Competitive protein binding assay of progesterone without chromatography

A competitive protein binding assay for the measurement of progesterone in human plasma without chromatographic separation of steroids and recovery evaluation in individual samples is described. It is based on the specificity of the progesterone binding plasma protein (PBP) of the pregnant guinea pig. A dried petroleum ether extract of plasma was incubated with ³H-progesterone and 1600 fold diluted pregnant guinea pig plasma. Bound radioactivity was measured with a dextran coated charcoal suspension technique. Plasma progesterone concentration was obtained by comparison with a standard curve and correction for extraction separately measured for each batch of petroleum ether. The sensitivity was 100 pg. Recovery experiments for progesterone and competing steroids added to plasma respectively showed the accuracy and the specificity of the method. However comparison of the results from assays with and without chromatographic separation of steroids, showed that in the latter-case the specificity was good only for plasmas containing more than 1ng/ml of progesterone. Concentration of progesterone in plasma from men was 0.46±0.14 ng/ml (mean ± S.D) and from post menopausal women 0.30± 0.13 ng/ml.Between days 1 and 13 and days 16 and 22 of the normal menstrualcycle the concentrations were respectively 0.81 ± 0.38 and 12.50 ± 2.96 ng/ml. The variations of the progesterone concentration during pregnancy are also shown.     

In vivo distribution of cryogenically preserved guinea pig granulocytes

Granulocytes were isolated from whole blood of guinea pigs by counterflow centrifugation and labeled with [¹⁴C]diisopropylfluorophosphate ([¹⁴C]DFP). One-half of the labeled cells was injected intravenously via the femoral vein into a guinea pig, while the other half was cryogenically preserved with 5% dimethyl sulfoxide (DMSO), 6% hydroxyethyl starch (HES), and 4% human albumin, at a rate of 4 °C per minute by storage at ?80 °C and then stored for 3 days at ?80 °C. Ninety percent of the isolated granulocytes were recovered after cryogenic preservation, thawing, and washing. Aliquots before injection all produced fluorescein from fluorescein diacetate and excluded ethidium bromide. Latex ingestion was 78% and yeast ingestion was 75%. The frozen-thawed-washed-resuspended labeled granulocytes were injected into a second guinea pig. Paired animals sacrificed 35 min after injection were examined in whole-body sections for distribution of radiolabeled granulocytes to the tissues. In two pairs of animals, activity was found in the lung, liver, spleen, and kidney. The technique does not permit a distinction between fresh and cryopreserved granulocytes although there was a greater deposition of fresh cells in the liver and spleen. No activity was found in the blood of the vena cava in animals with either fresh or frozen cells. An animal injected with free [¹⁴C]DFP revealed a vascular distribution with high activity in blood, lung, and kidney, and less activity in the liver and spleen. The data indicate that radiolabeled, cryogenically preserved guinea pig granulocytes exhibited a tissue distribution qualitatively similar to fresh granulocytes, and free [¹⁴C]DFP infused without granulocytes differed qualitatively and quantitatively from fresh and cryopreserved granulocytes.     

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia-Tolerant Reptiles Contrasted with Levels in Anoxia-Intolerant Species

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g^?1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g^?1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g^?1). Regionally distinct levels (2–4 µmol g^?1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2–3 µmol g^?1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.     

Association of 5′nucleotidase activity with immature granulocytes in the bone marrow of guinea pigs

