Immunologic characterization of chronic lymphocytic leukemia cells.

Immunologic characterization of the neoplastic cells in the circulation of patients with CLL suggests these cells show significant differences in membrane characteristics from normal B lymphocytes. Although the leukemic cells bear a homogenous membrane-associated immunoglobulin, they also react with an anti-human T cell serum. In all patients studied, 60-90% of the cells, were stained by this antiserum. This suggests that the leukemic cells share antigenic determinants with T lymphocytes. CLL cells, unlike normal B cells, showed a marked increase in mouse-complement receptors. No increase in receptors for guinea pig complement was observed in the leukemic cells. The population of SIg-bearing lymphocytes was significantly greater than that of complement-receptor bearing lymphocytes. The total number of E-rosetting cells was increased in all CLL patients. Mitogenic responses of the leukemic cells were depressed and delayed. These results suggest that neoplastic lymphocytes cannot be classified as T- or B-derived on the basis of criteria used to define normal lymphocytes.     

In vitro functional studies of mononuclear cells in patients with CLL: evidence for functionally normal T lymphocytes and monocytes and abnormal B lymphocytes.

In vitro functional studies of mononuclear cells from 34 patients with B-cell type CLL were investigated and the results of these studies were as follows: 1) The T lymphocytes from patients with CLL were capable of responding normally to PHA or PWM, of inducing allogeneic normal B lymphocytes to respond to these mitogens and of stimulating normally to allogeneic lymphocytes in "one-way" mixed lymphocyte reaction; 2) The monocytes from these patients were capable of enhancing the T lymphocyte response to mitogens and of stimulating normally to allogeneic lymphocytes; and 3) The leukemic B lymphocytes were incapable of responding to mitogens even in the presence of normal T lymphocytes and their enhancer cell activity on T lymphocyte response or their stimulating capacity on allogeneic lymphocytes was depressed. These observations suggest that the T lymphocytes and monocytes from patients with CLL are functionally normal while the leukemic B lymphocytes from these patients are functionally abnormal.     

Proliferation of leukemic B cells in response to SAC and anti-mu. Evidence for different modes of action and comparison to proliferation and differentiation induced by conditioned medium

We investigated the proliferative responses and immunoglobulin production of highly purified E-rosette negative largely leukemic B cells from patients with CLL to Staphylococcus aureus Cowan I (SAC) or to SAC in combination with anti-mu or conditioned medium (CM). The latter was derived by stimulating human peripheral blood mononuclear leukocytes with PHA. We observed: (1) that purified E-rosette negative largely leukemic B cells from 25% (five out of 20) of the patients exhibited proliferative responses to SAC; (2) inhibition of SAC-induced proliferation by anti-mu in certain patients, whereas synergism between SAC and anti-mu in inducing proliferative responses in others; (3) the lack of synergism between SAC and CM in inducing proliferative responses, which is in contrast to the strong synergism that was observed between anti-mu and CM in inducing proliferation; and (4) induction or enhancement by SAC alone of Ig production by largely leukemic B-cell populations from few patients with CLL and purified tonsillar B lymphocytes, but not peripheral blood B cells from normal donors. These results suggest that SAC and anti-mu induce proliferation of B cells by different mechanisms and that B-cell proliferation and differentiation is dependent not only on the mitogen but also on the activation state of the cells.     

Lymphocyte plasma membranes. VI. Surface antigens of lymphocytes in chronic lymphocytic leukemia.

Surface antigens from lymphocytes of patients with chronic lymphocytic leukemia (CLL) and from normal peripheral blood lymphocytes (PBL) were examined by radioimmunoassay; antisera to lymphocytes (ALS) were used to bind the labeled antigens. Cells from patients with CLL, normal PBL, thymus cells (THY), and cultured human lymphoblasts (CHL) were labeled by lactoperoxidase-catalyzed iodination. ALS (prepared against THY and CHL) were used to bind the labeled antigens solubilized in nonionic detergent. PBL resembled THY, but the CLL resembled CHL. Thus ALS(CHL) had greater potency for CLL antigens than for PBL antigens when compared to ALS(THY). Furthermore, the electrophoretic profiles of the immunoprecipitates from CLL cells revealed a peak of approximately 30,000-35,000 mol wt, which was not found for PBL or THY, but was associated with CHL.     

