Number Of Peripheral Blood Lymphocytes Research Articles

Bortezomib and IL-12 produce synergetic anti-multiple myeloma effects with reduced toxicity to natural killer cells

The aim of this study was to examine the hypothesis that a combination of proteasome inhibition by bortezomib and immune therapy with interleukin-12 (IL-12) can produce enhanced antitumor efficacy relative to the effects of either of these agents alone. A mouse xenograft model of myeloma was developed. The mice were randomly divided into saline control (NS), IL-12 (0.4 µg/animal; intraperitoneal), bortezomib (0.75 mg/kg; intravenous), and bortezomib+IL-12 groups. Effects of treatments on tumor growth were assessed by before and after treatment comparisons and group comparisons. The effects of various treatments on the number of peripheral blood lymphocytes and natural killer (NK) cells were assessed by complete blood count and flow cytometry analysis. The cell-killing function of NK cells in splenocytes was evaluated using the lactate dehydrogenase release assay. IL-12 treatment alone produced a mild decrease in tumor volume compared with control (P>0.05). Bortezomib alone resulted in substantial inhibition of tumor growth at varying time points, reaching ~65 and ~60% reduction in tumor volume after 15 and 21 days of therapy, respectively. At the same time points, the combination therapy produced ~75 and ~84% decreases in tumor growth, respectively, which were significantly greater than the reduction produced by bortezomib monotherapy. Tumors resumed growth upon termination of bortezomib treatment at 2 weeks, although the tumor volume was still significantly smaller than that in the time-matched NS and IL-12 animals. This rebound of tumor growth was completely prevented with the combination therapy, and tumor volume continued to decrease throughout the time course. The percentage and total number of NK cells were significantly decreased after bortezomib monotherapy and combination therapy; however, they remained unaltered after IL-12 treatment compared with no treatment. Further, combination therapy significantly restored the bortezomib-induced functional impairment of the cell-killing capability of NK cells, relative to bortezomib alone. We conclude that the bortezomib-IL-12 combination therapy offers superior antitumor efficacy over monotherapy with either bortezomib or IL-12 in a mouse model of myeloma. Restoration of bortezomib-induced functional impairment of NK cells by IL-12 may be a mechanism for the synergetic effects of the two agents. Therefore, a combination of the two agents may represent a more rational therapeutic approach for myeloma.

L-selectin deficiency decreases aortic B1a and Breg subsets and promotes atherosclerosis.

There is a significant recruitment of leucocytes into aortas during atherogenesis. L-selectin regulates leucocyte migration into secondary lymphoid and peripheral tissues and was proposed to play a role in leucocyte homing into aortas. Here, we determine the role of L-selectin in atherosclerosis. L-selectin-deficient Apoe-/- (Sell-/-Apoe-/-) mice had a 74% increase in plaque burden compared to Apoe-/- mice fed a chow diet for 50 weeks. Elevated atherosclerosis was accompanied by increased aortic leucocyte content, but a 50% reduction in aortic B cells despite elevated B cell counts in the blood. Follicular B cells represented 65%, whereas B1a and regulatory B cells (Breg) comprised 5% of aortic B cells. B1a and Breg cell subsets were reduced in Sell-/-Apoe-/- aortas with accompanied two-fold decrease in aortic T15 antibody and 1.2-fold decrease of interleukin-10 (IL-10) levels. L-selectin was required for B1 cell homing to the atherosclerotic aorta, as demonstrated by a 1.5-fold decrease in the migration of Sell-/-Apoe-/- vs Apoe-/- cells. Notably, we found a 1.6-fold increase in CD68hi macrophages in Sell-/-Apoe-/- compared to Apoe-/- aortas, despite comparable blood monocyte numbers and L-selectin-dependent aortic homing. L-selectin had no effect on neutrophil migration into aorta, but led to elevated blood neutrophil numbers, suggesting a potential involvement of neutrophils in atherogenesis of Sell-/-Apoe-/- mice. Thus, L-selectin deficiency increases peripheral blood neutrophil and lymphocyte numbers, decreases aortic B1a and Breg populations, T15 antibody and IL-10 levels, and increases aortic macrophage content of Sell-/-Apoe-/- mice. Altogether, these data provide evidence for an overall atheroprotective role of L-selectin.

