A Tandem-Locked Fluorescent NETosis Reporter for the Prognosis Assessment of Cancer Immunotherapy.

NETosis, the peculiar type of neutrophil death, plays important roles in pro-tumorigenic functions and inhibits cancer immunotherapy. Non-invasive real-time imaging is thus imperative for prognosis of cancer immunotherapy yet remains challenging. Herein, we report a Tandem-locked NETosis Reporter 1 (TNR1 ) that activates fluorescence signals only in the presence of both neutrophil elastase (NE) and cathepsin G (CTSG) for the specific imaging of NETosis. In the aspect of molecular design, the sequence of biomarker-specific tandem peptide blocks can largely affect the detection specificity towards NETosis. In live cell imaging, the tandem-locked design allows TNR1 to differentiate NETosis from neutrophil activation, while single-locked reporters fail to do so. The near-infrared signals from activated TNR1 in tumor from living mice were consistent with the intratumoral NETosis levels from histological results. Moreover, the near-infrared signals from activated TNR1 negatively correlated with tumor inhibition effect after immunotherapy, thereby providing prognosis for cancer immunotherapy. Thus, our study not only demonstrates the first sensitive optical reporter for noninvasive monitoring of NETosis levels and evaluation of cancer immunotherapeutic efficacy in tumor-bearing living mice, but also proposes a generic approach for tandem-locked probe design.

Multifactorial assessment of neutrophil chemotaxis efficiency from a drop of blood.

Following injury and infection, neutrophils are guided to the affected site by chemoattractants released from injured tissues and invading microbes. During this process (chemotaxis), neutrophils must integrate multiple chemical signals, while also responding to physical constraints and prioritizing their directional decisions to generate an efficient immune response. In some clinical conditions, human neutrophils appear to lose the ability to chemotax efficiently, which may contribute both directly and indirectly to disease pathology. Here, a range of microfluidic designs is utilized to test the sensitivity of chemotaxing neutrophils to various perturbations, including binary decision-making in the context of channels with different chemoattractant gradients, hydraulic resistance, and angle of approach. Neutrophil migration in long narrow channels and planar environments is measured. Conditions in which neutrophils are significantly more likely to choose paths with the steepest chemoattractant gradient and the most direct approach angle, and find that migration efficiency across planar chambers is inversely correlated with chamber diameter. By sequential measurement of neutrophil binary decision-making to different chemoattractant gradients, or chemotactic index in sequential planar environments, data supporting a model of biased random walk for neutrophil chemotaxis are presented.

Assessment of peripheral blood neutrophil respiratory burst, phagocytosis and apoptosis in obese non-insulin dysregulated horses.

Obesity is a highly prevalent condition in horses. Dysfunctional neutrophil activity has been reported in metabolically healthy obese humans, but minimal data exist regarding horses. The present study evaluated the effect of obesity on apoptosis, phagocytosis and oxidative burst activity of peripheral blood neutrophils from lean and obese non-insulin dysregulated horses. Seven lean (BCS, body condition score 4-6/9) and five obese (BCS 8-9) horses were enrolled in the study. All animals underwent two metabolic tests (OGT, oral glucose test; IRT, insulin response test) before their selection to ensure their metabolic status (non-insulin dysregulated). A single blood sample was obtained from each horse, and a discontinuous density gradient was carried out to isolate neutrophils. Phagocytosis, apoptosis and reactive oxygen species (ROS) production assays were performed for each animal. All statistical analyses were performed with unpaired two-tailed t-tests. Results indicate that neutrophils from obese non-insulin dysregulated horses have a significantly increased ROS production (P < .0001), with no changes observed on phagocytosis (P > .05) or apoptosis (P > .05) when compared to the control group. In conclusion, our results demonstrate that obesity per se, in absence of other endocrine disorders, alters neutrophil reactive oxygen species production. More research is needed to understand the role of obesity on the equine immune system of horses, and its role in the development of endocrine disorders.

Technical note: Assessment of neutrophil endocytosis and proteolytic degradation and its relationship with phagocytosis and oxidative burst in dairy cows.

