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PubMed1981

Hydrogen-tritium exchange and nuclear magnetic resonance titrations of the histidine residues in ribonuclease St and analysis of their microenvironment.

Ribonuclease St consists of 101 amino acid residues in a single polypeptide chain with one disulfide bond. It has two histidines located at positions 60 and 91 from the amino terminus. The pKa values of His-60 and His-91 were estimated by hydrogen-tritium exchange titration to be 8.0 and 6.3, respectively, and these values were confirmed by 1H NMR titration. The high pKa value of His-60 suggests that it interacts with a neighboring negative charge, presumably of a carboxylate. This is suggested by the presence of an inflection at pH 4.5 in the 1H NMR titration plot for His-60. The 1H-NMR titration plot for His-91 also suggests its interaction with a carboxylate, although the pKa of His-91 was close to that of unperturbed histidine residues. This suggests that a positively charged group is also located in the vicinity of His-91. It was concluded that His-91 is one of the active site residues of the enzyme. The pKa for His-91 was shifted to the alkaline side in the presence of 3'-GMP, a competitive inhibitor, in the titration plots observed by both hydrogen-tritium exchange and 1H NMR spectroscopy. The 31P NMR titration data suggest that in the 3'-GMP-RNase St complex the dianion form of the nucleotide participates in the interaction with the protonated form of His-91. The existence of another positively charged group with pKa of 7.0 was also suggested on the basis of the 31P NMR data.

Amino Acid Sequence↗
PubMed1993

Structural-functional correlates of the nicotinic acetylcholine receptor and its lipid microenvironment.

Among the proteins specialized in signal transduction, the nicotinic acetylcholine receptor (AChR) is the best-characterized member of the superfamily of ligand-gated ion channels. Since the early finding of a motionally restricted shell of lipids (annulus) encircling the protein in its native membrane, there is growing experimental evidence supporting the notion that this interface region is the locus of a variety of pharmacologically relevant processes, including some of the actions of local anesthetics, steroids, and other lipophilic compounds. Biochemical data on the composition and metabolism of AChR-rich membranes and cells, combined with biophysical data on their structure and dynamics, are beginning to provide information on the nature of the annulus itself. Patch-clamp data on cells expressing the AChR protein add another dimension to this knowledge, enabling correlations to be established between the chemical composition of lipid-modified cells and the functional properties of the receptor protein in situ at the single-channel level. The effect of free fatty acids, alcohols, phospholipids, cholesterol, and steroid glucocorticoid hormones on the function of the AChR is reviewed. In the case of steroids, their effects at the single-channel level bear a relationship to their use in pathological conditions of medical relevance, e.g., in the treatment of the disease myasthenia gravis.

Amino Acid Sequence↗
PubMedDate not supplied

An acidic microenvironment inhibits antitumoral non-major histocompatibility complex-restricted cytotoxicity: implications for cancer immunotherapy.

Local immunosuppression may explain the failure of an effective immune response against solid tumors. Although it is well known that the interstitial pH is significantly lower in solid tumors than in normal tissue, only a few studies in the mouse system have investigated the influence of this acidic milieu on the anti-tumoral cytotoxic response. Here the authors report the suppression of human non-major histocompatibility complex (MHC)-restricted cytotoxicity against tumor cells by an acidic extracellular pH (pHe). Unstimulated peripheral blood mononuclear cells, lymphokine-activated killer (LAK) cells, and natural killer cell clones were used as effector cells. According to pH measurements in solid tumors, representative pH values of 7.2 to 5.3 were chosen during the cytotoxic assays. Target cell lysis was measured using two nonradioactive fluorometric methods, namely two-color flow cytometry and a modified calcein-release assay, which allowed cell-mediated cytotoxicity to be measured and compared with that in adherent targets. Using K562, Daudi, or Raji as suspended target cell lines, the cytotoxic activity of unstimulated peripheral blood mononuclear cells and of LAK cells was markedly reduced by a decreasing pHe. An extracellular pH of 5.8 to 5.3 resulted in a nearly complete loss of the cytotoxic response. This pHe-dependent impairment of the killing activity could also be shown for killer cells stimulated with interleukins-7 and -12, phytohemagglutinin, or lipopolysaccharide. The lytic potential of homogeneous natural killer cell clones as effectors was also strictly influenced by the surrounding pH. The pHe dependence of the non-MHC-restricted killer cell functions against tumor cells seems to be a general phenomenon, because the cytolytic activity of LAK cells against six human adherent tumor cell lines (HeLa, HepG2, LS174T, LS174Te, MCF-7, and RT112) was also clearly reduced under acidic conditions. To initiate the killing process, adhesion molecules play an important role in recognition and binding of the target cell. However, flow cytometric analysis revealed that the expression pattern of relevant adhesion molecules was unaffected by acidic pHe. In conclusion, these data clearly indicate an inhibition of non-MHC-restricted cytotoxicity against tumor cells by an acidic pHe, which may contribute to the failure of immunosurveillance against solid tumors. Consequently, efforts to enhance the anti-tumoral cytotoxicity by immunotherapies may have limited success.

