chemical modification of the oligonucleotide. Phosphorothioate oligodeoxynucleotides displayed a greater stimulatory effect than partially modified phosphorothioate oligonucleotides. Second, we studied the effects of these chemically modified oligonucleotides after injection in mice. Massive splenomegaly and stimulation of cell proliferation were observed with some phosphorothioate oligonucleotides. These effects were minimized markedly by chimeric and hybrid oligonucleotides. We also demonstrate that in vitro the effects of oligonucleotides on murine lymphocytes were unaffected by T cell depletion, suggesting that oligonucleotides exert their effects mainly on the B cells.

Adjuvants, Immunologic↗
PubMed1996

Intrathoracic anastomosis after oesophageal resection for cancer.

Cervical anastomosis has been advocated to avoid the pulmonary complications and life-threatening anastomotic disruptions following intrathoracic oesophagogastric anastomosis. This is a retrospective review of 111 oesophageal resections followed by an intrathoracic anastomosis. These resections were performed between September 1993 and August 1994 within a residency training program. The left thoracoabdominal approach was used for distal tumours and the Ivor Lewis technique for more proximal tumours. Squamous cell carcinoma accounted for 72% patients (n = 80), adenocarcinoma for 25% (n = 28), and others for 2.7% patients (n = 3). Of the patients, 69% had pathologic Stage III tumours. Operative mortality rate was 1.8% (two patients). Perioperative complications occurred in 39 patients, including anastomotic leak in 10 patients and myocardial infarction in 2 patients. In the absence of a leak, there were no major pulmonary complications requiring intensive care or ventilatory support. Of those patients with anastomotic disruption, 80% were salvaged by early clinical diagnosis and appropriate treatment. We conclude that transthoracic oesophagectomy with an intrathoracic anastomosis is a safe procedure that can be performed with low mortality and acceptable morbidity.

Adenocarcinoma↗
PubMed1996

Histologic trends in thyroid cancer 1969-1993: a clinico-pathologic analysis of the relative proportion of anaplastic carcinoma of the thyroid.

BACKGROUND: It was observed that new presentations of anaplastic carcinoma of the thyroid had become infrequent in the last two decades. METHODS: All cases of thyroid cancer seen at our centre between 1969-1993 (n = 2921) were classified as papillary 49%, follicular 34%, medullary 7.5%, anaplastic 4.7%, and other 4.8%. The total number of thyroid cancers show a 3.5-fold rise. RESULTS: The differentiated thyroid cancers show a significant rising trend as against the relative proportion of anaplastic carcinoma, which shows a significant decline (P = 0.002). Clinicopathologic data on 124 patients of anaplastic carcinoma revealed 50% patients had either long-standing goitres, previous thyroid abnormalities, or associated differentiated thyroid carcinoma on histology. CONCLUSIONS: The decline in the relative proportion of anaplastic carcinoma may in part be explained by the clinicopathologic findings or it may be attributed to histological reclassification.

Adult↗