Quantitative buffy coat analysis in haematological patients compared to standard laboratory methods

We have tested a Quantitative Buffy Coat (QBC) instrument (Becton Dickinson, USA), and compared results obtained with this instrument to results obtained with standard methods in a haematology clinic. The basic principle of the method is a classical haematocrit centrifuge. The analysis provides a haematocrit value, platelet count, a total white blood count, and separates the white blood cells in granulocytes and mononuclear cells (lymphocytes plus monocytes). The instrument is easy to use but requires a trained observer. All results are available in 15 min. We have found the accuracy of the method good for all parameters. The precision of the instrument is good but for estimation of granulocytes and lymphocytes plus monocytes we did not find the high sensitivity claimed by the manufacturer (manufacturer's lower limit 0.02 x 10(9)/l; standard deviation for low levels of granulocytes: 0.300 x 10(9)/l and lymphocytes plus monocytes: 0.235 x 10(9)/l). A large fraction of samples (leukocytes 27%, platelets 40%) from a haematological clinic falls beyond the limits for reliable results set by the manufacturer, which reduces the utility of the instrument in such patients. Furthermore, in 21% and 10% of samples within the recommended range for leukocytes and platelets, respectively, QBC results could not be read owing to ill-defined boundaries. For granulocytes and lymphocytes plus monocytes 25% and 34% of the samples, respectively, could not be read owing to ill-defined boundaries. The instrument is not constructed to protect against blood contamination of the centrifuge and, therefore, the safety of the instrument is not satisfactory.     

Challenges for the translation of T cell costimulatory blockade therapies to the clinic

Ulcerative colitis (UC) and Crohn’s disease (CD) are debilitating idiopathic inflammatory bowel diseases (IBDs) with symptoms that impair ability to function and quality of life. The aetiology of IBD is inadequately understood and, therefore, drug therapy has been empirical instead of based on sound understanding of the disease mechanisms. This has been a major factor for poor drug efficacy and treatment-related side effects that often add to disease complications. The development of biologicals, notably infliximab, to block TNF-α reflects some progress, but there is major concern about their side effects and lack of long-term safety and efficacy profiles. However, IBD by its very nature is exacerbated and perpetuated by inflammatory cytokines, including TNF-α, IL-6 and IL-12, for which activated peripheral blood lymphocytes, monocytes/macrophages and granulocytes are major sources. Hence, activated leukocytes should be appropriate targets of therapy. At present, three strategies are available for removing excess and activated leukocytes by leukocytapheresis: centrifugation, Adacolumn^® and Cellsorba?. Centrifugation can deplete lymphocytes or total leukocytes, whereas Adacolumn selectively adsorbs granulocytes and monocytes together with a smaller fraction of lymphocytes (FcγR- and complement receptor-bearing leukocytes), and Cellsorba non-selectively removes all three major leukocyte populations. Efficacy has ranged from ‘none’ to an impressive 93% together with excellent safety profiles and downmodulation of inflammation factors. Furthermore, leukocytapheresis has shown strong drug-sparing effects and reduced the number of patients requiring colectomy or exposure to unsafe immunosuppressants, such as cyclosporin A. Leukocytapheresis removes from the body cells that contribute to IBD and, therefore, unlike drugs, it is not expected to induce dependency or refractoriness.     

