Biomedical subjects
Publications and source records attributed to A Guha.
A multi-disciplinary approach is described for the development of a microcomputer based on-line data collection system for blood flow experiments. Well designed software provides a flexible approach to data collection, which is particularly suited to blood flow research laboratories. The choice of well supported hardware ensures ease of development with minimum time and cost.
The value of decompression after spinal cord injury in patients is still an unresolved issue. It has previously been shown in our laboratory that functional recovery in rats after cord compression varied with both the force and time until decompression. However, the longest duration studied was only 15 minutes, which is far less than that usually encountered in clinical practice, and therefore, the present study was undertaken to determine the value of decompression after more prolonged periods of compression. A factorially designed experiment with five rats per cell was used with the clip compression injury model. Forces of 2.3, 16.9 or 53.0 gms were applied at C7-T1 until decompression was performed after 15, 60, 120, or 240 minutes of compression. Functional recovery was assessed weekly for 8 weeks using the inclined plane technique. Maximum and minimum performance limits were established in normal rats and rats with cord transection, respectively. Univariate analysis and multiple comparison tests were used to analyse the data. The major determinant of recovery was the force of the injury. For example, the animals injured by the 2.3 gm clip performed significantly better than those injured at higher forces for all times until decompression (p less than 0.0001), and there was a significant difference in recovery between the groups injured by the 16.9 and 53.0 gm clips, although only for the 15 minutes until decompression group (p less than 0.05). The time until decompression also affected recovery, but only for the lighter compression forces (2.3 and 16.9 gm). For example, animals decompressed after 60 minutes of 2.3 gm compression recovered significantly better than those decompressed after 240 minutes (p less than 0.05). Thus, if the initial injury force is small, decompression is beneficial even after prolonged injury.
Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).
The normal rat spinal cord blood flow (SCBF) has been shown to increase after administration of nimodipine, a calcium channel blocker. The present study investigates the capability of nimodipine to improve SCBF, as measured by the hydrogen clearance technique, after a 53.0-gm clip compression injury to the T-1 segment of the rat spinal cord. The profound drop in mean systemic arterial blood pressure (MSAP) after cervical cord injury precluded any improvement in posttraumatic SCBF by nimodipine alone. Hence, in a randomized controlled study with five rats per group, pressor agents (whole blood, angiotensin, or adrenaline) were infused to maintain MSAP between 100 and 120 mm Hg after injury. Control animals received only a saline infusion. Nimodipine at the optimal dose found in normal animals (1.5 microgram/kg/min) was added to the pressor agents. The MSAP and other physiological parameters were measured in rats receiving the pressor agents only and in those receiving pressor agents combined with nimodipine. In rats receiving whole blood, angiotensin, or adrenaline the posttraumatic MSAP improved to between 100 and 120 mm Hg, but there was no improvement in SCBF compared to the saline group. The addition of nimodipine decreased MSAP and SCBF in all groups except those animals also receiving adrenaline, where the MSAP was maintained at 109 +/- 5 mm Hg. In these animals a significant increase in posttraumatic SCBF from 16.5 +/- 2.1 to 20.2 +/- 2.3 ml/100 gm/min (mean +/- standard error of the mean) occurred at the site of injury with the addition of nimodipine. The maintenance of an adequate MSAP by a pressor agent was crucial for nimodipine to improve posttraumatic SCBF by its ability to dilate the spinal vascular bed. Adrenaline was the only pressor agent that could fulfill the above criteria, although other pressor agents need to be investigated. Experiments are underway with the combination of adrenaline and nimodipine to further verify these encouraging results demonstrating an improvement in posttraumatic ischemia of the spinal cord.
