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Protective headgear effects measured in the laboratory may not always translate to the field. In this study, we evaluated the impact attenuation capabilities of a commercially available padded helmet shell cover in the laboratory and on the field. In the laboratory, we evaluated the padded helmet shell cover's efficacy in attenuating impact magnitude across six impact locations and three impact velocities when equipped to three different helmet models. In a preliminary on-field investigation, we used instrumented mouthguards to monitor head impact magnitude in collegiate linebackers during practice sessions while not wearing the padded helmet shell covers (i.e., bare helmets) for one season and whilst wearing the padded helmet shell covers for another season. The addition of the padded helmet shell cover was effective in attenuating the magnitude of angular head accelerations and two brain injury risk metrics (DAMAGE, HARM) across most laboratory impact conditions, but did not significantly attenuate linear head accelerations for all helmets. Overall, HARM values were reduced in laboratory impact tests by an average of 25% at 3.5 m/s (range: 9.7 to 39.6%), 18% at 5.5 m/s (range: - 5.5 to 40.5%), and 10% at 7.4 m/s (range: - 6.0 to 31.0%). However, on the field, no significant differences in any measure of head impact magnitude were observed between the bare helmet impacts and padded helmet impacts. Further laboratory tests were conducted to evaluate the ability of the padded helmet shell cover to maintain its performance after exposure to repeated, successive impacts and across a range of temperatures. This research provides a detailed assessment of padded helmet shell covers and supports the continuation of in vivo helmet research to validate laboratory testing results., (© 2023. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Repeated head impact exposure and concussions are common in American football. Identifying the factors associated with high magnitude impacts aids in informing sport policy changes, improvements to protective equipment, and better understanding of the brain's response to mechanical loading. Recently, the Stanford Instrumented Mouthguard (MiG2.0) has seen several improvements in its accuracy in measuring head kinematics and its ability to correctly differentiate between true head impact events and false positives. Using this device, the present study sought to identify factors (e.g., player position, helmet model, direction of head acceleration, etc.) that are associated with head impact kinematics and brain strain in high school American football athletes. 116 athletes were monitored over a total of 888 athlete exposures. 602 total impacts were captured and verified by the MiG2.0's validated impact detection algorithm. Peak values of linear acceleration, angular velocity, and angular acceleration were obtained from the mouthguard kinematics. The kinematics were also entered into a previously developed finite element model of the human brain to compute the 95th percentile maximum principal strain. Overall, impacts were (mean ± SD) 34.0 ± 24.3 g for peak linear acceleration, 22.2 ± 15.4 rad/s for peak angular velocity, 2979.4 ± 3030.4 rad/s 2 for peak angular acceleration, and 0.262 ± 0.241 for 95th percentile maximum principal strain. Statistical analyses revealed that impacts resulting in Forward head accelerations had higher magnitudes of peak kinematics and brain strain than Lateral or Rearward impacts and that athletes in skill positions sustained impacts of greater magnitude than athletes in line positions. 95th percentile maximum principal strain was significantly lower in the observed cohort of high school football athletes than previous reports of collegiate football athletes. No differences in impact magnitude were observed in athletes with or without previous concussion history, in athletes wearing different helmet models, or in junior varsity or varsity athletes. This study presents novel information on head acceleration events and their resulting brain strain in high school American football from our advanced, validated method of measuring head kinematics via instrumented mouthguard technology., (© 2021. Biomedical Engineering Society.)

Background: Induction of nonshivering thermogenesis can be used to influence energy balance to prevent or even treat obesity. The pungent component of mustard, allyl-isothiocyanate (AITC), activates the extreme cold receptor transient receptor potential channel, subfamily A, member 1 and may thus induce energy expenditure and metabolic changes. Objective: The objective of our study was to evaluate the potential of mustard AITC to induce thermogenesis (primary outcome) and alter body temperature, cold and hunger sensations, plasma metabolic parameters, and energy intake (secondary outcomes). Design: Energy expenditure in mice was measured after subcutaneous injection with vehicle, 1 mg norepinephrine/kg, or 5 mg AITC/kg. In our human crossover study, 11 healthy subjects were studied under temperature-controlled conditions after an overnight fast. After ingestion of 10 g of capsulated mustard or uncapsulated mustard or a capsulated placebo mixture, measurements of energy expenditure, substrate oxidation, core temperature, cold and hunger scores, and plasma parameters were repeated every 30 min during a 150-min period. Subjects were randomly selected for the placebo and capsulated mustard intervention; 9 of 11 subjects received the uncapsulated mustard as the final intervention because this could not be blinded. After the experiments, energy intake was measured with the universal eating monitor in a test meal. Results: In mice, AITC administration induced a 32% increase in energy expenditure compared with vehicle (17.5 ± 4.9 J · min -1 · mouse -1 compared with 12.5 ± 1.2 J · min -1 · mouse -1 , P = 0.03). Of the 11 randomly selected participants, 1 was excluded because of intercurrent illness after the first visit and 1 withdrew after the second visit. Energy expenditure did not increase after ingestion of capsulated or uncapsulated mustard compared with placebo. No differences in substrate oxidation, core temperature, cold and hunger scores, or plasma parameters were found, nor was the energy intake at the end of the experiment different between the 3 conditions. Conclusion: The highest tolerable dose of mustard we were able to use did not elicit a relevant thermogenic response in humans. This trial was registered at www.controlled-trials.com as ISRCTN19147515.

