Biomedical subjects
Publications and source records attributed to M Ramanathan.
We highlight recent advances in flow cytometry that have potential applications in the pharmaceutical sciences, particularly in pharmacodynamics and drug delivery. These advances are discussed in the context of the preclinical development of anticancer agents, immunosuppressants and immunomodulators, and oligonucleotides and gene therapy.
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BACKGROUND: Abdominal tuberculosis (TB) is common. But the diagnosis of abdominal TB is fraught with difficulties as it is often not possible to get a microbiological confirmation of the infection. We therefore undertook this study to highlight those pertinent clinical and laboratory features which would enable one to make a provisional diagnosis of abdominal TB early, to pave way for a trial of anti-tuberculosis chemotherapy. METHOD: This is a retrospective study of 12 patients treated for abdominal TB in our department over a period of 2 years. FINDINGS: Seven of the patients suffered from chronic diarrhoea for periods ranging from 4 weeks to 12 months. Four patients had progressive abdominal distension (ascites). The last patient came in with multiple abdominal swellings. Seven patients had clinical and biochemical features of malabsorption. Another 9 patients also had persistent pyrexia. The ascitic fluid was exudative in the 4 patients mentioned earlier. A definitive diagnosis could not be established in any of these patients. The diagnosis of abdominal TB was thus one of exclusion in these patients who showed prompt response to anti-tuberculosis therapy. CONCLUSION: Our study justifies a trial of anti-TB chemotherapy in TB endemic areas in the following clinical situations: (a) patients with chronic diarrhoea of unknown aetiology and (b) patients with exudative ascitic fluid, after all other possible causes, have been excluded. A prompt response to anti-TB therapy should be accepted as sufficient ground for the diagnosis of abdominal TB even when histopathological or microbiological confirmation of the disease is not possible. Our study reflects the experience of other workers from Third World countries.
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Interferon (IFN)-gamma is an important mediator of transplant graft rejection. It induces endothelial cell expression of HLA-DR and intercellular adhesion molecule-1, which render transplant grafts more susceptible to rejection by the host. Oligonucleotide 5'-GGG GTT GGT TGT GTT GGG TGT TGT GT-RNH2 (oligo I) blocks multiple IFN-gamma effects in human K562 cell cultures. A systematic approach revealed that oligo I has a novel, and potentially important, mode of action--it blocks the binding of IFN-gamma to its receptor, thus preventing activation of the IFN-gamma signal transduction pathway. The results are consistent with an aptamer mechanism of action, because oligo I exerts its inhibitory effects by interacting with protein, not intracellular nucleic acid targets, such as mRNA or genomic DNA.
PURPOSE: Cytokine binding macromolecules such as antibodies and soluble receptors sometimes produce undesirable agonist-like activities instead of the expected antagonist-like effects when the cytokine binding macromolecule extends the half-life of a short-lived cytokine. The purpose of this paper is to identify the pharmacokinetic and physicochemical properties that can cause these agonist-like activities. METHODS: A simple pharmacokinetic model was used to determine whether a given cytokine binding macromolecule will function effectively as an antagonist in therapeutic situations in which cytokine is released chronically. RESULTS: The model proposed satisfactorily fits experimental data for soluble interleukin-4 receptor and for an anti-interleukin-4 monoclonal antibody under conditions in which agonist-like and antagonist activity are observed. CONCLUSIONS: We show that the unexpected agonist-like activities result only when there is nonlinearity in the cytokine-cytokine receptor interaction and the cytokine binding macromolecule prolongs the half-life of the cytokine.
In dogs, maximal O2 uptake (VO2max) per kilogram of body weight is two- to threefold that in humans; the difference cannot be explained solely by differences in structural features between species. We compared the functional recruitment of pulmonary diffusing capacity (DLCO) during exercise in dogs with that in humans to determine whether pulmonary gas exchange is matched to VO2max or the size of the lungs and to define the potential role of exercise-induced polycythemia in producing the superior aerobic capacity of the dogs. We compared the relationships of DLCO, membrane diffusing capacity (DMCO), and pulmonary capillary blood volume (Vc) with respect to pulmonary blood flow (Qc) by a rebreathing method during steady-state exercise in adult male human subjects and in conditioned adult male foxhounds. The slopes and intercepts of the relationships of DLCO and DMCO to Qc are significantly greater in dogs than in humans; the slopes of the relationship of Vc to Qc are similar. In dogs diffusive pulmonary gas transport is matched to the higher VO2max. The enhanced recruitment of DLCO and DMCO in dogs during exercise could potentially be explained entirely by the exercise-induced polycythemia, which is seen in dogs but not in humans.
