Changes In Surface Antigens Research Articles

Abstract 1040: Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry

Abstract The detection of liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have demonstrated promise as non-invasive assays for monitoring response to therapy for patients with cancer. While ctDNA cannot predict transcriptomic and proteomic changes in response to therapy, we can measure direct cell surface antigen and gene expression changes in CTCs. In pediatric settings, blood samples are often collected from multi-institutional trials. Therefore, to translate liquid biopsies into routine practice, it is important to explore variables most likely to impact the functionality of CTCs including time to process the sample, prospective cryopreservation, blood collection tube and fixative reagents. Cell stabilizer tubes such as Streck Cell-Free DNA, CellSave, CellRescue, or PAXgene ccfDNA have been designed to prevent cell lysis and release of genomic DNA into the plasma, often observed in standard EDTA tubes. In pediatrics, we are often limited by the volume of blood we can draw for research. To this end, we have established an innovative pipeline to collect ctDNA, CTCs, and matched germline DNA from a single blood sample. Blood samples were processed for plasma extraction and CTC isolation after 4h, 24h, and 48h. We isolated rare CTCs from whole blood by combining peripheral blood cell depletion with antigen-based CTC identification. We then evaluated CTC recovery rate and cell membrane integrity (cell-membrane permeabilization) across all blood collection tubes by flow or mass cytometry. Additionally, we integrated whole transcriptome analysis of RNA transcripts in individually sorted CTCs. In this study, we detected similar levels of cell-free DNA, germline DNA and ctDNA in healthy donors and patient samples collected in the DNA stabilizer tubes up to 48h after blood collection. Surprisingly, frequent clotting was observed in the cell layer of samples collected in PAXgene tubes only 24h after blood collection, preventing further analysis of CTC quality in these tubes. At 4h, EDTA tubes and stabilizer tubes seem to perform similarly in preserving CTC integrity. However, at later timepoints, CTC recovery rate, RNA quality and cell membrane integrity were significantly higher in CellRescue tubes. Finally, we showed that CTC count, and RNA quality were slightly lower in patient samples enumerated after cryopreservation compared to the paired-samples processed the same day in both EDTA and cell-stabilizing tubes. In conclusion, we validated the CellRescue tubes as the best candidate to reduce cell lysis and protect against cell membrane permeabilization and mRNA degradation for up to 48 hours, allowing sample collection from multi-institutional studies. To the best of our knowledge, this study is the first to compare the key steps in handling blood samples and how this affects transcriptomic-based assay performance. Citation Format: Anne-Florence Blandin, Sarah Finstuen-Magro, Samuel Abbou, Kelly Klega, Alejandra E. Aguilar, Mohammad Tanhaemami, Maria Ana Isabel C. De Los Santos, Joadly Duplan, Brian Crompton. Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1040.

Inhibition and eradication of Pseudomonas aeruginosa biofilms by secondary metabolites of Nocardiopsis lucentensis EMB25.

Millions of people worldwide have been impacted by biofilm-associated disorders, which are impregnable owing to frequent changes in surface antigens and gene expression. Globally, about 11% of nosocomial infections, including cystic fibrosis, chronic wound infections, and post-surgical infections, are caused by Pseudomonas aeruginosa, the most prevalent Gram-negative bacterial species. Moreover, biofilms are highly resistant to the host's immune system, and exhibit increased tolerance to stress factors such as starvation, dehydration, and antimicrobials. Here, we have isolated a rare halophilic actinobacteria, Nocardiopsis lucentensis EMB25, and utilized the secondary metabolites for inhibition and eradication of P. aeruginosa biofilm. For the first time, N. lucentensis EMB25 bacteria was explored to study the anti-effect of secondary metabolites on pre-established biofilm. The secondary metabolites targeted the quorum sensing pathway and were found to bind to LasR and RhlR, as confirmed via molecular docking. Also, the reduction in virulence factors, rhamnolipids and pyocyanin further supported the study as these two are regulated by LasR and RhlR. In addition, the downregulation of various QS system genes lasA, lasB, rhlA, rhlB, and pqsA confirmed that the secondary metabolites act on two main regulators of the quorum sensing pathway, LasR, and RhlR. The findings of this study support the bioprospecting of previously unknown and extreme-condition actinobacteria as a rich source of novel bioactives against infections caused by bacterial biofilms.

