Detection of small numbers of monoclonal B lymphocytes in the blood of patients with lymphoma.

The definition of complete remission and early relapse in patients with lymphoid malignancy is complicated by the difficulty of recognizing the presence of small numbers of malignant lymphocytes in a population of normal lymphocytes. We have used a method based on the cytofluorometric detection of lymphocytes having homogeneous amounts of surface immunoglobulin of one light-chain class to study blood from patients with lymphomas. The test is capable of reliably detecting 10 per cent or less of monoclonal B lymphocytes in normal blood, and it shows a high incidnece (30 to 40 per cent) of previously unsuspected monoclonal B lymphocytes in patients who were though to have no abnormal blood cells according to standard morphologic technics. The ability to estimate the number of such cells may facilitate the planning of therapy and make more meaningful the concept of complete remission.     

Increased numbers and functional activity of CD56+ T cells in healthy cytomegalovirus positive subjects

Mycobacterium indicus pranii (MIP) is an atypical mycobacterial species possessing strong immunomodulatory properties. It is a potent vaccine candidate against tuberculosis, promotes Th1 immune response and protects mice from tumours. In previous studies, we demonstrated higher protective efficacy of MIP against experimental tuberculosis as compared with bacillus Calmette–Guérin (BCG). Since macrophages play an important role in the pathology of mycobacterial diseases and cancer, in the present study, we evaluated the MIP in live and killed form for macrophage activation potential, compared it with BCG and investigated the underlying mechanisms. High levels of tumour necrosis factor-α, interleukin-12p40 (IL-12p40), IL-6 and nitric oxide were produced by MIP-stimulated macrophages as compared with BCG-stimulated macrophages. Prominent up-regulation of co-stimulatory molecules CD40, CD80 and CD86 was also observed in response to MIP. Loss of response in MyD88-deficient macrophages showed that both MIP and BCG activate the macrophages in a MyD88-dependent manner. MyD88 signalling pathway culminates in nuclear factor-κB/activator protein-1 (NF-κB/AP-1) activation and higher activation of NF-κB/AP-1 was observed in response to MIP. With the help of pharmacological inhibitors and Toll-like receptor (TLR) -deficient macrophages, we observed the role of TLR2, TLR4 and intracellular TLRs in MIP-mediated macrophage activation. Stimulation of HEK293 cells expressing TLR2 in homodimeric or heterodimeric form showed that MIP has a distinctly higher level of TLR2 agonist activity compared with BCG. Further experiments suggested that TLR2 ligands are well exposed in MIP whereas they are obscured in BCG. Our findings establish the higher macrophage activation potential of MIP compared with BCG and delineate the underlying mechanism.     

Myxofibrosarcomas contain large numbers of infiltrating immature dendritic cells

Myxofibrosarcoma is a malignant tumor with distinctive histologic features and is believed to be derived from fibroblasts. The function of infiltrating myeloid cells in myxofibrosarcoma is poorly understood. It previously has been shown that a combination of dendritic morphologic features and expression of the C-type lectin, dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN), is useful for identifying DC populations in tissue sections. In the present study, we found that 3% to 61% (median, 22%) of cells in myxofibrosarcomas express DC-SIGN and have dendritic morphologic features. These DC-SIGN--positive cells are not in cell cycle and are consistent with infiltrating DCs. The percentage of DCs in myxofibrosarcomas is independent of tumor grade. It previously has been shown that DC-SIGN--positive cells are either immature DCs or DCs that predominantly activate TH2 cells, both subsets likely to give rise to ineffective antitumor responses. The DC-SIGN--positive DCs that we have identified in myxofibrosarcoma may, therefore, be involved in the induction of ineffective immune responses or even tolerance to tumor antigens.     

