Lymphocyte Rosette Research Articles

The population dynamics of Т- and В-lymphocytes in blood of the cattle vaccinated against anthrax

Anthrax remains a serious problem for many countries in the world. Containment of major outbreaks of this disease is possible only with the help of specific preventive measures as vaccinating all susceptible livestock. Immunization of animals should create solid immunity in the organism, which should resist the development of the infection process when introducing field strains of an agent. Currently, the specific immunity of the organism to anthrax is studied in detail by Russian and foreign scientists. However, there are no data on the dynamics of the development of immunity in the literature. The purpose of this work is to study the cellular component of adaptive post-vaccinal immunity in animals at different stages after immunization against anthrax. The authors have studied the dynamics of the quantitative composition of T- and B-lymphocytes in cattle during a year. Isolation of T- and B-cells causes spontaneous rosetting of lymphocytes with erythrocytes of heterologous species of animals: sheep and mouse respectively. Recording of the reaction was carried out under a light microscope. 200 lymphocytes were counted, and the percentage of rosette-forming cells was calculated. It was found that immunization leads to an increase in the number of lymphoid cells and epiphylaixis. The increase of lymphocytes gives reason to talk about the enhanced immunity level of animals against this agent. The number of T- and B-lymphocytes in the blood of vaccinated animals has been observed for 35 days after immunization and has been maintained at the same level for two months; 3 months later the number of cells decreased, which shows weakening of the immune system and immune memory by the 6th month after immunization of animals.

Primary diagnosis of classic Hodgkin lymphoma by bone marrow aspirate and trephine – an unusual presentation with interesting morphology

An 82-year-old gentleman presented with breathlessness and a 3-month history of fevers and night sweats. Past history was remarkable for an unprovoked pulmonary embolism (PE) 10 months prior for which he had 6 months anticoagulation therapy with no specific malignancy diagnosed at that time. Further inpatient investigations revealed a recurrent PE and a subsequent positron emission tomography/computed tomography (PET/CT) scan showed retrocaval lymphadenopathy with fludeoxyglucose (FDG) avid nodes above and below the diaphragm with spleen, liver and marrow involvement. Staging bone marrow aspirate and trephine (BMAT) demonstrated large bilobed Hodgkin Reed-Sternberg (HRS) cells with lymphocyte rosetting. Trephine histology showed focal collections of large HRS cells with an accompanying inflammatory cell infiltrate. The HRS cells were CD15+/CD30+/PAX5(weak) on immunohistochemistry (IHC). A diagnosis of Classic Hodgkin Lymphoma (CHL) was established based on the marrow findings. Hodgkin lymphoma (HL) with bone marrow (BM) involvement is a relatively infrequent finding (∼5% cases of HL)1,2 and the presence of HRS cells in BM aspirate is rare. This case illustrates an unusual clinical presentation of CHL with primary diagnosis attained by BM biopsy. The patient was commenced on chlorambucil, procarbazine, prednisolone and vinblastine (Ch1VPP) chemotherapy and restaging PET/CT after 6 cycles showed marked interval improvement of FDG avid disease.

Isolation of B-1 subtype B lymphocytes from human cord blood

Introduction: B-1 lymphocytes with high levels of CD5 markers are capable of differentiation and transformation into macrophage-like cells, as B-1 cells. This study aims to isolate and purify this unique subset from umbilical cord blood cell. Materials and Methods: 14 umbilical cord samples from healthy neonates of natural or elective cesarean deliveries were used. White blood cells were separated from red blood cells using hydroxyethyl starch or gelatin 3%. Cell culture was performed to separate adhesive and non-adhesive cells. Umbilical cord lymphocytes subsets were separated from the remaining leukocyte contents by Ficoll densitygradient centrifugation. Lymphocyte rosette test was used to assess purification stage. Results: Despite lack of difference in cell count between the two blood dilution methods, cell vitality in hydroxylethyl starch method was less than 87%, while cell vital activity in the dilution method by gelatin 3% was measured between 90 and 95%. Lymphocyte rosette test results indicate B-1 cell purification rate of approximately 99% by using the above method. Conclusion: This process indicates most of the extracted B-cells from human umbilical cord blood belong to the B-1 cell subtype, and gelatin 3% procedure is the best and most appropriate method in terms of number and vitality of cells.

