John J. Turek

Modulation of macrophage cytokine production by conjugated linoleic acids is influenced by the dietary n-6:n-3 fatty acid ratio

Abstract Conjugated linoleic acids (CLA) are a mixture of 18:2 isomeric fatty acids that have conjugated double bonds. We undertook this study to determine if CLA altered spleen fatty acid composition, macrophage cytokine production, and liver prostaglandin E2 (PGE2) biosynthesis in the presence of varying ratios of (n-6) and (n-3) polyunsaturated fatty acids (PUFA). Groups of six rats were fed diets (AIN-93G) containing soybean oil (SBO) or menhaden oil and safflower oil (MSO), with CLA (10 g/kg) or without CLA. Spleens of rats consuming CLA contained the t9,c11 and t10,c12 isomers of CLA and a lower content of 18:1 fatty acids. CLA significantly reduced basal and lipopolysaccharide (LPS) induced levels of interleukin-6 (IL-6) by resident peritoneal macrophages in rats given SBO. CLA also reduced basal levels of tumor necrosis factor (TNF) production in both diets, but had no effect on LPS-induced levels of TNF. CLA had no effect on IL-1 production, but macrophage IL-1 production was increased in MSO fed rats compared with SBO fed rats. Rats given MSO had reduced PGE2 production in spleen compared with SBO fed rats. In SBO fed rats, CLA caused some reduction in PGE2, but the effect was not significant. The effect of CLA on PGE2 production may be dependent on the type of dietary fat consumed and the organ being studied.

Imaging of tumor necroses using full-frame optical coherence imaging

Holographic optical coherence imaging (OCI) has been used to acquire depth resolved images in tumor spheroids. OCI is a coherence-domain imaging technique that uses dynamic holography as the coherence gate. The technique is full-frame (en face) and background free, allowing real-time acquisition to a digital camera without motional reconstruction artifacts. We describe the method of operation of the holographic OCI on highly scattering specimens of tumor spheroids. Because of the sub-resolution structure in the sample, the holograms consist primarily of speckle fields. We present two kinds of volumetric data acquisition. One is uses fly-throughs with a stepping reference delay. Another is static holograms at a fixed reference delay with the coherence gate inside the tumor spheroids. At a fixed reference delay, the holograms consist of time-dependent speckle patterns. The method can be used to study cell motility inside tumor spheroids when metabolic or cross-linking poisons are delivered to the specimens.

Effect of age, breed and dietary omega-6 (n-6) : omega-3 (n-3) fatty acid ratio on immune function, eicosanoid production, and lipid peroxidation in young and aged dogs

The focus of this study was to examine the influence of age and diet on various parameters of immune function in young and old Fox Terriers and Labrador Retrievers. Eighteen young and old dogs were utilized for this study. Young and old dogs were fed a basal diet containing an (n-6):(n-3) ratio of 25:1 for sixty days (Phase I). Half of the dogs were then switched to a diet with an (n-6):(n-3) ratio of 5:1, and all were maintained on their respective diets for an additional sixty days (Phase II). Results from these studies revealed an age-associated decline in several immune parameters measured. Both these breeds demonstrated a reduction in sheep red blood cell titers, as well as in their ability to respond to different mitogens. Interestingly, this decline was greater in Fox Terriers, suggesting a decrease in cellular proliferative capacity in lymphocytes isolated from the larger breed. Neither cytokine production or DTH response was affected by age. Diet and breed interactions resulted in a significant increase in T- and B-cell mitogen responsiveness. In contrast, supplementation with n-3 fatty acids did not affect IL-1, IL-6 or TNF-alpha production. Supplementation with n-3 fatty acids resulted in increased PGE3 production from peritoneal macrophages but had no effect on PGE2 production from peripheral blood mononuclear cells or peritoneal macrophages. The n-3 fatty acid supplementation did not influence alpha-tocopherol status although older dogs had significantly lower serum alpha-tocopherol concentrations. Oxidative status of these dogs was assessed by serum levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Feeding an n-3-enriched diet did not affect 4-HNE levels but significantly decreased MDA levels in old dogs. In summary, this study indicates that feeding a diet containing an (n-6):(n-3) fatty acid ratio of 5:1 had a positive, rather than a negative, effect on the immune response of young or geriatric dogs.

