White Blood Cell Subsets Research Articles

Differential associations between white blood cell counts and fatigue in young and older adults.

The aims of this exploratory study were to study whether fatigue might be related to the cellular immune system by 1) analysing if the number of white blood cell subsets are related to fatigue and 2) if possible relationships vary in younger and older community-dwelling individuals. The participants were recruited from nine general practitioners in Aarhus County, Denmark and included 196 individuals aged 20-35 years and 314 individuals aged 70-85 years. The white blood cell counts included number of total leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. General fatigue was measured by a question from the SF-12 Vitality-Scale and mobility-related fatigue by the Avlund Mob-T Scale. Total number of lymphocytes was associated with fatigue in the old sample, both in the crude and adjusted analyses. Total number of leukocytes and netrophils were associated with fatigue in both age groups in the crude analyses. In the old sample the estimates were attenuated to non-significance when adjusting for physical activity and disability. In the young sample the estimates stayed statistically significant in the fully adjusted analyses regarding number of neutrophils, while the associations between fatigue and number of leukocytes were attenuated to non-significance when adjusting for depressive mood. Total number of leukocytes, lymphocytes and neutrophils were associated with fatigue in both age groups, while the explanatory factors for the associations differed by age group, in that the associations were partly explained by physical activity and disability in the old sample and partly by depressive mood in the young sample. The findings provide initial insight into the potential role of leukocyte, neutrophil and lymphocyte counts in the development of fatigue.

Folate-deficiency induced cell-specific changes in the distribution of lymphocytes and granulocytes in rats

Folate (vitamin B(9)) plays key roles in cell growth and proliferation through regulating the synthesis and stabilization of DNA and RNA, and its deficiency leads to lymphocytopenia and granulocytopenia. However, precisely how folate deficiency affects the distribution of a variety of white blood cell subsets, including the minor population of basophils, and the cell specificity of the effects remain unclear. Therefore, we examined the effects of a folate-deficient diet on the circulating number of lymphocyte subsets [T-lymphocytes, B-lymphocytes, and natural killer (NK) cells] and granulocyte subsets (neutrophils, eosinophils, and basophils) in rats. Rats were divided into two groups, with one receiving the folate-deficient diet (FAD group) and the other a control diet (CON group). All rats were pair-fed for 8 weeks. Plasma folate level was dramatically lower in the FAD group than in the CON group, and the level of homocysteine in the plasma, a predictor of folate deficiency was significantly higher in the FAD group than in the CON group. The number of T-lymphocytes, B-lymphocytes, and NK cells was significantly lower in the FAD group than in the CON group by 0.73-, 0.49-, and 0.70-fold, respectively, indicating that B-lymphocytes are more sensitive to folate deficiency than the other lymphocyte subsets. As expected, the number of neutrophils and eosinophils was significantly lower in the FAD group than in the CON group. However, the number of basophils, the least common type of granulocyte, showed transiently an increasing tendency in the FAD group as compared with the CON group. These results suggest that folate deficiency induces lymphocytopenia and granulocytopenia in a cell-specific manner.

Abstract 2512: PF-04605412 induces cytokine activation and marginalization of NK cells and macrophages in patients with solid tumor

