Tissue lipoprotein lipase in normal individuals and in individuals with exogenous hypertriglyceridemia and the relationship of this enzyme to assimilation of fat.

Lipoprotein lipase activity (LLA) was measured in the adipose tissue of six healthy subjects and five members of a family in whom the trait for familial exogenous hypertriglyceridemia was segregating. The lipase activity measured was characteristic of lipoprotein lipase: increased by feeding, dependent on the presence of serum, and inhibited by sodium chloride and protamine sulfate. When compared with lipase activity in healthy individuals, LLA was grossly deficient in two siblings with postabsorptive chylomicronemia and was intermediate in both parents and one sibling, who had normal postabsorptive triglycerides. These findings are compatible with autosomal recessive inheritance. The hormone-sensitive lipolytic enzyme responsible for mobilization of free fatty acids from adipose storage was normal in the hyperlipemic subjects. After a 104-g fat meal, the serum triglyceride increased more in subjects heterozygotic for LLA deficiency than in the healthy subjects, and there was a relatively greater increase in chylomicrons and very low density lipoproteins in the affected individuals. These observations demonstrate the physiologic importance of lipoprotein lipase in removal of these lipoprotein groups and further clarify the differences between endogenous and exogenous hypertriglyceridemia.

Cerebrovascular response to acute hypocapnic and eucapnic hypoxia in normal man.

Alterations in human cerebral blood flow and related blood constituents were studied during exposure to acute hypoxia. Observations were made during serial inhalation of decreasing O(2) concentrations with and without maintenance of normocarbia, during 8 min inhalation of 10% O(2), and after hyperventilation at an arterial P(O2) of about 40 mm Hg. In the range of hypoxemia studied, from normal down to arterial P(O2) of about 40 mm Hg, the magnitude of the cerebral vasodilator response to hypoxia appeared to be largely dependent upon the coexisting arterial CO(2) tension. The mean slope of the increase in cerebral blood flow with decreasing arterial O(2) tension rose more quickly (P < 0.05) when eucapnia was maintained when compared with the slope derived under similar hypoxic conditions without maintenance of eucapnia. When 12 subjects inhaled 10% oxygen, cerebral blood flow rose to more than 135% of control in four whose mean decrease in arterial CO(2) tension was - 2.0 mm Hg. The remaining eight had flows ranging from 97 to 120% of control, and their mean decrease in CO(2) tension was - 5.1 mm Hg. When mean arterial P(O2) was 37 mm Hg, hyperventilation was carried out in 10 subjects. Arterial P(O2) increased insignificantly, arterial P(CO2) declined from 34 to 27 mm Hg (P < 0.05), and cerebral blood flow which had been 143% of control decreased to 109%, a figure not significantly different from control.These data demonstrate the powerful counterbalancing constrictor effects of modest reductions in CO(2) tension on the vasodilator influence of hypoxia represented by arterial P(O2) reductions to about 40 mm Hg. Indeed, mild hyperventilation completely overcame the vasodilator effect provided by an arterial O(2) tension as low as 40 mm Hg. The effects of hypoxia on the control of the cerebral circulation must be analyzed in terms of the effects of any associated changes in CO(2) tension.

Differential sensitivity of tumor targets to liver macrophage-mediated cytotoxicity.

Liver macrophages activated in vivo with bacterially derived lipopolysaccharide (LPS) display enhanced chemotaxis, phagocytosis, and oxidative metabolism. To determine if LPS also activates these mononuclear phagocytes for tumor cell killing, we compared the cytotoxic activity of macrophages from livers of rats treated with LPS (5 mg/kg, i.v.) with resident Kupffer cells. We found that both macrophage cell types displayed cytotoxicity towards rat N1S1 hepatoma and RBL-1 basophilic leukemia cells. Cytotoxicity of resident and LPS-activated liver macrophages towards these targets increased with cocultivation time, was dependent on the effector:target cell ratio, and appeared to involve extracellular lysis. No direct correlation between macrophage activation and cytotoxicity was observed towards these targets. While liver macrophages from LPS treated rats were more cytotoxic towards N1S1 cells, resident Kupffer cells were more cytotoxic towards RBL-1 cells. In further studies, resident Kupffer cells were also found to display extracellular cytolytic activity towards mouse P815 mastocytoma cells. In contrast, LPS-activated liver macrophage-mediated killing of these targets involved phagocytosis of intact tumor cells, as evidenced by light and electron microscopy and by uptake of 51Cr-labeled cells. These results suggest that cytotoxicity mediated by liver macrophages depends on the type of macrophage and the nature of the tumor cell target. In addition, cytotoxicity towards tumor targets appears to involve at least two different mechanisms including extracellular cytolysis and phagocytosis.

Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol).

Eleutherobin is a novel natural product isolated from a marine soft coral that is extremely potent for inducing tubulin polymerization in vitro and is cytotoxic for cancer cells with an IC50 similar to that of paclitaxel. This compound is cross-resistant along with other multidrug-resistant agents against P-glycoprotein-expressing cells and is cross-resistant with paclitaxel against a cell line that has altered tubulin. In mechanistic studies, eleutherobin shares with paclitaxel the ability to induce tubulin polymerization in vitro and is most likely cytotoxic by virtue of this mechanism. Human colon carcinoma cells exposed to eleutherobin contain multiple micronuclei and microtubule bundles, and they arrest in mitosis, depending on concentration, cell line, and length of exposure. These morphological abnormalities appearing in cultured cells are indistinguishable from those induced by paclitaxel. Electron microscopy reveals that eleutherobin induces homogeneous populations of long, rigid microtubules similar to those formed by paclitaxel. Thus, eleutherobin is a new chemotype with a mechanism of action similar to that of paclitaxel and, as such, has promising potential as a new anticancer agent.

Blood flow in muscle groups and drug absorption.

Resting human muscle blood flow (MBF) was determined simultaneously in the usual intramuscular injection sites to resolve whether variance in MBF could account for differences in drug absorption. Three pairs of muscles (gluteus maximus, vastus lateralis, and deltoid) were studied in each of 20 adult subjects. Use of dual, matched linear rate meters allowed two muscles to be studied simultaneously, with the order of injection random within an incomplete block design. MBF was calculated from the 133xenon washout rate using a single exponential that the computer found to best fit the data. Deltoid MBF (11.6 ml/100 gm/min plus or minus 0.5) was significantly (p smaller than 0.05) greater than gluteus MBF (9.6 plus or minus 0.5), with vastus or between right and left sides for each muscle. These data indicate that there are consistent differencies in resting MBF among specific muscle groups of sufficient magnitude (19%) to affect the rate of absorption and peak serum levels following intramuscular administration of drugs.

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis.

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.

Diphenidol treatment of arrhythmias.

The antiarrhythmic activity of diphenidol, an antiemetic, has been demonstrated both in electrophysiologic studies of patiens and in experimental arrhythmias in animals. Accordingly, 18 patients with tachyarrhythmias were treated with intravenous diphenidol in doses of 0.5 to 1.5 mg/kg. In six patients with atrial arrhythmias, there was no notable effect. Similarly, 12 patients with premature ventricular contractions were treated and studied. In six of them, ectopic beats were abolished, at least transiently; in three the number of ventricular premature contractions decreased; in two there was no effect; and in one, the number of premature beats was increased. Of the total number of 18 patients, 14 suffered adverse effects related to the central nervous system. These adverse effects were of such severity as to suggest that further studies with diphenidol as an antiarrhythmic are not warranted.

Behaviour of fibroblasts and epidermal cells cultivated on analogues of extracellular matrix.

A porous collagen sponge can be used for supporting epidermal cells and fibroblasts in order to manufacture an artificial skin. Fibroblasts were grown on analogues of extracellular matrix containing collagen and glycosaminoglycans and/or glycoproteins. Cell replication, and also infiltration of fibroblasts, were enhanced by the presence of hyaluronic acid and/or fibronectin. Epidermal cells grown on a collagen sponge have been characterized by microscopic observations. Epidermal cells on the surface of the sponge showed an incomplete differentiation in comparison to normal skin; clumps of epidermal cells were found in the interior of the sponge. Epidermal cell replication was enhanced in the presence of collagen sponge seeded with fibroblasts.

