Immunohýstochemýcal staýnýng of cd3, cd79ácy and s-100 on bursa fabrýcýus, thymus and spleen of turkeys (Meleagrýs Gallapavo)

2018 ◽  
Author(s):  
Tolunay Kozlu ◽  
Ebru Karadað Sari ◽  
Yeþim Akaydin Bozkurt ◽  
Nevin Kurtdede
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
Positive Reaction ◽  
B Lymphocyte ◽  
Staining Method ◽  
General Structure ◽  
Bursa Of Fabricius ◽  
S 100 ◽  
Reticular Cells ◽  
Triple Staining

Distribution of CD3, CD79ácy and S-100, immunohistochemically, in the bursa of Fabricius, thymus and spleen has not been reported in turkeys. Therefore, we determined the localisation of anti-CD3 protein antibodies for mature T-lymphocytes, anti-CD79ácy antibodies for B-lymphocytes and anti-S-100 protein antibodies for follicular dentritic cells in the bursa of Fabricius, thymus and spleen, which are the lymphoid organs, in a study sample of turkeys. Triple staining method was applied to demonstrate the general structure. Moreover, in all the organs positive reactions were observed with the CD3, CD79ácy and S-100 antibodies. It was also found that similar areas had a positive reaction with CD79ácy and S-100 in all of the tested organs. It was remarkable that CD79ácy reacted positively on Hassall’s corpuscles (strongly) and the reticular cells (weakly) in the medulla of the thymus instead of the B-lymphocyte positive areas.

scholarly journals T lymphocyte-dependent B lymphocyte proliferative response to antigen. I Genetic restriction of the stimulation of B lymphocyte proliferation.

1981 ◽  
Vol 153 (4) ◽  
pp. 871-882 ◽  
Author(s):  
H Y Tse ◽  
J J Mond ◽  
W E Paul
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
T Lymphocyte ◽  
Lymphocyte Proliferation ◽  
B Lymphocyte ◽  
Antigen Specificity ◽  
Apparent Lack ◽  
The Face ◽  
Stimulation Of

For the purpose of examining more closely the interaction between T and B lymphocytes, we have developed an in vitro T lymphocyte-dependent B lymphocyte proliferation assay. Proliferation of B lymphocytes in response to antigen was found to depend on the presence of primed T lymphocytes; the B lymphocytes could be derived from nonprimed animals. It appears that these B cells were nonspecifically recruited to proliferate. This nonspecific recruitment, however, was found to be Ir-gene restricted in that B lymphocytes from B10.S mice, which are genetic nonresponders to the polymer Glu60-Ala30-Tyr10 (GAT), could not be stimulated by GAT-primed (responder X nonresponder) F1 T cells. The apparent lack of antigen specificity in the face of Ir gene-restricted T-B interaction may have important implications in our understanding of the recognition unit(s) on T lymphocytes.

scholarly journals B-lymphocyte-derived erythroid burst-promoting activity is distinct from other known lymphokines

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1814-1820 ◽  
Author(s):  
L Feldman ◽  
N Dainiak
Keyword(s):  
Growth Factors ◽  
T Lymphocytes ◽  
B Lymphocytes ◽  
Plasma Membranes ◽  
B Lymphocyte ◽  
Temperature Stability ◽  
Hematopoietic Growth Factors ◽  
Serum Free ◽  
Gm Csf ◽  
Lineage Specificity

Abstract Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

scholarly journals Development of suppressor T lymphocytes for Epstein-Barr virus-induced B-lymphocyte outgrowth during acute infectious mononucleosis: assessment by two quantitative systems

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 510-517 ◽  
Author(s):  
RT Schooley ◽  
BF Haynes ◽  
J Grouse ◽  
C Payling-Wright ◽  
AS Fauci ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
B Lymphocyte ◽  
Acute Stage ◽  
Cell Mediated Immunity ◽  
Barr Virus ◽  
Epstein Barr

