Number Of Neutrophils In Peripheral Blood Research Articles

Systemic Inflammatory Status of Patients with Obstructive Sleep Apnea Syndrome and the Predictive Value of Heparin-Binding Protein.

Objective: To investigate the alteration of the systemic inflammatory status of patients with obstructive sleep apnea syndrome (OSAS) and the predictive value of heparin-binding protein (HBP) for OSAS. Methods: Patients with OSAS who were hospitalized in our hospital from 2020 January to 2022 December and diagnosed by polysomnography (PSG) (OSAS group, n = 79) were retrospectively studied and their relevant examination findings and demographic characteristics were recorded. Sex- and age-matched non-OSAS patients hospitalized at the same time were selected as the control group (control group, n = 28). Differences in peripheral blood neutrophil counts, lymphocyte counts, neutrophil-to-lymphocyte ratio (NLR), procalcitonin (PCT), and HBP were compared between the 2 groups. The predictive value of these markers for OSAS was analyzed using the receiver operating characteristic curve, and independent risk factors for OSAS were determined using logistic regression analysis. Peripheral blood was drawn from all patients in the fasting state of the morning. Results: The number of peripheral blood neutrophils, lymphocytes, and HBP was higher in the OSAS group than in the control group, and the differences were statistically significant (P all <.05), while the differences in NLR and PCT between the 2 groups were not statistically significant (P all >.05). Plasma HBP level had an area under the curve (AUC) of 0.79 (P < .0001) in determining OSAS, with a sensitivity of 80.49% and a specificity of 70.83% and the best cutoff value was >10.73 ng/ml. Combining body mass index (BMI), neutrophil, lymphocyte, and HBP improved the predictive value of OSAS with an AUC of 0.89 (P < .0001), a sensitivity of 80.49%, and a specificity of 87.50%. Logistic regression analysis showed that both BMI and HBP were independent risk factors for OSAS (PBMI < .001, PHBP < .01), while neutrophils and lymphocytes were not (P all >.05). Conclusions: This is the first study to objectively examine HBP in OSAS patients, and HBP is an independent risk factor that may serve as a diagnostic biomarker in OSAS. Patients with OSAS have an altered systemic inflammatory state and may be more prone to severe bacterial infections.

Investigating HIV-1 TAT protein role in Atherogenesis

Abstract With the recent development of antiretroviral therapy, HIV patients can live a long and normal lifespan. Recent evidence demonstrates an increased incidence of developing cardiovascular diseases (CVD) specifically atherosclerosis in aged HIV-infected patients. The adaptive and innate immune responses play a complex specific role in atherosclerotic plaque burden. However, specific mechanisms of the implication of the immune system in HIV-accelerated atherosclerosis are unclear. The protein Trans-Activator of Transcription–TAT is strongly associated with HIV infection as it enhances the efficiency of viral transcription. To understand the contributions of TAT protein in HIV-associated atherogenesis, a doxycycline-inducible astrocyte-specific HIV-1 TAT transgenic mouse was used. An injection of recombinant adeno-associated virus vector (AAV) encoding a gain-of-function mutant PCSK9 into mice and feeding with high fat with added cholesterol (DDC) and doxycycline (Dox) was used to promote atherosclerosis and TAT expression. After 16 wks of feeding, various tissues were collected and used for flow cytometry analysis (FACS) for immune profiling. Our preliminary data suggest that TAT is involved in the regulation of peripheral blood neutrophil numbers and therefore can serve as a regulator of inflammation in HIV-associated atherosclerosis. Future analysis will provide insight into potential mechanisms of HIV TAT-dependent involvement in atherosclerosis development and progression. This work was supported by project –initiation funds from the Center for Integrative Neuroscience and Inflammatory Diseases at EVMS and NIH NHLBI R01HL142129-04S1 grant.

