Activation of peripheral blood monocytes results in more robust production of IL-10 in neonatal foals compared to adult horses

Sponseller, Brett A.; Macedo, M.M.A. de; Clark, Sandra K.; Gallup, Jack M.; Jones, Douglas E.

doi:10.1016/j.vetimm.2008.09.013

Cited by 27 publications

(13 citation statements)

References 37 publications

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“…In summary, these fi ndings support our claim that PREXCEL-Q-calculated nucleic acid sample and standard dilutions for qPCR, based on the "Stock I approach" (Grubor et al, 2004;Gallup et al, 2005;Gallup et al, 2006;Kawashima et al, 2006;Lazic et al, 2007;Gallup et al, 2008;Olivier et al, 2009;Sponseller et al, 2009), avoids qPCR inhibitory behavior in all fi nal reactions (Gallup et al, 2008;. Because of this and other aspects, we recommend the use of PREXCEL-Q in all laboratories performing qPCR of any kind.…”

Section: Spud Plasmid Spud Ampliconsupporting

confidence: 76%

“…PREXCEL-Q is a qPCR software program that, among its other functions, identifies dilution parameters for all samples and standards that avoid qPCR-inhibitory phenomena. The SPUD assay was thus used in this study to critically test and corroborate the reported ability of the PREXCEL-Q program to avoid inhibition in qPCR samples and standards (Grubor et al, 2004;Gallup et al, 2005;Gallup et al, 2006;Kawashima et al, 2006;Lazic et al, 2007;Gallup et al, 2008;Olivier et al, 2009;Sponseller et al, 2009). …”

Section: Introductionmentioning

confidence: 69%

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SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

Gallup

Sow²,

Geelen³

et al. 2010

Current Issues in Molecular Biology

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Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses. To address this, we invented the PREXCEL-Q software program to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD ampliconcontaining plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with ~15,500 SPUD amplicons yielded a C q of 27.39 ± 0.28 (at ~80.8% efficiency), while reactions spiked with ~7,750 SPUD plasmids yielded a C q of 23.82 ± 0.15 (at ~97.85% efficiency). This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.

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Section: Spud Plasmid Spud Ampliconsupporting

confidence: 76%

Section: Introductionmentioning

confidence: 69%

SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

Gallup

Sow²,

Geelen³

et al. 2010

Current Issues in Molecular Biology

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“…Previous studies on the induction of adaptive immune responses in foals and adult horses have focused on the expression of mRNA [5, 11, 16, 20, 35] but failed to provide detailed information about the cellular source of cytokine production. For example, IFN-γ mRNA up-regulation in PBMC could originate from several cell types such as Th cells, CTL or NK cells and would not necessarily represent a Th1 response.…”

Section: Discussionmentioning

confidence: 99%

“…Other cytokines such as IL-8, IL-12 and IL-23 were found to be increased or similarly expressed in PBMC from neonates compared to older foals [20]. Stimulation of PBMC from foals with lipopolysaccharide (LPS) and IFN-γ also resulted in increased IL-10 mRNA expression compared to PBMC from adult horses [35]. …”

Section: Introductionmentioning

confidence: 99%

Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory T_R1 cell activation and a delay of the Th2 cell response in equine neonates and foals

2010

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Cytokines produced by T helper (Th) cells are important in orchestrating the immune response during health and disease. Recent reports indicated that cytokine mRNA expression in foals is often quantitatively lower than that of adult horses suggesting that foal T cells are not fully mature. Here, peripheral blood mononuclear cells from foals and adult horses were stimulated with phorbol 12-myristate 13-acetate and analyzed for intracellular interferon-gamma (IFN-γ), interleukin-4 (IL-4) and IL-10 production, representing the Th1, Th2 and regulatory TR1 cell phenotypes respectively, by flow cytometry. In agreement with previous reports, all three cytokines were quantitatively reduced in foals compared to adults. However, the balance between Th1 and Th2 cytokines (IFN-γ/IL-4 ratio) showed a clear Th1-biased response in foals by 6 and 12 weeks of life, while similar IFN-γ/IL-10 ratios were found in foals and adult horses. By day 5 after birth, intracellular IFN-γ production by foal CD4+ and CD8+ T cells resembled that in adults. Overall, IL-4 production was low in foals. IL-4+ cells peaked at day 5 of age when IL-4 was mainly produced by IgE+ cells. Relative percentages of IL-4+ Th2 cells were significantly lower in foals at all time points. The data suggested that equine neonates and young foals have an impaired Th2 response, that the immune response of foals is Th1 biased, that IFN-γ production by Th and cytotoxic T cells is qualitatively similar to adult horses, and regulatory IL-10 production by T cells is developmentally mature in foals during the first three months of life.