Surface charge characteristics of peripheral blood lymphocytes in chronic lymphatic leukemia and malignant lymphoma

Peripheral blood lymphocytes from patients with chronic lymphatic leukemia (CLL) and malignant lymphoma (ML) were tested for their surface negative charge characteristics and compared with lymphocytes from normal subjects by use of measurements of lymphocyte agglutination with a positively charged poly-L-lysine (PLL) molecule and by use of electron microscopic observation of lymphocytes labeled with cationized ferritin (CF). Unfixed lymphocytes from CLL and ML patients exhibited clustering and patching of CF particles, whereas normal lymphocytes had a uniform, continuous CF-labeling pattern. Lymphocytes from CLL patients had significantly higher agglutination with PLL than did normal lymphocytes.     

Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture

Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal T lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.     

Relationship between bacterial binding to lymphocytes and clinical features in chronic lymphocytic leukemia.

In previous studies we showed that spontaneous bacterial adherence can be used to identify human lymphocyte subpopulations and to demonstrate variable binding patterns in chronic lymphocytic leukemia (CLL). In this study, 10 strains of bacteria of different genera and species were used in blood smears from 24 CLL patients to determine the percentages of lymphocytes that bind bacteria. From these percentages, binding indices were calculated. The symptoms and other laboratory tests were independently recorded and the stages determined. When the two sets of data were compared, relatively low binding indices were found in symptomatic patients or in Stages III and IV; relatively high binding indices were found in asymptomatic patients or in Stages I and II. We suggest that with progression of leukemia, lymphocytes with less "lectin" recognition potential are selected and escape any control mechanism of proliferation.     

Cytoplasmic IgM in leukemic B cells by flow cytometry

The presence of intracellular cytoplasmic immunoglobulin M (IgM) in leukemic cells from patients with acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL) was investigated by flow cytometry. The objective of the study was to develop a reproducible flow cytometric method. A Burkitt's lymphoma-derived B-cell line, Daudi, and a pre-B ALL, Nalm-6, served as prototypes. Normal B cells and cells from patients with chronic myelogenous leukemia (CML) were used as negative controls. Cytoplasmic mu was expressed in 77.3 +/- 7.5% (n = 10) of Nalm-6 cells. CALLA+ ALL and CML cells lacked cytoplasmic mu. The surface-membrane immunoglobulin on the viable B cells was blocked with purified goat anti-human IgM. Subsequently, the B cells were fixed in cold absolute methanol and stained with a fluorescein-conjugated goat anti-human IgM to demonstrate cytoplasmic IgM. After the surface-membrane IgM was blocked, normal B cells had no cytoplasmic IgM (0.3-0.5% positive cells) detectable by flow cytometry. However, the peripheral blood lymphocytes from five patients with CLL and the Daudi cells contained cytoplasmic IgM, ranging from 7.8 to 76.7% and 45.7 to 89.3%, respectively. We conclude that cytoplasmic mu in Nalm-6, CLL, and Daudi cells can be easily and rapidly demonstrated by flow cytometry.     

Interferon induces proliferation in leukemic and normal B-cell subsets

3H-thymidine incorporation following stimulation with interferon (IFN) in vitro was investigated in cell cultures from peripheral blood of patients with chronic lymphocytic leukemia (CLL), spleens from necro-kidney transplants and healthy blood donors. It was demonstrated, that IFN can induce a proliferative response in some normal as well as leukemic B lymphocyte subsets. The responses were not T-cell dependent. The results indicate, that B-cell subsets that proliferate in the presence of IFN, are present in higher proportions in spleen than in peripheral blood, and that they constitute a portion of the leukemic blood lymphocyte pool in some patients with CLL. We have previously demonstrated, that IFN induces varying degrees of transformation and differentiation in blood lymphocytes from a majority of CLL patients. The functional characteristics of different B-cell subsets, and their heterogeneous distribution in leukemia, may be important for the results of IFN treatment in various malignant B-cell disorders.     