Abstract 9852: T Lymphocyte Depletion Following Primary Percutaneous Coronary Intervention Predicts Infarct Size and Ischemia Reperfusion Injury

Background: We have previously demonstrated acute changes in lymphocyte numbers in peripheral blood following reperfusion with primary percutaneous coronary intervention (PPCI) for STEMI. The goal of this study was to investigate a potential role for T cells in ischemia reperfusion (I/R) injury. Methods: We analyzed blood from 22 STEMI patients undergoing PPCI. Blood was taken at the start of the procedure, and at 15, 30, 90 minutes, and 24 hours post reperfusion. In 5 patients with infarcts due to LAD occlusion, coronary sinus (CS) blood was also obtained 30-60min after reperfusion along with simultaneous aortic blood. 8-colour flow cytometric analysis was performed on the fresh blood to quantify lymphocyte populations. Cardiac MRI was performed at 1-7 days, and left ventricular ejection fraction, infarct size (measured by late gadolinium enhancement), microvascular obstruction (MVO), area at risk and salvage index quantified. Results: T-lymphocytes dropped between 0 and 90 minutes by a mean of 42% (0min mean 1767/μl, 90min 931/μl), CD4 T-helper cells by 31%, and CD8 cytotoxic T-cells by 55%. The change in total T-cells correlated with infarct size (r=-0.78, p<0.001), as did that of CD4 (r=-0.66, p<0.01) and CD8 T cells (r=-0.76, p<0.001). The T cell drop between 0 and 90 minutes also correlated strongly with MVO (r=-0.72, p<0.01), as did the CD4 (r=-0.66, p<0.01) and CD8 T cell drop (r=-0.77, p<0.001). Myocardial salvage index correlated strongly with the change in T cells (r=0.85, p<0.001), CD4 T cells (r=0.76, p<0.01) and CD8 T cells (r=0.76, p<0.01) between 30 and 90 minutes. Prior to reperfusion 6.4% of CD8 T cells were activated (CD69+), with progressive loss of CD69+ cells after 90 minutes (4.3%) and 24 hours (2.6%, 0min vs 24hr p<0.01). However, CS sampling showed an increase in CD69 expression between aortic and CS blood, suggesting activation of T cells as they pass through the myocardial vascular bed. Conclusion: Acute changes in T lymphocyte numbers following reperfusion after PPCI predict infarct size, MVO, and salvage index. The drop in these cells following reperfusion, and the dynamics of CD69 expression, suggest a potential role for CD69+ activated T cells in I/R injury. This requires further investigation and may represent a novel therapeutic target.

Brief Report: Deficient Thymic Output in Rheumatoid Arthritis Despite Abundance of Prethymic Progenitors

ObjectiveTo determine the frequencies of common lymphoid progenitors (CLPs) and recent thymic emigrants (RTEs) in patients with rheumatoid arthritis (RA) and healthy control subjects.MethodsFlow cytometry was performed to determine the frequencies of CLPs and RTEs in the peripheral blood of 101 control subjects and 51 patients with RA. Thirteen of these patients were also analyzed longitudinally for 6 months after initiation of treatment with a tumor necrosis factor (TNF) inhibitor.ResultsA significant correlation between the frequencies of CLPs and RTEs was observed in healthy control subjects. The frequencies of both CLPs and RTEs decreased with age and correlated inversely with absolute lymphocyte numbers in peripheral blood. In patients with RA, the frequencies of RTEs were significantly decreased compared with the frequencies in control subjects. Importantly, the frequencies of CLPs were significantly higher in patients with RA compared with control subjects. Therapeutic TNF blockade further increased the frequency of CLPs, thereby normalizing thymic output, as indicated by an increase in the number of RTEs.ConclusionThymic insufficiency in RA is not attributable to an inadequate supply of progenitor cells to the thymus. Thus, insufficient numbers of RTEs could result from inadequate thymic T cell neogenesis, or alternatively, could be a consequence of high CD4+ T cell turnover, homeostatic proliferation, and subsequent dilution of the RTE population.

