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Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated K D values of 0.5 to 0.7 μM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Shi et al.)

CD34 cells in the interstitial infiltrates in glomerulonephritis (GN) could be the turning point between regenerative processes and interstitial fibrosis. The aim of our study was to assess the presence of CD34+ cells in the interstitial infiltrates in GN. A cross-sectional study of 33 patients with glomerulonephritis, mean age: 43.3 ±11.31 years, 20 male and 13 female, was conducted. Conventional stains, as well as immunohistochemistry for the CD34 antigen were employed on kidney biopsies. Strength of immunohistochemical reaction was assessed semi-quantitatively. Regarding the percentage of cases with CD34+ cells in the interstitial infiltrates out of 33 patients: cells of interstitial infiltrates were 27.3% positive. The percentage of cases showing CD34+ cells at the level of interstitial infiltrates was: 44.4% in FSGS, 14.3% in membranoproliferative GN, 28.6% in membranous nephropathy, 20% in mesangial proliferative GN, 0% in minimal change disease, and 50% in crescentic GN. With the exception of minimal change disease, CD34+ cells were found in the interstitial infiltrates in all histopathological forms of GN. Some of these cells were spindle-shaped fibroblast-like cells. As inflammation in the tubulointerstitial compartment either resolves or proceeds to fibrosis, aims at reversing this process will benefit from analyses of the interstitial infiltrates harboring CD34+ cells.

Introduction and Aims: The HLA-DR antigen is a HLA class II molecule involved in the presentation of antigenic peptides to the T cell receptor, thus regulating the immune response. Renal expression of the HLA-DR antigen may indicate specific sites of immunologically-mediated kidney injury in glomerulonephritis (GN). The aim of our study was to assess the presence of the HLA-DR antigen along the nephron including the extraglomerular mesangium in GN., Methods: A cross-sectional study of 22 patients with glomerulonephritis, mean age: 46.59±10.77 years, 14 male and 8 female, was conducted. Conventional stains, as well as immunohistochemistry for the HLA-DR Antigen Alpha-Chain were employed on kidney biopsies. Immunohistochemistry was assessed using a semi-quantitative score: 0-absent, 1-mild, 2-moderate, 3-intense. Statistical analysis was performed using SPSS17., Results: Four patients presented Focal and Segmental Glomerulosclerosis (FSGS), 5 patients: membranoproliferative GN, 7 patients: membranous nephropathy, 3 patients: mesangial proliferative GN, 2 patients: minimal change disease (MCD), and 1 patient: crescentic GN. Regarding the percentage of cases with HLA-DR positive cells along the nephron out of 22 patients: glomerular endothelial cells were 100% positive, intraglomerular mesangium cells were 81.8% positive, podocytes were 36.4% positive, extraglomerular mesangium cells were 31.8% positive, proximal tubule cells were 95.5% positive, distal tubule cells were 68.2% positive, interstitial capillaries were 77.3% positive, and cells of interstitial infiltrates were 27.3% positive. The percentage of cases staining positively for the HLA-DR antigen in the extraglomerular mesangium was 25% in FSGS, 60% in membranoproliferative GN, 0% in membranous nephropathy, 33.3% in mesangial proliferative GN, 100% in minimal change disease and 0% in crescentic GN., Conclusions: A prominent HLA-DR antigen distribution was found on glomerular endothelial cells, intraglomerular mesangium cells and proximal and distal tubular cells. Extraglomerular mesangium cells and podocytes stained variably for the HLA-DR antigen, as did the cells of the interstitial infiltrates. The extraglomerular mesangium which serves as a portal of entry into the intraglomerular mesangium is endowed with antigen-presenting capabilities and is a region where induction of immune reactions could take place., (Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Introduction and Aims: N-Acetyl-β-D-glucosaminidase (NAG), a marker of renal tubular dysfunction, is increased in patients with lupus nephritis. In addition to the toxic effects of proteinuria, patients with lupus nephritis may exhibit other factors that contribute to tubular dysfunction, such as pathogenic antitubular basement membrane antibodies. The aim of our study was to assess urinary NAG, proteinuria, and glomerular filtration rate (GFR) before treatment and after 7 and 30 days of oral prednisone therapy in patients with lupus nephritis., Methods: Ten patients with lupus nephritis, all females, mean age: 29.4 ± 10.17 years, were enrolled into the study. All the patients received oral prednisone 1 mg/kg. Twenty healthy subjects served as controls. We measured urinary NAG before treatment and after 7 and 30 days of oral prednisone therapy. Proteinuria, GFR, blood pressure, and side effects of therapy were also followed up. Urinary NAG was measured using the colorimetrical method and expressed as units per gram of creatinine (U/gCr). Statistical analysis (Wilcoxon signed ranks test and Wilcoxon rank sum test) was performed using SPSS 17., Results: In the 10 patients with lupus nephritis, urinary NAG before treatment was 16.9 ± 13.39 U/gCr (P = 0.005 compared with controls). NAG in controls was 1.73 ± 0.51 U/gCr. Proteinuria before treatment was 3.84 ± 1.93 g/24 h. The GFR before treatment was 50.48 ± 11.98 mL/min/1.73 m². After 7 days of prednisone, urinary NAG was 23.55 ± 25.25 U/gCr (P = 0.878 compared with baseline, and P = 0.02 compared with controls). Proteinuria was 2.94 ± 1.3 g/24 h (P = 0.005 compared with baseline), and the GFR was 58.11 ± 13.64 mL/min/1.73 m² (P = 0.005 compared with baseline). After 30 days of prednisone, urinary NAG was 11.77 ± 12.18 U/gCr (P = 0.203 compared with baseline, P = 0.022 compared with the value after 7 days of prednisone, and P = 0.01 compared with controls). Proteinuria was 1.73 ± 0.68 g/24 h (P = 0.005 compared with baseline, and P = 0.005 compared with the value after 7 days of prednisone), and the GFR was 67.49 ± 16.42 mL/min/1.73 m² (P = 0.005 compared with baseline and P = 0.009 compared with the value after 7 days of prednisone). Blood pressure measurements did not show any significant changes. No major side effects of steroid therapy were noticed., Conclusions: Urinary NAG showed a significant reduction between 7 and 30 days of therapy. The reduction in urinary NAG set in later than the decline in proteinuria and the improvement in GFR. Further studies incorporating a longer follow-up are needed to observe whether the reduction in NAG persists upon continuation of prednisone therapy.