To lower the cost and improve accessibility of the rebreathing technique for measuring cardiopulmonary function during exercise, we implemented a fast-response infrared (IR) gas-analyzer system to simultaneously measure lung diffusing capacity, cardiac output lung tissue volume, and lung volume by a rebreathing technique in five healthy subjects at rest and during steady-state exercise. Interferences by water vapor and CO2 on the analyzer were determined and corrected for. During rebreathing, a gas mixture of 0.4% C2H2-0.3% CH4-9% He-30% O2, and either 0.3% C18O or 0.3% C16O in a balance of N2 was simultaneously sampled by both a mass spectrometer and the IR analyzer, permitting paired comparisons. Measurements obtained by the two devices were not significantly different. We conclude that this modified rebreathing technique using the IR analyzer is accurate for the measurement of cardiopulmonary function at rest and during exercise.
To determine the temporal progression and magnitude of functional compensation in immature dogs raised to maturity after extensive lung resection, we performed right pneumonectomy (R-Pnx) or right thoracotomy without pneumonectomy (Sham) in matched foxhounds at 2 mo of age. At 4, 8, 20, 40, and 60 wk after surgery, static transpulmonary pressure-lung volume relationships were determined. Lung diffusing capacity, membrane diffusing capacity, pulmonary capillary blood volume, pulmonary blood flow, septal lung tissue volume, and lung volumes were measured simultaneously by a rebreathing technique. During maturation, total lung capacity, lung volume at a given distending pressure, and compliance were lower in the R-Pnx group than in the Sham group (P < 0.05). Pulmonary viscous resistance at maturity was elevated after R-Pnx. There were no significant differences in total lung diffusing capacity, membrane diffusing capacity, pulmonary capillary blood volume, pulmonary blood flow, and septal lung tissue volume between groups. Compensation of alveolar-capillary gas exchange was complete by 4-8 wk after R-Pnx, but compensation of mechanical properties remained incomplete throughout maturation. Relative magnitude of compensation after R-Pnx was greater in immature dogs than in adult dogs studied previously by similar techniques.
A 43-year-old lady with long standing non-insulin dependent diabetes mellitus on glibenclamide presented with cholestatic liver disease. Initially she was thought to have developed primary biliary cirrhosis (PBC). When she made a spontaneous recovery following the withdrawal of glibenclamide, it became obvious that the patient had been suffering from drug-induced chronic cholestasis (DICC). The subtle differences between PBC and DICC are highlighted.
This report deals with a 54-year-old man with loss of memory. His impaired memory was found to be due to the atenolol he was on and he made a complete recovery on withdrawing the beta-blocker. This patient's experience stresses the need to consider beta-blockers as a potentially reversible cause of memory impairment.
In dogs during exercise, respiratory rate can reach 200 breaths/min, blood temperature can exceed 42 degrees C, and hematocrit can approach 60%. To determine whether these changes significantly affect the measurement of cardiac output by the acetylene rebreathing method (QcRB), we compared estimates of QcRB with those measured by thermodilution and Fick (QcFI) techniques in nine dogs at rest and during steady-state exercise on a treadmill up to near-maximal workloads. Solubility of acetylene in blood was corrected to the simultaneously measured blood temperature and hematocrit. Results were also adjusted for mixing efficiency. Up to a QcFI of 20 l/min, QcRB was not significantly different from QcFI (P > 0.05). However, cardiac output measured by thermodilution was consistently higher than those measured by the other techniques (P < 0.0001). We conclude that the overall agreement between QcRB and QcFI estimates supports the validity of the rebreathing technique under exercise conditions where body temperature and hematocrit are changing rapidly and the breathing pattern is unrestrained. Systematic error by the thermodilution technique may be related to a variety of methodological issues as well as possible dissipation of cooling into the myocardial tissue and subsequent incomplete washout.
Normal reference values of the pulmonary diffusing capacity for carbon monoxide (DLCO), diffusing capacity of the membrane (DMCO), and pulmonary capillary blood volume (VC) were derived by a rebreathing technique in 44 normal, healthy, nonsmoking individuals ranging from 17 to 68 yr of age. Simultaneous measurements of DLCO, lung volumes, pulmonary blood flow, and septal tissue volume were determined at rest and during steady-state exercise of 25, 50, and 80% of maximal workload and at two levels of alveolar O2 tension. DMCO and VC were calculated using the Roughton-Forster relationship. Using stepwise regression analysis, DLCO and DMCO (in ml CO/min/mm Hg) were found to be significantly related to pulmonary blood flow (Qc in L/min), age (in years), and body surface area (BSA in square meters). VC (in ml) was found to be significantly related to QC and BSA. Normalization of measured data with respect to these variables largely eliminated male versus female differences. We conclude that QC is the most important determinant of the recruitment of diffusing capacity. From rest to near-maximal exercise, DLCO, DMCO, and VC increase linearly with respect to QC without evidence of reaching a plateau.
