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Interleukin-10 (IL-10), an anti-inflammatory cytokine, may have therapeutic potential in the fetal inflammatory response syndrome and its sequelae such as bronchopulmonary dysplasia (BPD). Our aim was to compare the effects of IL-10 versusdexamethasone (DEX) on important PMN functions of the newborn. PMNs were isolated into culture medium from cord blood after elective cesarean section deliveries. IL-10 and DEX were compared on an equimolar basis corresponding to previously measured plasma levels of DEX from infants treated for BPD. The endotoxin (LPS)-stimulated release of the pro-inflammatory cytokines, tumor necrosis factor (TNF) and IL-1, were markedly inhibited equally by IL-10 and DEX; the anti-inflammatory cytokine IL-4 was not released and IL-1 receptor antagonist (IL-1ra) was released less with DEX compared with IL-10. PMNs exposed to LPS, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), or S. aureusdid not show a significant difference between control, DEX and IL-10 for apoptosis, respiratory burst, phagocytosis or killing respectively. Chemotaxis to fMLP or IL-8 was unaffected by DEX or IL-10. The principal effects of both IL-10 and DEX, on the PMN functions studied, are related to the control of pro- and anti-inflammatory cytokine release.

Interleukin-10 (IL-10), an anti-inflammatory cytokine, may have therapeutic potential in the fetal inflammatory response syndrome and its sequelae such as bronchopulmonary dysplasia (BPD). Our aim was to compare the effects of IL-10 versus dexamethasone (DEX) on important PMN functions of the newborn. PMNs were isolated into culture medium from cord blood after elective cesarean section deliveries. IL-10 and DEX were compared on an equimolar basis corresponding to previously measured plasma levels of DEX from infants treated for BPD. The endotoxin (LPS)-stimulated release of the pro-inflammatory cytokines, tumor necrosis factor (TNFa) and IL-1ß, were markedly inhibited equally by IL-10 and DEX; the anti-inflammatory cytokine IL-4 was not released and IL-1 receptor antagonist (IL-1ra) was released less with DEX compared with IL-10. PMNs exposed to LPS, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), or S. aureus did not show a significant difference between control, DEX and IL-10 for apoptosis, respiratory burst, phagocytosis or killing respectively. Chemotaxis to fMLP or IL-8 was unaffected by DEX or IL-10. The principal effects of both IL-10 and DEX, on the PMN functions studied, are related to the control of pro- and anti-inflammatory cytokine release.

An increase in polymorphonuclear leukocytes (PMNs) and proinflammatory chemokines, such as IL-8 and macrophage inflammatory protein-1 (MIP), are found in the airways during early stages of bronchopulmonary dysplasia. We determined whether IL-10 produces a dose-related inhibition of proinflammatory chemokine release from stimulated neutrophils of the newborn and whether the mechanism involves the pivotal transcription factor, nuclear factor-B. PMNs isolated from the cord blood of healthy newborns were stimulated submaximally with either lipopolysaccharide (n5) or tumor necrosis factor (n4), with and without IL-10 (0.01–1000 ng/mL). IL-8 and MIP release were measured in cell culture supernatants at 18 h. The presence or absence of nuclear factor-B activity and inhibitor-B degradation was measured at 30 min and 3 h after PMN stimulation began. During lipopolysaccharide stimulation, IL-10 significantly reduced IL-8 levels from 50 ± 16 ng/mL to 7 ± 3 ng/mL, and MIP levels from 14 ± 5 to 0.7 ± 0.1 ng/mL (mean ± SEM, p< 0.01). IL-10 produced an insignificant reduction in IL-8 and MIP levels after stimulation of PMNs with tumor necrosis factor. IL-10 did not inhibit nuclear factor-B activation and inhibitor-B degradation in PMNs stimulated with tumor necrosis factor or lipopolysaccharide for 30 min. After PMN stimulation for 3 h, inhibitor-B cytoplasmic levels were restored; however, they were unaffected by IL-10. We conclude that IL-10 is a potent inhibitor of lipopolysaccharide-stimulated release of IL-8 and MIP from neutrophils of the newborn via a mechanism not involving nuclear factor-B activity. Further work is needed to determine whether exogenous IL-10 may be useful for suppressing inflammation in bronchopulmonary dysplasia.