CD1c(+) immature myeloid dendritic cells are predominant in cord blood of healthy neonates

It has been suggested lately that some types of antigen presenting cells-myeloid dendritic (DC-1) cells can differentiate the immune response towards Th1 type immunity, whereas lymphoid cells (DC-2) can stimulate Th2 type immunity. It has been observed that neonates are deficient in Th1 response. The purpose of our study was to estimate the proportions of immature myeloid (CD1c(+)) and lymphoid (BDCA-2(+), BDCA-4(+)) dendritic cells and the CD1c(+):BDCA-2(+) cell ratio in cord blood of healthy neonates in comparison with dendritic cells of healthy adults. Thirty healthy neonates born from normal pregnancies and 30 healthy adults were included in the study. The dendritic cells were isolated from cord and peripheral blood, stained with anti-CD1c, anti-BDCA-2, anti-BDCA-4, anti-CD123 and anti-CD19 monoclonal antibodies and estimated using flow cytometry. The percentage of CD1c(+) dendritic cells in cord blood of healthy newborns did not differ significantly when compared to those in peripheral blood of healthy adults. The percentages of cord blood BDCA-2(+) and BDCA-4(+) dendritic cells of neonates were significantly lower when compared to lymphoid dendritic cells in peripheral blood of adults. The CD1c(+):BDCA-2(+) ratio was significantly higher in cord blood of neonates in comparison with CD1c(+):BDCA-2(+) ratio in adult's blood. Myeloid and lymphoid dendritic cells may be involved in the immune regulation during fetal development. Immature myeloid dendritic cells are predominant in cord blood of healthy neonates. Immature lymphoid dendritic cells are not the major population of dendritic cells in cord blood.     

Phospholipidosis in healthy subjects participating in clinical studies: Ultrastructural findings in white blood cells

Lipid storage disorders and phospholipidosis share similar morphologic characteristics displayed as lamellar bodies at ultrastructural level. More than 50 cationic amphiphilic drugs (CADs), including antidepressants, antianginal, antimalarial, and cholesterol-lowering agents, have been reported to induce phospholipidosis, however, the mechanism by which this occurs has not been extensively studied and is not well understood. Both the Food and Drug Administration (FDA) and the pharmaceutical industry recognized drug-induced phospholipidosis as a significant challenge for drug development. In a randomized, double-blind, placebo-controlled, active-controlled, ascending multiple-dose study to investigate the tolerability, safety, pharmacokinetics, and pharmacodynamics of a new investigational drug (an antihypertensive drug in early drug development) in healthy male subjects, possible drug-induced phospholipidosis was also explored ultrastructurally. Given the presence of these structures both pretreatment and following placebo treatment, it was concluded that the presence of phospholipid-like structures in individual volunteers could be a normal background finding in neutrophilic granulocytes thus emphasizing their role as natural phagocytic cells. Recommendations for the conduct of this type of studies are given.     

A method to assess the proliferative activity of small numbers of murine peripheral blood mononuclear cells

An economical, reproducible method was developed to assess the mitogenic response of small numbers of murine peripheral blood mononuclear cells (PBMC, 10(3)/ml) to phytohemagglutinin (PHA) stimulation in the presence of feeder layer cells (FLC) and interleukin-2 (IL-2). A linear cell dose-mitogenic response relationship was then demonstrated between the activity of PBMC and spleen cells of individual mice (slope = 1.054 +/- 0.037; r = 0.989 +/- 0.003). This would suggest that mice can be used for longitudinal studies because small blood volumes can be obtained frequently over a period of time from individual mice and the PBMC can be assessed for their T cell proliferative activity.     

正常人外周血单个核细胞端粒酶各组分基因的表达

端粒酶是一种具有特殊性质的逆转录酶 ,能以自身的RNA为模板合成约 10 kb的端粒重复序列。端粒酶在大多数肿瘤细胞、干细胞、淋巴细胞中有表达。我们以前的研究显示 :正常人外周血单个核细胞 ( PBMC)可表达端粒酶活性 [1 ]。人端粒酶主要由 3部分组成 ,即 ,端粒酶 RNA( h TR) ,端粒酶相关蛋白 ( TP1) ,端粒酶催化亚单位 ( h TERT) [2 ]。我们进一步研究了这 3个组分基因表达的状况 ,现报道如下。1　材料和方法1.1　正常人　 2 0例健康志愿者均系我院职工 ,年龄 2 7～5 0岁 ,平均 3 2岁 ,男性 11例 ,女性 9例。1.2　外周血单个核细胞…     

Lymphocyte cultures with small numbers of cells.

