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Biomedical subjects

R Raju

Publications and source records attributed to R Raju.

At least 19 recordsLinked to original sources
PubMed2001

Acetylcholine receptor peptide recognition in HLA DR3-transgenic mice: in vivo responses correlate with MHC-peptide binding.

HLA DR3 is an MHC molecule that reportedly predisposes humans to myasthenia gravis (MG). Though MG is an Ab-mediated autoimmune disease, CD4+ T cells are essential for the generation of high-affinity Abs; hence the specificities of autoreactive CD4+ T cells are important. In this study we report the HLA DR3-restricted T cell determinants on the extracellular region sequence of human acetylcholine receptor alpha subunit. We find two promiscuous determinants on this region 141-160 and 171-190 as defined by their immunogenicity in HLA DR3-, HLA DQ8-, and HLA DQ6-transgenic mice in the absence of endogenous mouse class II molecules. We also studied the minimal determinants of these two regions by truncation analysis, and the MHC binding affinity of a set of overlapping peptides spanning the complete sequence region of human acetylcholine receptor alpha subunit. One of the peptide sequences strongly immunogenic in HLA DR3-transgenic mice also had the highest binding affinity to HLA DR3. Identification of T cell determinants restricted to an MHC molecule known to predispose to MG may be an important step toward the development of peptide-based immunomodulation strategies for this autoimmune disease.

Amino Acid Sequence↗
PubMed2001

Glove reinforcement: an alternative to double gloving.

Gloves, worn by the surgical team to prevent transmission of infections from and to patients, are prone to tears and perforations. This study was done to determine the frequency and sites of unrecognized glove perforation during surgical procedures. The percentage of glove perforation was 14%. Of the punctures, 73% occurred in one of four contiguous locations on the glove. We recommend glove reinforcement at these locations to provide better protection, as well as to reduce the burden of double gloving.

Cross Infection↗
PubMed2001

Sequence requirements for Sindbis virus subgenomic mRNA promoter function in cultured cells.

The Sindbis virus minimal subgenomic mRNA promoter (spanning positions -19 to +5 relative to the subgenomic mRNA start site) is approximately three- to sixfold less active than the fully active -98 to +14 promoter region. We identified two elements flanking the -19 to +5 region which increase its transcription to levels comparable to the -98 to +14 region. These elements span positions -40 to -20 and +6 to +14 and act synergistically to enhance transcription. Nine different virus libraries were constructed containing blocks of five randomized nucleotides at various positions in the -40 to +14 region. On passaging these libraries in mosquito cells, a small subset of the viruses came to dominate the population. Sequence analysis at the population level and for individual clones revealed that in general, wild-type bases were preferred for positions -15 to +5 of the minimal promoter. Base mutagenesis experiments indicated that the selection of wild-type bases in this region was primarily due to requirements for subgenomic mRNA transcription. Outside of the minimal promoter, the -35 to -29 region contained four positions which also preferred wildtype bases. However, the remaining positions generally preferred non-wild-type bases. On passaging of the virus libraries on hamster cells, the -15 to +5 region again preferred the wild-type base but most of the remaining positions exhibited almost no base preference. The promoter thus consists of an essential central region from -15 to +5 and discrete flanking sites that render it fully active, depending on the host environment.

Amino Acid Sequence↗
PubMed2001

Interleukin-1, interleukin-2 and interleukin-10 expression in different techniques of saphenous vein harvesting.

BACKGROUND: Levels of the cytokines Interleukin-1 (IL-1), IL-2, and IL-10 are sensitive to the traumatic effect of saphenous vein harvesting. Their levels are compared between the endoscopic and traditional open techniques of harvesting. METHODS: Samples of human saphenous veins were harvested from 90 randomly selected patients undergoing coronary artery bypass surgery (CABG), using the open or endoscopic techniques. Endothelial cells collected from the vein samples retrieved through both techniques were cultured for 72 hours. Pre and postoperative sera, in addition to the supernatants from the cultures, were analyzed for IL-1, IL-2, and IL-10 using ELISA. RESULTS: Mean preoperative concentrations of IL-1, Il-2, and IL-10 were 0.11+/- 0.04, 0.09 +/- 0.04, and 0.09 +/- 0.04 pg/ml, respectively. Corresponding values for postoperative sera were 0.13 +/- 0.08, 0.12 +/- 0.10, 0.14 +/- 0.17 pg/ml, respectively. The differences between pre and postoperative means for each cytokine were not statistically significant (p = 0.13, 0.18, 0.05, respectively). Mean IL-1, IL-2, and IL-10 concentrations for endothelial cell culture supernatants did not differ significantly between the endoscopic (0.17 +/- 0.11, 0.11 +/- 0.05, and 0.32 +/- 0.40 pg/ml, respectively) and the open method (0.19 +/- 0.16, 0.11 +/- 0.05, and 0.46 +/- 0.80 pg/ml, respectively) (p = 0.48, 0.81, 0.30, respectively). CONCLUSION: Since endoscopic and open saphenectomies are technically comparable with respect to their effects on IL-1, Il-2, and IL-10 levels, we recommend the endoscopic method for its lower morbidity and the potential for earlier hospital discharge.