Serum Amyloid A: EVIDENCE FOR ITS ORIGIN IN POLYMORPHONUCLEAR LEUKOCYTES

In this study the presence of an amyloid A, antigenically related material was determined in four subpopulations of human leukocytes. Monocytes, granulocytes, thymus-derived lymphocytes, and bone marrow-derived and null lymphocytes were isolated from the peripheral blood of five apparently normal subjects, two patients with secondary amyloidosis, three patients with acute infections, and seven patients with metastatic cancer. Mononuclear leukocytes, isolated from the interface of a Ficoll-Hypaque gradient, were separated into monocytes, thymus-derived lymphocytes, and bone marrow-derived plus null lymphocytes by glass adherence and depletion of sheep erythrocyte rosette-forming lymphocytes. Granulocytes were isolated by sedimentation in 2% methyl cellulose from the erythrocyte-rich pellet formed at the bottom of the Ficoll-Hypaque gradient. The four isolated leukocyte subpopulations were cultured and, at varying intervals, the amyloid A content of the culture medium and of sonicated, 2 x 10(6) cells was determined by radioimmunoassay. Our results indicated a 2-14 times greater amount of amyloid A-related material in the sonicated granulocytes compared with the individuals' serum amyloid A levels. The mononuclear subpopulations showed a low or negligible amyloid A content. The amount of amyloid A antigenic material was further found to increase in cultured granulocytes, reaching a peak value between the 16th and 30th h of culture. The granulocytes of only two out of eight individuals tested released amyloid A antigenically related material into the culture medium. This release was found to be blocked by the presence of colchicine, vincristine, puromycin, or cycloheximide in the culture medium. In contrast, only the presence of puromycin or cycloheximide was shown to significantly inhibit the intracellular increase of amyloid A in the cultured granulocytes. Thus, it appears that among the circulating blood cells, the granulocytes produce amyloid A antigenically related material and could release it under conditions that remain to be further defined.     

Sonoporation of Immune Cells: Heterogeneous Impact on Lymphocytes,Monocytes and Granulocytes

Microbubble-mediated ultrasound (MB-US) can be used to realize sonoporation and, in turn, facilitate the transfection of leukocytes in the immune system. Nevertheless, the bio-effects that can be induced by MB-US exposure on leukocytes have not been adequately studied, particularly for different leukocyte lineage subsets with distinct cytological characteristics. Here, we describe how that same set of MB-US exposure conditions would induce heterogeneous bio-effects on the three main leukocyte subsets: lymphocytes, monocytes and granulocytes. MB-US exposure was delivered by applying 1-MHz pulsed ultrasound (0.50-MPa peak negative pressure, 10% duty cycle, 30-s exposure period) in the presence of microbubbles (1:1 cell-to-bubble ratio); sonoporated and non-viable leukocytes were respectively labeled using calcein and propidium iodide. Flow cytometry was then performed to classify leukocytes into their corresponding subsets and to analyze each subset's post-exposure viability, sonoporation rate, uptake characteristics and morphology. Results revealed that, when subjected to MB-US exposure, granulocytes experienced the highest loss of viability (64.0 ± 11.0%) and the lowest sonoporation rate (6.3 ± 2.5%), despite maintaining their size and granularity. In contrast, lymphocytes exhibited the lowest loss of viability (20.9 ± 7.0%), while monocytes had the highest sonoporation rate (24.1 ± 13.6%). For these two sonoporated leukocyte subsets, their cell size and granularity were found to be reduced. Also, they exhibited graded levels of calcein uptake, whereas sonoporated granulocytes achieved only mild calcein uptake. These heterogeneous bio-effects should be accounted for when using MB-US and sonoporation in immunomodulation applications.     

Transient monocyte release of interleukin-10 in response to traumatic brain injury.

A significant component of the immune response to trauma results in the systemic presence of cytokines which have the potential to suppress the patient's immune response to infection and contribute to post-injury complications. We assayed peripheral blood leukocytes obtained from 10 patients with head trauma to determine their production of interleukin (IL). Serum was assayed for the presence of IL-10, TGFbeta1, and IFNgamma by ELISA. Peripheral blood leukocytes were screened for intracellular IL-10 and IFNgamma by fluorescence-activated flow cytometry, and cytokine-specific mRNA was detected by the polymerase chain reaction. We detected an immediate, but transient, presence of IL-10 in the sera of all 10 patients who suffered head trauma. IL-10-specific intracytoplasmic immunofluorescence was also detected immediately after injury in peripheral blood monocytes, but not in lymphocytes or granulocytes. IL-10-specific mRNA was detected in peripheral blood leukocytes in only 50% of patients immediately after injury, when the highest serum levels of IL-10 were observed. Our data indicates that release of pre-formed IL-10 by monocytes contributes to the presence of IL-10 found in patient peripheral blood immediately after head injury.     

Comparison of lymphocyte depletion and clinical effectiveness on filtration leukocytapheresis in patients with rheumatoid arthritis.