Conventional assays of polymorphonuclear cell (PMN, neutrophil) function such as oxidative burst (OB) and phagocytosis (PC) are widely used to evaluate innate immunity in the transition period of dairy cows. Oxidative burst is commonly evaluated by measuring PMN median fluorescence intensity (MFI) involving the release of reactive oxygen species after in vitro stimulation. Phagocytosis can be measured by engulfment of fluorescent beads by PMN. DQ-ovalbumin (DQ-OVA) is a molecule suitable for the assessment of intracellular proteolytic degradation, so it might be informative about an additional pathway of pathogen handling by PMN. In this study, we evaluated the use of the DQ-OVA assay for the assessment of PMN function and the relationships among OB, PC, and DQ-OVA results in PMN isolated from blood of dairy cows between 5 and 21 d post partum. Results of the DQ-OVA validation assay were assessed with mixed linear regression models. Pearson correlation tests and kappa values for agreement were used to associate the MFI between each PMN function assay (OB, PC, and DQ-OVA). For the validation assay (9 cows in 3 replicates), PMN incubated with DQ-OVA were stimulated with IFN-γ or inhibited with cytochalasin D, and fluorescence was compared with untreated PMN. Stimulated and inhibited PMN had greater (970 ± 160 units) and lesser (593 ± 55 units) MFI relative to untreated PMN (791 ± 154 units), respectively, indicating that DQ-OVA fluorescence reflected enhanced or reduced endocytic and proteolytic function. To associate the MFI outcomes among OB, PC, and DQ-OVA, 153 samples from 40 cows were analyzed. Results showed significant, although weak association between DQ-OVA and PC MFI (Pearson r = 0.16). When values of MFI were categorized according to the first ("high" PMN functionality), second and third ("moderate" PMN functionality), or fourth ("low" PMN functionality) quartiles, agreement beyond chance (κ) was moderate: κ = 0.38 for DQ-OVA and OB, κ = 0.43 for DQ-OVA and PC, and κ = 0.43 for OB and PC. The DQ-OVA assay may complement traditional PMN functional assays because it provides additional information regarding the combination of endocytosis and proteolytic degradation, but it is not a substitute for assessment of OB or PC.

Transcription Factor T-bet Attenuates the Development of Elastase-induced Emphysema in Mice.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by peripheral airways inflammation and emphysema. Emerging evidence indicates a contribution of both innate and adaptive immune cells to the development of COPD. Transcription factor T-bet modulates the function of immune cells and therefore might be involved in the pathogenesis of COPD. To elucidate the role for T-bet in elastase-induced emphysema, pathological phenotypes were compared between wild-type and T-bet-/- mice. T-bet-/- mice demonstrated enhanced emphysema development on histological analyses, with higher values of mean linear intercept and dynamic compliance relative to wild-type mice. The number of neutrophils in BAL fluids, lung IL-6 and IL-17 expression, and the proportion of CD4+ T cells positive for IL-17 or retinoic acid receptor-related orphan receptor-γt were higher in T-bet-/- mice than in wild-type mice. Although T-bet downregulates cytokine expression in bone marrow-derived macrophages and MH-S cells, a murine alveolar cell line, depending on the surrounding environment, IL-6 expression in alveolar macrophages isolated from elastase-treated mice was not dependent on T-bet. Coculture of bone marrow-derived macrophages and CD4+ T cells revealed that T-bet regulation of IL-17 expression was dependent on CD4+ T cells. Neutralizing antibodies against IL-6R or IL-17 ameliorated the development of emphysema in T-bet-/- mice. In conclusion, we demonstrate that T-bet ameliorates elastase-induced emphysema formation by modulating the host immune response in the lungs.

Assessment of the neutrophil/lymphocyte ratio in patients with supraventricular tachycardia.