Analysis of Variance↗
PubMed2001

A cytokine analysis of the effect of cell saver on blood in coronary bypass surgery.

BACKGROUND: Increasing concern about the transmission of viral disease has generated greater interest in the use of salvaged blood as a means of alleviating the demand for homologous blood and expediting resuscitation during massive hemorrhage. Autologous blood processed by autotransfusion devices has become increasingly common in major surgery and is now largely viewed as safe and efficacious. However, there may be serious complications and sequelae associated with the use of processed blood, such as adult respiratory distress syndrome (ARDS) and renal failure. Complement cascade activation resulting from blood coming into contact with autotransfusion equipment leads to enrollment of leukocytes and release of large concentrations of cytokines, which may contribute to the development of organ failure. Our study evaluated cytokine release during cell saver (CS) blood salvage in the course of coronary artery bypass grafting (CABG) surgery. MATERIALS AND METHODS: Forty-five patients randomly selected for CABG were evaluated. All had received at least one unit of autotransfused blood by means of the Haemonetics Cell Saver System 5 (Haemonetics Corp., Braintree, MA). Each patient had four blood samples taken (pre-operative, CS container, autotransfusion from the blood bag, and one hour post-transfusion). These samples were then centrifuged and the sera were collected. An enzyme linked immunosorbent assay (ELISA) test, using the Biosource Cytoscreen solid phase "sandwich" ELISA kit (Biosource International, Camarillo, CA) was conducted to determine levels of the cytokines Interleukin (IL) 1, 2, 4, 6, 8, and 10, tumor necrosis factor (TNF), intracellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM). RESULTS: Significantly increased concentrations of the pro-inflammatory cytokines IL-1, 2, 4, 6, and 8, TNF, ICAM, and VCAM were noted throughout all time periods studied. The same effect was observed for the anti-inflammatory cytokine IL-10. CONCLUSION: Statistically significant increases in both the circulating levels of the pro-inflammatory and anti-inflammatory cytokines studied were recorded. It is our contention that the presence of IL-10, a down-regulator of inflammation, is responsible for attenuating the possible deleterious effects of the pro-inflammatory cytokines observed. However, morbidity and mortality, as well as the future patency of the bypass grafts, have not been correlated with the use of the autologous method of transfusion.

Adult↗
PubMed2001

A comparative study of endothelial cell injury during open and endoscopic saphenectomy: an electron microscopic evaluation.

BACKGROUND: The role of damaged endothelium in early graft occlusion has been extensively demonstrated. Seventy percent of early graft occlusions in coronary artery bypass were caused by thrombi overlying areas of endothelial loss. With the increased use of endoscopic vein harvesting, it becomes important to study the extent of endothelial damage by light and electron microscopy. In this study, we compared the degree of endothelial damage inflicted by the open and endoscopic techniques of vein harvesting using light, scanning, and transmission electron microscope. MATERIAL AND METHODS: Ninety samples of saphenous veins from 45 patients prepared for coronary artery bypass grafting (CABG) utilizing both endoscopic and standard open incision techniques were examined using light, scanning, and transmission electron microscopy. These vein samples were prepared in Plasma-lyte solution (Baxter) in combination with or without papaverine, at two distending pressures of 100 or 300 mmHg and at temperatures of either 4 degrees C or 28 degrees C in eight subgroups and one control group. The pathological alterations in the saphenous veins were graded either based on a scoring system (0 = none, 1 = < 10%, 2 = 10-25%, 3 = 25-50%, 4 = > 50%) to assess the degree of damage inflicted by these two different types of saphenectomies or by electron microscopic observed abnormalities, including endothelial cell (EC) separation, EC detachment, basement membrane (BM) exposure, collagen exposure, and EC edema. RESULTS: Using cross-tabulation and Chi-square statistical analysis, we found that the differences in the degree of endothelial damage using either of the techniques is not statistically significant (P > 0.05). CONCLUSION: Our findings indicate that endoscopic and open saphenectomies are technically comparable as far as structural damage is concerned, rendering the endoscopic technique of vein handling the preferred method for CABG.

