Biomedical subjects
R Steinman
Publications and source records attributed to R Steinman.
Maturation and migration of cutaneous dendritic cells.
Dendritic cells have been isolated from the epidermis, dermis, and lymphatics of skin. Cells from each cutaneous compartment can exhibit the distinct morphology, surface phenotype, and strong T-cell-stimulating activity of dendritic cells that are isolated from other organs. Of importance are the mechanisms by which the maturation and movement of dendritic cells are regulated within intact tissues. Epidermal dendritic cells turn over slowly in the steady state. Stimuli, including contact allergens and transplantation, perhaps by inducing a release of cytokines such as granulocyte macrophage-colony-stimulating factor, mobilize these dendritic cells into the dermis and lymph. This migration is accompanied by the maturation of dendritic cell functions; e.g., antigen-presenting major histocompatibility complex molecules and B7 costimulators increase markedly. On the other hand, there is a sizable, steady-state flux of dendritic cells in afferent lymph draining the skin, which suggests a constant traffic through the dermis that is independent of sessile epidermal dendritic cells. When explants of skin are placed in organ culture, dendritic cells emigrate into the medium for 1-3 d. The dendritic cells are mature and can bind tightly to small memory T cells that also migrate in these cultures. The emigrated mixtures of dendritic cells and T cells should be useful in the study of many clinical states. This is illustrated by recent experiments showing that migratory skin cells are readily infected with human immunodeficiency virus (HIV)-1. A strong productive infection takes place in the absence of exogenous cytokines, foreign sera, or mitogens or antigens. The dendritic cell-T-cell conjugates are the essential site for infection. This cellular milieu may model events during the sexual transmission of HIV-1, where relevant mucosal surfaces are covered by skin-like epithelia. The capture of CD4+ memory T cells by dendritic cells may explain the chronic drain of immune memory in HIV infection.
Presentation of a viral T cell epitope expressed in the CDR3 region of a self immunoglobulin molecule.
Synthetic peptides corresponding to microbial epitopes stimulate T cell immunity but their immunogenicity is poor and their half-lives are short. A viral epitope inserted into the complementarity-determining region 3 (CDR3) loop of the heavy chain of a self immunoglobulin (Ig) molecule was generated from the Ig context and was presented by I-Ed class II molecules to virus-specific, CD4+ T cells. Chimeric Ig-peptide was presented 100 to 1000 times more efficiently than free synthetic peptide and was able to prime virus-specific T cells in vivo. These features suggest that antigenized Ig can provide an improved and safe vaccine for the presentation of microbial and other peptides.
The urate oxidase gene of Drosophila pseudoobscura and Drosophila melanogaster: evolutionary changes of sequence and regulation.
The urate oxidase (UO) transcription unit of Drosophila pseudoobscura was cloned, sequenced, and compared to the UO transcription unit from Drosophila melanogaster. In both species the UO coding region is divided into two exons of approximately equal size. The deduced D. pseudoobscura and D. melanogaster UO peptides have 346 and 352 amino acid residues, respectively. The nucleotide sequences of the D. pseudoobscura and D. melanogaster UO protein-coding regions are 82.2% identical whereas the deduced amino acid sequences are 87.6% identical with 42 amino acid changes, 33 of which occur in the first exon. Although the UO gene is expressed exclusively within the cells of the Malpighian tubules in both of these species, the temporal patterns of UO gene activity during development are markedly different. UO enzyme activity, UO protein, and UO mRNA are found in the third instar larva and adult of D. melanogaster but only in the adult stage of D. pseudoobscura. The intronic sequences and the extragenic 5' and 3' flanking regions of the D. pseudoobscura and D. melanogaster UO genes are highly divergent with the exception of eight small islands of conserved sequence along 772 bp 5' of the UO protein-coding region. These islands of conserved sequence are possible UO cis-acting regulatory elements as they reside along the 5' flanking DNA of the D. melanogaster UO gene that is capable of conferring a wild-type D. melanogaster pattern of UO regulation on a UO-lacZ fusion gene.
Stability over time of variables measuring heart rate variability in normal subjects.
