Changes in human plasma essential fatty acid levels as a result of administration of linoleic acid and gamma-linolenic acid

Administration of doses of linoleic acid (LA) up to 36 g/d in humans raised blood levels of linoleic acid but not of its metabolites. This is probably because the conversion of LA to gamma-linolenic acid (GLA) is slow and rate-limiting. We have found that administration of relatively small amounts of GLA, up to 360 mg/d, raises human blood levels of dihomogammalinolenic acid (DGLA) and arachidonic acid (AA).     

Cellular basis of prolactin action: involvement of cyclic nucleotides, polyamines, prostaglandins, steroids, thyroid hormones, Na/K ATPases and calcium: relevance to breast cancer and the menstrual cycle.

The mechanisms of prolactin action are uncertain. Prolactin effects are characterised by great variability in the prolactin concentration required to produce a response and by the frequent occurrence of "bell-shaped" dose response curves. Relationships between prolactin and thyroid hormones and steroids are difficult to understand: both steroids and thyroid hormones may potentiate or inhibit prolactin effects in different situations. At the second messenger level, claims have been made that cyclic nucleotides, polyamines, Na/K ATPases and prostaglandins are involved. There is evidence that prostaglandin E1 may be an important second messenger and it is proposed that this is the clue to understanding the complexity. At low concentrations PGE1 enhances intracellular calcium release and at high concentrations PGE1 inhibits calcium release. The other second messengers proposed are all probably dependent upon calcium. Many prolactin effects may involve both PGE1 and another second messenger: at low prolactin concentrations PGE1 will potentiate the other messenger whereas at high concentrations the PGE1 effect will be inhibitory leading to a bell-shaped response. Thryoid hormones seem to enhance while glucocorticoids inhibit PGE1 synthesis. PGE1 dependent effects will thus be enhanced by thyroid hormone and blocked by cortisol. The reverse will be true of effects dependent on other second messengers.     

Gamma linolenic acid inhibits tyrosine phosphorylation of focal adhesion kinase and paxillin and tumour cell matrix interaction

Gamma linolenic acid (GLA) is an anti-cancer agent recently reported to inhibit tumour cell-matrix attachment. This study examined the effects of GLA on the adhesion of two tumour cell lines, HT115 (human colon) and MDA MB 231 (human breast), to an extracellular matrix, Matrigel. The action of GLA on focal adhesion kinase(FAK) and paxillin was also investigated. Following cell adhesion to Matrigel in control experiments, both FAK and paxillin were quickly tyrosine phosphorylated and become concentrated at focal adhesion areas. Inclusion of GLA resulted in an inhibition of the tyrosine phosphorylation of both FAK and paxillin leading to a reduced attachment of both cell types to Matrigel. FAK and paxillin were also less well distributed in the focal adhesions compared with the controls. It is concluded, therefore, that GLA inhibits tumour-matrix adhesion via the inhibition of FAK and paxillin tyrosine phosphorylation.     

Inhibition of membrane ruffling and ezrin translocation by gamma linolenic acid

Membrane ruffling of a tumour cell is correlated with its motile and metastatic behaviour. This study examined the effect of gamma linolenic acid (GLA), an anti-cancer agent, on HGF/SF induced membrane ruffling in the human cancer cell line, HT115. HGF induced a rapid appearance of membrane ruffling which was related to increased motility and the tyrosine phosphorylation and translocation of ezrin, a membrane-cytoskeleton linker protein. The presence of GLA significantly inhibited both the membrane ruffling and cell motility of the tumour cells, at sub-toxic concentrations. Western blotting revealed that the tyrosine phosphorylation of ezrin was inhibited by GLA. The translocation ezrin from cytosol and generalised areas of cell membrane to ruffled areas of the membrane induced by HGF/SF was also inhibited as shown by both indirect immunofluorescence and transmission electron microscopy. It is concluded that GLA inhibits HGF/SF induced membrane ruffling via its effect on ezrin, and this provides a further molecular explanation for the anti-tumour action of GLA.     

