GMP production and testing of Xcellerated T Cells for the treatment of patients with CLL

BACKGROUND: Pre-clinical studies suggest Xcellerated T Cells have the potential to produce a potent anti-tumor effect, restore broad immune function and reduce the risk of infectious complications in patients with CLL. Unlike other cancer settings, T cells constitute only a small fraction of CLL patients' PBMC. To generate large numbers of Xcellerated T Cells of high purity from CLL patients' PBMC, a reproducible, streamlined and cost-effective good manufacturing process (GMP) is required. METHODS: The 10-L volume Wave Bioreactor-based Xcellerate III Process using Xcyte Dynabeads in a single custom 20-L Cellbag container was adapted, qualified and implemented for GMP operations. RESULTS: For n=17 CLL patients, starting with approximately 1.34 x 10(9) CD3+ T cells at 6.8+/-7.5% purity in the PBMC leukapheresis products, using the 10-L volume Wave Bioreactor-based Xcellerate III Process, it was feasible to manufacture 137.0+/-34.3 x 10(9) Xcellerated T Cells at 98.5+/-1.0% CD3+ T-cell purity. An average 400-fold clearance of malignant B cells was documented during the manufacturing process. The Xcellerated T Cells produced from the Xcellerate III Process exhibited high in vitro biologic activity and have their T-cell receptor repertoire restored to a normal diversity. In-process T-cell activation was reproducibly robust, as measured by increase in cell size, up-regulation of CD25 and CD154 expression and the secretion of IL-2, IFN-gamma and tumor necrosis factor (TNF)-alpha. DISCUSSION: A low-volume, high-yield bioreactor-based process has been developed, qualified and implemented for the reproducible, GMP manufacture of high purity, biologically active Xcellerated T Cells for the treatment of CLL patients in clinical trials.     

The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes

Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.     

Quantitative ultrastructural study of nucleolus-organizing regions at some stages of the cell cycle (G0 period, G2 period, mitosis)

The quantitative characteristics of chromosomal nucleolus-organizing regions (NORs) and some other nucleolar components were studied on ultra-thin sections of pig embryo kidney cells (PK cells). It was shown that: 1) nucleoli-per-cell volumes were 3 times smaller in the G0 period than in the G2 period; 2) the number of fibrillar centers (FCs) per cell in the G0 period, the G2 period, and at metaphase was equal to 7, 33.7, and 8, respectively; 3) mean volumes of individual FCs in the G0 period (0.033 +/- 0.005 micron3), G2 period (0.014 +/- 0.001 micron3), and at metaphase (0.025 +/- 0.002 micron3) were significantly different; 4) the total volumes of FCs calculated per haploid set of chromosomes were practically the same in the G0 (0.105 micron3) and G2 (0.107 micron3) periods, but were twice as large as those at metaphase (0.04-0.05 micron3). These data show that partial activation and inactivation of ribosomal genes in interphase PK cells are not accompanied by a considerable change in the total volume of FCs and may be due to the fragmentation and fusion of individual FCs. Complete inactivation of ribosomal genes in mitosis results in a decrease of total volumes of FCs per cell; 5) in G0 and G2 periods the total volume of the dense fibrillar component per nucleolus is practically proportional to the nucleolus volume (r = 0.99); 6) in the G2 period, the nucleolus volume is also proportional to the number of FCs (r = 0.99; 7) the volume of the dense fibrillar component within individual fibrillar complexes is not a constant one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin resistance in myotonic dystrophy.

