The p38 mitogen-activated protein kinase pathway regulates interleukin-6 synthesis in response to tumor necrosis factor in osteoblasts.

The induction of interleukin-6 (IL-6), using a proinflammatory cytokine (tumor necrosis factor-alpha), was studied in a human osteoblast cell line (MG-63) in relation to p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB transcription factor. When added to MG-63 cells, tumor necrosis factor-alpha (TNF-alpha) had a stimulatory effect on the production of IL-6, and this elevation was significantly reduced by SB203580, a specific p38 MAPK inhibitor. In addition, the stimulation of IL-6 release was also reduced by pyrrolidine dithiocarbamate (PDTC) or NF-kappaB SN50, which has been reported to be a potent NF-kappaB inhibitor. Both the NF-kappaB inhibitors in the presence of SB203580 had a more inhibitory effect on IL-6 release. In this study, TNF-alpha stimulated NF-kappaB binding affinity as well as p38 MAP kinase activation, leading to the release of IL-6. However, the specific inhibitor of p38 MAPK, SB203580, had no effect on TNF-alpha-induced NF-kappaB activation and both NF-kappaB inhibitors failed to reduce the p38 MAPK activation in the TNF-alpha-stimulated osteoblasts. In addition, inhibition of p38 MAPK partially, but significantly, impaired TNF-alpha-regulated release of osteocalcin, an important differentiation marker in osteoblasts. These results strongly suggest that both p38 MAPK and NF-kappaB are required in TNF-alpha-induced IL-6 synthesis and that these two TNF-alpha-activated pathways can be primarily dissociated. Furthermore, p38 MAPK may play a significant role in differentiation in MG-63 cells.

Production of hydrogen peroxide by serum and its involvement in cell proliferation in ROS 17/2.8 osteoblasts.

The effects of hydrogen peroxide, which fetal bovine serum (FBS) releases, on proliferation have been studied in ROS 17/2.8 osteoblasts. Cell proliferation, when activated by FBS, was inhibited by catalase in ROS 17/2.8 osteoblasts, but did not in primary osteoblast-like cells. Serum-induced extracellular signal-regulated kinase(ERK) activity was reduced by the pretreated catalase in ROS 17/2.8 osteoblasts. In addition, the present studies demonstrate that addition of FBS led to an increase of fluorescence of dihydrorhodamine 123, indicating formation of free radicals including hydrogen peroxide in ROS 17/2.8 osteoblasts, but not in primary osteoblast-like cells. These phenomena may account for the generation of reactive oxygen species during cellular proliferation in ROS 17/2.8 osteoblasts.

Dexamethasone suppresses tumor necrosis factor-alpha-induced apoptosis in osteoblasts: possible role for ceramide.

Ceramide has been proposed as a second messenger molecule implicated in a variety of biological processes, including apoptosis. Recently, it has been reported that tumor necrosis factor-alpha (TNF-alpha) activates the release of ceramide and that ceramide acts as a mediator for the TNF-alpha-induced stimulation of the binding affinity of nuclear factor-KB (NF-KB), a ubiquitous transcription factor of particular importance in immune and inflammatory responses. In this study we demonstrate that dexamethasone, which reduces the production of ceramide, significantly inhibits TNF-alpha-induced activation of NF-KB, c-Jun N-terminal kinase, also known as stress-activating protein kinase, caspase-3-like cysteine protease, redistribution of cytochrome c, and apoptosis in MC3T3E1 osteoblasts. Compared with TNF-alpha-induced JNK activation, ceramide elicits a more rapid activation of JNK within 30 min. C2-ceramide activates NF-KB and caspase-3 like protease to the same degree and with kinetics similar to those of TNF-alpha. This study provides evidence that the release of ceramide may be required as a second messenger in TNF-alpha-induced apoptosis. These results also suggest a regulatory role for dexamethasone in TNF-alpha-induced apoptosis via inhibition of ceramide release. Therefore, our in vitro results suggest that therapies targeted at the inhibition of ceramide release may abrogate inflammatory processes in TNF-alpha-related diseases, including rheumatoid arthritis and periodontitis.