Acids↗
PubMed1997

The immune microenvironment of human fracture/soft-tissue hematomas and its relationship to systemic immunity.

The immune environment of human soft-tissue injury is unstudied. We studied fracture soft-tissue hematomas (FxSTH) in 56 patients with high-energy bony fractures. FxSTH serum and mononuclear cells (MNC) as well as fracture patient plasma and blood MNC were studied. Twenty healthy controls donated plasma and MNC. Soluble tumor necrosis factor (TNF)-alpha, interleukin (IL-1 beta, IL-2, 6, 8, 10, 12, and interferon-gamma were studied by enzyme linked immunosorbent assay. Cells were studied by flow cytometry after cell-membrane stains for CD-14, TNF-alpha (mTNF), and human leukocyte antigen-DR, or intracellular stains for TNF (icTNF) and IL-10. Thirty-six patients with Injury Severity Score < 15 were analyzed further to evaluate the effects of isolated fracture on systemic immunity. Cytokines were rarely detectable in control plasma. TNF-alpha, IL-1 beta, IL-2, and interferon-gamma were rarely found in FxSTH serum or fracture patient plasma. All FxSTH sera were rich in IL-6, peaking before 48 hours (12,538 +/- 4,153 vs. 3,494 +/- 909 pg/mL, p = 0.02, U test). In Injury Severity Score < 15, IL-6 was not detectable in most early fracture patient plasma, but rose after 48 hours (p = 0.028). FxSTH serum IL-8 peaked after 48 hours (440 +/- 289 vs. 4,542 +/- 1,219 pg/mL, p = 0.006) and circulating IL-8 appeared after 72 hours. IL-6 and IL-8 showed gradients from FxSTH serum to paired PtS (p < 0.05, Wilcoxon). IL-10 was abundant (884 +/- 229 pg/mL) in FxSTH serum < 24 hours old. FxSTH serum IL-12 peaked late (3,323 +/- 799 pg/mL, day 4-7) then fell (p < 0.001, analysis of variance). Only IL-12 was higher in fracture patient plasma (1,279 +/- 602 pg/mL) than FxSTH serum (591 +/- 327 pg/mL) during the first 48 hours (p = 0.032, U test). On flow cytometry, control monocytes expressed 201 +/- 31 mTNF sites/cell, but icTNF was absent. mTNF was up-regulated after injury more in FxSTH monocytes (3,202 +/- 870 sites/cell) than peripheral blood monocytes (584 +/- 186 sites/cell) (p < 0.05 vs. peripheral blood monocytes by Wilcoxon, p < 0.001 vs. control monocytes by U test). Intracellular IL-10 was abundant in all MNC, but varied widely after injury. Fracture and peripheral blood monocytes expressed far less human leukocyte antigen-DR than control monocytes. Fractures create an inflammatory local environment. Proximal mediators are cell-associated and relatively confined to the wound, but soluble IL-6, IL-8, and IL-10 are abundant and probably exported. Systemic MNC have complex responses to local injuries. These may reflect the combined impact of multiple soluble cytokines initially generated within the wound. FxSTH appear to be a potentially important source of immunomodulatory cytokines in trauma.