Holographic optical coherence imaging of tumor spheroids

We present depth-resolved coherence-domain images of living tissue using a dynamic holographic semiconductor film. An AlGaAs photorefractive quantum-well device is used in an adaptive interferometer that records coherent backscattered (image-bearing) light from inside rat osteogenic sarcoma tumor spheroids up to 1 mm in diameter in vitro. The data consist of sequential holographic image frames at successive depths through the tumor represented as a visual video “fly-through.” The images from the tumor spheroids reveal heterogeneous structures presumably caused by necrosis and microcalcifications characteristic of human tumors in their early avascular growth.

Omega-3 fatty acids enhance ligament fibroblast collagen formation in association with changes in interleukin-6 production.

Altering dietary ratios of n-3 and n-6 polyunsaturated fatty acids (PUFA) represents an effective nonpharmaceutical means to improve systemic inflammatory conditions. An effect of PUFA on cartilage and bone formation has been demonstrated, and the purpose of this study was to determine the potential of PUFA modulation to improve ligament healing. The effects of n-3 and n-6 PUFA on the in vitro healing response of medial collateral ligament (MCL) fibroblasts were investigated by studying the cellular coverage of an in vitro wound and the production of collagen, PGE2, IL-1, IL-6, and TNF. Cells were exposed to a bovine serum albumin (BSA) control or either eicosapentaenoic acid (EPA, 20:5n-3) or arachidonic acid (AA, 20:4n-6) in the form of soaps loaded onto BSA for 4 days and wounded on Day 5. AA and EPA improved the healing of an in vitro wound over 72 hr. EPA increased collagen synthesis and the overall percentage of collagen produced, but AA reduced collagen production and total protein. PGE2 production was increased in the AA-treated group and decreased in the EPA-treated group, but was not affected by wounding. IL-1 was not produced at the time point evaluated, but TNF and IL-6 were both produced, and their levels varied relative to the PUFA or wounding treatment. There was a significant linear correlation (r2 = 0.57, P = 0.0045) between IL-6 level and collagen production. These results demonstrate that n-3 PUFA (represented by EPA in this study) positively affect the healing characteristics of MCL cells and therefore may represent a possible noninvasive treatment to improve ligament healing. Additionally, these results show that MCL fibroblasts produce PGE2, IL-6, and TNF and that IL-6 production is related to MCL collagen synthesis.

Time-dependent speckle in holographic optical coherence imaging and the health of tumor tissue

Holographic optical coherence imaging acquires en face images from successive depths inside scattering tissue. In a study of multicellular tumor spheroids the holographic features recorded from a fixed depth are observed to be time dependent, and they may be classified as variable or persistent. The ratio of variable to persistent features, as well as speckle correlation times, provides quantitative measures of the health of the tissue. Studies of rat osteogenic sarcoma tumor spheroids that have been subjected to metabolic and cross-polymerizing poisons provide quantitative differentiation among healthy, necrotic, and poisoned tissue. Organelle motility in healthy tissue appears as super-Brownian laser speckle, whereas chemically fixed tissue exhibits static speckle.

Volumetric motility-contrast imaging of tissue response to cytoskeletal anti-cancer drugs

Microscopic imaging of cellular motility has recently advanced from two dimensions to three dimensions for applications in drug development. However, significant degradation in resolution occurs with increasing imaging depth, limiting access to motility information from deep inside the sample. Here, digital holographic optical coherence imaging is adapted to allow visualization of motility in tissue at depths inaccessible to conventional motility assay approaches. This method tracks the effect of cytoskeletal anti-cancer drugs on tissue inside its natural three-dimensional environment using time-course measurement of motility within tumor tissue.

Dietary polyunsaturated fatty acid effects on immune cells of the porcine lung.