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We obtained a hybridoma using immune spleen cells from a mouse injected with human brain tissue factor that had been purified on a factor VII-agarose affinity column. This monoclonal IgG1, HTF1-7B8, inhibits tissue factor procoagulant activity. The concentration of HTF1-7B8 producing half-maximal inhibition is influenced by the concentration of factor VIIa, suggesting that the antibody and enzyme compete for the cofactor. The antibody was successfully used to detect both human and bovine tissue factor on nitrocellulose dot blots, indicating that the epitope recognized by this antibody is conserved in both species. This antibody clearly reveals tissue factor on a Western blot. An HTF1-7B8 affinity column was used to purify tissue factor from both human brain and placenta. The electrophoretic mobilities in polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and the amino acid compositions of the purified tissue factor from brain and placenta are indistinguishable, as are their specific procoagulant activities in reconstituted systems. This antibody will be useful for immunopurification and characterization of tissue factor structure and mechanism.
The potential existence of down-regulation of cytotoxic immune response against an autologous human melanoma line was investigated as a possible explanation for cytotoxic unresponsiveness against the autologous melanoma cells. The melanoma cell line, PJ-M, was established and lymph node resident lymphocytes (LNL) were isolated from a lymph node which was partially infiltrated with the melanoma cells. Autologous peripheral blood lymphocytes (PBL) were sensitized in in vitro co-culture (IVC) against radiated PJ-M cells in the presence or absence of PJ-M-sensitized LNL and enriched suppressor (OKT8+) or inducer (OKT4+) LNL populations, and were assayed for cytotoxicity in a 4-hr 51Cr-release microcytotoxicity assay. Significant cytotoxic response against PJ-M could be generated in the PBL, but not in the LNL. The addition of sensitized, unfractionated LNL, OKT8+, or OKT4+ LNL populations abrogated cytotoxic response in the PBL against PJ-M. The suppression of cytotoxic response was induced selectively against the PJ-M targets, because IVC of PBL in the presence of the sensitized LNL did not affect the generation of polyclonal cytotoxic alloreactivities, nor did they abrogate the generation of cytotoxic response against allogeneic targets in IVC against the corresponding allogeneic targets. These results suggest the possibility that cytotoxic immune response against the autologous melanoma cells might have been suppressed by the individual's own immunoregulatory circuit.
The cytotoxic immune response in the peripheral blood lymphocytes (PBL) against an autologous malignant melanoma cell line, PJ-M, was found to be down-regulated in in vitro co-culture (IVC) selectively by unfractionated resident lymph node lymphocytes (derived from a lymph node infiltrated with the PJ-M melanoma cells) and T4+ as well as T8+ fractions of the resident lymph node-derived lymphocytes. In this study, the mechanism involved in, and the specificities of, cytotoxic immune response in this autologous system were examined at population and clonal levels. Resident lymph node lymphocytes were isolated from both involved and uninvolved lymph nodes from the same patient. Resident lymphocytes from both sources regulated the generation of cytotoxic immune response when both types of resident lymph node lymphocytes were further sensitized against the PJ-M cells in IVC and were expanded in interleukin 2 (IL 2). An IL 2-dependent homogeneous lymphocyte line (I-10:1) bearing the phenotype of a helper T cell (T4+) and a T4+ clone (I-10.3) of the I-10:1 line, established by limiting dilution culture, also down-regulated the generation of cytotoxic immune effector cells in the PBL in IVC against the PJ-M targets. The IL 2-dependent T4+ inducer line I-10:1 generated a functionally differentiated T8+ suppressor population(s) that, in turn, could abrogate cytotoxic response in fresh PBL in IVC against PJ-M cells. The inducer line I-10:1 and its subclone I-10.3 suppressed the generation of cytotoxic effector cells in the PBL in IVC selectively against the autologous PJ-M cells. Generation of cytotoxic allo-response in IVC was unaffected by the inducer lines. These results provide further evidence for the involvement of the regulatory network in cytotoxic immune response in an autologous human tumor system, and suggest a potential explanation for cytotoxic unresponsiveness against autologous melanoma cells.
