Mature Polymorphonuclear Leukocytes Research Articles

Delta Neutrophil Index as a New Marker of Purulent Inflammation in Men With Non-odontogenic Abscesses of the Neck.

Introduction If non-odontogenic abscesses and phlegmons (all purulent inflammations where the etiology is not a diseased tooth) of the neck are not promptly treated, they can lead to serious complications and even end in the death of the affected patient. Classical markers of inflammation such as plasma concentration of leukocytes (WBC), neutrophils (Neu), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) are elevated in inflammatory neck diseases, but none of them has been proven as a definite marker in the prediction of this type of pathology. This necessitates the search and analysis of new indicators that could be used in the diagnosis, follow-up, and prognosis of patients with purulent neck infections. Potentially, such a marker could be the delta neutrophil index (DNI), which is increasingly entering clinical practice as a prognostic indicator in critically ill patients with life-threatening illnesses, including sepsis and systemic inflammatory response syndrome (SIRS). In the world literature, there are no data that have been studied in patients with purulent diseases of the neck of non-odontogenic origin, which is the aim of this original article. Materials and methods This retrospective study included 40 men with an average age of 46 (18-87) years with non-odontogenic abscesses and phlegmons of the neck who were hospitalized and operated on. In all of them, the concentration of leukocytes, eosinophils (Eo), neutrophils, and mature polymorphonuclear neutrophil leukocytes (PMN) was examined on an automatic 5-Diff hematology analyzer, ADVIA 2120i (SiemensMedical Solutions USA, Malvern, PA). Thus, it was calculated according to the formula for calculating DNI (DNI% = (Neu%+Eo%) - PMN%). Retrospectively, 30 healthy men with an average age of 42 (18-81) years were used as a control group, in which the same indicators were examined during a preventive examination. Results and discussion Comparing the mean values of WBC, Neu, and DNI between the studied patients with non-odontogenic purulent neck infections (n=40) and the healthy male controls (n=30) it was found that all three indicators of inflammation are significantly higher in the ill men, and these differences are statistically significant (p<0.05): 10.19 ±2.68x103/L versus 7.37 ±1.93x103/L for leukocytes, 7.68 ±2, 76x103/L versus 4.13 ±1.48x103/L for neutrophils, and 1.11±0.83% versus -1.07±1.22% for DNI. Therefore, the high mean numbers of measured WBC and Neu in men with non-odontogenic purulent neck infections were associated with an increase in their mean calculated DNI. This gives us reason to think that while WBC and Neualone are not sufficient for definitive diagnosis, treatment follow-up, and prediction of disease outcome, in combination with DNI they become reliable indicators in purulent neck infections. Conclusions The DNI correlates well with other well-known and established indicators of inflammation, such as the concentration of leukocytes and neutrophils in the peripheral blood of patients. Its calculation is fast as an implementation procedure and is economically beneficial. Its independent use in the diagnosis and treatment of these diseases is about to be investigated and analyzed.

The Role of Zinc in GM-CSF-Induced Signaling in Human Polymorphonuclear Leukocytes.

Zinc is suggested to be necessary for functional signaling induced by certain growth factors. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key factor for differentiation and activation of myeloid cells. This report analyses the impact of different zinc concentrations on GM-CSF-induced signaling in mature polymorphonuclear leukocytes (PMN). As measured by flow cytometry, zinc increases surface GM-CSF receptor (GM-CSFR) in PMN, whereas monocytes respond with decreased GM-CSFR surface expression. Since total cellular GM-CSFR expression remains unaffected, the observed zinc-induced GM-CSFR surface dynamics may be explained by receptor redistribution. In PMN, zinc enhanced phosphorylation of mitogen-activated protein kinases (MAPK) in a dose-dependent manner as found in western blot. Zinc-induced MAPK phosphorylation is additionally augmented by moderate GM-CSF stimulation. The present study demonstrates the opposing influence of zinc on GM-CSFR surface expression in monocytes and PMN. Zinc and GM-CSF, use in optimized concentrations, augment MAPK signaling, and increase expression of MAPK-induced myeloid cell leukemia-1 (Mcl-1) in PMN. Thus, this study concludes that zinc strengthens growth factor-induced signaling. Hence, the study provides a basis for further in vivo studies, focusing on the therapeutic value of zinc in patients with a disturbed GM-CSF signaling.