Both time and frequency domain measures of heart rate (HR) variability have been used to assess autonomic tone in a variety of clinical conditions. Few studies in normal subjects have been performed to determine the stability of HR variability over time, or the correlation between and within time and frequency domain measures of HR variability. Fourteen normal subjects aged 20 to 55 years were studied with baseline and placebo 24-hour ambulatory electrocardiograms performed 3 to 65 days apart to assess the reproducibility of the following time domain measures of cycle length variability: the standard deviation of all normal cycle intervals; mean normal cycle interval; mean day normal cycle interval; night/day difference in mean normal cycle interval; root-mean-square successive cycle interval difference; percentage of differences between adjacent normal cycle length intervals that are greater than 50 ms computed over the entire 24-hour electrocardiographic recording (proportion of adjacent intervals greater than 50 ms); and the frequency domain measures of high (0.15 to 40 Hz), low (0.003 to 0.15) and total (0.003 to 0.40) power. The mean and standard deviations of these measures were virtually identical between placebo and baseline measurements and within the studied time range. Variables strongly dependent on vagal tone (high-frequency, low-frequency and total power, root-mean-square successive difference, and percentage of differences between adjacent normal cycle intervals greater than 50 ms computed over the entire 24-hour electrocardiographic recording) were highly correlated (r greater than 0.8). It is concluded that measures of HR variability are stable over short periods of time.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus.
Feline immunodeficiency virus (FIV) structural proteins were identified using sera obtained from experimentally inoculated cats. Proteins analysed by both radioimmunoprecipitation and Western blotting were specific for FIV infection and failed to cross-react with either antisera to feline leukaemia virus of feline syncytium-forming virus. Western blot analysis of purified virus revealed immunoreactive proteins with apparent Mr of 65K, 50K, 40K, 32K, 24K, 15K and 10K. The major core structural proteins of the virus were isolated by reverse phase HPLC and the aminoterminal sequences of p10 and p24 were determined. Monoclonal antibodies specific for p24 suggested the presence of a precursor protein that could be detected in 35[S]methionine/cysteine-labelled, virus-infected cell extracts. This putative precursor protein possessed an apparent Mr of 50K (Pr50gag). Further analysis revealed the presence of two additional proteins of 130K and 40K. Experiments utilizing tunicamycin, endoglycosidase H and glycopeptidase F revealed that p130 and p40 exhibited properties characteristic of glycoproteins. Our studies also indicated that FIV is immunologically related to other lentiviruses.
Social exchanges between older and younger gay male partners.
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Immunogenicity: role of dendritic cells.
In the development of the immune response, the dendritic cell subset of leukocytes plays a key role in enhnacing immunogenicity. Dendritic cells can pick up antigens in the tissues and move to lymphoid organs, through which T cells continually recirculate. It is proposed that dendritic cells at these sites express functions which have been identified in tissue culture models. These involve efficient binding to antigen-specific T lymphocytes, as well as the induction of the lymphokines and growth factor receptors required for immunity. The dendritic cell system, apparently under the control of cytokines, is a sentinel designed to signal T cells that a significant antigen burden is present, and to generate the activated T lymphoblasts that interact with many other cell types to bring about an immune response.
Development and evaluation of immunoassay for detection of antibodies to the feline T-lymphotropic lentivirus (feline immunodeficiency virus).
The feline T-cell lymphotropic lentivirus (feline immunodeficiency virus) is a recently described feline-specific retrovirus that can produce chronic immunodeficiency-like disorders in cats. A microdilution plate format enzyme-linked immunosorbent assay has been developed to detect the presence of antibody to the virus in feline serum or plasma. Temporal studies performed with experimentally infected animals show that seroconversion can be demonstrated 3 to 4 weeks after exposure to the virus. Results of a serosurvey (n = 1,556 samples) indicate that infection is fairly common in both clinic (5.2%) and sick cat (15.2%) populations. Western blot (immunoblot) and sodium dodecyl sulfate radioimmunoprecipitation assays were developed to confirm microdilution plate test results and to identify peptides specific for the feline immunodeficiency virus. All microdilution plate test positive results and selected negative results were confirmed by one or both of these procedures. These data demonstrate that this microassay plate enzyme-linked immunosorbent assay is a very sensitive and specific test for detection of antibody to the feline immunodeficiency virus.
An objective marker of lactate-induced panic.
There are a number of agents claimed effective in provoking panic attacks in the laboratory. For each case, however, the determination of whether an attack has occurred is largely subjective. An objective index of panic would be of great benefit in standardizing laboratory provocation studies. Using heart rate (HR) recordings during sodium lactate infusion, we developed a formula--the heart rate index (HRI)--which appears to correlate well with the subjective experience of panic. Of note is the fact that the HRI was most powerful in identifying attacks that occurred in the first 15 min of the 20-min infusions. In an accompanying study, we examined HR variability during the infusions using the successive difference mean square statistic. HR variability did not change during lactate-induced panic attacks. Therefore, tachycardia during lactate-induced panic probably does not result from decreased vagal tone to the heart.
Programmed electrical stimulation to determine the need for antiarrhythmic therapy in patients with complex ventricular ectopic activity.