A Mixture of Nucleosides and Nucleotides Increases Bone Marrow Cell and Peripheral Neutrophil Number in Mice Infected with Methicillin-Resistant Staphylococcus aureus

We studied the effects of a mixture of nucleosides and nucleotides on the peripheral neutrophil number and the proliferation of bone marrow cells in mice challenged with methicillin-resistant Staphylococcus aureus. BALB/c mice were fed a nucleotide-free 20% casein diet (control) or this diet supplemented with nucleosides and nucleotides orally (Expt. 1) or intraperitoneally (Expt. 2 and 3). On d 10, the mice were challenged intravenously with methicillin-resistant S. aureus (6.7 × 1012 colony forming units/L). In Expt. 1 and 2, numbers of total and differential counts of blood leucocytes were counted on d 0 (before), 1, 3 and 5 after the infection. In Expt. 3, 30 min before killing, bromodeoxyuridine (20 mg/kg), an analogue of thymidine, was administered intraperitoneally and its incorporation in the DNA synthetic phase of bone marrow cells was determined at 0 h (before), 3, 6 and 24 h after the infection. Mice fed the supplemented diet had higher (P < 0.05) leucocyte and neutrophil numbers on d 0 compared with the control group. The neutrophil numbers tended to be greater in the supplemented group at 1, 3 and 5 d after the infection. Intraperitoneal administration of nucleosides and nucleotides increased (P < 0.05) neutrophil numbers before and after the infection. Twenty-four h after the infection, incorporation of bromodeoxyuridine into the DNA synthetic phase of bone marrow cells in the group administered nucleosides and nucleotides was higher (P < 0.05) than in the control group. We conclude that, following methicillin-resistant S. aureus injection, intraperitoneal administration of a nucleoside-nucleotide mixture may stimulate bone marrow cell proliferation and increase the peripheral blood neutrophil numbers. Oral administration may elicit weaker effects.

Systemic inflammation in salivary gland cancer

Relevance. The inflammatory process and endogenous intoxication of the patient’s body has a negative impact on the course of many malignant neoplasms, including salivary gland cancer.The objective of the study was to determine the influence of some factors of systemic inflammation and endogenous intoxication in salivary gland cancer.Methods and materials. A prospective study included the data of 59 patients with salivary gland cancer. The influence of peripheral blood parameters and relative indicators characterizing systemic inflammation on overall and disease-free survival was assessed.Results. As a result of the multivariate analysis, it was revealed that an increase in the level of the relative number of peripheral blood neutrophils by more than 60.08 % increases the risk of death in patients from salivary gland cancer by 3.90 times (p=0.0456; HR 3.90: 95 % CI 1.03–14.79). The level of the absolute number of peripheral blood lymphocytes, not exceeding 1.49x109 /l, increases the risk of disease progression by 8.72 times (p=0.0002, R 8.72: 95 % CI 2.78–27.28).Conclusion. Individual factors of systemic inflammation and endogenous intoxication, it is advisable to evaluate at the stage of planning the primary treatment of patients with salivary gland cancer, in order to determine the prognosis of the disease and optimize the choice of tactics for the primary treatment of patients.

Patient Data Supports the Rationale of Low Dose Cyclophosphamide to Potentiate the Anti-Myeloma Activity of Daratumumab through Augmentation of Macrophage-Induced ADCP

