Digital self-aligned focusing schlieren.

In this Letter, a digital self-aligned focusing schlieren (D-SAFS) system is introduced. This system uses a digital transparent micro liquid crystal display (µLCD), in combination with a linear polarizer, to act on the linear polarization state of light transmitted in both the forward and reverse directions, essentially acting as both the source and cutoff grids. The use of the µLCD display allows for on-the-fly changes to the cutoff pattern type, spatial frequency, and orientation. This eliminates the need to physically access the source/cutoff grid in order to optimize the instrument's sensitivity, which is necessary with a conventional self-aligned focusing schlieren (SAFS) system.

Multi-point line focused laser differential interferometer for high-speed flow fluctuation measurements.

A multi-point focused laser differential interferometer (FLDI) has been developed to measure density fluctuations at 16 points along a line. A pair of cylindrical lenses on the transmitter side of a conventional single-point FLDI instrument form two closely spaced (≤200µm), orthogonally polarized, parallel laser lines at the instrument's focus. On the receiver side of the instrument, the interference of the beams on a 16-element photodiode array results in a single line of measurements. The further addition of a Nomarski prism creates two separate measurement lines, and the addition of a second photodiode array to the instrument enables simultaneous measurements of density fluctuations along the two lines separated by several millimeters. These two lines of measurement can be conveniently oriented at any azimuthal angle relative to the instrument's optical axis on the measurement plane, coinciding with the instrument's focus. Two experiments were performed to demonstrate the capabilities of the instrument. In the first experiment, a laser-induced breakdown spark generated a traveling spherical shock wave, and measurements of the resulting density disturbance and wave velocity were obtained. These results were compared to high-speed schlieren images of the shock wave acquired at 400 kHz. In the second experiment, the multi-point FLDI instrument was used to measure density disturbances in the boundary layer of a flat plate in a Mach 6 freestream flow. The measurements were made along two lines, both approximately 6 mm in length, extending from the surface of the plate through the boundary layer. High-speed schlieren images were acquired at 100 kHz during separate wind tunnel runs at matching unit Reynolds numbers to visualize the unsteady boundary layer flow and compare to the FLDI measurements.

Two-point, parallel-beam focused laser differential interferometry with a Nomarski prism.

A Nomarski polarizing prism has been used in conjunction with a focused laser differential interferometer to measure the phase velocity of a density disturbance at sampling frequencies ≥10MHz. Use of this prism enables the simultaneous measurement of density disturbances at two closely spaced points that can be arbitrarily oriented about the instrument's optical axis. The orientation is prescribed by rotating the prism about this axis. Since all four beams (one beam pair at each measurement point) propagate parallel to one another within the test volume, any bias imparted by density fluctuations away from the measurement plane on the disturbance phase velocity is minimized. A laboratory measurement of a spark-generated shock wave and a wind tunnel measurement of a second-mode instability wave on a cone model in a Mach 6 flow are presented to demonstrate the performance of the instrument. High-speed schlieren imaging is used in both cases to verify the results obtained with the instrument.

Severity of burn injury and sepsis determines the cytokine responses of bone marrow progenitor-derived macrophages.

BACKGROUND: Although thermal injury and sepsis result in enhanced monocytopoiesis, the functional characteristics of macrophages that develop in the microenvironment of burn and sepsis are unknown. Here we compare cytokine responses of bone marrow progenitor-derived macrophages (BMO) and peritoneal macrophages (PMO) after graded levels of thermal injury and sepsis. METHODS: Mice were randomly divided into sham (S), burn (B), and burn sepsis (BS) groups. The mild injury group received either a 7-second dorsal scald burn alone or in combination with 1,000 colony forming units (CFU) Pseudomonas aeruginosa at the wound site. The severe injury group was subjected to a 10-second burn with or without inoculation of 5,000 CFU P. aeruginosa. ER-MP12+ progenitors were separated from bone marrow cells 72 hour after injury. Macrophage colony stimulating factor (M-CSF) and Granulocyte-macrophage colony stimulating factor (GM-CSF) responsive clonogenic potentials, and lipopolysaccharide (LPS)-stimulated cytokine production were determined. RESULTS: In mild injury and sepsis, GM-CSF and M-CSF responsive clonal growth of ER-MP12+ progenitors was enhanced in the B and BS groups compared with the S group. M-CSF responsive colony growth in severe sepsis was significantly higher than that in all the other groups. LPS-stimulated tumor necrosis factor-alpha and Interleukin-6 levels were higher in the B and BS groups compared with the S group. Severe injury and sepsis attenuated this response significantly. The cytokine responses of PMO from both injury groups were similar to that of BMO. CONCLUSION: Severity of burn injury and the magnitude of sepsis influence the cytokine responses of BMO and PMO in a similar manner suggesting the microenvironment of burn injury and sepsis profoundly influence the functional phenotype of BMO.