A clinical observation of the influence of deep brain stimulation on peripheral blood lymphocytes in patients with Parkinson's disease

Objective To study the changing in number of peripheral blood lymphocyte (PBL) of patients with Parkinson's disease (PD) after deep brain stimulation (DBS), and to explore the mechanism of DBS in treating PD. Methods One hundred and thirty PD patients were divided into 2 groups, namely, non-DBS group [N = 105; 68 males and 37 females; mean age (61.54 ± 10.44) years; mean duration (7.29 ± 4.57) years], and DBS group [N = 25; 16 males and 9 females; mean age (59.20 ± 10.67) years; mean disease duration (12.16 ± 4.79) years]. There were 73 healthy subjects [37 males and 36 females; mean age (61.89 ± 12.20) years] in control group. The differences of the number of PBL among the 3 groups were analyzed. Spearman's rank correlation analysis was used to assess the relationship between PBL number and influenzing factors [gender, age, disease duration, Unified Parkinson's Disease Rating Scale (UPDRS) Ⅲ score, Hoehn-Yahr (H-Y) stage, and drug equivalent daily dose]. Results The number of PBL in non-DBS group was less than that in control group ( P = 0.000). There was significant correlation between UPDRS Ⅲ and PBL number ( rs = ⁃ 0.403, P = 0.031). No correlation was found between PBL number and gender, age, disease duration, H-Y stage or drug equivalent daily dose ( P > 0.05, for all). No difference was shown between PBL number in control group and in DBS group ( P = 0.137) and no correlations were found with clinical variables ( P > 0.05). The PBL number in non-DBS group was less than that in DBS group ( P = 0.006). With the same H-Y stage, PBL number in non-DBS group was also less than that in DBS group in Mann-Whitney U test (H-Y 2.5: Z = ⁃ 2.197, P = 0.043; H-Y 3: Z = ⁃ 1.875, P = 0.027; H-Y 4: Z = ⁃ 3.760, P = 0.016). Conclusion The changing in the number of PBL is the specific feature of PD and may be correlated with the immuno-inflammation of central nervous system, which may be relieved by DBS.

Влияние препарата Б-190 и интерлейкина-1β на динамику количества клеток периферической крови и функциональный статус нейтрофилов облученных мышей

The estimation of efficiency of consecutive application of radioprotector B-190 and interleukin-1beta administered at early times after acute irradiation by studying the changes of the number of peripheral blood leukocytes, neutrophils and lymphocytes, as well as cytochemical research into the contents of glycogen, myeloperoxidase and alkaline phosphatase in neutrophils of the irradiated mice was carried out. It is established that administration of radioprotector B-190 at a doze of 50 mg/kg for 15 minutes before irradiation and interleukin-1beta at a doze of 50 mkg/kg for 15 minutes after irradiation reduces the expressiveness of post-irradiation leukopenia, accelerates restoration of the number of neutrophils and lymphocytes in peripheral blood, normalizes the functional status of neutrophils of irradiated mice to a greater degree than their isolated introduction.

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.

PECULIAR IMMUNOLOGICAL PROPERTIES OF CHRONIC POSTTRAUMATIC OSTEOMYELITIS

A comparative study of cellular immunity parameters was performed in patients with long bone chronic posttraumatic osteomyelitis in regard to osteomyelitis stages. Thirty patients with the fistulous form of long bone chronic posttraumatic osteomyelitis and 13 patients with long bone chronic posttraumatic osteomyelitis in the remission stage were examined using laser flow citometry. It was revealed that a moderate leukocytosis, increased monocyte and neutrophile absolute number as well as increased CD25 and HLA-DR-positive T-lymphocyte number are typical for chronic osteomyelitis regardless of the pathological process stages. The remission stage in patients with chronic posttraumatic osteomyelitis is accompanied by moderate activation of humoral immunity (increased B-lymphocyte (CD19+) number and increased production of IgG). The decrease in the peripheral blood lymphocyte number and well as reduction of HLA-DR expression on monocytes creates prerequisites for the development of fistulous osteomyelitis form and can be an indication for administering the preparations of immunomodulating action.

From Genotypes of Immunoglobulin Constant Heavy G Chains (Fcγ) (GM) Genes (IGHG) to Phenotypes in Childhood Asthma