Delta-neutrophil index: a potential predictor of coronary artery involvement in Kawasaki disease by retrospective analysis.

DNI is the immature granulocyte fraction provided by a blood cell analyzer, which is determined by subtracting the fraction of mature polymorphonuclear leukocytes from the sum of myeloperoxidase-reactive cells. We aimed to evaluate the role of Delta-neutrophil index (DNI) in cardiac prognosis prediction in children with Kawasaki disease (KD). Medical records of 193 patients were retrospectively reviewed. The values of DNI, white blood cells, erythrocyte sedimentation rate, the percent of polymorphonuclear leucocytes, C-reactive protein, aspartate transaminase, alanine aminotransferase, total bilirubin data of children with KD were analyzed. Also, sex and age of children were compared. The value of DNI was higher in children with cardiac complications [median 0.8 (0-0.26) vs 5.3 (3.55-8.95); P < 0.001]. The ROC curves showed that DNI was a better predictor of cardiac complications than other parameters. The best cutoff value for DNI to predict cardiac complications was 5.55% with sensitivity of 80% and specificity of 82% (AUC 0.883, 95% confidence interval [CI] 0.807-0.959, P < 0.05). DNI could serve as a facile and useful marker to predict cardiac complications in children with KD, as it is included in a routine complete blood count.

Neonatal myeloid derived suppressor cells show reduced apoptosis and immunosuppressive activity upon infection with Escherichia coli.

Susceptibility to infection during the neonatal period and reduced control of inflammation in neonates are attributed to immunosuppression persisting from fetal life. Myeloid-derived suppressor cells (MDSCs) are immature myeloid progenitors with suppressive activity and increased numbers in cord blood. We hypothesized that MDSCs contribute to innate host defence in neonates, paralleled by anti-inflammatory signalling.Phagocytic activity, infection induced apoptosis, expression of B-cell lymphoma (Bcl)-2 family proteins, production of reactive oxygen species (ROS), cytokine production and T-cell suppression of neonatal granulocytic-MDSCs (G-MDSCs) after infection with Escherichia coli (E. coli) were compared to neonatal autologous mature polymorphonuclear leukocytes (PMNs). Phagocytic activity of G-MDSCs upon infection with E. coli was equal to that of mature PMNs, however, apoptosis of G-MDSCs was decreased. G-MDSCs showed enhanced Bcl-2-expression and lower ROS production compared to PMNs. Inhibition of Bcl-2 reduced apoptosis rates of G-MDSCs to that of mature PMNs. Induction of anti-inflammatory transforming growth factor beta (TGF-β) was enhanced, while pro-inflammatory IL-8 decreased in G-MDSCs compared to PMNs. Infected G-MDSCs strongly suppressed proliferation of T cells. We show a direct role of G-MDSCs for anti-bacterial host defence. Prolonged survival and anti-inflammatory capacity suggest that G-MDSCs are important for immune-regulation after bacterial infection.

Abstract 5497: Cabozantinib eradicates de novo castrate-resistant PTEN/p53 deficient murine prostate cancer via activation of neutrophil-mediated anti-tumor innate immunity