Murine hematopoietic stem cells and progenitors express adrenergic receptors.

Association between the nervous and immune system is well documented. Immune cells originate within the bone marrow that is innervated. Thermal injury induces adrenergic stimulation, augments monocytopoiesis and alters the beta-adrenergic receptor (AR) profile of bone marrow monocyte committed progenitors. This provides an impetus to study AR expression in hematopoietic progenitors along myeloid lineage. Using FACS analysis and confocal microscopy, we report the expression of alpha1-, alpha2- and beta(2)-AR in enriched populations of ER-MP209(+) and ER-MP12(+) myeloid progenitors, CD117(+) and CD34(+) multi-potential progenitors and more importantly pluripotent stem cells suggesting a plausible role for catecholamine in hematopoietic development.

Immunologic responses to critical injury and sepsis.

Almost 2 million patients are admitted to hospitals in the United States each year for treatment of traumatic injuries, and these patients are at increased risk of late infections and complications of systemic inflammation as a result of injury. Host response to injury involves a general activation of multiple systems in defending the organism from hemorrhagic or infectious death. Clinicians have the capability to support the critically injured through their traumatic insult with surgery and improved critical care, but the inflammatory response generated by such injuries creates new challenges in the management of these patients. It has long been known that local tissue injury induces systemic changes in the traumatized patient that are often maladaptive. This article reviews the effects of injury on the function of immune system cells and highlights some of the clinical sequelae of this deranged inflammatory-immune interaction.

Norepinephrine modulates the inflammatory and proliferative phases of wound healing.

BACKGROUND: Injury results in the massive release of norepinephrine (NE) into the peripheral circulation. Recent investigations have demonstrated functional adrenoreceptors on the cellular mediators of cutaneous wound healing and NE-induced phenotypic alterations in immune cells have been demonstrated in vitro. Despite this, there is little description of how NE might alter the phases of wound healing in vivo. The purpose of this study was to compare cutaneous wound healing in norepinephrine-intact and norepinephrine-depleted mice. METHODS: Norepinephrine-depleted (NED) mice were generated by chemical axotomy with 6-hydroxydopamine and compared with norepinephrine-intact (NEI) animals (n = 6-12 per group, per time point). Using an excisional wound model, neutrophil recruitment was measured by myeloperoxidase assay. Macrophage recruitment and angiogenesis were measured by immunohistochemistry and re-epithelialization was determined histologically. The development of incisional wound disruption strength was determined over time. Finally, macrophage scavenger function was assessed by an in vitro latex bead phagocytosis assay. RESULTS: Wounds from NEI mice demonstrated greater neutrophil infiltration than NED wounds (24, 72 hours; p < 0.05). Wound macrophage recruitment was initially higher in NEI animals (24 hours, p < 0.05), but was eventually surpassed by that of NED animals (120 hours, p < 0.05). Angiogenesis was decreased while re-epithelialization was accelerated in NEI animals (p < 0.05). Wound disruption strength and macrophage scavenger function were unaltered between NED and NEI mice. CONCLUSIONS: Norepinephrine modulates the inflammatory and proliferative phases of wound healing in a temporally defined, cell-specific manner. By increasing recruitment of innate immune cells and expediting wound closure, norepinephrine appears to play a protective role in defense against infection.

Thermal injury and sepsis modulates beta-adrenergic receptors and cAMP responses in monocyte-committed bone marrow cells.

We have previously reported that adrenergic stimulation enhances monocytopoiesis following experimental burn injury and sepsis (BI/S). In the present work we measured beta-adrenergic receptor number and affinity in bone marrow committed monocyte progenitor cells (CD59(+)) following BI/S. We find that BI/S treatment significantly decreased monocyte progenitor cell beta-adrenergic receptors but significantly increased receptor binding affinity and isoproterenol-stimulated cAMP production. CD14 expression in macrophages derived in vitro from CD59(+) cells following BI/S was significantly increased by epinephrine and this change was blocked by beta(2)-adrenergic receptor antagonist. PCR analysis suggests the presence of beta(2)- but not beta(1)-adrenergic receptors. Enhanced adrenergic receptor signaling in CD59(+) bone marrow cells following BI/S may be important in macrophage development.