Bone marrow leukocytes from adult strain 2 guinea pigs were found to have appreciable levels of 5′adenosine monophosphate hydrolytic activity (105 nmole/h/10⁶ cells). On the basis of substrate specificity studies, enzyme inhibition studies, and thin-layer chromatographic analysis of the reaction product, the activity is related to 5′nucleotidase (5′N). The enzyme activity was associated with the membrane-enriched particulate fraction of lysed bone marrow cells.The bone marrow cell-associated 5′N activity was consistently very high in all five strains of guinea pigs examined (77–127 nmole/h/10⁶ cells) and the range of activity was at least 10-fold greater than that observed for bone marrow cells obtained from mice, rabbits or rats. Furthermore, the bone marrow cell-associated 5′N activity in strain 2 guinea pigs was 5-fold greater than that observed for spleen and at least 13-fold greater than that of blood, mesenteric lymph nodes or thymus obtained from the same animal.Fractionation of strain 2 guinea pig bone marrow cells on Percoll density gradients showed that as the proportion of immature granulocytes increased in the various cell fractions, so did the 5′N activity. The cell fraction that sedimented at a density of 1.071 g/ml had the highest 5′N activity and the majority of the cells (94%) were immature granulocytes. The bone marrow compared to blood and spleen had the highest number of total granulocytes and the highest percentage of immature granulocytes. We conclude that the elevated bone marrow-derived 5′N activity in guinea pigs is associated with the resident population of immature granulocytes in that tissue.     

A simple and efficient method of labelling RBCs with 99mTc for spleen imaging

A new method of labelling human RBCs with ^99mTc by the use of Sn-α-d-glucose 1-phosphate (GP) is presented. It was tested for spleen imaging in 16 normal volunteers. The labelling was carried out during the heating (30 min at 49.5 °C) to damage the cells and cooling (10 min at R.T.) steps. The labelling efficiency was 95.5 ± 1.5% with Sn-GP and was better than Sn-PYP (61.5 ± 25.0%). The radioactivity retention of RBCs was > 96% after 6 washings. The spleen was delineated very well in all the subjects. The spleen activity reached a plateau at 20 min post-injection. Spleen-to-liver and spleen-to-cardiac blood pool concentration ratios were high (10.4 ± 2.2 and 10.1 ± 3.2, respectively) calculated at 30 min. The method is simple, rapid and efficient.     

Further isolation of endogenous factors in canine and human plasma that inhibit [3H]norepinephrine accumulation

We have previously shown that both homologous canine plasma and a crude extract of this plasma contain substances that inhibit accumulation of [³H]norepinephrine ([³H]NE) by the canine saphenous vein. The purpose of this study was to further purify these substances and to determine if similar factors were present in human plasma. Crude extracts of plasma were purified with a Folch extraction in which most of the biological activity was recovered in the bottom or organic phase. This phase significantly inhibited [³H]NE uptake by the canine saphenous vein (23.5 ± 7.6% by concentrate from 9.1 ml of original plasma/ml incubate solution) and increased development of tension following transmural electrical stimulation by 91.5 ± 23.3% (extract from 1 ml of plasma/ml bath solution). The Folch extracts obtained from 100ml of plasma were purified by column and thin layer (TLC) chromatography. Samples were applied to a silicic acid column and eluted with chloroform, acetone, and methanol. The [³H]NE uptake inhibitory activity was primarily recovered in the methanol fraction. TLC of the methanol fraction of canine plasma on silica gel G plates (with pre-absorbent) resulted in five zones which were then assayed for their ability to inhibit [³H]NE accumulation by the saphenous vein. In the first zone (concentrate from 27.5 ml plasma/ml bath solution) there was significantly greater inhibitory activity (55.4 ± 8.3%), than in the corresponding zone obtained from solvent blanks (20.7 ± 4.1%). These results indicate that there is a factor or possibly factors in canine and human plasma that have thin layer chromatographic properties of a polar lipid, which inhibit [³H]NE accumulation and enhance the contractile response of vascular smooth muscle to transmural electrical stimulation.     