Introduction:Daratumumab (Dara) is the first CD38-directed monoclonal antibody approved for the treatment of Multiple Myeloma (MM). Dara is thought to have pleiotropic mechanisms of activity, including antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). We focused our attention on potential mechanisms of enhancing macrophage (Mφ)-mediated ADCP and have highlighted that exposure of MM cells to low doses of Cyclophosphamide (Cy) led to a secretory response, which greatly augments Mφ-induced ADCP of Dara-coated MM cells1. Here, we sought to confirm this phenomenon in MM and establish the in-vivo relevance of these findings as seen in newly diagnosed MM patients enrolled in CyBorD-DARA phase 1b clinical trial (NCT02951819).Materials and Methods:Bone Marrow (BM) and peripheral blood (PB) samples were collected in EDTA vacutainers from newly diagnosed MM patients recruited to the CyBorD-DARA clinical trial both prior to- and 24hrs after Cy (150-300mg/m2) treatment. Total lymphocyte, monocyte and neutrophil counts were quantified by flow cytometry. Mononuclear cells were prepared and analysed by 8-colour flow cytometry to quantify monocyte, lymphocyte and plasma subsets as well as determine changes in surface expression profiles following treatment. As previously described, MM cells were conditioned with low-dose Cy for 24hrs. Media containing chemotherapy agents were removed and replaced with fresh media for 24hrs (TCM)1. THP-1 Mφ were conditioned with TCM for up to 48hrs and either characterised or incubated at 2:1 effector target ratio with CFSE labelled MM1S cells ± Cytochalasin D for 18hrs in the presence or absence of Dara/Isotype control. The % of Dara-specific tumour cell clearance was calculated to determine if the mechanism of tumour cell clearance was mediated by Mφ-mediated ADCP.Results:In patients receiving Cy for 24hrs, total BM and PB neutrophil numbers were increased (P<0.01, n=11), lymphocyte numbers were decreased (P<0.01, n=11) while monocyte numbers remain unchanged (P>0.05, n=11). More specifically, an increase in CD19+ B cells (P<0.05, n=6) and a decrease in natural killer cells (NK) (P<0.05, n=6) were observed. Interestingly, the number of Mφ precursor monocytes did not change following Cy treatment in both BM and PB of these patients. As innate immune cells are essential in immune surveillance, coupled with our finding that NK cells are reduced, this highlights a potential role for Mφ in Cy induced anti-tumour activity. Additionally, data previously presented highlighted that exposure of Mφ to TCM from Cy treated MM cells significantly enhanced % of Dara-specific clearance of MM cells) p<0.01). We also confirmed ADCP and ADCC as a likely mechanisms of tumour cell clearance in vitro.Furthermore, we observed a marked reduction in expression of the “don't eat me” antigen, CD47, on chemotherapy-treated MM cells in vitro, which we have now confirmed in MM cells isolated from PB and BM of patients (n=2) following Cy treatment, which may enhance phagocytosis. We also observed increased expression of other targetable markers including BCMA (p<0.05), SLAMF7 (p=0.053), PD-L1 (p<0.001) and PD-L2 (p<0.001) in both RPMI8226 or MM1S MM cell lines following low dose Cy treatment in vitro. To date, we can confirm similar findings for both BCMA (p<0.05, n=5) and SLAMF7 (p<0.05, n=3) in patient samples. Furthermore, our in vitro observation of induced CD64 Fc gamma receptor expression on conditioned Mφ following single-agent Cy treatment has been confirmed in Mφ isolated from both BM (P<0.05, n=11) and PB (P<0.01, n=11) of newly diagnosed MM patients following Cy treatment. This finding emphasizes the potential physiological relevance of our in vitro data, implicating Cy in the potentiation of Mφ mediated ADCP in Dara specific anti-tumour immunity.Conclusions:Observations in Mφ precursors isolated from patients' BM and PB following 24hrs Cy treatment, confirm our previous in vitro findings which suggest a role for Cy potentiation of Mφ mediated ADCP in anti-tumour efficacy of Dara. These findings provide a rationale for the efficacy observed to date when combining Cy with Dara in the ongoing CyBorD-DARA clinical trial. Additionally, increases in BCMA and SLAMF7 highlight the potential of other combination immune therapies to further enhance therapeutic efficacy in these patients.1. Rigalou et al, Blood 2016 128:2101 [Display omitted] DisclosuresChiu:Janssen: Employment. Sasser:Janssen Pharmaceuticals: Employment. Ryan:Janssen: Research Funding. O'Dwyer:Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlycoMimetics Inc: Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Nicotinamide (vitamin B3) treatment improves response to G‐CSF in severe congenital neutropenia patients

Nicotinamide (vitamin B3) treatment improves response to G‐CSF in severe congenital neutropenia patients

Decreased neutrophil levels in bronchopulmonary dysplasia infants

Previous studies have indicated that inflammation plays an important role in the occurrence and development of bronchopulmonary dysplasia (BPD); however, there were rare researches about the changes of neutrophils and their influence on the prognosis of BPD. Hence, we aimed to explore the changes in the number of peripheral blood neutrophils (PBNs), and the relationship between these changes and susceptibility to pulmonary infection among children with BPD. Firstly, the gene expression of lung tissues and the number of PBNs were respectively detected by RNA sequencing and complete blood count in the 85% O2-induced BPD model rats. Then it was analyzed the number of PBNs after birth and the incidence of pneumonia within 6 months of corrected age (CA) after discharge among full-term infants (FTIs: gestational age [GA] between 370/7 and 416/7 weeks, n=88), preterm infants with (PTIs-BPD: GA <32 weeks, n=35) or without BPD (PTIs-nBPD: GA <32 weeks, n=41). The levels of S100A8 and S100A9 mRNAs were significantly decreased in the lungs of BPD rats. Moreover, the number of PBNs was also decreased in BPD rats. The number of PBNs at birth in FTIs was significantly greater than that in PTIs-BPD or in PTIs-nBPD (p<0.001), while those between PTIs-BPD and PTIs-nBPD showed no significant difference (p>0.05). Although the peripheral blood neutrophils decreased overall after birth in both PTIs-nBPD and PTIs-BPD groups, only the reduction in the PTIs-BPD group was significant (p<0.001). Importantly, at 36-37 weeks of postmenstrual age (PMA), the number of PBNs in PTIs-BPD was significantly fewer than that in PTIs-nBPD (p<0.001). In addition, PTIs-BPD had a significantly higher incidence of pneumonia than PTIs-nBPD within 6 months of CA after discharge (p<0.01). The number of PBNs in PTIs-BPD decreased progressively when compared to that in PTIs-nBPD, which might contribute to their susceptibility to pulmonary infection.