Background: Mild cold exposure increases energy expenditure and can influence energy balance, but at the same time it does not increase appetite and energy intake., Objective: To quantify dermal insulative cold response, we assessed thermal comfort and skin temperatures changes by infrared thermography., Methods: We exposed healthy volunteers to either a single episode of environmental mild cold or thermoneutrality. We measured hunger sensation and actual free food intake. After a thermoneutral overnight stay, five males and five females were exposed to either 18°C (mild cold) or 24°C (thermoneutrality) for 2.5 h. Metabolic rate, vital signs, skin temperature, blood biochemistry, cold and hunger scores were measured at baseline and for every 30 min during the temperature intervention. This was followed by an ad libitum meal to obtain the actual desired energy intake after cold exposure., Results: We could replicate the cold-induced increase in REE. But no differences were detected in hunger, food intake, or satiety after mild cold exposure compared with thermoneutrality. After long-term cold exposure, high cold sensation scores were reported, which were negatively correlated with thermogenesis. Skin temperature in the sternal area was tightly correlated with the increase in energy expenditure., Conclusions: It is concluded that short-term mild cold exposure increases energy expenditure without changes in food intake. Mild cold exposure resulted in significant thermal discomfort, which was negatively correlated with the increase in energy expenditure. Moreover, there is a great between-subject variability in cold response. These data provide further insights on cold exposure as an anti-obesity measure., (© 2016 The authors.)

Objective: Huntington disease (HD) is a fatal autosomal dominant, neurodegenerative condition characterized by progressively worsening motor and nonmotor problems including cognitive and neuropsychiatric disturbances, along with sleep abnormalities and weight loss. However, it is not known whether sleep disturbances and metabolic abnormalities underlying the weight loss are present at a premanifest stage., Methods: We performed a comprehensive sleep and metabolic study in 38 premanifest gene carrier individuals and 36 age- and sex-matched controls. The study consisted of 2 weeks of actigraphy at home, 2 nights of polysomnography and multiple sleep latency tests in the laboratory, and body composition assessment using dual energy x-ray absorptiometry scanning with energy expenditure measured over 10 days at home by doubly labeled water and for 36 hours in the laboratory by indirect calorimetry along with detailed cognitive and clinical assessments. We performed a principal component analyses across all measures within each studied domain., Results: Compared to controls, premanifest gene carriers had more disrupted sleep, which was best characterized by a fragmented sleep profile. These abnormalities, as well as a theta power (4-7Hz) decrease in rapid eye movement sleep, were associated with disease burden score. Objectively measured sleep problems coincided with the development of cognitive, affective, and subtle motor deficits and were not associated with any metabolic alterations., Interpretation: The results show that among the earliest abnormalities in premanifest HD is sleep disturbances. This raises questions as to where the pathology in HD begins and also whether it could drive some of the early features and even possibly the pathology., (© 2015 The Authors Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.)