Evaluation of two 111In-oxinate formulations for labelling of white blood cells

This study compares the cell labelling characteristics of two ¹¹¹In-oxinate formulations. The two preparations differ by the solubilizing agent of the chelate and the total amount of oxine. White blood cell suspensions were obtained by standard separation techniques and were labelled with either of these formulations. The labelling efficiency was higher for ¹¹¹In-oxinate in aqueous solution (compound B) compared to the preparation where an organic solubilizer was added (compound A) (79.2 ± 7.7 vs 68.6 ± 17.6%, respectively, P = 0.03). Red blood cells contaminating the cell suspensions incorporated a higher fraction of ¹¹¹In if the cells were incubated with the aqueous ¹¹¹In-oxinate preparation (22.6 ± 4.6 vs 4.8 ± 4.6%, respectively, P < 0.0001). The uptake of activity by polymorphonuclear cells was reduced with compound B (46.1 ± 12.8 vs 63.8 ± 15.8%, respectively, P = 0.0002) whereas the fraction retained by mononuclear cells and platelets was similar (31.3 ± 13.9 vs 31.4 ± 15.0%, respectively). The recovery from the vial was higher for ¹¹¹In-oxinate in an organic solution (86.6 ± 1.82 vs 60.3 ± 14.3%, respectively, P < 0.0001). Twenty four hours after administration of the labelled cells, the vascular compartment was less frequently visualized if cells were labelled with compound A (8% of the scintigrams vs 62.5% respectively, P < 0.0001). High quality images were more often recorded after the administration of cells labelled with compound A (60.0% of the images vs 23.5%, respectively, P <0.02). The image quality of scintigrams was not related to any of the other cell labelling parameters. We conclude that ¹¹¹In-oxinate in an organic solubilizer was characterized by less uptake in the red blood cells contaminating the white blood cell suspensions. Good quality images were more often obtained with ¹¹¹In-oxinate in organic solubilizer.     

Does feeding area restriction inhibit social learning of toxic weed ingestion in cattle?

Social learning from peers can trigger herd-wide intoxication with white locoweed (Oxytropis sericea), an alkaloid-synthesizing herbaceous legume that grows on rangelands of western North America. We conducted an experiment to test the hypothesis that restriction of the area allocated to animals to feed in would inhibit social facilitation of locoweed ingestion in yearling heifers. Eight heifers that avoided white locoweed (LA) and eight heifers that readily consumed it (LE) were selected from a pool of 40 cross-bred heifers and were randomly assigned to the social facilitation or social interference treatment groups. We conducted 200 10-min feeding trials in three 5-day phases (pre-treatment, treatment, post-treatment) during which animals were presented with a set of bowls arrayed in a test arena, some of which contained ground wheat straw and others contained air-dried ground white locoweed. During the pre-treatment (days 1 to 5) and the post-treatment phases (days 11 to 15) non-social trials were conducted in which the feeding behavior of individual animals was investigated in an 80 m² arena containing 12 feeding bowls. During the treatment phase (days 6 to 10) social learning trials were conducted in which LA + LE pairs from the social interference group were exposed to 12 bowls of food distributed in an 80 m² arena intended to induce social interference, and LA + LE pairs from the social facilitation group were exposed to 36 bowls of food distributed in a 240 m² arena intended to permit social facilitation. During pre-treatment phase, LA heifers consumed detectably less locoweed and wheat straw and exhibited lower preference for locoweed than LE (P ⩽ 0.05) although wheat straw preference of LA and LE was similar. During social learning trials (treatment phase), LA in the social interference group visited similar number of locoweed bowls (mean ± s.e.m.: 0.2 ± 0.12) as they had during non-social learning (0.2 ± 0.20). Conversely, LA heifers in the social facilitation group visited detectably more locoweed bowls during social learning trials (1.6 ± 0.46) compared with the pre-treatment phase (0.2 ± 0.16). Correlation between daily number of locoweed bowls visited by LA and LE during social learning trials was detected in the social facilitation (r = 0.70; P < 0.01), but not in the social interference group (r = 0.15; P = 0.52). During testing trials (post-treatment phase), locoweed and wheat straw intake and preference of LA and LE in both treatment groups was similar. Manipulation of the feeding environment delayed, but did not inhibit social learning of toxic weed ingestion in this study.     