The aim of the present study was to obtain a comprehensive picture of the rate of insulin secretion and of tissue sensitivity to the endogenous hormone in myotonic dystrophy patients (MyD). The minimal model approach was utilized for the analysis of frequently sampled intravenous glucose tolerance test data (FSIGT). This method provided the characteristic parameters: SI, insulin sensitivity index; SG fractional glucose disappearance independent of dynamic insulin; n, fractional insulin clearance; phi 1 and phi 2 first and second phase insulin delivery sensitivities to glucose stimulation. In MyD patients SI was reduced (p less than 0.01) by 71% to 1.4 +/- 0.3 x 10(-4) min-1/(microU/ml), whereas in controls it was 4.85 +/- 0.77; SG was within the normal range: 0.044 +/- 0.012 min-1 in MyD patients and 0.036 +/- 0.017 min-1 in controls; phi 1 increased in MyD patients (7.4 +/- 1.3 min (microU/ml)/(mg/dl) versus 4.1 +/- 1.2 in controls); phi 2 increased in MyD patients (126 +/- 47 x 10(4) min-2/(microU/ml)/(mg/dl) versus 17 +/- 6 in controls; p less than 0.05). MyD patients showed a normal tolerance with the glucose disappearance constant, KG within the normal range: 2.75 versus 2.62% min-1 in controls. In MyD patients insulin resistance was associated with a higher than normal insulin delivery for both secretory phases, although the second phase was responsible for releasing a greater amount of hormone. In conclusion MyD patients try to compensate for overall insulin resistance by a more marked pancreatic response.     

Proliferation patterns of peripheral blood lymphocytes in CLL patients: cytophotometric and microfluorimetric study

Peripheral blood lymphocytes of 19 patients with CLL, 9 patient with LS and 10 healthy donors were studied by Feulgen cytophotometry, 3HTdR autoradiography, A0 microfluorimetry and PHA stimulated cultures. In CLL the bulk of cells are in G0 (80.6 +/- 3.7%) the rest are in G1 (16.3 +/- 3.6%) and S + G2 (3.0 +/- 1.0%). Thymidine LI values were two orders lower (0.098 +/- 0.04). In five cases combined autoradiographic and cytophotometric study on the same cells revealed 6-14% of cells arrested in S. In peripheral blood of LS patients G0 cells also predominate, and only in 3 cases cytophotometry revealed hyperdiploid (S + G2) cells. In normal lymphocytes 1.5 hrs after PHA stimulation A0 binding increases on the average by 80% compared to unstimulated cultures and remains at this level during 12 hrs. CLL and LS cells behave nearly the same with the only difference: the 80% increase is observed only after 3-4.5 hrs in culture. G0----G1 flow rate in case of normal lymphocytes is higher than for neoplastic cells but both are recruited into cell cycle during all the period in culture. G1----S transition is delayed in case of LS lymphocytes and strongly inhibited in CLL lymphocyte cultures compared to normal cells. The possible mechanisms of these features are discussed.     

Actin and tubulin of normal and leukaemic lymphocytes.

Double labelling and the isolation of actin- and tubulin-derived peptides were used to determine the amounts of these proteins in peripheral lymphocytes from normal donors and from patients with chronic lymphocytic leukaemia. As a precaution against proteolysis, samples were boiled before assay. The actin content of chronic-lymphocytic-leukaemia (CLL) lymphocytes was 8.1 +/- 2.1% of total protein, which was lower (P less than 0.05) than the amount (12.8 +/- 3.0%) of actin found in normal lymphocytes. The tubulin content of CLL lymphocytes was 4.4 +/- 1.5% of total protein, which was also significantly less (P less than 0.05) than that of normal lymphocytes, which was found to be 6.1 +/- 1.1%.     

Evaluation of the capacity of the liver for phenazone biotransformation in patients with uremia on peritoneal dialysis

This work aimed at establishing whether liver ability to biotransformation of drugs expressed by antipyrine kinetics is disturbed in peritoneally dialysed patients with end-stage renal failure. The investigations were carried out in 10 uraemic patients using the antipyrine test and comparing antipyrine kinetics with those obtained in 13 healthy individuals. At the time of investigations, standard clinical tests of liver function were normal and HBs antigen was absent in all patients. It was shown that peritoneally dialysed patients with end-stage renal failure had not significantly changed antipyrine elimination as compared with the group of healthy controls: t0.5 = 13.2 +/- 6.8 v. 11.8 +/- 8.1 h, plasma clearance = 50 +/- 30 v. 34 +/- 21 ml/min (x +/- SD). The obtained results indicate that antipyrine kinetics is within normal range in uraemic patients regularly dialysed suggesting cytochrome P-450 in microsomes not being markedly reduced.     