An increase in polymorphonuclear leukocytes (PMNs) and proinflammatory chemokines, such as IL-8 and macrophage inflammatory protein-1a (MIP), are found in the airways during early stages of bronchopulmonary dysplasia. We determined whether IL-10 produces a dose-related inhibition of proinflammatory chemokine release from stimulated neutrophils of the newborn and whether the mechanism involves the pivotal transcription factor, nuclear factor-?B. PMNs isolated from the cord blood of healthy newborns were stimulated submaximally with either lipopolysaccharide (n = 5) or tumor necrosis factor (n = 4), with and without IL-10 (0.01–1000 ng/mL). IL-8 and MIP release were measured in cell culture supernatants at 18 h. The presence or absence of nuclear factor-?B activity and inhibitor-?Ba degradation was measured at 30 min and 3 h after PMN stimulation began. During lipopolysaccharide stimulation, IL-10 significantly reduced IL-8 levels from 50 ± 16 ng/mL to 7 ± 3 ng/mL, and MIP levels from 14 ± 5 to 0.7 ± 0.1 ng/mL (mean ± SEM, p < 0.01). IL-10 produced an insignificant reduction in IL-8 and MIP levels after stimulation of PMNs with tumor necrosis factor. IL-10 did not inhibit nuclear factor-?B activation and inhibitor-?Ba degradation in PMNs stimulated with tumor necrosis factor or lipopolysaccharide for 30 min. After PMN stimulation for 3 h, inhibitor-?Ba cytoplasmic levels were restored; however, they were unaffected by IL-10. We conclude that IL-10 is a potent inhibitor of lipopolysaccharide-stimulated release of IL-8 and MIP from neutrophils of the newborn via a mechanism not involving nuclear factor-?B activity. Further work is needed to determine whether exogenous IL-10 may be useful for suppressing inflammation in bronchopulmonary dysplasia.

Background:Zinc deficiency is associated with chronic diarrhea. This condition is generally linked to an overproduction of nitric oxide (NO), which induces secretion and cellular damage as a free radical. Use of oral rehydration solutions (ORS) is an important part of diarrhea treatment, especially early in infancy and for patients with cholera. The presence of zinc in an ORS could be a positive factor in recovery from diarrheal disease.Objective:This study was undertaken to determine whether zinc added to an ORS could regulate the synthesis of NO metabolites in the lumen of zinc deficient rat intestine, acting as a gastrointestinal protector and thus accelerating normalization of intestinal function and zinc status.Methods:The effects of zinc on NO metabolism were studied in young male rats fed a zinc deficient diet for three weeks to mimic the condition of patients with recurrent diarrheal episodes. During the fourth week of the zinc deficient feedings, experimental diarrhea was induced using cathartics (magnesium citrate plus phenolphthalein) that exacerbate NO production. A standard ORS with or without 1 mM zinc was given to the rats for the last two days. A control group received a zinc sufficient diet. Rats were killed at each stage. Intestinal nitric oxide synthase (NOS) was assayed, cecal fluid contents were analyzed for nitrates and nitrites, intestinal histology was examined, and activation of nuclear factor NF-κB DNA binding activity was determined.Results:Rats fed the zinc-deficient diet for three weeks gained less weight than rats fed a normal zinc content diet and had a lower plasma zinc than controls (51.6 ± 5.4 [n = 10] vs. 143.6 ± 7.2 μg/dL [n = 11], p< 0.05). Recovery with ORS+Zn resulted in a higher plasma zinc than with the ORS-Zn (ORS+Zn: 186.5 ± 12.2; ORS−Zn: 57.5 ± 6.6 μg/dL, p< 0.05). The zinc-deficient diet did not alter mucosal NOS, as compared to the values of rats fed a normal diet. However, those animals which received five days of cathartic fluids had a small intestinal NOS higher than that of all other groups. Either ORS+Zn or ORS−Zn normalized NOS activity, regardless of cathartic treatment. The rats fed the zinc deficient diet had generally a higher content of NO metabolites in the cecum than rats fed a normal diet. After recovery with either type of ORS, rats given the cathartic remained with higher cecal NO metabolite concentrations than rats that had no induced diarrhea. After recovery with ORS+Zn, intestinal villi showed significant expansion of the lamina propria, an indication of greater absorption of fluid, while with ORS−Zn this was not present. Small intestinal homogenates of rats recovering with ORS+Zn had a decreased NF-κB DNA binding activity than tissues from rats consuming ORS−Zn.Conclusions:The results are consistent with the hypothesis that addition of Zn to an ORS may contribute to improving the physiologic status of the small intestine and potentially reduce the risks of recurrent diarrhea episodes.

