E-rosette Test Research Articles

Strange blood — The Rise and Fall of Lamb Blood Transfusion in 19th Century Medicine and Beyond Medical Humanities, Volume 5, BoelBerner, (Transcript), 2020. ISBN‐978‐3‐8376‐5163‐8, 214 pages, including notes, sources and literature and an index

This is an interesting little volume seen from several angles. It concerns transfusion (part of the BJH repertoire), but notably it describes how transfusion of lamb's blood became a short-lived fancy among doctors, gaining traction in the 1870s and disappearing within a mere decade Though initially introduced by Riva in Rome in 1667, transfusion of lamb's blood was largely disregarded until the 1870s, when a treatise by Gesellius sought to summarize the literature. Concurrent with this, a series of wars had raised the wish in army doctors to attempt transfusions, most notably in the German Dr. Hasse from Nordhausen, who had served during the war with Denmark in 1864 and against France in 1870. Though blood from lambs had been used for humans every now and then, largely due to its easy availability and the fact that sheep red blood cells are smaller than their human counterparts, Dr. Hasse did significant scaling up and wrote his own book about it. The latter initiated a wave of lamb transfusions for a number of diseases in many Western countries. This book entertainingly describes these developments, both with respect to indications, techniques and actual procedures. In these troubled times, where nostrums for COVID-19 gain rapid and sometimes premature application, it is instructive to see how fast the idea and application of lamb blood transfusions spread. It is salutary in turn to find how quickly the practice disappeared. Although the author has a non-medical background, she has succeeded in amply describing the nature of the transfusion reactions seen. My first research project, in the mid-1970s, was to improve the E-rosette test, which measured the binding of human lymphocytes to sheep red blood cells and served as gold standard for identifying T cells. Reading about the sometimes dramatic reactions to even small volumes of lamb blood, I could not help wondering whether these were due to in vivo E-rosetting! Surprisingly, xenogeneic transfusion got a further but very brief renaissance in the 1890s as part of the serotherapy revolution, which culminated in Behring's tetanus antisera. It would have been instructive if the book had bridged that event and finished with Landsteiner's discoveries of blood groups in 1901 and 1909 which became the true foundation for today's transfusion medicine. Nevertheless, this is a good yarn, showing us how new treatments, also in the 19th century, were touted and ultimately ridiculed.

Polysaccharides Extracted from Rhizoma Pleionis Have Antitumor Properties In Vitro and in an H22 Mouse Hepatoma Ascites Model In Vivo.

Malignant ascites is a highly severe and intractable complication of advanced or recurrent malignant tumors that is often immunotherapy-resistant. Rhizoma Pleionis is widely used in traditional medicine as an antimicrobial and anticancer agent, but its effectiveness in treating malignant ascites is unclear. In the current study, we investigated the effect of polysaccharides isolated from Rhizoma Pleionis (PRP) on murine hepatocarcinoma H22 cells in an ascites model. We have found that the main components of PRP, that presented a relative molecular weight of 383.57 kDa, were mannose and glucose. We also found that PRP reduced the occurrence of abdominal ascites and increased survival in our mouse model. An immune response in the ascites tumor model was observed by performing a lymphocytes proliferation experiment and an E-rosette test. The ratios of CD8+ cytotoxic T cells and NK cells in the spleen were examined by flow cytometry, and the mRNA expression of Foxp3+in CD4+CD25+ (T regulatory Tregs) was measured by RT-PCR (reverse transcription-polymerase chain reaction). The levels of the cytokines TNF-α (tumor necrosis factor), VEGF (vascular endothelial growth factor), IL-2 (interleukin), and IFN-γ (interferon) in the serum and ascites supernatants were measured by ELISA. The expression of Foxp3 and Stat3 in peritoneal cells in the mouse model was measured by immunocytochemistry. The results indicated that PRP increased H22 tumor cell apoptosis in vivo by activating and enhancing the immune response. Furthermore, the effects of PRP on the proliferation of H22 cells were assessed by the CCK8 assay, Hoechest 33258, and TUNEL staining in vitro. We found that PRP suppressed the proliferation of H22 tumor cells but had no effect on BRL (Big rat liver) -3A rat hepatoma normal cells in vitro. Next, we investigated the underlying immunological mechanism by which PRP inhibits malignant ascites. PRP induced tumor cell apoptosis by inhibiting the Jak1–Stat3 pathway and by activating Caspase-3 and Caspase-8 to increase the Bax/Bcl-2 ratio. Collectively, our results indicate that PRP exhibits significant antitumor properties in H22 cells in vivo and in vitro, indicating that PRP may be used as a new therapeutic drug for cancer treatment.

