Genetic Knockout Models Research Articles

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models.

The targeted nuclease revolution (TALENs, CRISPR/Cas9) now allows Xenopus researchers to rapidly generate custom on-demand genetic knockout models. These novel methods to perform reverse genetics are unprecedented and are fueling a wide array of human disease models within the aquatic diploid model organism Xenopus tropicalis (X. tropicalis). This emerging technology review focuses on the tools to rapidly generate genetically engineered X. tropicalis models (GEXM), with a focus on establishment of genuine genetic and clinically relevant cancer models. We believe that due to particular advantageous characteristics, outlined within this review, GEXM will become a valuable alternative animal model for modeling human cancer. Furthermore, we provide perspectives of how GEXM will be used as a platform for elucidation of novel therapeutic targets and for preclinical drug validation. Finally, we also discuss some future prospects on how the recent expansions and adaptations of the CRISPR/Cas9 toolbox might influence and push forward X. tropicalis cancer research.

Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML

Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.

GPER/GPR30 Knockout Mice: Effects of GPER on Metabolism.

Endogenous estrogens, predominantly 17β-estradiol (E2), mediate various diverse effects throughout the body in both normal physiology and disease. Actions include development (including puberty) and reproduction as well as additional effects throughout life in the metabolic, endocrine, musculoskeletal, nervous, cardiovascular, and immune systems. The actions of E2 have traditionally been attributed to the classical nuclear estrogen receptors (ERα and ERβ) that largely mediate transcriptional/genomic activities. However, more recently the G protein-coupled estrogen receptor GPER/GPR30 has become recognized as an essential mediator of certain, and particularly rapid, signaling events in response to E2. Murine genetic knockout (KO) models represent an important approach to understand the mechanisms of E2 action in physiology and disease. Studies of GPER KO mice over the last years have revealed functions for GPER in the regulation of obesity, insulin resistance and glucose intolerance, among other areas of (patho)physiology. This chapter focuses on methods for the evaluation of metabolic parameters in vivo and ex vivo with an emphasis on glucose homeostasis and metabolism through the use of glucose and insulin tolerance tests, pancreatic islet and adipocyte isolation and characterization.

Targeting VLA-4 to Reduce GvHD

▪The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies are primarily derived from anti-leukemia effect that is mediated by T cells in donor grafts. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT.We have recently reported that blockade of alloreactive donor T cell trafficking to the GvHD target organs significantly reduces GvHD in both a MHC fully-mismatched (B6 (H-2b) → Balb/c (H-2d)) (75% vs 0% overall survival) and a minor-mismatched (B6 (H-2b) → B6x129 (H-2b)) allo-HSCT models (100% vs 10% overall survival) compared to WT T cells (Choi et al., Blood 2012). In this study, we examined if inhibition of VLA-4, which is required for transendothelial migration and access to GvHD target organs, could reduce GVHD. VLA-4 consists of two subunits α4 (CD49d) and β1 (CD29). To genetically eliminate VLA-4 from allogeneic donor T cells, we generated Tie-2 cre+ α4-/- mice (B6, H-2b, CD45.2+) in which α4 is knocked out by Tie-2 cre, which is expressed in hematopoietic cells. Splenic pan T cells were isolated from these mice and T cell-depleted bone marrow cells (TCD BM) from congenic B6 mice (CD45.1+). 5x106 TCD BM and 5x105 splenic pan T cells were transplanted into lethally irradiated allogeneic Balb/c recipient mice (H-2d, CD45.2+). We found that VLA-4 deficient T cells significantly reduced GvHD compared to WT T cells (Fig. A). VLA-4 deficient T cells had no significant effect on donor engraftment and achieved a complete donor chimerism. In addition, recipients transplanted with VLA-4 deficient T cells had significantly better histopathology score (Fig. B). Similar results were observed when donor T cells were infused at day 11 after allo-HSCT.While we speculate defective T cell trafficking to GvHD target organs would be the primary reason for the reduced GvHD in the recipients of VLA-4 deficient T cells, we examined other possible mechanisms as well, such as T cell alloreactivity and proliferation. We found that VLA-4 deficient T cells proliferated at the same rate as WT T cells in the presence of anti-CD3/CD28 antibody-coated beads. However, VLA-4 deficient T cells proliferate less in the presence of allogeneic antigen presenting cells (APCs) compared to WT T cells. Interestingly, VLA-4 deficient T cells upregulate CTLA-4 and GZMB significantly more than WT T cells after in vitro activation. These data suggest that not only T cell trafficking to GvHD target organs but also altered T cell phenotype and function might help explain the observed reduced GvHD. Genetic deletion of α4 in T cells will also alter expression of α4b7. Since antibodies to b7 have also been shown to reduce GvHD (Waldman et al., Blood, 2006) our current studies will focus on determining which of these pathways (α4b1 vs. α4b7) is most important in mitigating GvHD by using genetic knockout models, antibodies to b1 and b7 and small molecule inhibitors of α4b1 vs. α4b7.In conclusion, our data suggests that VLA-4 represents a promising therapeutic target for future efforts to mitigate GvHD after allo-HSCT. In addition, this strategy can be exploited in other diseases and settings in addition to allo-HSCT, such as solid organ transplantation, chronic inflammatory diseases and autoimmune diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Indoleamine 2,3-dioxygenase 2, a novel mediator of pathogenic autoantibodies in autoimmune arthritis (BA12P.110)

