Leukocyte Common Antigen CD45 Research Articles

Dual targeting negative enrichment strategy for highly sensitive and purity detection of CTCs.

Circulating tumor cells (CTCs) have significant clinical value in early tumor detection, dynamic monitoring and immunotherapy. CTC detection stands out as a leading non-invasive approach for tumor diagnostics and therapeutics. However, the high heterogeneity of CTCs and the occurrence of epithelial-mesenchymal transition (EMT) during metastasis pose challenges to methods relying on EpCAM-positive enrichment. To address these limitations, a method based on negative enrichment of CTCs using specific leukocyte targets has been developed. In this study, aiming to overcome the low purity associated with immunomagnetic beads targeting solely the leukocyte common antigen CD45, we introduced CD66b-modified immunomagnetic beads. CD66b, a specific target for neutrophils with abundant residues, was chosen as a complementary approach. The process involved initial collection of nucleated cells from whole blood samples using density gradient centrifugation. Subsequently, magnetically labeled leukocytes were removed by magnetic field, enabling the capture of CTCs with higher sensitivity and purity while retaining their activity. Finally, we selected 20 clinical blood samples from patients with various cancers to validate the effectiveness of this strategy, providing a new generalized tool for the clinical detection of CTCs.

Protein Tyrosine Phosphatase CD45 As an Immunity Regulator and a Potential Effector of CAR-T therapy.

The leukocyte common antigen CD45 is a receptor tyrosine phosphatase and one of the most prevalent antigens found on the surface of blood cells. CD45 plays a crucial role in the initial stages of signal transmission from receptors of various immune cell types. Immunodeficiency, autoimmune disorders, and oncological diseases are frequently caused by gene expression disorders and imbalances in CD45 isoforms. Despite extensive research into the structure and functions of CD45, the molecular mechanisms behind its role in transmitting signals from T-cell receptors and chimeric antigen receptors remain not fully understood. It is of utmost importance to comprehend the structural features of CD45 and its function in regulating immune system cell activation to study oncological diseases and the impact of CD45 on lymphocytes and T cells modified by chimeric antigen receptors.

Identification of Reed-Berezovsky-Sternberg cells in lymphatic nodes in classic Hodgkin's lymphoma by flow cytometry: a clinical case series

Despite their B cell origin, Reed-Berezovsky-Sternberg tumor cells (RBS) in classic Hodgkin's lymphoma (cHL) demonstrate an absolutely unique phenotype. Immunohistochemistry of RBS cells is positive for CD15 antigen in most of cases, CD30, PAX-5; they do not express the T cell antigen CD3, В cell CD19, and in most cases are negative for the B cell antigen CD20, as well as for common leukocyte antigen CD45. Taking into account such unequivocal immunophenotype, RBS cells can be identified by multiparameter flow cytometry. Thus, J.R. Fromm et al. (2006, 2014) have convincingly shown the possibility to identify RBS cells in a puncture and/or biopsy sample of lymphatic nodes in cHL and were of the fair opinion that such rather simple and reproducible technique as flow cytometry could be an additional diagnostic instrument in cHL.
 We have tested the technique proposed by J.R. Fromm et al. for the assessment of lymphatic node involvement in cHL and used 8 to 10-parameter flow cytometry for detection RBS cells in cHL in 8 biopsy samples of a lymphatic node, and confirmed the feasibility to identify RBS cells by high performance flow cytometry. We also performed morphological and immunohistochemical assessment of the biopsy samples of lymphatic nodes from patients with suspected cHL. The study included clinical cases with immunohistochemically confirmed cHL (n = 8), and the control samples were from those with other diagnoses than Hodgkin's lymphoma. In all cases of cHL we found RBS cells. In future we plan to analyze larger case samples by flow cytometry.