Abstract The majority of kinase inhibitors in cancer clinical trials have been evaluated in the context of cell autonomous induction of apoptosis. However, apoptosis can result in protumorigenic immunosuppression, which limits the durability of an anti-tumor response. Recent clinical trial data shows striking clinical and radiographic responses with kinase inhibitor cabozantinib (XL-184) in metastatic solid tumors, particularly in the context of bone metastases. However, the mechanism responsible for this robust antitumor response remain unknown. Here we show that cabozantinib eradicates poorly differentiated invasive cancer that develop in the context of prostate-specific PTEN and p53 loss, respectively, within 48 hours of cabozantinib treatment. This rapid tumor clearance was temporally associated with infiltration of mature polymorphonuclear leukocytes into the tumor bed. These anti-tumor effects of cabozantinib appear to be MET-independent, since the MET inhibitor PF-04217903 did not phenocopy the effects of cabozantinib in the prostate-specific PTEN/p53 deficient mouse model in vivo. Unexpectedly, in vitro treatment of PTEN/p53 deficient cell lines derived from murine tumors showed insignificant apoptosis, but robust extracellular release of HMGB1, a neutrophil chemoattractant, and marker of immunogenic cell death. To elucidate the relevance of an immunogenic anti-tumor mechanism in vivo, we performed RNA-seq profiling and quantitative RT-PCR analysis, which showed an acute increase in anti-tumor inflammatory cytokine gene expression signature and neutrophil activation/chemotaxis markers following acute cabozantinib treatment. Critically, blockade of neutrophil chemotaxis/trafficking with dexamethasone or depletion with anti-Ly6G antibody, reversed the effects of cabozantinib towards eradication of advanced PTEN/p53 deficient tumors. Finally, cytokine array profiling of supernatant from bone marrow metastases from castrate-resistant prostate cancer patients showed increased neutrophil markers and decreased IL-6 levels within the bone microenvironment following 6 weeks of cabozantinib treatment., thus polarizing the neutrophils into” N1” effector cells. Collectively, these results shed light on a novel anti-tumor immunogenic mechanism for cabozantinib within the tumor microenvironment of PTEN/p53 deficient tumors, which unleashes a profound innate anti-tumor immune response. These findings suggest the possibility of combination therapies of cabozantinib with T-cell checkpoint blockade or vaccine-based approaches, to augment immunologic responses in advanced cancers. Citation Format: Akash Patnaik, Kenneth Swanson, Katja Helenius, Thornley Thomas, Athalia Pyzer, Vilmosne Csizmadia, Sabina Signoretti, Todd Morgan, Yugang Wang, Olivier Elemento, Lily Wang, Elena Levantini, John Clohessy, John Asara, David Smith, Jacalyn Rosenblatt, David Avigan, Steven Balk, Lewis Cantley. Cabozantinib eradicates de novo castrate-resistant PTEN/p53 deficient murine prostate cancer via activation of neutrophil-mediated anti-tumor innate immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5497. doi:10.1158/1538-7445.AM2015-5497

False positivity of magnetic resonance imaging under the effect of granulocyte-colony stimulating factor in a child with leukemia

Granulocyte-colony stimulating factor (G-CSF) increases the proliferation and maturation of committed polymorphonuclear leukocyte precursors, as well as the function of mature polymorphonuclear leukocytes. It has previously been shown in pediatric patients that G-CSF induces reconversion of fatty bone marrow to hematopoietic bone marrow in the pelvis and lower extremities that is detectable by magnetic resonance imaging (MRI). Here, we report a 13-year-old Burkitt leukemia patient with bone pain while he was in remission. He was on G-CSF after cessation of high-dose and low-dose cytarabine chemotherapy. He was suspected to have a leukemia relapse. Pelvic MRI was consistent with leukemic infiltration. However, the pathology of bone marrow biopsy resulted in normal findings. Thus it was suggested that concurrent administration of G-CSF could be the causative agent for both bone pain and false-positive MRI findings. The control MRI after interruption of G-CSF revealed normal findings. In conclusion, radiologists should be informed about the type of therapy, including G-CSF administration, in order to overcome misinterpretation of bone marrow MRI.

