Mesenchymal stem cells in peripheral blood of severely injured patients.

PURPOSE: Mesenchymal stem cells (MSCs) are primarily stromal cells present in bone marrow and other tissues that are crucial for tissue regeneration and can be mobilized into peripheral blood after different types of organ damage. However, little is known about MSC appearance in blood in the setting of polytrauma. METHODS: We conducted a monocentered and longitudinal observational clinical study in 11 polytraumatized patients with an injury severity score (ISS) ≥ 24 to determine the numbers of MSCs in peripheral blood. Blood was collected from healthy volunteers and patients after polytrauma in the emergency room and 4, 12, 24, 48 h, 5 and 10 day later, and cells carrying MSC-surface markers (negative for CD45, positive for CD29, CD73, CD90, CD105, and CD166 in different combinations also employing the more stringent markers STRO1 and MSCA1) were detected and characterized using flow cytometry. Relative numbers of MSC-like cells were correlated with clinical parameters to evaluate if specific injury patterns had an influence on their presence in the blood cell pool. RESULTS: We were able to detect MSC marker-positive cells in both cohorts; however, the percentage of those cells present in the blood of patients during the first 10 day after injury was mostly similar to healthy volunteers, and significantly lowers starting at 4 h post trauma for one marker combination when compared to controls. Furthermore, the presence of a pelvis fracture was partly correlated with reduced relative numbers of MSC-like cells detectable in blood. CONCLUSIONS: Polytrauma in humans was associated with partly reduced relative numbers of MSC-like cells detected in peripheral blood in the time course after injury. Further studies need to define if this reduction was due to lower mobilization from the bone marrow or to active migration to the sites of injury.

Complement C5a-Induced Changes in Neutrophil Morphology During Inflammation.

The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl-/HCO3--exchanger or the Cl- -channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP-P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.

Quantitative determination of lysophosphatidic acids (LPAs) in human saliva and gingival crevicular fluid (GCF) by LC-MS/MS.

Lysophosphatidic acid (LPA) is a phospholipid mediator that plays multiple cellular functions by acting through G protein-coupled LPA receptors. LPAs are known to be key mediators in inflammation, and several lines of evidence suggest a role for LPAs in inflammatory periodontal diseases. A simple and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify LPA species (LPA 18:0, LPA 16:0, LPA 18:1 and LPA 20:4) in human saliva and gingival crevicular fluid (GCF). LPA 17:0 was used as an internal standard and the LPA species were extracted from saliva by liquid-liquid extraction using butanol. Chromatography was performed using a Macherey-Nagel NUCLEODUR® C8 Gravity Column (125 mm × 2.0 mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5 mM ammonium formate (mobile phase A) and methanol/water: 99/0.5 (v/v) containing 0.5% formic acid and 5mM ammonium formate (mobile phase B) at a flow rate of 0.5 mL/min. LPAs were detected by a linear ion trap-triple quadrupole mass spectrometer with a total run time of 8.5 min. The limit of quantification (LOQ) in saliva was 1 ng/mL for all LPA species and the method was validated over the range of 1-200 ng/mL. The method was validated in GCF over the ranges of 10-500 ng/mL for LPA 18:0 and LPA 16:0, and 5-500 ng/mL for LPA 18:1 and LPA 20:4. This sensitive LC-MS/MS assay was successfully applied to obtain quantitative data of individual LPA levels from control subjects and patients with various periodontal diseases. All four LPA species were consistently elevated in samples obtained from periodontal diseases, which supports a role of LPAs in the pathogenesis of periodontal diseases.

Protective effects of anti-C5a peptide antibodies in experimental sepsis.

We evaluated antibodies to different peptide regions of rat C5a in the sepsis model of cecal ligation and puncture (CLP) for their protective effects in rats. Rabbit polyclonal antibodies were developed to the following peptide regions of rat C5a: amino-terminal region (A), residues 1-16; middle region (M), residues 17-36; and the carboxyl-terminal region (C), residues 58-77. With rat neutrophils, the chemotactic activity of rat C5a was significantly inhibited by antibodies with the following rank order: anti-C > anti-M >> anti-A. In vivo, antibodies to the M and C (but not A) regions of C5a were protective in experimental sepsis, as determined by survival over a 10-day period, in a dose-dependent manner. The relative protective efficacies of anti-C5a preparations (in descending order of efficacy) were anti-C > anti-M >> anti-A. In CLP rats, a delay in infusion of antibodies, which were injected at 6 or 12 h after CLP, still resulted in significant improvement in survival rates. These in vivo and in vitro data suggest that there are optimal targets on C5a for blockade during sepsis and that delayed infusion of anti-C5a antibody until after onset of clinical evidence of sepsis still provides protective effects.