OBJECTIVE: The neutrophil/lymphocyte ratio (NLR) has been evaluated as a new predictor of cardiovascular risk. Inflammation has been shown to be associated with various arrhythmias including supraventricular tachycardias (SVTs). In this study, we aimed to investigate the relation between NLR and SVT in patients with a documented atrial tachyarrhythmia. METHODS: The study used a retrospective cross-sectional design. Patients who had SVT but were otherwise healthy were included. The exclusion criteria included drug use (except antiarrhythmic agents), morbid obesity, acute or chronic infection, inflammatory diseases, systemic diseases, and cancer. Total and differential leukocyte counts and routine biochemical tests were performed before the ablation procedure. RESULTS: The study included 150 patients with SVT and 98 healthy controls. The biochemical and hematological parameters were comparable between the groups, except neutrophil and lymphocyte counts. The neutrophil count was significantly higher (4.7±1.5x103/µL versus 4.1±1.0x103/µL; p<0.001) and lymphocyte count was significantly lower (2.2±0.6x103/µL versus 2.5±0.6x103/µL; p=0.001) in the SVT group than in the control group. As a result, the SVT group had significantly higher NLR values than the control group (2.2±0.9 versus 1.7±0.5; p<0.001). In addition, NLR values were higher in patients in whom tachycardia was induced during an electrophysiological study (EPS) (2.3±0.9 versus 2.0±0.8; p=0.02). The association between NLR and SVT remained significant after multivariate analysis (odds ratio: 1.5, 95% confidence interval: 1.001-2.263, p=0.049). CONCLUSION: Our study indicated that NLR values were significantly higher in patients with documented SVT than in control subjects. Inducibility of SVT during EPS was associated with higher NLR values.

Optofluidic device for label-free cell classification from whole blood.

A unique optofluidic lab-on-a-chip device that can detect optically encoded forward scattering signals is demonstrated. With a unique design of a spatial mask that patterns the intensity distribution of the illuminating light, the position and velocity of each travelling cell in the flow can be measured with submicrometer resolution, which enables the generation of a cell distribution plot over the cross section of the channel. The distribution of cells is highly sensitive to its size and stiffness, both being important biomarkers for cell classification without cell labelling. The optical-coding technique offers an easy route to classify cells based on their size and stiffness. Because the stiffness and size of neutrophils are distinct from other types of white blood cells, the number of neutrophils can be detected from other white blood cells and red blood cells. Above all, the enumeration of neutrophil concentration can be obtained from only 5 µL of human blood with a simple blood preparation process saving the usual steps of anticoagulation, centrifugation, antibody labelling, or filtering. The optofluidic system is compact, inexpensive, and simple to fabricate and operate. The system uses a commodity laser diode and a Si PIN photoreceiver and digital signal processing to extract vital information about cells and suppress the noise from the encoded optical scattering signals. The optofluidic device holds promise to be a point-of-care and home care device to measure neutrophil concentration, which is the key indicator of the immune functions for cancer patients undergoing chemotherapy.

Assessment of neutrophil apoptosis.

Timely neutrophil apoptosis and cell clearance by surrounding phagocytes are essential components of the resolution phase of acute inflammation. Programmed cell death by apoptosis occurs with maintenance of an intact cell membrane in order to prevent the release of histotoxic intracellular products such as proteases and reactive oxidant species into the extracellular surroundings as occurs during necrosis. Macrophage phagocytosis results in attenuation of toll-like receptor-driven proinflammatory mediator production further promoting inflammation resolution. Failures in this cascade of events can result in tissue damage, chronic inflammation and disease. By studying human neutrophil apoptosis and phagocytic clearance in vitro, it is possible to delineate key control mechanisms in the regulation of these processes and therefore also identify potential therapeutic targets. Apoptotic signalling pathways are well described in the literature using a variety of laboratory techniques. In this paper, we outline the key in vitro assays used to assess neutrophil apoptosis, activation of key components of the apoptotic machinery, and phagocytic clearance of these cells.

Assessment of priming of the human neutrophil respiratory burst.

Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce reactive oxygen species (ROS) such as superoxide anion (O2 (∙-)), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl), a process referred to as the respiratory burst. The enzyme responsible for this process is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox), and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. The NADPH oxidase is in a resting state in circulating neutrophils, and its activation can be induced by a large number of soluble and particulate agents such as the formylated peptide, formyl-methionyl-leucyl-phenylalanine (fMLF). This activation can be enhanced or "primed" by pro-inflammatory cytokines, LPS and other agents. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS that can damage surrounding tissues and participate to inflammation. In this chapter, we describe the techniques used to measure priming of the NADPH oxidase in human neutrophils.

Critical assessment of the value of sputum neutrophils.

Neutrophils are central to the pathogenesis of COPD, releasing a range of pro-inflammatory and tissue destructive mediators. Sputum neutrophil numbers are elevated in COPD patients compared to healthy controls. We critically appraise the potential of sputum neutrophils as a biomarker in COPD. We show that there is insufficient evidence to support the use of this biomarker to define a phenotype of patients with more severe disease characteristics or a different prognosis. However, sputum neutrophil measurements can be used to measure the effects of anti-inflammatory drugs for the treatment of COPD.