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“…31 Antibodies utilized consisted of mouse monoclonal anti-cluster of differentiation (CD)172a, b which specifically stain equine monocytes and granulocytes, and cyanine (Cy)2-conjugated goat anti-horse IgG. c, 34,36 R-phycoerythrin-labeled rabbit anti-mouse polyclonal antibodies d were used as the secondary detection reagent for CD172a staining. After final staining washes, cells were transferred to flow tubes and kept in the dark at 4°C until flow cytometric analysis.…”

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confidence: 99%

Alloimmune neonatal neutropenia and neonatal isoerythrolysis in a Thoroughbred colt

et al. 2011

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A 3-day-old, 59-kg Thoroughbred colt was presented to the Lloyd Veterinary Medical Center (Ames, Iowa) for suspected neonatal isoerythrolysis (NI). The colt's dam was a 5-year-old primiparous mare without reported medical disorders during gestation or parturition. Upon initial examination, the colt was quiet and lethargic but still responsive, with a rectal temperature of 38.0°C, heart rate of 160 beats/min, and respiratory rate of 80 breaths/min. Marked jaundice of the mucous membranes and sclera was observed. interval: 0.5-3.9 mg/dl). Agglutination of red cells was not observed on the blood smear. After identification of an appropriate donor via major and minor cross-match, a blood transfusion consisting of 1.5 liters of packed red cells was performed over 6 hr; no adverse reactions were observed. Administration of amikacin (25 mg/kg, intravenously [IV], every 24 hr) and ceftiofur (5 mg/kg, IV, every 12 hr) also was initiated. The colt rapidly appeared much brighter and energetic with the hematocrit rising to 19% shortly after blood transfusion was complete. The hematocrit slowly increased during hospitalization and was 29% at the time of discharge on day 30 (Fig. 1). Blood typing of the mare identified the Aa, Ca, Qa, Qb, and Qc antigens on maternal red blood cells; Ca, Ka, Qa, Qb, and Qc antigens were identified on the colt's red blood cells, suggesting the Ka erythrocyte antigen as the cause of NI in this colt.Despite the gradually increasing hematocrit, leukopenia characterized by marked neutropenia was identified on both automated a and manual evaluation of blood smears during the initial 5 days of hospitalization (Fig. 1). A thorough diagnostic investigation to identify a possible source of infection that could result in persistent neutropenia was conducted including repeated physical examinations and thoracic and abdominal ultrasonography. The colt was consistently bright and alert and maintained a normal body temperature; no abnormal findings were observed via ultrasonography, and blood cultures did not yield bacterial growth. Because an obvious source of infection could not be identified in conjunction with continued and profound neutropenia along with the absence of immature or toxic neutrophils, an immune-mediated neutropenia was considered. On day 5 of hospitalization, Abstract. A 3-day-old Thoroughbred colt was originally presented for treatment of neonatal isoerythrolysis, which was treated with a blood transfusion. However, persistent neutropenia was observed despite the absence of detectable infection. Subsequently, a granulocyte agglutination test was performed by incubating the colt's neutrophils with the mare's serum; results were positive, leading to a clinical diagnosis of alloimmune neonatal neutropenia. The diagnosis was further supported via flow cytometric analysis. The colt was hospitalized and treated prophylactically with antimicrobials and 4 separate doses of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 1.4-3.5 µg/kg, subcutaneously) in attempts to maintain the...

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Activation of peripheral blood monocytes results in more robust production of IL-10 in neonatal foals compared to adult horses

Cited by 27 publications

References 37 publications

SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory T_R1 cell activation and a delay of the Th2 cell response in equine neonates and foals

Alloimmune neonatal neutropenia and neonatal isoerythrolysis in a Thoroughbred colt

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Activation of peripheral blood monocytes results in more robust production of IL-10 in neonatal foals compared to adult horses

Cited by 27 publications

References 37 publications

SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals

Alloimmune neonatal neutropenia and neonatal isoerythrolysis in a Thoroughbred colt

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Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory T_R1 cell activation and a delay of the Th2 cell response in equine neonates and foals