Introduction: HBV and HCV chronic hepatitis can be accompanied by secondary renal disease. In addition, these patients receive antiviral drugs with potential nephrotoxicity. It is known that interferon (IFN) therapy in HCV-infected kidney transplant recipients is followed by rejection of the transplant in 50% of the cases. Ribavirin is contraindicated in hemodialyzed patients and in patients with a GFR <50 ml/min/1.73 m(2). IFN therapy requires dosage reduction and close monitoring in patients with a GFR <50 ml/min/1.73 m(2) and in patients with end stage renal disease. The aim of our study was to assess the nephrotoxicity of antiviral drugs in patients with chronic hepatitis by measuring three renal biomarkers: urinary albumin, N-acetyl-β-D-glucosaminidase (NAG) and α 1-microglobulin, as well as glomerular filtration rate (GFR-MDRD4) before and at 6 months of therapy., Methods: Fifty-five patients (28 male and 27 female, with a mean age of 47.85 ± 12.03 years) with chronic hepatitis (40 patients with HCV, 13 patients with HBV, 1 patient with HBV+HCV, and 1 patient with HBV+HDV) were enrolled into the study. Different antiviral drug associations were used on a case-by-case basis. The 40 patients with HCV chronic hepatitis received either Peg-IFN-α 2a+Ribavirin (37 patients) or Peg-IFN-α 2b+Ribavirin (3 patients). The 13 patients with HBV chronic hepatitis received Peg-IFN-α 2a (9 patients), Lamivudine (2 patients), Entecavir (1 patient), or Adefovir (1 patient). The patient with HBV+HCV chronic hepatitis received Peg-IFN-α 2a+Ribavirin. The patient with HBV+HDV chronic hepatitis received IFN-α 2a. Urinary albumin (ELISA), NAG (colorimetrical method), α 1-microglobulin (ELISA), and serum creatinine were measured before and at 6 months of antiviral therapy. Urinary markers were expressed as either mg/gCr (for albumin and α 1-microglobulin) or U/gCr (for NAG). Statistical analysis (Pearson's correlation coefficient, paired t-test and χ(2)-test) was performed., Results: At 6 months of therapy urinary albumin/gCr did not increase significantly: 16.58 ± 23.39 vs. 15.85 ± 24.96 mg/gCr before therapy, p = 0.87. Urinary NAG/gCr did not increase significantly: 4.21 ± 3.37 vs. 3.83 ± 3.2 U/gCr before therapy, p = 0.53. Urinary α 1-microglobulin/gCr was almost unchanged: 4.38 ± 4.47 vs. 4.38 ± 3.57 mg/gCr before therapy, p = 0.99. The GFR did not decline significantly: 92.41 ± 22.21 vs. 94.59 ± 36.1 ml/min/1.73 m(2) before therapy, p = 0.7. Ten patients (18.18%) were albuminuric before therapy, and 14 patients (25.45%) were albuminuric at 6 months of therapy, a non-significant increase (p = 0.35). We found a correlation between urinary albumin/gCr and NAG/gCr and between urinary albumin/gCr and α 1-microglobulin/gCr both at baseline and at 6 months of therapy: r = 0.54, p = 0.0005; r = 0.29, p = 0.03; r = 0.51, p = 0.0005; and r = 0.4, p = 0.002, respectively. In the patient receiving Adefovir, a known nephrotoxic drug, two of the three biomarkers (urinary albumin/gCr and NAG/gCr) increased, most notably NAG/gCr. Both HCV and HBV chronic hepatitis therapy were associated with non-significant changes in renal biomarker excretion and GFR., Conclusions: With the exception of Adefovir, all of the drug associations used in this study were safe.