Background/objectives: The objective of this study was to develop approaches to expressing resting energy expenditure (REE) and lean body mass (LM) phenotypes of metabolic disorders in terms of Z-scores relative to their predicted healthy values., Subjects/methods: Body composition and REE were measured in 135 healthy participants. Prediction equations for LM and REE were obtained from linear regression and the range of normality by the standard deviation of residuals. Application is demonstrated in patients from three metabolic disorder groups (lipodystrophy, n=7; thyrotoxicosis, n=16; and resistance to thyroid hormone (RTH), n=46) in which altered REE and/or LM were characterised by departure from the predicted healthy values, expressed as a Z-score., Results: REE (kJ/min) = -0.010 × age (years)+0.016 × FM (kg)+0.054 × fat-free mass (kg)+1.736 (R2 = 0.732, RSD = 0.36 kJ/min). LM (kg)=5.30 × bone mineral content (kg)+10.66 × height2 (m)+6.40 (male). LM (kg)=0.20 × fat (kg)+14.08 × height2 (m)-2.93 (female).(male R2=0.55, RSD = 3.90 kg; female R2 = 0.59, RSD=3.85 kg).We found average Z-scores for REE and LM of 1.77 kJ/min and -0.17 kg in the RTH group, 5.82 kJ/min and -1.23 kg in the thyrotoxic group and 2.97 kJ/min and 4.20 kg in the LD group., Conclusion: This approach enables comparison of data from individuals with metabolic disorders with those of healthy individuals, describing their departure from the healthy mean by a Z-score.

Haemodynamic and hormonal effects of two oral isovolaemic, isoosmotic solutions of 0.5 g/kg and 1.0 g/kg glucose were studied in 10 humans with sympathetic denervation due to primary autonomic failure (AF). Measurements were made supine for 60 min, and also after 5 min 45 head-up tilt, before and 60 min after glucose. There was a similar fall in blood pressure (BP) after each dose, after 0.5 g/kg from 160+/-12 / 87+/-6 to 143+/-13 / 76+/-6 mm Hg, P < 0.05 and after 1.0 g/kg from 160+/-13 / 90+/-6 to 136+/-9 / 76+/-5 mm Hg, P < 0.05. Heart rate, cardiac index and forearm muscle blood flow did not change after either dose. After 0.5 g/kg, superior mesenteric artery blood flow was unchanged but rose significantly after 1.0 g/kg, from 243 (169-395) to 722 (227-982) ml/min, P < 0.05, 15 min after ingestion. BP fell further on tilt 60 min after each dose, but there was no difference between doses. Plasma glucose was higher after 1.0 g/kg but plasma insulin was similar after each dose. Thus, in AF with sympathetic denervation there was no dose-related effect of glucose on supine or postural hypotension. Supine hypotension after glucose was not attributable solely to increased splanchnic blood flow; other factors, including dilatation in other vascular beds may have contributed.

The haemodynamic and catecholamine responses to supine exercise, and the effect on standing blood pressure (BP), were studied in three groups with peripheral autonomic dysfunction; insulin-dependent diabetes mellitus (IDDM), familial amyloid polyneuropathy (FAP) and pure autonomic failure (PAF). Healthy normal subjects were studied as controls. With exercise, BP increased in controls, was unchanged in IDDM and FAP, and fell in PAF. Heart rate (HR) increased more in controls than IDDM, FAP or PAF. Cardiac index (CI) increased less in IDDM than controls, FAP or PAF. Systemic vascular resistance (SVR) fell similarly in controls and IDDM, with a greater fall in FAP and PAF. Plasma noradrenaline increased in controls and IDDM only; plasma adrenaline did not change and plasma dopamine was undetectable in all groups. On standing, BP was unchanged in controls; BP fell pre- and post-exercise in IDDM, FAP and PAF, with a significantly greater fall post-exercise in FAP and PAF. In conclusion, the haemodynamic responses to supine exercise and to standing after exercise differed in the three groups with peripheral autonomic dysfunction. These differences, and also the similarities, between different forms of peripheral autonomic dysfunction, may be of relevance to the clinical assessment and therapy of these patients.

Systemic and regional haemodynamics and hormonal responses to two isovolaemic, iso-osmotic solutions of 0.5 and 1.0 g/kg of glucose were compared in ten normal young subjects (mean age 24 +/- 3 years). Measurements were made while subjects were supine before glucose and every 15 min for 60 min after ingestion of each solution. Superior mesenteric artery (SMA) blood flow rose similarly after each dose. There were corresponding reductions in SMA vascular resistance after each dose but no difference between doses. Pulsatility index of the SMA was lower after 1.0 g/kg. There was no change in blood pressure, heart rate, cardiac index or forearm muscle blood flow after either dose. Plasma glucose levels rose after each dose and were higher after 1.0 g/kg. There was no difference in the plasma insulin rise between doses. Plasma levels of noradrenaline and adrenaline did not change after either dose. These results suggest that with glucose loads within the ranges we used, changes in SMA blood flow are not dose-related and larger increases in SMA blood flow or in plasma insulin than we observed are needed to reflexly activate cardiac output and raise plasma noradrenaline levels in young normal subjects.