Ultrastructure of human autologous macrophage lymphocyte rosettes in Chagas disease

Ultrastructure of human autologous macrophage lymphocyte rosettes in Chagas disease

Lymphocyte Rosettes in an Aspirate of Bone Marrow Involved by T-Cell-Rich B-Cell Lymphoma (TCRBCL)

Lymphocyte Rosettes in an Aspirate of Bone Marrow Involved by T-Cell-Rich B-Cell Lymphoma (TCRBCL)

Effect of dietary ghee--the anhydrous milk fat on lymphocytes in rats.

Lymphocytes are important components of the immune system. Dietary lipids affect the functioning of the immune system. Changes in the lipid composition of the lymphocyte membrane is a case in point. Membrane structural changes are reflected in the altered function of the cell. Lymphocyte proliferation and lymphocyte rosetting are membrane associated phenomena. Ghee, is a clarified butter product, commonly used in the Indian diet. It is rich in saturated fatty acids and also contain oxysterols which are generated on prolonged heating of ghee. Male weanling rats were fed 2.5% (of the total fat levels) of fresh or thermally oxidized ghee for a period of 8 weeks. The control rats were fed groundnut oil. Lipid composition of lymphocytes in ghee fed rats showed changes. In vitro lipid peroxidation of lymphocyte membranes increased by 26% in oxidized ghee fed rats. Na+K+ ATPase activity was decreased in oxidized ghee fed rats (18%). Lymphocyte proliferation was reduced in ghee fed rats (32%), compared to the controls, irrespective of the mitogens used (Con-A or PHA), or the tissue (splenocytes or peripheral blood lymphocytes). Oxysterols present in oxidized ghee are the likely agents inhibiting lymphoproliferation. Rosetting of lymphocytes decreased in the fresh ghee fed rats by 16% and in oxidized ghee fed rats by 25%. Membrane fluidity declined in the oxidized ghee fed rats. It is concluded that feeding ghee results in decreased proliferation of lymphocytes. Also, feeding oxidised ghee results in decreased proliferation of lymphocytes through alterations in the structure of the lymphocyte membranes in the rat.

Expression of the complement regulatory protein CD59 on human spermatozoa: characterization and role in gametic interaction.

Protectin (CD59) is a complement regulatory protein which blocks the membrane attack complex during complement activation. CD59 was identified on the human sperm surface by means of H19, an IgG1 anti-protectin mouse monoclonal antibody. Using indirect immunofluorescence, flow cytometry and immunoperoxidase, CD59 was found to be present on the whole plasma membrane including the head and tail of fresh ejaculated, capacitated and acrosome-reacted spermatozoa. Immunoperoxidase staining of normal testicular sections indicated that this protein was already present on intraluminal germ cells. Analysis of this sperm protein by gel electrophoresis and immunoblotting revealed that its molecular weight of 20 kDa was comparable to that of CD59 expressed on peripheral blood cells (erythrocytes, lymphocytes) and that it was bound to the membrane through a glycophospholipid tail which could be released after treatment with phosphatidylinositol-specific phospholipase C. Associated to membrane cofactor protein (CD46) and decay accelerating factor (CD55) located in the acrosomal membranes, CD59 may participate to the protection of male gametes against complement-mediated damage as they travel through the female genital tract. Moreover CD59, known as an adhesion molecule involved in lymphocyte rosettes, may also participate in cell to cell adhesion during gametic interaction since H19 inhibited sperm binding and reduced the penetration rate and index during the hamster egg penetration test.

Modulatory effects of ceruloplasmin on lymphocytes, neutrophils and monocytes of patients with altered immune status.