Experimental induction of caprylate-dependent albumin antibodies.

A normal donor with the caprylate-dependent albumin agglutinins volunteered to receive 50 ml. of 25% caprylate-stabilized human albumin to determine the clinical significance of the antibodies. The donor manifested no clinical sign of a reaction. Samples of blood and urine collected before, during and after administration of albumin were subjected to extensive laboratory investigation. Biochemical, immunologic, hematologic, hemostatic and immunohematologic variables investigated showed only insignificant variations in all blood and urine samples. Results indicated that the infusion of albumin was safe, for the donor showed no sign of a hemolytic, febrile, allergic or anaphylactic transfusion reaction. However, since this is the first and only reported experiment of its kind, it is deemed advisable to exercise caution when administering albumin to patients who have caprylate-dependent albumin agglutinins until sufficient supportive data attesting to the complete safety of this procedure have accumulated.

Differential production of interleukin 1 on the surface of biomaterials.

The production of cytokines on the surface of surgical biomaterials plays a major role in their biocompatibility. Membrane-associated interleukin 1 (mIL-1) is a cytokine found on the surface of macrophages activated by biomaterials. To better understand the host-foreign body interaction, we quantitated the production of mIL-1 on the surface of two materials commonly used in surgery, expanded polytef (ePTFE) and silicon elastomer (SE). The mean (+/- SD) level of mIL-1 produced by adherent cells to ePTFE significantly decreased from day 2 (13,746 +/- 3630 cpm per disk) compared with day 7 (2828 +/- 1304 cpm per disk). However, the level of mIL-1 produced by ePTFE-adherent cells was still markedly greater than the level of mIL-1 produced by cells adherent to SE (1877 +/- 1028 vs 1595 +/- 822 cpm per disk). These results indicate that ePTFE and SE elicit a differential host response in terms of cytokine production. This study may enhance our understanding of the cellular events on the surface of biomaterials that underlie clinical observations.

An apomorphine-induced vomiting model for antiemetic studies in man.

Apomorphine-induced vomiting is often used for preclinical efficacy testing of putative antiemetics in normal volunteers. The usual technique of individual intravenous titration for finding the threshold emitic dose of apomorphine in each subject is slow and tedious. We used a uniform dose of 0.05 mg/kg apomorphine given subcutaneously to test the antiemetic action of metoclopramide and votracon in ten healthy, young male volunteers. All ten subjects vomited in response to this dose of apomorphine when pretreated with placebo. Pretreatment with metoclopramide prevented vomiting in all subjects, and votracon prevented vomiting in two. Apomorphine, 0.05 mg/kg, subcutaneously appears to be an appropriate challenge dose for testing compounds for antiemetic activity in normal human volunteers.

Ultrastructural analysis of beta-amyloid-induced apoptosis in cultured hippocampal neurons.

Following treatment with the beta-amyloid (A beta) 25-35 analog, scanning and transmission electron microscopy were used to investigate the morphological changes in cultured hippocampal neurons during the course of degeneration. Ultrastructural analysis revealed focal cell surface blebbing and rapid condensation of nuclear chromatin. Changes in cytoplasmic morphology included prominent vacuole formation, dispersal of polyribosome rosettes and the disappearance of the golgi complex, smooth endoplasmic reticulum and microtubules with increased cytoplasmic electron density. Mitochondria and limited rough endoplasmic reticulum remained intact throughout the process of cell death. These results provide additional evidence suggesting A beta-induced cell death in vitro occurs via an apoptotic mechanism.

Didanosine (ddl) and stavudine (d4T): absence of peripheral neurotoxicity in rabbits.

Some 20 male New Zealand White rabbits (five/group) were given either didanosine (ddl) or stavudine (d4T) at 750 and 1500 mg/kg body weight/day by oral intubation for 24 wk. An additional group was given 300 mg/kg body weight/day zidovudine (AZT) as a negative control. After 13 weeks the high dose of ddl was lowered from 1500 to 1000 mg/kg body weight/day following the death of one rabbit and continued inappetence in the dose group. The rabbits were observed daily, plasma drug levels were monitored, and electrophysiological measurements of peripheral nerve conduction were performed during the study. Additionally, body weight and food intake were recorded, and clinicopathological parameters were evaluated. Sections of selected peripheral nerves, and dorsal and ventral spinal nerve roots were examined by light and transmission electron microscopy. Although peripheral neuropathy has been reported in rabbits with the nucleoside analogue zalcitabine (ddC), based on clinical observations, electrophysiological measurements, and light and electron microscopy, no evidence of peripheral neurotoxicity was observed in rabbits given either ddl of d4T.