Delta Neutrophil Index

Delta neutrophil index (DN) is the immature granulocyte fraction provided by a blood cell analyzer (ADVIA 2120; Siemens Healthcare Diagnostics, Deerfield, Ill), which is determined by subtracting the fraction of mature polymorphonuclear leukocytes from the sum of myeloperoxidase-reactive cells. The purpose of this study was to define the role of DN in differential diagnosis and prognosis prediction of patients with sepsis. Hospital records of 273 patients were retrospectively collected: 47 with systemic inflammatory response syndrome, 78 with sepsis, 51 with severe sepsis, and 97 control subjects. Delta neutrophil index and C-reactive protein data on the day of the first blood culture were compared among the groups, and 28-day mortality associated with sepsis was assessed. Median values of DN were 0.0% (interquartile range, 0.0%-0.0%) in the control group, 0.8% (0.0%-1.7%) in the systemic inflammatory response syndrome group, 3.4% (1.5%-5.3%) in the sepsis group, and 18.6% (9.3%-24.7%) in the severe sepsis group. Furthermore, there were significant differences among the groups. The receiver operating characteristic curves showed that DN was a better predictor of sepsis than C-reactive protein. The best cutoff value for DN for predicting sepsis was 2.7%. Delta neutrophil index was significantly higher in those who died than in the survivors (median [interquartile range], 11.5% [3.5%-25.0%] vs. 4.7% [2.2%-10.6%], P = 0.008) and was identified to be an independent predictor for 28-day mortality in patients with sepsis by Cox proportional hazards model. Delta neutrophil index may serve as a facile and useful marker for early diagnosis and prognostic assessment of patients with sepsis, as it is included in a routine complete blood count.

Neutrophilic Dermatoses

Sweet syndrome, pyoderma gangrenosum, and subcorneal pustular dermatosis are neutrophilic dermatoses - conditions that have an inflammatory infiltrate consisting of mature polymorphonuclear leukocytes. The neutrophils are usually located within the dermis in Sweet syndrome and pyoderma gangrenosum; however, in subcorneal pustular dermatosis, they are found in the upper layers of the epidermis. Sweet syndrome, also referred to as acute febrile neutrophilic dermatosis, is characterized by pyrexia, elevated neutrophil count, painful erythematous cutaneous lesions that have an infiltrate of mature neutrophils typically located in the upper dermis, and prompt clinical improvement following the initiation of systemic corticosteroid therapy. Classical, malignancy-associated, and drug-induced variants of Sweet syndrome exist. Pyoderma gangrenosum is characterized by painful, enlarging necrotic ulcers with bluish undermined borders surrounded by advancing zones of erythema; its clinical variants include: ulcerative or classic, pustular, bullous or atypical, vegetative, peristomal, and drug-induced. Subcorneal pustular dermatosis is an uncommon relapsing symmetric pustular eruption that involves flexural and intertriginous areas; it can be idiopathic or associated with cancer, infections, medications, and systemic diseases. Since Sweet syndrome, pyoderma gangrenosum, and subcorneal pustular dermatosis share not only the same inflammatory cell but also similar associated systemic diseases, it is not surprising that the concurrent or sequential development of these neutrophilic dermatoses has been observed in the same individual. Also, it is not unexpected that several of the effective therapeutic interventions - including systemic drugs, topical agents, and other treatment modalities - for the management of these dermatoses are the same. The treatment of choice for Sweet syndrome and idiopathic pyoderma gangrenosum is systemic corticosteroids; however, for subcorneal pustular dermatosis, dapsone is the drug of choice. Yet, tumor necrosis factor-alpha antagonists are becoming the preferred choice when pyoderma gangrenosum is accompanied by inflammatory bowel disease or rheumatoid arthritis. Potassium iodide and colchicine are alternative first-line therapies for Sweet syndrome and indomethacin (indometacin), clofazimine, cyclosporine (ciclosporin), and dapsone are second-line treatments. Cyclosporine is effective in the acute management of pyoderma gangrenosum; however, when tapering the drug, additional systemic agents are necessary for maintaining the clinical response. In some patients with subcorneal pustular dermatosis, systemic corticosteroids may be effective; yet, systemic retinoids (such as etretinate and acitretin) have effectively been used for treating this neutrophilic dermatosis - either as monotherapy or in combination with dapsone or as a component of phototherapy with psoralen and UVA radiation. Topical agents can have an adjuvant role in the management of these neutrophilic dermatoses; however, high-potency topical corticosteroids may successfully treat localized manifestations of Sweet syndrome, pyoderma gangrenosum, and subcorneal pustular dermatosis. Intralesional corticosteroid therapy for patients with Sweet syndrome and pyoderma gangrenosum, hyperbaric oxygen and plasmapheresis for patients with pyoderma grangrenosum, and phototherapy for patients with subcorneal pustular dermatosis are other modalities that have been used effectively for treating individuals with these neutrophilic dermatoses.

