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Biomedical subjects

S Sundaram

Publications and source records attributed to S Sundaram.

At least 73 records · Page 4Linked to original sources
PubMed1991

Modification of the influence of biomaterials on contact activation.

Knowledge of the influence of biomaterials on the contact phase activation of blood is important. Consequently, it is important to examine if the biomaterial influence is modified by the action of anticoagulants. Contact activation was determined by measuring FXII activity (FXIIA) by a chromogenic substrate assay. Cuprophan in the absence of anticoagulants and in the presence of heparin, low molecular weight heparin (LMWH) and hirudin was compared with controls. The controls were a polystyrene incubation test cell (PS) and two polyamide membranes, NR (zeta potential -28.6 mv and NRZ (zeta potential--18.0 mv). The investigation has confirmed that measurement of FXIIA is material dependent and demonstrated that contact activation can be mediated by antithrombotic agent.

Anticoagulants↗
PubMed1990

Counseling strategies for blood cholesterol screening programs: recommendations for practice.

Blood cholesterol screening programs offer an important venue for nutrition counseling aimed at lowering blood cholesterol levels. This paper presents a screening, counseling and referral protocol that has been developed by the Pawtucket Heart Health Program (PHHP). A review of general considerations for counseling strategies in the screening context highlights the necessity for brief, focused, behaviorially oriented tactics. The PHHP Summary and Referral protocol includes a review of multiple risk factors for heart disease, a discussion of blood cholesterol levels, recommendations for eating pattern changes based on response to a food frequency questionnaire, and a final summary and referral as needed. The training of lay volunteers and health professionals in delivery of the program is also reviewed.

Adult↗
PubMed1989

The Lec4A CHO glycosylation mutant arises from miscompartmentalization of a Golgi glycosyltransferase.

Two CHO glycosylation mutants that were previously shown to lack N-linked carbohydrates with GlcNAc beta 1,6Man alpha 1,6 branches, and to belong to the same genetic complementation group, are shown here to differ in the activity of N-acetylglucosaminyltransferase V (GlcNAc-TV) (UDP-GlcNA: alpha 1,6mannose beta-N-acetylglucosaminyltransferase V). One mutant, Lec4, has no detectable GlcNAc-TV activity whereas the other, now termed Lec4A, has activity equivalent to that of parental CHO in detergent cell extracts. However, Lec4A GlcNAc-TV can be distinguished from CHO GlcNAc-TV on the basis of its increased sensitivity to heat inactivation and its altered subcellular compartmentalization. Sucrose density gradient fractionation shows that the major portion of GlcNAc-TV from Lec4A cells cofractionates with membranes of the ER instead of Golgi membranes where GlcNAc-TV is localized in parental CHO cells. Other experiments show that Lec4A GlcNAc-TV is not concentrated in lysosomes, or in a post-Golgi compartment, or at the cell surface. The altered localization in Lec4A cells is specific for GlcNAc-TV because two other Lec4A Golgi transferases cofractionate at the density of Golgi membranes. The combined data suggest that both lec4 and lec4A mutations affect the structural gene for GlcNAc-TV, causing either the loss of GlcNAc-TV activity (lec4) or its miscompartmentalization (lec4A). The identification of the Lec4A defect indicates that appropriate screening of different glycosylation-defective mutants should enable the isolation of other mammalian cell trafficking mutants.

Animals↗
PubMed1984

Transferable plasmid-mediated drug resistance among non-O1 Vibrio cholerae and rough strains of Vibrio cholerae from Tamilnadu, India.