The effects of various dietary polyunsaturated fatty acids (PUFAs) on the function of immune cells of the porcine lung was studied. Groups of six pigs were fed diets containing 10.5% corn oil [CO; enriched in linoleic acid (18:2, n‐6)], linseed oil (LO; enriched in α‐linolenic acid (18:3, n‐3)], menhaden oil (MO; enriched in eicosapentaenoic (20:5; n‐3) and docosahexaenoic (22:6; n‐3) acids], linseed + corn oil (1:1; LC), and menhaden + corn oil (1:1; MC) for 28–30 days. Basal levels of alveolar macrophage (mφ) tumor necrosis factor‐α (TNF‐α) production were higher (P < .05) for LC‐ and MC‐fed pigs than for CO‐ and LO‐fed pigs. Lipopolysaccharide (LPS)‐stimulated LC and MC mφs produced more TNF than mφs from pigs fed CO, LO, and MO diets. Macrophages from pigs receiving the CO and LC diets had higher (P < .05) levels of leucine aminopeptidase than mfs from the other dietary groups. Lipopolysaccharide did not increase mφ nitrite production over basal levels except in the MO diet group. However, LPS‐stimulated mφs from the CO, MO, and LC dietary groups produced more nitrite than mfs from MC‐fed pigs. Alveolar lymphocytes from pigs receiving the MC diet produced more T cell growth factors than LO and MO mφs. Alveolar mφs from the different dietary groups did not differ in their capacity for non‐immune‐mediated phagocytosis of fluorescent latex beads. These results indicate that dietary PUFAs can modulate some functions of porcine alveolar immune cells and that this may prove significant for host response to respiratory disease agents. J. Leukoc. Biol. 56: 599–604; 1994.

Fourier-domain digital holographic optical coherence imaging of living tissue

Digital holographic optical coherence imaging is a full-frame coherence-gated imaging approach that uses a CCD camera to record and reconstruct digital holograms from living tissue. Recording digital holograms at the optical Fourier plane has advantages for diffuse targets compared with Fresnel off-axis digital holography. A digital hologram captured at the Fourier plane requires only a 2D fast Fourier transform for numerical reconstruction. We have applied this technique for the depth-resolved imaging of rat osteogenic tumor multicellular spheroids and acquired cross-section images of the anterior segment and the retinal region of a mouse eye. A penetration depth of 1.4 mm for the tumor spheroids was achieved.

Linoleate Impairs Collagen Synthesis in Primary Cultures of Avian Chondrocytes

Abstract The effects of supplemental fatty acids, vitamin E (VIT E), and iron-induced oxidative stress on collagen synthesis, cellular injury, and lipid peroxidation were evaluated in primary cultures of avian epiphyseal chondrocytes. The treatments included oleic and linoleic acids (0 or 50 μM) complexed with BSA and dl-α-tocopheryl acetate (VIT E at 0 or 100 μM). After 14 days of preculture, the chondrocytes were enriched with fatty acids for 8 days then cultured with VIT E for 2 days. The chondrocytes were then treated with ferrous sulfate (0 or 20 μM) for 24 hr to induce oxidative stress. Collagen synthesis was the lowest and the activity of lactate dehydrogenase (LDH) was the highest in chondrocyte cultures treated with 50 μM linoleic acid and 0 VIT E. In contrast, VIT E supplemented at 100 μM partially restored collagen synthesis in the chondrocytes enriched with linoleic acid and lowered LDH activity in the media. The iron oxidative inducer significantly increased the values of thiobarbituric acid-reactive substances (TBARS) in the culture medium. The data showed that linoleic acid impaired chondrocyte cell function and caused cellular injury but that VIT E reversed these effects. Results from a previous study demonstrated that VIT E stimulated bone formation in chicks fed unsaturated fat, and the present findings in cultures of epiphyseal chondrocytes suggest that VIT E is important for chondrocyte function in the presence of polyunsaturated fatty acids. VIT E appears to be beneficial for growth cartilage biology and in optimizing bone growth.