Defective polymorphonuclear leukocyte functions in children receiving chemotherapy for cancer are partially restored by recombinant human granulocyte colony-stimulating factor in vitro.

Granulocyte colony-stimulating factor (G-CSF) has important direct and priming effects on different functions of normal mature polymorphonuclear leukocytes (PMNL). Previous study has shown an alteration in respiratory burst and bactericidal activities of PMNL harvested from children with cancer treated with chemotherapy. The present study evaluates the possibility that recombinant human (rh) G-CSF could correct these defective functions in vitro. Free radical formation in defective PMNL was enhanced by rhG-CSF to a level similar to that found in normal PMNL primed by rhG-CSF. The defective bactericidal activity against Escherichia coli and Staphylococcus aureus was also corrected. This bactericidal activity was not different from that observed in normal PMNL primed by rhG-CSF. In conclusion, correction of the altered free radical-formation pathway by rhG-CSF in these cells contributed to the restoration of normal bactericidal activity against both gram-positive and gram-negative microorganisms.

Mature polymorphonuclear leukocytes express high-affinity receptors for IgG (FcγRI) after stimulation with granulocyte colony-stimulating factor (G-CSF)

The high-affinity receptor for the constant region of immunoglobulin G IgG (Fc gamma RI; CD64) is virtually undetectable on mature polymorphonuclear neutrophils (PMNs) in healthy individuals but is expressed on PMNs in patients with certain infections and in patients treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF). The induction of Fc gamma RI by rhG-CSF has previously been reported to result from effects on immature granulocyte progenitors. To evaluate the G-CSF effect on mature PMNs, we studied the correlation between G-CSF plasma concentration and expression of Fc gamma RI on PMNs in vivo as well as the effect of G-CSF on Fc gamma RI expression on mature PMNs in vitro. Fc gamma RI expression on PMNs correlated (R = 0.79; p < .001) with plasma concentrations of endogenous or recombinant G-CSF in healthy volunteers and in patients undergoing high-dose chemotherapy and autologous bone marrow transplantation. PMNs exhibited a unimodal distribution for elevated Fc gamma RI expression, suggesting that G-CSF induced increased expression of Fc gamma RI on mature as well as on immature PMNs. In vitro, incubation of mature PMNs with G-CSF induced mRNA for Fc gamma RI. Significant Fc gamma RI surface expression was induced in a time- and dose-dependent manner. Thus, G-CSF can act on mature PMNs to increase Fc gamma RI expression and may be useful for stimulating antibody mediated immune functions of PMNs in vivo.

Inflammatory and Procoagulant Mediator Interactions in an Experimental Baboon Model of Venous Thrombosis

Theoretic and in vitro evidence suggests that thrombosis and inflammation are interrelated. The purpose of the present study was to define the relationship between inflammation and deep venous thrombosis (DVT) in an in vivo model. Initiation of DVT was accomplished by administration of antibody to protein C (HPC4, 2 mg/kg) and tumor necrosis factor (TNF, 150 micrograms/kg); stasis; and subtle venous catheter injury. Thrombosis was assessed by thrombin-antithrombin assay (TAT), 125I-fibrinogen scanning (scan) over both the proximal and distal iliac veins, and ascending venography. Cytokines TNF, interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8) were measured along with differential white blood cell counts, platelet counts, fibrinogen (FIB), and erythrocyte sedimentation rates (ESR). Baboon pairs were sacrificed on day 3 (T + 3d), T + 6d, and T + 9d and veins removed. All animals developed inferior vena cava and left iliofemoral DVT by venography; no right DVT was found. TAT was elevated by T + 1hr and peaked at T + 3hrs. Left iliofemoral DVT was found at T + 1hr by scan and reached a 20% uptake difference between the affected left and nonaffected right side at T + 3hrs. TNF peaked at T + 1hr; MCP-1 peaked at T + 6hrs; IL-8 and IL-6 peaked on T + 2d; all cytokines declined to baseline. TNF and TAT elevations were found to correlate with all cytokines; elevations in IL-8 were correlated with elevations in MCP-1 and IL-6 (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The serine-protease inhibitor of cartilage matrix is not a chondrocytic gene product.