Neutrophil-to-Lymphocyte Ratio and Intratumoral CD45RO-Positive T Cells as Predictive Factors for Longer Survival of Patients with Colorectal Liver Metastasis after Hepatectomy.

Colorectal cancer is the fourth most common malignancy across the world, and over 50% of patients had colorectal liver metastases (CLM). Activated neutrophils and tumor-infiltrating lymphocytes (TILs) are considered to interrupt progression of primary colorectal cancer; however, immunological host reactions to CLM have not been fully elucidated. We thus aimed to explore the prognostic implication of neutrophil-to-lymphocyte ratio (NLR) in peripheral blood and TILs in resected metastatic cancer tissues of 29 patients with CLM who underwent hepatectomy. To evaluate local immunological responses in CLM, we examined the infiltration of CD66b+ neutrophils and TILs, such as CD8+ T cells, CD45RO+ T cells, and forkhead box P3+ (FOXP3+) T cells. The presence of fewer than 4 tumors (p = 0.0005), the absence of distant metastasis (p = 0.018), adjuvant anti-cancer chemotherapy (p = 0.0013), and elevated NLR over 4.1 (p = 0.026) were found to be significant parameters related to longer survival after hepatectomy. Further, high numbers of infiltrated CD45RO+ T cells in CLM were significantly associated with longer patient survival (p = 0.020). The numbers of CD45RO+ T cells were correlated with those of CD8+ T cells (p = 0.008). The numbers of peripheral blood neutrophils were negatively correlated with those of CD45RO+ T cells (p = 0.038) and of CD66b+ neutrophils (p = 0.008) in CLM. The present data indicate that elevated peripheral blood NLR and high numbers of intratumoral CD45RO+ T cells are predictive of longer CLM patient survival after hepatectomy among current biomarkers.

A cohort study of immune and hematopoietic functionality changes in severe aplastic anemia patients treated with immunosuppressive therapy.

To investigate if variations in immune and hematopoietic parameters correlated with immunosuppressive therapy (IST) in severe aplastic anemia (SAA) patients.A total of 115 SAA patients who received IST were included. Their immune and hematopoietic functionality changes had been evaluated at 0, 0.5, 1, 2, and 3-year(s) IST.For SAA patients with complete remission (CR), the CD4+/CD8+T cell ratio continued to increase after a year of IST. The T helper (Th)1/Th2 ratio continued to decrease after 6 months of IST, as did the activated CD8+ T cell percentage. The myeloid dendritic cell (mDC)/plasmacytoid dendritic cell (pDC) ratio after 3 years of IST was significantly lower compared to that of untreated patients. The mDC/pDC and Th1/Th2 ratios exhibited positive correlation. The activated CD8+ T cell percentage and the number of peripheral blood neutrophils showed inverse correlation. For SAA patients with partial remission (PR), the CD4+T cell percentage increased at 1-year post-IST, but the later changes were not statistically significant. The other immune indexes of patients in partial remission group and nonremission (NR) group showed no obvious recovery. For all SAA patients, the percentage of T regulatory cells in CD4+ lymphocyte was higher in post-IST group compared to the pretreatment group.For SAA patients responded well to IST, increase in peripheral neutrophils and improvement in bone marrow myeloid cells were first observed followed reduction in the activated CD8+ T cell percentage, Th1/Th2 ratio, CD4+/CD8+T ratio, along with mDC/pDC ratio, all of which negatively correlated with the hematopoietic parameters. This demonstrates that IST prompts improvements of hematopoietic functionalities of the SAA patients by regulating their immune functionalities.