Abstract A system of 3H-thymidine incorporation by lymphocytes in culture for 3 wk has been utilized for quantitative assessment of the ability of T lymphocytes to inhibit outgrowth of autologous Epstein-Barr virus (EBV) transformed B lymphocytes. Lymphocytes from EBV-seronegative individuals lack the ability to suppress outgrowth of autologous EBV- transformed B lymphocytes. This capability appears during the course of primary EBV-induced infectious mononucleases (IM) as the atypical lymphocytosis is subsiding and persists for years after recovery from primary EBV infection. The ability of T lymphocytes from EBV- seropositive subjects or convalescent IM patients to inhibit B- lymphocyte outgrowth is not HLA restricted. Thus, T lymphocytes capable of inhibition of in vitro EBV-induced B-cell outgrowth emerge during the acute stage of IM and may represent an important control mechanism of EBV-induced B-lymphocyte proliferation in vivo. The system provides a highly sensitive quantitative means for in vitro assessment of cell- mediated immunity to EBV.

CD39/NTPDase-1 Expression Is Associated with Activation in T-Lymphocytes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1733-1733
Author(s):  
Dianne Pulte ◽  
Marinus Johan Broekman ◽  
Joan Drosopoulos ◽  
Kim E. Olson ◽  
Naziba Islam ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Enzymatic Activity ◽  
B Lymphocytes ◽  
T Lymphocyte ◽  
B Lymphocyte ◽  
Inflammatory Responses ◽  
Geometric Mean ◽  
Compensatory Mechanism ◽  
Nucleotidase Activity ◽  
Cd8 Cells

Abstract CD39/NTPDase-1 is an ecto-ATP/ADPase expressed on leukocytes and endothelial cells. CD39 is the main control system for blood fluidity. CD39 on lymphocytes was first reported in 1991 by Kansas et al. However, studies of CD39 expression and activity on leukocytes have not been done. We characterized levels of CD39 expression and enzymatic activity on neutrophils (PMN), lymphocytes and lymphocyte subsets. Since inflammatory responses occur in arterial vascular disease, we also examined expression of CD39 on naive versus activated and memory lymphocytes. Lymphocytes were isolated by a histopaque procedure, and PMN by dextran gradient. B-lymphocytes were isolated using the RosetteSep B-cell kit. All cell types were confirmed to have purities of >90%. CD39 activity was assayed via our radio-thin-layer chromatographic system. CD39 expression was measured on leukocytes via FACS. PMN, monocytes, and lymphocytes were identified by their forward and side-scatter characteristics. Subsets of lymphocytes were examined via double staining for CD39 and antibodies against specific sub-types. CD39 localized to the surface of greater than 95% of neutrophils, monocytes, and B-lymphocytes. It was also present on a minority (~8%) of T-lymphocytes with no difference in frequency of expression between CD4+ and CD8+ cells. Geometric mean (GM) expression of CD39 per cell was greatest in B-lymphocytes and monocytes, lower in CD4+ cells, and lowest in CD8+ cells and PMN. Interestingly, incubation of T- lymphocytes with PHA up-regulated CD39 in CD8+ cells both in terms of number of cells expressing and GM, with expression rising to 65%. The GM increased 4-fold after 6d of stimulation with PHA. A similar but less dramatic increase was seen with LPS. This is the first time we have accomplished up-regulation of CD39 expression and enzymatic activity. Radio-TLC measurement of nucleotidase activity showed B-lymphocytes>PMN>T-lymphocytes. B-lymphocyte ADPase and ATPase activities (in pmol/min/50K cells) were 75 and 43, respectively. PMN displayed 39 (ADPase) and 22 (ATPase), while T-lymphocytes had enzymatic activity of 16 and 11.5, respectively. ADPase:ATPase ratios were similar for B-lymphocytes and PMN, but lower for T-lymphocytes (1.8 for B-lymphocytes and PMN, vs 1.45 for T-lymphocytes, p=0.03). Lymphocytes stimulated with PHA demonstrated an increase in enzyme activity of 10–20X baseline that peaked at 7–10d. ADPase:ATPase ratio was unchanged. FACS measurement showed that CD39+ lymphocytes were more often activated than CD39− lymphocytes in both CD3+ (p=0.06) and CD4+ (p=0.02) subgroups. Preliminary experiments indicated that >85% of CD39+ T-lymphocytes are CD45RO+. Importantly, this suggests that CD39 is expressed primarily on activated or memory cells in the T-lymphocyte population. Thus, CD39 is expressed on a broad variety of leukocytes. T-lymphocyte expression can be induced by stimulation with mitogens. Moreover, CD39 is present primarily on CD45RO+ T-lymphocytes. We conclude that CD39 expression can be induced by activation of the immune system. The up-regulation of CD39 on activated and memory T-lymphocytes may be a compensatory mechanism for protection from thrombosis as a consequence of inflammation. It may serve as a mechanism for metabolizing extracellular ATP and therefore decreasing the inflammatory stimulus. Abnormalities in CD39 may result in decreased nucleotidase activity and increased vulnerability to thrombosis as a consequence of inflammation.