Phytochemical screening, antibacterial, anti-biofilm and quorum sensing inhibiting activity of Alpinia nigra leaf extract against infectious pathogen Pseudomonas aeruginosa PAO1

The recurrent cycles of several bacterial diseases are an endorsement of the formation of biofilms that are intractable to antibiotic therapy. Biofilm-associated diseases have affected millions of people worldwide and are unassailable due to frequent changes of surface antigens and gene expression. Therefore, there is a need to find out an alternative approach to cure biofilm infections. Plant-derived bioactive compounds and their derivative have shown a significant role in combatting bacterial infection. In this scenario, we performed phytochemical screening of ethyl acetate leaf extract (L-EtAc) of Alpina nigra and antibacterial, anti-biofilm activity towards Pseudomonas aeruginosa PAO1. Antibacterial and growth curve analysis of bacterial cells confirmed the significant bacterial cell death in L-EtAc treated sample. Relatively less biofilm formation was observed in the treated sample as revealed through fluorescence microscopy. Further, quorum-sensing inhibition was observed by applying a similar concentration of L-EtAc on Chromobacterium violaceum bacteria. The interaction between the biofilm-forming protein and phytochemical constituent was validated through in-silico analysis and further confirmed with qRT-PCR. The qRT-PCR data revealed reduced lasI, lasR, lasB, rhlI, rhlR and rhlA gene expression, which may be directly associated with biofilm and extracellular matrix formation. This study might be helpful for the development of new natural product for curing biofilm-associated disease.

Abstract 3146: Heterogeneity of the antigen-expression pattern in primary and recurrent glioblastoma patients

Abstract Introduction: Immunotherapy leads to promising results in the treatment of glioblastoma (GBM) patients. Recent clinical trials revealed a survival benefit of patients treated with monospecific CAR-T-cells. However, antigen escape mechanisms due to heterogeneous surface expression of target antigens still constitute a critical hurdle in the immunotherapeutic treatment of GBM and are a major reason for non-response or relapse. Therefore, we aim to characterize GBM's heterogeneity, the intratumoral distribution and changes of surface antigens in relapse. Methods: We performed immunohistochemical staining of the ten in vitro and in vivo most targeted antigens in CAR-T cell therapy (HER2, EGFRvIII, CD70, B7H3, Il13Rα2, NY-ESO1, GD2, CD133, CSPG4 and EphA2). We analyzed 35 patients, who underwent surgery for primary (PT) as well as recurrent GBM (RL). To address intrapersonal heterogeneity, we examined five fields of view per slide. As controls, we stained cerebral, cerebellar and brain stem slides of three healthy specimens. We quantified the percentage of low, medium and high antigen expressing cells on each slide by applying a specialized macro in ImageJ. We further weighted this expression score by multiplication with 1 (low), 2 (medium) or 3 (high expression). Finally, we calculated the median expression values of all tumors combined for each antigen. Summary: Median values of EGFRvIII (PT: 0.14; RL: 0.73), HER2 (PT: 0.67; RL: 4.00) and NY-ESO1 (PT: 0.00; RL: 0.16) expression were very low. Antigen expression of CSPG4 (PT: 4.67; RL: 2.78), Il13Rα2 (PT: 3.84; RL: 2.78), GD2 (PT: 7.50; RL: 27.03) and B7H3 (PT: 8.81; RL: 16.62) was mild. CD133 (PT: 52.16; RL: 56.13), CD70 (PT: 19.18; RL: 25.02) and EphA2 (PT: 60.65; RL: 55.46) showed moderate antigen expression. None of the evaluated antigens displayed high expression. Median values of HER2 (p=0.005, Cohen's r=0.48), EGFRvIII (p=0.042, Cohen's r=0.344), and GD2 (p=0.013, Cohen's r=0.42) were significantly increased in RL compared to PT. We found significant intrapersonal expression differences for each antigen. However, none of the healthy brain tissue specimens expressed any of the ten antigens. Conclusions: Inter- and intrapersonal heterogeneity constitutes a major obstacle for the implementation of GBM immune therapies which avoid antigen escape mechanisms. Citation Format: Vera Dufner, Moritz Meyer-Hofmann, Jonas Feldheim, Katja Maurus, Camelia Monoranu, Thomas Nerreter, Michael Hudecek, Carsten Hagemann, Ralf-Ingo Ernestus, Mario Löhr. Heterogeneity of the antigen-expression pattern in primary and recurrent glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3146.