Background: IgE-mediated allergy is associated with immunoglobulin heavy constant G chain (Fcγ) (GM) genes (IGHG) on chromosome 14q32.3. Investigation of the alternative GM allotypes of γ3, γ1 and γ2 chains has disclosed new structural and functional IgG subclasses and B-cell variants, with possible effects on childhood asthma. Objective: To investigate different IGHG (GM) gene complexes in a childhood asthma population for allergy parameters. Methods:IGHG alleles and correlated allotypic (allelic) IgG subclass levels were analyzed with a sensitive indirect competitive ELISA in 10-year-old children with bronchial asthma. Individual IGHG diplotype-, genotype- and haplotype-related B cells were compared for allelic IgG subclass levels, IgE sensitization, IgE, IgA and IgM levels, and numbers of peripheral blood eosinophils and lymphocytes. Results: The group with homozygous IGHG*bfn/*bfn (B1/B1 cells) demonstrated low IgG1*f levels (p < 0.001) but increased IgG2*n levels (p < 0.001) together with increasd IgE and IGHG2*n gene dose-dependent IgE sensitization (atopic phenotype). The IGHG*bf–n/*bf–n (B2/B2 cells) demonstrated low IgG1*f (p < 0.05) and IgG2*–n (p < 0.001) and the IGHG*ga–n/*ga–n (B4/B4 cells) low IgG1*a (p < 0.001) and IgG2*–n (p < 0.02) together with low IgE sensitization (non-atopic phenotype). B*^bfn (B1) and B*^bf–n (B2) demonstrated increased numbers of peripheral blood eosinophils, compared to B*^gan (B4) cells, which demonstrated increased peripheral blood CD8 lymphocytes instead. Conclusion:IGHG diplotypes present different phenotypes in childhood asthma. The IGHG2*n dose relationship to IgE sensitization and increased IgG2*n levels in IgE sensitized are risk markers for IgE-mediated asthma. The opposite IGHG2*–n presents non-IgE-mediated asthma and IgG subclass deficiencies.

The use of IKK inhibitor as a lymphocyte depleting agent (47.4)

Abstract Purposes: Transcription factor NF-κB and its activating kinase IκB Kinase β (IKKβ) are critical for inflammation and innate immunity. In our previoius report, MLN120B, an IKKβ inhibitor, would cause neutrophilia in mice but their peripheral blood lymphocyte numbers were not affected. We tried other IKK inhibitors; however, we found that feeding with BMS-345541, a highly selective IKKβ inhibitor, would cause lymphopenia in mice. We explored the possibility of BMS-345541 as a therapeutic agent in depleting lymphocytes. Results: To characterize how BMS-345541 affects lymphocyte homeostatsis, C57BL/6 mice received vehicle, or BMS-345541 oral gavage once daily at different dose (10, 25, 50, and 100 mg/kg body weight in 3% Tween 80), and mice were sacrificed on day 3 or day 5. We collected the peripheral blood for blood cell counts and also collected spleen and lymphnodes for histology examination. The lymphocytes, but not neutrophils, were significantly decreased in mice received BMS-345541. Our preliminary results showed that the effect of BMS-345541 on lymphocytes were through apoptosis rather than trapping the lymphocytes in lymphoid organs. BMS-345541 affected both T cells and B cells. Conclusion: We conclude that oral administration of BMS-345541 would reduce lymphocyte counts and could be helpful in depleting lymphocytes in circulation, which would be useful as a therapeutic agent for lymphoproliferative disorder.

Changes in Lymphocyte Count Induced by Repeated Cycles with Low-Dose Interleukin-2 and Interferon-α in 146 Patients with Renal Cell Carcinoma

The exact mechanism by which recombinant interleukine-2 and interferon-α modulate the immunological response, inducing long-term responses in metastatic renal cell carcinoma, is still not clear. The aim of the study was to analyze the modifications in peripheral blood lymphocytes during cycles of low-dose immunotherapy as a marker of the biological response to the treatment in 146 patients with renal cell carcinoma (advanced and localized disease). Peripheral blood lymphocytes were evaluated before and after every cycle of treatment. We found a statistically significant overall difference between pre- and post-cycle values (mean increase of 39%). The differences between pre- and post-cycle lymphocyte counts for each cycle were significant. Also, the post-cycle lymphocyte count of each cycle remained higher than the baseline value. Furthermore, pre-cycle lymphocyte counts of each cycle were still higher than the baseline value, with no difference between a pre-cycle lymphocyte mean value and the other one (except that between the first and second cycle). From the end of each cycle, but before starting the next one, the absolute value of lymphocytes fell on the average by 15-30%, concurring with the fact that, even starting from pre-cycle values higher than baseline, the immune system remains sensitive to chronically repeated stimulation by immunotherapy. We also found that non-metastatic patients had a higher number of peripheral blood lymphocytes than metastatic patients, whereas the latter had a lower immune response to therapy. The results support the idea that "maintenance" immunotherapy may not develop resistance over time in terms of biological response and thus may have a role as chronic therapy in combination with other drugs such as target therapy. We suppose that the immune system of patients with metastases is in a state of relative impairment, resulting in less sensitivity to immunostimulating agents.