Age dependent change in size distribution of blood cells in fetal and young mice

The mean corpuscular volume (MCV) and the size distribution of circulating blood cells were determined in the fetal mice of C3H/He strain by using a new electric cell size analyser, Coulter Channelyzer C-256. On the 12th day of gestation, the volume of circulating blood cells was distributed between approximately 240-820 fl (mode at 470 fl), and the MCV was 534.9 +/- 30.1 fl, i. e., ten to eleven times that of adult one. On the 14th day, two types of cell population were observed; one with smaller cell volume and another with larger one corresponded to that of the blood cells on day 12. Therefore, two peaks were observed to be at 140 and 501 fl in the size distribution curve. The cell population with large volume observed on day 12-14 had been almost disappeared by the 16th day of gestation, and the small blood cells became dominant. The MCV of blood cells was then decreased with the development of fetus, from 188.2 +/- 19.3 fl on day 16, to 135.1 +/- 7.3 fl on day 18, and 117.5 +/- 7.2 fl on day 20. The size of blood cells continued to decrease gradually after birth, and became adult range by 8 weeks after birth. The MCV values of the blood cells were 120.9 +/- 8.6 fl, 87.5 +/- 6.2 fl, and 48.7 +/- 0.8 fl for the newborns of 1 day and 7 days old, and the adult mice, respectively. White blood cells were not separated from the blood samples in this study. However, the size distribution and MCV presented above were appeared to be related essentially to the red blood cells, since the number of white blood cells are negligible small compared with that of the red cells.     

Contribution of new methods of quantitative cytology and immunology to the understanding of lymphoproliferative syndromes]

Seventy one patients with a lymphoid hemopathy, three with agammaglobulinemia and six normal controls were investigated with regard to their blood lymphocyte membrane-associated light chains. Detection and quantitation of antigenic determinants were performed by means of peroxidase-labeled antibodies. Compared to normal controls, values found in chronic lymphocytic leukemia (CLL) were very low (tenfold decrease). The number of antigenic determinants on lymphoid cells from patients with blast crises supervening in CLL, prolymphocytic leukemia, Waldenstrom's macroglobulinemia and Burkitt cell acute leukemia were significantly higher than those seen in patients with CLL. The data obtained in this investigation through quantitative immunocytology constitutes a new parameter for the classification of lymphoid hemopathies and for an approach to their pathogenesis, in particular if the quantity of membrane immunoglobulin correlates with the stage of cell maturation.     

Inspiratory muscles during exercise: a problem of supply and demand

The capacity of inspiratory muscles to generate esophageal pressure at several lung volumes from functional residual capacity (FRC) to total lung capacity (TLC) and several flow rates from zero to maximal flow was measured in five normal subjects. Static capacity was 126 +/- 14.6 cmH2O at FRC, remained unchanged between 30 and 55% TLC, and decreased to 40 +/- 6.8 cmH2O at TLC. Dynamic capacity declined by a further 5.0 +/- 0.35% from the static pressure at any given lung volume for every liter per second increase in inspiratory flow. The subjects underwent progressive incremental exercise to maximum power and achieved 1,800 +/- 45 kpm/min and maximum O2 uptake of 3,518 +/- 222 ml/min. During exercise peak esophageal pressure increased from 9.4 +/- 1.81 to 38.2 +/- 5.70 cmH2O and end-inspiratory esophageal pressure increased from 7.8 +/- 0.52 to 22.5 +/- 2.03 cmH2O from rest to maximum exercise. Because the estimated capacity available to meet these demands is critically dependent on end-inspiratory lung volume, the changes in lung volume during exercise were measured in three of the subjects using He dilution. End-expiratory volume was 52.3 +/- 2.42% TLC at rest and 38.5 +/- 0.79% TLC at maximum exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Size and Surface Charge Properties of Myelin Vesicles from Normal and Diseased (Multiple Sclerosis) Brain