Patients with complex ventricular ectopy (greater than or equal to Lown grade III) and organic heart disease (OHD) are at increased risk for sudden cardiac death. Despite this fact, many such patients will remain free of symptomatic ventricular arrhythmia and thus are unnecessarily exposed to antiarrhythmic drug toxicity and arrhythmic potentiation. Programmed stimulation (PS) was used to direct therapy in 88 patients with asymptomatic ventricular ectopy complicating OHD. Thirty-three had inducible ventricular tachycardia (VT) and underwent treatment. The 55 patients without inducible VT (less than or equal to 6 repetitive ventricular responses) are the focus of this study. Three patients required treatment for persistent cardiac awareness. The remaining 52 have been followed for 22 months off antiarrhythmic drugs and all have remained free of subsequent major arrhythmic events. Therefore, in patients with complex ventricular ectopy, OHD, and absence of prior symptomatic ventricular arrhythmia, PS identifies patients at low risk for future disabling or life-threatening arrhythmic episodes and patients with absence of inducible VT can usually be managed without antiarrhythmic drugs.
The pharmacokinetics and serum and urine bactericidal activity of ciprofloxacin.
Ciprofloxacin is an investigational quinolone agent possessing an impressive antibacterial spectrum. Its pharmacokinetics were studied in six volunteers after 250-mg and 500-mg single oral doses, and its bactericidal activity compared to that of trimethoprim-sulfamethoxazole given to the same volunteers. Mean peak serum levels were 1.45 micrograms/mL and 2.46 micrograms/mL for 250-mg and 500-mg doses, and time to peak was 1 and 1.3 hours. The 12-hour levels were 0.12 micrograms and 0.22 microgram. Half-life (T1/2)alpha were 0.32 and 0.43 with T1/2 beta were 3.97 and 4.15 and volume of distribution (area) were 80L and 90L, respectively. Area under the concentration curve (AUC) was 5.65 h X micrograms/mL and 10.37h X micrograms/mL. Serum clearance was 23L for both doses. Approximately 49% of the 250-mg dose and 43% of the 500-mg dose was recovered in the urine. Bactericidal levels were determined against clinical isolates. Sera at 1.5 hours after the 500-mg dose averaged bactericidal levels of 1:20 or better for an Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and beta-lactamase producing Haemophilus influenzae and Branhamella catarrhalis. Urinary bactericidal levels at eight to 12 hours were greater than or equal to 1:157 for E coli, K pneumoniae, gentamicin-piperacillin resistant P aeruginosa, Staphylococcus aureus, and 1:20 for Streptococcus faecalis. Serum bactericidal levels were superior, and urine bactericidal levels were superior or equal to the bactericidal levels obtained with trimethoprim-sulfamethoxazole.(ABSTRACT TRUNCATED AT 250 WORDS)
The pharmacology of orally administered ciprofloxacin.
Ciprofloxacin pharmacokinetics were studied in 6 volunteers after 250 and 500 mg single oral doses. Mean peak serum levels were 1.45 micrograms/ml and 2.5 micrograms/ml for 250 and 500 mg doses. The 12-h levels were 0.12 micrograms and 0.22 micrograms. T1/2 alpha values were 0.32 and 0.43 h; T1/2 beta was 4 h and Vd (area) values were 80L and 90L for the two doses respectively. AUC was 5.65 h. micrograms/ml and 10.37 h. micrograms/ml. Serum clearance was 23L for both doses. Approximately 49% of the 250 mg dose and 43% of the 500 mg dose was recovered in the urine. Ciprofloxacin's in vitro activity and human pharmacology should permit a twice or once-daily dosing schedule for systemic infections due to most Enterobacteriaceae, Haemophilus, Branhamella and Pseudomonas and S. aureus, and once-daily doses for urinary and gastrointestinal infections.
The pharmacology of clavulanic acid and ticarcillin combined.
The pharmacokinetics of ticarcillin and clavulanic acid were studied in normal volunteers. Ticarcillin at 50 mg/kg was combined with clavulanic acid at 1.7 and 3.4 mg/kg and infused over 30 minutes. Peak serum levels of ticarcillin were 325 micrograms/ml and its pharmacokinetic parameters were similar when combined with either dose of clavulanic acid. Peak serum levels of clavulanic acid were 8 micrograms/ml for 1.7 mg/kg and 15.8 micrograms/ml for 3.4 mg/kg. Serum concentrations of clavulanic acid were greater than or equal to 1 microgram/ml for 2 h with the 1.7 mg/kg dose and for 3 h with the 3.4 mg/kg dose. Serum half-lives of ticarcillin and clavulanic acid were similar, 1.2 hours. Urinary concentrations of ticarcillin exceeded 100 micrograms/ml and clavulanic acid concentrations exceeded 1 microgram/ml for 6 h. The serum and urine levels were such that many beta-lactamase producing organisms would be inhibited by this combination.