We investigated the effect of plasma ceruloplasmin (Cp) on the different types of lymphocyte rosetting, and phagocytosis of polystyrene particles and culture Candida albicans by peripheral blood neutrophils and monocytes. Lymphocytes, neutrophils, and monocytes were isolated from the blood of patients with elevated immuno-status (n = 9), healthy donors (n = 21), and patients with reduced immuno-status (n = 21). The ability of Cp to decrease the number of lymphocytes forming E- and EAC-rosettes and rosettes with auto-erythrocytes was shown for both patients and healthy donors. The maximal decrease of the number of E-rosettes (by 35%) and EAC-rosettes (by 57%) was shown for lymphocytes of the patients with elevated immuno-status. Cp had an effect on rosetting only when lymphocytes were preincubated with it, suggesting that Cp binding to lymphocytes was responsible for these effects. The decrease in all types of rosetting caused by Cp was dose-related, with a maximum effect at physiological concentration of Cp (300 micrograms/ml). We demonstrated an enhancing effect of Cp on phagocytosis of Candida albicans and polystyrene particles by neutrophils (with a maximum enhancement by 180% for neutrophils of the patients with decreased immuno-status) and monocytes (with a maximum of 89% for monocytes of healthy donors). Cp enhances phagocytosis of neutrophils and monocytes by binding these cells, not by opsonizing ingested particles as a conventional opsonin (ie. lipopolysaccharide from E.coli). The stimulating effect of Cp on phagocytosis was three times higher than that of LPS from E.coli.

Characteristics of interleukin-6-enhanced lymphokine-activated killer cell function

To study the effect of IL-6 on the development of cytotoxic cells, we examined lymphokine-activated killer (LAK) activity generated from human nonadherent PBL. Addition of rIL-6 at the initiation of 5-day PBL cultures significantly increases LAK activity in the presence of low concentrations (between 5 and 25 u/ml) of rIL-2. RIL-6 alone induces no PBL LAK activity but at doses as low as 0.8 u/ml rIL-6 enhances LAK activity with optimal enhancement of LAK at 5.0 u/ml of rIL-6. This enhancement is independent of effects on cells growth as rIL-6 did not affect the cell recovery of PBL cultured in rIL-2. RIL-6-enhanced LAK is mediated by the same type of effector cells as those of LAK from rIL-2 alone with effector cells primarily generated from large granular CD3-negative E rosetting lymphocytes. RIL-6 does not change the time course of LAK development and pretreatment of PBL with rIL-6 has no effect on the PBL response to subsequent rIL-2 induction of LAK. Addition of rIL-6 to LAK cultures 2 hr before the cytotoxicity assay shows equal enhancement as addition at the initiation of the culture. However, rIL-6 requires the presence of both rIL-2 and another factor in the supernatant from LAK cultures in order to enhance LAK. Our results indicate that IL-6 can modulate LAK activity at a very late stage of LAK development, and that the enhancement by IL-6 is dependent on the presence of IL-2 and another soluble factor generated during rIL-2 culture.

L428 cells derived from Hodgkin's disease produce E rosette-inhibiting factor

Diminished rosetting capacity of T cells is a well-known phenomenon in Hodgkin's disease, and inhibitors of E rosette formation have been reported to be present in the plasma of patients with Hodgkin's disease. The cell line L428, representing an in vitro counterpart of Hodgkin and Sternberg-Reed cells, could be shown to release a factor capable of suppressing the binding of sheep red blood cells (SRBC) to normal peripheral-blood T lymphocytes or to a T-cell line (L735). At maximally effective concentrations, RIF (rosette inhibiting factor) inhibited T lymphocyte rosetting by approximately 40% (mean from 184 healthy controls). The diminished E rosetting of T lymphocytes from Hodgkin's patients was not further suppressed by added RIF. This factor inhibited binding of SRBC to their target cells at 37 degrees C but not at 4 degrees C. The factor could be stored lyophilized at -20 degrees C and was stable at 56 degrees C (30 minutes). RIF was inactive below pH 6 and above pH 9 or after trypsin digestion. Purification by affinity, ion exchange, and molecular sieve chromatography showed activity peaks at 12.5 Kd, 25 Kd, 50 Kd, and 100 Kd.