Adolescent↗
PubMed1985

Alteration of neuroma formation by manipulation of its microenvironment.

In a primate model a histologic assessment of neuroma formation is reported. Three experimental groups were defined. Transected sensory nerves left adjacent to the incisional wound in an area of movement (wrist) were considered the control group. In the "proximally cut" group the same sensory nerves were positioned well proximal to the incisional wound. In the "muscle-implantation" group these nerves were placed in adjacent muscles. At 6 months a histologic assessment of the neuroma formation in the three experimental groups was carried out. Implantation of the sensory nerve into muscle significantly altered the regenerative potential of that nerve. The muscle completely surrounded the sensory nerve. The minimal neuroma that formed had significantly less scar tissue and contained nerve fibers that were of a smaller diameter and decreased density than either the control or the proximally cut group. There were no histologic differences between these latter two groups. However, regeneration into the overlying skin that was noted in the control neuromas was not seen in those nerves which had been proximally cut.

Animals↗
PubMed1999

Gut microenvironment and immune function.

Overreaction of the acute phase response is responsible for the two major complications to surgery, sepsis and thrombosis, but also most likely for the leading sequela to surgery, adhesion formation. The gastrointestinal tract, especially the colon, is a major player in the acute phase response and responsible for important immune functions with important interactions between the commensal flora, mucosal cells and the mucosa/gut associated lymphoid tissues. These responses can effectively be modulated by enteral nutrition, provided it is properly composed and administered. There is increasing evidence that the important clinical effects sometimes observed in enteral nutrition are more related to immunostimulatory effects than to reduction in microbial translocation. It is suggested that in order to be effective enteral nutrition should be instituted if possible before the operation, but always at least immediately after. Furthermore, much supports that the formula given should contain what has been called colonic food, e.g. plant fibres, and have a low content of saturated fat. Use of antibiotics with deleterious effects on the commensal flora should also be limited as much as possible. Lack of compliance with these requests seems to explain the lack of consistency in clinical experience of enteral nutrition, when tried in connection with trauma and clinical surgery.

Acute-Phase Reaction↗
PubMed2005

The microenvironment around total hip replacement prostheses.

The metal stem of the totally replaced hip carries load and resists fatigue, but it is electrochemically corroded. Metallic atoms act as haptens, induce type 1 T-helper cells/Th1-type immune responses and enhance periprosthetic osteolysis. Stiff metal implants, which do not have the same elasticity as the surrounding bone, cause stress shielding. Cyclic loading and lack of ligamentous support lead to mechanical and ischemia reperfusion injury and particle formation from bone, polymethylmethacrylate, and porous implant surfaces, which accelerate third-body polyethylene wear. Surgical injury and micromotion induce the formation of a fibrous capsule interface. Type-B lining cells produce lubricin and surface-active phospholipids to promote solid-to-solid lubrication but may loosen the implant from bone. The pumping action of the cyclically loaded joint and synovial fluid pressure waves dissect the implant-host interface and transports polyethylene particles and pro-inflammatory mediators to the interface. Hyaluronan induces formation of a synovial lining like layer. Because of its localization close to bone, foreign body inflammation at the interface stimulates osteoclastogenesis and peri-implant bone loss. Metal-on-metal and ceramic-on-ceramic pairs might minimize third body wear, but can lead to high-impact load of the acetabulum. Diamond coating of a metal-on-polyethylene couple might solve both of these problems. The basic biomaterial solutions allow good mechanical performance and relatively long life in-service, but surface modifications (porous coating, hydroxyapatite, diamond, bioglass, and others) may facilitate performance of the implant and improve the biomaterial and body interfaces.

Arthroplasty, Replacement, Hip↗
PubMed2006

The pulmonary and hepatic immune microenvironment and its contribution to the early systemic inflammation following blunt chest trauma.