This paper deals with two patients on warfarin in whom the use of topical methylsalicylate preparations led to clinically significant bleeding problems. The first patient required fresh frozen plasma to tide over the crisis while the second patient recovered spontaneously on stopping the warfarin temporarily. The possible mechanisms by which salicylates potentiate the anticoagulant effect of warfarin are briefly outlined.
The major histocompatibility complex (MHC) Class I and II genes and intercellular adhesion molecule-1 (ICAM-1) are regulated by interferon-gamma in a variety of cell types. We have previously shown that the oligodeoxynucleotide 5'-GGG GTT GGT TGT GTT GGG TGT TGT GT-RNH2 (oligo I) inhibits the interferon-gamma-mediated enhancement of MHC Class I and ICAM-1 proteins in the K562 cell line. We have now investigated the mechanism of action of oligo I and report that it acts by inhibiting the binding of interferon-gamma to cells. We also show that the dose-response curves, the selectivity profile, and the kinetics of oligo I are consistent with this novel mechanism of action. The dose-response curves for oligo I, obtained using antibodies against the MHC Class I heavy chain, beta 2-microglobulin, or ICAM-1, are almost superimposable at each observation time. MHC Class I induction by 6400 units/ml interferon-alpha or interferon-beta or ICAM-1 enhancement by 800 units/ml tumor necrosis factor-alpha is not inhibited by oligo I. However, the synergistic induction of MHC Class I by mixtures of tumor necrosis factor-alpha and interferon-gamma is inhibited. Oligo I belongs to a class of active oligodeoxynucleotides that inhibits interferon-gamma-induced MHC Class I and ICAM-1 in K562 cells. The activity and potency is sequence-dependent, but remarkably different sequences can have comparable effects. The activity of oligo I in the HeLa S3 cell line inhibits the interferon-gamma-mediated enhancement of both ICAM-1 and MHC Class II DR and the interferon-gamma-mediated reduction in transferrin receptor expression. Thus, oligo I appears to specifically inhibit interferon-gamma-induced changes in protein expression, which is consistent with oligo I acting at an early step(s) in the induction process. Taken together, our results show that oligo I exerts its effects by inhibiting the association of interferon-gamma with the cell surface, which is a novel mechanism of action for oligodeoxynucleotides.
We report that certain oligonucleotides are capable of inhibiting cell surface induction of the major histocompatibility complex class I (MHC-I) proteins by interferon-gamma in K562 cells. The inhibition by oligodeoxy-nucleotide I 5' GGG GTT GGT TGT GTT GGG TGT TGT GT-RNH2 is dose-dependent, with an EC50 24 hr after dosing of approximately 4 microM for 800 U/ml interferon-gamma. The reverse complement II 5' AC ACA ACA CCC AAC ACA ACC AAC CCC-RNH2 did not show activity. Oligodeoxynucleotide I inhibits induction of MHC-I by interferon-gamma, but does not inhibit induction by either interferon-alpha or interferon-beta. Four other oligodeoxynucleotides were also evaluated, and three showed activity against interferon-gamma at 25 microM.
A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using total DNA obtained from normal swine feces inoculated with decreasing concentrations of S. hyodysenteriae cells, the sensitivity of the PCR assay was calculated to be between 1 and 10 organisms per 0.1 g of feces. The PCR assay was 1,000 times more sensitive than conventional culture of dysenteric feces on selective medium. There was complete agreement between the results of PCR assays and anaerobic culture on selective agar medium with diagnostic specimen (n = 9) obtained from six farms on which there were cases with clinical signs suggestive of swine dysentery. Detection of S. hyodysenteriae by PCR amplification of DNA has great potential for rapid identification of S. hyodysenteriae in diagnostic specimens.
Static and dynamic mechanical characteristics of the respiratory system were measured in five adult foxhounds 6-15 mo after right pneumonectomy (R-PNX) and in five matched foxhounds that underwent right thoracotomy without pneumonectomy (Sham). In R-PNX dogs, elastic recoil was lower than that in the left lung of Sham dogs. On exercise, absolute ventilatory power requirements of the lung and its components were measured, i.e., power requirements to overcome elastic and viscous resistances of the lung as well as power requirements during inspiration and expiration. All components were higher for a given minute ventilation in R-PNX dogs than in both lungs of Sham dogs. Ventilatory power requirements after R-PNX were also higher than in three adult foxhounds after left PNX studied previously by the same techniques. After R-PNX, the mass of the right costal diaphragm and total mass of inspiratory muscles were greater than in Sham dogs. There were no significant differences in ultrastructural features of the costal diaphragm. The unilateral increase in muscle mass is likely the result of chronic elevation and stretch of the right costal diaphragm after R-PNX.