Automated assessment of the neutrophil and platelet activation status in patients with essential thrombocythemia.

Neutrophil and platelet activation are consistently found in essential thrombocythemia (ET), but the techniques employed to demonstrate such abnormalities are complex. To ascertain whether the ADVIA 120 analyzer can be employed to assess neutrophil and platelet activation status in ET, 55 such patients and the same number of matched healthy individuals were studied and the results correlated with neutrophil CD11b and platelet P-selectin expressions measured by flow cytometry. Compared with controls, ET patients had significantly higher values of neutrophil myeloperoxidase index (MPXI), mean platelet volume (MPV), platelet distribution width (PDW), and platelet component distribution width, and significantly lower values of neutrophil lobularity index and mean platelet component (MPC). Patients with the JAK2 mutation had significantly lower values of MPC and higher values of MPV and PDW than those with wild-type allele. A positive correlation was observed between MPXI and neutrophil CD11b expression and a negative correlation between MPC and platelet P-selectin expression. The intensity of the agreement between the variables obtained by the two methods was moderate. These results support the possible value of MPC as surrogate parameter of platelet activation in ET.

Flow cytometric assessment of leukocyte function in sickle cell anemia.

Sickle cell anemia is associated with susceptibility to infection due to hyposplenism and the reduced ability of neutrophils to kill pathogenic organisms. In this study, blood samples from sickle cell anemia patients were divided into two groups: the painful crisis group and the steady state group. Flow cytometric assessment of phagocytosis and burst formation of neutrophils and monocytes as well as basophil function were performed, and these were compared to those of age- and sex-matched normal control subjects. Neutrophils and monocytes in sickle cell anemia patients were significantly different from those in the normal control subjects in the areas of weaker phagocytosis, fewer ingested bacteria and reduced burst formation. Basophil degranulation was normal. This pilot study using flow cytometry explains in part the susceptibility to infection of sickle cell anemia patients despite their high neutrophil and monocyte counts.

Assessment of Streptococcus pneumoniae capsule in conjunctivitis and keratitis in vivo neuraminidase activity increases in nonencapsulated pneumococci following conjunctival infection.

PURPOSE: The pneumococcal capsule is required for pathogenesis in systemic infections, yet reports show most conjunctivitis outbreaks are caused by nonencapsulated pneumococci, while keratitis infections are caused by encapsulated strains. This study aims to determine the effect of capsule in pneumococcal keratitis and conjunctivitis in rabbit models of infection. METHODS: A capsule-deficient isogenic mutant was created using homologous transformation. Parent and mutant strains were injected within the upper bulbar conjunctiva (conjunctivitis) or into the corneal stroma (keratitis) of New Zealand white rabbits. Clinical examinations were performed 24 and 48 hr post-infection at which time corneas or conjunctivae were removed, homogenized, and plated to determine the recovered bacterial load. Whole eyes were removed for histological examination. The neuraminidase activity was determined following in vitro and in vivo growth. RESULTS: There were no significant differences in clinical scores between the eyes infected with the parent or mutant for either infection, nor was there a difference in the amount of bacteria recovered from the cornea. In the conjunctivae, however, the mutant strain was cleared by the host faster than the parent strain. Histological examination showed slightly more infiltrating polymorphonuclear leukocytes (PMN) and macrophages in the conjunctivae infected with the parent strain. The neuraminidase activity of both strains was not significantly different when the strains were grown in vitro. However, the neuraminidase activity of the parent was significantly less than that of the mutant at 3 and 12 hr post conjunctival infection. CONCLUSIONS: Although more outbreaks of pneumococcal conjunctivitis are tied to nonencapsulated S. pneumoniae strains, this study showed that an encapsulated strain was capable of establishing conjunctivitis in a rabbit injection model and survive attack by the host immune system longer than its nonencapsulated isogenic mutant. Nonetheless, the nonencapsulated pneumococci had an increased neuraminidase activity level in vivo when compared to the parent strain.

Randomized phase III trial of pegfilgrastim versus filgrastim after autologus peripheral blood stem cell transplantation.