L428 Cells Derived From Hodgkin's Disease Produce E Rosette-Inhibiting Factor

Diminished rosetting capacity of T cells is a well-known phenomenon in Hodgkin's disease, and inhibitors of E rosette formation have been reported to be present in the plasma of patients with Hodgkin's disease. The cell line L428, representing an in vitro counterpart of Hodgkin and Sternberg-Reed cells, could be shown to release a factor capable of suppressing the binding of sheep red blood cells (SRBC) to normal peripheral-blood T lymphocytes or to a T-cell line (L735). At maximally effective concentrations, RIF (rosette inhibiting factor) inhibited T lymphocyte rosetting by approximately 40% (mean from 184 healthy controls). The diminished E rosetting of T lymphocytes from Hodgkin's patients was not further suppressed by added RIF. This factor inhibited binding of SRBC to their target cells at 37 degrees C but not at 4 degrees C. The factor could be stored lyophilized at -20 degrees C and was stable at 56 degrees C (30 minutes). RIF was inactive below pH 6 and above pH 9 or after trypsin digestion. Purification by affinity, ion exchange, and molecular sieve chromatography showed activity peaks at 12.5 Kd, 25 Kd, 50 Kd, and 100 Kd.

Enhancement of mononuclear cell oxidative burst by early post-transfer sera from human patients after unsuccessful embryo transfer

Early pregnancy sera were earlier shown to modulate T lymphocyte rosetting with sheep erythrocytes. We set out to investigate whether early pregnancy sera also modulate the state of activation of another immunologically relevant cell type, the monocyte. As an index of monocyte activation, we measured phorbol ester-triggered oxidative burst activity by a highly sensitive chemiluminescence method. Contrary to our expectations, incubation of mononuclear cells with sera taken early after embryo transfer from patients with a successfully developing pregnancy had no effect. Unexpectedly, such sera from patients from a control group of patients in whom no pregnancy developed after embryo transfer caused enhancement of mononuclear cell chemiluminescence. Stimulatory activity of these sera appeared between days 4 and 6 and was maximal between days 7 and 9 post embryo transfer. Whether this phenomenon is causally related to, or represents a consequence of the failure of embryo transfer, can currently not be decided.

(Avid) rosette-forming cells as evidence for cellular immunity following measles virus vaccination in monkeys: correlation with the presence of haemagglutination inhibitory antibodies

Lymphocytes characterized by in vitro rosette formation with sheep erythrocytes were enumerated in blood of normal and measles virus-vaccinated monkeys. Rosetting lymphocyte counts increased significantly as a result of vaccination. Concomitantly, HAI-antibody, undetectable before vaccination, became detectable, suggesting that the simple and inexpensive technique of enumerating rosetting lymphocytes can be used as an alternative to other more cumbersome and expensive methods for evaluation of development of cell-mediated immunity following measles virus vaccination.

A subset of human rosetted lymphocytes contains previously unidentified histamine

By sequential solid-phase immunoadsorption (panning) steps, we have isolated a subset of lymphocytes (comprising 3–7% of rosetted cells) that contains high concentrations of histamine. We have used a radioenzymatic assay for the determination of histamine and have located 117 ng of histamine/1 × 10 6 cells in Leu-5 + (OKT-11), Leu-15 + cells. This subset did not contain basophils and was negative for Leu-4 (OKT-3), Leu-3 (OKT-4), Leu-2 (OKT-8), and 9.3 antigens. The function of this subset of rosetted cells has not been determined.

Suppressive characteristics of the cultured umbilical cord blood lymphocytes: Enhanced suppression of non specific MLR by short term cultured peripheral blood and rosetted lymphocytes

We have used the mixed leukocyte reaction assay (MLR) to compare the non-specific suppressor activity of fresh and short-term cultured peripheral blood rosetted lymphocytes from human umbilical cord blood. To a one way MLR between adult responders and irradiated adult stimulator cells, we added adult or cord blood mononuclear cells. Cord-blood derived mononuclear cells were more potent suppressors of the MLR than the adult peripheral blood mononuclear cells. When the fresh cord blood T cell fraction was separated from non-T cells by rosetting, the frequency and capacity of suppression fell, suggesting a role of non-T cells in this phenomenon. However, short-term culture (up to nine days) of cord blood derived T cells in the absence of mitogen resulted in a significant increase in their suppressive characteristics. Maternal cells were not used in any of these assays and the suppression was non specific. There was no HLA restriction to the response. The cord blood lymphocytes responded poorly to alloantigen when compared to the adult lymphocytes. In addition, there was a correlation between the allogeneic proliferative response and the suppressive capacity of the cord blood cells. Such a correlation was not found with the adult periheral blood lymphocytes.