Differences have been observed between myelin vesicles prepared from normal human central nervous system and from white matter of patients who died with multiple sclerosis (MS). The mean cross-sectional area of the vesicles was 5.69 +/- 0.17 micron 2 from normal myelin and 3.71 +/- 0.28 micron 2 for diseased myelin. Vesicle size was reduced to 4.08 +/- 0.21 micron 2 when normal myelin vesicles were prepared in the presence of 0.1 mM EDTA. The presence of Ca2+ during the preparation of the vesicles had no effect on the mean cross-sectional area. In the case of MS myelin vesicles, 0.1 mM EDTA had no effect on vesicle size, whereas the presence of Ca2+ increased the vesicle size from 3.71 +/- 0.28 to 5.40 +/- 0.31 micron 2. Electrokinetic analysis revealed that the electrophoretic mobility of normal myelin vesicles was -5.169 +/- 0.193 X 10(-8) compared with -6.093 +/- 0.202 X 10(-8) m2 s-1 V-1 for the MS myelin vesicles. The presence of 0.1 mM EDTA increased the electrophoretic mobility of the normal vesicles to -6.483 +/- 0.151 X 10(-8) m2 s-1 V-1 but did not significantly affect that of the MS vesicles. Addition of 0.1 mM Ca2+ decreased the electrophoretic mobility of both normal and MS vesicles to similar mobilities. From these data, the surface charge densities were calculated for both normal and MS myelin vesicles and found to be -2.93 and -5.39 mV m-1, respectively. The phase transition temperature determined by wide-angle x-ray diffraction studies was 63 degrees C for normal myelin vesicles and 43 degrees C for MS myelin vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibin B levels in adolescents and young adults with type 1 diabetes

OBJECTIVE/METHODS: To assess exocrine and endocrine testicular function in subjects with diabetes, we evaluated serum inhibin B, gonadotrophins and testosterone levels in 33 male adolescent and young adult patients affected by type-1 diabetes (age 21.0 +/- 5 years; range 14.2-33.3), with a mean disease duration of 12.7 +/- 5.8 years (range 1.5-25.3) and various metabolic control (HbA1c 7.8 +/- 1.5%; range 5.5-13.2) and compared them with those of an age-matched group of 36 healthy control subjects (age 19.5 +/- 4.1 years; range 13.6-28.1). Both patients and controls had a testicular volume >or=15 ml. Inhibin B was measured by ELISA method. RESULTS/CONCLUSION: Diabetics and controls had comparable inhibin B (203 +/- 74 vs. 221 +/- 69 pg/ml, respectively) and follicle-stimulating hormone (FSH) levels, while luteinizing hormone (LH) and testosterone levels were significantly higher in the diabetic group. Inhibin B was negatively correlated both in patients and controls with FSH, while a negative correlation with LH was found only in the diabetic group. We conclude that our young diabetic males, after a mean disease duration of 12 years and various metabolic control, had inhibin B and FSH levels comparable to those of normal subjects. Therefore, they seem to have a regular testicular function and in particular a normal seminiferous tubule/Sertoli cell activity despite sustained hyperglycemia.     

Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy

Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac hypertrophy in rats after supravalvular aortic constriction. I. Size and number of cardiomyocytes, endothelial and interstitial cells

The ascending aorta of 22 adult male Sprague-Dawley rats was constricted with a silver ring, and 25 animals were subjected to a sham-operation. The hearts, including the main arteries, were fixed by retrograde perfusion 3, 7, 14, 21 and 35 days after the operation. The cross-sectional area of the aorta was reduced by the constriction to an average of 20% of the values found after sham-operation. Twenty-one days after the constriction the weight of the left ventricular myocardium including the septum was increased 1.7-fold compared with controls. No further increase in weight was observed 35 days after the operation. The relative volumes of the tissue components remained largely constant in the subepicardial myocardium. In the subendocardial myocardium, however, the volume fraction of interstitial and, to a lesser extent, of endothelial tissue was significantly increased. Twenty-one days after constriction the estimated total volumes of the different myocardial components per left ventricle were increased 1.7-fold for heart muscle parenchyma, 1.8-fold for endothelial tissue, 2.9-fold for interstitial tissue, and 1.3-fold for capillary lumina compared with controls. At 35 days, only the interstitial tissue showed a further increase to 4.8-fold of control values. The mean cardiomyocyte volume was increased after aortic constriction in proportion to the increase in left ventricular weight, i.e. 1.7-fold over controls at 21 days. After 35 days its value was 29,500 +/- 790 micron 3 in rats subjected to aortic constriction compared with 16,800 +/- 640 micron 3 in controls. At this time the estimated number of cardiomyocytes per left ventricle showed no significant differences between experimental animals (2.9 X 10(7)) and controls (3.1 X 10(7)). Endothelial and interstitial cells were not only increased in average single cell volume (1.3-fold and 2.0-fold, respectively), but also in number per left ventricle (1.4-fold and 2.7-fold, respectively). Two-dimensional parameters indicated that during hypertrophy the capillary supply lagged behind the overall mass increase but achieved control levels on termination of hypertrophic growth at 35 days. These results show that even in pronounced hypertrophy the increase in mass of the myocardial parenchyma in the rat is due exclusively to an enlargement of cardiomyocytes (hypertrophy), whereas in endothelial and interstitial tissues enlargement of cells as well as increase in cell number (hyperplasia) also plays a role.     

Catecholamines and pituitary function. VI. Effect of different dopamine doses on TRH-induced prolactin release in women with pathological hyperprolactinemia

The present study was designed to examine the effect of low-dose dopamine (DA) infusion rates (0.02 and 0.1 microgram/kg X min) on both basal and TRH-stimulated prolactin release in normal and hyperprolactinemic individuals. Sixteen normally menstruating women in the early follicular phase of a cycle and 23 hyperprolactinemic patients were studied. 0.1 microgram/kg X min DA was infused in 8 normal women and 15 patients with pathological hyperprolactinemia, while 8 normal controls and 8 patients received 0.02 microgram/kg X min DA TRH (200 micrograms, i.v.) was administered alone and at the 180th min of the 5-hour DA infusion in all controls and patients. A significant reduction in serum PRL levels, which was similar in normal women (-59.5 +/- 4.0%, mean +/- SE) and hyperprolactinemic patients (-48.2 +/- 5.5) was observed in response to 0.1 microgram/kg X min DA. In normal cycling women DA infusion significantly (P less than 0.02) reduced the PRL response to TRH with respect to the basal TRH test (delta PRL 45.0 +/- 7.0 vs. 77.9 +/- 15.4 ng/ml). On the contrary, the PRL response to TRH was significantly higher during 0.1 microgram/kg X min DA than in basal conditions in hyperprolactinemic patients, both in absolute (delta PRL 91.8 +/- 17.6 vs. 38.4 +/- 6.8, P less than 0.03) and per cent (198.5 +/- 67.6 vs. 32.1 +/- 7.5, P less than 0.02) values. A normal PRL response to TRH, arbitrarily defined as an increase greater than 100% of baseline, was restored in 11 out of 15 previously unresponsive hyperprolactinemic patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical function of dystrophin in muscle cells

We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.     

