Validation of real-time RT-PCR assays for mRNA quantification in baboons.

Real-time RT-PCR has been used widely, both in fundamental research and in clinical diagnostics, for instance for quantification of RNA levels in human tissues and tissue biopsies. In the present study we provide a strategy to validate primers/probes for real-time RT-PCR quantification of baboon samples. The method is based on the TaqMan system and uses primers/probes that have been designed and validated for human real-time RT-PCR. A prerequisite for the accuracy of this strategy is a similar amplification efficiency between human and baboon PCR reactions. We propose two different methods, i.e. by calculating PCR efficiencies from the slope of a dilution curve or by using the linear regression method, to compare the amplification efficiency between human and baboon samples. In conclusion, by performing a simple validation experiment, real-time PCR assays based on human sequences, which are easily available, can be applied for analysis of baboon samples.

1Alpha,25-dihydroxyvitamin D3 restores thymocyte apoptosis sensitivity in non-obese diabetic (NOD) mice through dendritic cells.

AIMS/HYPOTHESIS: Resistance of NOD thymocytes to apoptosis-inducing signals is restored by 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3), a therapy preventing diabetes in NOD mice. We studied whether modulation of thymocyte apoptosis is due to direct effects on thymic T lymphocytes or indirect effects via thymic dendritic cells, since both cell types constitute known targets for 1alpha,25OH2D3. METHODS AND RESULTS: Female NOD mice were treated with 1alpha,25OH2D3 (5microg/kg/2d) from 21 to 70 days. Vehicle-treated NOD and NOR mice served as controls. Analysis of thymic T lymphocytes from 1alpha,25OH2D3)-treated mice revealed a decrease in number of apoptosis-resistant CD4+CD8+ and CD4+CD8-HSA(high) T lymphocyte subsets, higher pro-apoptotic IL-2 and FasL, and lower anti-apoptotic Bclx-L mRNA expression levels. Thymic dendritic cells from 1alpha,25OH2D3-treated NOD mice had increased CD8alpha+FasL+ and CD80+/86+ expression compared to control NOD mice. In a syngeneic co-culture system of thymocytes and thymic dendritic cells, apoptosis levels were 20% higher only in co-cultures where both T cell- and dendritic cell-compartments originated from 1alpha,25OH2D3-treated mice. Activation-induced cell death-sensitivity in peripheral T lymphocytes was comparable to levels present in NOR mice, confirming better thymic selection in 1alpha,25OH2D3-treated mice. CONCLUSION/INTERPRETATION: We conclude that 1alpha,25OH2D3 needs both thymic T cell- and dendritic cell-compartments to exert its apoptosis-restorative effects in NOD thymocytes.

The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression.

Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is becoming a widely used method to quantify cytokines from cells, tissues, or tissue biopsies. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in a single step. Using TaqMan chemistry (Applied Biosystems, Foster City, CA) and the ABI Prism 7700 Sequence Detection System (Applied Biosystems), we validated a large panel of murine and human cytokines, as we as other factors playing a role in the immune system, such a chemokines and apoptotic markers. Although the method allows fast, sensitive, and accurate quantification, different control assays are necessary for the method to be reliable. By construction of complementary DNA (cDNA) plasmid clones, standard curves are generated that allow direct quantification of every unknown sample. Furthermore, the choice of a reliable housekeeping gene is very important. Finally, co-amplification of contaminating genomic DNA is avoided by designing sets of primers located in different exons or or intron-exon junctions. In conclusion, the real-time RT-PCF technique is very accurate and sensitive, allows high through put, and can be performed on very small samples. The development of real-time RT-PCR has resulted in an exponential increase in its use over the last couple of years, and the method has undoubtedly become the standard for quantifying cytokine patterns, clarifying many functional properties of immune cells and their associated diseases.

In vitro and in vivo analysis of the immune system of vitamin D receptor knockout mice.