The haemodynamic and catecholamine responses to supine leg exercise were studied in vasovagal syncope (n = 10), pure autonomic failure (n = 10) and in control (n = 10) subjects. With exercise, blood pressure increased in controls; with a smaller rise in vasovagal syncope, and a substantial fall in pure autonomic failure. Heart rate increased similarly in controls and vasovagal syncope, but less in pure autonomic failure. The increase in cardiac index was less in controls and pure autonomic failure than vasovagal syncope; the fall in systemic vascular resistance was greatest in pure autonomic failure, but also fell more in vasovagal syncope than controls. Plasma noradrenaline levels increased in controls; with a smaller rise in vasovagal syncope and no increase in pure autonomic failure. Plasma adrenaline levels increased in vasovagal syncope only. The blood pressure responses to standing before and after exercise were similar in controls and vasovagal syncope, with no postural blood pressure fall; in pure autonomic failure there was a greater postural blood pressure fall post exercise. In conclusion, with supine exercise, blood pressure rose in controls and vasovagal syncope, and fell in pure autonomic failure. Systemic vascular resistance fell more in vasovagal syncope and pure autonomic failure, than controls. Noradrenaline responses differed and adrenaline rose in vasovagal syncope only. Standing post exercise did not induce syncope in vasovagal syncope, but increased postural hypotension in pure autonomic failure. There are clear differences in response to exercise in vasovagal syncope and pure autonomic failure. The differences between vasovagal syncope and control subjects suggest an underlying abnormality which may predispose to vasodepression in subjects with vasovagal syncope.

The neurohumoral, peptidergic and biochemical responses to supine leg exercise were studied in two groups with primary autonomic failure: Shy-Drager syndrome (SDS, n = 15) and pure autonomic failure (PAF, n = 15), to determine if these accounted for exercise-induced hypotension and the greater blood pressure (BP) fall in PAF. Responses were compared to normal subjects (controls, n = 15), in whom BP rose with exercise. Resting plasma noradrenaline (NA) was higher in controls than SDS, and was lowest in PAF. With exercise, NA increased in controls, with a small rise in SDS, but no change in PAF. Resting plasma adrenaline (A) was higher in controls and SDS than PAF, with no change during exercise. Plasma dopamine was unrecordable at all stages in all groups. Resting plasma renin activity (PRA) was higher in controls than SDS and PAF, and was unchanged with exercise in all groups. Plasma insulin, C-peptide and serum growth hormone (GH) were similar at rest and with exercise in the three groups. Plasma glucose was higher at rest in SDS and PAF, and increased with exercise in all three groups. In conclusion, neither exercise-induced hypotension, nor the differences between SDS and PAF could be related to abnormalities in the release of A, PRA, insulin, glucose or GH. The abnormal NA response to exercise was consistent with the BP fall being due to inadequate compensatory sympathetic activity. In SDS, the small NA increase, in the presence of supersensitivity, may have reduced their BP fall as compared to PAF. These results suggest that impaired sympathetic neural activity is a key factor in exercise-induced hypotension.

1. In autonomic failure, supine exercise lowers blood pressure and worsens postural hypotension. The somatostatin analogue, octreotide, reduces post-prandial and postural hypotension, but its effects on exercise-induced hypotension and on postural hypotension post-exercise are unknown. 2. Eighteen subjects with chronic sympathetic denervation were studied; 12 had pure autonomic failure and six had additional neurological features of the Shy-Drager syndrome. Haemodynamic, hormonal and biochemical changes were measured before, during and after incremental supine leg exercise on two occasions: on no treatment and after subcutaneous octreotide. Exercise was performed 120 min after octreotide in eight subjects and 60 min after octreotide in ten subjects. 3. Octreotide did not improve exercise-induced hypotension; the blood pressure fall was greater during exercise, but the blood pressure level was no different than without treatment. Heart rate, stroke distance, cardiac index and systemic vascular resistance were similar at rest and changed to the same degree with exercise on and off octreotide. After octreotide, resting levels of serum growth hormone, plasma noradrenaline, adrenaline and renin were unchanged, but glucose was higher and insulin was lower. There was no change in biochemical and hormone levels during exercise either off or on octreotide. 4. After octreotide, although the rate of blood pressure recovery was similar post-exercise, the levels of blood pressure were higher than in the non-treatment phase and postural hypotension was improved before and after exercise. 5. In conclusion, in primary autonomic failure, octreotide did not improve exercise-induced hypotension in the supine position, suggesting that octreotide-sensitive vasodilatory peptides do not contribute to the blood pressure fall.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The cardiovascular and catecholamine responses to supine leg exercise were measured in fifteen normal subjects (controls) and in three groups with sympathetic dysfunction: fifteen with central failure (Shy-Drager syndrome; SDS), fifteen with peripheral failure (pure autonomic failure; PAF) and two with isolated dopamine beta-hydroxylase deficiency (DBH deficiency). 2. With exercise, blood pressure increased in controls, fell markedly in SDS and PAF and was unchanged in DBH deficiency. After exercise, blood pressure rapidly returned to baseline in controls, but remained low in SDS and PAF. With exercise, heart rate increased more in controls than SDS or PAF; the response varied in DBH deficiency. 3. With exercise, cardiac output increased similarly in controls, SDS and PAF, with a larger increase in DBH deficiency. Vascular resistance fell less in controls than SDS, PAF and DBH deficiency. 4. With exercise, plasma noradrenaline increased in controls only; plasma adrenaline remained unchanged in all groups. In DBH deficiency, plasma noradrenaline and adrenaline were undetectable, but plasma dopamine was elevated and rose further with exercise. 5. Supine exercise substantially lowered blood pressure in sympathetic failure due to SDS and PAF. In DBH deficiency blood pressure was unchanged; this lack of fall may have been due to vasoconstriction induced by dopamine and other substances released from otherwise intact sympathetic terminals, or to preserved cardiac vagal function.