Human articular cartilage contains significant amounts of antileukoprotease, a cationic low-molecular-mass serine-protease inhibitor, which was originally purified from mucous secretions (synonym: secretory leukocyte proteinase inhibitor). As it was not known whether the inhibitor molecule is also synthesized locally, we investigated antileukoprotease gene expression in chondrocytes. No antileukoprotease-specific mRNA was detected in adult or foetal human chondrocytes by in situ hybridization, Northern-blot analysis or polymerase chain reaction. Concurrently, the chondrocytes remained unstained on immunohistology, whereas immunoreactive antileukoprotease was demonstrated in the cartilage matrix. By Northern-blot analysis, the antileukoprotease message was detected in the promyelocytic cell line HL60, the myelomonocytic cell line U937 and even in mature polymorphonuclear leukocytes from the peripheral blood of healthy donors. Immunoperoxidase staining of polymorphonuclear leukocytes for the antileukoprotease protein indicated that this cell is likely to be the physiological source of the inhibitor in serum. The results further suggest an accumulation of the inhibitor in the cartilage matrix.

Clinical applications of granulocyte colony stimulating factor (G-CSF).

AbstractThe production of neutrophil granulocytes is a complex and dynamic process during which a small number of limited stem cells give rise to lineage specific progenitors that proliferate in the bone marrow subsequently entering the blood as mature polymorphonuclear leukocytes. One hematopoietic growth factor which appears to specifically control the proliferation and terminal maturation of this specific myeloid lineage is granulocyte colony stimulating factor.

CD 10 (CALLA, common acute lymphoblastic leukemia antigen)/neutral endopeptidase 24.11 (NEP, “nkephalinase”) : molecular structure and role in regulating met-enkephalin mediated inflammatory responses

Summary CD10 (common acute lymphoblastic leukemiaantigen, CALLA) is a 100 kDa cell surface glycoprotein (Brown et al., 1975; Ritz et al., 1980; Metzgar et al., 1981; Cossman et al., 1983; Graves et al., 1983; Shipp et al., 1988, 1989), expressed by acute lymphoblastic leukemias (Brown et al., 1975; Ritz et al., 1980), normal lymphoid progenitors (Greaves et al., 1983), mature polymorphonuclear leukocytes (Cossman et al., 1983) and certain non-hematopoietic cells (Metzgar et al., 1981). Molecular cloning and expression studies have shown that CD10 is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”) (LeTarte et al., 1988; Shipp et al., 1988, 1989). CD 10/NEP hydrolyzes a number of naturally occurring peptides including the endogenous opioid pentapeptides met- and leu- enkephalin. Hence, the enzyme in brain has been termed “enkephalinase” (Malfroy et al., 1978). In invertebrate organisms such as the mollusc M. edulis , met-enkephalin triggers inflammatory responses by inducing morphological changes, directed migration, and aggregation of hemocytes (Stefano et al., 1989a,b). We recently found that M. edulis hemocytes express a CD10/NEP related structure and that abrogation of CD10/NEP enzymatic activity reduces the amount of met-enkephalin required for hemocyte activation by five orders of magnitude (Shipp et al., 1990). Human CD 10+ polymorphonu clear leukocytes are similarly responsive to met-enkephalin and inhibition of CD 10/NEP enzymatic activity dramatically potentiates met-enkephalin effects in these cells (Shipp et al., 1990). Therefore, CD10/NEP related structures modulate enkephalin-mediated inflammatory responses in organisms that are ≈500 million years divergent in evolution (Meglitsch, 1967). These results suggest that in hematopoietic, and perhaps other cells, enkephalin signals are regulated via co-expression of cell surface opioid receptors and the CD10/NEP hydrolytic enzyme and hat CD10/NEP functions to down-regulate induced responses to peptide hormones that are CD10/NEP substrates.