Abstract Introduction: Integrin Δ5α1 is a fibronectin receptor involved in cell adhesion, proliferation, and survival expressed by cancer cells in addition to tumor-associated macrophages, fibroblasts, and endothelial cells. PF-04605412 is a fully human IgG1 mAb against integrin α1Δ5, with enhanced FcαRIII binding capacity, currently in Phase 1. Pre-clinical studies indicate this antibody has anti-angiogenic properties and is capable of inducing antibody dependent cellular cytotoxicity. We report on correlative biological studies designed to understand the underlying mechanisms for infusion reactions which occurred to some patients during the dose escalation. Methods: This sub-study includes 9 patients (5 female, 4 males; median age 58, ECOG PS 0-1) who received doses of PF-04605412 from 7.5 mg to 34 mg as 2 hrs infusion. Cytokine-like infusion reactions was observed at the first dose level (7.5 mg). To mitigate symptoms, subsequent patients were pre-medicated and the infusion time was extended to two hours. An extensive cytokine panel was assessed at time points corresponding to pharmacokinetic (PK) analysis. Circulating monocytes (CD14+/CD16+) and NK (CD56+/CD16+) cells were evaluated by flow cytometry. Cell-bound PF-04605412 was detected using Fab’2 anti-human antibody. PK analysis has been conducted in four patients to date (7.5 mg (n=3) and 11.25 mg (n=1). Results: Infusion reactions with premedications were mild and included chills, fever, rigors and hypotension (grades 1-2). A decline in white blood cell subsets occurred at the onset of fevers and chills, independent of the dose administered, and normalized within 24 hours post infusion. Activation of cell populations was evident by an increase in percentage of cells expressing CD16 at 24 hour post infusion compared to baseline. Persistent (up to 24 hours) in vivo cell-bound PF-04605412 was observed with the Fab’2 anti-human antibody in all patients. Cmax values for the 7.5 mg and 11.25 mg doses were 671 ng/mL and 933 ng/mL, respectively. PF-04605412 serum concentrations were below quantitation limit 24 hours post infusion at these two doses. Pro inflammatory mediators such as IL 6, IL 8, and TNF alpha typically increased within 1-1.5 hours post infusion and normalized over 10-24 hours. Marked increases were seen in several patients with the first infusion while this was reduced with subsequent doses. There was not a direct relationship between adverse events and the magnitude of cytokine increase. Conclusions: PF-04605412 leads to robust cellular activation and cytokine induction resulting from Fc- FcαRIII engagement of effector cells. Biopsies of metastatic sites pre- and post- treatment will be examined for evidence of effector cell accumulation at the tumor site and will be presented at the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2512. doi:1538-7445.AM2012-2512

Diallyl Disulfide Reduced Dose-Dependently the Number of Lymphocyte Subsets and Monocytes in Rats

Diallyl disulfide (DADS) is a major sulfur compound of garlic, and exerts anti-inflammatory, immune-modulatory, and enhancing sympathetic activity effects. However, it still remains unclear how DADS affects the distribution of white blood cell subsets, which is essential to execute effective immune responses and partially regulated by adrenal glucocorticoids. Therefore, we examined the dose-dependent effects of DADS administration on the circulating number of white blood cells (WBCs) and lymphocyte subsets, and plasma corticosterone concentration in rats. Male 10-wk-old Sprague Dawley rats were divided into the DADS-free and DADS-orally administered (dose=10, 20, and 40 mg/kg BW) groups. Blood samples were collected from the tail vein at 0, 1, 2, 4, and 6 h after the administration. DADS administration decreased dose- and time-dependently the circulating number of total WBCs, total lymphocytes, and monocytes. Within the lymphocyte subsets, the circulating number of T-lymphocytes and B-lymphocytes was significantly reduced 4 h after DADS administration in a dose-dependent manner, although that of natural killer (NK) cells was not affected. On the other hand, although DADS administration did not significantly change the circulating number of neutrophils, the circulating number of eosinophils and basophils showed a decreasing tendency after DADS administration. In contrast, plasma corticosterone concentration was increased 2 h after DADS administration in a dose-dependent manner. These results suggest that DADS administration reduces the circulating number of monocytes and lymphocytes, including especially acquired immune cells, via the action of corticosterone, and the effects are induced in a dose-dependent manner.

Leukopenia in Patients with Iron Deficiency Anemia

Abstract 5279 Background:There is a lack of clinical research regarding the effect of iron deficiency anemia on white blood cells (WBC).This study presents the incidence of leukopenia in patients with iron deficiency anemia and the relationship between the white blood cell count and the level of hemoglobin(Hb). Patients and Method:Medical records of patients over 12 years of age with iron deficiency anemia were reviewed. Patients with concomitant disease that may alter the WBC count such as hematologic malignancy, solid tumor, history of gastrectomy, vitamin B12 or folate deficiencies and active inflammation were excluded. Results:From January 2006 to December 2010, a total of 516 eligible patients were included. Median age was 47 years with female predominance (80.6%). Leukopenia was found in 91 patients (17.6%). The incidence of leukopenia was significantly higher in patients with hemoglobin level of less than 8 g/dL than those with higher level of hemoglobin (44/183, 24.0% vs. 47/333, 14.1%, P=0.005, Table 1). [Display omitted] The levels of WBC count were found to decrease in accordance with the decrease in hemoglobin level (Pearson’s coefficient: 0.178, P<0.001, Figure 1). The decrease in WBC count was seen more often in younger patients. (R=0.104, P=0.018) Also, lymphocytes were found to decrease in both total number(R=0.282, P<0.001) and percentage (R=0.122, P=0.006) within total WBCs in proportion to the decrease in Hb level. [Display omitted] Conclusion:This study shows the high incidence of leukopenia in patients with iron deficiency anemia. The severity of leukopenia correlates with the decrease of hemoglobin in dose-dependent manner. Among the WBC subsets, lymphocyte count was significantly affected. Disclosures:No relevant conflicts of interest to declare.

