Androstenedione conversion in human peripheral blood lymphocytes

Indigenous Indian groups comprise approximately 20% of Ecuador's population, the third largest percentage in all of Central or South America, yet immunogenetic data on these groups are lacking in the literature. In the course of population migration studies, sera collected from 65 Ecuadorians living in the northern province of Esmeraldas were typed for six GM and two KM markers. The study population consisted of 47 Cayapa Indians and 18 blacks of African origin, descendants of slaves imported into the area during the seventeenth century. The Cayapa demonstrated three GM phenotypes, two of which are common to other South American Indian tribes. The frequency of KM1 positive Cayapa Indians (63%) is similar to other South American Indian tribes, but is significantly greater than the Huaorani of eastern Ecuador (2%), the only other Ecuadorian Indian group for whom limited immunoglobulin allotype data are available (chi 2 = 35.8, P < 0.0001).     

Calcineurin protein phosphatase activity in peripheral blood lymphocytes

The protein phosphatase activity of peripheral blood T lymphocytes (PBLs) was examined to quantify the contribution of calcineurin and other members of the family of serine/threonine protein phosphatases. Using selective phosphatase inhibitors, the fractional phosphatase activities of calcineurin, protein phosphatases 1 (PP1), 2A (PP2A), and 2C (PP2C) were determined. Okadaic acid was used to inhibit the activity of both PP1 and PP2A while cyclosporin A/cyclophilin or trifluoperazine were used as a specific inhibitors of the calmodulin-dependent phosphatase calcineurin. Using a [32P]labeled 19-residue phosphopeptide substrate, RII peptide, it was found that PP1 and PP2A comprise the majority of the total phosphatase activity in PBLs with okadaic acid inhibiting 80% of the phosphatase activity. The remaining 20% of the phosphatase activity can be attributed primarily to calcineurin since it was Ca2+ dependent, sensitive to inhibition by the calmodulin antagonist trifluoperazine, and inhibited by the complex of cyclosporin A (CsA) and cyclophilin. These results indicate that PBL extracts contain little PP2C activity. In addition, PBLs treated with CsA had measurably lower calcineurin activity in cell lysates. The measurement of calcineurin activity may provide a useful means of assessing the extent of immunosuppression during drug therapy.     

Phenotyping of peripheral blood lymphocytes in adult coeliac disease

OBJECTIVE: To evaluate nerve fiber density in vestibular specimens from women operated upon for vulvar vestibulitis. METHODS: Forty-seven women with vulvar vestibulitis syndrome underwent modified posterior vestibulectomies. Vestibular specimens were analyzed after being stained for S-100 neural tissue protein. Women were followed up for 2 years. RESULTS: In specimens from 44 of 47 patients, the densities and numbers of nerve fibers per square unit in the preparations were greater than those in specimens from six control women. In the patients, a statistically significant linear correlation was found between inflammation and nerve bundle density in the preparations (Spearman rank correlation coefficient rs=.41; P=.005). There were no signs of infectious etiology in any preparation. No or slight postoperative dyspareunia was reported by 38 of 42 women after 6 months, 36 of 39 after 12 months, and 26 of 28 after 24 months. CONCLUSION: Vestibular neural hyperplasia may provide a morphologic explanation of the pain in vulvar vestibulitis syndrome.     

Detection of cytogenetic effects in peripheral lymphocytes of students exposed to formaldehyde with cytokinesis-blocked micronucleus assay

A range of specific and unusual biological pathways are found in Gram-negative bacteria. It is possible to express the genes involved in these processes in Escherichia coli, however, some genes prove lethal when cloned into high copy number vectors in common usage. Conversely, various genetic functions remain silent in E. coli and require to be transferred into their original host for expression and subsequent analysis. To facilitate the cloning and the characterisation of bacterial genes, we have constructed CcdB 'positive-selection' vectors that possess one or more of the following properties: (i) low or medium copy number; (ii) narrow or broad replication host range; (iii) conjugational mobilisation. In this communication, we illustrate the use of these new cloning tools and analyse the CcdB toxicity in different bacterial species.     

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.     