Protection from arabinofuranosylcytosine and n-mustard-induced myelotoxicity using hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys monomer and dimer

We have previously shown that the synthetic peptide pGlu-Glu-Asp-Cys- Lys (pEEDCK monomer) inhibits the cytostatic drug-induced proliferation of hematopoietic stem cells CFU-S. Keeping CFU-S quiescent by pEEDCK treatment renders them insensitive to cycle-specific cytostatic drugs and leads to reduced toxicity. Here we show that pEEDCK application during repeated (twice) administration of clinically relevant (nonlethal) 1-beta-D-arabinofuranosylcytosine (Ara-C) doses reduced the percentage of CFU-S in S-phase from 60%-70% to 25%-30% and led to a sustained stem cell number in the bone marrow (BM), whereas unprotected mice had lost about 75% of their CFU-S population. Owing to its cysteine content, the pEEDCK monomer is easily oxidized. The resulting dimer (pEEDCK)2 is a potent stimulator of hematopoiesis. As we show, it can be used for postchemotherapy acceleration of hematologic recovery, similar to the use of recombinant hematopoietic growth factors. A single injection of 30 micrograms/kg pEEDCK monomer to mice 2 hours before the second Ara-C injection retarded onset of neutropenia (by 2 to 3 days) and improved recovery after depression. The quantitative degree of neutropenia was not changed. Postchemotherapy (Ara-C administered twice, followed by N-mustard) infusion of the stimulatory (pEEDCK)2 dimer (1.4 micrograms/kg/d) produced a 4.6-fold increase of progenitor levels (6.7 CFU-GM/1,000 BM cells v 1.45 CFU-GM/1,000 in normal mice) 2 days after the end of the cytostatic treatment when CFU- GM were not detectable in unprotected mice. This increase was followed after several days by strongly elevated granulocyte counts, which remained high for approximately 1 week. Up to 75% of the peripheral leukocytes were mature polymorphonuclear leukocytes (PMN) during this phase. Ara-C (twice) and monomer treatment as above followed by dimer infusion resulted in the complete protection of hematopoiesis. Mice treated with the protective pEEDCK monomer plus stimulatory dimer did not develop the leukocyte depression noted in unprotected animals. The inhibitory monomer appears to keep the stem cell population numerically and qualitatively intact, thus providing optimum target cell conditions for the subsequent stimulator (dimer) treatment. Our results show that the hemoregulatory peptide monomer and dimer can be used for improving the hematologic status of mice treated with clinically relevant doses of cytostatic drugs (antimetabolite and alkylating, alone and in combination). Combining both peptides can prevent occurrence of neutropenia completely. Both peptides can be obtained easily by chemical synthesis and are also active on human cells. They are thus highly promising candidates for application as multilevel hemoprotectors in cancer chemotherapy.

Downregulation of enkephalin-mediated inflammatory responses by CDl0/neutral endopeptidase 24.11

The antigen CD10 (common acute lymphoblastic leukaemia antigen), which is the zinc metalloprotease, neutral endopeptidase 24.11 (also known as NEP or 'enkephalinase'), is expressed by acute lymphoblastic leukaemias, normal lymphoid progenitors, mature polymorphonuclear leukocytes and certain nonhaematopoietic cells. CD10/NEP hydrolyses several naturally occurring peptides, including the endogenous opioid pentapeptides Met- and Leu-enkephalin. In invertebrate organisms such as the mollusc Mytilus edulis, Met-enkephalin triggers inflammatory responses by inducing morphological changes, directed migration and aggregation of haemocytes. We report here that a structure related to CD10/NEP is expressed by M. edulis haemocytes and that abrogation of CD10/NEP enzymatic activity reduces the amount of Met-enkephalin required for haemocyte activation by five orders of magnitude. Similar results are obtained with CD10+ human polymorphonuclear leukocytes, indicating that CD10/NEP related structures regulate enkephalin-mediated inflammatory responses in organisms whose ancestors diverged approximately 500 million years ago.