Integration-Mediated Activation of PRDM16 and HMGA2 in Multiple Clones without Adverse Hematopoietic Consequences Following Transplant of Autologous MGMTP140K Gene-Modified CD34+ Cells

Abstract 2053▪▪This icon denotes a clinically relevant abstractDrug resistance gene therapy with mutant MGMTP140K gene-modified hematopoietic CD34+ cells has been proposed to circumvent myelosuppression associated with alkylating agent chemotherapy; however, the safety of this approach has not yet been demonstrated. We have achieved successful engraftment of MGMTP140K gene-modified hematopoietic progenitor cells in 3 glioblastoma patients following BCNU (600mg/m2) conditioning, as part of a Phase I/II clinical study which includes post-transplant combination O6-benzylguanine (O6BG) and temozolomide (TMZ) chemotherapy. Two of these patients, one of whom remains alive with no evidence for disease progression at more than 2 years since diagnosis, received a total of 9 and 4 cycles of O6BG/TMZ chemotherapy, respectively. We observed transient increases in the number of circulating gene-modified white blood cells (WBCs) and granulocytes in these patients following each cycle. Analysis of CD34+ colony forming cells (CFCs) in peripheral blood revealed increases in the number of gene-modified CFCs over time and with multiple cycles of chemotherapy. Given this, we have conducted a longitudinal retroviral integration site (RIS) analysis in an attempt to identify any selective advantage for gene-modified clones following repeated O6BG/TMZ regimens. In the first patient, at day 200 following transplant, 2 separate RISs were identified, each mapping to the intronic region between exons 1 and 2 of the human PRDM16 gene, previously identified as being associated with insertional activation and clonal expansion in a gene therapy trial for X-linked chronic granulomatous disease (Ott et al 2006). Semi-quantitative capture site frequency analysis revealed that these 2 clones with PRDM16 integrations constituted >20% of the gene-modified cell pool, which included hundreds of RISs at this time point in this patient. Overall gene marking at this same time point was 39.9% of peripheral blood WBCs, indicating that as many as 8% of circulating WBCs arose from these two clones. In the second patient, 3 distinct RISs were identified mapping to the 3’untranslated region (UTR) of the HMGA2 gene, within a cluster of let-7 microRNA binding domains known to be responsible for post-transcriptional regulation of this gene product. While HMGA2-associated RISs have been reported in 2 other gene therapy trials for β-thalassemia (Cavazzana-Calvo et al 2010) and X-linked severe combined immunodeficiency syndrome (Wang et al 2010), the genomic loci of these RISs mapped to the third intron of this genomic sequence. Capture frequency analysis indicated that overall contribution of the most abundant HMGA2-associated clone identified in this study reached 5.2% of gene-modified peripheral blood WBCs at day 300 after transplant. Analysis of PRDM16 and HMGA2 transcripts in each patient’s cells by reverse-transcriptase real-time PCR indicated increased transcription of these genes in peripheral blood and bone marrow WBCs. Analysis of WBC subsets in the first patient found PRDM16 RISs present in granulocyte and CD3+ blood cell lineages, suggesting these clones originated from multipotential gene-modified hematopoietic stem cells capable of differentiating despite PRDM16 transactivation. Furthermore, since discontinuation of chemotherapy, we have observed a steady decline in levels of both PRDM16 clones, with complete disappearance of one clone. Extended analysis of HMGA2 expression and clone tracking in WBC subsets of the second patient is currently under way. Importantly, analysis of both patients bone marrow demonstrated normal cytogenetics, with no evidence for MDS or leukemia by hematopathology and flow cytometry. We believe that these data, in combination with other clinical gene therapy studies identifying similar clones, indicate the PRDM16 and HMGA2 gene sequences as “hot-spots” for either initial retrovirus integration in CD34+ hematopoietic progenitor and stem cells, or as genomic loci where aberrant gene expression, as a result of retrovirus integration, is associated with increased cell proliferation. At this time, these observations suggest that in the absence of underlying genomic disease, in vivo fluctuations in PRDM16- and HMGA2-associated clones following transplant may be a common, clinically inconsequential phenomenon in retrovirus-mediated gene therapy targeting CD34+ cell populations for modification. Disclosures:No relevant conflicts of interest to declare.