A total of 289 non-O1 Vibrio cholerae (NVC) strains and 20 rough V. cholerae (RVC) strains isolated in an endemic area were tested for antibiotic resistance and for transferable R-plasmids. Twenty three per cent of NVC and 40% of the RVC isolates were found to be resistant to one or more drugs. Eight NVC and four RVC strains possessed multiple drug resistance, varying from four to eight drugs. The common spectrum found in NVC isolates were chloramphenicol and streptomycin (CS) or chloramphenicol, streptomycin, tetracycline and ampicillin (CSTA). Resistance to sulphamethoxazole (Su) and to trimethoprim (Tm) was encountered infrequently. In RVC isolates in addition CSTASuTm determinants, resistance markers to aminoglycosides kanamycin, gentamicin and neomycin were also found. Eighteen of the 27 V. cholerae strains with two or more resistance determinants transferred them en bloc to Escherichia coli K12. The level of resistance in the recipient strain was equal to or greater than that of the donor vibrio strains. Most of the strains possessing solitary resistance markers were unable to transfer them. beta-lactamase production could be demonstrated in 92.8% of the ampicillin resistant strains. None of the strains was resistant to nalidixic acid or furazolidone. The results emphasize the importance of antimicrobic susceptibility determination of V. cholerae isolates, regardless of the serotypes, before commencing chemotherapy.

Anti-Bacterial Agents↗
PubMed1981

Serological diagnosis of pulmonary aspergillosis.

Methods for the preparation of antigens from clinically isolated cultures of Aspergillus were standardized. Sera from 25 suspected cases of pulmonary aspergillosis were tested against antigens prepared by us, from 4 strains of A. fumigatus and one strain of A. flavus, using the Ouchterlony double diffusion and immunoelectrophoretic techniques. Of the 25 sera, 18 reacted positively with antigens of A. fumigatus, one with A. flavus and 2 with both these species. Antigens of two non-pathogenic Aspergilli included in the study failed to react with any of the sera. Our antigen preparations gave more numerous as well as sharper precipitin lines than the commercial Bencard antigens which were used for comparison. Moreover, mycelial antigens from 48 to 96 h old cultures revealed precipitin lines comparable to that of the routine, 4 week old culture filtrate antigens, thus suggesting that the incubation period for obtaining antigens could be cut down considerably.

Antigens, Fungal↗
PubMedDate not supplied

Population data on the forensic genetic markers: phosphoglucomutase-1, esterase D, erythrocyte acid phosphatase and glyoxylase I.

Blood specimens from white and black sample populations from Baltimore, Maryland, were analyzed for the four most forensically important, polymorphic red cell enzyme systems-phosphoglucomutase-1, esterase D, erythrocyte acid phosphatase and glyoxalase I. The distributions of the phenotypes for each marker in each racial group were in Hardy-Weinberg equilibrium. The population data were similar to previously reported data for Whites and Blacks from different geographical locations within the United States.

Acid Phosphatase↗
PubMedDate not supplied

Immunoadsorption: strategies for antigen elution and production of reusable adsorbents.

Immunoadsorption is a powerful and generalizable method for protein purification that exploits the fine specificity of antigen-antibody interactions. In spite of its potential utility, the more widespread process scale use of immunoadsorption has been limited by the high cost of the antibody and the lack of gentle elution schemes that completely preserve the activity of both the immunoadsorbent and the eluted product. In this report, we review common chemical elution strategies such as pH, ionic strength, chaotropic salts, denaturants, and organic solvents as well as physical techniques such as pressure, electrokinetics, and temperature. In general, selection of elution strategies has largely been an empirical art, balancing stability of the immunoadsorbent and the eluted product and efficiency. The limitations of the available choices demonstrate that more attention must be placed on the antibody. Techniques which assist in the identification or creation of new antibodies with improved binding properties and resistance to degradation, e.g., screening and/or rational protein engineering, are also discussed.

Antigens↗
PubMedDate not supplied

Pressure-induced dissociation of antigen-antibody complexes.

Pressures on the order of 1000-4000 bar have been reported to reversibly dissociate a number of oligomeric protein complexes without gross changes in protein structure. Here, we report that hydrostatic pressure can also dissociate some antigen-antibody complexes in solution. The association of fluorescent-labeled antigens with monoclonal antibodies was monitored via increases in the fluorescence anisotropy upon binding. Previously, we had found that pressures of 2000 atm were able to dissociate bovine serum albumin (BSA) from immunoadsorbents formed from certain antibodies but not others. In this study, we have found that the sensitivity to pressure in solution is present for the interaction of BSA with MAb 9.1 and absent for the interaction of BSA with MAb 6.1; this behavior is consistent with the immunoadsorbent study. The interaction of hen egg white lysozyme with two monoclonal antibodies was also measured. Interestingly, the complex with the greater electrostatic character (HyHEL-5) did not exhibit pressure sensitivity, as would be expected due to electrostriction effects, whereas the more hydrophobic complex (HyHEL-10) exhibited a strong pressure sensitivity. In each of the systems displaying pressure sensitivity, the free energy of association was found to increase linearly with pressure, indicating a constant change in volume between the free and bound states. Overall, these results indicate that some antigen-antibody complexes exhibit significant sensitivity to pressure, whereas others do not; the mechanisms that discriminate between these cases remain unresolved. Understanding and manipulation of this phenomenon may prove useful in a variety of processes involving the recovery from antigens of antibodies.