Nylon-fiber-induced neutrophil fragmentation

We have investigated the effects of mechanical elution of neutrophils from nylon-wool fiber (NWF) using the scanning electron microscope and biochemical analysis of elution fractions. We have determined that mechanical removal of neutrophils from nylon-wool fiber disrupts neutrophils adherent to nylon-wool fiber and augments release of granules, release of peripheral cytoplasmic fragments, and release of lactic dehydrogenase, a soluble cytoplasmic enzyme. Mechanical shearing of the adherent cell, and not adherence per se, causes the fragmentation. The extent of fragmentation is proportional to the NWF surface area available to neutrophils and is maximal at the temperature for optimal adherence and spreading. Agents that decrease cell spreading (n-ethylmaleimide and cold) diminish fragmentation. Cytochalasin B, an agent that destabilizes the neutrophil cortex, increases fragmentation. Fragmentation may be an important contributing cause of the abnormal morphology, function, and in vivo survival of nylon-wool-fiber procured human neutrophils. The prevention of fragmentation would appear to be necessary to insure the procurement of optimally functioning cells. Elution of NWF-adherent neutrophils in the cold might be a practical way to diminish neutrophil damage during clinical filtration leukapheresis.     

Alloimmunization to platelets in heavily transfused patients with sickle cell disease

Bone marrow transplantation (BMT) is now an option for some patients with sickle cell disease (SCD). Many SCD patients are multiply transfused with red blood cells (RBCs), and may be immunized to alloantigens other than erythrocyte antigens. Because platelet refractoriness is a significant complication during BMT, we wished to determine the prevalence of alloimmunization to platelets in transfused SCD patients. Sera collected from 47 transfused and 14 untransfused SCD patients were screened for HLA and platelet-specific antibodies. Transfusion and RBC antibody histories were reviewed. A subset of the patients were rescreened 1 year later. Eighty-five percent of patients with at least 50 RBC transfusions (22 of 26), 48% of patients with less than 50 transfusions (10 of 21), and none of 14 untransfused patients demonstrated platelet alloimmunization (P < .05). Platelet alloimmunization was more prevalent than RBC alloimmunization (20% to 30%). Half of the platelet reactivity was chloroquine-elutable. Eighteen of 22 patients (82%) on chronic RBC transfusion remained platelet-alloimmunized 11 to 22 months after initial testing. In summary, 85% of heavily transfused SCD patients are alloimmunized to HLA and/or platelet-specific antigens. These patients may be refractory to platelet transfusion, a condition that would increase their risk during BMT. Leukodepletion in the transfusion support of SCD patients should be considered to prevent platelet alloimmunization.     

Immunohistochemical localization of membrane and alpha-granule proteins in human megakaryocytes: application to plastic-embedded bone marrow biopsy specimens

Using a new technique for antigen localization, we have demonstrated platelet proteins in megakaryocytes in plastic-embedded biopsy specimens of normal human bone marrow. In a series of 25 specimens, megakaryocytes showed labeling with antibodies to the integral membrane glycoproteins IIIa, IIb, and the IIb-IIIa complex; granule membrane protein 140; and five alpha-granule matrix proteins: thrombospondin, factor VIII-related antigen, beta-thromboglobulin, platelet factor 4, and fibrinogen. The antibodies to the membrane glycoproteins IIIa, IIb, and IIb-IIIa produced diffuse cytoplasmic staining and heavier staining on the plasma membrane, whereas the antibodies to the alpha-granule matrix proteins produced a distinct granular staining within the cytoplasm. Staining for granule membrane protein 140 was also granular in distribution. Rare mononuclear cells consistent with megakaryocyte precursors were labeled with these markers. Other enzyme histochemical and lectin-binding studies showed that the enzyme alpha-naphthyl acetate esterase, the lectin Ulex europaeus I, and the periodic-acid Schiff reaction were consistent, but not specific, markers of megakaryocytes. This immunohistochemical technique should facilitate the examination of qualitative and quantitative changes in megakaryocytes in a variety of physiologic and pathologic processes.