Prior experience with cultures of cerebrospinal fluid lymphocytes indicated a need to develop methods for culturing small numbers of cells. Peripheral blood lymphocytes (PBL) were obtained from 20 normal volunteers. Standard microcultures using 100 X 10(3) PBL/0.2 ml and cultures with 50, 25 and 12.5 X 10(3) in 0.1 ml or 0.2 ml were established in RPMI 1640 with autologous plasma. These cultures were incubated with PHA (1--30 microgram) for 3, 4 and 5 days, pulsed with [3H]thymidine and harvested. In unstimulated cultures, cpm declined linearly with decreasing cell numbers. Standard cultures (100 X 10(3) PBL/0.2 ml) had maximal PHA stimulation (80,916 +/- 6394) at day 3 with 30 microgram PHA. Other 0.2 ml cultures had lower cpm. By culturing 25 X 10(3) PBL in 0.1 ml for 3 days cpm were 82,874 +/- 6875 with 30 microgram PHA and 77,153 +/- 6022 with 15 microgram PHA and were similar to standard cultures. Similar cpm were seen with 12.5 X 10(3) PBL in 0.1 ml after 4 days with 30 micrograms of PHA (80,838 +/- 6674) and with 15 micrograms of PHA (72,860 +/- 6243), and also after 5 days with 30 micrograms of PHA (86,703 +/- 6732) and with 15 micrograms of PHA (74,066 +/- 6388). The maximal response (126,578 +/- 6580) was seen with 25 X 10(3) PBL/0.1 ml at day 4 with 30 micrograms of PHA. By decreasing culture volume to 0.1 ml and increasing time, the number of cells necessary to give PHA responses similar to standard cultures can be reduced by 75--88%.     

Arterial blood gas standards for healthy young nonsmoking subjects

Arterial blood gas analysis has become indispensable for precise physiologic assessment of many lung and heart conditions. Previous studies have related the level of arterial oxygenation to age, smoking habits, and the severity of lung or heart dysfunction. However, no study has reported complete normal blood gas values under all conditions most commonly used by cardiopulmonary laboratories to assess patients. Therefore, we assessed blood gas values in 20 nonsmoking volunteers (ten men, ten women) between 20 and 28 years of age who were healthy (negative heart-lung history and normal results on physical examination, chest radiography, and lung function testing). Blood samples were drawn from the radial artery and measured immediately on a blood gas analyzer. The findings were no significant different (P less than 0.05) in blood gas values among rest (supine), rest (sitting), and exercise (supine) conditions within sex groups; significantly lower mean PCO2 for women than for men under all conditions (except for subjects breathing 100% O2), and a higher pH for women in the rest (supine) position than under other conditions; and a lower mean PCO2 and higher pH for both groups breathing 100% O2. This study provides valid normal arterial blood gas reference standards for routine cardiopulmonary function testing.     

Minimum numbers of fresh whole blood and plasma samples from patients and healthy subjects for ISI calibration of CoaguChek and RapidPointCoag monitors

The international sensitivity index (ISI) calibration of point-of-care-test (POCT) prothrombin time (PT) whole blood monitors is complex, requiring manual PT testing of 60 patients' and 20 healthy subjects' plasma samples. The possibility of reducing these numbers was studied by a Monte Carlo Bootstrap study for 2 POCT PT systems. For reduced sample numbers, this consisted of 50,000 calibrations using whole blood and plasma samples tested on the monitors with manual PT testing of plasma samples from the same blood donations. There was little effect on mean ISI by reduction of sample numbers to a total of 7, but there was progressively less certainty regarding the reliability of the calibration. Precision of the calibrations and international normalized ratio deviation were not affected markedly by reducing numbers to half As ISI calibration with the 2 POCT systems was less precise than conventional manual testing, for maximum confidence, reduction of numbers is not advised.     

Detection of tumor cells in the blood stream

The effectiveness of trypan blue, acridine orange, tetracycline, and oxytetracycline for the detection of tumor cells injected into the bloodstream was investigated in experiments on rats. Cells were detected in the microvessels of the mesentery by intravital microscopy. Luminescence of the fluorochromed cells was observed in blue-violet (λ_Max 400 nm) and ultraviolet (λ_Max 365 nm) rays of a mercury vapor lamp and in laser radiation (λ 337 nm). The intensity of luminescence of cells stained with acridine orange was higher, and their structure could be distinguished better, than in cells containing tetracyclines. Identification of the cells by trypan blue was difficult. The luminescence method is sufficiently simple, and it can be used to discover neoplastic cells actually in the bloodstream.     