The involvement of proteoglycans in hair growth has been recognized through the observation of increased hair growth in diseases such as the mucopolysaccharidoses and pre-tibial myxedema, which involve an increase in skin proteoglycan content. In an attempt to understand this, we have examined the distribution of chondroitin 6 sulphate (C6S), unsulphated chondroitin (COS), dermatan sulphate (DS), and heparan sulphate proteoglycans (HSPG) in frozen tissue sections of normal scalp by immunostaining. Results show that during anagen, the thick connective tissue sheath around the follicle strains strongly for C6S, COS, and DS. COS is uniquely associated with this region and is not found beneath the epidermis or infundibular epithelium. HSPG is, however, localized in the basement membrane zone adjacent to the outer root sheath. In addition, all of these proteoglycans are localized in the dermal papilla. In mid-catagen, we observed significant loss of C6S and COS staining from both the dermal papilla and the connective tissue sheath, but no decrease in staining for HSPG. In late catagen, very little staining of C6S and COS was observed. In early anagen, we observed that C6S was again present in the connective tissue sheath and dermal papilla; however, COS staining appeared to be weaker and less closely associated with the follicle. HSPG staining was observed in early anagen in a pattern very similar to that found for other basement membrane components. Results for DS were not obtained for catagen or early anagen. These results provide further evidence that hair growth is associated with the presence of chondroitin proteoglycans in the follicle environment and that the cessation of growth is associated with their removal. Further studies are underway to characterize the relationship between hair growth and proteoglycans.

The binding of NK cells to a target cell appears to be a necessary step for NK cell-mediated cytolysis. In this report, we demonstrated effector-target binding by immunoelectron microscopy by using monoclonal antibodies against NK cells (Leu-7, Leu-11a) and T-cell subsets (Leu-2a/T8, Leu-3a/T4). The surfaces of NK and K562 cells were characterized by antitransferrin receptor antibody and various lectins. In addition, the controversial phagocytic activity of NK cells was studied by incubation of peripheral blood mononuclear cells with opsonized Staphylococcus aureus and labeling with anti-Leu-7 or anti-Leu-11a antibody. Results showed that only Leu-11a+ cells displayed a broad cell-to-cell contact with the target by a shallow intercellular interdigitation of cytoplasmic projections, while Leu-7+, Leu-2a+, or Leu3a+ cells showed only a partial contact with target without interdigitation. The Leu-11a+ cells were frequently observed in small clusters and in close association with monocytes. Cluster formation and association with monocytes were not observed in other NK and T-cell immunophenotypes. In Leu-11a+ cells conjugated with target cells, membrane-bound granules, small vesicles, parallel tubular arrays, Golgi apparatus, endoplasmic reticulum, and small vacuoles were evident and concentrated toward the target. The surface of NK cells was intensely stained for glycoprotein by chromic acid-phosphotungstic acid, whereas target cells were not stained. Transferrin receptors were stained only on the surface of target cells. Only the lectins RCA and UEA labeled the surfaces of both NK and target cells. Phagocytic vacuoles containing cell debris or fragments and ingested bacteria were found in the cytoplasm of Leu-11a+ cells but not in Leu-7+ cells. NK cells were also found within the cytoplasm of K562 target cells. All these findings suggest that Leu-11a+ cells are the true functional NK cells involved in NK cell-mediated cytolysis, phagocytosis, and emperipolesis. Therefore, the NK cell is probably "a phagocyte in lymphocyte's clothing." The presence of peroxidase in the small vesicles of NK cells and endocytotic vesicles of target cells at the effector-target contact area indicates that cytolytic enzymes or factors derived from NK cells may be transported into the target by endocytosis.