A Mechanistic Proof-of-concept Clinical Trial With JX-594, a Targeted Multi-mechanistic Oncolytic Poxvirus, in Patients With Metastatic Melanoma

JX-594 is a targeted and granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing oncolytic poxvirus designed to selectively replicate in and destroy cancer cells through viral oncolysis and tumor-specific immunity. In order to study the mechanisms-of-action (MOA) of JX-594 in humans, a mechanistic proof-of-concept clinical trial was performed at a low dose equivalent to ≤10% of the maximum-tolerated dose (MTD) in other clinical trials. Ten patients with previously treated stage IV melanoma were enrolled. Tumors were injected weekly for up to nine total treatments. Blood samples and tumor biopsies were analyzed for evidence of transgene activity, virus replication, and immune stimulation. The β-galactosidase (β-gal) transgene was expressed in all patients as evidenced by antibody induction. Six patients had significant induction of GM-CSF-responsive white blood cell (WBC) subsets such as neutrophils (25-300% increase). JX-594 replication and subsequent shedding into blood was detectable in five patients after cycles 1-9. Tumor biopsies demonstrated JX-594 replication, perivascular lymphocytic infiltration, and diffuse tumor necrosis. Mild flu-like symptoms were the most common adverse events. In sum, JX-594 replication, oncolysis, and expression of both transgenes were demonstrated; replication was still evident after multiple cycles. These findings have implications for further clinical development of JX-594 and other transgene-armed oncolytic viruses.

Long-term Regulation of Genetically Modified Primary Hematopoietic Cells in Dogs

We report long-term results from a large animal model of in vivo selection. Nine years ago, we transplanted two dogs (E900 and E958) with autologous marrow CD34(+) cells that had been transduced with a gammaretrovirus vector encoding a conditionally activatable derivative of the thrombopoietin receptor. Receptor activation through administration of a chemical inducer of dimerization (CID) (AP20187 or AP1903) confers a growth advantage. We previously reported responses to two 30-day intravenous (i.v.) courses of AP20187 administered within the first 8 months post-transplantation. We now report responses to 5-day subcutaneous (s.c.) courses of AP20187 or AP1903 at months 14, 90, and 93 (E900), or month 18 (E958), after transplantation. Long-term monitoring showed no rise in transduced cells in the absence of drug. Retroviral insertion site analysis showed that 4 of 6 (E958) and 5 of 12 (E900) transduced hematopoietic cell clones persisted lifelong. Both dogs were euthanized for reasons unrelated to the gene therapy treatment at 8 years 11 months (E958) and 11 years 1 month (E900) of age. Three clones from E900 remained detectable in each of two secondary recipients, one of which was treated with, and responded to, AP1903. Our results demonstrate the feasibility of safely regulating genetically engineered hematopoietic cells over many years.

White blood cell subsets are associated with carotid intima-media thickness and pulse wave velocity in an older Chinese population: the Guangzhou Biobank Cohort Study

Cross-sectional associations between white blood cell (WBC) count, lymphocyte and granulocyte numbers, and carotid intima-media thickness (IMT) and brachial-ankle pulse wave velocity (PWV) were examined in a novel older Chinese community sample. A total of 817 men and 760 women from a sub-study of the Guangzhou Biobank Cohort Study had a full blood count measured by an automated hematology analyzer, carotid IMT by B-mode ultrasonography and brachial-ankle PWV by a non-invasive automatic waveform analyzer. Following adjustment for confounders, WBC count (β=0.07, P<0.001) and granulocyte (β=0.07, P<0.001) number were significantly positively related to PWV, but not lymphocyte number. Similarly, WBC count (β=0.08, P=0.03), lymphocyte (β=0.08, P=0.002) and granulocyte (β=0.03, P=0.04) number were significantly positively associated with carotid IMT, but only the association with lymphocyte count survived correction for other cardiovascular risk factors. In conclusion, higher WBC, particularly lymphocyte and granulocyte, count could be used, respectively, as markers of cardiovascular disease risk, measured through indicators of atherosclerosis and arterial stiffness. The associations for WBC count previously observed by others were likely driven by higher granulocytes; an index of systemic inflammation.