Animals↗
PubMedDate not supplied

Removal of cytokine inducing substances by polymyxin-B immobilized polystyrene-derivative fibers during in vitro hemoperfusion of 10% human plasma containing Staphylococcus aureus challenge.

Staphylococcus aureus (S. aureus) is frequently isolated from blood cultures in the hospital setting. The pathogenesis of S. aureus bacteremia probably replicates mechanisms implicated in gram negative bacterial infections. Cell wall components, such as peptidoglycans and lipoteichoic acids (LTA), can trigger cytokine production. Polymyxin-B (PMX-B) is a cationic peptide that binds endotoxin (ET) and inhibits its activity. Based on this principle, PMX-B was incorporated in polystyrene-derivative fibers, creating a hemoperfusion column (PMX-20R) that removes ET. The authors assessed whether S. aureus possesses PMX-B suppressible cytokine-inducing substances, and whether LTA, an anionic molecule, is one such substance. Heparinized blood was obtained from healthy volunteers, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque separation, and 10% human plasma prepared. PBMC were incubated with 1, 5, or 10 microg/ml of S. aureus LTA, with and without 10 microg/ml of PMX-B. Also, using PMX-20R, in vitro hemoperfusion (IVH) was performed with 10% human plasma containing a 1:1,000 dilution of S. aureus challenge at 100 ml/min for 2 hours at 37 degrees C, and plasma obtained before and after IVH was incubated with PBMC. After a 24 hour incubation at 37 degrees C, PBMC were subjected to three freeze-thaw cycles, and total TNFalpha was measured by radioimmunoassay. TNFalpha production by PBMC incubated with LTA was 164+/-4 pg, 324+/-54 pg, 657+/-55 pg, and 1143+/-215 pg in control, and LTA 1, 5, and 10 microg/ml, respectively. The addition of PMX-B resulted in a 40+/-12% (p = 0.02), 61+/-6% (p = 0.002), and 62+/-14% (p = 0.02) decrease in TNFalpha production, respectively. Before IVH, TNFalpha production by PBMC incubated with 10% plasma containing S. aureus challenge was 1275+/-70 pg. After 2 hours of IVH, the decrease in TNFalpha production was 20+/-4% (p = 0.002). In conclusion, S. aureus LTA induces TNFalpha production that is significantly suppressed by PMX-B. Consequently, S. aureus cytokine-inducing substances are removed during IVH with PMX-20R, and this may be due to stoichiometric binding of LTA to PMX-B.

Anti-Bacterial Agents↗
PubMedDate not supplied

Endotoxin removal by polymyxin-B immobilized polystyrene-derivative fibers during in vitro hemoperfusion of 10% human plasma.