Expression of leucocyte-common antigen and large sialoglycoprotein on leukemic cells in B-cell chronic lymphocytic leukemia and non-Hodgkin's lymphoma

Patterns of leucocyte-common antigen (L-CA) and large sialoglycoprotein (LSGP) expression on leukemic peripheral blood lymphocytes of 13 patients with chronic lymphocytic leukemia (CLL), 17 with non-Hodgkin's lymphoma (NHL) in leukemic phase and one with hairy cell leukemia (HCL) have been examined by means of surface labelling and electrophoresis in 5% polyacrylamide gels. The 13 CLL, 10 of the 11 diffuse NHL and the six nodular poorly differentiated lymphocytic lymphoma (PDLL) patients fell into three groups according to expression of 210, 198 and 185k forms of L-CA. Group 1 (210 less than 198 less than 185k L-CA) included eight CLL and one diffuse NHL; Group 2 (210 greater than or equal to 198 and 185k L-CA) included four CLL, three diffuse NHL and four nodular PDLL; Group 3 (mainly 210k L-CA) included one CLL, six diffuse NHL and two nodular PDLL. A patient with diffuse large cell lymphoma and the HCL patient both had patterns of multiple, diffuse, very high Mr labelled glycoproteins. LSGP on these cells varied from nil to very high and levels were not related to L-CA patterns. Lymph node cells from five patients were also studied and were found to express larger numbers of L-CA forms and less LSGP than corresponding peripheral blood lymphocytes. Possible relationships of L-CA forms and LSGP to lymphocyte function and disease patterns are discussed.     

Distinct α-L-Fucosidase Isoenzyme Profiles in Human Leukemic Cells

-L-Fucosidase (EC 3.2.1.51; FUS) activity and isoenzyme characteristics were analyzed in normal lymphocytes, normal (polymorphonuclear leukocytes, PMNs), and myeloid and lymphoid leukemic cells. Chronic lymphocytic leukemia (CLL) lymphocytes had a lower mean specific activity than normal lymphocytes (2.5 vs. 4.0, p 0.05). Acute lymphoblastic leukemia (ALL) blasts had a higher mean specific activity compared to normal lymphocytes (9. 7 vs. 4.0; p 0.001), CLL lymphocytes (9.7 vs. 2.5; p 0.001), and acute myeloid leukemic (AML) blasts (9.7 vs. 7.6; p = NS). Normal PMNs had a higher mean specific activity than normal lymphocytes (7.0 vs. 4.0; p 0.05) but similar activity when compared to CML cells or AML blasts. Blasts from acute myelomonocytic leukemia (AMMoL) patients had higher activity than normal PMNs (9.0 vs. 7.0; p 0.05). The isoenzyme patterns of normal and leukemic granulocytes and lymphocytes were obtained by automated chromatofocusing on PBE-94 microcolumns with normal and leukemic lymphocyte lysates. With normal and leukemic lymphoid lysates two major isoenzyme components (B and A) were isolated. The isoenzyme patterns of PMN, AML, CML, and AMMoL revealed three major peaks (B, A, I), totally different from those seen in lymphoid cells. The patterns of AML, CML, and PMN appeared to be similar to each other; however, the isoenzyme pattern obtained from AMMoL cells could be distinguished from the others by a prominent I peak. Thus, the FUS isoenzyme profile distinguishes the blasts of AMMoL from AML; and AMMoL and AML from ALL.     