Myeloperoxidase involvement in PMA-induced chemiluminescence in retinoic-acid-differentiated HL-60 cells

The luminol-dependent chemiluminescent (CL) response of retinoic-acid (RA)-differentiated cells to PMA was studied. HL-60 cells were differentiated into apparently mature polymorphonuclear leukocytes (PMNs) by exhibiting segmented nuclei and increased capacity to reduce NBT which reaches maximum 96 h post RA treatment. Sodium azide, an inhibitor of myeloperoxidase (MPO), strongly decreased the PMA-induced luminol-dependent CL response. The MPO activity as assessed by cytochemical staining was found to be most intense prior to differentiation and gradually decreased to undetectable levels after 96 h post RA treatment. CL response however reached maximum 48 h post RA treatment and gradually decreased thereafter. The lack of correlation between MPO activity and CL response in RA-differentiated HL-60 cells indicates a defect in release of MPO from azurophilic granulae.

Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade.

Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in children is reliably associated with the presence of dengue antibody--actively or passively acquired--before the onset of illness. Limited observations by electron microscopy and fluorescent antibody testing and the recovery of virus from tissues obtained at autopsy show that dengue viruses are consistently associated with cells of mononuclear phagocyte lineage. In particular, virus is associated with Kupffer cells, pulmonary macrophages, and mononuclear cells in skin and blood. Endothelial cells fail to demonstrate necrosis or inflammatory changes. Since acute vascular permeability, shock, and hemorrhage occur late in illness, a plausible hypothesis is that phlogistic factors, resulting from interactions with elements of the immune response, are released from virus-infected mononuclear phagocytes. Such phenomena as generalized depression of mitotic activity of bone marrow cells, destruction of mature polymorphonuclear leukocytes, complement activation, and abnormal hemostasis may serve as markers of these phlogistic factors. It will be of interest to establish whether other viral hemorrhagic fevers involve the same target cells as in DHF/DSS and are mediated by similar effector mechanisms.

Defective chemiluminescence response in differentiated HL60 cells due to impaired degranulation

In the presence of dimethyl sulfoxide, the promyelocytic leukemic cell line, HL60, differentiates into apparently mature polymorphonuclear leukocytes. When correlating the superoxide production from HL60 cells with the number of phagocytozing and NBT-positive cells, no difference was observed in comparison with normal peripheral blood leukocytes. In contrast, the luminol-dependent chemiluminescence was greatly impaired in the differentiated HL60 cells. Analysis of degranulation, i.e., release of myeloperoxidase and N-acetyl- β-glucosaminidase- and myeloperoxidase-mediated iodination by HL60 cells, suggested that the defective chemiluminescence response observed in HL60 cells may be due to impaired release of myeloperoxidase from azurophilic granulae. This may lead to impaired microbicidal activity in these cells.

Expression of cytolytic functions in HL-60 leukaemic cells after induction of polymorphonuclear leukocyte differentiation.

Mature polymorphonuclear leukocytes (PMN) are capable of mediating phorbol myristate acetate (PMA)- and antibody (A)-dependent cellular cytotoxicity (DCC) against ox red blood cells (ORBC) by using oxidative means. The purpose of the present study was to investigate the acquirement of these cytotoxic functions during PMN ontogeny, using the promyelocytic HL-60 cell line as a model for PMN differentiation. HL-60 cells were induced to differentiate along the PMN pathway by exposure to dimethyl sulfoxide (DMSO). Uninduced HL-60 cells were found to be completely devoid of PMA-DCC and ADCC activity. DMSO-induced cells progressively acquired the capacity to kill ORBC and to undergo the activation of oxidative metabolic burst when triggered by PMA. Despite approximately 40% of them also were capable of binding IgG-sensitized ORBC, no ADCC activity and respiratory burst activation was observed: this finding indicates that maturing HL-60 cells require a more complete maturation than that induced by DMSO to actually exert ADCC. Together the results suggest that: a. the acquirement of both PMA-DCC and ADCC potential is a post-promyelocytic event; b. the cytotoxicity activating stimuli, PMA and IgG-coated targets, follow different post-receptor transductional pathways to trigger the effector cell lytic systems: only the PMA receptor-linked pathway develops during DMSO-driven differentiation of HL-60 cells.