Effect of ionizing radiation on the differentiation of ROS 17/2.8 osteoblasts through free radicals.

Although the acceleration of bone regeneration by radiation has been reported, the mechanisms of action of radiation on bone are unclear. The present results indicate that ionizing radiation-stimulated differentiation could result from the generation of reactive oxygen species during radiation exposure. The free radical release is considered as the most important mechanism of bone effect by radiation treatment. In addition, we report that radiation induced transient activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation and the transcription factor, AP-1. The JNK and AP-1 activation is mediated with radiation-released free radicals in ROS 17/2.8 osteoblasts. These results indicate that ionizing radiation at a single dose of up to 5 Gray stimulates differentiation of ROS 17/2.8 osteoblasts via free radial release which may affect JNK/SAPK and AP-1 activities.

Nitric oxide is a regulator of bone remodelling.

Nitric oxide (NO) is known to be implicated in the metabolism of bone, especially as a mediator of cytokine effects on the remodelling of bone tissue. In this study we examine whether NO affects the osteoblast activation or the osteoclast differentiation of primary mouse osteoblast-like and osteosarcoma ROS 17/2.8 cell lines. Primary osteoblast and ROS 17/2.8 cells released NO upon stimulation of interleukin-1 beta, tumour necrosis factor-alpha, and interferon-gamma. Sodium nitroprusside, a donor of nitric oxide, increased the activity of alkaline phosphatase in ROS 17/2.8 cells as well as the number of calcified nodule formations in primary mouse osteoblast-like cells. Sodium nitroprusside also completely inhibited 1 alpha, 25-(OH)2D3-induced osteoclast generation in a high concentration (100 microM). However, a low concentration of sodium nitroprusside (3-30 microM) significantly increased the generation of osteoclasts. These results indicated that NO appears to be an important regulatory molecule in the processes of bone formation and resorption. Hence, NO may be involved in the pathogenesis of bone loss in diseases associated with cytokine activation, such as periodontal disease and rheumatoid arthritis.

Effect of SO2 on nasal secretory cells.

Quantitative histochemical analysis of the secretory cells producing the different types of glycoproteins were examined in the chicken nasal cavity with and without moderate levels of SO2 exposure for 14 days. On the nasal maxillary concha of chickens, the number of acinar gland cells containing glycoproteins was significantly reduced on the 1st, 7th and 14th days of exposure to 11.8 ppm of SO2, but not on the 5th day of exposure. There was no histochemical modification in the intracellular glycoproteins of the acinar cells. The number of goblet cells in the same region increased greatly and showed a change of intracellular glycoproteins from neutral to acid between the 5th and 7th day of exposure.

Mechanism of slowing of mucociliary transport induced by SO2 exposure.

Responses of nasal mucociliary transport mechanisms to exposure to 6 ppm SO2 were studied in chickens in vivo. This model takes advantage of the natural cleft palate which exposes the mucociliated base of the nasal septum. Exposure to 6 ppm SO2 decreased the mucociliary transport rate along the base of the nasal septum. The minimum force required to move an iron particle along this area of mucous membrane by use of a magnetic field in vivo increased significantly after SO2 exposure, while the minimum force required to move an iron particle on a pool of mucus collected from the same chicken and tested in vitro showed no change after SO2 exposure. The elastic recoil distance of mucus was measured both in vivo and in vitro. The in vivo recoil distance decreased significantly after SO2 exposure, while SO2 exposure did not change recoil distance in vitro. It is proposed that exposure of chickens to SO2 results in the formation of multiple points of adhesion of strands of mucus between the acinar gland cells and the emergent extracellular mucus or adhesion of a mucous blanket to the cilia, causing mucociliary transport to be retarded or static.