alpha-L-Fucosidase activity and isoenzyme characteristics analyzed by chromatofocusing in normal and leukemic lymphocytes

alpha-L-Fucosidase (EC 3.2.1.51) activity and isoenzyme characteristics were analyzed in normal lymphocyte subpopulations, chronic lymphocytic leukemia subpopulations, and acute lymphoblastic leukemia blasts. Similar pH activity profiles revealed that pH 5.0 was optimal in normal and leukemic cells. Unfractionated CLL lymphocytes had a lower specific activity than normal unfractionated lymphocytes (2.5 +/- 1.0 u/10(6) cells v. 4.0 +/- 1.1). CLL B cells and T-cells had lower specific activity than their respective normal counterparts (1.8 +/- 0.2 v. 5.9 +/- 2.0) (B-cells); (2.2 +/- 0.5 v. 3.7 +/- 1.0) (T-cells) suggesting T and B cells in CLL are abnormal. ALL blasts had a higher specific activity compared to unfractionated normal lymphocytes (9.7 +/- 3.0 v. 4.0 +/- 1.1; p less than 0.001). The isoenzyme pattern of normal, CLL and ALL lymphocytes were obtained by automated chromatofocusing on PBE 94 microcolumns using 0.025 M histidine and polybuffer 74. Two major isoenzyme components (B and A) were isolated. The activity ratio of B/A was different in normal, ALL, and CLL cells.     

Influence of human ascitic fluid on the in vitro antibacterial activity of moxifloxacin

We investigated the in vitro influence of HAF on the antibacterial activity of moxifloxacin against Escherichia coli ATCC 10798, Escherichia coli K-12, Proteus rettgeri (Sanelli), Staphylococcus aureus ATCC 25923, Staphylococcus aureus NCTC 1808 and Staphylococcus epidermidis ATCC 12228. Human ascitic fluid was obtained from 6 cirrhotic patients by paracentesis. The interaction effect was evaluated by the checkerboard technique. Our results indicate the ability of human ascitic fluid to reduce minimum inhibitory concentrations of moxifloxacin against Gram-negative bacteria, but not against Gram-positives.     

Role of myeloid cell factor-1 (Mcl-1) in chronic lymphocytic leukemia

The primary abnormality in chronic lymphocytic leukemia (CLL) is a defect in apoptosis, probably related to alterations in the expressions of Bcl-2 family members. In transgenic mice over expressing the anti-apoptotic Bcl-2 family member, myeloid cell factor-1 (Mcl-1), B cell lymphomas occur. Moreover, mice conditional for the loss of Mcl-1 display a profound reduction in B and T lymphocytes. This suggests that Mcl-1 is an essential survival factor in lymphocytes. In the present study, we have evaluated the role of Mcl-1 in CLL. Mcl-1 protein expression was measured by Western blot analysis in the CLL cells of 45 patients and correlated with clinical variables and survival. Mcl-1 levels were similar in 29 patients to normal B and T lymphocytes, were decreased in 8 patients and increased in 12 patients. An inverse correlation was found between Mcl-1 expression and Rai stage (P = 0.001). When assessed by flow cytometry, Mcl-1 expressions were normally distributed among CLL cells in individual patients and the mean levels correlated with those obtained by Western blotting. To evaluate the role of Mcl-1 in drug resistance, Mcl-1 levels were sequentially measured in the leukemic cells of 4 CLL patients during therapy with fludarabine (Flu). The Mcl-1 levels were found to increase in 2 patients while the peripheral blood lymphocyte counts dropped, suggesting that the residual drug-resistant cells had the highest Mcl-1 levels. Primary CLL cells were also treated with chlorambucil (CLB) or Flu in vitro and the Mcl-1 levels decreased correlating with the sensitivity of these cells to undergo apoptosis. Drug sensitivities of the CLL cells to CLB and Flu were also measured by MTT assay and the concentrations of drug required to decrease cell viability by 50% (IC50) varied from 1.9 to 9.27 microM for Flu (median, 9.4 microM) and 10 to 32.5 microM (median, 5.5 microM) for CLB. The sensitivities of the leukemic cells to CLB correlated inversely with Mcl-1 levels (P < 0.05). These results suggest that Mcl-1 may contribute to cell survival in CLL.     