Immune cells carry receptors for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; vitamin D receptor (VDR)] and individuals with severe vitamin D deficiency have immune abnormalities. The aim of this study was to investigate the role of vitamin D in the immune system by studying VDR-knockout (VDR-KO) mice. VDR-KO mice had the same metabolic phenotype as rachitic animals with severe hypocalcemia. Leukocytosis, lymphocyte subset composition in different immune organs, and splenocyte proliferation to several stimuli were normal, except for a lower response to anti-CD3 stimulation (simulation index [SI] of 13 +/- 4 vs. 24 +/- 9 in wild-type mice; p < 0.01). Macrophage chemotaxis was impaired (41 +/- 19% vs. 60 +/- 18% in wild-type mice; p < 0.01) but phagocytosis and killing were normal. In vivo rejection of allogeneic (31 +/- 12 days vs. 45 +/- 26 days of survival in wild-type mice, NS) or xenogeneic (10 +/- 2 days vs. 16 +/- 9 days of survival in wild-type mice, NS) islet grafts was comparable with wild-type mice. Surprisingly, VDR-KO mice were protected from low-dose streptozotocin-induced diabetes mellitus (LDSDM; 5% vs. 65% in wild-type mice; p < 0.001). Correcting hypocalcemia by use of lactose-rich or polyunsaturated fat-rich diets fully restored the immune abnormalities in vitro and the sensitivity to diabetes in vivo. On the other hand, treatment with 1,25(OH)2D3 protected wild-type mice against diabetes but did not protect normocalcemic VDR-KO mice. We conclude that immune defects observed in VDR-KO mice are an indirect consequence of VDR disruption because they can be restored by calcium homeostasis normalization. This study proves that although 1,25(OH)2D3 is a pharmacologic and probably a physiological immunomodulator, its immune function is redundant. Moreover, we confirm the essential role of calcium in the immune system.

An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.

The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.

Early graft failure of xenogeneic islets in NOD mice is accompanied by high levels of interleukin-1 and low levels of transforming growth factor-beta mRNA in the grafts.

Early graft failure, graft rejection, and autoimmune recurrence remain unresolved issues in islet xenotransplantation in type 1 diabetes. The first aim of this study was to examine the existence of early graft failure in spontaneously diabetic autoimmune NOD mice after rat islet transplantation under technically controlled circumstances. The second aim was to examine the mediators of this early xenograft dysfunction. First, we demonstrated a higher percentage of early xenograft failure (48%) in spontaneously diabetic NOD mice as compared with chemically diabetic old NOD (13%, P < 0.05) and C57Bl/6 (7%, P < 0.01) mice. In addition, in spontaneously diabetic NOD mice, xenogeneic islets displayed early graft failure more frequently than allogeneic (23%, P < or = 0.05) or isogeneic islets (7%, P < 0.01). No early graft failure was observed in allotransplantation or isotransplantation in chemically diabetic mice. Reverse transcriptase-polymerase chain reaction analysis of cytokine mRNA in islet xenografts 8 h after transplantation showed higher levels of interleukin (IL)-1 mRNA in autoimmune diabetic mice compared with chemically diabetic old NOD mice (1.40 +/- 0.32 vs. 0.90 +/- 0.14 IL-1 copies/beta-actin copies, P < 0.05). In contrast, mRNA levels of transforming growth factor (TGF)-beta were lower in spontaneously diabetic NOD mice than in chemically diabetic old NOD mice (0.67 +/- 0.16 vs. 1.36 +/- 0.50 TGF-beta copies/beta-actin copies, P < 0.05). No differences in tumor necrosis factor-alpha, IL-6, and inducible nitric oxide synthase were seen between autoimmune and nonautoimmune diabetic mice. T-cell cytokines (IL-2, IL-4, IL-10, and gamma-interferon) were absent in all mice until 48 h after transplantation. These data suggest that early islet xenograft failure is more common in spontaneously diabetic NOD mice and could be due to a nonspecific inflammatory reaction locally in the grafts.

1alpha,25-dihydroxyvitamin D3 induces an autoantigen-specific T-helper 1/T-helper 2 immune shift in NOD mice immunized with GAD65 (p524-543).

Prevention of type 1 diabetes in NOD mice by 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is accompanied by a T-helper (Th) 1/Th2 cytokine shift in the pancreas. The aim of this study was to investigate whether this immune shift also occurs outside of the pancreas and whether it is limited to autoantigen-specific immune responses. NOD mice treated with 1alpha,25(OH)2D3 (5 microg/kg every 2 days) or control vehicle were immunized with GAD65 (p524-543) or ovalbumin (OVA) in the rear footpads. First, we examined T-cell proliferation and cytokine production (via enzyme-linked immunosorbent assay) of draining lymph node cells in vitro with or without peptide rechallenge. Although no differences in proliferation were measured between control and 1alpha,25(OH)2D3-treated mice after in vitro GAD65 rechallenge, a marked shift in cytokine secretion profile was seen in 1alpha,25(OH)2D3-treated mice: interleukin-4 was increased (37 +/- 5 vs. 21 +/- 12 pg/ml in controls, P < 0.005), whereas gamma-interferon levels were decreased (6 +/- 3 vs. 9 +/- 3 ng/ml in controls, P < 0.05). This shift was absent in OVA-primed mice. Second, we measured cytokine profiles by reverse transcriptase-polymerase chain reaction in popliteal lymph nodes at different time points after priming with GAD65 or OVA in vivo. A marked Th1/Th2 shift occurred in 1alpha,25(OH)2D3-treated mice after in vivo priming with GAD65. Again, this shift was absent after OVA immunization. Finally, we measured cytokine profiles after rechallenge with a panel of autoantigens (GAD65, heat shock protein 65, insulin B-chain) and control antigens (OVA, keyhole limpet hemocyanine, myelin proteolipid protein, tetanus toxin) and confirmed the Th1/Th2 shift in autoantigen-injected mice but not in control antigen-injected mice. In conclusion, the immune deviation induced by 1alpha,25(OH)2D3 in NOD mice can also be induced in the peripheral immune system but is limited to pancreatic autoantigens.