Minor populations of paroxysmal nocturnal hemoglobinuria-type cells in patients with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells under the influence of pro-inflammatory mediators and oligoclonal/monoclonal T-lymphocytes. Because patients with immune-mediated BM failure display frequently paroxysmal nocturnal hemoglobinuria (PNH)-type cells in the peripheral blood (PB), we investigated the possible existence of PNH-type cells in 91 patients with CIN using flow cytometry. The patients displayed increased proportions of PNH-type glycophorin A+ /CD59dim and glycophorin A+ /CD59- red blood cells (RBCs), FLAER- /CD24- granulocytes, and FLAER- /CD14- monocytes, compared to controls (n = 55). A positive correlation was found between the proportions of PNH-type RBCs, granulocytes, and monocytes and an inverse correlation between the number of PB neutrophils and the proportions of PNH-type cell populations. The number of patients, displaying percentages of PNH-type cells above the highest percentage observed in the control group, was significantly increased among patients with skewed compared to those with normal T-cell receptor repertoire suggesting that T-cell-mediated immune processes underlie the emergence of PNH-type cells in CIN. Our findings suggest that patients with CIN display PNH-type cells in the PB at a high frequency corroborating the hypothesis that CIN belongs to the immune-mediated BM failure syndromes.

Survival, but not maturation, is affected in neutrophil progenitors from GSD‐1b patients

Glycogen storage disease type 1b (GSD 1b) is caused by mutations in the Glucose-6-phosphate transporter and is characterized by impaired glucose homeostasis. In addition, GSD-1b is associated with chronic neutropenia resulting in recurrent infections and inflammatory bowel disease. It is unclear whether the neutropenia is solely due to enhanced apoptosis of mature neutrophils or whether aberrant neutrophil development may also contribute. Here we demonstrate that hematopoietic progenitors from GSD-1b patients are not impaired in their capacity to develop into mature neutrophils. However, optimal survival of neutrophil progenitors from GSD-1b patients requires high glucose levels (> 200 mg dl(-1)), suggesting that even under normoglycemic conditions these cells are more prone to apoptosis. Furthermore, analysis of cytokine levels in peripheral blood suggests an inflammatory state with an inverse correlation between the level of inflammation and the number of neutrophils. Finally, in some patients, with low numbers of peripheral blood neutrophils, high numbers of neutrophils were observed in the intestine. Together, these results suggest that the neutropenia observed in GSD-1b patients is not caused by impaired maturation, but may be caused by both increased levels of apoptosis and egress of neutrophils from the blood to the inflamed tissues.

The Nod2 Sensor Promotes Intestinal Pathogen Eradication via the Chemokine CCL2-Dependent Recruitment of Inflammatory Monocytes

The intracellular sensor Nod2 is activated in response to bacteria, and the impairment of this response is linked to Crohn's disease. However, the function of Nod2 in host defense remains poorly understood. We found that Nod2-/- mice exhibited impaired intestinal clearance of Citrobacter rodentium, an enteric bacterium that models human infection by pathogenic Escherichia coli. The increased bacterial burden was preceded by reduced CCL2 chemokine production, inflammatory monocyte recruitment, and Th1 cell responses in the intestine. Colonic stromal cells, but not epithelial cells or resident CD11b+ phagocytic cells, produced CCL2 in response to C. rodentium in a Nod2-dependent manner. Unlike resident phagocytic cells, inflammatory monocytes produced IL-12, a cytokine that induces adaptive immunity required for pathogen clearance. Adoptive transfer of Ly6C(hi) monocytes restored the clearance of the pathogen in infected Ccr2-/- mice. Thus, Nod2 mediates CCL2-CCR2-dependent recruitment of inflammatory monocytes, which is important in promoting bacterial eradication in the intestine.

BKT140 Is a Novel CXCR4 Antagonist with Stem Cell Mobilization and Antimyeloma Effects: An Open-Label First Human Trial In Patients with Multiple Myeloma Undergoing Stem Cell Mobilization for Autologous Transplantation