Immunological study of the T- and B-lymphocytes in normal cows and in chronic lympholeukemia

T- and B-lymphocyte populations from lymphoid organs and tissue of normal cattle and cattle with chronic lymphocytic leukaemia (CLL) were studied. Comparative studies of surface properties, quantitative parameters and heterogeneity of main T- and B-cell populations were performed. It must be noted that proliferation of B-lymphocytes, bearing surface IgM in blood, lymph nodes and spleen is closely connected with the progression of leukaemic process. An increased number of B-lymphocytes (2-3 times) with the receptors for complement was found. The proportion of T mu and T gamma cell subsets in CLL cows is distorted. The T gamma cell subset in T-cell suspensions from blood and spleen in CLL cows prevails in comparison with that in controls. The number of T mu cells in blood and lymph nodes in CLL is decreased.     

Mitogen-stimulated phospholipid synthesis in normal and immune-deficient human B cells

Eight patients with common variable panhypogammaglobulinemia were shown in the in vitro Ig biosynthesis assay to have defective B cell responses to pokeweed mitogen (PWM). Phospholipid synthesis was assessed in the B cell plus monocyte fraction (MB) and irradiated T cells (T*) of patients and paired normal controls. Cell populations were studied separately and in the four possible combinations (1:1), with and without PWM, to reveal the effect of cell interactions. At 16 to 20 hr the mean stimulation index (SI) +/- standard error for MB cells alone was 1.01 +/- 0.02 for eight patients and 0.99 +/- 0.02 for the paired normals; the T* cell SI was 1.25 +/- 0.04 for patients and 1.28 +/- 0.05 for normals. Combinations of normal MB cells with normal T* cells showed significantly higher SI when compared with the combinations of normal MB cells with patient T* cells (p less than 0.005). However, the combination of patient MB cells with patient T* cells and the combination of patient MB cells with normal T* cells were not significantly different in SI (0.05 less than p less than 0.1). Isolation of patient and normal B cells, T* cells, and monocytes after the choline pulse showed that patient B cells gave a higher SI with normal T* help than with patient T* help. Of greatest interest is the finding that patient B cells that were defective in PWM-stimulated Ig production nevertheless showed a phospholipid synthesis response to PWM in the normal range, suggesting that the maturation defect in these B cells occurs later than the phospholipid synthesis acceleration step, or on a different pathway.     

Quantitative deposition of ultrafine stable particles in the human respiratory tract

Theoretical models of particle deposition in the respiratory tract predict high fractional deposition for particles of less than 0.1 micron, but there are few confirming experimental data for those predictions. We have measured the deposition fraction of a nonhygroscopic aerosol in the human respiratory tract. The aerosol had a count mean diameter of 0.044 micron SD of 1.93, as measured with an electrical aerosol analyzer, and was produced from a 0.01% solution of bis(2-ethylhexyl) sebacate using a condensation generator. Subjects inhaled the aerosol using a controlled respiratory pattern of 1 liter tidal volume, 12/min. Deposition was calculated as the difference in concentration between inhaled and exhaled aerosol of five size fractions corrected for system deposition and dead-space constants. Three deposition studies were done on each of five normal male volunteers. Means (+/- SE) for the five size fractions were 0.024 micron, 0.71 +/- 0.06; 0.043 micron, 0.62 +/- 0.06; 0.075 micron, 0.53 +/- 0.05; 0.13 micron, 0.44 +/- 0.04; and 0.24 micron, 0.37 +/- 0.06. These data demonstrate that deposition of inhaled particles in the 0.024- to 0.24-micron size range is high and increases with decreasing size. These observations agree with and validate predictions of mathematical models.     

Evaluation of the nucleolar organizer activity of tumor cells in patients with esophageal and stomach cancers

Silver impregnation findings of normal and cancer cell nucleoli are given for 30 cancer patients with esophageal (16) and gastric (14) carcinoma and 5 controls. It has been shown that the content of silver granules per cell (35.3 +/- 2.4) and per nucleolus (13.3 +/- 0.7) in tumor cells were significantly higher than in normal ones (11.4 +/- 1.9 and 5.3 +/- 0.5, respectively). The importance of these findings for clinical oncology is discussed.