IN VITRO INCORPORATION OF 3H-THYMIDINE, 3H-URIDINE, 3H-LEUCINE AND 3H-MELPHALAN BY PLASMACYTOMA PLASMA CELLS

Bone marrow plasma cells from fifteen cases of multiple myeloma, immunologically typed, were incubated with different tritiated compounds. The labelling index with tritiated thymidine is generally low, while the mean grain count is fairly normal in the active cells. The labelling index of ³H-uridine and ³H-leucine was very high, while the mean grain count per cell lies within the normal range. The results obtained with ³H-phenylalanine-mustard (melphalan), which is a drug used in the treatment of the plasmacytoma, show also incorporation values roughly comparable to those of ³H-leucine. The present data seem to support the clinical use of melphalan as a compound that is actively incorporated into the plasma cells of plasmacytoma although inhibition of protein synthesis due to specific binding to protein was not demonstrated.     

Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [³H]DNA in alkaline sucrose gradients or by direct assay of the amount of [³H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.     

Metabolic coupling between cultured pancreatic B-cells

The transfer of [³H]uridine nucleotides from donor (uridine-loaded) to recipient (thymidine-prelabelled) pancreatic endocrine islet cells in monolayer culture was qualitatively and quantitatively assessed by light and electron microscope autoradiography. Recipient cells showed a labelled cytoplasm only when they were in contact with donor cells or positive recipients. Controls indicated that the labelling was not due to the incorporation of [³H]uridine, ³H nucleotides or nucleic acids lost by donor cells in the medium. Quantitation showed that cytoplasmic labelling of positive recipient cells was higher than cellular background, but lower than the cytoplasmic labelling of donor cells. These data indicate that label in recipient cells was derived from donors by direct intercellular transfer of ³H nucleotides. Differentiated insulin-producing cells (B cells) were involved in the exchange.     

[3H]Inositol incorporation into phosphoinositides of pig reticulocytes

Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) of pig reticulocytes were extensively labelled when these cells were incubated with [³H]inositol. In marked contrast, a total lack of [³H]inositol labelling of phosphoinositides was observed in mature erythrocytes. Phosphoinositides of both reticulocytes and mature erythrocytes were labelled with ³²P but the labelling in reticulocytes was several-fold higher than in mature erythrocytes. Inclusion of Ca²⁺ (2 mM) + ionophore A23187 (2 μg/ml) during the labelling experiments substantially reduced the radioactivity incorporation into phosphoinositides of reticulocytes. When [³H]inositol-prelabelled reticulocytes were treated with Ca²⁺ + A23187 the levels of radioactive PI and PIP₂ did not change significantly. However, the PIP pool exhibited a remarkable sensitivity to Ca²⁺ as shown by a 75% increase in its radioactivity over the control. The ability to incorporate [³H]inositol into phosphoinositides remains transitorily intact in the reticulocyte stage. Thus, pig reticulocytes offer a suitable model in which to explore the physiological role of phosphoinositides in relation to cellular maturation process.     

Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization,cellular ATP and associated membrane changes in D+ and D− cells

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D⁺ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D^? myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D⁺ than in D^? cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D^? than in D⁺ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D⁺ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D⁺ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D^? cells, were found before most of the D⁺ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D^? cells to differentiate and the acquisition of the metabolic properties of normal granulocytes by differentiating D⁺ cells, were associated with differences in the organization of the cell surface membrane.     

HeLa cell plasma membranes : IV. Iodination of membrane proteins in synchronized cells

Intact HeLa cells and isolated HeLa cell plasma membranes were subjected to lactoperoxidase-catalysed iodination. The ¹²⁵I-labelled proteins were separated by SDS-polyacrylamide gel electrophoresis. Six protein species with apparent molecular weights from 32 000 to 200 000 were accessible to labelling from the outer cell surface, while most of the proteins present in the plasma membrane were labelled when isolated plasma membranes were iodinated. Iodination of synchronized intact cells revealed that the labelling obtained was cell cycle dependent with maximal labelling at mitosis. No changes in the distribution of radioactivity among the labelled proteins were observed when cells from different phases were iodinated.     