Characterization of a lectin from goat peripheral blood lymphocytes

A D-glucose specific lectin was isolated from goat peripheral blood lymphocytes by affinity chromatography on N-acetyl D-glucosamine agarose gel. The fluorescence intensity of 4 methyl umbelliferyl D-glucose was quenched to about 62% on addition of the lectin. This lectin gave a single band corresponding to 112 kDa in SDS-PAGE irrespective of treatment with 2-mercaptoethanol. The molecular weight and the Stoke's radius of the lectin in the native conditions were found to be 114 kDa and 4.54 nm, respectively, as determined by gel filtration on Sephacryl S 500 column. The lectin was found to be a glycoprotein with 5.6% of neutral hexose content and 5.5% of sialic acid. The lectin agglutinated trypsinized rabbit erythrocytes and human type A erythrocytes. The hemagglutinating activity was dependent on the presence of divalent cations like Mn2+ and Ca2+. Optimum pH, ionic strength and temperature for rebinding of lectin to acid treated Sephadex G200 were found to be 7.5, 0.16 and 30-37 degrees C, respectively.     

Spontaneous chromosome aberrations in the lymphocytes of human peripheral blood]

The authors carried out cytogenetic studies on lymphocytes from human peripheral blood of 105 persons--49 women and 56 men in order to examine spontaneous frequency of the structural chromosomal abberations in the somatic cells. There were 191 cells with abberations (1,17%), out of 16,267 analyzed metaphasic plates. They found 0,39% of chromatid fragments, 0,71% of chromosomal fragments, 0,06% dicentricets, 0,01% of symetric and asymetric chromatid exchanges and 0,006% of rings. There were no statistically significant differences in the discovery of the spontaneous abberations in the examined persons at various age and sex.     

Studies on the peripheral blood and the in vitro response of peripheral blood lymphocytes to common mitogens in Macaca speciosa

In a longitudinal study on 13 adult female stumptailed macaques, Macaca speciosa, peripheral blood samples were analyzed hematologically and the in vitro response of the lymphocytes to the common mitogens concanavalin A, phytohemagglutinin and pokeweed mitogen were investigated. The results showed that the total leukocyte and differential counts in each animal remained quite stable throughout the 3-month experiment. The responses in culture to the mitogens showed that there were differences in the stimulation obtained by each mitogen and that the animals may be placed into high, medium and low groups based on differences in their level of response.     

High sensitivity of leukemic peripheral blood lymphocytes to triethyllead action

In a previous study we reported that triethyllead (Et3Pb+) inhibits cell proliferation of normal human lymphocytes. To further characterize this interaction, we studied herein the effects of Et3Pb+ on the cell viability of normal and leukemic human lymphocytes and analysed the expression and dynamics of the monomer/polymer equilibrium of tubulin in these cells. Short- and long-term cell culture experiments demonstrated significantly different dose-dependent effects of Et3Pb+ on cell viability of leukemic compared to normal lymphocytes. Indeed, in the presence of increasing concentrations of Et3Pb+ (10(-12)-10(-5) M), primary cultures of chronic lymphocytes (CLL) and acute lymphoblastic (ALL) leukemic human Lymphocytes were much more sensitive to Et3Pb+ treatment when compared to normal peripheral blood lymphocytes (PBL). The IC50 values were approximately 5 x 10(-6) M for PBL and 8 x 10(-10) M for both CLL and ALL respectively, when cells were preincubated for 3 h with this agent. These experiments revealed a 1000-fold higher responsiveness of leukemic cells to Et3Pb+ treatment. Quantitative immunoblot analysis showed that leukemic cells express up to 4-fold higher total tubulin amounts. However, the proportion of polymerized tubulin in leukemic compared to normal lymphocytes increased only slightly (up to 1.4-fold). These findings reveal a significant decrease in the polymeric to total tubulin ratio in leukemic lymphocytes, indicating important modifications in tubulin dynamics and reorganization of the microtubular structures. Our results demonstrate that leukemic cells are much more sensitive than normal lymphocytes to Et3Pb+ action. This effect may be due to the altered monomer/polymer dynamic equilibrium of tubulin shown in leukemic cells. It is, therefore, worthwhile exploring future applied uses of Et3Pb+ as a potential suppressor of leukemic cell growth.     

Cytotoxic lymphocytes in the peripheral blood of patients with B cell lymphomas

In order to analyze systemic immune surveillance in patients with B cell non-Hodgkin's lymphomas (B-NHL), we investigated circulating lymphocytes using two-color flow cytometry. The proportions of CD3-CD56+ natural killer (NK) cells and CD8++(bright) S6F1++ killer-effector T cells corresponding to activated cytotoxic T lymphocytes (aCTL) were studied in the peripheral blood of 26 patients with indolent lymphoma (IL) and 24 with aggressive lymphoma (AL). The AL patients with both limited disease and advanced disease had an increased proportion of NK cells. However, this feature was not evident in IL patients with either limited or advanced disease. In contrast, an increased proportion of aCTL was observed only in IL patients with advanced disease. These findings indicate that IL may differ from AL in terms of immune surveillance against neoplastic B cells.     