Effect of mechanical stimulation on mucociliary clearance of chicken sinus.

The hypothesis that homeostatic control mechanisms control mucociliary function in ciliated mucous membranes was induced artificially by means of mechanical stimulation. The edge of right palatine cleft was stimulated mechanically by gentle touching with a dissecting needle, and sinus clearance time was recorded as soon as mechanical stimulation was initiated. Mechanical stimulation caused acceleration of mucociliary flow of the sinus; sinus clearance time was accelerated on the side adjacent to the mechanically stimulated side of the palatine cleft, but not on the opposite side. Therefore, the reflex may be effective only on the stimulated side. We investigated the effect of nerve blockers on mechanical stimulation. Mucociliary clearance in the chicken sinus was not affected by parasympatholytic agents, but was decelerated by the beta-adrenergic blocker. The effect of nerve blockers on the mechanical stimulation showed that parasympatholytic agents blocked mechanical stimulation, while sympatholytic agents did not completely block the response. These data suggest that mucociliary clearance may be regulated by the reflex of parasympathetic and partially sympathetic nerve fibers.

Effect of infection and SO2 exposure on nasal and paranasal mucociliary clearance in intact chickens.

The hypothesis that homeostatic control mechanisms control mucociliary function in ciliated mucous membrane was tested. Nasal mucociliary transport rates were recorded in chickens in vivo at successive intervals during exposure to SO2 or after inoculation with Newcastle disease virus (NDV), or both. Either agent alone caused deceleration of the turbinate clearance. However, when SO2 exposure was limited to one nasal fossa and turbinate mucociliary rates were determined in the unexposed and infected side, the two acted antagonistically and yielded approximately normal rates. Exposure of the nasal mucosae to SO2 caused decreased rates of sinus clearance, while NDV infection of nasal membranes induced increased rates of sinus clearance. Exposure of nasal mucosae to both agents acted antagonistically to effect rates of sinus clearance in normal ranges. These data support the idea of homeostasis.

Differences in mucus-stimulating serum fractions of cystic fibrosis patients and controls.

In nature, the urn cell complexes which swim in the coelomic fluid of the marine invertebrate, Sipunculus nudus, produce "tails" of mucus in response to bacterial pathogens. Since they produce measurable tails of mucus in vitro, suspensions of urn cell complexes provide a bioassay for mucus-stimulating substances (MSS) in biological fluids, including several human body fluids. Heat-activated seawater dilutions of human serum contain MSS. Serum from 87 cystic fibrosis (CF) homozygotes, 60 obligate heterozygotes, and 45 controls were fractionated on a Sephadex G-200 gel filtration column. After subsequent heating for 4 min at 85 degrees C, the fractions of all normal sera showed two characteristic peaks of MSS activity. The pattern differed in heated serum fractions of CF patients, in that the second peak was lacking in 59% of individual tests. The pattern was intermediate in heterozygote sera. Of the 36 CF serum fractions which did have two peaks of activity, 89% had the predominant activity in peak 1. The frequency of single peaks of activity increased with patient age, from 33% in those under 10 years to 75% in those over 16. The molecular weight of peak 1 is about 75,000 daltons, of peak 2 about 30,000. One may speculate that the frequent lack of peak 2 serum components may be associated with the inability of most CF patients to produce normal mucus following respiratory infection.

Effect of SO2 exposure on nasal mucociliary clearance in intact chickens.