We evaluated the relationship between the clinical benefit of filtration leukocytapheresis (LCP) and the number of removed leukocytes in patients with rheumatoid arthritis (RA). LCP was performed in 31 drug-resistant RA patients. LCP was carried out 3 times with 1 week separating each session. Assessment of RA before and after LCP showed a substantial and rapid improvement in tender joint counts, swollen joint counts, and patients' and physicians' assessments. Careful analysis indicated that 19 of 31 patients with RA showed > or = 20% improvement following LCP therapy. The number of leukocytes in the peripheral blood significantly decreased during each session of LCP. However, there was no significant decrease in the number of circulating blood cells during the study period. No adverse reactions or complications were noted. There was no significant difference in any indices of clinical activity and the removal rates of leukocytes between responders and nonresponders. The total numbers of removed lymphocytes in responders were significantly higher than those in nonresponders (responders 64.1 x 10(8) versus nonresponders 50.7 x10(8), p < 0.05). The relationship between clinical effectiveness and the number of removed granulocytes and monocytes was not statistically significant. Our results suggest that filtration LCP to remove leukocytes from the peripheral blood, especially lymphocytes, exerts an immunomodulatory effect in patients with RA.     

Therapeutic cytapheresis for inflammatory bowel disease.

Cytapheresis therapy has recently been investigated as a treatment for several diseases, especially autoimmune related diseases such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. The removal of leukocyte components has been performed by the centrifugal method; however, using fiber technology or column technology, leukocyte components can be removed simply, and these technologies are more effective than the centrifugal method in removing numbers of cells. Each of 3 types of leukocytapheresis methods removes a different kind of cell in its therapeutic principle. Thus, if we understand what kind of cells should be removed, we can choose the best method for removing leukocytes. For this reason, the authors propose an international standard for unifying names. The therapy that makes use of a centrifuge to selectively remove about 40% of neutrophils and more than 60% of lymphocytes may be called a lymphocyte removal therapy, lymphocytapheresis (LCA). Using cellulose acetate beads in a G-1 granulocyte removal column, granulocytes and monocytes are removed but not lymphocytes, so we suggest calling this granulocytapheresis (GCAP). In addition, using a leukocyte removal filter, the Cellsorba leukocyte removal filter, 99% of both granulocytes and monocytes and about 70% of lymphocytes are removed. We propose calling this leukocytapheresis (LCAP). In the near future, we hope that we will be able to select one of these methods for cytapheresis for each disease pathogenesis or cellular immune abnormality. Presently, a lot of research is on-going to analyze how cytapheresis is effective for the immune related diseases. The mechanism of cytapheresis will be clarified by investigators. We strongly believe that cytapheresis therapies offer good news to those patients suffering from incurable diseases as well as their physicians.     

Receptors for complement of leukocytes

Sheep red blood cells sensitized by 7S, but not by 19S rabbit anti-Forssman antibodies, adhere and form rosettes on mouse macrophages and on a few monocytes and polymorphonuclear cells (PMN). When, however, C'' factors from mouse serum are added to the antigen-19S antibody complex (EAC''), rosettes are formed on most mouse peritoneal macrophages and PMN and on a few monocytes. In addition EAC'' also adheres to 10–25% of lymph node lymphocytes but not to thymus lymphocytes. EAC'' prepared with 7S anti-Forssman antibodies has identical properties. The adherence of red cells induces an increase in the membrane activity of the leukocytes and causes injury to the red cells which rapidly become deformed and fragmented. Adherence of EAC'' occurs at 37°C and is minimal at 4°C. Probably only the first four C'' components are involved in this phenomenon as mouse serum deficient in C''5 or rabbit serum, deficient in C''6 can be used as a source of C'' components. Treatment of EAC'' with EDTA does not modify its leukocyte-adherence properties. The adherence of EAC'' to the leukocytes is not inhibited in the presence of serum. The receptors for C'' on macrophages, PMN, and monocytes differ from those found on lymphocytes. Rosette formation by EAC'' on macrophages, PMN, and monocytes depends on divalent cations (Mg⁺⁺) and can be reversed by Na₃H EDTA, while adherence to lymphocytes is independent of these ions and occurs in the presence of 0.01 M Na₃H EDTA. Both types of receptors for C'' components are destroyed by trypsin treatment of the leukocytes, in contrast with the receptors for 7S antibodies on the same cells which persist after enzyme treatment.     