Factors affecting the stability of cryogenically preserved granulocytes

Human granulocytes free of other cell types were obtained by counterflow centrifugation, cryogenically preserved, and studied for stability and function after thawing.Isolation of granulocytes by counterflow centrifugation was optimal at reduced temperatures (4–10 °C) in phosphate-buffered saline (or Ca²⁺-free buffers) at pH 7.1. A stabilizing protein, or HES was required. Routinely, 1.2% human or bovine serum albumin was used. Hyperosmolar (310 m0sm) buffers and post isolation handling in ice water baths was optimal for cryogenic preservation. Addition of DMSO at 22 °C produced transient shrinkage initially which depended on the rate of addition, concentration, and temperature. Within 10–15 min granulocytes returned to volume, but continued to swell, equilibrating for 1 hr at 20% larger volume. Ethidium uptake gradually increased. After 24 hr, extreme swelling, lysis, and ethidium uptake was observed at the highest concentration (10%) of DMSO. DMSO-induced swelling was prevented with HES.Granulocytes (30 × 10⁶ ? 50 × 10⁶) were frozen in 2.0-ml volumes in plastic tubes. The combination of 5% DMSO, 6% HES, 4% albumin, 0.056 M glucose in NormosolR at pH 7.1 produced the best yields. Granulocytes were first cooled to 4 °C, then to ?80 °C (approx rate 4 °C per min) in a mechanical freezer and finally stored in liquid nitrogen. Storage varied from days to months. Granulocytes were thawed at 42 °C by manually twirling the freezing tubes and they were subsequently maintained in ice water. They were diluted 3:1 dropwise with a room temperature solution of 7% HES, 1.2% albumin, and 0.026 M glucose in Normosol. Particle ingestion tests were conducted by incubation at room temperature for forty minutes with yeast or zymosan opsonized with autologous serum. Particles ingested were counted by microfluorimetry after two washings at 150g.Granulocytes could not be cryogenically preserved in plasma or serum. Heating or prefreezing of serum was ineffective, but dialysis or addition of EDTA overcame the destructive effect of serum. Neither treatment was an improvement over the standard freeze procedure using buffered albumin and cryoprotective components. β-mercaptoethanol added to the freezing medium caused the production of a single homogeneous population of osmotically inert, nonviable, ethidium-reactive granulocytes. This suggests that osmoregulation by granulocyte membranes is a critical requirement for cryopreservation.Preservation efficiency is species dependent, increasing in the order of human, baboon, guinea pig, and dog. Dog granulocytes can be stored for at least 8 months in liquid nitrogen with small loss of cells and functionality.The present efficiency of preservation of human granulocytes for 3–4 weeks of liquid nitrogen storage is 90–100% morphological and 40% functional recovery. Attempts to increase stability of thawed granulocytes with other additions to our current procedure have so far proved fruitless. These have consisted of inosine, adenine, pyruvate, gluconate, vitamin C, β-mercaptoethanol, para-phenylmethyl-sulfonylfluoride, and mannitol.     

Metabolic studies of rat liver plasma membranes using d-[l-C]glucosamine

The metabolism of plama membranes of rat liver cells was studied using d-[l-¹⁴C]glucosamine. The labelling of plasma membranes occurred more slowly than that of microsomes, reaching a maximum at about 3 h after injection compared to 1.5 h for microsomes, and the radioactivity decayed with a half-life of 37 h which is close to the value obtained using [guanidino-¹⁴C]arginine to label proteins. Hexosamine and sialic acid of plasma membranes were found to metabolize at practically equal rates.     