Prolonged Azacitidine Exposure Alters Immunophenotype in Myeloid Leukemia Cell Lines

Hypomethylating agents (HMA), namely azacitidine and decitabine, are used for treatment of MDS and AML. Although HMAs have demonstrated efficacy in a subset of patients, their mechanism of action is not well established. Some studies indicate that HMAs induce expression of specific target proteins, thus enhancing the efficacy of targeted therapies. For example, studies of HMAs used in combination with gemtuzumab ozogamicin (GO) suggest that they can apply additional epigenetic stress to AML cells and increase surface CD33 expression, thereby increasing the efficacy of GO. Despite suggestions of epigenetic modulation of target antigens, data on comprehensive response to HMAs is lacking. We proposed to evaluate the full extent of surface antigen expression in different AML cell lines after prolonged exposure to azacitidine.The dose and duration of azacitidine therapy used for prolonged exposure was established in Kasumi-1 cells (t(8;21), N822K KIT mutation). A daily dose of 250 nM allowed continued cellular proliferation for 18 days. We then treated two cell lines, Kasumi-1 and MV4-11 (t(4;11), FLT3 -ITD+) as above for 14-18 days. Cells were submitted for multi-dimensional flow cytometry (MDF) every 2-4 days.Prolonged azacitidine exposure resulted in broad changes in hematopoietic cell surface antigens, as demonstrated by MDF analysis. CD33, the premier cell surface target for AML immunotherapy, increased significantly in Kasumi-1 cells, approximately doubling by day 14 of treatment (p=0.003, Fig. 1). C -kit, or CD117, which is overexpressed in Kasumi-1 cells and is a potential target of tyrosine-kinase inhibitor (TKI) therapy in t(8;21) AML, was significantly decreased with azacitidine exposure (p <0.0001, Fig. 1). In contrast, treatment of MV4-11 cells resulted in significant decrease in CD33 with a corresponding increase in CD117 (Fig. 1).Kasumi-1 and MV4-11 both exhibited significant increases in surface CD38, CD13, and CD36, and significant decreases in CD64, CD123, and CD11b. Cell marker changes observed in only one cell line include significantly increased CD34 in Kasumi-1 and significantly increased CD56 in MV4-11. Many of the observed changes peaked after day 10 of treatment. No significant changes were seen in either line in CD7, CD14, CD16, CD19, CD45, and HLA-DR. Notably, the shared pattern of surface antigen changes in both Kasumi-1 and MV4-11 indicates that prolonged azacitidine exposure may induce a less mature myeloid phenotype, as typified by increased CD38 and decreased CD11b and CD123 (Fig. 1).The results above were validated in an experiment in which 4 leukemia cell lines were exposed to azacitidine as above for 10 days, with MDF analyses on days 5 and 10. Cell lines included Kasumi-1, MV4-11, K562 (t(9;22)), and the ALL cell line REH (t(12;21)). Similar trends in surface antigens for Kasumi-1 and MV4-11 were again observed. K562 cells exhibited increased CD38, CD36, CD7, CD14, and CD19, and decreased CD33, CD56, CD64, and CD45 (Fig. 1). No change was seen in K562 in CD117, CD13, CD11b, CD16, CD34, CD123, and HLA-DR (Fig. 1). Of these myeloid markers tested, REH cells had only a significant increase in CD13, a marker also seen in very early B-lymphocytes. Thus, HMAs do not appear to promote lymphoid to myeloid phenotype shift in REH.We observed broad changes in cell surface antigen expression in myeloid leukemia cell lines treated with prolonged azacitidine, which may signify changes in gene expression induced by hypomethylation. Among AML cell lines, prolonged azacitidine exposure resulted in a less mature immunophenotype. We observed significant changes in CD33 and CD117, both of which are considered potential therapeutic targets in AML. CD33 expression was increased by azacitidine exposure in Kasumi-1 cells, and reduced in MV4-11 and K562. C -kit was also noted to variably change in response to azacitidine. Downregulation of cell surface antigens is a known resistance mechanism to immunotherapeutic agents, including antibody-drug conjugates such as GO. Resistance to c -kit-targeted agents has been proposed to occur through a variety of mechanisms, including cell surface expression, therefore changes to c -kit levels induced by HMAs may be an important avenue of investigation in averting TKI resistance. The use of prolonged, low-dose hypomethylating agents warrants further investigation as a potential means to modulate surface protein levels for therapeutic benefit. [Display omitted] DisclosuresPardo:Hematologics Inc: Employment. Loken:Hematologics Inc: Employment, Equity Ownership. Brodersen:Hematologics Inc: Employment.