Modulation of natural killer-mediated lysis by red blood cells

Natural killer (NK)-mediated cytotoxicity is significantly enhanced in the presence of red blood cells (RBC). The enhancement is dose dependent on the number of RBC present and can be induced by autochthonous, allogeneic, or xenogeneic RBC. The enhancement was demonstrated in cytotoxicity against two different NK-sensitive tumor target cell lines, K562 and U937, and by three different assay systems, chromium release, lactate dehydrogenase release, and inhibition of thymidine incorporation. RBC directly enhance the cytotoxic activity of NKH-1/Leu19 + large granular lymphocyte NK cells. Intact RBC have to be present during the cytotoxicity assay to induce the enhancement, which probably occurs at a postbinding, preprogramming phase. The anti-CD2 antibody Leu5 cannot block the enhancement at a concentration inhibitory to lymphocyte rosetting with sheep RBC, suggesting that the enhancement is not induced by interaction through the CD2 antigen. These results indicate that RBC are a potent modulator of NK cytotoxicity and suggest that in vitro NK studies using purified lymphocytes may not always truly reflect NK activity in the blood stream.

A novel method for the purification of sheep red cell rosetting lymphocytes

A rapid, direct method for the purification of sheep red cell rosetting lymphocytes (ERFC) was developed. The whole procedure, including rosette formation, density separation and hemolysis could be completed within 10 min. A mixture of human peripheral blood mononuclear cells (PBMC) and 2-aminoethylisothiouronium bromide hydrobromide-treated sheep erythrocytes (E AET) was layered onto Ficoll-Paque without any pretreatment and centrifuged at 600 × g for 2.5 min. The pellet was then immediately treated with an NH 4Cl solution containing 10% FCS and hemolysis was completed within 1 min. The purity of ERFC separated in one cycle of the procedure was 98%, the viability 99% and the yield 56% of the initial lymphocyte count. The re-rosetting ability of the prepared cells, after hemolysis, was 95%. The lymphocytes in the fraction prepared by the same method contained 94.3% CD2(OKT11) + cells, 90% of which were CD3(OKT3) + cells (T cells) and 9% were CD16(Leulla) + cells (NK cells).

Cytoadherence of lymphocytes from type I diabetic subjects to insulin-secreting cells. Marker of anti-beta-cell cellular immunity.

We studied the ability of lymphocytes from type I (insulin-dependent) diabetic patients to adhere to murine beta-cells. Lymphocytes from 17 recent-onset type I diabetic subjects (less than 6 mo) displayed enhanced ability to form rosettes with RINm5F cells (P less than .001) compared with lymphocytes from 27 healthy subjects forming background rosettes, whereas the number of RIN cytoadherent lymphocytes was unimpaired in 12 type II (non-insulin-dependent) diabetic subjects. This phenomenon tended to decline in 21 subjects with long-standing diabetes (greater than 1 yr) who taken as a group presented a normal number of RIN rosetting lymphocytes. The islet specificity of these diabetic rosettes was confirmed because, compared with controls, lymphocytes from recent-onset type I diabetic subjects also displayed a greater intensity of adherence to normal mouse islets but not to unrelated K562 and TS cell lines. As demonstrated by indirect immunofluorescence studies, these diabetic rosettes contained 54% of T-lymphocytes (OKT3+, OKT4+, or OKT8+), whereas only 20% of T-lymphocytes were found in background rosettes. The high percentage (66%) of la+ cells found in diabetic rosettes suggests that at least some of the cytoadherent T-lymphocytes from recent-onset type I diabetic subjects are activated. Natural killer (NK) cells do not seem to be the major cell type implicated in this phenomenon, because Leu 11+ cells were less represented in diabetic rosettes (25%) than in background rosettes (53%).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of human T lymphocyte E rosette formation by neutrophils and hydrogen peroxide. Differential sensitivity between helper and suppressor T lymphocytes.