Increased expression of tyrosine protein phosphatase (CD45) on the surface of human blood granulocytes under the influence of the plague microbe vaccine strain <I>Yersinia pestis</I> EV NIIEG <I>ex vivo</I> and <I>in vivo</I>

Tyrosine protein phosphatase (common leukocyte antigen CD45) regulates FcᵧR-mediated cell signaling and secretory function of neutrophilic granulocytes (NG) when interacting with antigen-antibody immune complexes. The aim of the work is to study changes in the expression of CD45 on the surface of human granulocytes in ex vivo modeling of bacteremia by live cells of the plague microbe vaccine strain Yersinia pestis EV NIIEG and to evaluate the priming effect of the live plague vaccine (LPV) in vivo in terms of this parameter. The expression density of CD45 on NG was determined by flow cytometry in conventional units of fluorescence intensity (MFI) after staining the cells with the CD45-FITC labeled mouse antibody reagent (Backman Coulter, USA) during immunophenotyping of blood leukocytes according to the Lyse/No Wash protocol. In donors not previously vaccinated against plague (group 1), the value of the indicator was assessed before and 30 min, 2 h, 6 h after the addition of Y pestis EV cells to whole blood at a dose of 108 mc/ml, as well as 1 month and 6 months after the primary anti-plague vaccination. In individuals who had previously been repeatedly vaccinated with LPV in the territory of the natural plague focus (group 2), CD45 expression on blood granulocytes was determined one year after the last annual vaccination, and then 1 month and 6 months after revaccination. Getting into human blood, living cells of the vaccine strain Y pestis EV of the plague microbe induced a change in the NC phenotype already after 30 minutes, associated with a 3.5-fold increase in the surface expression of CD45, which remained at an elevated level for 6 hours. The studied indicator depended ex vivo on the degree of resistance of plague microbes to phagocytosis and killing of NG. Plague vaccination had a similar stimulating effect on human peripheral blood NG in vivo. Under the influence of HPV, CD45 expression increased on blood NG in groups 1 and 2 one month after vaccination, and the changes persisted in volunteers for 6 months. The experimental data obtained in the work may reflect the result of NG priming with lipopolysaccharide and other Y. pestis antigens. The registered functional activation of NG by expression of tyrosine protein phosphatase probably indicates the formation of “immune memory” at the level of innate immunity cells under the influence of LPV, the functioning of which explains the development of a faster and more intense antigen-specific immune response to repeated introduction of the plague vaccine into the body.

Morphological and phenotypical characteristics of porcine satellite glial cells of the dorsal root ganglia.

Satellite glial cells (SGCs) of the dorsal root ganglia (DRG) ensure homeostasis and proportional excitability of sensory neurons and gained interest in the field of development and maintenance of neuropathic pain. Pigs represent a suitable species for translational medicine with a more similar anatomy and physiology to humans compared to rodents, and are used in research regarding treatment of neuropathic pain. Knowledge of anatomical and physiological features of porcine SGCs is prerequisite for interpreting potential alterations. However, state of knowledge is still limited. In the present study, light microscopy, ultrastructural analysis and immunofluorescence staining was performed. SGCs tightly surround DRG neurons with little vascularized connective tissue between SGC-neuron units, containing, among others, axons and Schwann cells. DRG were mainly composed of large sized neurons (∼59%), accompanied by fewer medium sized (∼36%) and small sized sensory neurons (∼6%). An increase of neuronal body size was concomitant with an increased number of surrounding SGCs. The majority of porcine SGCs expressed glutamine synthetase and inwardly rectifying potassium channel Kir 4.1, known as SGC-specific markers in other species. Similar to canine SGCs, marked numbers of porcine SGCs were immunopositive for glial fibrillary acidic protein, 2',3'-cyclic-nucleotide 3'-phosphodiesterase and the transcription factor Sox2. Low to moderate numbers of SGCs showed aquaporin 4-immunoreactivity (AQP4) as described for murine SGCs. AQP4-immunoreactivity was primarily found in SGCs ensheathing small and medium sized neuronal somata. Low numbers of SGCs were immunopositive for ionized calcium-binding adapter molecule 1, indicating a potential immune cell character. No immunoreactivity for common leukocyte antigen CD45 nor neural/glial antigen 2 was detected. The present study provides essential insights into the characteristic features of non-activated porcine SGCs, contributing to a better understanding of this cell population and its functional aspects. This will help to interpret possible changes that might occur under activating conditions such as pain.