AbstractAbstract 2260 Background:BKT140 is a high affinity CXCR4 inhibitor with an extended K off-rate. Pre-clinical studies in animal models with BKT140 showed a robust mobilization of white blood cells (WBC) and hematopoietic stem cells (HSC). Furthermore, BKT140 also showed a direct anti-tumor effect against human-derived multiple myeloma (MM), lymphoma and primary leukemia cells and cell lines in vitro and in vivo, causing significant apoptosis. Aims:To assess BKT140 toxicity (primary endpoints), the mobilization capacity of CD34+ hematopoietic progenitors and CD138 MM cells, and pharmacokinetic (PK) and pharmacodynamic (PD) (secondary endpoints). Methods:16 MM patients in first CR/PR were included in a phase I/IIa study, in which escalating doses of BKT140 (30, 100,300,900 μg/kg) were administered together with a high-dose cyclophosphamide (Cy) (2 g/m2) and G-CSF (5 μg/Kg) for stem cell mobilization. G-CSF was started on day 5 post Cy and BKT140 was injected subcutaneously (SC) once on day 10. Toxicity, PK, and mobilization capacity (assessed by serial measurements of number of WBC and CD34+ and CD138+ cells) were measured pre- and post BKT140 administration. Results:BKT140 was well tolerated at all doses and none of the patients developed grade II-IV toxicity. BKT140 was rapidly absorbed with no observed lag time, with peak plasma concentrations occurring 0.5h after administration. Clearance was rapid, with a median terminal half-life of 0.69h. BKT140 administration resulted in a significant dose-dependent increase in the number of peripheral blood neutrophils, monocytes, lymphocytes, and CD34+ cells compared to the G-CSF/Cy individual patient baseline. The maximum increase in the number of WBC from baseline was observed within 8h following BKT140 injection, 2.5-, and 3.0-, 4.1- and 4.8-fold, for the 4 BKT140 doses, respectively. Furthermore, BKT140 administration resulted in a significant increase in the mean absolute PB CD34+ cells mobilized (6.6, 7.5, 11.2 and 20.6 ×106/kg) for the 4 BKT140 administered doses, respectively. Moreover, the number of aphaeresis was reduced from 2.25 procedures at the first two BKT140 doses to 1.25 and 1 aphaeresis at the highest BKT140 doses, respectively. An increase in the number of CD138+ cells was observed in 6 out of 6 pts that had CD138+ cells in their blood and were treated with lower doses of BKT140 (30 and 100 μg/kg). Interestingly, in pts that were treated with the highest doses of BKT140 (300 and 900 μg/kg) a reduced number of CD138+ cells was observed in 3 out of 7 pts that had CD138+ cells in their blood, whereas in 4 pts, an increase in the number of CD138+ cells was shown. Three pts who did not have CD138+ cells in their blood were not affected by BKT140. The BKT140 mobilized grafts were used for AutoSCT following 200 mg/m2 melphalan conditioning. Pts received an average of 5.3×106 CD34+ cells/kg. All transplanted pts rapidly engrafted (n=15). The median day for neutrophil (>500/mm3) and platelet (>20,000/mm3, >50,000/mm3,) was on day 11 (range, 0–13), day 11 (range, 0–14), and day 14 (range, 0–23), respectively. Conclusions:The current data suggests that BKT140 can safely be added to G-CSF-based harvesting regimens, can increase CD34+ cell mobilization and reduce the number of collection days. Furthermore, due to its ability to release MM cells from the bone marrow and stimulate their cell death, additional studies are warranted to further evaluate the effect of BKT140 as an anti-MM agent. Disclosures:Nagler: Biokine Therapeutics Ltd: Consultancy. Abraham: Biokine Therapeutics Ltd: Employment, Equity Ownership, Patents & Royalties. Wald: Biokine Therapeutics Ltd: Employment. Shaw: Biokine Therapeutics Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Eizenberg: Biokine Therapeutics Ltd: Employment, Equity Ownership, Patents & Royalties. Peled: Biokine Therapeutics Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Infection withAnaplasma phagocytophilumInduces Multilineage Alterations in Hematopoietic Progenitor Cells and Peripheral Blood Cells

Infection with Anaplasma phagocytophilum, a gram-negative, lipopolysaccharide (LPS)-negative, obligate intracellular bacterium, results in multiple peripheral blood cytopenias. We hypothesized that infection with this organism would result in decreased bone marrow (BM) function and shifts in hematopoietic progenitor cells (HPCs) and lineage-committed cells in a well-established murine model of infection. HPCs and lineage-committed progenitors were enumerated in the BM and spleen during acute infection. BM cytokine production and BM CXCL12 expression were determined. Infection resulted in peripheral blood bicytopenia, marked decreases in the number of lineage-committed HPCs in the BM along with concurrent increases in the number of lineage-committed HPCs in the spleen, and a mixed, predominantly myelosuppressive BM cytokine environment. There was significant downregulation of CXCL12 in BM cells that may have been partially responsible for changes in HPC trafficking observed. Changes occurred in the absence of direct pathogen infection of BM cells. Hematopoietic lineage assessment demonstrated that there was loss of erythrocytes and B lymphocytes from the BM along with increased granulopoiesis. These changes were accompanied by splenomegaly due to lymphoid hyperplasia and increased hematopoiesis, most notably erythropoiesis. These changes largely mimic well-described inflammation and endotoxin-mediated effects on the BM and spleen; however, the numbers of peripheral blood neutrophils appear to be independently modulated as granulocytic hyperplasia does not result in neutrophilia. Our findings highlight a well-conserved series of events that we demonstrate can be instigated by an LPS-negative pathogen in the absence of an endotoxin-mediated acute proinflammatory response.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Evaluation of the Immune System in Rats and Mice Administered Linear Ammonium Perfluorooctanoate