Chi-Square Distribution↗
PubMed2000

Alphavirus RNA genome repair and evolution: molecular characterization of infectious sindbis virus isolates lacking a known conserved motif at the 3' end of the genome.

The 3' nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3'CSE). The 3'CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3'CSEs were studied in detail to evaluate the role of the 3'CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3'CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3' terminal motifs of the SIN genome; iii) the 5' 1-kb regions of all SIN genomes, regardless of the differences in 3' terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3'CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3' terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3'CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.

3' Untranslated Regions↗
PubMed2000

Intercellular and vascular cell adhesion molecule levels in endoscopic and open saphenous vein harvesting for coronary artery bypass surgery.

BACKGROUND: Numbers of intercellular and vascular cell adhesion molecules (ICAM and VCAM) and major ligands on endothelial cells for adherence of activated polymorphnuclear leukocytes, macrophages, and lymphoid cells increase in many inflammatory disorders and after trauma to different tissues. METHODS: Samples of human saphenous veins were harvested from 90 randomly selected patients who underwent coronary artery bypass graft (CABG) surgery, utilizing two different techniques (open and endoscopic). Endothelial cells were collected from the vein samples and cultured for 72 hours. Pre- and postoperative sera, in addition to the supernatants from the cultures, were analyzed for ICAM-1 and VCAM-1 using enzyme-linked immunosorbent assay. RESULTS: Mean preoperative levels of ICAM-1 and VCAM-1 (0.95 +/- 0.58 ng/mL and 1.81 +/- 1.03 ng/mL, respectively) did not differ significantly from that of postoperative sera (0.98 +/- 0.451 ng/mL and 1.74 +/- 1.05 ng/mL, respectively) (p = 0.77 and p = 0.73, respectively). Mean ICAM-1 and VCAM-1 levels in endothelial cell culture supernatants did not differ significantly between the endoscopic (0.16 +/- 0.05 ng/mL and 0.23 +/- 0.10 ng/mL, respectively) and the open method (0.18 +/- 0.08 ng/mL and 0.30 +/- 0.27 ng/mL, respectively) (p = 0.19 and 0.13, respectively). CONCLUSION: Our findings indicate that endoscopic and open saphenectomies are technically comparable in their effects on ICAM-1 and VCAM-1 synthesis during saphenous vein harvesting for CABG. We recommend the endoscopic method for its low morbidity and earlier hospital discharge.

Angioscopy↗
PubMed1999

In vivo addition of poly(A) tail and AU-rich sequences to the 3' terminus of the Sindbis virus RNA genome: a novel 3'-end repair pathway.

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5' end and polyadenylated at its 3' end. The 3' nontranslated region (3'NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3'CSE) and a poly(A) tail. This 3'CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693-2704, 1997). Here, we demonstrate that the 3'-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3'NTR, we generated a battery of mutant genomes with mutations in the 3'NTR and tested their ability to generate infectious virus and undergo 3' polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3'CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3'CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3'NTR. These results indicate the ability of alphavirus RNAs to undergo 3' repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3' end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.

3' Untranslated Regions↗
PubMed1998

Polymorphism at the HLA-DQ locus determines susceptibility to experimental autoimmune myasthenia gravis.