Isolation and identification of a mesenchymal stem/stromal cell-like population from pediatric urethral tissue.

Mesenchymal stem cells (MSCs) are important seed cells for cell therapy and tissue engineering because of their multidirectional differentiation potential, high proliferative capacity, low immunogenicity, and immunomodulatory ability. In this study, we successfully isolated and cultured a population of mesenchymal stem-like cells from pediatric urethra (PU-MSLCs). The cells had a spindle-shaped fibroblast-like morphology, similar to MSCs derived from other tissues. The PU-MSLCs highly expressed MSC surface markers CD29, CD73, CD90, and CD105 but were negative for leukocyte common antigen CD45, and MHC class II-encoded molecule HLA-DR. After in vitro induction, the PU-MSLCs had the potential to differentiate into adipocytes, osteocytes, and chondrocytes. The PU-MSLCs maintained a normal karyotype and showed no tumorigenicity during long-term cultivation. We thus demonstrated that the mesenchymal stem/stromal cell-like population obtained from pediatric urethra tissue is capable of self-renewal and multidirectional differentiation, has promising application prospects for cell therapy and tissue engineering, and is expected to contribute to urethral tissue reconstruction.

Natural Antisense Transcript PEBP1P3 Regulates the RNA Expression, DNA Methylation and Histone Modification of CD45 Gene.

The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.

Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images

We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.

Morphological and immunofenotypic features of the monoclonal population of B-lymphocytes in chronic lymphocytic leukemia

Purpose of the study. To evaluate the features of morphological and immunophenotypic characteristics of the lymphoid population with different restriction of light chains of immunoglobulins in patients with chronic lymphocytic leukemia (CLL).Materials and methods. The study included 30 CLL patients aged 47–79 years (20 men and 10 women). All patients underwent a General clinical blood test (SysmexXE 2100, Japan), morphological examination of the bone marrow (BioVision; Micros, Austria), immunophenotyping of bone marrow and peripheral blood by flow cytofluorometry (Navios10/3, Beckman Coulter, USA). B-cell clonality established by detection of restriction of light chains of surface immunoglobulins kappa or lambda. Morphological analysis of lymphocytes that differ in the expression of light chains of surface immunoglobulins: kappa (k) — group I (22 people — 73,3%), lambda (λ) — group II (8 people — 26,7%).Results. Determination of cell types by values of direct (FSC) and lateral (SSC) light scattering during immunophenotyping of peripheral blood and bone marrow samples showed that in patients of group I (CD19k+/CD5+/CD23+) on the light scattering diagram, the lymphoid population had low parameters: on the FSC scale — from 200 to 400, on the SSC — from 10 to 160 units, which indicates morphological uniformity of cells. In group II (CD19λ+/CD5+/CD23+), on the contrary, on the light scattering sketogram, the lymphoid zone was heterogeneous and stretched: on the FSC scale — from 200 to 1000, on the SSC — from 10 to 400 units, which indicates morphological polymorphism of cells. There were also differences in the expression of the common leukocyte antigen CD45. In group I, the expression is higher: the population of B-lymphocytes in terms of fluorescence intensity is on the dot graph on the CD45 scale in the second half of the third decade and in the fourth decade — to the right, than in group II, in which B-lymphocytes lie in the third decade. The data indicate that the CD19k+/CD5+/CD23+ population is represented by Mature cells, while the Cd19k+/CD5+/CD23+ population is represented by less Mature and / or intermediate forms. Significant morphological differences in lymphocyte populations were also observed in microscopic studies of blood and bone marrow preparations.Conclusion. The established immunophenotypic and morphological differences in lymphoid populations expressing either kappa — or lambda-light chains of immunoglobulins may be important for identifying risk groups among patients with biologically heterogeneous variants of chronic lymphocytic leukemia.