中晚期肿瘤患者导尿管伴随尿路感染的细菌学监测及抗菌药物应用

目的:探讨中晚期肿瘤患者导尿管伴随尿路感染的细菌分布以及抗菌药物的敏感性。方法:选取我院收治的中晚期肿瘤患者留置导尿期间发生尿路感染者183例,取尿液进行细菌培养和药物敏感试验。结果:183例患者共分离得到细菌菌株177株,以革兰阴性菌为主,检出104株,占58.76%,其中大肠埃希菌、克雷伯杆菌分别检出49株（27.68%）和31株（17.51%）;革兰阳性菌检出较少,检出73株,占41.24%,其中金黄色葡萄球菌和表皮葡萄球菌分别检出29株（16.38%）和21株（11.86%）。以大肠埃希菌、克雷伯菌为代表的革兰阴性菌普遍对头孢哌酮/舒巴坦、亚胺培南、哌拉西林/他唑巴坦敏感,敏感性分别为100.00%、97.12%、97.12%,而对氨苄西林、哌拉西林以及头孢唑林的敏感性较低,仅为9.62%、23.08%和27.88%。以金黄色葡萄球菌和表皮葡挞球菌为代表的革兰阳性菌普遍对万古霉素、丁胺卡那敏感性较高,分别达到98.63%、91.78%,而对庆大霉素、红霉素敏感性较低,仅为6.85%、26.03%。结论:引起中晚期肿瘤留置导尿患者发生尿路感染的细菌多样,在早期可根据经验给予氨苄西林和万古霉素联合应用,并根据随后的药物敏感试验对治疗方案及时进行调整,合理应用抗菌药物。     

Characterization of chronic lymphocytic leukemia (CLL) cells with low density of surface membrane bound immunoglobulins (smIg)

The study consists of 6 CLL patients with leukemic blood lymphocytes lacking T-cell characteristics and with smIg on only a very small fraction of the cells detected by microscopy or FACS analysis after direct immunofluorescence (IFL) staining. Using B-cell specific monoclonal antibodies all leukemias were found to be of the B-cell type. SmIg, mainly of IgD-class and the monotypic light chain, was detected on a large number of cells in all cases when using direct IFL. No lymphocyte clone was judged to be of the pre-B-cell type or represented fully differentiated terminal B-cells. CLL cells from the individual cases probably represent intermediate B-cell maturation steps. The results lend no support to the suggestion that this subtype of CLL necessarily belongs to an early stage of differentiation just beyond the pre-B-cell level.     

Monitoring of antibiotic resistance in bacterial isolates from bacteremic patients

The aim of the study was to monitor the prevalence of pathogens and development of resistance in bacteria isolated from bacteremic patients. Five University Clinics and/or Regional Hospitals in the Slovak Republic participated in the study and a total of 421 isolates were collected in the second half of the year 2002. The most prevalent organisms were coagulase-negative staphylococci (CONS) (19%), Staphylococcus aureus (18.3%), among Gram-negative bacteria Escherichia coli (13.3%), Klebsiella pneumoniae (11.4%) and Pseudomonas aeruginosa (7.8%) followed by enterococci, Acinetobacter baumannii and Enterobacter sp. All CONS and S. aureus were susceptible to vancomycin; resistance to oxacillin was observed for 55% of the CONS and only for 4% of S. aureus isolates. A higher prevalence of resistance to erythromycin, clindamycin, gentamicin and ofloxacin was found in CONS in comparison to S. aureus. Enterococcus sp. isolates were fully susceptible to vancomycin and teicoplanin. Gentamicin, amoxicillin/clavulanate, third generation cephalosporins and ciprofloxacin showed good activity against E. coli. Although 17% of K. pneumoniae isolates were resistant to ciprofloxacin, it was the most effective drug against K. pneumoniae; the prevalence of resistance to other antibiotics was rather higher. Gentamicin and ciprofloxacin were the most active against Enterobacter sp. isolates and ceftazidime and meropenem against P. aeruginosa.     