A phase II study of temozolomide in hormone-refractory prostate cancer.

INTRODUCTION: Hormone-refractory disseminated prostate cancer is a major oncological problem. Preclinical studies with temozolomide, an oral alkylating agent, in prostate cancer have shown encouraging results. In phase I studies the safety of temozolomide in non-prostate cancer patients has been demonstrated. Based on these results, a phase II study of temozolomide in patients with metastatic disease who had developed progressive symptomatic disease while on antiandrogen therapy, was initiated. METHODS: A group of 18 patients started a 5-day temozolomide regimen, with a 28-day treatment cycle. Response parameters (prostate-specific antigen, bone scan, quality of life questionnaire) and toxicity (common toxicity criteria for international studies) were recorded at regular intervals. RESULTS: Of the 18 patients, 16 were evaluable by completing two or three cycles. All patients developed progressive disease within two cycles, except one who had progressive disease at the end of cycle 3. Of the 16 evaluable patients, 11 developed new bone metastases (bone scan), 1 developed lung metastases, 4 had progressive disease as reflected by a 25% increase in serum PSA together with subjective progression, and 7 and 5 had progressive disease as reflected by decreased quality of life and increased pain score, respectively. Toxicity was limited to nausea and vomiting, which was effectively treated with antiemetic medication, and anemia and thrombocytopenia, which returned to normal values within 1 week. DISCUSSION: Treatment with temozolomide was generally well tolerated, with occasionally moderate toxicity. As all patients developed progressive disease the results are rather discouraging. Temozolomide is ineffective for the treatment of patients with symptomatic progressive hormone-refractory prostate cancer.

Immunotherapy with monoclonal antibodies directed against the immunosuppressive domain of p15E inhibits tumour growth.

Immunosuppressive retrovirus-related proteins, like p15E, are involved in tumour-associated immunosuppression. In the present study we investigated whether such proteins could be used as targets in tumour immunotherapy using MoAbs. Immunotherapy was performed in mice inoculated with the Rauscher virus-transformed myeloid cell line RMB-1. RMB-1 cells express retroviral antigens at their cell surface. In order to obtain constant serum titres of MoAbs over a prolonged period of time during therapy, anti-p15E antibody-producing hybridoma cells were encapsulated in alginate and injected intraperitoneally in tumour-bearing mice. Using this technique, serum antibody titres of 50-100 micrograms/ml were obtained, which remained constant over a period of at least 3 weeks. Therapy experiments were performed using anti-p15E antibodies 19F8, which recognizes both cell surface-associated as well as circulating p15E, and ER-IS5, which did not react with surface-bound p15E beyond background, but which neutralizes circulating p15E. Inoculation of alginates containing anti-p15E hybridoma cell lines in RMB-1 tumour-bearing mice showed inhibition of tumour cell growth. In survival experiments, 19F8 cured eight of 23 tumour-bearing mice. The p15E neutralizing antibody ER-IS5 caused a significant longer survival, but therapy with this MoAb alone was not sufficient to cure the animals of the RMB-1 tumour.

New monoclonal antibodies against the putative immunosuppressive site of retroviral p15E.

Both retroviral infections as well as human tumors may cause immunosuppression. One of the factors involved in immunosuppression in patients with squamous cell carcinoma of the head and neck (SCC-HN) is a protein related to the retroviral protein p15E. A conserved, 17-amino acid sequence represents the immunosuppressive epitope of retroviral p15E. In order to study the relationship between SCC-HN associated immunosuppression and retroviral p15E, we produced three new monoclonal antibodies (MAbs; ER-IS1, ER-IS2, and ER-IS5) directed against the immunosuppressive synthetic CKS-17 peptide. These MAbs react with the immunosuppressive peptide (in enzyme-linked immunosorbent assay), with human tumor cell lines (in FACScan analysis), with retroviral p15E (on Western blot), and with cryostat sections of SCC-HN tumor tissue. In addition, the MAbs neutralize the immunosuppressive low molecular weight factors present in sera of patients with SCC-HN. These results show that retroviral p15E and the immunosuppressive factors associated with SCC-HN share a conserved immunosuppressive epitope and that MAbs against this epitope can be used for detection and neutralization of the tumor-associated immunosuppressive protein(s).