A Novel Type of Stem Cells Double-Positive for SSEA-3 and CD45 in Human Peripheral Blood

Peripheral blood (PB) contains several types of stem/progenitor cells, including hematopoietic stem and endothelial progenitor cells. We identified a population positive for both the pluripotent surface marker SSEA-3 and leukocyte common antigen CD45 that comprises 0.04% ± 0.003% of the mononuclear cells in human PB. The average size of the SSEA-3(+)/CD45(+) cells was 10.1 ± 0.3 µm and ∼22% were positive for CD105, a mesenchymal marker; ∼85% were positive for CD19, a B cell marker; and ∼94% were positive for HLA-DR, a major histocompatibility complex class II molecule relevant to antigen presentation. These SSEA-3(+)/CD45(+) cells expressed the pluripotency markers Nanog, Oct3/4, and Sox2, as well as sphingosine-1-phosphate (S1P) receptor 2, and migrated toward S1P, although their adherence and proliferative activities in vitro were low. They expressed NeuN at 7 d, Pax7 and desmin at 7 d, and alpha-fetoprotein and cytokeratin-19 at 3 d when supplied to mouse damaged tissues of the brain, skeletal muscle and liver, respectively, suggesting the ability to spontaneously differentiate into triploblastic lineages compatible to the tissue microenvironment. Multilineage-differentiating stress enduring (Muse) cells, identified as SSEA-3(+) in tissues such as the bone marrow and organ connective tissues, express pluripotency markers, migrate to sites of damage via the S1P-S1P receptor 2 system, and differentiate spontaneously into tissue-compatible cells after homing to the damaged tissue where they participate in tissue repair. After the onset of acute myocardial infarction and stroke, patients are reported to have an increase in the number of SSEA-3(+) cells in the PB. The SSEA-3(+)/CD45(+) cells in the PB showed similarity to tissue-Muse cells, although with difference in surface marker expression and cellular properties. Thus, these findings suggest that human PB contains a subset of cells that are distinct from known stem/progenitor cells, and that CD45(+)-mononuclear cells in the PB comprise a novel subpopulation of cells that express pluripotency markers.

Lysosomal Associated Membrane Protein Correlates with Breast Cancer Recurrence CD68 and CD45 leukocyte common antigen in Iraqi women patients

Lysosomal Associated Membrane Protein Correlates with Breast Cancer Recurrence CD68 and CD45 leukocyte common antigen in Iraqi women patients

The influence of chestnut wood and flubendazole on morphology of small intestine and lymphocytes of peripheral blood, spleen and jejunum in broiler chickens

SummaryThe study examined subpopulations of lymphocytes in peripheral blood, spleen, and jejunum including morphology of that segment in broiler chicken farm after treatment with flubendazole (Flimabend) and natural extract from chestnut wood (Farmatan). A total of 24 forty-day-old Kalimero-Super Master hybrid chickens were divided into 4 groups (n=6): the Fli group received Flimabend per os, 100 mg/g suspension in 1.43 mg of active substance/kg body weight during 7 day of experiment, Far group received Farmatan per os at 0.2 % concentration for 6 hours per day during 5 day (experimental days – from 3 to 7); the Far+Fli group received a combination of doses administered in the same way as for the first two groups; and control –C group with no active substance administration. The results demonstrated mild increase of leukocytes, lymphocytes, monocytes, leucocyte common antigen CD45, IgM+ and IgA+ cells in peripheral blood after administration of Flimabend. Similarly, subpopulations of followed lymphocytes (CD3+, CD4+, CD8+, IgM+) were increased in the jejunum after application of that drug. On the other hand, administration of Farmatan revealed opposite effect on determined immunocompetent cells what proves anti-inflammatory effect. Morphology of villi was also negatively influenced by administration of Flimabend. Administration of Farmatan suggests also its preventive administration in chickens. This tanin-containing drug as plant natural product may be used due to its antibacterial activity and as promising alternative to conventional drug with possible antihelminthic effect.