During gram-negative bacterial sepsis, lipid A, the biologically active moiety of endotoxin (ET), activates monocytes and induces the release of cytokines. PMX-B, a cationic peptide, binds to lipid A and inhibits its activity. Based on this principle, PMX-B was incorporated in polystyrene-derivative fibers, creating a hemoperfusion column (PMX-20R) that removes ET. After in vitro characterization of the cytokine inducing potency of three gram-negative bacterial challenges, the authors evaluated the in vitro efficacy of PMX-20R in a model using 10% human plasma. Cytokine production by peripheral blood mononuclear cells (PBMC) incubated with plasma before and after in vitro hemoperfusion (IVH) was used as the index of ET removal. One hundred forty milliliters of heparinized blood were obtained from healthy volunteers. Forty milliliters were used to harvest PBMC at baseline, and 10% plasma prepared from the rest, was challenged with: 1) 0.01, 1, or 100 ng/ml of purified Escherichia coli ET; or 2) 1:1,000 dilution of E. coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae. IVH was performed at 100 ml/min at 37 degrees C for up to 6 hours. One half milliliter samples, drawn before and at designated time intervals after the start of IVH, were mixed with a 0.5 ml suspension of 5 x 10(6) PBMC/ml from the same donor, and incubated for 24 hours at 37 degrees C. PBMC were subjected to three freeze-thaw cycles, and total tumor necrosis factor alpha (TNFalpha) was measured by radioimmunoassay. Before IVH, TNFalpha production by PBMC incubated with 10% plasma containing 0.01, 1, or 100 ng/ml of purified E. coli ET was 1905+/-391 pg, 2076+/-552 pg, and 5304+/-1001 pg, respectively. After 2 hours of IVH, the respective decrease in TNFalpha production was 82+/-5% (p = 0.005), 78+/-10% (p = 0.01), and 95+/-1% (p = 0.002). Before IVH, TNFalpha production by PBMC incubated with 10% plasma containing 1:1,000 dilution of E. coli, P. aeruginosa or K. pneumoniae was 2896+/-273 pg, 1816+/-122 pg, and 1131+/-125 pg, respectively. After 2 hours of IVH, the respective decrease in TNFalpha production was 83+/-4% (p < 0.001), 53+/-4% (p < 0.001), and 70+/-5% (p < 0.001). When IVH was extended to 6 hours, the further decrease in TNFalpha production was not statistically significant. These results suggest an impressive in vitro removal of ET by PMX-20R from 10% human plasma containing either purified E. coli ET or E. coli, P. aeruginosa, or K. pneumoniae. Further in vitro studies are required, using whole blood challenged with gram-negative bacteria.

Anti-Bacterial Agents↗
PubMedDate not supplied

Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes.

In this report, we present results of a recent investigation in our laboratories demonstrated the effect of process conditions and/or drug product composition on the ability of 0.2 micron and 0.22 micron sterilizing grade filters to fully retain Ralstonia (formerly Burkholderia, formerly Pseudomonas) pickettii. R. pickettii is a opportunistic pathogen widely distributed in nature as well as clinical specimens and there have been several reports of nosocomial infections due to intrinsic manufacture-related R. pickettii contamination in filter-sterilized parenteral fluids. This study documents the penetration of 0.2 micron nylon 66 and 0.22 micron modified PVDF sterilizing grade filters by R. pickettii (grown and challenged) in a drug solution under conditions that simulated a pharmaceutical filling operation. Penetration was not observed for every filter disc tested, and this may be explained, in part, by the stochastic nature (i.e., governed by the rules of probability) of the retention mechanisms involved. Scanning electron microscopy revealed significant changes in the microorganism's size and morphology as a result of exposure to the drug solution; these changes are consistent with those reported for bacteria subjected to nutrient deprivation. The SEM analyses of R. pickettii challenge suspensions in the drug solution showed that the average cell length decreased from 1.25 +/- 0.27 microns to 0.84 +/- 0.17 micron between zero and 24 hours. In addition, significant changes were observed in the size (length) distributions, with approximately 35% of the cells at 24 hours being smaller than any cell observed at the start of the challenge. These data suggest that the significant reduction in bioburden size and morphology that occurred as a result of exposure to the drug solution may play a role in the reduced ability of the 0.2 micron and 0.22 micron filters tested in this study to retain these organisms. Under the same test conditions where penetration of 0.2/0.22 micron filters was observed, 0.1 micron rated membrane filters qualified with both B. diminuta and Acholeplasma laidlawii mycoplasma consistently provided sterile effluent. Bacterial penetration of 0.2 (or 0.22) micron sterilizing grade filters was not observed under identical test conditions with either R. pickettii in a standardized solution (saline lactose broth) routinely used in challenge testing filters, or with the standard test organism, B. diminuta, in the drug solution. This study thus supports the renewed emphasis on both product- and process specific validation as well as routine bioburden monitoring expressed by regulatory agencies, and the use of enhanced bacterial removal efficiency 0.1 micron rated filters to provide enhanced sterility assurance in pharmaceutical processes.