New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment

The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of <10kDa, extracted from leukocytes of healthy donors. There is significant clinical evidence of improvement using DLE during treatment of allergies, cancer,immunodeficiencies, and in mycotic and viral infections. Nevertheless, the DLE exact nature and mechanism of action have been elusive for more than 50 years. DLE biological activity testing is necessary in DLE production and quality control. Both in vitro and in vivo assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (−Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between −DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.

Bioactivity Assay of Chitosan Microspheres Containing Pig Thymosin&lt;i&gt; In Vitro&lt;/i&gt;

Thymosin, composed of many peptides, is distributed in animal and has immunomodulating effects. However, many factors could affect its bioactivity. Chitosan microspheres containing pig thymosin (CMPT) were prepared through W/O emulsion cross-linking method. In this study, thymosin activity between pig-thymosin and CMPT was investigated through E-rosette test and mouse spleen lymphocyte proliferation (MPLP) experiment in vitro. Results showed that CMPT has the same immunomodulatory activity as pig thymosin. When pig-thymosin was 1.5 μg / ml, E-rosette binding rate was 63%, and E-rosette binding rate of CMPT reached 60%。MPLP rate was 73.3% and 68.5%, respectively. CMPT have the capacity to keep the E-rosette binding and MPLP activity of pig thymosin. This may help to improve traditional formulation and bioavailability of thymosin, and expand its scope of application in clinic.

Production and characterization of highly purified recombinant thymosin beta 4 in Escherichia coli

Thymosin β4 (Tβ4) is a small peptide composed of 43 amino acids. It has many important biological functions, such as promoting cardiac repair and wound healing, and therefore has great potential in clinical applications. In this report, we describe a novel and efficient way to produce highly purified and active Tβ4. It was expressed in a soluble form using a DsbA and hexahistindine tag in Escherichia coli (E. coli). Using high cell density cultivation, the final biomass concentration was about 50gL−1 dry cell weight with the expression level of the fusion protein being 40%. To obtain highly purified protein, a purification process involving a five-step column procedure was implemented. The purity of Tβ4 was above 98% and all the host cell related impurities, such as endotoxin, host cell protein and residual DNA levels, were within the permissible range listed in the Chinese Pharmacopoeia. The E-rosette test demonstrated that the bioactivity of purified Tβ4 was consistent with other published work. This is the first report producing highly purified Tβ4 from genetically engineered sources.

T lymphocytes in peripheral blood from patients with neurological diseases

Absolute and relative numbers of T lymphocytes in peripheral blood were determined in 19 different groups of patients with neurological diseases, using the E-rosette test. A significantly decreased total number of T lymphocytes was found in the following groups: acute Guillain-Barré syndrome (GBS), active multiple sclerosis (MS), and malignant cerebral tumor. A less pronounced and not significant reduction was observed in patients with amyotrophic lateral sclerosis (ALS). In all other groups of patients normal total numbers of T lymphocytes were found, thus indicating that the decrease in T lymphocyte counts in GBS, MS and malignant cerebral tumors is not due to nonspecific injury of nerve tissue.

Detection of T-lymphocytes in the peripheral blood and lymphoid tissues of cattle using a variant of the E-rosette test and monoclonal antibodies.