Atypical Plasmablastic Lymphoma Presenting as a Perianal Mass in a 19-Year-Old Male

Abstract Objectives To describe a case of plasmablastic lymphoma with an atypical immunophenotype in an unusual location. Methods We received this specimen for routine processing after the patient underwent an incision and drainage surgery. The specimen was sent to hematopathology for workup of lymphoma. We then ordered several special stains and molecular tests to confirm our diagnosis of plasmablastic lymphoma. Results Histologic examination of the tissue reveals sheets of large plasmablastic cells with eccentrically located round nuclei, prominent nucleoli, and cartwheel chromatin. Frequent apoptotic bodies and mitotic figures are present. Immunohistochemistry shows strong, diffuse positivity for leukocyte common antigen CD45, CD10, plasmacytic markers (MUM1, CD79a) and negativity for B-cell markers (CD20, PAX5) and CD138. The Ki-67 proliferation index is very high (>90%) and Epstein-Barr virus encoded small RNA (EBER) in situ hybridization is diffusely positive. Molecular FISH testing for aggressive B-cell lymphoma genetic abnormalities is positive for MYC gene rearrangement in 68% of nuclei and the loss of one copy of the BCL2 gene on chromosome 18 in 64% of nuclei. Conclusion This case of plasmablastic lymphoma is unique due to its atypical presentation as a perianal mass in a young patient with undiagnosed HIV. Usually, this lymphoma presents as an oral lesion in an older patient with HIV. In addition, immunohistochemistry showed an unusual immunophenotype (CD138–, MUM1+, CD45+, CD10+). Most plasmablastic lymphomas are CD138+, MUM1+, and CD45– but this specimen is CD138–, MUM1+, and CD45+. Further testing shows EBER positivity, a positive HIV test, and MYC gene rearrangement, which confirms our diagnosis of a plasmablastic lymphoma. Of note, genetic analysis also reveals a loss of one copy of the BCL2 gene on chromosome 18 in 64% of nuclei.

Combined Sabal and Urtica Extracts (WS® 1541) Exert Anti-proliferative and Anti-inflammatory Effects in a Mouse Model of Benign Prostate Hyperplasia.

WS® 1541 is a phytopharmaceutical drug combination containing a lipophilic extract from fruits of Sabal serrulata (WS® 1473) and an aqueous ethanolic extract from roots of Urtica dioica (WS® 1031). It is approved in several countries worldwide for the treatment of lower urinary tract syndrome (LUTS) linked to benign prostate hyperplasia (BPH). Clinical studies have demonstrated the efficacy of this unique combination in the treatment of BPH-related LUTS. However, its mechanisms of action in vivo remain partly uncharacterized. The aim of this study was to take advantage of a validated mouse model of BPH to better characterize its growth-inhibitory and anti-inflammatory properties. We used the probasin–prolactin (Pb-PRL) transgenic mouse model in which prostate-specific overexpression of PRL results in several features of the human disease including tissue hypertrophy, epithelial hyperplasia, increased stromal cellularity, inflammation, and LUTS. Six-month-old heterozygous Pb-PRL male mice were randomly distributed to five groups (11–12 animals/group) orally treated for 28 consecutive days with WS® 1541 (300, 600, or 900 mg/kg/day), the 5α-reductase inhibitor finasteride used as reference (5 mg/kg/day) or vehicle (olive oil 5 ml/kg/day). Administration of WS® 1541 was well tolerated and caused a dose-dependent reduction of prostate weight (vs. vehicle) that was statistically significant at the two highest doses. This effect was accompanied by a reduction in prostate cell proliferation as assessed by lower Ki-67 expression (qPCR and immunohistochemistry). In contrast, finasteride had no or only a mild effect on these parameters. The growth-inhibitory activity of WS® 1541 was accompanied by a strong anti-inflammatory effect as evidenced by the reduced infiltration of cells expressing the leukocyte common antigen CD45. In sharp contrast, finasteride significantly increased the prostate inflammatory status according to this readout. Molecular profiling (qPCR) of 23 selected pro-inflammatory genes confirmed the strong anti-inflammatory potency of WS® 1541 compared to finasteride. Since treatment of WS® 1541 did not interfere with transgene expression and activity in the prostate of Pb-PRL mice, the effects observed in this study are entirely attributable to the intrinsic pharmacological action of the drug combination.

