Impaired interleukin 12 production in human immunodeficiency virus-infected patients.

Peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients, asymptomatic or with acquired immunodeficiency virus, produced 10-fold less interleukin 12 (IL-12) free heavy chain and fivefold less biologically active IL-12 heterodimer than PBMC from uninfected healthy donors when challenged in vitro with the common human pathogen Staphylococcus aureus. In contrast, PBMC from HIV-infected individuals and uninfected control donors produced similar levels of tumor necrosis factor alpha, IL-1 beta, and IL-10, and PBMC from HIV-infected individuals produced three- to fourfold more IL-6 compared with PBMC from uninfected control donors. The defect in IL-12 production is not due to hyperproduction of IL-10, a cytokine exerting an autocrine-negative feedback on IL-12 production, but was directly related to HIV infection, as suggested by the reduced ability of monocytes infected in vitro with HIV to produce IL-12. IL-12 deficiency may be an important component of the immunodeficiency associated with HIV infection.

Energy-dependent intracellular translocation of proparathormone.

We previously suggested that after synthesis, proparathormone is transferred from rough endoplasmic reticulum to the Golgi region where its conversion to parathormone occurs. We have attempted to define more closely this transfer process. In the first type of study, bovine parathyroid slices were incubated with [3H]leucine for 10 min and then radioisotope labeling was restricted by addition of a large excess of nonradioactive leucine. Under these conditions, more than 90% of the initially labeled proparathormone was converted to parathormone in 40 min. Lowered temperature in the chase period markedly inhibited the conversion. Several chemical agents were employed individually in the chase period to examine their effect on the conversion process. Antimycin A, dinitrophenol, oligomycin, and anaerobiosis (N2) inhibited the conversion, whereas sodium flouride and cycloheximide had no effect. In the second type of study, parathyroid slices were incubated with [3H]leucine for the entire incubation period. Lowered temperature and inhibitors of energy metabolism and microtubular function all lengthened the interval (lag) between the initial synthesis of [3H]parathormone. Cycloheximide, Tris, and chloroquine decreased the rates of protein synthesis and conversion, respectively, but none had any effect on the lag. We interpret the lag to represent the time of transit for proparathormone from rough endoplasmic reticulum to the Golgi region. We conclude that this transfer process is independent of the synthesis of the prohormone and its conversion to the hormone. Moreover, this translocation requires metabolic energy and appears to be mediated by microtubules.

Mapping human brain monoamine oxidase A and B with 11C-labeled suicide inactivators and PET.

The regional distributions of monoamine oxidase (MAO) types A and B have been identified in human brain in vivo with intravenously injected 11C-labeled suicide enzyme inactivators, clorgyline and L-deprenyl, and positron emission tomography. The rapid brain uptake and retention of radioactivity for both 11C tracers indicated irreversible trapping. The anatomical distribution of 11C paralleled the distribution of MAO A and MAO B in human brain in autopsy material. The corpus striatum, thalamus, and brainstem contained high MAO activity. The magnitudes of uptake of both [11C]clorgyline and L-[11C]deprenyl were markedly reduced in one subject treated with the antidepressant MAO inhibitor phenelzine. A comparison of the brain uptake and retention of the 11C-labeled inactive (D-) and active (L-) enantiomers of deprenyl showed rapid clearance of the inactive enantiomer and retention of the active enantiomer within MAO B-rich brain structures, in agreement with the known stereoselectivity of MAO B for L-deprenyl. Prior treatment with unlabeled L-deprenyl prevented retention of L-[11C]deprenyl. Thus, suicide enzyme inactivators labeled with positron emitters can be used to quantitate the distribution and kinetic characteristics of MAO in human brain structures.

Induced sputum improves the diagnosis of pulmonary tuberculosis in hospitalized patients in Gaborone, Botswana.

SETTING: Low sensitivity of acid-fast bacilli (AFB) sputum smears and absence of productive cough are obstacles to the diagnosis of pulmonary tuberculosis (PTB) in hospitals that lack access to bronchoscopy. OBJECTIVES: To evaluate induced sputum, gastric content, blood and urine specimens to improve PTB diagnosis in patients not diagnosed by expectorated sputum AFB smears. DESIGN: Patients admitted to the medical wards of a large public hospital in Gaborone, Botswana, were prospectively enrolled if they had symptoms consistent with PTB, an abnormal chest radiograph, were treated empirically with anti-tuberculosis chemotherapy or had no improvement on antibiotics, and had a non-productive cough or AFB smear-negative sputum. Induced sputum was stained for AFB and Mycobacterium tuberculosis cultures were performed on induced sputum, gastric contents, urine and blood. RESULTS: Of 140 patients meeting the enrollment criteria, 113 (81%) were human immunodeficiency virus (HIV) positive. Fifty-seven (41%) had PTB based on positive cultures from one or more sites, including 48 (84%) from induced sputum, 17 (30%) urine, 13 (23%) gastric contents and 7 (12%) blood. AFB smears were positive in only 18 (32%) culture-proven PTB cases. CONCLUSION: Induced sputum cultures greatly enhanced M. tuberculosis detection in patients with a high prevalence of HIV/AIDS in a hospital without access to bronchoscopy.