Bacterial lipopolysaccharide (LPS) is known to be implicated in the pathogenesis of endotoxemia and septic shock. The liver is the first vital organ to exhibit pathological alterations in shock. The present studies include immunoelectron microscopic localization of tissue fibronectin and cytochemical localization of calcium and enzymes in hepatocytes of animals with LPS-induced endotoxemia or cecal ligation-induced septic shock. The results showed increased staining of fibronectin in the basal (perisinusoidal) surfaces and in the cisternae of rough endoplasmic reticulum and the Golgi complex of hepatocytes in rats with endotoxemia or septic shock. Intracellular calcium content was significantly increased in the LPS-treated or septic rats. Calcium pyroantimonate precipitate was deposited predominantly on the outer surfaces of the RER of hepatocytes. In addition, diminution or depletion of glycogen, reduction of catalase-containing peroxisomes, increase of G-6-Pase activity, and depletion of cytochrome c oxidase in many mitochondria were also observed in hepatocytes of experimental animals. The overall results suggest that LPS stimulates: (a) hepatic synthesis and secretion of fibronectin; (b) uptake of calcium by hepatocytes; and (c) G-6-Pase activity. LPS treatment also leads to reduced numbers of peroxisomes and depletion of cytochrome c oxidase.

Bacterial endotoxin (lipopolysaccharide, LPS) and interleukin-2 (IL-2) are known to stimulate NK cell mediated cytotoxicity against tumor cells. In the present report we sought to correlate the stimulatory effect of LPS and IL-2 on NK cell activity with ultrastructural changes which occurred as a result of such stimulation. Peripheral blood mononuclear cells (PBMC) were purified from healthy donors by a Ficoll-Hypaque density gradient technique. Leu-11a+ NK cells were isolated by flow microfluorometry using a monoclonal FITC conjugated anti-Leu-11a antibody and a FACS II cell sorter. The PBMC were incubated, respectively, with E. coli LPS or recombinant IL-2 (IL-2) for various time periods. Sorted Leu-11a+ NK cells were incubated with LPS for 24 hours. The NK cytotoxicity in the PBMC and sorted Leu-11a+ cells was assessed by a 51Cr release technique using K562 tumor cells as targets. Leu-11a+ NK cells were identified by immunoelectron microscopy using anti-Leu-11a antibody and labeling with horseradish peroxidase or colloidal gold. Results showed that both LPS and IL-2 significantly enhanced the cytotoxic activity of PBMC. The cytotoxicity of sorted Leu-11a+ cells was augmented by LPS. Recombinant IL-2 induced a significant increase in the number of dense granules, hypertrophy of Golgi apparatus and rough endoplasmic reticulum, and mitosis of Leu-7+ cells and Leu-11a+ cells 4 or 7 days after stimulation. These data indicate that: (1) the effect of LPS on the enhancement of NK cytotoxicity in PBMC may be a direct and/or indirect process involving production of lymphokines; (2) LPS has a direct effect on sorted Leu-11a+ cells; (3) IL-2 stimulates mitosis of Leu-7+ cells and Leu-11a+ cells; and (4) the LPS or IL-2 induced ultrastructural changes in Leu-11a+ cells are consistent with the enhanced NK cytotoxicity.

Trypanosoma duttoni exhibits the typical trypanosome morphology in that it is bounded by a unit membrane which included its anterior flagellum; it is surrounded by a framework of suppellicular fibrils, and it contains a nucleus and the posteriorly located kinetoplast-blepharoplast structures along with cytoplasmic organelles (mitochondria, Golgi, and enoplasmic reticulum). Very prominently displayed in T. duttoni is the contractile vacuole, which has not previously received wide-spread recognition. The kinetoplast is clearly continuous with mitochondria in some cases. Inclusion bodies are categorized into three distinct types, none of which can be conclusively designated as the very popular volutin granules, without cytochemical evidence. The existence of a third tubule among peripheral tubule doublets of the flagellum is observed.