ABSTRACTWe have observed elevated NF-?B DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-?B activity could be correlated with a reduced level of cytoplasmic I?Bß and could be associated with the overexpression of p21CIP1/WAF1in papilloma cells. Increased NF-?B activity and cytoplasmic accumulation of p21CIP1/WAF1might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.

We have observed elevated NF-kappaB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-kappaB activity could be correlated with a reduced level of cytoplasmic IkappaBbeta and could be associated with the overexpression of p21(CIP1/WAF1) in papilloma cells. Increased NF-kappaB activity and cytoplasmic accumulation of p21(CIP1/WAF1) might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.

Polymorphonuclear leukocytes (PMN) of the newborn, to a greater extent than those of the adult, have the ability to amplify PMN recruitment to an inflammatory site by their own release of IL-8, and this process is inhibited by dexamethasone. The aim of the present study was to determine whether the regulation of nuclear factor-B (NF-B) could explain the previous observations. NF-B is a transcription factor pivotal for expression of genes encoding inflammatory cytokines such as IL-8, but NF-B has not been previously studied in the PMN of the newborn. NF-B activation was measured by an electrophoretic mobility shift assay in nuclear extracts prepared from PMN isolated from adults and cord blood from newborns. Two distinct molecular forms of NF-B were identified after tumor necrosis factor- stimulation; this included the previously characterized p50/65 heterodimer and a newly identified p50/50 homodimer. Both NF-B dimers were activated by tumor necrosis factor- to significantly higher levels in the neutrophil of the newborn versusadult. An additional new finding was that pretreatment of PMN with dexamethasone (10-7M, therapeutic range) inhibited activation of both NF-B complexes in both the newborn and the adult PMN. We conclude that the increased activation of NF-B by the PMN of the newborn may play an important role in neonatal inflammatory reactions. Eventually, specific targeting of NF-B activation in the neutrophil may be an effective molecular approach for the treatment of neutrophil-mediated disorders in the newborn.

Polymorphonuclear leukocytes (PMN) of the newborn, to a greater extent than those of the adult, have the ability to amplify PMN recruitment to an inflammatory site by their own release of IL-8, and this process is inhibited by dexamethasone. The aim of the present study was to determine whether the regulation of nuclear factor-?B (NF-?B) could explain the previous observations. NF-?B is a transcription factor pivotal for expression of genes encoding inflammatory cytokines such as IL-8, but NF-?B has not been previously studied in the PMN of the newborn. NF-?B activation was measured by an electrophoretic mobility shift assay in nuclear extracts prepared from PMN isolated from adults and cord blood from newborns. Two distinct molecular forms of NF-?B were identified after tumor necrosis factor-a stimulation; this included the previously characterized p50/65 heterodimer and a newly identified p50/50 homodimer. Both NF-?B dimers were activated by tumor necrosis factor-a to significantly higher levels in the neutrophil of the newborn versus adult. An additional new finding was that pretreatment of PMN with dexamethasone (10-7M, therapeutic range) inhibited activation of both NF-?B complexes in both the newborn and the adult PMN. We conclude that the increased activation of NF-?B by the PMN of the newborn may play an important role in neonatal inflammatory reactions. Eventually, specific targeting of NF-?B activation in the neutrophil may be an effective molecular approach for the treatment of neutrophil-mediated disorders in the newborn.