Conservation of Myeloid Surface Antigens on Primate Granulocytes

Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

Comparison of HBsAg changes between HBeAg-negative patients who discontinued or maintained entecavir therapy.

The study compared the hepatitis B surface antigen (HBsAg) changes between hepatitis B e antigen (HBeAg)-negative non-cirrhotic chronic hepatitis B patients who discontinued or maintained entecavir therapy. A total of 250 HBeAg-negative, non-cirrhotic patients who were treated with entecavir previously and had stopped treatment for at least 12months (discontinued group) and 231 HBeAg-negative, non-cirrhotic patients who had received entecavir treatment for at least 4years (maintained group) were recruited. In the discontinued group, 71 had a persistent virological suppression (Group I), 35 experienced virological relapse but no clinical relapse or retreatment (Group II), 26 experienced clinical relapse without retreatment (Group III), and 118 experienced HBV relapse and retreatment (Group IV). Patients in Groups I, II, and III, but not in Group IV, experienced a significantly larger drop in HBsAg levels' post-treatment than during entecavir treatment. Patients in Groups I and III exhibited a greater post-treatment HBsAg decline than patients in Groups II and IV (p < 0.001). Discontinued group experienced a significantly larger drop in HBsAg levels (p < 0.001) and higher HBsAg loss rate (p = 0.001) than maintained group after adjusting for clinical features and HBsAg levels in propensity score matched patients. Patients in maintained group exhibited a smaller drop in HBsAg and lower HBsAg loss rate than patients in groups I, II, and III, but not in Group IV. Patients who discontinued entecavir therapy and achieved persistent virological suppression exhibited a greater HBsAg decline and higher HBsAg loss rate compared with patients who maintained entecavir therapy.

Lower platelet count with increased density of platelet antigens in granulocyte colony-stimulating factor mobilized peripheral blood stem cell donors