Repeated high doses of ammonium perfluorooctanoate (APFO) have been reported to affect immune system function in mice. To examine dose-response characteristics in both rats and mice, male CD rats and CD-1 mice were dosed by oral gavage with 0.3-30 mg/kg/day of linear APFO for 29 days. Anti-sheep red blood cell (SRBC) IgM levels, clinical signs, body weights, selected hematology, and lipid parameters, liver weights, spleen, and thymus weights and cell number, selected histopathology, and serum corticosterone concentrations were evaluated. In rats, linear APFO had no effect on production of anti-SRBC antibodies. Ten and 30 mg/kg/day resulted in systemic toxicity as evidenced by decreases in body weight gain to 74 and 37%, and increases in serum corticosterone levels to 135 and 196% of control, respectively. In mice dosed with 10 and 30 mg/kg/day, marked systemic toxicity and stress were observed, as evidenced by a loss in body weight of 3.8 and 6.6 g, respectively (despite a tripling of liver weight), approximately 230% increase in serum corticosterone, and increases in absolute numbers of peripheral blood neutrophils and monocytes with an accompanying decrease in absolute lymphocyte numbers. Immune-related findings at 10 and 30 mg/kg/day that likely represent secondary responses to the systemic toxicity and stress observed at these doses include: decreased IgM antibody production at 10 (20% suppression) and 30 mg/kg/day (28% suppression); decreased spleen and thymus weights and cell numbers; microscopic depletion/atrophy of lymphoid tissue at 10 (thymus) and 30 mg/kg/day (spleen). In summary, no immune-related changes occurred in rats, even at doses causing systemic toxicity. In mice, immune-related changes occurred only at doses causing significant and profound systemic toxicity and stress.

Exacerbated tissue destruction in DSS‐induced acute colitis of OPN‐null mice is associated with downregulation of TNF‐α expression and non‐programmed cell death

Osteopontin (OPN), a pro-inflammatory mediator, is constitutively expressed in normal gut and is upregulated in inflammatory colitis. To determine the significance of OPN in inflammatory bowel disease, we studied the development of acute, experimental colitis induced by dextran sulfate sodium (DSS) in OPN-null and wild-type (WT) mice. OPN expression was markedly increased in WT diseased colons, while a higher disease activity index, including spleen enlargement, bowel shortening, and mucosal destruction, was observed in OPN-null mice. Although peripheral blood neutrophil numbers were lower in DSS-treated OPN-null mice, tissue myeloperoxidase levels, reflecting enhanced neutrophil activity, were increased in the diseased colons. In comparison, lymphocyte numbers in peripheral blood were increased earlier than in DSS-treated WT mice. Despite a significantly greater spleen enlargement, flow cytometric analysis of splenocytes from the DSS-treated OPN-null mice revealed lower numbers of differentiated macrophages and (CD4+ and CD8alpha+) lymphocytes. Whereas pro-inflammatory cytokines, including G-CSF, RANTES, MIP1alpha, and TNF-alpha, were increased < 10-fold in DSS-treated WT splenocytes, expression of these cytokines was dramatically suppressed in the DSS-treated OPN-null splenocytes as well as gut tissues. The suppressed TNF-alpha response in OPN-null mice was reflected in a marked increase in non-apoptotic cell death in diseased colons. Collectively, these studies demonstrate that OPN is required for mucosal protection in acute inflammatory colitis.

Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen-induced arthritis.

Annexin 1 (Anx-1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx-1 in experimental arthritis and in GC-mediated inhibition of inflammation, using antigen-induced arthritis (AIA) in Anx-1 knockout (Anx-1(-/-)) mice. Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti-mBSA IgG were determined by enzyme-linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction. A significant exacerbation of arthritis was observed in the Anx-1(-/-) mice compared with wild-type (WT) mice. This was associated with increased mRNA expression of synovial interleukin-1 beta, tumor necrosis factor alpha, interleukin-6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx-1(-/-) mice, and also significantly reduced the serum levels of anti-mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx-1(-/-) mice. Anx-1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.