Treatment of hypercholesterolemia in black patients.

The treatment of hypercholesterolemia in the black patient will be the next public health challenge facing physicians in the black community. Cost-effective care of hypercholesterolemia will be necessary and is possible, but it will require skill in the use of available therapies, extensive patient education, and excellent communication between patients and health care providers.

Analysis of mammalian connective tissue: relationship between hierarchical structures and mechanical properties.

It is widely accepted that the mechanical properties of implants must match those of the surrounding connective tissue to prevent stress concentration and premature failure. The purpose of this paper is to review the structure-mechanical property relationships that exist for connective tissue. The mechanical properties of connective tissue depend on the content of collagen, elastic tissue, and proteoglycans, as well as the geometric arrangement of the fibrous components, age, and location of the specimen. To a first approximation the geometry and loading pattern of the collagen networks in these tissues dominate the mechanical response at high strains. The behavior of the elastic fiber networks dominate the low-strain mechanical response in tissues where energy and shape recovery are critical. Proteoglycans are involved in resisting tissue compressive forces. Since the stiffness of connective tissue increases with age, it is necessary to attempt to match this property by designing implants that have similar behaviors to insure that stress concentration does not occur at the interface between the implant and host.

Central and peripheral neurosecretory pathways to an insect flight motor nerve.

Ultrastructural examination of the IIN1b nerve to the dorsal longitudinal flight muscle of Manduca sexta L. verified the presence of neurosecretory processes. Subspherical and irregular vesicles were found where the nerve enters the muscle, while spherical vesicles were found in the proximal region only. A dorsal unpaired median (DUM) cell, the median nervous system, and two or more peripheral cells are the sources of these neurosecretory inclusions. Light the electron microscopy CoCl2 backfills of the transverse nerve produced intensification of a peripheral neuron (#1) and processes in nerves IIN1a and IIN1b. Similar backfills of nerve IIN1b produced intensification of a DUM cell, a second peripheral neuron (#2), and processes in the transverse nerve and nerve IIN1a. Neuron #1 contained large spherical electron-dense vesicles while neuron #2 contained smaller subspherical vesicles. These cells were situated upon the link and/or transverse nerves. Based on these results, we suspect central and peripheral neurosecretory processes reach nerve IIN1b as follows: the link nerve projects prothoracic median nervous system and neuron #2 processes, nerve IIN1a projects neuron #1 processes, and nerve IIN1 projects mesothoracic DUM cell processes, although this latter pathway was less clear.

Liver macrophage-mediated cytotoxicity toward mastocytoma cells involves phagocytosis of tumor targets.

Macrophage-mediated cytotoxicity toward tumor cells usually involves extracellular lysis of the targets. In this study, we report that liver macrophages from rats treated with lipopolysaccharide (5 mg/kg, intravenous) also kill certain tumor cell targets by phagocytosis. Liver macrophages were coincubated with P815 mouse mastocytoma cells for 24 to 72 hr at an effector/target ratio of 10:1. Macrophage phagocytosis was characterized by flow cytometry and by light and electron microscopy. For flow-cytometric studies, P815 cells were prelabeled with the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate. We found that coincubation of macrophages with labeled targets resulted in a time-dependent increase in macrophage-associated fluorescence, reaching a maximum at 72 hr. This correlated with light-microscopic observations of increased numbers of tumor cells in the macrophages and enhanced macrophage surface area and density. Electron microscopic studies revealed that the initial event in the phagocytic process involved the capture of P815 cells by the pseudopodia of the macrophages. Target cells were then surrounded by lamellipodia, internalized in phagosomes and destroyed. These data, together with previous studies, provide evidence for multiple mechanisms of cytotoxicity mediated by activated liver macrophages.