HIV-1 rev antisense phosphorothioate oligonucleotide binding to human mononuclear cells is cell type specific and inducible.

A fluorescein-conjugated, antisense phosphorothioate oligonucleotide with specificity for HIV-1 rev sequence (FAM-anti-rev) was investigated for its ability to bind to specific subsets of human peripheral blood mononuclear cells. Oligonucleotide binding by CD4+ and CD8+ T cells, B cells, and monocytes isolated from 15 normal and 15 HIV-infected individuals was evaluated on both nonstimulated mononuclear cells and after 24-hr activation with phytohemagglutinin (PHA). In both normals and HIV-infected individuals, we found a significantly higher percentage of monocytes and B cells binding oligonucleotide in comparison to T cells. Oligonucleotide binding by both T cells and B cells was enhanced by 24-hr PHA stimulation while monocyte uptake was unchanged. In comparison to normal controls, HIV-1-infected patients showed slightly higher percentages of both unstimulated and PHA activated CD4+, CD8+, and CD25+ T cells binding oligonucleotide. The propensity for a high percentage of monocytes, which may act as an HIV-1 reservoir, to bind the anti-rev oligonucleotide and the enhanced binding by T cells in the HIV-1-infected patient samples provides some optimism for potential in vivo therapy of HIV-1 infection using antisense oligonucleotides.

OMA-AML-1: a leukemic myeloid cell line with CD34+ progenitor and CD15+ spontaneously differentiating cell compartments.

OMA-AML-1 was established from a patient with acute myelomonocytic (M4) leukemia at fifth relapse when blasts were greater than 85% CD34+, CD15-. Leukemic cells were established in suspension culture and independently grown as subcutaneous tumors in SCID mice. Cells growing in suspension culture underwent differentiation by phenotypic and morphologic criteria. In contrast, cells grown as subcutaneous solid tumors in SCID mice maintained progenitor cell characteristics with high-density CD34 expression and lack of morphologic differentiation. A tendency toward differentiation to CD15+, CD34- cells in vitro and self-renewal of CD34+, CD15- cells in vivo was consistently demonstrated regardless of whether cells were initially grown in vitro or in vivo. The cell line maintains both a CD34+, CD15- progentitor cell pool and a non-overlapping, CD15+, CD34- differentiating cell compartment after more than 1 year in continuous culture. Cell cycle analysis and cloning experiments were consistent with terminal differentiation occurring in the CD15+, CD34- population. The cell line shows concentration-dependent proliferative responses to interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6, but not to granulocyte CSF (G-CSF). OMA-AML-1 appears to mimic several features of normal myeloid hematopoiesis and should prove useful for the study of normal and malignant myeloid differentiation.

Differential expression of CD24-related epitopes in mycosis fungoides/Sézary syndrome: a potential marker for circulating Sézary cells.

In the hematopoietic system, the B-cell associated antigen CD24 is expressed at high density on B cells, B-cell precursors, and B-cell malignancies as well as at low density on peripheral blood polymorphonuclear leukocytes. The 42-Kd sialoglycoprotein has not been previously demonstrated to be expressed on T cells, thymocytes, or T-cell malignancies. We identified three patients with mycosis fungoides/Sézary syndrome that showed low density expression of the CD24-related epitope recognized by antibody BA-1 on circulating T cells. All three patients had Sézary cells by morphologic assessment and clonal T-cell populations in the peripheral blood by gene rearrangement studies. In two of these patients, indirect immunofluorescence with a panel of six anti-CD24 monoclonal antibodies demonstrated reactivity for two of six antibodies in one case and only one of six antibodies in the other. The biologic significance of CD24-related epitope expression on circulating T cells in mycosis fungoides/Sézary syndrome is unclear. However, these findings suggest that differential, low density expression of CD24-related epitopes (BA-1+, OKB2-) may be a useful phenotypic marker for identifying circulating Sézary cells.