Linear and nonlinear analysis of blood flow in healthy subjects and in subjects with Raynaud's phenomenon.

The paper presents analyses of the dynamics contained in the blood flow signals measured on healthy subjects and on subjects with primary Raynaud's phenomenon. Different signal processing methods are presented and discussed. The dynamics was evaluated in the time and frequency domains and in phase space. Additionally, changes in the basal value during temperature provocation were studied using multiresolution analysis. The analyses demonstrate differences between the blood flow dynamics in healthy subjects and subjects with Raynaud's phenomenon. Moreover, the observed decrease in the amplitude of oscillation in regions approximately 0.04 Hz and approximately 0.1 Hz suggests an impairment in the neurogenic and the myogenic regulation of the blood flow. The administration of nifedipine in subjects with Raynaud's phenomenon results in an increase in the basal value and in the amplitude of the blood flow component oscillating with the heart rate. However, it does not restore the dynamics to that found in healthy subjects.     

Effects of acupuncture on skin and muscle blood flow in healthy subjects

In 14 healthy female subjects, the effects of needle stimulation (acupuncture) on skin and muscle blood flow were investigated using a non-invasive custom-designed probe and photoplethysmography (PPG). In randomised order, 2–7 days apart, three modes of needle stimulation were performed on the anterior aspect of the tibia: superficial insertion (SF), insertion into the anterior tibial muscle (Mu), and insertion into the muscle including manipulation of the needle in order to elicit a distinct sensation of distension, heaviness or numbness (DeQi). Before intervention, the subjects rested for 30 min. After the intervention, the needle was left in situ for 20 min. Blood flow recordings were performed intermittently from 10 min prior to the intervention to the end of the trial. In a fourth session, serving as control, corresponding measurements were performed without any needle stimulation. Area under curve was calculated for 5-min periods prior to and after stimulation, respectively, and for the remaining 15-min period after stimulation. Compared to the control situation, muscle blood flow increased following both Mu and DeQi for 20 min, with the latter being more pronounced for the initial 5 min. Skin blood flow increased for 5 min following DeQi. However, no increase was found following SF. The DeQi stimulation was preceded by higher visual analogue scale ratings of anxiety prior to stimulation, which might have influenced skin blood flow to some extent. The results indicate that the intensity of the needling is of importance, the DeQi stimulation resulting in the most pronounced increase in both skin and muscle blood flow.     

Strenuous exercise increases late outgrowth endothelial cells in healthy subjects

Endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) seem to play an important role in vessel formation. While EPCs seem to exert their function mainly through a paracrine effect, the OECs can develop into mature endothelial cells and form tubular structures. Exercise is known to increase angiogenic factors that can mobilize EPCs; however, the effect on OECs is not known. We investigated the response to a single session of strenuous exercise on OECs, vascular endothelial growth factor (VEGF) and inflammatory cell levels in the healthy. Eleven healthy subjects performed 1 h of spinning exercise. Blood samples were collected at 1, 6, 24 and 48 h post-exercise for cell culture and biochemical analysis. OEC colonies doubled one hour after the spinning session (baseline 4.5 ± 4.3 vs. 9.0 ± 3.7, P < 0.05). Serum VEGF increased from 194 ± 107 pg/ml at baseline to 224 ± 111 pg/ml after 1 h, p = ns and neutrophilic granulocytes increased from 3.73 ± 1.38 at baseline to 9.08 ± 10.5 at 1 h (P < 0.01). The increased levels of OECs, VEGF and neutrophilic granulocytes declined gradually at the following time points. VEGF levels and neutrophilic granulocytes were highly correlated to OEC levels, r = 0.903 (VEGF) and r = 0.85 (neutrophilic granulocytes), respectively. Strenuous physical activity increases OEC colonies and is correlated to serum VEGF and neutrophilic granulocytes levels. An acute exercise-induced inflammatory response might be responsible for the VEGF release and subsequent increase of OECs. The clinical importance of these findings remains to be elucidated.