Studies on the activities and properties of lysosomal hydrolases in fractionated populations of human peripheral blood cells

Studies have been carried out on the activities and properties of the isozymes of alpha-mannosidase, alpha-glucosidase and beta-glucosidase in granulocytes, monocytes, lymphocytes and platelts from peripheral blood of heatlhy adult donors. The findings reveal the differences in activities as well as a characteristic distribution of the different molecular forms of these lysosomal hydrolases in specific cell types. Therefore, the results obtained with unfractionated total leukocyte smples from different subjects may vary according to the distribution of cell types in the circulation. Granulocytes and monocytes show only the acid alpha-mannosidase activity whereas lymphocytes and platelets show both acid and neutral activities. The specific activity of acid alpha-mannosidase in granulocytes and monocytes is higher than in lymphocytes and platelets. By DEAE-cellulose chromatography, the acid alpha-mannosidase in granulocyte and monocyte extracts elutes as two peaks, but only one peak is seen in lymphocytes. All cell types show both acid and neutral alpha-glucosidase activities. The specific activities of both isozymes are higher in granulocytes and monocytes than in lymphocytes and platelets. Monocytes show a higher acid than neutral activity. All other cell types show a higher neutral activity. Beta-Glucosidase in all cell types is mainly membrane-bound and it can be released by Triton X-100 and sodium taurocholate. Taurocholate also stimulates the beta-glucosidase activity of granulocytes, monocytes and lymphocytes whereas it inhibits the activity of this enzyme in platelets. These results indicate that variations in the total number of leukocytes and in the relative proportion of the various cell types in health and disease may yield inconsistent or unreliable values for enzyme activity in the diagnosis of lysosomal storage disease and in carrier detection.     

A rapid and simple radiometric assay for thymidine phosphorylase of human peripheral blood cells

A radiometric method is described for measuring thymidine phosphorylase activity in human peripheral blood cells. The substrates [¹⁴C] thymidine or [¹⁴C]thymine are converted to the base or deoxynucleoside, respectively, and two alternative Chromatographic methods to isolate the products of the reaction have been employed. With the described methods the specific activity for thymidine phosphorylase in human lymphocytes is 0.21 ± 0.08; monocytes 0.21 ± 0.16 and granulocytes 0.17 ± 0.02 μmol δ h^?1 δ mg^?1 protein. For human T- or null lymphoblasts, thymidine phosphorylase activity was found to be approximately 10% of that of B lymphoblasts.     

PEROXIDASE-MEDIATED MAMMALIAN CELL CYTOTOXICITY

Lactoperoxidase, in the presence of hydrogen peroxide and iodide is cytotoxic for human and mouse lymphoid cells, and human erythrocytes. Myeloperoxidase, in amounts equivalent to 1.5 x 10⁶ neutrophils, readily replaces lactoperoxidase, and allows the substitution of the iodide ion by chloride. The myeloperoxidase-mediated reaction is rapid, and highly efficient, leading to 85–90% cell death in 90 min, as measured by ⁵¹chromium release and dye exclusion. The mixture of granulocytes. monocytes, and lymphocytes present in an inflammatory exudate, and the intimate cell-to-cell association characteristic of cytotoxic phenomena may provide the in vivo requirements for such a system.     