The time required for mucociliary clearance from the chicken nasal turbinate and from the maxillary sinus was investigated in individual animals by using a newly designed plastic holder for the experimental animals. Determined in this way were: 1) the effect of SO2 exposure on sinus and turbinate clearance time, 2) the effect of the nerve blocking drugs atropine, scopolamine, reserpine, and propranolol on turbinate clearance time, and 3) the effect of these nerve blockers on clearance rates in chickens exposed to SO2. Turbinate mucociliary clearance was measured at 5 intervals per day, during 1 to 7 hr after exposure, for 7 consecutive days. Sinus clearance time was measured twice daily 1 to 4 hr after exposure. Turbinate clearance time in birds exposed to 6 ppm, and sinus clearance time in birds exposed to 40 ppm intermittently for 2 consecutive days both increased strikingly as a direct effect of SO2 exposure. However, continuous exposure to 6 ppm of SO2 during 16 hr per day for 7 consecutive days produced double peaks of increased turbinate clearance time with intervening recovery periods, suggesting an intranasal mucociliary homeostatic response. In individual animals, 26 of 35 animals (75%) exposed to 5 ppm, and 5 of 10 animals (50%) exposed to 20 ppm continuously during 16 hr per day for 7 consecutive days showed the same patterns. Reserpine and propranolol, which are sympatholytic agents, produced decelerated intranasal transport rates. Atropine and scopolamine, which are parasympatholytic agents, did not affect clearance rates. These nerve blockers, however, blocked the biphasic recovery pattern due to SO2 exposure. This blocking effect was statistically significant for atropine and reserpine 1 hr after injection.

Mucus-stimulating factor in tears.

Mechanisms responsible for regulation of tear film mucus are poorly understood. Humoral factors responsible for stimulation of mucus secretion can be studied in vitro by using the free-swimming urn cell, a normal component of the coelomic fluid of the marine invertebrate Sipunculus nudus. With this system, a tear mucus-stimulating factor was found in normal human tears but was markedly decreased in patients with dry eye syndromes. It is suggested that a mucus-stimulating factor exists in normal human tears and that a decrease in this substance may be instrumental in the pathophysiology of certain dry eye syndromes.

Mucus-stimulating substances in human body fluids assayed in an invertebrate mucous cell system.

An in vitro cell system has been shown to respond differentially to body fluids from normal subjects and from those with disorders of mucus secretion. The urn cell complex of the marine invertebrate Sipunculus nudus responds to mucus-stimulating substances (MSS) in normal human lacrimal fluids and stool filtrates by producing mucus. The process of mucus secretion can be directly observed, and the amount produced can be measured, in a calibrated light microscope. MSS are decreased in lacrimal fluids of patients with dry-eye conditions, while they are periodically increased in filtered stools of patients with acute Shigella dysentery and acute cholera. MSS are remarkably increased isotonic dilutions of sera of rabbits with acute mucoid enteritis, but are absent from sera of normal rabbits. MSS are present in isotonic dilutions of normal human sera which are heated to 85 degrees C for 4 minutes, but are absent from similarly processed sera of immunosuppressed patients. Mean MSS values of heated sera of children with cystic fibrosis are higher than those of controls. The active factor in tears and serum is a large molecule and is heat-stable.

Quantitative histological changes produced in the tracheal mucosa of young chickens by the inhalation of sulfur dioxide in low concentrations.

Chickens were exposed to SO2 in relatively low concentrations (3.4 to 18.5 parts per million (ppm)) for 1 to 14 days. A portion of their tracheas was embedded in water-soluble methacrylate, cut at 2 micrometer and stained with hematoxylin and eosin, Wright's stain, methyl green-pyronin, Alcian blue - periodic and Schiff, and for acid phosphatase. An increase was found in (a) the mucosa to wall ratio; (b) the number of mucosal cells in mitosis; (c) the number of macrophages, lymphocytes, plasma cells, and neutrophils in the epithelium and lamina propria; and (d) the number of these infiltrating cells which contained acid phosphatase. The number of mucus- and seromucus- secreting cells and vasoamine-containing cells were sometimes increased, but not consistently. The percentage of cells containing sialidase-sensitive sialomucins was elevated, and percentage of cells containing neutral mucins was reduced. These changes were only partly related to the SO2 concentration and the duration of SO2 exposure, in that increasing amounts of SO2 did not always cause increasing changes in the mucin composition. Evidently, the altered mucins sometimes protected against further mucin modification.