Circulating lymphocyte is an important determinant of the effectiveness of preoperative radiotherapy in advanced rectal cancer

BackgroundAlthough preoperative radiotherapy (RT) is widely used as the initial treatment for locally advanced rectal cancer (RC) in the neoadjuvant setting, factors determining clinical response have not been adequately defined. In order to find other factors possibly related with radiosensitivity, we evaluated the relationships between circulating blood cell counts and RT effects.MethodsIn 179 cases with advanced RC, we retrospectively examined hemoglobin (Hb) levels and counts of white blood cells (WBC), platelets and WBC subsets before and after RT and investigated their associations with the complete response (CR) rate together with other clinicopathological factors.ResultsThe ratio of lymphocytes in WBC taken before RT was significantly greater in 15 CR cases as compared with those in non-CR cases. Patients with high lymphocyte percentages (25.7%) showed better outcome than the counterparts. Conversely, the ratio of neutrophiles was reduced in CR cases. The lymphocyte ratio showed an independent association with CR with multivariate analysis, and tended to be maintained at relatively high levels in CR cases.ConclusionsIn RC patients, peripheral blood lymphocytes have a significant impact on the CR rate in response to RT. Lymphocyte-mediated immune reactions are supposed to have positive roles on clinical response in radiotherapy for RC.

Central memory CD4+ T cells dominate the normal cerebrospinal fluid

To use cerebrospinal fluid (CSF) immune phenotyping as a diagnostic and research tool, we have set out to establish reference values of white blood cell (WBC) subsets in CSF. We assessed the absolute numbers and percentages of WBC subsets by 6-color flow cytometry in paired CSF and blood samples of 84 individuals without neurological disease who underwent spinal anaesthesia for surgery. Leukocyte (i.e., lymphocytes, granulocytes, and monocytes), lymphocyte (i.e., T [CD4(+) and CD8(+) ], NK, NKT and B cells), T cell (i.e., naïve, central memory, effector memory, and regulatory) and dendritic cell subsets (i.e., myeloid and plasmacytoid) were studied. CSF showed a predominance of T cells, while granulocytes, B and NK cells were relatively rare compared to blood. The majority of T cells in CSF consisted of CD4(+) T cells (∼70%), most of them (∼90%) with a central memory phenotype, while B cells were almost absent (<1%). Among the small population of dendritic cells in CSF, those of the myeloid subtype were more frequent than plasmacytoid dendritic cells (medians: 1.7% and 0.4% of leukocytes, respectively), whilst both subsets made up 0.2% of leukocytes in blood. This study reports reference values of absolute numbers and percentages of WBC subsets in CSF, which are essential for further investigation of the immunopathogenesis of neuro-inflammatory diseases. Furthermore, the relative abundance of CD4(+) T cells, mainly with a central memory phenotype, and the presence of dendritic cells in CSF suggests an active adaptive immune response under normal conditions in the central nervous system (CNS).

유도선수들의 체중감량이 면역기능 및 염증성 사이토카인에 미치는 영향

The aim of this research is to find out the influence on immune function(a white blood cell subset, immunity globulin) and inflammatory Cytokine (IL-1β, IL-6, TNF-α) based on Judo masters` weight loss in short-term & long-term. 8 Judo master-subjects had gone through weight loss to the 5% of original body weight in short term(for 5 days) afterward, in long term(for 14 days) for this research. Diet and exercise were performed in order to the weight loss. It revealed that body water, percent body fat, body fat mass, fat free mass, BMI got reduced meaningfully after the weight loss in short term(for 5 days) (p.001) and the rest of variables except for percent body fat got reduced meaningfully after the diet in long term(for 14days). There was a meaningful difference in body water, percent body fat, body fat mass, fat free mass, BMI(p001) between two groups. There was no meaningful change of white blood cell subset(neutrophil, lymphocyte, monocyte,) in case of immune function in the short & long term weight loss, but there was a meaningful increase in IgA in the long term diet even though there was no change of immunity globulin(IgA, IgG, IgM) in the short term diet. There was a meaningful difference in IgA, IgG, IgM in case of inter groups. In case of inflammatory Cytokine, there was no meaningful difference in IL-1β, IL-6, TNF-α based on the result of intra groups and inter groups after the short & long term weight loss. In result, it revealed that short & long term weight loss resulted in the decline of bodily composition, the partial change in immunity globulin(IgA) and no change in immune function & inflammatory cytokine. Considering the change of the decrease in immune function and the increase in inflammatory cytokine, it would be possible to provoke the inflammation and the decrease of immune function with the continuous short term weight loss.