Propranolol antagonizes the anti-inflammatory effect of alcohol and improves survival of infected intoxicated rabbits.

We studied the effects of alcohol and propranolol on the course of peritonitis in rabbits. Induction of sterile peritonitis with normal saline led to a 50% augmentation of granulocyte adherence in normal rabbits, and a mean cumulative granulocyte count of 27,000/mm(3) in peritoneal exudate by 8 h. Rabbits intoxicated with alcohol at the time of peritonitis induction maintained a granulocyte adherence below pretreatment values, and only delivered a cumulative mean of 12,000 granulocytes/mm(3) into the peritoneal fluid. When intoxicated rabbits received propranolol intravenously at the time of intoxication, adherence increased above preperitonitis levels, and stayed significantly above values for animals given alcohol alone. In addition, the defect in granulocyte delivery was prevented by propranolol, resulting in a mean cumulative granulocyte count in peritoneal fluid of 24,000/mm(3).When peritonitis was induced with live pneumococci instead of a sterile inflammatory stimulus, 14/18 normal animals survived the infection and were culture-negative when sacrificed at 2 wk. In contrast, 17/18 intoxicated animals died of the infection, in a mean of 2.8 days. 9 of 18 intoxicated animals who also received propranolol survived, and those who died lived a mean of 7.5 days. The survival rates and the time-to-death among the nonsurvivors given propranolol were both significantly greater than in the animals intoxicated without propranolol. Thus, propranolol prevents the granulocyte adherence and delivery defects induced by alcohol intoxication, and significantly improves survival from infection.

Comparative adherence of granulocytes to endothelial monolayers and nylon fiber.

Adherence of granulocytes to tissue culture monolayers of endothelium averaged 26.2 +/- 1.3% SEM, which was similar to their adherence on 50-mg nylon fiber columns (27.7 +/- 3.6%). In contrast, adherence to epithelial cells, fibroblasts, kidney cells, and plastic Petri dishes without monolayers was only 12.4, 9.9, 11.1, and 4.3%, respectively. Cyclic nucleotides and adherence-modifying plasma factors induced changes of adherence to endothelium similar to those in nylon fiber columns. Adherence of granulocytes in whole blood was the same as for purified granulocytes in Hank's balanced salt solution. Exposure of endothelial monolayers to 0.18% trypsin for 10 min reduced subsequent granulocyte adherence to 25.2% of control values. Incubation of trypsin-treated monolayers with nutrient medium for 4 h did not improve adherence, but values returned to normal or above by 24 h, with or without serum proteins present in the nutrient medium. The similarity of granulocyte adherence to nylon fiber and to endothelial monolayers in vitro suggests that results with the nylon fiber assay reflect in vivo granulocyte-endothelium interaction. Furthermore, the endothelial monolayer offers a new model for studying this cell-cell relationship in vitro.

The induction of augmented granulocyte adherence by inflammation. Mediation by a plasma factor.

The adherence of granylocytes to surfaces, measured in vitro in nylon fiber columns, is inhibited by in vivo administration of anti-inflammatory agents. Therefore, the effect of inflammation itself was assessed in blood from patients with acute inflammatory diseases. Mean adherence in these patients was twice normal (56.4 +/- 5.6% vs. 29.4 +/- 5.2%); their plasma contained a factor that augmented adherence of normal cells to 47.5 +/- 5.6% whereas the patient's cells showed a normal level of adherence (34.0 +/- 6.8%) when resuspended in normal plasma. Although exudate fluid from exprimental inflammation also contained the augmenting factor, cells from the exudate maintained their high level of adherence after washing and suspension in normal plasma. The augmenting factor detected in plasma from patients with inflammation was not present in serum and was inactivated by heating plasma to 56 degrees C for 30 min; restoration of augmenting activity was accomplished by addition of 20% guinea pig serum to the heat-treated plasma. Because the guinea pig serum itself did not increase adherence when added to normal plasma, it appears that the augmenting factor is heat-stable, but requires a heat-labile cofactor like complement. Sephadex G-200 fractionation of inflammatory plasma showed adherence-augmenting activity in the majority of fractions, with peak activity in the fractions corresponding to approximate molecular wts of 30,000, 160,000 and 400,000.

Virus infection of endothelial cells increases granulocyte adherence.