OBJECTIVE: Blunt chest trauma is accompanied by an early increase in plasma cytokine concentrations. However, the local sources of these mediators are poorly defined. We investigated the impact of blunt chest trauma on the inflammatory mediator milieu in different compartments (lung tissue, bronchoalveolar lavage, liver tissue, Kupffer cells, plasma) along with the time course of trauma-induced pulmonary endothelial barrier dysfunction to elucidate potential relationships. In addition, the correlation between intratracheally instilled interleukin-6 and its systemic release were studied. DESIGN: Prospective, randomized, controlled animal study. SETTING: Basic science laboratory of a university affiliated level 1 trauma center. SUBJECTS: Male C3H/HeN mice, 8-9 wks old, n = 141. INTERVENTIONS: Blunt chest trauma induced by a focused blast wave, intravenous injection of Evans blue, and intratracheal instillation of recombinant human interleukin-6. MEASUREMENTS AND MAIN RESULTS: Two hours after blunt chest trauma, plasma interleukin-6 was markedly increased. Simultaneously, interleukin-6, tumor necrosis factor-alpha, macrophage inflammatory protein-2, monocyte chemotactic polypeptide-1 and neutrophil/monocyte accumulation in bronchoalveolar lavage and interleukin-6, monocyte chemotactic polypeptide-1, and myeloperoxidase activity in lung tissue were significantly increased. This was accompanied by a coinciding elevation in the Evans blue lung-plasma ratio. Recombinant human interleukin-6, instilled intratracheally before blunt chest trauma, was detected in a dose-dependent manner in the plasma of the mice. Additionally, Kupffer cell interleukin-6, tumor necrosis factor-alpha, and interleukin-10 production was significantly augmented as early as 30 mins after the insult. CONCLUSIONS: These results indicate that early increased cytokine concentrations in the lung, particularly interleukin-6, are important mediator sources as their local peak coincides with the systemic inflammatory response and is accompanied by a simultaneous impaired function of the pulmonary endothelial barrier. A direct relationship between their local and systemic concentrations can be established. Furthermore, this is the first study to show that Kupffer cells are activated early after blunt chest trauma.

Animals↗
PubMed2007

Modeling the cholesteatoma microenvironment: coculture of HaCaT keratinocytes with WS1 fibroblasts induces MMP-2 activation, invasive phenotype, and proteolysis of the extracellular matrix.

BACKGROUND: Increased keratinocyte proliferation, increased keratinocyte migration, elaboration of proteases resulting in proteolysis of the extracellular matrix (ECM), and destruction of surrounding tissues all typify the course of cholesteatoma growth. The contribution of stromal fibroblasts to these behaviors remains relatively unexplored. OBJECTIVES: Our objective for the current studies was to create a simple model with which to study these cholesteatoma behaviors, specifically, cell migration, invasion, and proteolysis of the extracellular matrix as well as the role of fibroblasts in the activated keratinocyte phenotype of cholesteatoma. DESIGN: The authors conducted an in vitro culture model. METHODS: The resulting model consists of activated keratinocytes (HaCaT cells) cocultured with normal dermal fibroblasts (WS1 cells) within a three-dimensional reconstituted ECM. We used a confocal imaging assay and software analysis to quantify total functional proteolysis of the ECM in monotypic and organotypic cocultures. This was accomplished by growing cells on an artificial ECM comprised of Matrigel and DQ-collagen IV. DQ-collagen is a "quenched" fluorescent peptide whose fluorescence is unmasked by proteolytic cleavage. RESULTS: Organotypic cocultures of keratinocytes and fibroblasts exhibited increased cell migration, increased cell invasion, increased matrix metalloproteinase-2 secretion and activation, and increased proteolysis of type IV collagen in three-dimensional ECM. Exposure to NSC27366, inhibitor of the small GTPase, Rac, resulted in reduction in both cell invasion and ECM proteolysis. CONCLUSIONS: Stromal fibroblasts may stimulate the invasive phenotype of keratinocytes, including ECM proteolysis. Increased cell invasion and proteolysis are dependent on the Rac pathway in this model. This simple culture model may help further our understanding of these destructive behaviors in cholesteatoma keratinocytes.

Cell Communication↗