Nonrandomized trials suggest that pegfilgrastim, a pegylated granulocyte colony-stimulating factor, could be used in lieu of filgrastim after autologus peripheral blood stem cell transplantation. This phase III, randomized, double-blinded, placebo-controlled trial compared the efficacy, costs, and safety of single-dose pegfilgrastim (single 6 mg dose) versus daily filgrastim (5 microg/kg/day) for this indication. Seventy-eight patients, matched for age, sex, underlying disease, stage, and CD34/kg transplant dose were enrolled. Cytokines were started on day +1 posttransplant and continued to an absolute neutrophil count (ANC) of 5x10(9)/L for 3 days or 10x10(9)/L for 1 day. The median time to neutrophil engraftment (ANC >1.5x10(9)/L for 3 days or 5x10(9)/L for 1 day) was the same in both groups (12 days). No differences in platelet engraftment (11 versus 13 days), number of platelet transfusions (5 versus 4), percent with positive cultures for bacterial pathogens (23% versus 15%), days of fever (1 versus 2), deaths prior to engraftment (1 versus 1), or duration of hospital stay (19 versus 19 days) were seen between the pegfilgrastim and filgrastim groups, respectively. Using the average wholesale price for doses used in this trial, there was a per-patient savings of $961 for the pegfilgrastim group (P < .001). This phase III study failed to demonstrate a difference in time to neutrophil engraftment or any clinical sequelae between pegfilgrastim and filgrastim when given post-APBSCT, with pegfilgrastim achieving a cost savings over filgrastim.

Inflammatory mechanisms and treatment of obstructive airway diseases with neutrophilic bronchitis.

Obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are major global health issues. Although considered as distinct diseases, airway inflammation is a key underlying pathophysiological process in asthma, COPD and bronchiectasis. Persistent neutrophilic airway inflammation (neutrophilic bronchitis) occurs with innate immune activation and is a feature of each of these airway diseases. Little is known about the mechanisms leading to neutrophilic bronchitis and few treatments are effective in reducing neutrophil accumulation in the airways. There is a similar pattern of inflammatory mediator release and toll like receptor 2 expression in asthma, COPD and bronchiectasis. We propose the existence of an active amplification mechanism, an effector arm of the innate immune system, involving toll like receptor 2, operating in persistent neutrophilic bronchitis. Neutrophil persistence in the airways can occur through a number of mechanisms such as impaired apoptosis, efferocytosis and mucus hypersecretion, all of which are impaired in airways disease. Impairment of neutrophil clearance results in a reduced ability to respond to bacterial infection. Persistent activation of airway neutrophils may result in the persistent activation of the innate immune system resulting in further airway insult. Current therapies are limited for the treatment of neutrophilic bronchitis; possible treatments being investigated include theophylline, statins, antagonists of pro-inflammatory cytokines and macrolide antibiotics. Macrolides have shown great promise in their ability to reduce airway inflammation, and can reduce airway neutrophils, levels of CXCL8 and neutrophil proteases in the airways. Studies also show improvements in quality of life and exacerbation rates in airways diseases.

Costs of hematopoietic cell transplantation: comparison of umbilical cord blood and matched related donor transplantation and the impact of posttransplant complications.

Allogeneic hematopoietic cell transplantation (HCT) is a complex and costly procedure. Unrelated umbilical cord blood (UCB) is an alternative graft source for patients without matched related donors (MRD); however, costs of UCB HCT have not been described. We compared the costs of HCT within the first 100 days among recipients of MRD (myeloablative = 67, nonmyeloablative = 54) or UCB (myeloablative = 63, nonmyeloablative = 110) HCT. Cost and hospitalization data were obtained from the institutional accounting department. The 100-day probabilities of overall survival (OS) and cumulative incidence of treatment-related mortality (TRM) were comparable among 4 transplant types; however, neutrophil recovery was delayed and graft failure was more likely in UCB recipients. The median cost per day survived (excluding costs of graft acquisition) was $1016 for myeloablative MRD, $2082 for myeloablative UCB, $612 for nonmyeloablative MRD, and $1156 for nonmyeloablative UCB recipients, respectively (P < .001). In multivariate analysis, adjusting for important patient, disease, and HCT-related characteristics, as well as major post-HCT complications, factors associated with higher costs within the first 100 days were myeloablative UCB HCT (relative risk 1.3 [95% confidence intervals, 1.1-1.5] versus myeloablative MRD HCT), graft failure (1.8 [1.7-1.9]), need for dialysis (1.3 [1.1-1.5]) or mechanical ventilation (1.3 [1.2-1.4]) and total hospital stay in the highest tertile (>48 days; 2.1 [1.9-2.3]). The median cost per day survived for patients with graft failure was $6976 (versus $1105 for no graft failure), dialysis was $4764 (versus $1102 for no dialysis), and $5099 for mechanical ventilation (versus $977 for no mechanical ventilation). Within the first 100 days, the absolute costs of myeloablative and nonmyeloablative UCB are higher than myeloablative and nonmyeloablative MRD transplantation. These costs are primarily driven by severe posttransplant complications, graft failure, and prolonged inpatient stay. Strategies to enhance engraftment will decrease the costs of UCB transplantation.