scholarly journals B-lymphocyte-derived erythroid burst-promoting activity is distinct from other known lymphokines

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1814-1820
Author(s):  
L Feldman ◽  
N Dainiak
Keyword(s):  
Growth Factors ◽  
T Lymphocytes ◽  
B Lymphocytes ◽  
Plasma Membranes ◽  
B Lymphocyte ◽  
Temperature Stability ◽  
Hematopoietic Growth Factors ◽  
Serum Free ◽  
Gm Csf ◽  
Lineage Specificity

Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

scholarly journals Cellular immunity of rabbits incase of parasite association (Treponema cuniculi and Eimeria sp.)

2019 ◽  
Vol 21 (96) ◽  
pp. 8-13
Author(s):  
Y. V. Duda
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
Cellular Immunity ◽  
B Lymphocyte ◽  
Absolute Number ◽  
Blood Parameters ◽  
Control Group ◽  
The Absolute ◽  
T Helpers ◽  
Experimental Group

Despite a huge number of studies, the uniqueness of antiparasitic immunity is so great that there is still insufficient knowledge of the factors contributing to the manifestation of the characteristics of immunity in mixed parasitic diseases of rabbits. Therefore, the question of the influence of the association of pathogens Treponema cuniculi and Eimeria sp. on indicators of cellular immunity of rabbits is relevant. The study was conducted on 59 male rabbits age 3–5 months of the Californian breed, selected by analogy. Animal were separated into two groups: healthy animals (control group) and sick animals (research group). Intensity of invasion was determined by the method of the Mac-Master. It has been established that the level of damage of rabbits by spirochetosis and eimeriosis was, on average, 1155.17 ± 184.87 and 6668.97 ± 284.16 pathogens in 1 g of feces. The count of T- and B-lymphocytes was determined by the method of spontaneous rosette-formation with sheep erythrocytes. Parasitizing the association of pathogens Treponema cuniculi and Eimeria sp. was revealed a high number of leukocytes (1.22 times, P < 0.001), which increased mainly due to lymphocytes, which were 1.45 times higher (P < 0.001), as well as neutrophilic metamyelocytes – 1.48 times (P < 0.05), eosinophils – 1.68 times (P < 0.001) and basophils – 1.57 times (P < 0.001) compared with similar blood parameters of healthy animals. In the blood of sick rabbits, the absolute number of T-lymphocytes (1.56 times, P < 0.001) and B-lymphocytes (3.02 times, P < 0.001) was significantly higher in comparison with a low number of O-lymphocytes (3.46 times, P < 0.001) compared with the control. This indicates the redistribution of lymphocytes to cells that carry T and B lymphocyte receptors on the plasma membrane. The absolute number of T-lymphocytes became high due to T-helpers, which in these animals were higher both in absolute (1.87 times, P < 0.001) and percentage (by 9.18%, P < 0.001) compared to control. Moreover, the percentage of T-suppressors in the blood of rabbits of the experimental group was significantly lower on 5.46% (P < 0.05) compared with the same blood count of healthy animals. Such a redistribution of the T-cell population in the peripheral blood of this group of rabbits led to an increase in the immunoregulatory index by 1.64 times (P < 0.01) than in healthy ones. High IRI and the number of T-active lymphocytes (by 28.23%, P < 0.05) in the blood of rabbits with parasitism of the association of pathogens Treponema cuniculi and Eimeria sp. indicate increased immune system tension.

Whole-Exome Sequencing Links CARD11 Inactivation with SCID

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 258-258
Author(s):  
Johann Greil ◽  
Tobias Rausch ◽  
Thomas Giese ◽  
Obul Reddy Bandapalli ◽  
Volker Daniel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Lymphocytes ◽  
Exome Sequencing ◽  
B Lymphocytes ◽  
Whole Exome Sequencing ◽  
B Lymphocyte ◽  
Whole Exome ◽  
Life Threatening

Abstract Abstract 258 Primary immunodeficiencies represent model diseases for the mechanistic understanding of the human innate and the adaptive immune response and are per se clinically highly relevant, because in SCID patients infections by opportunistic pathogens are typically life-threatening early in life. We identified an infant of consanguineous parents suffering from a novel form of SCID, who presented with a life-threatening Pneumocystis jirovecii pneumonia. This entity was characterized by agammaglobulinemia and profoundly deficient T-cell function despite quantitatively normal T- and B-lymphocytes. Lymphocyte proliferation was strongly inhibited after stimulation of PBMCs with T-cell mitogens such as PHA, Con A, or anti-CD3 monoclonal antibody. The expression of several T-cell response associated cytokines upon stimulation with PMA/ionomycin was dramatically reduced in comparison to normal controls. By contrast, proliferation induced by the classical B-cell mitogen PWM was almost comparable to healthy controls. Immunophenotyping revealed a predominantly naïve phenotype (CD45RA+ CCR7+) in CD4+ and CD8+ T-lymphocytes, whereas central memory T-lymphocytes (CD45RA− CCR7+) were nearly absent. B-lymphocytes from peripheral blood were mainly naïve B-cells (CD27−) with a uniformly immature transitional B-lymphocyte phenotype (CD24++, CD38++). Patient B-lymphocytes retained the ability to proliferate and differentiate in response to BCR-independent stimuli, while their response to BCR activation was defective. Our findings thus revealed a combined defect of TCR-mediated T-lymphocyte functions and BCR-mediated B-lymphocyte functions but did not enable us to link the immunological phenotype with one of the known molecularly defined categories of SCID. Diagnostic whole-exome sequencing and systematic variant categorization revealed a single pathogenic homozygous nonsense mutation of the caspase recruitment domain 11 (CARD11) gene. CARD11 is a scaffold protein that is known to be required for the assembly and activation of the NF-kB complex. In reconstitution assays we demonstrated that the patient derived truncated CARD11 protein is defective in antigen receptor signaling and NF-kB activation. Several lines of evidence substantiate the involvement of the identified CARD11 mutation in the new form of SCID that we report here. First, PCR and Sanger re-sequencing validated the truncating CARD11 mutation to be homozygous in the patient and heterozygous in the parents, in agreement with the recessive transmission of the mutation through the healthy consanguineous parents. Second, CARD11 is a scaffold protein required for TCR- and BCR-induced NF-kB activation as well as lymphocyte activation and proliferation, which is specifically expressed in hematopoietic cells, consistent with a causative role of CARD11 mutations in the context of an immune disorder. Third, the GUK domain of CARD11, which is missing in the mutated form of CARD11 due to truncation, was previously reported to be necessary for NF-kB activation by PMA/ionomycin treatment, further supporting the presumed damaging nature of the homozygous CARD11 mutation observed in the female patient reported here. Finally, the immunological findings in this patient are compatible with the phenotype of a previously described Card11 −/− k.o. mouse, which shows a selective defect in NF-κB activation leading to diminished antigen receptor or PKC mediated proliferation and defective cytokine production in T-cells and B-cells. Thus, we have identified an inactivating CARD11 mutation linking defective NF-kB signaling with a novel cause of autosomal recessive SCID, which must be considered in the diagnostic assessment of patients with suspected SCID but with quantitatively normal T-cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals SURFACE MARKERS ON HUMAN B AND T LYMPHOCYTES