The transcription factor NF-κB is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-κB activation in human neutrophils in response to stimulation with tumor necrosis factor-α (TNFα) are undefined. Since recent studies implicated activation of NF-κB as well as protein kinase C-δ (PKCδ) in neutrophil apoptosis, we investigated involvement of PKCδ in the activation of NF-κB in TNFα-stimulated neutrophils. Specific inhibition of PKCδ by rottlerin prevented IκBα degradation and NF-κB activation in TNFα-stimulated neutrophils. This regulation of NF-κB activation by PKCδ was specific only for TNFα signaling, since lipopolysaccharide- or interleukin-1β-induced NF-κB activation and IκBα degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IκBα localizes in significant amounts in the nucleus of unstimulated cells, and the amount of IκBα in the nucleus, as well as in the cytoplasm, correlates with the NF-κB DNA binding. These results suggest that in human neutrophils, the presence of IκBα in the nucleus may function as a safeguard against initiation of NF-κB dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-κB regulation.

Glucose absorption from the small intestine is largely mediated via the sodium-coupled glucose transporter (SGLT1). The goal of this study was to investigate the ontogenesis of the SGLT1, using the rat as an animal model at three stages of development: during lactation, at weaning, and at physiologic maturity. The techniques involved upper small intestinal perfusions with solutions containing 200 mM glucose and 50 mM NaCl, with or without 1 mM phloridzin (Phl), as an inhibitor of SGLT1. Molecular expression of the SGLT1 was also investigated via Western blot analysis from intestinal specimens of the three growth periods. Glucose absorption in weanling rats, in the absence of Phl, was several times higher than in sucklings and approximately double that of mature animals, and the effects of Phl were the greatest in weanlings. Furthermore, the physiologic data correlate to the molecular analysis of the SGLT1 which showed an increase in expression of the SGLT1 in both the weanlings and the adults compared to the sucklings. At all three stages of development Phl abolished Na absorption, and in sucklings there was a net outflow of Na. Due to the coupling between Na and water transport, net water absorption and the influx/efflux ratio, a more sensitive indicator of changes in unidirectional fluid movement, were similarly affected by Phl at the three stages of development. Net water absorption was highest in weanling animals. These findings are consistent with an early development of SGLT1 in rat small intestine and an apparent burst of activity at weaning. Less than complete maturity of other absorptive mechansims is occurring at this time.

Phosphatidylinositol (4)P 5-kinase (PtdIns(4)P 5-kinase) catalyzes the last step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic. To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2must be stringently regulated. In this paper we describe extraction, purification, and characterization of PtdIns(4)P 5-kinase from the plasma membranes of Schizosaccharomyces pombe.We also provide evidence that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S. pombehomolog of casein kinase I. Phosphorylation by Cki1 in vitrodecreases the activity of PtdIns(4)P 5-kinase. In addition, and most importantly, overexpression of Cki1 in S. pomberesults in a reduced synthesis of PtdIns(4,5)P2and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane. These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S. pombeand that Cki1 is involved in regulation of PtdIns(4, 5)P2synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase.

We constructed mutants of the prototypical, nuclear-accumulating protein nucleoplasmin and used them in bothin vivoandin vitronuclear transport assays to search for transport-influencing domains distinct from this protein's recognized nuclear localization sequence. We identified the polyglutamic acid tract on the amino flank of the nuclear localization sequence as being involved in two stages of nuclear transport. This poly-glu tract is required for the facilitated translocation of nucleoplasmin through the nuclear pore complex, and it also enhances the subsequent binding of nucleoplasmin within the nucleus.

Nucleoplasmin is a phosphorylated nuclear-accumulating protein. We report herein that the kinetics of its cytoplasm r nucleus transport are affected by its degree of phosphorylation. Therefore, we sought to identify any protein kinase which specifically associates with nucleoplasmin. We discovered that nucleoplasmin co-isolates by two independent methods (immunoabsorption and chromatography) in a complex including a kinase which phosphorylates nucleoplasmin. The co-purifying kinase is casein kinase II-like because: (i) it phosphorylates casein; (ii) its phospho-transferase activity can be competed out by GTP; (iii) it is stimulated by polylysine; and (iv) it is inhibited by heparin. Moreover, a polyclonal antibody to the α (38 kDa) and α′ (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin’s nuclear transport. Additionally, we found that phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones and that, in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II.