中剂量rhG-CSF动员对供者外周血免疫细胞组成的影响

本研究观察12例健康供者在使用10μg/(kg·day)rhGCSF动员前后白细胞总数变化。应用外周血涂片瑞氏染色对白细胞进行形态学分类,使用流式细胞术分析动员前后外周血单个核细胞中T细胞、B细胞、NK细胞和单核细胞比例的变化。结果发现,动员前1天外周血白细胞计数中位数为6.25(4.7-7.8)×109/L,其中淋巴细胞中位数为2.07(1.63-3.1)×109/L,单核细胞中位数为0.163(0.078-0.414)×109/L;动员第5天外周血白细胞计数中位数为37.47(24-72.57)×109/L,其中淋巴细胞中位数为3.22(1.46-5.31)×109/L,单核细胞中位数为1.2(0.706-3.627)×109/L。供者外周血白细胞的增加为动员前的6.26±2.14倍(P<0.01),其中淋巴细胞的增加为动员前的1.45±0.76倍(P<0.05),单核细胞数增加为动员前的7.48±4.41倍(P<0.01)。流式细胞术分析发现,动员前CD3 T淋巴细胞占外周血单个核细胞(PBMNC)比例的中位数为46.96%[(32.36-57.45)%],动员后为40.94%[(25.31-48.9)%];动员前CD4 /CD8 淋巴细胞比例为1.27±0.46,动员后为1.36±0.51;动员前CD4 CD8 T淋巴细胞占PBMNC比例的中位数为0.41%[(0.16-1.51)%],动员后为0.49%[(0.09-2.0)%];动员前CD16 CD56 NK细胞占PBMNC比例的中位数为13.98%[(4.08-25.08)%],动员后为16.65%[(12.06-33.05)%];动员前CD3 CD16 CD56 NK-T细胞占PBMNC比例的中位数为2.75%[(0.37-6.38)%],动员后为3.13%[(0.46-5.95)%];动员前CD20 B淋巴细胞占PBMNC比例的中位数为9.28%[(5.97-16.33)%],动员后为9.94%[(7.36-20.41)%];动员前CD14 单核细胞占PBMNC比例的中位数为12.48%[(3.54-19.35)%],动员后为29.52%[(16.51-36.76)%]。动员后CD14 单核细胞在PBMNC中的比例比动员前增加2.87±1.51倍(P<0.05);动员前后T淋巴细胞、NK细胞、NK-T细胞、B淋巴细胞在PBMNC中的比例以及动员前后CD4 /CD8 淋巴细胞比均无显著变化(P>0.10)。结论:rhGCSF动员引起的单核细胞增加可能在异基因外周血造血干/祖细胞移植的相关事件中发挥着重要作用。     

Selective Granulocyte and Monocyte Apheresis as a Non-Pharmacological Option for Patients with Inflammatory Bowel Disease

Ulcerative colitis and Crohn''s disease are the two most prevalent inflammatory bowel diseases. In both cases, the medically refractory and steroid-dependent type presents a therapeutic challenge. To help resolve this problem, a mainly Japanese team developed a new therapeutic option. There are two systems, both of which are able to selectively remove the main mediators of the disease, namely the activated pro-inflammatory cytokine-producing granulocytes and monocytes/macrophages, from the patient''s blood circulation (GMA = granulocyte monocyte apheresis). One of the two systems is the Adacolumn^® (Immunoresearch Laboratories, Takasaki, Japan) consisting of the ADA-monitor and a single-use column, which contains approximately 35,000 cellulose acetate beads. The exact mode of action is not yet sufficiently understood, but however, a modulation of the immune system takes place. As a result, less pro-inflammatory cytokines are released. Furthermore, the production of anti-inflammatory interleukin-1 receptor antagonist is increased, and the apoptosis of granulocytes boosted. The decreased LECAM-1-expression on leukocytes impedes the leukotaxis to the inflamed tissue, and CD10-negative immature granulocytes appear in the peripheral blood. Another effect to be mentioned is the removal of the peripheral dendritic cells and the leachate of regulatory T cells (T-regs). The second system is the Cellsorba^® FX Filter (Asahi Medical, Tokyo, Japan). The range of efficiency, the indication, and the procedure are very similar to the Adacolumn. Solely the additional removal of lymphocytes can possibly limit the implementation since lymphopenia can increase the risk of autoimmune disease. Both systems provide a low-risk therapy with few adverse reactions. ASFA recommendations for GMA in inflammatory bowel disease are 2B due to the fact that not enough randomized double-blind studies are available to proof the efficacy of this treatment.     

The use of dextran as an adjunct to granulocyte collection with the continuous-flow blood cell separator

High molecular weight dextran (dextran 110 or 150 in saline) was added to the input line of the continuous-flow blood cell separator during a series of procedures for the collection of granulocytes from normal donors. Compared with procedures in which dextran was not used, there was enhanced sedimentation of red blood cells within the centrifuge bowl, and analysis of serial samples taken from the white blood cell line showed that contamination of the buffy coat by red cells was reduced by 88 per cent at the point where the granulocyte count was highest. The total leukocyte yields were 1.0 × 10¹⁰ cells when dextran was not used (five procedures), 1.6 × 10¹⁰ cells with dextran 110 (28 procedures), and 2.3 × 10¹⁰ cells with dextran 150 (20 procedures), and there were corresponding increases in the numbers of granulocytes collected. The technique has proved safe and simple, and the addition of dextran is recommended as a means of increasing the leukocyte yield from normal donors while reducing the number of red blood cells collected.     

THE INTERACTION IN VITRO BETWEEN POLYMORPHONUCLEAR LEUKOCYTES AND MYCOPLASMA

The interaction, between mycoplasma (PPLO) and human or rabbit leukocytes was examined in vitro. Upon incubation of M. hominis or M. arthritidis for 2 hr with rabbit peritoneal exudate granulocytes or leukocytes from human peripheral blood, no killing of mycoplasma was observed either in the presence or absence of type-specific antiserum. However, ¹⁴CO₂ production from glucose-1-¹⁴C was stimulated up to 10-fold in the presence of live or heat-killed PPLO. The extent of stimulation depended upon the number of organisms and the presence of type-specific antiserum. The stimulation of ¹⁴CO₂ production seems not because of tight adherence of PPLO to the leukocytes, since PPLO were quantitatively recovered in the medium after sedimenting the granulocytes. The enhanced conversion of medium lysolecithin to cellular lecithin that accompanies phagocytosis of polystyrene particles was significantly reduced when PPLO were also present. Mycoplasma alone elicited no stimulation of lecithin formation. Killing of E. coli, a microorganism readily engulfed and killed by leukocytes in vitro, was diminished when the leukocytes were preincubated with mycoplasma. These findings indicate that M. hominis and M. arthritidis are not ingested by granulocytes to any detectable extent, but that these organisms affect the leukocytes' metabolism and also impair phagocytosis of E. coli.     

The logics of leukocytapheresis as a natural biological therapy for inflammatory bowel disease

Ulcerative colitis (UC) and Crohn's disease (CD) are debilitating idiopathic inflammatory bowel diseases (IBDs) with symptoms that impair ability to function and quality of life. The aetiology of IBD is inadequately understood and, therefore, drug therapy has been empirical instead of based on sound understanding of the disease mechanisms. This has been a major factor for poor drug efficacy and treatment-related side effects that often add to disease complications. The development of biologicals, notably infliximab, to block TNF-alpha reflects some progress, but there is major concern about their side effects and lack of long-term safety and efficacy profiles. However, IBD by its very nature is exacerbated and perpetuated by inflammatory cytokines, including TNF-alpha, IL-6 and IL-12, for which activated peripheral blood lymphocytes, monocytes/macrophages and granulocytes are major sources. Hence, activated leukocytes should be appropriate targets of therapy. At present, three strategies are available for removing excess and activated leukocytes by leukocytapheresis: centrifugation, Adacolumn and Cellsorba. Centrifugation can deplete lymphocytes or total leukocytes, whereas Adacolumn selectively adsorbs granulocytes and monocytes together with a smaller fraction of lymphocytes (FcgammaR- and complement receptor-bearing leukocytes), and Cellsorba non-selectively removes all three major leukocyte populations. Efficacy has ranged from 'none' to an impressive 93% together with excellent safety profiles and downmodulation of inflammation factors. Furthermore, leukocytapheresis has shown strong drug-sparing effects and reduced the number of patients requiring colectomy or exposure to unsafe immunosuppressants, such as cyclosporin A. Leukocytapheresis removes from the body cells that contribute to IBD and, therefore, unlike drugs, it is not expected to induce dependency or refractoriness.     

Receptor-mediated Modulation of Human Monocyte, Neutrophil, Lymphocyte, and Platelet Function by Phorbol Diesters

The tumor promoting phorbol diesters elicit a variety of responses from normal and leukemic blood cells in vitro by apparently interacting with cellular receptors. The biologically active ligand [20-³H] phorbol 12,13-dibutyrate ([³H]PDBu) bound specifically to intact human lymphocytes, monocytes, polymorphonuclear leukocytes (PMN), and platelets, but not to erythrocytes. Binding, which was comparable for all four blood cell types, occurred rapidly at 23° and 37°C, reaching a maximum by 20-30 min usually followed by a 30-40% decrease in cell associated radioactivity over the next 30-60 min. The time course for binding was temperature dependent with equilibrium binding occurring after 120-150 min at 4°C, with no subsequent loss of cell-associated radioactivity at this temperature. Bound [³H]PDBu could be eluted by addition of unlabeled PDBu. Scatchard analysis of data from 4°C binding studies revealed linear plots with high affinity receptors in these cell types with dissociation constants and receptors per cell of 60 nM and 7.8 × 10⁵/cell for lymphocytes, 51 nM and 15.5 × 10⁵/cell for monocytes, 38 nM and 4.0 × 10⁵/cell for PMN, and 19 nM and 2.9 × 10⁴/cell for platelets. Structure-activity studies using unlabeled phorbol-related compounds demonstrated a close correlation between their abilities to inhibit binding of [³H]PDBu to cells and their abilities to induce cellular responses (monocyte and PMN H₂O₂ secretion, lymphocyte ³HTdR incorporation, and platelet tritiated serotonin release); phorbol and 4-alpha phorbol were inactive while phorbol 12-myristate 13-acetate (PMA), PDBu, mezerein, and phorbol 12,13-diacetate (in decreasing order of potency) inhibited [³H]PDBu binding and elicited the various responses. Thus, these high affinity, specific receptors for the phorbol diesters, present on monocytes, lymphocytes, PMN, and platelets, mediate the pleiotypic effects induced by these ligands.     

Type‐2 diabetes down‐regulates glucose transporter proteins and genes of the human blood leukocytes

Objective. White blood cells are essential in mediating immune and inflammatory responses. A prominent feature of these cells during activation of the immune function is increased glucose utilization, and this is dependent on the functioning of specific glucose transporter (GLUT) isoforms. The few data available on leukocyte glucose transporter expression are limited to type‐2 diabetes mellitus, and nothing is known about its regulation. Material and methods. Peripheral blood was drawn from 35 healthy controls and 35 diabetic subjects. Expression of GLUT1, GLUT3 and GLUT4 was determined in the leukocytes of healthy individuals and diabetic patients by flow cytometry, Western blot and semi‐quantitative RT‐PCR. Results. GLUT 3 was decreased in granulocytes, lymphocytes and monocytes from diabetic patients. In monocytes, GLUT3 and GLUT4 were reduced in type‐2 diabetic patients. In leukocytes of diabetic patients, GLUT1 and GLUT4, protein and mRNA were unchanged, but GLUT3 protein and mRNA levels were down‐regulated compared to those of healthy controls. Conclusion. Elevated glucose concentration affects leukocyte GLUT expression. Decreased expression of GLUT isoforms in leukocytes may be responsible for diminished activation of diabetic leukocytes. These situations possibly contribute to a predisposition to infection and to a decreased immune response in diabetes.     

Enrichment of dog leukocyte subpopulations using density gradients

Density gradient methods have been used for the enrichment of leukocyte subpopulations from human blood. We have adapted a three step method utilizing centrifugation on Hypaque-Ficoll (HF), double density HF (ddHF) and Percoll density gradients to separate lymphocytes, monocytes and basophils from dog blood. After HF separation, both Basenji-Greyhound (BG) and mongrel dogs had similar percentages of lymphocytes and monocytes, but BG dogs had significantly greater (p greater than 0.025) numbers of granulocytes. When cells from HF gradients were spun directly on Percoll, the presence of granulocytes decreased the purification of leukocyte subpopulations. An intervening separation on a ddHF gradient removed granulocytes without a selective loss of lymphocytes or monocytes. When cells from ddHF gradients were further separated on Percoll, 2 distinct cell layers resulted. Layer 1 was monocyte rich with 72.4 +/- 6.5% monocytes, 26.6 +/- 6.2% lymphocytes, and 0.8 +/- 1.8% granulocytes. Layer 2 was lymphocyte rich with 74.3 +/- 11.1% lymphocytes, 21.4 +/- 7.8% monocytes and 3.9 +/- 4.2% granulocytes. Viability as determined by Trypan blue exclusion was above 90%. Using cAMP-Phosphodiesterase (PDE) as a marker, higher PDE activity was present in the monocyte rich layer. This was similar to that reported in humans. Basophil numbers were enhanced by the use of a 1-step separation on a ddHF gradient. The percentage of basophils was increased 10-fold over baseline levels, with a viability of 90%. These techniques can be used to purify various canine leukocyte subpopulations and provide cells for biochemical analysis.