Single Cell Network Profiling (SCNP): Mapping Drug and Target Interactions

Measuring target coverage of small molecule inhibitors is paramount-first, for selection of molecules to progress through the drug development process and second, once a candidate drug moves to clinical testing, for guiding dose/schedule selection. Single cell network profiling (SCNP) using multiparameter flow cytometry can measure compound effects on multiple signaling cascades in a cell-type-specific manner. We applied SCNP to a panel of compounds with reported inhibitory effects on Jak/Stat signaling using a novel system where modulation of multiple signaling cascades are simultaneously measured in discrete cell subsets in whole (ie, unfractionated) blood. Jak2 vs. Jak3 selectivity as well as "off-target" effects on other cell signaling pathways were measured using a combination of cytokines that target different white blood cell subsets, namely GM-CSF (monocytes/granulocytes), IL-2 (T cells), and CD40L (B cells). The compounds were then rank-ordered by potency and selectivity against the different pathways tested. Notably, SCNP performed in whole unfractionated blood compared to fractionated peripheral blood mononuclear cells (PBMC) from the same donors revealed potency loss for all compounds, with one exception. These studies show that SCNP can be used to efficiently measure a drug candidate's potency and selectivity in a physiologically relevant environment (eg, whole blood) and that robust IC(50) are attainable from rare subpopulations (<100 cells). The ability to generate in vitro IC(50) measurements in whole blood can be used not only for the preclinical selection of lead molecules, but also to determine the target plasma concentration for clinical studies and to measure target coverage after drug administration in early phase clinical trials. Knowledge of the compound plasma concentration necessary to achieve biochemical coverage permits rational design of clinical trials based on biologically active dose vs. the traditional maximum tolerated dose (MTD) design, which is better suited for cytotoxic, nontargeted drugs.

Relationship of lymphocyte count to effectiveness of preoperative radiotherapy in advanced rectal cancer.

e14101 Background: Although preoperative radiotherapy (RT) is widely used as the initial treatment for locally advanced rectal cancer (RC) in the neoadjuvant setting, factors determining clinical response have not been adequately defined. In order to find other factors possibly related with radio sensitivity, we evaluated the relationships between circulating blood cell counts and RT effects. Methods: In a total of 165 cases with advanced RC who received 50.4 Gy radiation before surgery, we retrospectively examined hemoglobin (Hb) levels and counts of white blood cells (WBC), platelets and WBC subsets before and after RT and investigated their associations with the complete response (CR) rate together with other clinicopathological factors. Results: After radiotherapy, Hb, WBC, and platelets were significantly reduced from pre-RT levels. In particular, the post-RT lymphocyte counts were markedly reduced to less than half of the pre-RT values (1,570 ± 676 to 605 ± 258, p < 0.001; average 42.4% ± 22.1%). Fifteen of the 165 patients (9%) showed CR clinically. Among these laboratory data, only lymphocyte counts were significantly greater in CR than in non-CR cases both pre-RT (1,990 ± 503 vs. 1,640 ± 662, p < 0.05) and post-RT (742 ± 198 vs. 567 ± 262, p < 0.01). CR rate showed positive associations with nodal metastasis, the circumferential extent of the tumor, and combination with chemotherapy (CRT). However, on multivariate analysis, only lymphocyte counts showed an independent association with CR. Conclusions: In RC patients, peripheral blood lymphocyte counts have a major impact on the CR rate in response to RT. This raises the possibility of lymphocyte-mediated immune reactions being essential for achieving complete eradication of tumor cells. Enhancement of immune function during RT may critically improve the effectiveness of RT for advanced RC. No significant financial relationships to disclose.

Factors influencing cord blood viability assessment before cryopreservation

Cord blood (CB) viability determines product quality and varies with time and temperature of exposure before cryopreservation. Global viability assessment may not reflect viability of white blood cell (WBC) subsets, CD34+ cell viability, or hematopoietic stem/progenitor cells function. We compared trypan blue (TB) and acridine orange/propidium iodide (AO/PI) staining with flow-cytometric (7-aminoactinomycin D [7-AAD]) viability in total WBCs (Tot-AAD), granulocytes, monocytes, lymphocytes, and CD34+ cells and total nucleated cell, CD34+, and colony-forming cell (CFC) recovery as a function of time and temperature (4, 24, and 37 degrees C) before cryopreservation. TB, AO/PI, and Tot-AAD viability was concordant up to 72 hours (4 degrees C) and 48 hours (24 degrees C) postcollection; however, CD34+ viability was significantly higher due to loss of viable granulocytes. In contrast, at "physiologic" temperature (37 degrees C), the decline in TB, AO/PI, and Tot-AAD viability was significantly lower than the rate of viable CD34+ and CFC loss. At all times and temperatures, CFC recovery correlated best with CD34+ viability and recovery. CB cell populations exhibit differential time- and temperature-dependent susceptibility to in vitro cell death; consequently, global viability measurements using TB, AO/PI, or 7-AAD (Tot-AAD) significantly underestimate (4-24 degrees C) or overestimate (24-37 degrees C) CD34+ viability and CFC recovery. Our results demonstrate the limitations of global viability assessment with TB, AO/PI, and total AAD; endorse the routine use of CD34+ cell viability measurements; emphasize the importance of temperature control during shipment; and have implications with regard to establishing acceptable "cutoff" values for viability measurements and CB collection through processing time.