Co-incubation of human mononuclear cells with small numbers of autologous polymorphonuclear leukocytes resulted in a ratio-dependent inhibition of T lymphocyte rosette formation with sheep erythrocytes (SRBC). Inhibition by polymorphonuclear leukocytes was reversed with catalase, implicating H2O2 or associated products as the suppressive agent(s). Exposing T lymphocytes directly to micromolar concentrations of H2O2 also inhibited subsequent rosette formation. Inhibition was dose-dependent between 40 and 320 microM H2O2 and was not due to cytotoxicity at those H2O2 concentrations. Kinetic studies demonstrated that after exposing T lymphocytes to H2O2 a proportion of cells appeared to be relatively resistant in that they retained their ability to form E rosettes. T cell phenotype analysis of these cells showed that, in contrast to untreated T cells, H2O2-treated T rosette-forming cells were almost exclusively of the helper/inducer phenotype. Analysis with the monoclonal antibody 4B4 revealed that H2O2-resistant T rosette-forming cells contained significantly fewer 4B4-positive cells than predicted for the T helper population, indicating that H2O2-resistant T cells might be a subset of helper/inducer T lymphocytes. The interaction between H2O2 and T cells was rapid but did not lead to loss of Leu-5b binding sites. Preliminary functional analysis indicates that interleukin 2 production and phytohemagglutinin-induced proliferation by the relatively H2O2-resistant T cells is similar to untreated T cells. In contrast, H2O2-treated T cells which lost their ability to form E rosettes produced undetectable levels of interleukin 2 and proliferated poorly in response to phytohemagglutinin. Finally, mononuclear cells pretreated with H2O2 and subsequently stimulated with mitogens demonstrated a preferential expansion of the T helper population with concomitant loss of T suppressors. Our results show that, although neutrophil-released H2O2 inhibits effective interactions between SRBC and T lymphocyte sheep erythrocyte receptors, there appears to be a population of T helper cells which is relatively resistant to H2O2 both in terms of SRBC rosette formation and response to mitogens. These data suggest that at sites of inflammation phagocyte-released H2O2 could exert immunoregulatory effects on T cells by altering T cell subset survival and allowing the function of a particular lymphocyte population to predominate.

Placental proteins in the diagnosis and evaluation of the "elusive" early pregnancy.

For many many years much time and effort has been invested to identify, purify, and quantify markers of significant reproductive events. The search for such markers has been recently intensified with the widespread acceptance of IVF and ET as a treatment regimen for the management of certain causes of infertility. Now it is possible to monitor ovarian folliculogenesis and time of ovulation, successful fertilization, and subsequent nidation and the ensuing pregnancy. Following successful fertilization and zygote development, EPF, a protein complex which suppresses in vitro lymphocyte rosette formation can be detected within 24 hours. Shortly after implantation hCG can be measured in the maternal circulation. The developing trophoblastic tissue continues to synthesize and secrete into the maternal circulation many varied proteins including SP1, PAPP-A, and PP5. To date hCG is the mainstay of rapid and early pregnancy diagnosis and is an accepted biochemical marker of successful implantation and trophoblastic activity. Combination of hCG and EPF measurement offers the clinician new insight into his infertile patient for now it is possible to monitor successful fertilization and implantation as separate independent events. Such diagnostic assays should also provide new insight into early embryo mortality rate at the pre- and postimplantation stage and hence indicate the selection pressures acting on an embryo. It is the application of these assays which has led to the definition of subclinical abortions and to the description of biochemical pregnancies. The value of these proteins for the management of compromised early pregnancies has to be more fully examined. Although the differentiation between a normal and abnormal pregnancy is more readily ascertained by serial sampling and analysis of the time taken for circulating levels to double (3). Preliminary data suggest the potential of EPF and PAPP-A measurement in such clinical conditions is enormous. Our studies suggest that PAPP-A, at present, is the only biochemical marker which can predict pregnancy abortion, even in the presence of normal ultrasound scans, many weeks prior to the event, thus implying a vital biological role for PAPP-A in normal pregnancy (52). Despite the need for more intensive evaluation of these early pregnancy indicators the search for that "demmed elusive" marker has more than "Those Frenchies" earnestly looking for him here and there.