This tutorial paper describes techniques for processing microorganisms for scanning electron microscopy (SEM). For ease of discussion, the subject is divided into two sections: A. General Processing Requirements, and B. Specific Processing Techniques. The objective of the first section is to outline processing requirements, explain their purpose, and point out where variations are possible. The following basic steps are discussed: (1) specimen selection, (2) prefixation treatment, (3) chemical fixation, (4) dehydration, (5) critical-point drying and freeze-drying, (6) coating, and (7) viewing. The second section describes methods of manipulating microorganisms through the processing steps. Instructions will cover microorganisms processed for SEM (1) in suspension, (2) in tissues. (3) in tissue culture, and (4) on agar. We are relying heavily on our own experiences in the laboratory and the results are illustrated by use of bacteria, mycoplasmas, rickettsiae, fungi, free-living protozoa, and parasitic protozoa. This tutorial is intended to be a general reference for processing microorganisms for study with the scanning electron microscope. Although we have described basic requirements and several specific techniques, it must be emphasized that there is a wide range of preparation flexibility permissible, depending upon the objectives of the study.

Lipopolysaccharides (LPS) of gram-negative bacteria are known to augment the ability of macrophages and natural killer (NK) cells to lyse susceptible target cells. In the present studies, we sought correlations between the ultrastructural changes and function of Leu-11a+ cells from the peripheral blood mononuclear cells which occurred as a result of their incorporation of LPS. We also studied the effect of LPS on the NK activity of purified Leu-11a+ cells. LPS samples from E. coli and Pseudomonas aeruginosa were utilized. A double-immunolabeling technique employing immunogold and immunoperoxidase markers was used to identify the Leu-11a+ cells with incorporated LPS. Pinocytosis, phagocytosis, and direct insertion into membrane bilayers appeared to be the routes for incorporation of LPS by Leu-11a+ cells. Subsequent ultrastructural effects included dilation of intracellular membrane compartments, formation of tubuloreticular inclusions and increased acid phosphatase activity. Opsonized Staphylococcus aureus were ingested both by control and LPS-treated Leu-11a+ cells; however, the number of Leu-11a+ cells with bacteria increased significantly as a result of LPS treatment. Autoradiography combined with immunolabeling showed no [3H] thymidine incorporation by either control or LPS-stimulated Leu-11a+ cells. NK cell-mediated cytotoxicity was significantly increased as compared with the control (p less than or equal to 0.02) when isolated Leu-11a+ cells were treated with LPS for 24 hours. This result suggests that LPS may have direct effect on the NK cell activity. Tests of peripheral blood mononuclear cell samples after LPS treatment showed up to a 2-fold increase in NK cytotoxicity and a dose-related increase of in vitro interferon production. This latter finding together with the ultrastructural observations of tubuloreticular inclusions in Leu-11a+ cells suggests that, in addition to any direct effect of LPS, cytotoxic activity could be indirectly augmented as a result of autocrine or paracrine interferon, or lymphokine production.

Human natural killer (NK) cells have been reported to express various surface antigens. The majority and the most functionally potent NK cells are Leu-11a (NKP-15) positive cells. Only a small number of functional NK cells express Leu-7 (HNK-1) antigen. In the present study, we have established techniques for immunoelectron microscopic identification of NK cells by mouse monoclonal FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies. Ficoll-Hypaque-isolated peripheral blood lymphocytes (PBL) were reacted with the specific antibodies before or after fixation in a 1% glutaraldehyde/1% paraformaldehyde fixative. Prefixation labeling of viable cells with the antibodies was carried out at 4 degrees C or 37 degrees C. Cells prelabeled with anti-Leu-11a antibody were reacted with secondary antibodies either before or after fixation. Anti-Leu-7 antibody was stained directly via an avidin-biotin-peroxidase (ABC) system, anti-Leu-11a antibody was stained indirectly by the ABC immunoperoxidase procedure via a biotinylated anti-mouse IgG secondary antibody or by a 10 nm or 40 nm colloidal gold-labeled anti-mouse IgG antibody. Results indicate that Leu-7 antigen could be localized by incubation with the specific antibody either before or after 20 min fixation; however, Leu-11a antigen was totally abrogated following the same fixation procedure. The Leu-11a antigen was well stained by the methods of prefixation labeling of cells with anti-Leu-11a antibody and incubation with a biotinylated secondary antibody and the ABC system after fixation. With respect to colloidal gold labeling, better results were obtained when cells were reacted with the gold-labeled antibodies immediately after incubation with anti-Leu-11a antibody but before fixation. Ultrastructurally both Leu-7 positive (+) and Leu-11a positive (+) cells shared common ultrastructural features associated with large granular lymphocytes. Using the above described techniques, we found approximately 2-5% Leu-7+ and 9-15% Leu-11a+ cells in the PBL of healthy donors. The overall results suggest that Leu-11a antigen is more sensitive to glutaraldehyde/paraformaldehyde fixation than Leu-7, since it can be localized only by prefixation labeling procedures; the ABC immunoperoxidase procedure is an ideal technique for labeling NK cells for light and electron microscopic enumeration; the immunogold method provides an adequate technique for labeling NK cells which are designated for ultracytochemical studies.