Abstract Several autoimmune disorders, including rheumatoid arthritis (RA), are associated with altered activity of the immunomodulatory enzyme indoleamine-2,3-dioxygenase (IDO). However, the precise contributions of IDO function to autoimmunity remain unclear. We examined the effect of two different IDO enzymes, IDO1 and IDO2, on the development of autoimmune arthritis in the KRN preclinical model of autoimmune arthritis by comparing the effects of both genetic knockout models and chemical inhibitors of IDO on joint inflammation. We find that IDO2, but not IDO1, is critical for arthritis development, providing the first direct evidence of separate in vivo functions for IDO1 and IDO2. Specifically, IDO2 knockout mice display decreased joint inflammation relative to wild-type mice due to a reduction in pathogenic autoantibodies and antibody secreting cells. Notably, IDO2 appears to specifically mediate autoreactive, but not normal B cell responses, as total serum Ig levels are not altered and IDO2 ko mice are able to mount productive antibody responses to model antigens in vitro and in vivo. Reciprocal adoptive transfer studies confirmed that autoantibody production and arthritis are modulated by IDO2 expression in a cell type extrinsic to the T cell. Future work will define the relevant cell types for IDO2 activity. Our results provide the first insights into the previously unknown function of IDO2 by defining its pathogenic contributions to autoantibody-mediated autoimmunity.

CRF and urocortin peptides as modulators of energy balance and feeding behavior during stress.

Early on, corticotropin-releasing factor (CRF), a hallmark brain peptide mediating many components of the stress response, was shown to affect food intake inducing a robust anorexigenic response when injected into the rodent brain. Subsequently, other members of the CRF signaling family have been identified, namely urocortin (Ucn) 1, Ucn 2, and Ucn 3 which were also shown to decrease food intake upon central or peripheral injection. However, the kinetics of feeding suppression was different with an early decrease following intracerebroventricular injection of CRF and a delayed action of Ucns contrasting with the early onset after systemic injection. CRF and Ucns bind to two distinct G-protein coupled membrane receptors, the CRF1 and CRF2. New pharmacological tools such as highly selective peptide CRF1 or CRF2 agonists or antagonists along with genetic knock-in or knock-out models have allowed delineating the primary role of CRF2 involved in the anorexic response to exogenous administration of CRF and Ucns. Several stressors trigger behavioral changes including suppression of feeding behavior which are mediated by brain CRF receptor activation. The present review will highlight the state-of-knowledge on the effects and mechanisms of action of CRF/Ucns-CRF1/2 signaling under basal conditions and the role in the alterations of food intake in response to stress.

Differentiating connexin hemichannels and pannexin channels in cellular ATP release

Adenosine triphosphate (ATP) plays a fundamental role in cellular communication, with its extracellular accumulation triggering purinergic signaling cascades in a diversity of cell types. While the roles for purinergic signaling in health and disease have been well established, identification and differentiation of the specific mechanisms controlling cellular ATP release is less well understood. Multiple mechanisms have been proposed to regulate ATP release with connexin (Cx) hemichannels and pannexin (Panx) channels receiving major focus. However, segregating the specific roles of Panxs and Cxs in ATP release in a plethora of physiological and pathological contexts has remained enigmatic. This multifaceted problem has arisen from the selectivity of pharmacological inhibitors for Panxs and Cxs, methodological differences in assessing Panx and Cx function and the potential compensation by other isoforms in gene silencing and genetic knockout models. Consequently, there remains a void in the current understanding of specific contributions of Panxs and Cxs in releasing ATP during homeostasis and disease. Differentiating the distinct signaling pathways that regulate these two channels will advance our current knowledge of cellular communication and aid in the development of novel rationally-designed drugs for modulation of Panx and Cx activity, respectively.