Introduction: Because dysfunction of the B-cell compartment is thought to be important in the pathogenesis of systemic lupus erythematosus (SLE), there has been a recent focus on therapies that target humoral immunity via multiple mechanisms. The aim of this paper was to demonstrate the importance of immunomonitoring in two cases with class II lupus nephritis on steroids who presented with a flare-up of disease. After a thorough work-up for infectious triggers of disease activity, conversion to another histopathological class of lupus nephritis was suspected. Deterioration of the patients' clinical condition made kidney biopsy impossible, and as B-cell targeted therapy was considered, we decided to perform an immunophenotypic analysis and to tailor therapy to the results of the lymphocyte profile. As we incidentally found extremely low B-cell counts, any B-cell-targeted therapy was prohibited, and cyclophosphamide (Cy) was considered a viable therapeutic option., Methods: We performed flow-cytometric lymphocyte (Ly) phenotyping (CD19, CD3, CD3CD4, CD3CD8, CD56/16) on two patients with class II lupus nephritis before and after two intravenous (i.v.) Cy pulse administrations. During all this time, patients were on steroids., Results:  Both patients showed extreme B-cell lymphopenia, a marker of active SLE, which was not greatly impacted by the treatment over the follow-up period., Conclusions: As current therapies are aimed at targeting the B cell, an important component of adaptive immunity, caution must be exercised before their use. In addition, monitoring of Ly subsets is essential due to the occurrence of extreme B-cell lymphopenia.