Granulocyte colony-stimulating factor (G-CSF) is widely used for prophylaxis and treatment of neutropenia in cancer patients and also for peripheral blood stem cells (PBSC) mobilization. The aim of this study is to evaluate the possible changes of platelet surface antigens after G-CSF injection in PBSC donors compared with healthy control. Between January 1st and December 31st, 2014, 48 healthy voluntary PBSC donors were eligible for this study. Donors received G-CSF (Filgrastim) subcutaneously for five days, and then their whole blood was collected for complete blood count. Analysis of platelet antigens was performed by flow cytometry. Sixteen healthy controls were also included for comparison. Lower platelet counts were found in PBSC donors after G-CSF use and in comparison with health controls. The platelet size evaluated by forward scattering (FSC) showed smaller platelets in PBSC donors after G-CSF use compared with healthy controls (39.3 vs 46.7 mean fluorescence intensity, P=0.015). CD31 were higher in PBSC donor (203.2 vs. 120.7, P<0.001). Except CD31, other platelet surface antigens were not different between PBSC donors and healthy controls. After adjusting by FSC data, the mean antigen intensity/FSC of CD31, CD41a, CD42a, CD42b and CD61 showed 5.45 vs 2.78 (P<0.001), 4.35 vs 3.47 (P=0.007), 3.87 vs 3.17 (P=0.015), 20.45 vs 16.94 (P=0.045), and 5.98 vs 4.88 (P=0.018) respectively. We noted higher density of platelet surface antigens, lower platelet count and smaller platelet size after G-CSF injection.

Hyperosmotic Condition Reduces Nucleus Pulposus Growth in Monolayer Culture and 3D Alginate Beads Culture

Introduction The intervertebral disc (IVD) is a specialized structure composed of mainly collagens and proteoglycans to hold water and to accommodate applied mechanical forces. The high glycosaminoglycans side chains of the aggrecan raise the osmolarity of the nucleus pulposus, therefore, nucleus pulposus cells (NPC) are under hyperosmotic condition inside the disc. Recent studies raised contradictory results in the NPC responses to isosmotic and hyperosmotic culture conditions, while one proposed hyperosmotic culture condition could preserve the matrix production activity of NPC. However, another study suggested that a hyperosmotic condition reduced NPC viability and matrix production. This study aimed to monitor cell growth and changes of the NPC phenotype in isosmotic and hyperosmotic culture condition by monitoring a selection of their important NPC surface antigens by flow cytometry. Material and Methods Five human NPC were isolated from human nucleus pulposus tissue (aged 54–65 years) by enzymatic digestion with hyaluronidase and liberase (Roche). Primary cells were expanded in monolayer culture to passage 1 and then seeded at 10,000 cells per 6—well in duplicates and cultured in—MEM containing 10% FSC either in isosmotic medium (300 Osm/L), or in hyperosmotic medium (400 Osm/L) adjusted with 1% 5 M NaCl and 0.4 M KCl. Cells were trypsinized after 7 days culture and stained with the following three NPC surface markers: antihuman disialoganglioside GD2 (GD2) (BD Pharmingen; 14; G2a) mAb and FITC-conjugated antimouse Ig goat (BD Biosciences), allophycocyanin-conjugated antihuman Tie2 (R&amp;D Systems, clone 83,715) mAb and PE-conjugated antihuman CD24 (BD Biosciences; clone ML5) mAb for flow cytometry characterization for percentage of cells expressing NPC surface markers by FACS Calibur. Cumulative population doubling and changes of NPC cell surface antigens were monitored at days 0, 7, 14, 21 and 28. To culture the cells in alginate beads, NPC were mixed with 1.2% alginate solution in a density of 10,000 cells/mL. Alginate beads were formed by adding the alginate solution into 102 mM CaCl2 solution through a 23G needle. Beads were cultured in isosmotic or hyperosmotic medium as described earlier. After 28 days culture, cells were released from the alginate by incubating in 55 mM sodium citrate, 0.9% NaCl2 for 10 minutes followed by trypsinization for 10 minutes. Two-way ANOVA analysis was performed to analyze the difference between isosmotic and hyperosmotic culture conditions. Results Cumulative population doubling time indicated that NP cell growth was inhibited in hyperosmotic condition over the 28-day culture, which was 50% lower in hyperosmotic condition than isosmotic condition. Hyperosmotic medium reduced NPC growth even stronger in 3D alginate beads culture. Total cell number recovered from the beads was four times reduced in hyperosmotic medium (71,000 ± 48,432) than in isosmotic medium (15,000 ± 7,906).