Adrenergic modulation of cytokine release in bone marrow progenitor-derived macrophage following polymicrobial sepsis.

Catecholamines may impact on the pathophysiology of sepsis by attenuating proinflammatory cytokine and augmenting antiinflammatory cytokine production by macrophages. We tested this premise in bone marrow monocyte progenitor-derived macrophages. Polymicrobial sepsis was induced in mice through cecal ligation and puncture. ER-MP 12 monocyte progenitors were isolated and differentiated into macrophages in vitro 72 hr later. Lipopolysaccharide (LPS)-stimulated cytokine production was measured with and without epinephrine, IL-10 and anti-IL-10 antibody. Epinephrine significantly increased IL-10 production, but attenuated TNF-alpha release exclusively through beta2 adrenergic receptors, and is independent of IL-10 production. Together, these results suggest that epinephrine can promote a potent antiinflammatory response in sepsis.

Bone marrow norepinephrine mediates development of functionally different macrophages after thermal injury and sepsis.

OBJECTIVE: We sought to determine the influence of thermal (burn) injury with sepsis and norepinephrine on the clonogenic potential and functional cytokine response to lipopolysaccharide (LPS) stimulation in nonmyeloid committed (CD117) and myeloid committed (ER-MP12) bone marrow progenitor cells. SUMMARY AND BACKGROUND DATA: We have previously demonstrated that norepinephrine stimulated myelopoiesis after burn injury and sepsis, but the site of this stimulation in monocyte development is unknown. In the present study the influence of norepinephrine on the developmental hierarchy of bone marrow cells after thermal injury and sepsis was determined by assessing the clonogenic potential and LPS-stimulated cytokine responses of mature macrophages derived from CD117 and ER-MP12 bone marrow progenitor cells. METHODS: Tissue and bone marrow norepinephrine content was ablated by chemical sympathectomy with 6-hydroxydopamine treatment. CD117 and ER-MP12 bone marrow cells were isolated using antibody-linked magnetic microbeads. Clonogenic potential in response to colony-stimulating factors was determined. Both progenitor cell types were differentiated to mature macrophages in vitro and tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 cytokine responses to LPS provocation were determined. RESULTS: The macrophage- and granulocyte-macrophage colony-stimulating factor responsive clonogenic potential was increased with burn sepsis, suggesting an expansion of both progenitor populations. Such increases were greatly reduced with prior depletion of norepinephrine. TNF-alpha and IL-6 cytokine responses to LPS were markedly influenced by the specific progenitor cells involved as well as the injury conditions and the status of norepinephrine prior to injury. In burn sepsis the depletion of norepinephrine resulted in a dramatic decrease in both IL-6 and TNF-alpha production by both progenitor-derived macrophages. CONCLUSIONS: Depletion of norepinephrine attenuated burn and burn sepsis-induced bone marrow progenitor clonal growth in response to macrophage- and granulocyte-macrophage colony-stimulating factor. Functional phenotypes of bone marrow progenitor-derived macrophages are greatly influenced by norepinephrine and the milieu created by thermal injury and sepsis.

Adrenergic modulation of splenic macrophage cytokine release in polymicrobial sepsis.

Enhanced adrenergic stimulation and catecholamine release are important components of the pathophysiology of sepsis. Under physiological conditions, adrenergic stimulation has been shown to be a negative regulator of proinflammatory cytokine production through increasing IL-10 production. Here we have investigated if adrenergic stimulation similarly inhibits TNF-alpha and IL-6 production by splenic macrophages isolated from a polymicrobial sepsis model. Male B(6)D(2)F(1) mice were subjected to sham (S), laparotomy (Lap), and cecal ligation and puncture (CLP) under anesthesia. Splenic macrophages were isolated 72 h after the initial injury and were stimulated with endotoxin (LPS) in the presence and absence of epinephrine. Compared with S and Lap, splenic macrophages from the CLP group produced significantly less TNF-alpha and IL-6 and more IL-10 when stimulated with LPS. Macrophage cultures from CLP animals incubated with either epinephrine or IL-10 for 2 h had significantly reduced TNF-alpha and IL-6 release in response to LPS. However, similar cultures pretreated with IL-10 antibody before the addition of exogenous epinephrine failed to reverse the attenuation of LPS-stimulated cytokines. Pretreatment of macrophage cultures with beta(2)- (ICI-118551) but not beta(1)-adrenergic (atenolol) receptor antagonists reversed the epinephrine-mediated cytokine attenuation following LPS treatment. Data are also presented that demonstrate the involvement of protein kinase A activation with adrenergic agonist but not with IL-10 stimulation. Taken together, these findings suggest that adrenergic mechanisms may influence peripheral tissue macrophage inflammatory cytokine response following trauma and sepsis, independent of the effects of IL-10.