The interactions of osteoblasts with one another and with the extracellular milieu are of vital importance for cell function. These interactions are mediated by cell membrane-associated components. In the present work, we studied the distribution of several mediators known to be associated with the cell surface, using ultrastructural cytochemistry, to characterize the three cell membrane domains (osteoid, lateral, and vascular) of osteoblasts. Osteoblasts in neonatal rat calvariae were studied for cell surface distribution of alkaline phosphatase (APase), calcium-activated adenosine triphosphatase (Ca2+-ATPase), calcium, soybean agglutinin (SBA)-reactive sites, and peanut agglutinin (PNA)-reactive sites. APase was absent in the osteoid domain but was evenly distributed in the other domains. Ca2+-ATPase was found to be concentrated mainly in the lateral domains. In contrast, calcium was present in all cell membrane domains. Using lectins conjugated to horseradish peroxidase, we demonstrated that SBA binding sites were evenly distributed along the osteoblast cell membrane, whereas PNA binding sites were absent or minimally present in the osteoid and lateral domains but were evenly distributed in the vascular domain. These results suggest that the various functions of osteoblasts may be facilitated by specialized cell membrane domains which are cytochemically distinct. Previous reports have failed to demonstrate the cytochemical differences between the three domains of the osteoblast cell membrane.

In pursuing the mechanism of endotoxin action, we examined the effect of lipopolysaccharide (LPS) and its chemically defined components, lipid A and lipid X on cultured bovine endothelial cells. We report that LPS and lipid A caused detachment and altered morphology of endothelial cells while lipid X did not. Phorbol myristate acetate, a compound known to activate protein kinase C, also caused endothelial cell detachment. Morphologic changes were readily apparent in the endothelial cells after 6 hours of exposure to lipopolysaccharide (1 microgram/ml); at that time many of the cells had contracted and formed bleblike structures on the surface. Large vacuoles, dense bodies, and pyknotic nuclei were found in the detaching cells, indicating necrosis or cell death. Preceding the morphologic changes and actual detachment, endothelial cell DNA and RNA synthesis was impaired by LPS. The changes in DNA and RNA synthesis occurred within 4 hours of exposure to 1 microgram/ml of LPS when the cells were still able to maintain normal levels of ATP. In addition to the inhibition of nucleic acid synthesis, protein synthesis was inhibited after 6 and 8 hours of LPS exposure. DNA, RNA, and protein synthesis returned to control levels after 24 hours of exposure. Investigation on the cultured bovine endothelial cells as a model for LPS action was useful in that these cells are sensitive to relatively low levels of LPS and the endothelium may be an important target in sepsis.

The splenic B-lymphocyte population of C57BL/6 mice was analyzed by flow microfluorometry to determine the relative density of surface immunoglobulin (sIg) during the course of infection with Trypanosoma musculi and after the injection of T. musculi derivatives. Cells were stained with fluorescent-conjugated antisera directed against 7S mouse immunoglobulins or the major sIg components, IgM and IgD. Evaluation of relative density of sIg was made through observation of histograms plotted by the Fluorescence Activated Cell Sorter (FACS). The FACS also provided the percentage of fluorescence-positive cells detected with each stain. The relative density of sIg was altered in all experimental groups as evidenced by abnormal histograms. These alterations in relative density of sIg persisted only on B cells of the trypanosome-infected and not the T. musculi-derivative-treated animals. The changes in sIg presumably resulted from sIgD modulation, because the histograms resulting from cells stained with anti-IgM remained unchanged. In the trypanosome-infected animals, there also was a reduction in the percentage of sIgD-positive cells near the end of the parasitemia. Observations of spleen sizes showed the same pattern of change with splenomegaly as with relative density of sIg. These data may be the result of the persistence of the parasite in host tissues after clearance of the blood infection.