The roles of membrane surface antigens on neutrophil during sepsis

Background Surface antigens on neutrophil may be associated with the occurrence,development and deterioration of inflammation.Objective To find out the underlying biological effects and significance of surface antigens on neutrophil.Content Research progress of changes in surface antigens related to adhesion,chemotaxis and phagocytosis of neutrophil.Trend Further studies on mechanism underlying changes in surface antigen on neutrophil would give insight into diagnosis and treatment for patients with sepsis. Key words: Sepsis; Neutrophil; CD16; CD64; CXCR2; CCR2; CD11a; CD54; CD62L

CD5-Positive Diffuse Large B Cell Lymphoma Infiltrating the Central Nervous System Presenting Guillain-Barr^|^eacute;-Like Syndrome after Chemotherapy

An 83-year-old woman was admitted to our hospital with abdominal pain. Examination revealed mediastinal lymphoadenopathy, hepatosplenomegaly, and infiltration of abnormal cells into the bone marrow with hemophagocytosis, and CD5-positive diffuse large B cell lymphoma was diagnosed. Chemotherapy was administered and progressive weakness of the limbs, resembling a Guillain-Barré-like syndrome, subsequently appeared. Cerebrospinal fluid examination indicated lymphoma cell infiltration. Although immune globulin and steroid therapies were not effective, intrathecal injection of methotrexate, predonisolone, and cytarabine improved these symptoms. Subsequent to chemotherapy, cell surface antigen changes were observed in the cerebrospinal fluid relative to those in bone marrow.

Trichobilharzia regenti: Antigenic structures of intravertebrate stages

Abstract Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.

Age-related changes in surface antigens on peripheral lymphocytes of healthy children.

The age-related changes in proportion of various subsets within lymphocytes were investigated in cord blood and peripheral blood from healthy children and adults. The percentages of T and B cells did not show age-related changes, whereas natural killer (NK) cells increased significantly with age. Within lymphocytes or the CD3+ T cell population the proportion of CD45RAbright+ lymphocytes decreased and that of CD45RO+ cells increased, while that of CD45RAdim+ cells showed no age-related change. Within lymphocytes, the percentage of CD45RAbright+ CD4+ cells decreased, together with a decline of that of CD4+ cells. The proportions of CD45RAbright+ CD8+ cells and S6F1bright+ CD8+ cells increased with age, and the age-dependent increase of the proportion of CD8+ cells seems to be mainly attributable to the increases in these subsets. The CD45RAdim+ CD4+ and CD45RAdim+ CD8+ cells co-expressing CD45RO at a low level nevertheless showed no age-related changes. In gamma delta T cells, both delta TCS1+ and delta TCS1- T cells increased with age, but the delta TCS1- gamma delta T cells increased more than the delta TCS1+ subset. Among lymphocytes, the percentages of CD20+, CD21+ and CD22+ cells remained similar, with no age-related changes, but the proportion of CD5+ cells within lymphocytes or B cells decreased. The proportions of CD16+ NK cells among lymphocytes increased with age, and this change was attributable to the increase of CD56+ cells.

Expression of the surface antigens of lymphocytes and the levels of cytokines in mice infected with Aspergillus fumigatus

Aspergillus fumigatus (A. fumigatus) is an opportunistic fungus that causes invasive aspergillosis. Determining the immune changes during A. fumigatus infection and the factors leading to such changes clearly will make it possible to prevent the spread of the infection and to provide new strategies in the treatment of infection. Thus, the present study aims at determining the changes of lymphocyte surface antigens which develop during A. fumigatus infection and the role of cytokines in immune response. The expression of the surface antigens of lymphocytes was analysed by flow cytometry and the cytokine levels were determined by ELISA in a mouse model of aspergillosis. It was observed that in mice infected by A. fumigatus the percentage of CD19+ B cells and the levels of IL-4 and IL-10 increased when compared to those in noninfected mice cells. These results suggest that Th2 type cytokines are important in the pathogenesis of A. fumigatus infection. Humoral immunity is considered to be effective during A. fumigatus infection because of the increase in Th2 type response.