Reply from Aihua Li, Charles C. T. Hindmarch, Eugene E. Nattie and Julian F. R. Paton

The authors appreciate the interest of Silvani et al. in our recent publication (Li et al. 2013). The key points of our paper are: (1) treating spontaneously hypertensive rats (SHR) with a single dose of a dual orexin receptor antagonist, almorexant, significantly decreased blood pressure for 8 h in wakefulness and sleep during both the light and dark period of the diurnal cycle; (2) in the normotensive control (WKY) rats the same treatment did not significantly affect blood pressure in either wakefulness or sleep in any light cycle. Our experimental design includes: (a) having the rats surgically implanted with EEG, EMG and blood pressure radio-telemetry at least 7 days before testing; (b) placing the rats in a light- and temperature-controlled recording chamber for 12 h with food and water available ad libitum; and (c) measuring blood pressure, ECG, EEG and neck EMG before and after administration of a dual orexin receptor antagonist. Silvani et al. express concern, not about our major findings, but about our ‘… claim … that the effect of Almorexant on BP did not differ significantly between wakefulness and non-rapid-eye-movement sleep (NREMS) in SHR either in the light (rest) or dark (activity) period of the day.’ They raise four ‘issues’. First, our recording protocol ‘…may not have been sensitive enough to detect changes in BP related to sleep states and the day–light cycle…’. As stated above, we used radio-telemetry of blood pressure (BP) with long uninterrupted recording periods. Figure 2B of our study (Li et al. 2013) shows that in normotensive WKY rats, this protocol does detect sleep–wake differences. In SHR, we reported that mean arterial blood pressure was slightly higher in wakefulness compared with NREM sleep, a difference that reached statistical significance in the light period. In the reference cited by Silvani et al., Kuo & Yang (2005) reported that ‘The WKY had significant changes in arterial pressure … with the sleep–wake transitions … The sleep-related changes …, however, were not as evident in the SHR’. The smaller BP difference (∼5 mmHg) between wakefulness and sleep that we observed in SHRs are in fact within the range reported in SHR by Kuo & Yang (2005). Second, we did not report the effects of almorexant on sleep structure in SHR rats. Silvani et al. suggest that improvement of disrupted sleep in SHR by almorexant could ‘…confound the observed results on BP.’ As noted by Silvani et al., we (and others) have reported the effects of almorexant on sleep in non-SHR and we agree with their prediction that SHR may sleep ‘better’ after almorexant, but this requires robust validation. However, we do not believe that this explains the lowering of BP since Lee et al. (2013) demonstrated that intracerebroventricular or intra-rostral ventrolateral medulla blockade of orexin 2 receptors significantly reduced arterial pressure in anaesthetized SHRs but not WKY rats. Third, Silvani et al. state that we ‘…did not indicate whether Almorexant modified the difference in BP between wakefulness and NREM sleep in SHR and WKY rats.’ Figure 4A and B of our paper (Li et al. 2013) showed the maximum changes in BP before and after almorexant; the largest decrease of BP after blocking orexin receptors was in wakefulness during the dark period (−37 mmHg) and smallest change in NREM sleep during the light period (−25 mmHg) relative to the pretreatment baseline. In our experiments the observed difference between day and night, quiet wakefulness and sleep is around 10 mmHg. Further, Silvani et al. compare our results to human narcolepsy and animal models thereof in which the absence of orexin has been present for much longer time periods than the orexin receptor blockade induced by using almorexant as we performed. This difference between genetic knockout models and acute, reversible orexin receptor inhibition is a key part of our approach. Fourth, we did not report the effects of our almorexant treatment on blood pressure in REM sleep. This is true and is an important omission from the paper, which we fully acknowledged. In summary, we agree with Silvani et al. that antagonism of orexin receptors has a significant anti-hypertensive effect in unanaesthetized SHR rats, which suggests that modulation of the orexin system could be a potential target in treating hypertension. We thank Silvani et al. for their interest in our work.