Adherence of human granulocytes was measured on endothelial monolayers of human and bovine origin, grown in 35-mm Diam petri dishes and in cluster wells. Adherence to human endothelium in petri dishes using 1.0 ml of whole blood averaged 17.9+/-3.7%, and to bovine endothelium was 20.3+/-3.7%. Cluster wells required only 1/5 the endothelial cells needed for petri dishes, and 0.25 ml of whole blood yielded average adherence of 26.2+/-3.4 to human cells and 28.0+/-3.7 to bovine in the wells. The impact of infection of the endothelium by different viruses on subsequent granulocyte adherence was measured. Polio virus produced an acute lytic infection of human endothelial cells, with associated increased adherence to 185.4% of control 24 h after inoculation. Significantly increased adherence was noted at 6 h, before detectable cytopathic effect. Herpes simplex type I caused a similar rapidly lytic infection of bovine endothelium associated with increased adherence to 213.7% of control 6 h after inoculation. This augmented adherence could be demonstrated when granulocytes were suspended in physiologic saline solution, showing that antibody and complement need not be present. Trypsin treatment of infected monolayers did not prevent the augmentation, and supernate from infected monolayers increased the adherence of polymorphonuclear leukocytes to normal, uninfected monolayers. Chronic, slowly lytic infections, lasting 7 d or more, were induced with adenovirus in human endothelium and with measles virus in bovine cells. Adherence increased as virus was noted in the cell cultures on day 4, several days before cytotoxicity was seen. Thus, chronic viral infection of the endothelium appears possible, and results in increased granulocyte adherence. In naturally occurring disease, such an infection may act synergistically with adherent granulocytes to damage the endothelium, and may represent an in vitro model of vasculitis.

Biosynthesis of proparathyroid hormone and parathyroid hormone by human parathyroid glands.

Human parathyroid glands obtained at autopsy were incubated with [(3)H]leucine and [(3)H]lysine. After incubation, nonradioactive parathyroid tissue of either human or bovine origin was added. Radioactive parathyroid hormone and proparathyroid hormone were isolated from the gland and medium by organic solvent and salt fractionation, trichloroacetic acid precipitation, Sephadex G-100 gel filtration, and carboxymethyl cellulose column chromatography. The human hormonal peptides were identified in the ion-exchange column eluates by their relatively high levels of radioactivity, their elution positions, and their immunoreactivity to anti-PTH antiserum. The time-course of radioactive amino acid incorporation into these peptides and a brief incubation of the gland with radioactive amino acids, followed by various lengths of incubation with nonradioactive amino acids, indicated that a precursor-product relationship exists for the two peptides. An alternate method for isolation of the hormone and prohormone, which involves separation of peptides by urea-polyacrylamide gel electrophoresis, confirmed the identities of the human parathyroid hormone and proparathyroid hormone.

The release of parathyroid hormone and the exocytosis of a proteoglycan are modulated by extracellular Ca2+ in a similar manner.

Secretion of parathyroid hormone (PTH) is regulated in part by a classical "stimulus-secretion" pathway responsive to catecholamines. The primary physiological modulator of PTH exocytosis in parathyroid cells, however, is extracellular free Ca2+. Ca(2+)-modulated PTH release exhibits several characteristics suggestive of constitutive secretion. The aim of this work was to obtain further information about the possible intracellular origins of Ca(2+)-modulated exocytosis in parathyroid cells. Freshly dissociated bovine parathyroid cells labeled with [35S]sulfate synthesized a soluble chondroitin/dermatan sulfate proteoglycan (M(r) approximately 90-150 K) that was secreted into the medium. The export of [35S]sulfated proteoglycan satisfied several criteria that generally define constitutive release: 1) export is detected in the medium shortly (7-15 min) after a 5-min pulse, 2) there is minimal intracellular storage after equilibrium labeling (because of combined processes of rapid release and intracellular degradation), and 3) there is insensitivity to stimulation with isoproterenol, a known secretagogue in parathyroid cells. Nevertheless, the increase in extracellular Ca2+ from 0.5 to 2.0 mM reduced the export of the [35S]sulfated proteoglycan from 60% of total labeled to 30%. In addition, a secreted pool of immunoreactive PTH and [35S]sulfated proteoglycan was modulated by external Ca2+ to the same degree and sensitivity, although isoproterenol was more effective in stimulating the release of PTH than that of proteoglycan. Together, our experimental results show that in the parathyroid cell extracellular Ca2+ modulates negatively the export of both PTH and proteoglycan, a putative marker for constitutive secretion. We further suggest that a portion of newly synthesized PTH also enters this pathway, whereas another portion proceeds to an isoproterenol-releasable compartment from which the proteoglycan is largely excluded.