Epstein-Barr Virus–Positive CD20- and CD45-Negative Diffuse Large B-Cell Lymphoma: A Diagnostic Challenge

Diffuse large B-cell lymphoma (DLBCL) is characterized by medium- to large-sized neoplastic cells that express a wide range of B-cell markers including CD19, CD20, CD22, and CD79a. Also, as this is a hematopoietic malignancy, there is expression of the leukocyte common antigen CD45. Lack of CD20 expression occurs in a specific rare heterogeneous subgroup of DLBCL including primary effusion lymphoma, plasmablastic lymphoma, ALK-positive large B-cell lymphoma, and large B-cell lymphoma arising in HHV8+ multicentric Castleman disease. In this article, we report a rare case of CD20- and CD45-negative Epstein-Barr virus-positive DLBCL in which the entities listed above were ruled out, thereby posing a significant diagnostic challenge. Arriving at the correct diagnosis of Epstein-Barr virus-positive DLBCL was supported by immunoreactivity for the B-cell transcription factor Oct-2 and the pan-B cell marker CD79a.

A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 co-existed with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signaling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics.

Molecular features of ascitic ovarian cancer cells detected by immunofluorescence assay using flow cytometry

Peritoneal dissemination and growth of tumor cells in the ascitic fluid in stages III and IV of recurrent ovarian cancer is difficult to treat; resistance to many anticancer drugs used in the treatment of solid ovarian cancer is observed at this stage. The author’s hypothesis that the differences in the molecular phenotype of tumor cells for various types of the disease could be an explanation for this. A comparative evaluation of the expression and coexpression of a number of molecular markers (immunofluorescence assay with flow cytometry) was performed using solid and ascitic ovarian cancer cells. In contrast to solid ovarian cancer, ascitic ovarian cancer cells present leukocyte common antigen CD45 and mesenchymal marker vimentin in addition to epithelial marker cytokeratin. In addition to the inhibition of anoikis (specific mechanism of epithelial cell death in a liquid medium in the absence of contact with the substrate), ascitic ovarian cancer cells are characterized by (1) emperipolesis (intracellular migration of leukocytes without damage to the tumor cell), and (2) phenotype of epithelial-mesenchymal transition. Thus, the data on clinically significant molecular differences between solid and recurrent ascitic ovarian cancer was first obtained, opening up opportunities for anticancer therapy, which previously was not used in the treatment of ovarian cancer.

A Depleting Anti-CD45 Monoclonal Antibody as Isolated Conditioning for Bone Marrow Transplantation in the Rat.