1973 ◽  
Vol 138 (6) ◽  
pp. 1365-1378 ◽  
Author(s):  
Mikael Jondal ◽  
George Klein
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
T Lymphocyte ◽  
B Lymphocyte ◽  
Surface Markers ◽  
Barr Virus ◽  
Human Peripheral Lymphocytes ◽  
Epstein Barr ◽  
Viral Determinants ◽  
B And T Lymphocytes

Human peripheral lymphocytes were investigated for receptors binding Epstein-Barr virus (EBV) because of the regular association of this virus with infectious mononucleosis and Burkitt's lymphoma. This was done by a cytoadherence technique where virus-producing cells, displaying fresh viral determinants in their cytoplasmatic membrane, were mixed with lymphocytes. Unfractionated lymphocytes were found to adhere to these cells in contrast to column-purified T lymphocytes. The specificity of the binding was confirmed by blocking experiments that showed that sera containing high titers of antibodies directed against the virus could partially inhibit the adherence in contrast to low-titer sera. It is concluded that B lymphocytes, in contrast to T lymphocytes, have receptors for EBV. In a second line of experiments it was found that established human lymphoblastoid lines that carry the EBV genome had receptors characteristic for B lymphocytes and did not form T-lymphocyte rosettes. In contrast, a line of known T-lymphocyte origin that did not carry the EBV genome had receptors characteristic for T lymphocytes. EBV-transformed simian lymphoblastoid lines had surface markers indicating a B-lymphocyte origin in contrast to HVS-transformed simian lines that lacked surface immunoglobulin but carried receptors for sheep red blood cells.

scholarly journals Anhydroretinol: a naturally occurring inhibitor of lymphocyte physiology.

1993 ◽  
Vol 178 (2) ◽  
pp. 675-680 ◽  
Author(s):  
J Buck ◽  
F Grün ◽  
F Derguini ◽  
Y Chen ◽  
S Kimura ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
B Lymphocyte ◽  
Interleukin 2 ◽  
Fk 506 ◽  
Intracellular Signaling Pathway ◽  
Insect Cell Lines

Vitamin A (retinol) is an essential cofactor for growth of B lymphocytes in culture and for activation of T lymphocytes by antigen receptor-mediated signals. 14-hydroxy-4,14-retro-retinol (14-HRR) a metabolite of retinol, has been implicated as the intracellular mediator of this effect. Anhydroretinol (AR) is a retinol derivative with retro structure produced in activated human B lymphocytes and the insect cell lines SF 21 and Schneider S2. AR reversibly inhibits retinol- and 14-HRR-dependent effects and blocks B lymphocyte proliferation as well as activation of resting T lymphocytes. The intracellular signaling pathway blocked by AR in T cell activation is distinct from the calcineurin/interleukin 2 pathway inhibitable by cyclosporine A or FK-506.

scholarly journals Control of t-lymphocyte and B-lymphocyte activation by two complementing Ir-GLphi immune response genes.

1976 ◽  
Vol 143 (4) ◽  
pp. 906-918 ◽  
Author(s):  
D H Katz ◽  
M E Dorf ◽  
B Benacerraf
Keyword(s):  
T Lymphocytes ◽  
B Lymphocytes ◽  
B Lymphocyte ◽  
Lymphocyte Activation ◽  
Inbred Strains ◽  
Cell Type ◽  
T And B Cells ◽  
Cooperative Interactions ◽  
Inbred Strains Of Mice ◽  
B Lymphocyte Activation

The possibility that the two complementing alpha- and beta-Ir-GLphi genes are independently responsible for controlling events in T lymphocytes and B lymphocytes, respectively, has been tested in double adoptive transfer experiments utilizing cells from appropriate inbred strains of mice. The results of these studies show that the functions of T lymphocytes and B lymphocytes and the cooperative interactions between T and B cells require the presence of both alpha- and beta-genes in each respective cell type. Moreover, evidence has been obtained in these studies that indicates a preference for the alpha- and beta-Ir-GLphi genes in the cis position to obtain the most effective T-B-cell interactions. The possible implications of these findings are discussed.

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