Assessment of the hemorheological profile of koala and echidna

Koala, a marsupial, and echidna, a monotreme, are mammals native to Australia. Blood viscosity (62.5–1250 s^?1), red blood cell (RBC) deformability, RBC aggregation, aggregability and surface charge, and hematological parameters were measured in blood samples from six koalas and six echidnas and compared to adult human blood. Koala had the largest RBC mean cell volume (107.7±2.6 fl) compared to echidna (81.3±2.6 fl) and humans (88.4±1.2 fl). Echidna blood exhibited the highest viscosity over the entire range of shear rates. Echidna RBC were significantly less deformable than koala RBC but more deformable than human RBC. Echidna RBC had significantly lower aggregability (i.e., aggregation in standardized dextran medium) than koala or human RBC, while aggregation in autologous plasma was similar for the three species. Erythrocyte surface charge as indexed by RBC electrophoretic mobility was similar for human and echidna cells but was 40% lower for koala RBC. Data obtained during this preliminary study indicate that koala and echidna have distinct hemorheological characteristics; investigation of these properties may reveal patterns relevant to specific behavioral and physiological features of these animals.     

Does ex vivo labelling of proliferating cells in colonic and vaginal mucosa reflect the S-phase fraction in vivo?

Summary The ex vivo labelling of DNA-synthesizing epithelial cells in colonic and vaginal mucosa was compared with in vivo labelling. For this purpose, in vivo S-phase cells were labelled with [³H]thymidine (Tdr) and ex vivo labelling was continued by culturing tissue specimens in bromodeoxyuridine (BrdU). Various methods of tissue culture were employed in order to improve diffusion of medium (and BrdU) in the tissue. BrdU and ³H-TdR labelling were evaluated by immunohistochemistry and autoradiography respectively. Ex vivo labelling resulted in a patchy distribution of labelled cells, which did not correspond with the ³H-TdR labelling pattern obtained in vivo. Under the described conditions ex vivo labelling does not appear to be a reliable for estimation of the proliferative activities in vivo.     

Labelling of leukocytes with 99mTc-HMPAO for scintigraphy of inflammatory lesions and abscesses

A simplified and efficient procedure for ^99mTc-HMPAO-labelling of leukocytes is described. For this purpose, the pH and concentration of the ^99mTc-HMPAO preparation was modified. Leukocytes were isolated from a 20 mL mixture of patient blood, 5 mL ACD and 0.8 mL methylcellulose after 1 h sedimentation of erythrocytes and centrifugation (at 400 g) of the obtained plasma layer. Simultaneously, ^99mTc-HMPAO was prepared (one single-dose kit for two patients) by adding 2.2 mL ^99mTc-generator eluate and, after 10 min, 0.3 mL of phosphate buffer to lower the pH to 7. The isolated WBCs were then labelled by the addition of 1-1.2 mL of ^99mTc-HMPAO solution and incubated for 20 min. The unbound tracer was then discarded, the labelled WBC washed and finally resuspended in autologous cell-free plasma. Leukocytes labelled by this procedure were used for scintigraphic localization of inflammatory lesions and abscesses in the gastro-intestinal tract.The labelling efficiency was 60 ± 9%, with a separation yield of 55 ± 11%.     

Schistosoma mansoni: Inhibition in vitro of granulocyte cytotoxicity against schistosomula by autologous mononuclear cells from patients

Granulocytes and mononuclear cells from normal subjects are able to kill Schistosoma mansoni schistosomula in the presence of human complement in vitro. In contrast, mononuclear cells from chronic schistosomiasis patients failed to kill the parasites. The relative efficiencies expressed in terms of the mean percentage killing of schistosomula for 15 experiments were 50.5 ± 3.2 and 49.7 ± 3.5 for granulocytes from normal and infected patients, and 50.8 ± 2.8 against 23.0 ± 3.2 for mononuclear cells from normal and infected patients, respectively. The killing effect of granulocytes dropped from 48.7 ± 2.8 to 22.1 ± 2.2 when autologous mononuclear cells from chronically infected patients were added to the system. Similar inhibitory effect of granulocyte function was obtained when these cells were incubated with normal mononuclear cells precultured with concanavalin A. Extracts prepared from mononuclear cells obtained from infected patients had the same inhibitory effect of intact cells on the complement-dependent granulocyte cytotoxicity.