Interactions with apoptotic but not with necrotic neutrophils increase parasite burden in human macrophages infected with Leishmania amazonensis.

Neutrophils are involved in the initial steps of most responses to pathogens. In the present study, we evaluated the effects of the interaction of apoptotic vs. necrotic human neutrophils on macrophage infection by Leishmania amazonensis. Phagocytosis of apoptotic, but not viable, neutrophils by Leishmania-infected macrophages led to an increase in parasite burden via a mechanism dependent on TGF-beta1 and PGE2. Conversely, infected macrophages' uptake of necrotic neutrophils induced killing of L. amazonensis. Leishmanicidal activity was dependent on TNF-alpha and neutrophilic elastase. Nitric oxide was not involved in the killing of parasites, but the interaction of necrotic neutrophils with infected macrophages resulted in high superoxide production, a process reversed by catalase, an inhibitor of reactive oxygen intermediate production. Initial events after Leishmania infection involve interactions with neutrophils; we demonstrate that phagocytosis of these cells in an apoptotic or necrotic stage can influence the outcome of infection, driving either parasite survival or destruction.

Effect of feeding yeast culture on performance, health, and immunocompetence of dairy calves.

Objectives were to determine effects of feeding a culture of Saccharomyces cerevisiae on performance, health, and immunocompetence of calves in the first 70 d of age. Holstein calves (n = 512) at 2 +/- 1 d of age were randomly assigned to yeast culture (YC, 218 females and 37 males) or control (223 females and 34 males). Yeast culture was fed at 2% of the grain dry matter. All calves received colostrum during the first 24 h, pasteurized milk thereafter until 60 d of age, and grain was fed ad libitum for the first 70 d of age. Calves were housed in individual hutches, and grain intake was measured 5 d/wk. Body weight was measured at 5, 30, and 68 d of age, and attitude and fecal consistency were scored daily. Incidence and duration of health disorders and treatments were recorded. Neutrophil phagocytic and killing activities and antibody response to immunization with ovalbumin were measured. Concentrations of glucose and 3-hydroxybutyrate were measured in plasma. Grain intake did not differ between treatments and averaged 908 g/d throughout the study. Body weight change, concentrations of glucose, and 3-hydroxybutyrate did not differ between YC and control. Minor effects on neutrophil function were observed, and YC tended to increase the number of phagocytized bacteria and killing of phagocytized bacteria but did not influence humoral immune response. Attitude scores were similar between treatments throughout the study. Almost all calves experienced mild diarrhea during the study, but feeding YC improved fecal scores, reduced days with watery feces, incidence of fever and diarrhea, and risk of health disorders. Because of the high incidence of diarrhea, mortality preweaning was also high, but YC improved survival of calves by decreasing mortality rate past 13 d of age. Income at the end of the study was improved by $48/calf with YC. Feeding yeast culture in grain improved health, minimized frequency of health treatments, and reduced risk of morbidity and mortality in dairy calves.

Flexibility of single microvilli on live neutrophils and lymphocytes.

We measured the flexural stiffness of single microvilli on live human neutrophils and lymphocytes using 40-nm fluorescent beads. The beads were bound to the tips of the microvilli by anti-L-selectin antibodies. Digital bead images were acquired with an exposure time of 3 s at high magnification. Using a Gaussian point spread function, we obtained an analytical expression that relates the image profile to the flexural stiffness. We found that the flexural stiffnesses were 7 and 4 pN/microm for single microvilli on human neutrophils and lymphocytes, respectively. We also verified with live cells that 75% of neutrophil L-selectin and 72% of lymphocyte L-selectin were on the microvillus tips. Our results indicate that the leukocyte microvilli in contact with the endothelium or other surfaces will bend easily under physiological shear stresses.