A simple and efficient variant of the E-rosette test based on addition of dextran to the incubation media is described. This variant 1) does not include B cells 2) involves some CD2+ null cells as described 3) is not inhibited by anti-CD5 antibody 4) correlates with CD2 expression 5) detects specific changes in the relative proportion of T-lymphocytes under the different conditions. In a group of calves the mean percentage of RFC in the peripheral blood lymphocytes was 53.59 +/- 8.70 and in cows there was 72.57 +/- 3.85. The proportion of RFC detected in bovine leukemia virus (BLV)--infected cows with lymphocytosis was less than one third of that in BLV--negative animals and vice versa in B (MHC class II+) lymphocytes.

Evaluación de las subpoblaciones de linfocitos T en pacientes tuberculosos empleand la modulación con teofilina

T cells and T cells subsets in peripheral blood of patients with different forms of pulmonary tuberculosis were evaluated to explain some aspects of the immunocompromised state of these subjects. Diagnosis was made by baciloscopy (BAAR), chest roentgenography i.d.r. Mantoux, and other clinical analysis. Spontaneous E-Rosette test (RE) was used to quantify Total (RET 4 degrees C) and Active T cells (REA 37 degrees C) and the same test after incubation with Theophylline (The) for helper cells (The-resistant cells: RETR) and suppressor cells (The-sensitive cells: RETS). Patients were followed for at least 4 months after therapy. The data demonstrate a significant decrease of relative and absolute numbers of Total T-cells and a diminished T helper/T suppressor subset ratio (RETR/RETS) which dropped to less than 1 in untreated patients. Treated patients with a favourable evolution showed a restoration of Total active and helper T cells. RETR/RETS ratio was also significantly increased. In patients with highly positive BAAR, low on negative i.d.r. Mantoux, a decreased level of immunocompetent cells was observed. The 3 aspects were associated. Nutritional condition of the patients was also associated with the predisposition to acquire this disease.

Clear cell lymphoma: A clinicopathological study of four cases

Four cases of clear cell lymphoma were studied by means of light and electron microscopy, enzyme-histochemistry, and E- and EAC-rosette formation. On light microscopic examination the tumors were seen to be composed mainly of round, oval, or slight irregular cells with water-clear, abundant cytoplasm. The neoplastic cells has round, oval, or convoluted nuclei with fine, evenly dispersed chromatin and one or more small but distinct nucleoli. On electron microscopic examination the clear cell lymphoma were characterized by lymphoid cells with striking electron lucent cytoplasm with few organelles. According to E-rosette test and enzyme-histochemical findings, the investigators proposed that the clear cell lymphoma may be derived from T-cell lineages. Differentiated diagnoses of clear cell lymphoma from B-immunoblastic sarcoma, pleomorphic cell lymphoma, clear cell sarcoma, and clear carcinomas derived from lung, ovary, or kidney were discussed.

Immunoregulating peptides II. In vitro effects of TP5 analogs on E-rosette formation and cell division.

The effects of seventeen synthetic analogs of thymopentin (TP-5) have been studied in the active and azathioprine-inhibited E-rosette tests. Thymopentin was gradually shortened from the C terminus to peptides and single amino acids. Thymopoietin 32-34 (Arg-Lys-Asp-RGH-0205-TP-3) (II) and thymopoietin 32-35 (Arg-Lys-Asp-Val-RGH-0206-TP-4) (I) were the most active peptides. Dipeptide Arg-Lys produced significant stimulatory effect on azathioprine (ED75) inhibited E-receptor. Treatment of azathioprine (ED75)-inhibited E-rosette forming cells (ERFC) with arginine or especially lysine increased the number of ERFC. Some of TP-4 analogs decreased further the number of ERFC decreased by azathioprine ED30. These "suppressive" peptides as well as TP-3 caused a partial arrest of K 562 cell proliferation up to 96 hours. Results suggest that TP-5 is not the smallest active fragment of thymopoietins, since peptides (TP-3 and TP-4) exhibit similar or higher T-cell membrane activation on E-receptor. Arginine, lysine, and acidic aspartyl residue seem to be a necessary basic structure to produce a cumulative chemical signal on the activity of T-lymphocytes.