Purine metabolic cycle in normal and leukemic leukocytes.

Purine metabolism and reutilization pathways were studied as they applied to normal and leukemic leukocytes. The enzyme activities were expressed in terms of the quantity of protein extracted and per 10(10) cells. Whereas the protein extracted and the enzyme activities from normal lymphocytes were relatively constant, considerable variation was noted in cases of chronic lymphocytic leukemia (CLL). This variability in the properties of the leukemic cells suggests that the difference may be useful in the subclassification of the leukemias. The studies of the complete enzyme system were done with 300 million cells. The extraction of 350,000 normal lymphocytes/mul gave a soluble protein concentration of 1.46+/-0.16 mg protein per ml, and the yield from the same number of CLL lymphocytes varied between 0.72 and 8.32 mg protein per ml. The 5'-nucleotidase activity gave an inverse correlation with the amount of extractable protein. In individual cases of CLL, the protein concentrations and the 5'-nucleotidase activities were found on either side of the normal values. In most cases, the adenosine deaminase of CLL lymphocytic cell extracts was lower than normal, and the adenosine kinase was higher; in the CLL cells, these two enzymes gave a positive correlation with one another. Little or no difference was observed in the activities of the purine nucleoside phosphorylases in extracts of normal or leukemic lymphocytes and granulocytes. The hypoxanthine-guanine and adenine phosphoribosyltransferase activities increased in the leukemic granulocytes but almost always showed a decrease in the CLL lymphocytes when compared with the normal cells. Most of the leukemic cells had greater than normal activities of the enzymes synthesizing phosphoribosyl pyrophosphate when tested with the purines. The total nucleotide produced from adenine and guanine with adenine- and hypoxanthine-guanine phosphoribosyltransferase was about equal in normal and leukemic lymphocytes, but the proportion of the adenosine 5'-triphosphate in the product was much greater with the leukemic cells. This suggested that the ribosyltransferase activities were the same in both types of cells, but the nucleoside kinases and the nucleoside diphosphate kinases were more active in the leukemic cells. Inosine monophosphate dehydrogenase was less active than normal in the CLL cell extracts and was not directly related to the amount of inosine monophosphate generated from hypoxanthine.     

Flow-cytometry detection of minimal DNA aneuploidy in mature lymphoid leukemias - comparison with metaphase and interphase cytogenetics

The relative DNA content of peripheral blood cells from 79 cases with lymphoid leukemias was analyzed by a dual-parameter flow cytometric analysis. The leukemia samples corresponded to: chronic lymphocytic leukemia (CLL) 40, CLL with more than 10% prolymphocytes (CLL/PL) 12, CLL mixed 9, prolymphocytic leukemia (PLL) 5, and B-cell lymphoma in leukemic phase 13. DNA aneuploidy was found overall in 26 (32.9%) of the cases and these corresponded to: 7 (17.5%) with CLL, 7 (58.3%) with CLL/PL, 4 (44.4%) with CLL mixed, 2 (40%) with PLL and 6 (46.2%) with B-cell lymphoma. There was a good correlation between DNA content and cytogenetics/fluorescent in situ hybridization in all but 2 cases as follows: 6 of 7 cases with diploid DNA had normal karyotype and only one had trisomy 12: 4 of 6 cases with hyperdiploid DNA had trisomy 12, one had tetraploidy and only one had a normal karyotype. Two cases were hypodiploid both by DNA and cytogenetic analysis. Our findings demonstrate a higher incidence of DNA aneuploidy in B-cell lymphoma in leukemic phase, PLL, and atypical CLL in comparison with typical CLL and a good correlation with cytogenetics. We conclude that flow cytometric DNA analysis represents a useful, sensitive, and rapid method to detect and monitor minimal changes of DNA content in leukemic lymphocytes without the need of short-term cultures.