Autologous T-cell clones or lines, established from peripheral blood lymphocytes (PBLs) and lymphocytes from tumor-affected lymph nodes, were used to examine the immunoregulatory circuitry that might influence cell-mediated cytotoxic responses against human cancers. In a chromium 51-release microcytotoxicity assay, PBLs, when activated in vitro against autologous tumor cells in interleukin-2, generated marked cytotoxicity against the autologous targets. In four solid-tumor systems, such generation of cytotoxicity in the PBLs could be suppressed by T-cell lines or clones derived from lymphocytes from tumor-affected lymph nodes (LNLs). The suppressions, mediated by both T8+ suppressor and T4+ suppressor-inducer cells, were restricted against only the autologous tumors in two systems. In the other cases, the suppressions were nonspecific. Clarification of the receptors through which these types of regulation are mediated might provide a new approach for immunotherapeutic manipulations in cancer.
Tissue factor, a known initiator of blood coagulation, was found to be active in Triton X-100. A system consisting of tissue factor, factor VIIa, calcium ions, and coagulation factor X generated activated factor X at an appreciable rate. Based on this observation, we coupled human and bovine factor VII to a solid support. Each column bound tissue factor, solubilized in Triton X-100, in a species-specific manner. These interactions required calcium ions; when the columns were washed with detergent containing calcium ions, no tissue factor was eluted. When calcium ions were omitted from the eluant, tissue factor emerged as a sharp peak. Human tissue factor was extracted from an acetone brain powder into 2% Triton X-100. This extract, made 10 mM in CaCl2, was passed over a factor VII column. Human factor VII (1.2 mg) was coupled to 30 ml of Affi-Gel 15. This column bound approximately equal to 15 micrograms of human tissue factor. The eluted material was approximately equal to 25% pure. Final purification was achieved by gel filtration after chymotryptic digestion of contaminants. The tissue factor activity was stable to this treatment. The molecular weight determined by sodium dodecyl sulfate/PAGE (approximately equal to 46,000) was also unchanged by chymotrypsin. The final material was a single band on PAGE, demonstrated similar resistance to tryptic and chymotryptic digestion as bovine tissue factor, and had approximately the same specific coagulant activity as the previously purified bovine material. Tissue factor was also purified from human placenta, yielding a similar protein. A partial 28-residue sequence of the latter has been obtained.
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Nimodipine, a calcium channel blocker, is known to increase cerebral blood flow. In the present study, the authors investigated the effect of nimodipine on spinal cord blood flow in normal rats. Cardiovascular parameters, including mean systemic arterial blood pressure, cardiac output, and heart rate, were recorded during infusion of nimodipine in a dose-response fashion. The experiment was a randomized blind study in which four groups of five rats received different doses of nimodipine (0.001, 0.01, 0.05, and 0.10 mg/kg) intravenously over 30 minutes, and a control group of five rats received only the diluent. The hydrogen clearance and thermodilution techniques were used to measure spinal cord blood flow and cardiac output, respectively. The 0.05-mg/kg dose of nimodipine caused the largest increase in spinal cord blood flow, with a 40% increase over the preinfusion level, although there was a 25% reduction in mean arterial pressure. The 0.10-mg/kg dose did not increase spinal cord blood flow more than the 0.05-mg/kg dose, most likely due to the concomitant 37% reduction in mean arterial pressure. Cardiac output was significantly increased by the 0.05- and 0.10-mg/kg doses secondary to the drop in total peripheral resistance. The increase in spinal cord blood flow produced by nimodipine lasted approximately 20 minutes after the termination of the infusion. Thus, nimodipine at a dose of 0.05 mg/kg markedly increased blood flow in the normal spinal cord even though there were major changes in mean systemic arterial pressure and cardiac output. Further research is required to determine whether this drug might be beneficial in treating ischemic states of the spinal cord, such as posttraumatic ischemia.