Prevalence of Human Metapneumovirus Infection in Hospital Wards Handling Patients with Severe Motor and Intellectual Disabilities

Epidemics of infectious diseases often occur in hospital wards handling patients with severe motor and intellectual disabilities. However, the causative pathogens are not identified in half of these epidemics. Recently, an epidemic occurred in 2 wards consecutively in the National Hospital Organization Ehime Hospital in March-April, 2010. In this epidemic, 18 of 40 patients in one ward and 14 of 60 patients in another ward showed symptoms, and human metapneumovirus (HMPV) was detected from pharyngeal and/or nasal swabs of 4 patients. Phylogenetic analysis of the viral genomes showed that the virus belonged to subgroup B2, and the same virus had spread consecutively in 2 wards. High fever lasted for 5.3 days on average in the 32 inpatients, was mostly over 38 degrees C and was accompanied by productive coughs. In the 4 patients in whom human HMPV was detected, the number of peripheral blood lymphocytes decreased but that of monocytes increased. Pneumonia developed in two patients as shown by chest radiography, and an increased number of peripheral blood neutrocytes and increased CRP levels were noted. Of the 151 inpatients, anti-human HMPV antibody was found in the sera of 143 (95%), and the relationship between high fever and antibody titer was not significant, indicating that HMPV infection could recur in spite of the presence of specific antibodies. Because human HMPV is highly prevalent, and causes high fever and pneumonia, hospital staff should be vigilant for any signs of this virus in hospital wards and take steps to prevent infection in long-term inpatients.

Functional polymorphism rs7072793 C &gt; T affect individual susceptibility to breast cancer by modulating CD25 transcription activity

Substantial evidence has demonstrated immune defects in breast cancer patients. They have decreased numbers of peripheral blood lymphocytes, but higher numbers of functionally suppressive CD4(+) CD25(+) Treg in both peripheral blood and tumor microenvironment. Constitutive high expression of CD25 is a pivotal characteristic of natural Treg cells. This study aims at investigating if CD25 variability affects breast carcinogenesis. Two polymorphisms (rs7072793 C > T, rs3118470 C > T) in the promoter of CD25 were selected and analyzed by a multiple independent case-control study to assess the association between CD25 genotypes and breast cancer risk. Genotyping a total of 1110 patients and 1060 healthy controls in Chinese populations showed that rs7072793 CT genotype had an odd ratio of 1.49 (95% confidence interval, 1.23-1.89) for developing breast cancer compared with CC genotype, the rs7072793 TT carriers had a further increased risk of breast cancer (OR = 2.11; 95% CI = 1.66-2.87). Furthermore, our transient transfection which focused on reporter gene expression modulated by CD25 promoter demonstrated that the presence of an rs7072793 T allele led to greater transcriptional activity than the C allele. Similarly, rs13347 T carriers were shown to have larger proportion of CD4(+) CD25(+) Tregs in the PBMCs than C carriers in the flow cytometry analysis. However, no significant differences were found in genotype frequencies at rs3118470 C > T site between cases and controls. Our findings suggest that rs7072793 C > T genetic variation in CD25 genes may be genetic modifier for developing breast cancer.

Chickenpox in childhood is associated with decreased atopic disorders, IgE, allergic sensitization, and leukocyte subsets

Wild-type varicella zoster infection (WTVZV) up to 8 yr of age has been shown to protect against atopic dermatitis (AD) and asthma. We sought to determine whether WTVZV in childhood protects against atopic disorders, allergic sensitization or decreases serum Immunoglobulin E (IgE) levels. We conducted a retrospective, practice-based study of outpatient pediatric practices in NY. One hundred children with WTVZV up to 8 yr of age and 323 children who received varicella vaccine (VV) were randomly selected. WTVZV up to 8 yr of age is associated with decreased odds of subsequent asthma (exact logistic regression; OR = 0.12, 95% CI = 0.03-0.57, p = 0.003), allergic rhinoconjunctivitis (OR = 0.16, 95% CI = 0.05-0.49, p = 0.0003), and AD (OR = 0.57, 95% CI = 0.33-0.96, p = 0.02), but not food allergies (p = 0.78); decreased total serum IgE levels [mixed linear model, LSM (95% CI): 129.09 (33.22-501.63) vs. 334.21 (102.38-1091.04) IU/ml; p = 0.02] remained significant at all time intervals after WTVZV (<5, 5-10, and >10) compared with VV (p = 0.003-0.03). WTVZV was associated with decreased allergic sensitization (logistic regression, OR = 0.11, 95% CI = 0.03-0.38, p = 0.0004). WTVZV is also associated with persistently decreased numbers of peripheral blood lymphocytes (p < 0.0001) for up to 12 yr (p = 0.0003-0.047), monocytes (p = 0.002) for up to 16 yr (p < 0.001) and basophils at ages 4-6, 10-12, and 14-16 (p < 0.03). WTVZV up to 8 yr of age protects against atopic disorders, which is likely mediated by suppression of IgE production and allergic sensitization, as well as altered leukocyte distributions.