Endothelial cells (ECs) are active participants of an inflammatory process in glomeruli. EC damage has been shown to play an important role in the progression of glomerulonephritis (GN). The degree of glomerular and peritubular capillary loss in models of progressive renal disease correlates with the severity of glomerulosclerosis and interstitial fibrosis. The aim of our study was to analyze the association of vWF, CD31 and CD34 immunoreactivity with the morphological indices of glomerular sclerosis, interstitial fibrosis, activity and chronicity in GN. A cross-sectional study of 22 patients with GN was conducted. Conventional stains (hematoxylin-eosin, periodic acid Schiff and Trichrome Gömöri stains) and immunohistochemistry (vWF, CD31 and CD34) were employed on kidney biopsies. Activity and chronicity of GN, as well as glomerular segmental sclerosis and interstitial fibrosis, were evaluated according to a scoring system initially used for lupus nephritis and antineutrophil-cytoplasmic-antibody-associated vasculitis. Immunohistochemistry was assessed using a semi-quantitative score. Statistical analysis was performed using EpiInfo 6.04. The mean patient age was 46.68+/-14.09; 14 patients were male, and eight were female. Performing Spearman's rank correlation test, no correlation was found between each marker and glomerular segmental sclerosis, interstitial fibrosis, activity and chronicity, which suggests a loss of these markers and microvasculature involvement.

Purpose: To present the study regarding Ig E tears dosation in cases with allergic conjunctivitis., Method: The Ig E tear dosation was done on two study groups--group A with 124 patients presenting allergic conjunctivitis symptoms (58 with seasonal allergic conjunctivitis, 43 with atopic allergic ocular symptoms, 23 treated with olopatadina 0.1% for 10 days) and compare group B consisting of 40 healthy patients (no local or general allergic symptoms) evaluated in the the Ophthalmology Clinic Arad between 2002-2008. The biological material used in the study was that of tears samples. For the determination of Ig E we used ELISA method, HUMAN IMUNO IG E QUANTITATION (Bethyl Laboratoires, Montgomery, TX) test., Results: Significant statistic high levels of IgE concentration were observed in study group A in patients with allergic seasonal conjunctivitis (331.11 +/- 125.22 ng/ml) and patients with atopic terrain (177.18 +/- 56.70) compared to group B (67.25 +/- 8.89 ng/ml) with healthy patients. In the study group with allergic seasonal conjunctivitis there have been significant diminished IgE levels (71.70 +/- 9.94) after treatment., Conclusion: The method of IgE tears dosation proved to be efficient in establishing the ethiology of ocular allergies and in the screening of new cases with allergic conjunctivitis.

CD34, traditionally a marker of hematopoietic stem cells (HSCs), was found on endothelial cells and fibroblasts as well. At the level of the extraglomerular or intraglomerular mesangium, CD34 may signal either the presence of HSCs or, conversely, may be a marker of transdifferentiation. CD34-positive cells of the extraglomerular mesangium could migrate into the intraglomerular mesangium and participate in reparative processes at this level. The aim of our study was to analyze the presence of CD34 at the level of the extraglomerular and intraglomerular mesangium and its relationship with histological markers of activity and chronicity, as well as with other immunohistochemical markers in glomerulonephritis (GN). A cross-sectional study of 36 patients with GN was conducted. Conventional stains: hematoxylin-eosin, periodic acid Schiff, and Trichrome Gömöri, as well as immunohistochemistry: CD34, alpha smooth muscle actin (alpha SMA), vimentin, and proliferating cell nuclear antigen (PCNA) were employed. Activity and chronicity of GN were evaluated according to a scoring system initially used for lupus nephritis and antineutrophil-cytoplasmic-antibody-associated vasculitis. Immunohistochemistry was assessed using a semiquantitative score. The mean age was 46.44 +/- 12.97 years; 22 were male and 14 were female. The extraglomerular mesangium was visible on specimens in 30 patients. CD34 was present in the extraglomerular mesangium in 15 patients: 11 of these patients showed concomitant intraglomerular and extraglomerular mesangial CD34 immunostaining, while four showed only extraglomerular mesangial immunostaining. In three patients, CD34 immunostaining was present only in the intraglomerular mesangium. Twelve patients showed negative immunostaining in both the extraglomerular and the intraglomerular mesangium. Overall, there was a fair degree of relationship, which did not reach statistical significance between CD34 in the extraglomerular mesangium and CD34 in the intraglomerular mesangium across the 36 patients. In the intraglomerular mesangium, CD34 did not significantly correlate with mesangial alpha SMA, vimentin, PCNA, and activity or chronicity index. In the extraglomerular mesangium, CD34 did not show a significant correlation with alpha SMA, vimentin, or PCNA. The activity index and the chronicity index showed a good correlation with serum creatinine. Mesangial cell proliferation correlated well with the mesangial matrix increase, while interstitial vimentin showed a good correlation with interstitial alpha SMA. We demonstrated the presence of CD34 in the extraglomerular mesangium, which could be related to transdifferentiated mesangial cells or to HSCs in the absence of blood vessels at this level. Our study shows the value of histological indices for evaluating GN but cannot assign significance to CD34 immunolabeling for the assessment of GN.