Burn injury and pulmonary sepsis: development of a clinically relevant model.

BACKGROUND: Despite improvements in the early resuscitation of the critically injured, mortality from multiple organ failure has remained stable, with the lung often the first organ to fail. Early intubation and mechanical ventilation predispose patients to the development of pneumonia and respiratory failure. Our objective was to establish a murine model of combined injury, consisting of burn/trauma and pulmonary sepsis with reproducible end-organ responses and mortality. METHODS: Male B6D2F1 mice were divided into four groups: burn/infection (BI), burn (B), infection (I), and sham (S). Burned animals had a full-thickness 15% dorsal scald burn. BI and I groups were inoculated intratracheally with Pseudomonas aeruginosa (3-5 x 103 colony-forming units). S and B animals received saline intratracheally. All animals were resuscitated with 2 mL of intraperitoneal saline. Mortality was recorded at 24, 48, and 72 hours. Bacterial sepsis was confirmed by tissue Gram's stain of the lungs and positive organ and blood cultures for Pseudomonas aeruginosa. Femoral bone marrow cells were collected at 72 hours from surviving animals. Clonogenic potential was assessed by response to macrophage (M) colony-stimulating factor (CSF) and granulocyte-macrophage (GM) CSF in a soft agar assay and the data were represented as colonies per femur. Isolated alveolar macrophages and whole lung tissue were assayed for levels of the inflammatory cytokines tumor necrosis factor-alpha and interleukin-6. RESULTS: Mortality at 72 hours was 30% in BI, 12% in I, and <10% in B and S groups. Pneumonia was documented in all infected animals at 24 hours by Gram's stain and positive tissue cultures for Pseudomonas aeruginosa. Systemic sepsis as confirmed by blood, and remote organ cultures was seen in BI animals only. Significantly increased responsiveness to M-CSF stimulations was noted in all groups (BI, 8,291 +/- 1,402 colonies/femur; B, 6,357 +/- 806 colonies/femur; and I, 8,054 +/- 1,112 colonies/femur; p < 0.05) relative to sham (3,369 +/- 883 colonies/femur, p < 0.05). Maximal responsiveness to GM-CSF stimulation was noted in the BI group (11,932 +/- 982 colonies/femur, p < 0.05), and similar GM responsiveness was noted in all other groups (B, 7,135 +/- 548 colonies/femur; I, 7,023 +/- 810 colonies/femur; and S, 6,829 +/- 1,439 colonies/femur). Alveolar macrophage release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 increased in all animals, but the magnitude of increase was not proportional to the strength of the inciting stimulus. CONCLUSION: Although minimal perturbations were seen after burn or pulmonary infection alone, the combined insult of burn and pulmonary sepsis resulted in statistically significant hematopoietic changes with increased monocytopoiesis. Only the combined injury resulted in systemic sepsis and significantly increased mortality. We have developed a clinically relevant model of trauma and pulmonary sepsis that will allow further clarification of the inflammatory response after injury and infection.

Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression.

Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis.

High-efficiency solid-phase capture using glass beads bonded to microcentrifuge tubes: immunoprecipitation of proteins from cell extracts and assessment of ras activation.

We have bonded glass microbeads (425-600 microm diameter) to the inner walls of polypropylene microcentrifuge tubes. In addition to increasing the surface area of the tubes manyfold, the beads provide surface Si groups which can be reacted with a silane compound such as aminopropyltriethoxysilane, yielding a free amino group. The amino group is reacted with another cross-linking reagent, for example, the homobifunctional compound dimethyl suberimidate, which can form a covalent bond with amine groups of proteins. After binding protein A or G to the dimethyl suberimidate, the beads were used to immunoprecipitate proteins from cell extracts; we show that the protein A/G-coated glass beads yield similar amounts of immunoprecipitated proteins as a standard method using protein A- or G-agarose beads, but with fewer contaminating proteins. In addition, we show that when immunoprecipitating Ras from cell extracts and measuring the amounts of Ras-bound GTP and GDP, the new method yielded higher guanine nucleotide levels than protein G-agarose beads, suggesting that it caused less denaturation of Ras. Because the glass beads are bonded to the walls of the tubes, the immunoprecipitates can be washed rapidly and efficiently, and we show that 20-30 tubes can be washed in 1/10 the time required to wash immunoprecipitates on protein A- or G-agarose beads.