GRP78 as a regulator of liver steatosis and cancer progression mediated by loss of the tumor suppressor PTEN

Glucose-regulated protein 78 (GRP78), a molecular chaperone widely elevated in human cancers, is critical for endoplasmic reticulum (ER) protein folding, stress signaling and PI3K/AKT activation. Genetic knockout models of GRP78 revealed that GRP78 maintains homeostasis of metabolic organs, including liver, pancreas and adipose tissues. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the most common liver cancers. There is a lack of effective therapeutics for HCC and CC, highlighting the need to further understand liver tumorigenic mechanisms. PTEN, a tumor suppressor that antagonizes the PI3K/AKT pathway, is inactivated in a wide range of tumors, including 40–50% of human liver cancers. To elucidate the role of GRP78 in liver cancer, we created a mouse model with biallelic liver-specific deletion of Pten and Grp78 mediated by Albumin-Cre-recombinase (cPf/f78f/f). Interestingly, in contrast to PTEN, deletion of GRP78 was progressive but incomplete. At 3 months, cPf/f78f/f livers showed hepatomegaly, activation of lipogenic genes, exacerbated steatosis and liver injury, implying that GRP78 protects the liver against PTEN-null mediated pathogenesis. Furthermore, in response to liver injury, we observed increased proliferation and expansion of bile duct and liver progenitor cells in cPf/f78f/f livers. Strikingly, bile duct cells in cPf/f78f/f livers maintained wild-type (WT) GRP78 level while adjacent areas showed GRP78 reduction. Analysis of signaling pathways revealed selective JNK activation, β-catenin downregulation, along with PDGFRα upregulation, which was unique to cPf/f78f/f livers at 6 months. Development of both HCC and CC was accelerated and evident in cPf/f78f/f livers at 8–9 months, coinciding with intense GRP78 expression in the cancer lesions, and GRP78 expression in adjacent normal areas reverted back to the WT level. In contrast, c78f/f livers showed no malignancy even at 14 months. These studies reveal GRP78 is a novel regulator for PTEN-loss mediated liver injury and cancer progression.

Abstract C61: GRP78 as a regulator of liver steatosis and cancer progression mediated by loss of the tumor suppressor PTEN

Abstract Glucose-regulated protein 78 (GRP78), a molecular chaperone widely elevated in human cancers, is critical for endoplasmic reticulum (ER) protein folding, stress signaling, and PI3K/AKT activation. Genetic knockout models of GRP78 revealed that GRP78 maintains homeostasis of metabolic organs, including liver, pancreas, and adipose tissues. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the most common liver cancers. There is a lack of effective therapeutics for HCC and CC, highlighting the need to further understand liver tumorigenic mechanisms. Phosphatase and tensin homolog (PTEN), a tumor suppressor that antagonizes the PI3K/AKT pathway, is inactivated in a wide range of tumors, including 40-50% of human liver cancers. To elucidate the role of GRP78 in liver cancer, we created a mouse model with biallelic liver-specific deletion of Pten and Grp78 mediated by Albumin-Cre-recombinase (cPf/f78f/f). Interestingly, in contrast to PTEN, deletion of GRP78 was progressive but incomplete. At 3 months cPf/f78f/f livers showed hepatomegaly, activation of lipogenic genes, exacerbated steatosis, and liver injury, implying that GRP78 protects the liver against PTEN-null mediated pathogenesis. Furthermore, in response to liver injury, we observed increased proliferation and expansion of bile duct and liver progenitor cells in cPf/f78f/f livers. Strikingly, bile duct cells in cPf/f78f/f livers maintained wild-type GRP78 level while adjacent areas showed GRP78 reduction. Analysis of signaling pathways revealed selective c-Jun N-terminal protein kinase activation, β-catenin downregulation, along with platelet-derived growth factor receptor α upregulation, which was unique to cPf/f78f/f livers at 6 months. Development of both HCC and CC were accelerated and evident in cPf/f78f/f livers at 8-9 months, coinciding with intense GRP78 expression in the cancer lesions, and GRP78 expression in adjacent normal areas reverted back to the WT level. In contrast, livers of Albumin-Cre; Grp78f/f (c78f/f) mice showed no malignancy even at 14 months. These studies reveal GRP78 is a novel regulator for PTEN-loss mediated liver injury and cancer progression. Citation Format: Wan-Ting Chen, Genyuan Zhu, Kyle Pfaffenbach, Gary Kanel, Bangyan Stiles, Amy S. Lee. GRP78 as a regulator of liver steatosis and cancer progression mediated by loss of the tumor suppressor PTEN. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C61.