Effects of protected fish oil in the diet of periparturient dairy goats on phenotypic variation in blood and milk leukocytes

The goal of this study was to evaluate the effects of dietary protected fish oil (FO) on phenotypic variation in blood, milk leukocytes, and some productive and metabolic parameters in periparturient dairy goats. About 12 Alpine goats, selected from a larger group of second-parity animals, were fed from 15 days before kidding until the 15th day of lactation with the same basal diet that had been supplemented with either 47 g/head per day of FO or 47 g/head per day hydrogenated palm oil (PO). Dry matter intake, live body weight (LBW), body condition score (BCS), and productive performance were evaluated in 2 weeks after kidding. On days 15, 7, and 2 before kidding and days 2, 7, and 15 after kidding, plasma samples were collected for evaluation of alanine aminotransferase, aspartate aminotransferase, non-esterified fatty acids, glucose, beta-hydroxybutyrate, cholesterol, and urea levels. White blood cell and blood leukocyte subsets were counted in whole blood samples on the kidding day, as well as at 1, 4, and 15 days after kidding. In addition, milk somatic cell count, intramammary infection (IMI), and milk leukocyte subsets were evaluated on days 4 and 15 after kidding. No differences were observed in dry matter intake and BCS, while LBW was higher in FO-fed animals. Milk production and composition, plasma metabolites, and liver enzymes were similar in both experimental groups. Blood CD4 positive cells increased constantly (P = 0.05) in FO-fed group, while CD8 and CD14 cell counts significantly increased 4 days after kidding (P < 0.01). Milk leukocyte subsets showed a significant (P < 0.01) decrease in PO-fed group and a non-significant increase (P = 0.34) in FO-fed group, despite the presence of coagulase negative staphylococci IMI. The results of the productive performance evaluation agreed with those of many other studies, which did not find any significant differences between dairy goats fed diets enriched with FO or PO supplements. The administration of FO to dairy goats in transition appeared to affect the variation in blood leukocytes with a constant increase in CD4- and CD8-positive cells in comparison with a PO fat-supplemented diet.

Correlation of Telomere Length in Blood, Buccal Cells, and Fibroblasts From Patients with Inherited Bone Marrow Failure Syndromes.