Coculture of Pediatric Acute Lymphoblastic Leukemia (ALL) Blasts with CD40 Ligand Transfected Cells Leads to Changes of Surface Antigens and RNA Expression.

Although multi-agent chemotherapy is remarkably successful in the treatment of pediatric acute lymphoblastic leukemia (ALL), about 25% of patients experience relapse. A possible way to increase the cure rate for leukemia could be to modulate the immune response against leukemic cells.It is known that ALL blasts are poor stimulators of cellular immune responses. This is believed to be mainly the result of a reduced presentation of costimulatory molecules on the surface of the ALL blasts.For B cells, CD40 is an essential molecule which can be targeted therapeutically through its ligand, CD40 ligand or CD152. Normal B cells can induce cytotoxic T cell responses following stimulation with a soluble trimeric CD40 ligand.We therefore hypothesized that by coculture with CD40 ligand transfected HeLa cells for four days, pediatric ALL blasts could be induced to upregulate costimulatory molecules, apoptosis inducing ligands and cytokines. The expression of the surface antigens CD40, CD40 ligand, CD80, CD86, CD95/fas, HLA-ABC and HLA-DR was evaluated by flow cytometry and the RNA expression of CD40, CD80, CD86, Fas ligand (FasL), TRAIL, IL-4, IL-6, IL-10, TGF-β was assessed by semiquantitative RT-PCR.We found that coculture with CD40 ligand transfected HeLa cells significantly increased the expression of CD40 ligand, CD80, CD86 and CD95/fas while the expression of HLA-ABC, HLA-DR and CD40 remained unchanged. We found a significantly increased RNA expression of IL-6, TGF-β and CD80, a small but not significant increase of CD86, but no change in expression for IL-4, IL-10, FasL, TRAIL and CD40 after coculture. Addition of IL-4 to the culture did not influence the results.In summary CD40 ligand mediated stimulation lead to increased expression of costimulatory molecules CD80 and CD86 and the pro-apoptotic CD95/fas, while the anti-apoptotic cytokines IL-6 and TGF-β were also upregulated.Our results show that stimulation through CD40 ligation upregulates costimulatory molecules on pediatric ALL blasts, while it has differential effects on apoptosis inducers. This might have functional consequences which could be therapeutically exploited to modulate the immune system in an attempt to increase cure rates in pediatric ALL.

Flow cytometric study of neutrophilic granulopoiesis in normal bone marrow using an expanded panel of antibodies: correlation with morphologic assessments.

Flow cytometry studies of surface markers of neutrophils have been performed mostly on peripheral blood, and for a limited spectrum of diseases. Studying maturation defects on developing neutrophils in the bone marrow (BM) may be helpful in BM diseases, such as myelodysplastic syndromes and Shwachman-Diamond syndrome. We applied an expanded panel of antibodies to examine normal maturation patterns in 26 control samples of BM together with microscopic correlation. Promyelocytes correlated well with the CD24(-) and CD11b(-) populations, and metamyelocytes correlated well with the CD16(+) population (intermediate positivity). An excellent correlation was also identified between the sum of bands and segmented neutrophils and each of the following: CD16(++) (strong positivity), CD35(+), CD87(+), and CD64(-). Although visually identified segmented neutrophils paralleled CD10 positivity, there was an appreciable difference between both methods. We conclude that neutrophilic granulocyte maturation in the BM is accompanied by a change in surface antigens that reflects certain stages of development. A successful strategy for detecting maturation defects is to include several antibodies that are known to be expressed or absent at the same stage of maturation, such as CD16, CD35, CD64, and CD87.

Surface Antigen Changes during Normal Neutrophilic Development: A Critical Review

ABSTRACTNeutrophil surface molecules function in part as biological sensors. Surface antigens undergo several changes during neutrophilic maturation to accommodate the cell's function. Surface antigens may appear with neutrophilic maturation, such as CD16b, CD35, and CD10; disappear with maturation, such as CD49d and CD64; be maintained during maturation, such as CD32, CD59, and CD82; or disappear with maturation but reappear after neutrophilic extravasation, such as CD49b. This article reviews the alterations in surface antigen expression during normal neutrophilic granulopoiesis.