Hepatitis B viral (HBV) DNA was detected in a hepatoma cell line which produces hepatitis B surface antigen (HBsAg) and in patients with acute hepatitis B. The serum of one patient with acute hepatitis B was found to be infectious when injected i.v. into a chimpanzee up to a dilution of 10(-8). Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were detectable in the same serum sample by radioimmunoassay up to a dilution of 10(-5) and of 10(-3), respectively. Using DNA: DNA hybridization on nitrocellulose membranes, HBV DNA sequences were detectable up to 10(-8) dilution corresponding to the infectivity level. Based on this finding, it appears that DNA: DNA hybridization is the most sensitive method for detecting hepatitis B virus (HBV) infection. In situations with low virus levels it may be the only indicator of the presence of infectious hepatitis B virus. The use of a tritium-labelled probe makes the method economical and adaptable to hospital laboratories.
The establishment of a spontaneously transformed tumorigenic human fibroblast line, VIP-F:T, is described. This line was developed from a primary culture of normal skin from a donor from whom a separate nontransformed fibroblast line, Pen-F2, also was established. The transformed line VIP-F:T exhibited aneuploid karyotype with a marker chromosome, showed anchorage-independent growth, and produced progressively growing tumors with morphologic characteristics of sarcoma in CD-1 (nu/nu) nude mice. The normal fibroblast line Pen-F2 exhibited diploid karyotype, showed no anchorage-independent growth, and produced no tumors in the nude mice. The spontaneously transformed fibroblast line VIP-F:T and its normal counterpart Pen-F2 will be valuable in studies of oncogene expression and in other investigations relevant to neoplasia.
A patient with acute myelomonocytic leukemia (AML) developed nephrotic syndrome. The renal biopsy showed focal glomerulosclerosis by light microscopy. Electron microscopy and immunofluorescence revealed electrondense deposits, IgG and C'3 in the glomerular mesangium. A 21S circulating immune complex (CIC) present in the patient's serum and the renal biopsy eluate contained immunochemically identical materials. The isolated antibodies from the 21S CIC and the eluate showed restricted reactivity against autologous AML cells. Immunodiffusion studies demonstrated common antigenicity between the 21S CIC antigen, the eluted antigen and between autologous AML cell membrane antigens.
The nature of divergent transcription from the bioABFCD gene cluster of Escherichia coli K-12 has been studied in vitro by using DNA restriction fragments as templates. The in vitro RNA transcript initiated at the promoter pa was found to be similar to the in vivo bioA transcript, whereas the transcription initiated at pB produced a small piece of RNA less than 200 nucleotides long. Substituting ITP for GTP or using an S100 cell-free extract as a source of antiterminator permitted transcription of the bioBFCD gene cluster initiating at the promoter pB to be read through. The possible site for in vitro transcription termination in the bioB region is discussed.
The nucleotide sequence of the 5'-terminal oligonucleotides produced by pancreatic RNase digestion of bacteriophage T3 RNA polymerase (EC 2.7.7.6) transcripts of T3 DNA has been determined. The sequence determination is based upon a simple isolation procedure for the 5'-terminal oligonucleotides. This procedure involves treatment of pancreatic RNase digests of alpha 32P-labeled T3 RNA polymerase transcripts with bovine brain exoribonuclease to remove oligonucleotides with free 5'-hydroxyl termini and then chromatographing the products on hydroxylapatite to resolve the remaining oligonucleotides having 5'-phosphate termini. By application of standard two-dimensional separation and sequence techniques, the major 5'-end sequences deduced were pppGpGpGpApGpApGpApY(Y = pyrimidine nucleoside) and pppGpGpGpApGpApCp. In addition, the sequences of other minor 5'-terminal oligonucleotides observed on homochromatograms were also determined. The sequences of these 5'-oligonucleotides were pppGpGpGpApApCpY, pppGpGpGpApApUpY, pppGpGp(2-4 Gp, 2-3 ApGp)..., and pppGpGpGp.... These results demonstrate that T3 phage-induced RNA polymerase possesses a high degree of specificity in the initiation of RNA chains.