Modulation of taste responsiveness by the satiation hormone peptide YY

It has been hypothesized that the peripheral taste system may be modulated in the context of an animal's metabolic state. One purported mechanism for this phenomenon is that circulating gastrointestinal peptides modulate the functioning of the peripheral gustatory system. Recent evidence suggests endocrine signaling in the oral cavity can influence food intake (FI) and satiety. We hypothesized that these hormones may be affecting FI by influencing taste perception. We used immunohistochemistry along with genetic knockout models and the specific reconstitution of peptide YY (PYY) in saliva using gene therapy protocols to identify a role for PYY signaling in taste. We show that PYY is expressed in subsets of taste cells in murine taste buds. We also show, using brief-access testing with PYY knockouts, that PYY signaling modulates responsiveness to bitter-tasting stimuli, as well as to lipid emulsions. We show that salivary PYY augmentation, via viral vector therapy, rescues behavioral responsiveness to a lipid emulsion but not to bitter stimuli and that this response is likely mediated via activation of Y2 receptors localized apically in taste cells. Our findings suggest distinct functions for PYY produced locally in taste cells vs. that circulating systemically.

Cold Exposure Promotes Atherosclerotic Plaque Growth and Instability via UCP1-Dependent Lipolysis

SummaryMolecular mechanisms underlying the cold-associated high cardiovascular risk remain unknown. Here, we show that the cold-triggered food-intake-independent lipolysis significantly increased plasma levels of small low-density lipoprotein (LDL) remnants, leading to accelerated development of atherosclerotic lesions in mice. In two genetic mouse knockout models (apolipoprotein E−/− [ApoE−/−] and LDL receptor−/− [Ldlr−/−] mice), persistent cold exposure stimulated atherosclerotic plaque growth by increasing lipid deposition. Furthermore, marked increase of inflammatory cells and plaque-associated microvessels were detected in the cold-acclimated ApoE−/− and Ldlr−/− mice, leading to plaque instability. Deletion of uncoupling protein 1 (UCP1), a key mitochondrial protein involved in thermogenesis in brown adipose tissue (BAT), in the ApoE−/− strain completely protected mice from the cold-induced atherosclerotic lesions. Cold acclimation markedly reduced plasma levels of adiponectin, and systemic delivery of adiponectin protected ApoE−/− mice from plaque development. These findings provide mechanistic insights on low-temperature-associated cardiovascular risks.

Molecular mechanisms of liver ischemia reperfusion injury: Insights from transgenic knockout models

Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery. Understanding the mechanisms of liver ischemia reperfusion injury (IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation, as well as expanding the potential pool of usable donor grafts. The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes, increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis. Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury. IRI involves a complex interplay between neutrophils, natural killer T-cells cells, CD4+ T cell subtypes, cytokines, nitric oxide synthases, haem oxygenase-1, survival kinases such as the signal transducer and activator of transcription, Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ pathways. Transgenic animals, particularly genetic knockout models, have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies. Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein. This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11. Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Unmet Expectations: miR-34 Plays No Role in p53-Mediated Tumor Suppression In Vivo

Unmet Expectations: miR-34 Plays No Role in p53-Mediated Tumor Suppression In Vivo

The atypical PKCs in inflammation: NF‐κB and beyond

From the very early days of nuclear factor-κB (NF-κB) research, it was recognized that different protein kinase C (PKC) isoforms might be involved in the activation of NF-κB. Pharmacological tools and pseudosubstrate inhibitors suggested that these kinases play a role in this important inflammatory and survival pathway; however, it was the analysis of several genetic mouse knockout models that revealed the complexity and interrelations between the different components of the PB1 network in several cellular functions, including T-cell biology, bone homeostasis, inflammation associated with the metabolic syndrome, and cancer. These studies unveiled, for example, the critical role of PKCζ as a positive regulator of NF-κB through the regulation of RelA but also its inflammatory suppressor activities through the regulation of the interleukin-4 signaling cascade. This observation is of relevance in T cells, where p62, PKCζ, PKCλ/ι, and NBR1 establish a mesh of interactions that culminate in the regulation of T-cell effector responses through the modulation of T-cell polarity. Many questions remain to be answered, not just from the point of view of the implication for NF-κB activation but also with regard to the in vivo interplay between these pathways in pathophysiological processes like obesity and cancer.