Bacteria↗
PubMedDate not supplied

Retention of water-borne bacteria by membrane filters. Part III: Bacterial challenge tests on 0.1 micron rated filters.

Clear performance differences were observed between different 0.1 micron rated filters in terms of their microbial removal efficiency when challenged with naturally occurring waterborne bacteria from a water source. Penetration occurred with three 0.1 micron rated "sterilizing grade" filter types tested, from three different filter manufacturers, that did not have a specific high titer reduction claim for Acholeplasma laidlawii. Bacteria shown to penetrate these 0.1 micron rated filters were quite similar to those recovered downstream of 0.2.0.22 micron rated filters (described in Part II). All of the isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. In contrast, four different 0.1 micron rated "sterilizing grade" filter types from two different manufacturers, which had been qualified with both B. diminuta and A. laidlawii, consistently produced sterile effluents under similar test conditions. This study thus highlights the need for an industry or regulatory standard method of defining the microbial removal performance of 0.1 micron rated filters, and supports the use of functionally qualified 0.1 micron rated filters as sterilizing grade filters in pharmaceutical operations for enhanced sterility assurance.

Bacteria↗
PubMedDate not supplied

Retention of water-borne bacteria by membrane filters. Part I: Bacterial challenge tests on 0.2 and 0.22 micron rated filters.

The results of bacterial challenge tests conducted on several 0.2 and 0.22 micron rated "sterilizing grade" filter cartridge types with bacteria from a natural water source are presented. Eight different 0.2/0.22 micron rated "sterilizing grade" filter types from four different filter manufacturers, claimed to be capable of retaining Brevundimonas diminuta at a challenge level of 10(7) CFU/cm2, were tested. The filters tested included nylon 6.6 and polyamide filters from two manufacturers, modified or hydrophilic PVDF filters from two manufacturers, modified or asymmetric PES filters from three manufacturers, and cellulose acetate filters from a single manufacturer. Consistent bacterial penetration was observed, over the 18-24 h challenge period, for all twenty-five integral 0.2 and 0.22 micron rated filter cartridges tested, at challenge levels of about 10(1)-10(4) CFU/cm2, indicating that natural waterborne bacteria were more penetrative than B. diminuta. The observed penetration was thus qualitatively independent of filter media type or manufacturer. These results add to the growing body of evidence that shows 0.2 and 0.22 micron rated filters may not remove all microorganisms under all conditions. These results further establish that bacterial penetration of 0.2/0.22 micron rated filters is not limited just to (1) specific membrane types, or (2) extended duration challenges (>> 24 h), or (3) extremely high challenge levels, or (4) bacteria that can only exist in a penetrative state in an artificial laboratory setting.

Culture Media↗
PubMedDate not supplied

Retention of water-borne bacteria by membrane filters. Part II: Scanning electron microscopy (SEM) and fatty acid methyl ester (FAME) characterization of bacterial species recovered downstream of 0.2/0.22 micron rated filters.

The results of scanning electron microscopic (SEM) and fatty acid methyl ester (FAME) characterization of the bacterial species shown to penetrate conventional 0.2/0.22 micron rated "sterilizing grade" filters are presented. SEM data suggest that retention of bacteria by these filters appears to be strongly influenced by the morphology, and especially the width of bacteria and less so by length. When the bacterial cell width is small, less than 0.3 micron or so, the cell length does not appear to limit the ability to penetrate 0.2/0.22 micron rated filters. As the bacterial width increases, there is also a strong, almost exponential, decrease in the allowable length for penetration, with most penetrative cells tending to be coccoid beyond a width of 0.5 micron. Significant percentages of the bacteria (40-50%) that were observed downstream of these filters were larger than B. diminuta, the standard organism used to qualify 0.2/0.22 micron rated filters. The average sizes of natural waterborne bacteria that penetrated the filters tested were 20-40% larger in width, and 40-70% larger in length, compared to B. diminuta. These results indicate that size exclusion is not the sole mechanism governing bacterial retention. All isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. Most of the bacteria recovered downstream of 0.2/0.22 micron rated filters were gram negative, oxidase positive, motile, nonfermentors.

Bacteria↗