The effects of a single injection of dexamethasone-21-isonicotinate on the lymphocyte functions of dairy cows at two weeks post partum.

Dexamethasone is a potent therapeutic for treatment of the fatty liver syndrome or primary ketosis in post partum dairy cows. Reservations exist, however, among practitioners with respect to the risk of immunosuppression induced by corticosteroids. The aim of this study was to investigate the effect of a single injection of dexamethasone-21-isonicotinate on distinct immune functions of postpartum dairy cows because only scarce information is available on the effects of corticosteroid preparations when administered at a dosage and frequency for treatment of the fatty liver syndrome or primary ketosis. Sixteen Swedish red-pied dairy cows, between days 9 and 15 post partum, were allotted to either a control group (n = 8) or a treatment group (n = 8). The cows in the treatment group received a single intramuscular injection of a dexamethasone-21-isonicotinate suspension at a dosage of 0.02 mg/kg i.m. at the start of the experiment. White blood cell counts and selected lymphocyte functions (lymphocyte proliferation, expression of lymphocyte markers and the b2 and a4 chain of adhesion molecules belonging to the integrin family) and some parameters of the energy metabolism (glucose, insulin) were determined before the administration of corticosteroids (day 0) and subsequently at days 2, 4, 7 and 9 of the experiment. Changes in glucose and insulin were within the target range for treatment of the fatty liver syndrome or primary ketosis. Significant (P < 0.05) increases in the number of circulating white blood cells were observed in treated cows on the second day following treatment which was exclusively caused by an increase in the number of circulating neutrophils. Lymphocyte blastogenesis in response to ConA and the percentages of lymphocytes positive for CD2, CD4, CD8, CD49d and CD18 as well as the intensity of CD49d expression did not differ between the treatment and control groups. There was, however, a significant reduction (P < 0.01) in the intensity of CD18 expression on lymphocytes in the treated animals on the fourth day after treatment. In conclusion, a single administration of dexamethasone-21-isonicotinate in a dosage of 0.02 mg/kg i.m. at two weeks post partum in healthy cows had a significant but highly transient effect on CD18 expression on lymphocytes and the number of peripheral blood neutrophils, but did not affect lymphocyte blastogenesis or lymphocyte subpopulation patterns in peripheral blood.

Proteolytic enzyme levels are increased during granulocyte colony-stimulating factor-induced hematopoietic stem cell mobilization in human donors but do not predict the number of mobilized stem cells.

Previous studies from our laboratory indicate that functional, mature neutrophils are essential for interleukin-8 (IL-8)-induced stem cell mobilization. To study a possible role of neutrophils in granulocyte colony-stimulating factor (G-CSF) induced hematopoietic mobilization, we assessed the number of circulating CD34+ cells in healthy allogeneic stem cell donors on days 3, 4, and 5 of mobilization for comparison with the number of peripheral blood neutrophils and the plasma levels of IL-8, Flt3 ligand (FL), matrix metalloproteinase-9 (MMP-9), and human neutrophil elastase (HNE). Thirty-seven of 45 donors required 1 day of apheresis to obtain 5 x 10(6) CD34+/kg recipient body weight (high responders), the remaining 8 donors required 1 extra day of apheresis on day 6 (low responders). On day 5, CD34+ numbers in the blood were significantly highe in high responders (116 x 10(3) +/- 10.4/ml) than in low responders (54.1 x 10(3) +/- 10.3, p < 0.001). In all donors, MMP-9 and HNE levels were increased compared to nonmobilized individuals, but in high responders, plasma MMP-9 levels on days 3-5 of mobilization were substantially higher than in low responders (p < or = 0.02 for MMP-9 and p = 0.89, p = 0.05 and p = 0.52 for HNE on days 3, 4, and 5, respectively). These results are in accordance with the hypothesis that neutrophils play a role in G-CSF-induced mobilization through the release of proteases such as MMP-9 and elastase. No change in plasma levels of IL-8 or Flt3 ligand was observed, suggesting that these cytokines do not play a role in stem cell mobilization. However, because stem cell numbers could not be predicted by proteolytic enzyme levels and/or neutrophil numbers, other undefined factors may be more important.