Alteration of signal-transducing molecules in tumor-infiltrating lymphocytes and peripheral blood T lymphocytes from human colorectal carcinoma patients

An analytical method for the rapid isolation and recovery of the homologous series of 2-aminoethanols, a class of organic compounds of importance to wood preservative treatment, is successfully developed. The method is applied to an aqueous solution of copper amine (copper[II] hydroxide complexed monoethanolamine) and to copper-amine-treated sawdust. The method incorporates a gas chromatograph-ion-trap mass spectrometer. A discussion of the secondary equilibrium effects involved when ionizable analytes are extracted from an aqueous phase with respect to organic bases is presented. Using 2-propanol as the extractant coupled to a salt-saturated aqueous phase results in recoveries of 63% for 2-aminoethanol, 51% for N,N-dimethyl-2-aminoethanol, and 56% for N-methyl-2-aminoethanol for a single liquid-liquid extraction. The choice of 2,2,2-trifluoroethanol as an internal standard is found to be quite suitable. A comparison of the precision and accuracy for an external versus an internal mode of instrument calibration demonstrates that the internal standard mode is preferable for this manual injection.     

Changes in corticosteroid sensitivity of peripheral blood lymphocytes after strenuous exercise in humans

Although plasma corticosteroid concentrations can be measured accurately, the biological effect on the target tissue is uncertain. The availability of an accurate measure of corticosteroid sensitivity would potentially clarify the putative roles of endogenous glucocorticoids in illnesses such as inflammatory disease and obesity and allow evaluation of an additional regulatory level of glucocorticoid action. To measure corticosteroid sensitivity, we developed an assay based on the inhibition by dexamethasone (Dex) of lipopolysaccharide (LPS)-induced Interleukin-6 (IL-6) production and release in whole unseparated blood in vitro. LPS induced a dose-dependent increase in IL-6 concentrations up to 34 +/- 6.6 ng/mL, reaching plateau levels after 8 h, whereas Dex dose dependently inhibited LPS-induced IL-6 production. Involvement of the glucocorticoid receptor in this response was supported by abrogation of Dex (10(-7) mol/L) inhibition of IL-6 production by the glucocorticoid receptor antagonist RU 38486. To determine whether corticosteroid sensitivity is a dynamic phenomenon, we subjected healthy males to a graded quantifiable exercise associated with increases in plasma ACTH and cortisol. Before exercise, 3 x 10(-8) mol/L Dex inhibited LPS-induced IL-6 production in vitro; after exercise, 3 x 10(-8) and 10(-7) mol/L Dex were unable to inhibit IL-6 production. We conclude that Dex suppression of LPS-induced IL-6 production is an effective means of determining corticosteroid sensitivity, and that corticosteroid sensitivity in human subjects is a dynamic, rather than a static, phenomenon.     

Kinetics of cell division in peripheral blood lymphocytes of stainless steel welders]

The management of fracture within previously shortened extremity is presented. Three patients with femoral fracture and one with crural fracture were treated. All fractures were displaced. Modified Ilizarov ring distractor was used to stabilize the fracture within 3 days from injury. Distraction was commenced on third postoperative day at speed of 0.75-2 mm/h with progressive reduction of fragments and alignment of mechanical axis of the limb. Lengthening required to equalize extremities was achieved in all cases. The author claims fracture within previously shortened extremity to be one more indication for external osteosynthesis as a method of choice.     

Estrogenic xenobiotics affect the intracellular activation signal in mitogen-induced human peripheral blood lymphocytes: immunotoxicological impact

The present study was an attempt to elucidate the effect of estrogenic xenobiotics on the proliferation of mitogen-stimulated human peripheral blood lymphocyte (PBL). Our findings follow: (a) the proliferation of PBL in response to phytohemagglutinin (PHA) was mediated by protein kinase C activity, but estrogenic xenobiotics had a strong inhibitory effect on protein kinase C activity of PHA-stimulated PBL; (b) cytoplasmic extracts from PHA-stimulated PBL greatly activated DNA replication, but estrogenic xenobiotics had a strong inhibitory effect on these activities. The results suggest that the cytoplasmic signal-generating system in mitogen-treated PBL is inhibited by estrogenic xenobiotics, and that the defect occurs at all stages in the sequence of events leading to DNA synthesis and cell proliferation.