Steroids are still the mainstay of therapy in primary chronic glomerulonephritis (PCGN), regardless of underlying disturbance or pathology. Moreover, relationship between known abnormalities and disease manifestation is stochastic, therefore treatment continues to be empirical. It is not known whether responsiveness is related to immune phenotype. We performed flowcytometric lymphocyte (Ly) phenotyping (CD19, CD3, CD3CD4, CD3CD8, CD56/16) on 16 patients (pts) (12M, 4F), mean age 37.6+/-13 years with primary chronic glomerulonephritis (PCGN): minimal change disease (MCD)--6 pts, focal and segmental glomerulosclerosis (FSGS)--4 pts, mesangial proliferative glomerulonephritis--5 pts, mesangiocapillary glomerulonephritis--1 pt, before and at 7 days of oral Prednisone 1 mg/kg/day (in 2 divided doses). Before steroids: 4/16 pts(25%) had elevated BP; 9/16(56.2) showed nephrotic proteinuria. Serum creatinine was >1.2 mg% in 6/16(37.5%). At 7 days WBC count increased (13,079.37+/-4966.4/microl vs. 8021.25+/-2077.4/microl; p=0.0007), Ly percentage (%) decreased (20.30+/-9% vs. 29.9+/-10.4%; p=0.0095), while absolute (abs.) Ly count remained unchanged. Both CD19 Ly% and CD19 Ly abs. count increased (16.13+/-6.5% vs. 9.52+/-3.7%; p=0.0015, and 410.012+/-29.7/microl vs. 223.56+/-123.8/microl; p=0.0077, respectively). NK (natural killer)% decreased (9.15+/-5.2% vs. 14.19+/-7.1%; p=0.0296). CD3, CD3CD4, CD3CD8 Ly subsets and CD4/CD8 ratio showed no change. Variation in proteinuria (2.88+/-2.1 g/24 h vs. 3.45+/-1.7 g/24 h; p=0.4) did not reach statistical significance (Wilcoxon-Mann-Whitney). In 11 pts we performed an additional analysis at 1 month. Compared to levels before steroids, there was an increase in WBC, CD19 Ly% and CD19 Ly abs. count and a decrease in NK% and NK abs. count. Other Ly subsets and CD4/CD8 ratio remained unchanged. Variation in clinical parameters (proteinuria, serum Creatinine, BP) did not reach statistical significance. Changes in Ly profile precede changes in clinical parameters and thus are divergent. While our patients proved to be early non-responders, further studies to elucidate whether profile changes provide for response specification are warranted.

The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging.