Studies in myasthenia gravis (MG) patients demonstrate that polymorphism at the HLA-DQ locus influences the development of MG. Several studies using the mouse models also demonstrate the influence of class II molecules, especially the H2-A, which is the mouse homologue of HLA-DQ, in experimental autoimmune myasthenia gravis (EAMG). We used transgenic mice expressing two different DQ molecules, DQ8 (DQA1*0301/B1*0302) and DQ6 (DQA1*0103/B1*0601), to evaluate the role of HLA-DQ genes in MG. These mice do not express endogenous mouse class II molecules since they contain the mutant H2-A beta0 gene. The mice were immunized with Torpedo acetylcholine receptor, and EAMG was assessed by clinical evaluation and was confirmed by electrophysiology. Clinical scores for EAMG were highest in HLA-DQ8 transgenic mice, whereas the scores of HLA-DQ6 mice rarely exceeded grade 1. There was no incidence of EAMG in class II-deficient (H2-A beta0) mice. These results demonstrate that polymorphism at the HLA-DQ locus affects the incidence and the severity of EAMG. The manifestation of susceptibility to EAMG in the context of human class II molecules underscores the important roles of these molecules in the initiation and perpetuation of EAMG.

Animals↗
PubMed1997

TCR V beta usage by acetylcholine receptor-specific CD4+ T cells in myasthenia gravis.

In myasthenia gravis the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. AChR epitopes recognized by CD4+ T cells in myasthenic patients have been identified. AChR-specific CD4+ cell lines can be propagated by stimulation of blood lymphocytes with synthetic or biosynthetic AChR sequences. We analysed, using a semi-quantitative PCR assay, the T cell receptor (TCR) V beta usage of 16 anti-AChR polyclonal CD4+ T cell lines of known epitope specificity, propagated from myasthenic patients using pools of overlapping peptides corresponding to the sequence of an AChR subunit, or individual synthetic AChR sequences. Twelve lines had been propagated for less than 2 months, four lines for 3.5-5 months. Most lines had limited V beta usage, but in most cases different V beta regions were used for different epitopes in the same patient, and for the same epitope in different patients. In a few patients, the same V beta regions were used for recognition of different epitopes. The V beta 4 and V beta 6 regions were used most frequently. These findings suggest that the potentially autoimmune T cells that survive clonal deletion have a limited TCR repertoire. Although the present data do allow conclusions on the role of a superantigen in triggering the anti-AChR autoimmune response, the finding that different V beta regions were used in different patients does not support an important role of a superantigen in the maintenance of the CD4+ response in myasthenia gravis.

Amino Acid Sequence↗
PubMed1997

TCR vbeta usage of TSH receptor-specific CD4+ T cells in Graves' disease patients and healthy humans.

Healthy humans have CD4+ T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+ cells present in healthy persons. We studied CD4+ cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves' disease (GD) patients. We used short-term CD4+ cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject. We determined their TCR Vbeta usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vbeta region family, in conjunction with a constant region primer. Six lines preferentially used one Vbeta family (42-94%), different for each line. In all lines, three or less Vbeta families accounted for approximately 60% or more of the Vbeta usage. Different Vbeta regions were used by each subject. There was no obvious difference between the Vbeta usage of the lines from GD patients and healthy controls. These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4+ cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4+ cells specific for the same autoantigen in healthy subjects.

Adult↗
PubMed1997

T cell recognition of human pre-proinsulin peptides depends on the polymorphism at HLA DQ locus: a study using HLA DQ8 and DQ6 transgenic mice.

HLA DQ8 (DQ A1*0301/DQB1*0302) molecule is implicated in the susceptibility to insulin dependent diabetes mellitus whereas, HLA DQ6 (DQ A1*0103/DQB1*0601) molecule may have a protective effect. In this study we used mice transgenic to HLA DQ8 and HLA-DQ6 to elucidate the T cell determinants on a putative islet cell target antigen, insulin. These mice do not express endogenous mouse class II heterodimers on cell surface. Using overlapping synthetic peptides spanning the complete sequence of huma pre-proinsulin, we identified the sequences recognized by T cells in DQ8 transgenic mice and compared these to those in DQ6 transgenic mice. We observed a differential pattern of recognition of epitopes on human pre-proinsulin (HPI) polypeptide presented by the HLA DQ8 allele as compared to HLA DQ6. The sequences 1-24 and 44-63 were immunodominant in DQ8 transgenic mice while DQ6 transgenic mice primarily recognized sequences 14-33 and 74-93 of HPI. We found that the immune response generated in HLA DQ8 transgenic mice against HPI 1-24 cross-reacted to the mouse pre-proinsulin sequence 1-24. The T cell response were specifically inhibited using anti-CD4 and anti-DQ8 monoclonal antibodies. This cross-recognition of self sequences raises the possibility of modulation of experimental diabetes using this peptide.

Amino Acid Sequence↗