Ultrastructure of the mucociliary interface in the nasal mucosa of the chicken.

The ultrastructure of the extracellular mucous blanket at the nasal cavities of the chicken was preserved by a method that stabilizes primarily carbohydrate moieties. The cilia were apparently fixed as if "frozen" in the act of beating. The blanket was markedly heterogeneous, with a basic fibrous structure, and it contained membrane remnants. The lumenal surface of the blanket was smooth, but the surface in contact with the cilia penetrated to varying depths between the ciliary shafts. The findings are discussed in terms of the assumptions made by others on the basis of indirect evidence.

Mucociliary transport in chickens infected with Newcastle disease virus and exposed to sulfur dioxide.

Mucociliary transport was studied in the nasal mucous membranes and sinuses of 3-week-old chickens which were either exposed to sulfur dioxide (SO2), infected intranasally with the mesogenic strain of Newcastle disease virus (NDV), or exposed to SO2 after NDV infection. A newly developed apparatus was used to follow intranasal transport rates over time in the same animal, and to follow sinus transport rates over time in a separate group of animals. Intermittent exposure to concentrations of 1.4-66.0 ppm SO2 produced peaks of increased intranasal transport time, with intervening recovery periods. This suggests a homeostatic mechanism. Transport was also decelerated in the sinus when concentrations of SO2 were above 10 ppm. NDV infection produced decelerated intranasal transport rates but did not decelerate sinus rates. Combined NDV and SO2 interacted to produced persistent deceleration of the intransal transport rate. In the sinus, the combination seemed to conteract the decelerating effect of SO2 alone, suggesting a separate mechanism of homeostasis.

T and B lymphocyte rosetting in undernourished children.

T and B lymphocyte rosetting values were obtained for 18 children with kwashiorkor, marasmus, or nutritional edema. T cell values were subnormal in all malnutrition classes, but were lowest in children with kwashiorkor. Four of five malnourished children who were sensitized with 2,4-dinitrochlorobenzene (DNCB) before refeeding failed to respond to repeated subsequent challenges; five of six children who were sensitized after refeeding responded strongly to the first challenge.

Ecology of respiratory virus transmission: a comparison of three communities in West Bengal.

Respiratory virus transmission in children was studied comparatively in three ecologically different low-income communities in West Bengal: an isolated village, a suburban village, and a crowded urban community. Continued use of contaminated pond water for bathing, irrigation of nasal passages, post-defecation washing of the anus, and washing of food vessels was common to all, as was intense crowding of indoor sleeping quarters during cold and wet seasons. Intensity of infection was highest (26%) in the most crowded urban area, the variety of virus types least in the most isolated village. Sources of drinking water differed but seemed unrelated to virus transmission. Toxigenic diphtheria organisms were found in nonspecific skin lesions in children in each area.

Acute newcastle viral infection of the upper respiratory tract of the chicken. II. The effect of diets deficient in vitamin A on the pathogenesis of the infection.

Keratotic and squamous changes characteristic of vitamin A deficiency were minimal even in chicks which were malnourished and growth stunted and had no vitamin A in their diet. However, when these chicks were infected with Newcastle disease virus (NDV), keratotic changes appeared, most markedly in areas regenerating after infection. In chicks raised on full nutrient diets lacking only vitamin A, keratotic changes appeared in several areas of nasal mucosa but were absent from the mucosa of the inner (under) surface of the maxillary turbinate. Following NDV infection, such changes did appear in the inner lining epithelia. It is suggested that depletion of vitamin A causes regenerating epithelial cells to keratinize. Other effects of combined lack of vitamin A plus NDV infection were exhaustion of lymphoid cells from cranial bone marrow and exhaustion of lymphoid cell systems locally from the nose and paranasal glands.