Lymphocyte Subtypes CD3 +, CD19 +, CD16 +CD56 +, CD4 +, CD8 +, and CD3 +HLA-DR + in Peripheral Blood Obtained From Patients After Thoracic Organ Transplantation

Introduction The aim of this study was to assess peripheral blood lymphocyte subtypes (CD3 +, CD19 +, CD16 +CD56 +, CD4 +, CD8 +, and CD3 +HLA-DR +) obtained from thoracic organ recipients at various periods after transplantation. Material and Methods Seventeen patients after lung transplantation (LT) and 5 patients after heart transplantation (HT) included 13 males (76.5%) and 4 females (23.5%) of overall mean age at the time of transplantation of 46.7 ± 11.55 years and mean body mass index of 21.1 ± 4. Lymphocyte phenotypes were estimated using Simultest IMK Plus. Results A significant decrease in lymphocytes of the majority of subtypes was observed at 1 year posttransplantation compared with normal ranges: CD19 + B lymphocytes in 56% of patients, CD8 + T cells among 48% and CD16 +CD56 + natural killer elements, 56%. In contrast, there were increased numbers of activated lymphocytes (CD3 +HLA-DR +). Beyond the 1-year observation, we observed a trend to normalize parameters among the majority of subjects. Conclusion A clear tendency to a decrease number of peripheral blood lymphocytes of various subtypes was observed among thoracic organ recipients in the first year posttransplantation with the exception of activated HLA-DR + cells. After the first year, there was slow restoration of lymphocytes.

Correlation between lymphocyte counts and response to outpatient intravenous interleukin-2 preceded by famotidine in metastatic melanoma.

8585 Background: Increased numbers of peripheral blood lymphocytes have been seen in patients receiving high-dose continuous and bolus infusion Interleukin-2 (IL-2) regimens. The toxicity of high-dose IL-2 due to a “capillary leak” syndrome requires in-patient administration of such regimens. Daily intravenous doses of IL-2 of 18-21.6 MIU/m2 over 15-30 minutes have been developed to attempt to lessen toxicity of this therapy. Famotidine may increase Interleukin-2 internalization by the IL-2 receptor on lymphocytes (Tsunoda, 1992). Previously, we have shown that patients with metastatic melanoma may be treated safely with outpatient intravenous Interleukin-2 preceded by intravenous famotidine daily for 3-5 days (Quan et al, 2003; Quan et al, 2010). The current study involves 17 patients with metastatic melanoma. Methods: Thirteen patients with stage IV melanoma received IL-2 18 MIU/m2 intravenously (IV) preceded by famotidine 20 mg IV daily for 3 days for six consecutive weeks. Four patients with metastatic melanoma received IL-2 21.6 MIU/m2 IV preceded by famotidine 20 mg IV daily for four days every 3 weeks. Peripheral blood lymphocyte counts were measured on the first day of each week of IL-2 therapy for all patients. Student’s t-test was used to compare the highest lymphocyte counts in responding patients vs. non-responders. Results: Four patients experienced either a complete (2) or partial (2) response. Thirteen patients were non-responders. The median lymphocyte count achieved in the responding patients was 4750/mm3 (range: 3100-5314/mm3) compared to the median of 2400/mm3 (range: 1500-4949/mm3) in the non-responders (p = 0.008). Conclusions: Peripheral blood lymphocyte numbers are significantly higher in patients with metastatic melanoma responding to outpatient intravenous Interleukin-2 preceded by famotidine than in non-responders. Further studies using flow cytometry and lymphocyte cytoxicity assays are currently being done.