Initial characterization of the Streptococcus gordonii htpX gene.

Examination of the Streptococcus gordonii chromosomal region, which lies immediately upstream of the glucosyltransferase positive regulatory determinant rgg, revealed two open reading frames. Based on nucleotide sequences, these genes were similar to the Listeria monocytogenes lemA gene, which is involved in antigen presentation, and the Escherichia coli htpX heat shock gene, which has an unknown function. Northern hybridization analysis indicated that S. gordonii lemA and htpX genes were associated with a ca. 1.7-kb polycistronic transcript. Although levels of the lemA/htpX transcript did not increase in response to heat to levels seen with dnaK controls, insertional inactivation of htpX resulted in changes in adhesiveness, cellular morphology and detergent-extractable surface antigens in cells grown at 41 degrees C, implying that htpX may be involved in surface protein expression. Insertional inactivation of lemA and htpX indicated that, despite their proximity to rgg and the structural gene, gtfG, these upstream genes do not affect S. gordonii glucosyltransferase activity.

Maturational Changes of Lymphocyte Surface Antigens in Human Blood: Comparison between Fetuses, Neonates and Adults

The objective of this study was to investigate the maturational changes of lymphocyte surface antigens during ontogeny from fetuses to adults using the proven whole blood lysis technique. Two-color flow cytometric analysis of lymphocyte surface markers was performed on 20 fetal blood samples obtained by cordocentesis, 70 cord blood and 30 adult blood samples. The leukocyte count and all T-cell subsets were highest in neonates compared to fetuses and declined into adulthood. In contrast, the percentage of CD2+ T cells (fetal blood 57% versus adult blood 82%; p ≤ 0.001), CD3+ T cells (fetal blood 52% versus adult blood 77%; p ≤ 0.001), CD4+ T cells (fetal blood 39% versus adult blood 50%; p ≤ 0.05) and CD8+ T cells (fetal blood 15% versus adult blood 24%; p ≤ 0.001) showed a significant increase from fetuses to adults. The absolute and relative amounts of B cells were highest in fetuses (fetal blood 547 × 10⁶/l, 18%) followed by a steady decline to adulthood (adult blood 243 × 10⁶/l, 13%; p ≤ 0.001). The discordance between the absolute and relative size of lymphocyte subpopulations emphasizes the consideration of both variables in the assessment of lymphocyte maturation. The presented data may contribute to a better knowledge of the maturation of the immune system.

Changes of Platelet Surface Antigens in Patients Suffering from Abdominal Septic Shock

Sepsis and related syndromes account for a high morbidity and mortality caused by the development of multiorgan failure. Pathogenesis of sepsis is complex, involving humoral as well as cellular factors. Since the role of platelets is still undefined in this concern, we investigated CD63, CD62P, CD36, and CD31 expression on platelets of patients in septic shock ( n=18) using a flow cytometric assay in whole blood. Samples were drawn within 24 hours of onset. We found thrombocytopenia accompanied by a significantly higher expression of CD63, CD62P, and CD31 and a significant downregulation of CD36 in comparison to healthy volunteers ( n=18). Changes in CD63 and CD62P expression indicates platelet activation. Because CD62P, CD36, and CD31 mediate interaction of platelets with leukocytes, subendothelial matrix and probably endothelial cells as well as platelet adhesion/aggregation, our findings suggest an involvement of platelets in leukocyte/endothelial cell interaction in septic shock. We suspect that thrombocytopenia is not due to bone marrow depression, but rather is due to consumption of highly activated platelets in the microcirculation. We feel that our observations may offer a rationale for potentially beneficial effects of antiplatelet therapy in sepsis; however, further studies have to evaluate its beneficial impact as well as its potential risk for bleeding complications.