Dendritic cells (DCs) have the capacity to prime tumor-specific T cell responses and are considered as potentially effective vaccines for immunotherapy of cancer. Critical parameters in the development of DC vaccines are the source of tumor antigen (TA) and the mode of DC-loading. Whole tumor cells contain complex assortments of TA, which has been exploited to enhance cross-presentation to CD8 T cells by DCs loaded with anti-syndecan mAb-opsonized myeloma cells. This approach may be broadly improved by targeting the MHC class I chain-related protein A (MICA), which is frequently and abundantly expressed on most if not all types of epithelial cancers but not in normal tissues except intestinal mucosa. Loading of DC with anti-MICA mAb-coated breast, melanoma, or ovarian tumor lines or uncultured ovarian cancer cells efficiently promoted TA cross-presentation and priming of multivalent anti-tumor CD8 and CD4 T cell responses. These were of substantially greater breadth and magnitude than those of T cells primed by peptide-pulsed or apoptotic tumor cell-loaded DCs. These results may advance DC vaccine development and provide a platform for adoptive T cell therapy and TA discovery. These results further suggest that antibody targeting of MICA might be applicable to elicit T cell immunity against tumors of diverse tissue origins in cancer patients.

We evaluated cells from 24 patients with B cell chronic lymphocytic leukemia (B-CLL) to determine apoptosis induced by CD5 hypercross-linking. Following the CD5 hypercross-linking with anti-CD5 monoclonal antibodies (MoAbs), we identified 10 patients where CD5 hypercross-linking induced apoptosis (group A) and 14 patients whose cells were resistant to the anti-CD5 MoAbs (group B). The programmed cell death pathway of the cells from patient group A was caspase-3 and poly (ADP-ribose) polymerase (PARP)-dependent, involved a reduction of the mitochondrial transmembrane potential DeltaPsi and a down-regulation of the anti-apoptotic Bcl-2, Mcl-1 and iNOS proteins. Early activation-associated molecules such as CD25 and CD69 were expressed at higher levels than in controls after 6 h of culture with anti-CD5 MoAb. The expression of CD5 and of CD72, the ligand for CD5, were significantly lower in group A compared with group B. Anti-CD20 MoAb had similar activity with anti-CD5 MoAb and the combination of the two MoAbs seemed to be additive. In this study, it is suggested that the cells from some B-CLL patients can be induced into programmed cell death by CD5 hypercross-linking with anti-CD5 MoAbs.

We explored the mechanism through which patients sometimes show immunosuppression after cardiac surgery. To test the hypothesis that commonly used drugs could cause apoptosis of immune cells, the proapoptotic effects of heparin and catecholamines (dopamine and dobutamine) on peripheral blood lymphocytes were evaluated. Peripheral blood lymphocytes were purified from blood samples of normal healthy volunteers. These cells were cultured in the presence of heparin, dobutamine or dopamine. The apoptosis was quantified by Annexin V fluorescent assay, by DNA content and by morphological assessment. Lymphocytes did not show significant levels of apoptosis induction after 24 hours of incubation with heparin. Both dopamine and dobutamine demonstrated a clear apoptosis inducing effect on lymphocytic population after 24 and 48 hours of culture, in concentrations comparable with the clinically used levels. Apoptosis was time and concentration dependent for both catecholamines. The dopamine and dobutamine effect on lymphocyte viability was due, at least partially, to lymphocyte beta receptor engagement, as proved by blocking the receptor with propranolol. These results suggest that catecholamines could induce apoptosis of lymphocytes. This finding may be associated with immunosuppression observed in patients undergoing cardiac surgery.

Jaspamide, a natural peptide isolated from the marine sponge Hemiastrella minor, was used in the study of polyploidy in HL-60 cells. Jaspamide at 5 x 10(-8) M concentration exhibited antiproliferative activity and an increased CD4 and CD14 surface expression. After 2 days of cultivation, 56.3% of the exposed cells became multinuclear compared with 2.4% in controls. Moreover, the size and the number of nuclei of the cells increased in a time-dependent manner. An increased number of metaphase chromosomes was observed by microscopical enumeration after colcemid treatment for 60 min. The analysis of the DNA content of these cells, measured by propidium iodide staining, revealed a significant increase in the cells percentage with increased DNA content. Taken together, these findings indicate that the jaspamide treatment induces polyploidization in the HL-60 cell line.