ObjectiveA monoclonal antibody (mAb) against the leukocyte common antigen CD45 (RT7 in rats) could facilitate bone marrow transplantation (BMT). This study in rats evaluates a depletive rat anti-RT7a mAb as isolated tool for BMT conditioning without using irradiation or any chemotherapeutic / immunosuppressive agent.MethodsThe model used a CD45 di-allelic polymorphism (RT7a/RT7b). The anti-RT7a mAb was intravenously administered to LEW.1W rats (RT1uRT7a) at 5, 10 and 15 mg/kg. 1x108 BM cells of MHC syngeneic (RT1u), MHC disparate (RT1l) or MHC haploidentical (RT1u/l) donors were transplanted. All BM donor strains carried the RT7b allele so that their CD45+ cells were not affected by the anti-RT7a mAb. Recipients were monitored for reconstitution and donor-chimerism in blood leukocytes.ResultsmAb dosages of 5 or 10 mg/kg were myelosuppressive, whereas 15 mg/kg was myeloablative. Multi-lineage donor-chimerism at day 100 indicated engraftment of MHC syngeneic BM after any used mAb dosage (5 mg/kg: 46+/-7%; 10 mg/kg: 62+/-5%; 15 mg/kg: 80+/-4%). MHC disparate BM resulted in autologous reconstitution after conditioning by 10 mg/kg of the mAb and caused transient chimerism ending up in death associated with aplasia after conditioning by 15 mg/kg of the mAb. MHC haploidentical BM (F1 to parental) engrafted only after conditioning by 15 mg/kg (chimerism at day 100: 78+/-7%). Abandonment of α/β TCR+ cell depletion from BM grafts impaired the engraftment process after conditioning using 15 mg/kg of the mAb in the MHC syngeneic setting (2 of 6 recipients failed to engraft) and the MHC haploidentical setting (3 of 6 recipients failed).ConclusionThis depletive anti-RT7a mAb is myelosuppressive and conditions for engraftment of MHC syngeneic BM. The mAb also facilitates engraftment of MHC haploidentical BM, if a myeloablative dose is used. RT7b expressing, BM-seeded α/β TCR+ cells seem to impair the engraftment process after myeloablative mAb conditioning.

Optimization of a diagnostic algorithm for benign breast nodules

The results of examinations were studied in 76 reproductive-aged patients with benign breast nodules (BBNs). The determination of the leukocyte common antigen CD45 in the trephine biopsy specimens, as well as proteinase activity in the solid tumor homogenates and in the cyst puncture samples in the preoperative stage or the postpuncture period can identify groups of patients with BBNs in relation to a tissue inflammatory response. BBNs with a tissue inflammatory response showed increases in the level of nonspecific proteinases and in the expression of the leukocyte common antigen CD45, which was accompanied enhanced proliferative activity (Ki-67) and elevated levels of apoptosis proteins (p53 and Bcl-2), and estrogen and prolactin receptors.

Myeloid-derived suppressor cells (MDSC) facilitate distant metastasis of malignancies by shielding circulating tumor cells (CTC) from immune surveillance

The mechanisms of distant metastasis of malignancies largely remain unknown. Circulating tumor cells (CTC) derived from the primary cancer initiate distant metastasis by entering and traversing the bloodstream. Current methods to detect CTC are based on the notion that CTC do not express the common leukocyte antigen CD45. However, these methods neglect the fact that CTC can directly adhere to platelets and immune cells and therefore appear to be CD45-positive. The potential effects of interactions between CTC and adhesive immune cells have been largely overlooked, despite the fact that most CTC are killed by immune effector cells and only those that evade immune surveillance result in clonal expansion and metastatic lesions. It is crucial to define the characteristics that allow a select CTC population to escape immune surveillance; particularly, it must be determined whether interactions between CTC and adhesive immune cells provide a protective effect on CTC survival. If interactions between CTC and adhesive immune cells offer a selective advantage to those CTC cells, the next consideration is which characteristics of a CTC–immune cell population allow sufficient protection to facilitate immune evasion. Myeloid-derived suppressor cells (MDSC) are a large heterogeneous population of immature myeloid cells that accumulate during cancer progression to induce extensively systemic and local immunosuppression, a phenomenon that has been demonstrated to facilitate cancer distant metastasis. We hypothesize, therefore, that CTC populations interacting with adhesive immune cells will have different biological behavior than CTC populations alone. Further, we hypothesize that CTC can create a defensive shield consisting of adhesive MDSC, which allows evasion of immune surveillance and therefore facilitates distant metastatic lesions. This possibility highlights the importance of direct interactions between CTC and adhesive immune cells and suggests the potential target that the CTC–MDSC cluster represents for prevention and treatment of distant metastasis of malignancies.