Defective gamma interferon production in leprosy. Reversal with antigen and interleukin 2.
Antigen and mitogen-induced gamma interferon (gamma-IFN) production was studied in peripheral blood mononuclear cells from 34 leprosy patients. 17 of 18 lepromatous leprosy and borderline lepromatous patients (LL and BL) failed to release gamma-IFN in response to specific antigen (Mycobacterium leprae) and displayed reduced responses to mitogen (concanavalin A) stimulation. In contrast, cells from six tuberculoid and borderline tuberculoid patients (TT and BT) produced considerable levels of gamma-IFN under the same experimental conditions. Normal controls failed to respond to M. leprae and most displayed good responses to concanavalin A. Mid-borderline patients (BB) showed intermediate levels of gamma-IFN release. gamma-IFN release by lepromatous patients could be partially restored with purified interleukin 2 and M. leprae antigen but not with interleukin 2 alone.
Energetics of citrulline synthesis by rat liver mitochondria.
The energetics of flux through carbamyl phosphate synthetase and of citrulline formation from added ammonia, bicarbonate, and ornithine have been investigated in liver mitochondria from rats fed a high protein diet. In the presence of an oxidizable substrate, but in the absence of ornithine, carbamyl phosphate accumulated as a function of the medium phosphate concentration (K'm approximately 1.5 mM) up to values of 30 nmol/mg of protein. Upon addition of ornithine, citrulline was produced at the rate of 70 nmol/mg/min, and the carbamyl phosphate content fell to below 1 nmol/mg. The intramitochondrial ATP/ADP ratio decreased after ornithine addition, indicating that release of inhibition of carbamyl phosphate synthetase by carbamyl phosphate predominated over the expected inhibition due to the fall of the ATP/ADP ratio. Under partially uncoupled conditions in the presence of ornithine, citrulline formation decreased linearly with a fall of the calculated intramitochondrial MgATP/MgADP ratio. Changes of the thermodynamic parameters of mitochondrial phosphorylation potential, delta Gp(m), proton electrochemical gradient, delta mu H+, and oxidation-reduction potential difference between NAD+ and cytochrome c, delta Eh, were measured under conditions of enhanced respiration induced by citrulline synthesis and compared with ADP-stimulated respiration. Under both conditions, delta Gp(m) decreased and delta Eh also decreased due to a net oxidation of NADH and reduction of cytochrome c. However, delta mu H+ showed no change after citrulline addition although it decreased during ADP-stimulated respiration. The average H+/2e stoichiometry over the first two phosphorylation sites calculated from the delta Eh/delta mu H+ ratio ranged from 3.0 to 3.5, while the H+/ATP stoichiometry calculated from the delta Gp(m)/delta mu H+ ratio ranged from 2.0 to 2.5. The calculated ratios of H+/2e and H+/ATP both increased as delta mu H+ was lowered by addition of an uncoupling agent. The overall data are apparently not in accordance with the commonly held view that delta mu H+ is an obligatory intermediate between the oxidation-reduction pumps of the respiratory chain and ATP synthase.
Inhibition of Ca2+ of carbamoylphosphate synthetase (ammonia).
The effect of Ca2+ ions on carbamoylphosphate synthetase (ammonia) (EC 6.3.4.16) of rat liver mitochondria was studied. In lysed mitochondria, carbamoylphosphate synthetase (ammonia) was inhibited by Ca2+ ions. In intact rat liver mitochondria, the rate of citrulline production from added ammonia, ornithine, and bicarbonate was inversely correlated with the magnitude of the intramitochondrial concentration of Ca2+. This effect was found at concentrations of Ca2+ which are within the physiological range. The inhibition was reversed by added Mg2+ and was not due to effects of Ca2+ on mitochondrial energy production. It is proposed that the activity of carbamoylphosphate synthetase (ammonia) may be modulated in vivo by changes in the concentration of Ca2+ ions in the mitochondrial matrix.
Disseminated gonorrhea: diagnosis through contact tracing.
Over a 1-year period four patients were seen at the Jewish General Hospital with presumed disseminated gonococcemia. The patients' histories, clinical findings and responses to therapy strongly supported this diagnosis; however, cultures from various sites were negative for Neisseria gonorrhoeae in all four patients. Sexual contacts of these patients were traced and appropriate cultures were found to be positive, demonstrating the importance of tracing sexual contacts to help confirm the diagnosis in such patients.