Abstract 1083Poster Board I-105 IntroductionPatients with dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS), have defects in telomere biology. Measurement of telomere length (TL) by flow fluorescence in situ hybridization (FISH) in white blood cell (WBC) subsets is a very important diagnostic tool in DC. Quantitative PCR (Q-PCR) is a high-throughput method of TL measurement that is currently used in large epidemiologic studies. It measures telomere length as a ratio of TTAGGG repeat copy number to a single copy gene number (T/S), and is often used on DNA derived from blood or buccal cells. There are limited data on tissue-specific correlations of TL and on the comparability of studies using different methods. In order to better understand tissue TL variability, we evaluated intra-individual correlations of DNA extracted from frozen blood, fibroblasts, and buccal cells by Q-PCR; and flow-FISH TL in WBC subsets. Patients and MethodsWe studied 21 patients: 5 Diamond-Blackfan Anemia (DBA), 6 DC, 5 Fanconi anemia (FA), and 4 Shwachman-Diamond Syndrome (SDS), enrolled in the National Cancer Institute's IBMFS study who contributed blood, buccal cells, and fibroblasts, and also had WBC flow-FISH TL measured. Genomic DNA was extracted from either whole blood (n=4) or the WBC pellet remaining after ficoll separation (n=17, primarily granulocytes) by manual Gentra Puregene. DNA was isolated from fibroblasts and buccal cells by phenol-chloroform extraction. TL was measured by Q-PCR of DNA from the three tissue types, and by flow-FISH in WBC subsets (lymphocytes and cell types matched to the types of WBC used in DNA extraction). We used the Wilcoxon signed-rank test to compare the median ranks of paired TL, and Spearman rank correlation coefficient to measure the strength of the associations between these measurements. ResultsTL in patients with DC was significantly (p<0.01) shorter than in patients with other IBMFS in all Q-PCR measurements of DNA from blood, fibroblasts, buccal cells; and flow-FISH WBC lymphocytes and matched cell types of WBC used in DNA extraction (86% were from granulocytes). Across all disorders, the median Q-PCR TL was longer in fibroblast and buccal cell DNA when compared with peripheral blood DNA (overall T/S ratio= 1.42 and 1.16 vs. 1.05, p=0.0002, 0.002, respectively). Although the absolute values varied, we observed in all IBMFS overall statistically significant (p≤0.001) intra-individual correlations in TL measured by Q-PCR in blood and fibroblast (r=0.67), blood and buccal cells (r=0.77), as well as fibroblast and buccal cells (r=0.67). When stratifying by disease subtype, statistically significant (p<0.05) correlations of Q-PCR in blood and buccal cells (r=0.9), and fibroblast and buccal cells (r=0.9) were observed in DC, and Q-PCR in blood and fibroblasts (r=1.0) in SDS. None of the Q-PCR tissue correlations reached statistical significance in FA or DBA. In addition, overall statistically significant (p≤0.01) correlations between TL in flow-FISH WBC subsets and Q-PCR in blood, fibroblast and buccal cells were observed. The correlations were driven mainly by the correlations with Q-PCR in blood and buccal cells in DC patients and with Q-PCR in fibroblasts in SDS patients. ConclusionsOverall Q-PCR TL was correlated between blood, buccal cells and fibroblasts and in comparison with flow-FISH TL, but there was some variability within different IBMFS. The poor correlation between tissues in FA and DBA might reflect blood-specific TL attrition in response to bone marrow stress. Most important, the correlation profile observed in DC suggests that the heritability of TL in DC is tissue independent. Blood or tissue Q-PCR TL appear to be useful in identifying individuals with DC who are unable to provide a fresh blood sample (e.g. epidemiologic studies or patients after bone marrow transplantation), although larger studies are needed to confirm or refute these findings. When possible, flow-FISH TL in subsets of leukocytes remains the modality of choice for the diagnosis of DC. DisclosuresLansdorp:Repeat Diagnostics Inc.: Equity Ownership.

The Mind-Body Connection

The field of psychoneuroimmunology has witnessed an explosion of empirical findings during the last two decades. Research has documented the mechanisms through which stressful emotions alter white blood cell function. Stress diminishes white blood cell response to viral infected cells and to cancer cells. Moreover, vaccination is less effective in those who are stressed and wounds heal less readily in those who are stressed. While stress decreases the activity of some white blood cells, stress does not compromise the function of all types of white blood cells. Indeed, some types of autoimmune disease, which involve particular subsets of white blood cells, are exacerbated by stress. The literature documents the efficacy of talk-therapy interventions in altering immune system parameters and enhancing the body's ability to combat disease. The literature also documents the impact of the chronic stress of poverty on immune system function.

Effects of Gender on White Blood Cell Populations and Neutrophil-Lymphocyte Ratio Following Gastrectomy in Patients with Stomach Cancer

Alterations of absolute number or percentage of circulating white blood cell (WBC) subsets are associated with psychological and physical stress. Gender effects on the changes of circulating WBC subsets following surgical treatment have not been determined. Therefore, the current study aimed to determine whether circulating neutrophils, lymphocytes and monocytes, and neutrophil-lymphocyte ratio (N/L) are different following major surgery according to the gender. We studied 409 male patients and 212 female patients who underwent total or subtotal gastrectomy due to stomach cancer, from 1 January to 31 December in 2005. The WBC count and percentage of its subsets were obtained from database and N/L was directly calculated from the full blood count preoperatively, immediate postoperatively, and postoperative day 1, 3, 5 in a retrospective manner. Compared to preoperative values, neutrophilia, lymphopenia, monocytopenia, and increased N/L were associated with gastrectomy in all patients. In the comparison study between genders, there were significantly increased proportion of neutrophils, decreased lymphocytes and monocytes, and higher N/L in female patients than in male patients after gastrectomy. These findings indicate that female patients showed more immune-compromised response to gastrectomy than male patients.

A microfluidic device for practical label-free CD4(+) T cell counting of HIV-infected subjects.

Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4(+) T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4(+) T lymphocytes with high efficiency directly from 10 microliters of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R(2) = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings.