Technique and Practical Problems: Comparative Studies of the Value of Two Cyanide-Xitroprusside Methods ix the Diagnosis of Cystixuria: Cystinuria in Sweden,Part ix

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 0.02x 10⁹/1; standard deviation for low levels of granulocytes: 0.300×10⁹71 and lymphocytes plus monocytes: 0.235×10⁹/1).

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.     

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。     

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.     

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.     

Evaluation of the automated haematology analyser Sysmex M-2000

The automated Sysmex M-2000 was evaluated according to the ICSH (International Committee for Standardization in Haematology) protocol. After dilution of packed cells with cell-free plasma, blood cell counts were linear. The overall precision of the measured parameters was good; the CV's ranged between 0.64% and 2.06%. The carry-over was negligible; platelets showed the biggest carry-over with 0.25% in the Whole Blood Mode, while red blood cells (RBC) showed a carry-over of 0.55% in the Prediluted Mode. 300 clinical samples were measured on the Sysmex M-2000 and the Sysmex CC-700 with PL-100, and the results were compared. The coefficients of correlation for white blood cells (WBC), red blood cells (RBC), haemoglobin and haematocrit were greater than 0.99; platelets showed an r of 0.982. Comparison of the results from the Sysmex M-2000 trimodal leukocyte distribution with a manual 100 cell differentiation showed a close correlation for lymphocytes (r = 0.948), and neutrophils (r = 0.931). The middle cell fraction corresponding to monocytes, eosinophils and basophils showed a correlation with r = 0.703. Pathological samples showed no interference with the blood count. Leukocyte counts less than 1000 x 10(9)/l did not effect the measurement of haemoglobin. During the period of evaluation, no instrument malfunctions occurred. Because of its precision and reliability, the Sysmex M-2000 is well suited for routine work and stat analysis in medium-sized laboratories.     

Diagnostic value of an automatic hematology analyzer in patients with hematologic disorders

The Sysmex SE-9000 is a newly designed automatic hematology analyzer that provides complete blood counts and white-cell differential counts. Few reports in the literature, however, discuss the sensitivity and specificity of this type of automatic analyzer, especially in patients with hematologic illnesses. This study compared differences between hematologist assessments and measurements made by the SE-9000 analyzer of differential counts, immature granulocytes, atypical lymphocytes, nucleated red cells, and platelet counts. Significant differences were found between manual and apparatus counting of neutrophils, lymphocytes, and monocytes but not of eosinophils and basophils. Atypical lymphocytes and nucleated red cell could not be counted accurately, and when platelet counts fell below 20 x 10(9)/L, accurate assessments were not possible. We conclude that not even the Sysmex SE-9000 can provide unflawed results and any suspicious blood report should be rechecked by an experienced hematologist.     

Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 X 10(4)/cell), followed by monocytes (7.3 X 10(4)/cell) and lymphocytes (4.0 X 10(4)/cell). Compared with conventional analysis of binding of 125I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a "docking" type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias.     

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.     

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.     

Changes in immunological and virological parameters in HIV-1 infected subjects following leukapheresis

In order to assess immune responses during HIV-1 therapeutic immunization, a large number of blood mononuclear cells (PBMC) are needed. Clinical tolerance and safety, as well as changes in immunological and virological parameters, were assessed, following leukapheresis in HIV-1 infected subjects with CD4(+) cell count >200 x 10(6)/l. PBMC were collected using a Fenwal CS3000 cell separator in 29 subjects with mean CD4(+) cell counts of 503 x 10(6)/l (range 172-1,119) and viral load of 2.5 log(10) copies/ml (range <1.7-5.4). Twenty-four (83%) subjects were on antiretroviral therapy while 5 (17%) were untreated. The blood volume processed was 7 L over a period of 3 hours. A mean value (+/- standard error) of 82 +/- 26 x 10(9)/l lymphocytes was collected by a single apheresis in a mean volume of 200 +/- 1.8 ml, containing 9.0 +/- 1.3 x 10(9)/l CD4(+) and 10.2 +/- 1.3 x 10(9)/l CD8(+) cells. The leukapheresis procedures were well tolerated and no immediate or delayed side effects were observed within 90 days of follow-up. No changes from blood pre-leukapheresis values were detected for white blood cells, lymphocytes, monocytes, CD8(+), CD34(+), naive and memory CD4(+) cell counts immediately after, 1 h, 7 days, or within 90 days after leukapheresis. However, absolute CD4(+) cell counts and percentage significantly increased from pre-leukapheresis values after 1 h (530 +/- 43 vs. 700 +/- 75 cell x 10(6)/l; 32.6 +/- 1.6 vs. 36.9 +/- 1.9%; P < 0.001 for both paired t-tests) before returning to pre-leukapheresis levels on day 7. No significant changes in viral load from pre-leukapheresis levels in treated or untreated subjects were detected at any time points. We conclude that leukapheresis in HIV-1 infected subjects with CD4(+) cell counts >200 x 10(6)/l is safe and induces a transient increase in the absolute and percentage of CD4(+) cell count without enhancing viral replication.     

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.     

Leukocytes in peritoneal dialysis effluents. Danish Study Group on Peritonitis in Dialysis (DASPID)

The concentration of leukocytes and the fraction of neutrophil granulocytes are two important criteria in the diagnosis of peritonitis in continuous ambulatory peritoneal dialysis (CAPD). We have found that leukocytes are unstable in dialysis effluents, resulting in false low leukocyte concentrations if not counted immediately. At 25 degrees C the leukocyte count decreases 25%-30% in 4-6 hours. Sampling in tubes containing EDTA and storage at 4 degrees C make the leukocyte concentration stable for 6 hours, while the combination of EDTA and storage at 4 degrees C ensures stability for 24 hours. When samples are handled accordingly, concentrations as high as 2 x 10(8)/L are observed without any clinical signs of peritonitis, especially within the first months of CAPD-treatment. Thus, we suggest a leukocyte-concentration of 2 x 10(8)/L as the diagnostic limit for peritonitis. Concerning fraction of neutrophils a diagnostic limit of 0.50 still seems relevant.     

Haematological phenotypes in relation to the C1797T beta-adducin polymorphism in a Caucasian population

beta-Adducin plays a role in maintaining the structural integrity of the red blood cell (erythrocyte) membrane. Moreover, beta-adducin-deficient knock-out mice show a phenotype characterized by mild anaemia and compensated haemolysis. We therefore investigated whether, in humans, common haematological phenotypes of red blood cells were associated with a polymorphism in exon 15 of the human beta-adducin gene (C1797T). We studied 802 unrelated individuals and 294 families (459 parents and 609 offspring) randomly selected from a Caucasian population. We employed generalized estimating equations to allow for the non-independence of the observations within families, while controlling for co-variables. In 917 men, with adjustments applied for age, body mass index, serum total cholesterol, smoking and alcohol intake, CC homozygotes had significantly ( P =0.02) lower values for red blood cell count (4.93 x 10(12)/l compared with 4.86 x 10(12)/l), haemoglobin level (9.30 compared with 9.18 mmol/l) and haematocrit (45.0% compared with 44.4%) than T allele carriers. In the 329 men who consumed alcohol, the differences between CC homozygotes and T allele carriers were 0.13 x 10(12)/l ( P =0.02) for red blood cell count, 0.23 mmol/l ( P =0.005) for haemoglobin and 1.08% ( P =0.02) for haematocrit. In 953 women, none of these associations was significant ( P >/=0.06), regardless of alcohol intake [13.3% of women ( n =127) consmued alcohol]. In conclusion, in men consuming alcohol, the beta-adducin CC genotype was associated with lower red blood cell count, haemoglobin level and haematocrit. We hypothesize that, in CC homozygotes, alcohol consumption may unveil the greater fragility of the red blood cell membrane. This genotype may slightly potentiate the structural and functional haematological disturbances associated with alcohol intake.     

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.     

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.     

Time course changes in the count of peripheral granulocytes in children with acquired aplastic anemias treated with antithymocyte globulin,cyclosporin A and granulocytic colony stimulating factor

AIM: To examine the pattern of changes in the count of peripheral granulocytes in children with aplastic anemias (AA), receiving a combined immunosuppressive therapy with antithymocytic globulin (ATG) and cyclosporin A in combination with granulocytic colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 31 children (17 boys and 14 girls) aged 2-15 years (median 9 years) with newly diagnosed severe and very severe acquired AA took a combined immunosuppressive therapy with ATG and cyclosporin A in combination with G-CSF in an initial dose of 10 micrograms/kg a day. RESULTS: A three-linear and response was recorded in 19 (61%) children, an isolated granulocytic response was in 26 (84%). The interval median before the recovery of granulocytes to 1.5 x 10(9)/l and 5 x 10(9)/l was 19 and 38 days, respectively. CONCLUSION: Use of G-CSF may increase the count of granulocytes in the vast majority of patients with AA, without dramatic influence on the frequency of a three-linear response. Intermittent use of G-CSF may maintain the count of granulocytes long at the safe level and reduce the cost of treatment.     

Pyruvate and acetate oxidation by leukocytes in vitro. Activation of the pyruvate dehydrogenase complex by uncoupling of oxidative phosphorylation

Pyruvate oxidation by normal intact leukocytes has been systematically studied to define optimal conditions for detection of enzymatic defects in this process. Leukocytes were isolated by dextran sedimentation and lymphocytes by Ficoll centrifugation. Cells were incubated for 2 h with [1-14C]-pyruvate, [2-14C]-pyruvate or [1-14C]-acetate as substrate. The specific oxidative capacity of lymphocytes was almost three times higher than that of granulocytes from the same blood. Oxidation of both pyruvate and acetate was highly dependent on the substrate concentration in the medium reaching a plateau between 0.5 and 1.0 mmol/l. Addition of succinate (1 mmol/l) stimulated oxidation of [1-14C]-pyruvate by 30%. Uncoupling of phosphorylation by addition of carbonyl cyanide chlorophenylhydrazone (CCCP) (0.1 mumol/l) increased oxidation of [1-14C]-pyruvate by 200% and of [1-14C]-acetate by 70%. Addition of CCCP plus succinate caused further stimulation of pyruvate oxidation (+40%), but not of acetate oxidation. It is therefore concluded that: (1) Lymphocytes are better than mixed leukocytes for oxidative studies. (2) Unlabelled substrate should be added at optimal concentrations. (3) The pyruvate dehydrogenase complex is normally only partially active in lymphocytes. (4) Stimulation of oxidation by CCCP greatly enhances the flux through the PDH step thus facilitating the detection of defects in pyruvate oxidation.     

Adenosine receptors on human leukocytes. IV. Characterization of an A1/Ri receptor.

Adenosine (10(-9)-10(-6) mol/l) and R-phenylisopropyladenosine (10(-9)-10(-7) mol/l) partially inhibited the intracellular accumulation of cyclic AMP induced by isoproterenol, prostaglandin E1, histamine and 5'-N-ethylcarboxamidoadenosine in lymphocytes. In contrast, S-phenylisopropyladenosine, which is a poor agonist of the adenosine A1/Ri receptor, had essentially no inhibitory effect. 8-Phenyltheophylline, in low concentrations that do not inhibit cyclic AMP phosphodiesterase, completely blocked the inhibitory effect of R-phenylisopropyladenosine on the increase in cyclic AMP induced by prostaglandin E1. R-Phenylisopropyladenosine (10(-8)-10(-6) mol/l) also inhibited the cyclic AMP accumulation in lymphocytes induced by forskolin (10(-5) mol/l), which activates adenylate cyclase through direct interaction with the enzyme. We also investigated the presence of the adenosine A1/Ri receptor on human polymorphonuclear leukocytes. R-Phenylisopropyladenosine (3 x 10(-9)-10(-7) mol/l) abolished the stimulating effects of prostaglandin and forskolin on cyclic AMP accumulation in polymorphonuclear leukocytes. This effect was blocked by 8-phenyltheophylline and was not observed with the stereoisomer S-phenylisopropyladenosine. The results support the existence of an A1/Ri receptor that regulates cyclic AMP metabolism of human lymphocytes and polymorphonuclear leukocytes.     

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.     

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.