Malignant lymphoma of convoluted lymphocytes: diagnosis by fine-needle aspiration cytology and cytochemistry.

The technique of fine-needle aspiration cytology (FNAC) was utilized in the diagnosis of 31 cases of malignant lymphoma of convoluted lymphocytes. The age of the patients ranged from 5 to 70 yr (median, 24 yr). Clinical history was available in 29 cases. All patients had peripheral lymphadenopathy. There was radiological evidence of mediastinal adenopathy in 62% of the cases, and 41% had pleural effusion; 52% of the patients presented with mediastinal compression syndrome. The diagnostically crucial convoluted cells comprised 47.3 +/- 23.73% of all lymphoid cells. Nuclear convolutions were appreciated in a significant number of cells in paraffin sections of 17 of 21 cases (85.7%) having subsequent histopathology. Cytochemical and E-rosette tests performed in 17 cases confirmed the T cell nature of the neoplasms. On an average, 72.3% of cells showed focal positive reaction for acid phosphatase, and 73.8% of cells formed E-rosettes.

Partial defects of cell-mediated immunity in patients with epidermodysplasia verruciformis

Different parameters of cell-mediated immunity, including natural cytotoxic reactions, were studied in nine patients with epidermodysplasia verruciformis with or without cutaneous malignancy. We found decreased total number of T lymphocytes and T-helper cells in peripheral blood of the patients, and normal T-suppressor cell number, as detected by monoclonal antibody typing and functional E-rosette test with the use of theophylline. This decrease was found both in active and in late rosette-forming cell subpopulations. Natural killer cell activity of peripheral blood mononuclear cells was found to be increased in four of nine patients with epidermodysplasia verruciformis, whereas antibody-dependent cellular cytotoxicity was within the normal range in all patients studied. Lymphocyte-induced angiogenesis assay, which is a sensitive test for the estimation of the immunocompetence of lymphoid cells, revealed increased angiogenic capability of peripheral blood mononuclear cells in the majority of the patients. Our results suggest that cellular defects in patients with epidermodysplasia verruciformis did not relate to all functions of the immune system.

Study of T cell subsets in patients with cutaneous leishmaniasis.

The proportion of T lymphocyte populations was assessed using monoclonal antibodies (OKT3, OKT4, OKT8) and spontaneous E-rosette tests with SRBC (for total and active TE cells) in 36 patients with cutaneous leishmaniasis subdivided according to the clinical variety. The T cell profile was normal in patients with simple cutaneous leishmaniasis of early ulcerative type whereas active TE cells and OKT4 positive cells were reduced in some cases of late ulcerative type. A significant reduction of active TE cells and of OKT4 positive cells (helper/inducer phenotype) and increase of OKT8 positive cells (suppressor/cytotoxic phenotype) was observed in peripheral blood of all patients with persistent active lesions and leishmaniasis recidivans. In patients with highly ulcerated persistent lesions a low proportion of active TE cells was also demonstrated in cellular extracts from dermal tissue. The results support the data obtained in experimental leishmaniasis that lack of helper/inducer cells and generation of suppressor T cells may be responsible for the development of chronic leishmaniasis.

Lymphocyte subpopulations in Spanish parenteral drug addicts.

Lymphocyte subpopulations were studied in 29 symptom-free Spanish prisoners who were active parenteral drug abusers. In 16/29 drug addicts (55%) the helper-inducer/suppressor-cytotoxic ratio (Leu 3a/Leu 2a) was less than 1. None of these 16 patients had lymphopenia and only 2 had a reduction in the number of Leu 3a cells. In drug addicts the number of lymphocytes (p less than 0.01) and the number and percentage of Leu 2a cells (p less than 0.001) were significantly raised. The addicts also had a higher absolute number of T cells when measured by their ability to react with the Leu 4 antibody (p less than 0.01). However, the E-rosette forming cells were significantly reduced. Thus the E-rosette test may lead to an overestimation of "null" cells. Furthermore drug addicts had a higher percentage (p less than 0.001) of Leu 7 positive cells (NK and K cells), but a normal percentage of B and phagocytic cells. Five drug addicts had generalized persistent lymphadenopathies, and 2 had skin anergy. We believe that most of the immunologic abnormalities seen in these apparently healthy drug abusers can be explained by an antigenic overload.