Identification and immune regulation of 25-hydroxyvitamin D-1-alpha-hydroxylase in murine macrophages.

Receptors for 1,25(OH)2vitaminD3 are found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to examine the presence and nature of the enzyme responsible for final activation of the molecule, 1-alpha-hydroxylase, in murine macrophages and to analyse its regulation and possible role in the immune system. Peritoneal macrophages from C57Bl/6 mice were incubated with lipopolysaccharide (LPS; 100 microg/ml), interferon-gamma (IFN-gamma; 500 U/ml) or a combination of both. By quantitative reverse transcriptase-polymerase chain reaction, using primers based on the murine renal cDNA sequence, low levels of 1-alpha-hydroxylase mRNA were detected in freshly isolated cells (18 +/- 7 x 10-6 copies/beta-actin copies). Analysis of the cDNA sequence of the gene revealed identical coding sequences for the macrophage and renal enzymes. mRNA levels rose three-fold with LPS (NS), but a six-fold increase was seen after IFN-gamma stimulation (P < 0.05). Combining LPS and IFN-gamma did not result in a major additional increase, but addition of cyclosporin A further increased levels 2.5-fold both in IFN-gamma- and combination-stimulated cells (P < 0.05). Time course analysis revealed that up-regulation of 1-alpha-hydroxylase was a late phenomenon, preceded by the up-regulation of activating macrophage products such as IL-1 and tumour necrosis factor-alpha. Finally, a defect in 1-alpha-hydroxylase up-regulation by immune stimuli was found in autoimmune non-obese diabetic mice. In conclusion, we propose that the up-regulation of 1-alpha-hydroxylase in activated macrophages, resulting in the synthesis of 1,25(OH)2D3, might be a negative feedback loop in inflammation. A defect in this system might be an additional element in tipping the balance towards autoimmunity.

Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.

Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.

1,25-Dihydroxyvitamin D3 restores sensitivity to cyclophosphamide-induced apoptosis in non-obese diabetic (NOD) mice and protects against diabetes.

The activated form of vitamin D, 1,25(OH)2D3, and its analogues can prevent type I diabetes in NOD mice. Protection is achieved without signs of systemic immunosuppression and is associated with a restoration of the defective immune regulator system of the NOD mice. The aim of the present study was to investigate whether this restoration of regulator cell function is the only mechanism in the prevention of diabetes by 1,25(OH)2D3. We tested therefore if 1,25(OH)2D3 could prevent cyclophosphamide-induced diabetes, since diabetes occurring after cyclophosphamide injection is believed to be due to an elimination of suppresser cells. NOD mice treated with 1,25(OH)2D3 (5 microg/kg every 2 days) from the time of weaning were clearly protected against diabetes induced by cyclophosphamide (200 mg/kg body wt at 70 days old) (2/12 (17%) versus 36/53 (68%) in control mice, P < 0.005). By co-transfer experiments it was demonstrated that cyclophosphamide had indeed eliminated the suppresser cells present in 1,25(OH)2D3-treated mice. Since cyclophosphamide injection did not break the protection offered by 1,25(OH)2D3, it was clear that diabetogenic effector cells were affected by 1,25(OH)2D3 treatment as well. This was confirmed by the finding that splenocytes from 1,25(OH)2D3-treated mice were less capable of transferring diabetes in young, irradiated NOD mice, and by the demonstration of lower Th1 cytokine levels in the pancreases of 1,25(OH)2D3-treated, cyclophosphamide-injected mice. This better elimination of effector cells in 1,25(OH)2D3-treated mice could be explained by a restoration of the sensitivity to cyclophosphamide-induced apoptosis in both thymocytes and splenocytes, in normally apoptosis-resistant NOD mice. Altogether, these data indicate that the protection against diabetes offered by 1,25(OH)2D3 may be independent of the presence of suppresser cells, and may involve increased apoptosis of Th1 autoimmune effector cells.