Sensitivity of porcine peripheral blood leukocytes to gamma irradiation in vivo, in vitro and ex vivo

Purpose: The large white pig is a useful experimental model to compare in vivo, in vitro and ex vivo sensitivity of peripheral blood leukocytes to ionising radiation. Such studies are impossible to perform in humans and laboratory rodents due to ethical reasons and body size, respectively. We analysed dose- and time-dependent changes of lymphocyte and granulocyte absolute numbers in porcine peripheral blood after either whole-body irradiation (in vivo and ex vivo experiments) or exposure of porcine whole blood to γ-irradiation (in vitro experiments).Materials and methods: CytoCount™ absolute counting beads and light scatter analysis using a flow cytometer were used to determine major leukocyte subpopulation numbers in blood samples after red cell removal.Results: Similar to other species, lymphocyte numbers significantly decreased in pigs both in vivo and in vitro in a dose-dependent manner. Most importantly, our data clearly show that reduction of lymphocyte numbers after irradiation in vivo proceeds much faster than after irradiation in vitro and that granulocyte changes depend only on the time of analysis after irradiation.Conclusions: All three tested experimental arrangements demonstrated the radiosensitivity of lymphocytes and the radioresistance of peripheral blood granulocytes. These in vivo and in vitro approaches, as well as the newly introduced ex vivo observations, appear to be relevant to biodosimetry.

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.

Sphingosine 1-phosphate receptor type 1 regulates egress of mature T cells from mouse bone marrow

Sphingosine 1-phosphate (S1P) and its receptor, S1P receptor type 1 (S1P(1)), are essential for lymphocyte egress from secondary lymphoid organs (SLO). Fingolimod (FTY720), the S1P receptor modulator, inhibits lymphocyte egress from SLO and decreases circulating lymphocytes; however, it also induces a significant decrease in the number of peripheral blood lymphocytes in alymphoplasia (aly/aly) mice lacking SLO. In this study, we demonstrated that the administration of FTY720 induced sequestration of mature lymphocytes, particularly T cells, into the bone marrow (BM) in aly/aly mice, implying that the reduction of circulating lymphocytes in these mice by FTY720 was due to inhibition of lymphocyte egress from the BM. Since sequestration of mature T cells into the BM was also induced in normal mice by selective S1P(1) agonist or S1P lyase inhibitor, it is suggested that S1P(1) expression and the S1P gradient play an important role in egress of mature T cells from the BM. Prophylactic administration of FTY720 to ovalbumin (OVA)-immunized mice significantly inhibited footpad swelling induced by OVA challenging with a marked reduction of OVA-specific T(h) cells in the BM, indicating that immunomodulation by FTY720 is likely due to reduced circulation of antigen-specific T(h) cells. On the other hand, OVA-specific T(h) cells, like naive T cells, were also sequestered into the BM and SLO of OVA-immunized mice by a short exposure of FTY720 after OVA challenging. These results suggest that the S1P-S1P(1) axis plays a regulatory role in egress of mature T cells including antigen-specific T(h) cells from the BM.

Removal of Sphingosine 1-Phosphate Receptor-3 (S1P3) Agonism is Essential, But Inadequate to Obtain Immunomodulating 2-Aminopropane-1,3-diol S1P1 Agonists with Reduced Effect on Heart Rate

A series of 2-substituted 2-aminopropane-1,3-diols having a biphenyl moiety and their phosphate esters were synthesized to obtain sphingosine 1-phosphate receptor-1 (S1P(1)) receptor agonists with potent immunomodulatory activity accompanied by little or no effect on heart rate. Many of the synthesized compounds sufficiently decreased the number of peripheral blood lymphocytes. Some of the phosphates had potent agonism at S1P(1) but no agonism at S1P(3), which had been reported to be a receptor responsible for heart rate reduction. Although high S1P(1)/S1P(3) selectivity was considered to be favorable to reduce the effect on heart rate, almost all the phosphates showed a remarkable heart rate lowering effect in vivo. The results suggest that other factors in addition to S1P(3) agonism should be responsible for the heart rate reduction caused by S1P(1) agonists. Only 2-amino-2-[2-[2'-fluoro-4'-(4-methylphenylthio)biphenyl-4-yl]ethyl]propane-1,3-diol (6d) was identified as a desired S1P(1) receptor agonist having both the immunomodulatory activity and an attenuated effect on heart rate by a unique screening flow using in vivo evaluating systems primarily.