Synthesis of blood serum thymic factor and its analogs

The blood serum thymic factor (FTS) and its two analogs (desGlp1)-FTS and (Gln1)-FTS were synthesized by a novel procedure. The biological activity of these compounds was assayed by the E-rosette test. The activity of (desGlp1)-FTS is decreased and (Gln1)-FTS shows an inhibitory effect.

The binding of SRBC by human peripheral blood monocytes.

In a population of E-rosette forming human peripheral blood cells, about 20% of the rosettes were formed by monocytes. About 60% of the peripheral blood monocytes bound SRBC in a standard E-rosette test.

Enumeration of T-lymphocytes and T-lymphocyte subsets in rheumatoid arthritis using monoclonal antibodies

The number of T-lymphocytes and T-lymphocyte subsets was measured in peripheral blood of 51 patients with rheumatoid arthritis. T-lymphocytes were counted by E-rosette tests and by the immunogold staining method with OKT3.PAN monoclonal antibody. Helper and suppressor T-lymphocytes were determined by the immunogold staining method with OKT4.IND and OKT8.SUP monoclonal antibody. The relative and absolute numbers of T-lymphocytes and helper T-lymphocytes in peripheral blood of patients with RA did not differ significantly from those in the blood of healthy subjects. However, the relative and absolute numbers of suppressor T-cells were significantly lower in patients with RA than in healthy subjects. The decrease of suppressor T-cells in the blood of patients with RA dit not correlate with the activity of the disease nor the presence of the rheumatoid factor.

Effect of medroxyprogesterone acetate upon PHA, Con A and PWM stimulated lymphocytes and on E-rosette function.

We evaluated if Medroxyprogesterone acetate (MPA) had an immunosuppressive action as progesterone and other steroid hormones. Lymphocyte function was evaluated by the blastogenic response to mitogens and by the E-rosette test. MPA was tested at 7 ng/ml and 100 ng/ml concentrations. Cells were either preincubated for 30 min., 90 min., 180 min. and 12 hr or incubated with MPA for 72 hr, 18 hr and 16 hr. MPA showed a significative inhibitory effect only when used at the concentration of 100 ng/ml.

Multi-sialo brain gangliosides are powerful stimulators of active E-rosetting lymphocytes from multiple sclerosis patients

Peripheral blood T lymphocytes from all of 14 patients with clinically definite multiple sclerosis (MS) were significantly stimulated by MS brain gangliosides in the active rosetting of sheep erythrocytes. Fractionated mono- and disialogangliosides were devoid of any stimulating effect on MS lymphocytes whereas the trisialoganglioside G T1 and to a greater extent the tetrasialoganglioside G Q1b were fully effective at a dose as low as 2 × 10 −18 moles. Gangliosides extracted from MS brains or from MS brain myelin were far more effective than gangliosides derived from control human brains or from bovine and mouse brains, suggesting the importance of highly sialylated gangliosides occurring to a greater extent in MS brain as previously reported. Lymphocytes from only 3 out of 24 other neurological patients were stimulated by the slow migrating gangliosides in the same way, but none of 32 healthy subjects responded to these gangliosides in the active E-rosette test. Lymphocytes from 5 of 8 patients with unilateral optic neuritis reacted positively to brain gangliosides by rosette formation, several weeks before a similar reaction to myelin basic protein was evident. Our data are compatible with a release of gangliosides during demyelination or other CNS degenerative processes occurring in multiple sclerosis.

An enzyme immunoassay for the enumeration of peripheral human T-lymphocytes with OKT3.PAN monoclonal antibody

An enzyme immunoassay is described for the enumeration of peripheral human T-lymphocytes with a monoclonal antibody OKT3.PAN defining an antigenic determinant present on all mature T-lymphocytes. The method is compared with the indirect immunofluorescence technique and with an E-rosette test.