The role of APP and APLP for synaptic transmission, plasticity, and network function: lessons from genetic mouse models

APP, APLP1, and APLP2 form a family of mammalian membrane proteins with unknown function. APP, however, plays a key role in the molecular pathology of Alzheimer's disease (AD), indicating that it is somehow involved in synaptic transmission, synaptic plasticity, memory formation, and maintenance of neurons. At present, most of our knowledge about the function of APP comes from consequences of AD-related mutations. The native role of APP, and even more of APLP1/2, remains largely unknown. New genetic knockout and knockin models involving several members of the APP/APLP family may yield better insight into the synaptic and systemic functions of these proteins. Here, we summarize recent results from such transgenic animals with special emphasis on synaptic plasticity and coherent patterns of memory-related network activity in the hippocampus. Data from APP knockout mice suggest that this protein is needed for the expression of long-term potentiation (LTP) in aged, but not in juvenile mice. The missing function can be rescued by expressing part of the protein, as well as by blocking inhibition. Double knockout mice lacking APP and APLP2 die shortly after birth indicating that different members of the APP/APLP family can mutually compensate for genetic ablation of single proteins. Recent techniques allow for analysis of tissue with combined defects, e.g., by expressing only part of APP in APLP2 knockout mice or by growing stem cells with multiple deletions on normal slice cultures. Data from these experiments confirm that APP and APLP2 do indeed play an important role in synaptic plasticity. Much less is known about the role of APP/APLP at the network level. Coherent patterns of activity like hippocampal network oscillations are believed to support formation and consolidation of memory. Analysis of such activity patterns in tissue from mice with altered expression of APP/APLP has just started and may shed further light on the importance of these proteins for cognitive functions.

What Knock-Out Animals Tell Us About the Effects of Caffeine

Caffeine is well known for its complex pharmacological actions, in part reflecting the multiple molecular targets of caffeine. The adenosine receptors are the primary extracellular targets of caffeine. Since caffeine has similar affinity for several adenosine receptors, it has been difficult to determine which receptor subtypes mediate caffeine's effects using pharmacological tools. The development of genetic mutant mice deficient in adenosine receptors and other signaling molecules has allowed targeted inquiry into the molecular targets by which caffeine elicits its biological effects on behavior and gene expression. This review summarizes recent work using genetic knockout models to elucidate the mechanisms of caffeine action in the brain. This review focuses on insights into caffeine action from genetic knockout models on: (1) the molecular basis for caffeine's effects on psychomotor activity; (2) the involvement of adenosine receptors in caffeine-mediated arousal and cognitive effects; and (3) a novel approach using knockout animals coupled with microarray profiling to validate multiple molecular targets of caffeine in striatal gene expression.

As nature did not predict dialysis--what we can learn from FGF23 in end-stage renal disease?

The fibroblast growth factor 23 (FGF23)/klotho system has raised a lot of attention in the past 10 years. Genetic knockout models demonstrated that failure of this system produces a phenotype of premature ageing and especially a failure to excrete phosphate resulting in significant hyperphosphataemia and vascular calcification [1,2]. FGF23 is synthesized in the bone, and osteocytes increase FGF23 production in response to elevated phosphate and calcitriol [3]. Therefore, FGF23 may be a key adaptive factor preventing early hyperphosphataemia in progressive chronic kidney disease (CKD). In the preterminal phases of CKD, FGF23 may become a valuable biomarker of phosphate load and phosphate exposure, perhaps analogous to the predictive value of HbA1C in the evaluation of diabetes control. The central target organ of FGF23 appears to be the kidney, where tubular phosphate reabsorption and 1-alphahydroxylase expression are suppressed. These features raised the question which role FGF23 might play in dialysis patients (CKD stage 5D), the CKD stage where end-stage kidney failure is firmly established and neither substantial phosphaturic effects can be caused nor an already almost lost calcitriol synthesis can be substantially further suppressed. The European study by Jean and colleagues in this issue of the journal came to similar results as the US analysis by Gutierrez et al., using a somewhat, but not substantially, different clinical study approach [4,5]. While the latter study had exclusively focussed on incident dialysis patients not on active vitamin D treatment employing a ‘nested’ analysis, the current cross-sectional study reports on a 2-year follow-up of prevalent dialysis patients independent of their active vitamin D therapy. Both investigations found FGF23 serum levels to be extremely high and remaining closely correlated to serum phosphate levels. Thus, FGF23 regulation is apparently regulated in a similar way in dialysis patients as in progressive non-terminal CKD, despite an obvious loss of its intended biological actions on kidney tissue. However, this does not come as a total surprise, since nature could not predict the invention of renal replacement therapy when establishing such a potent antiphosphataemic protection system to protect against premature hyperphosphataemia on the route to renal failure. The key finding, however, is that in the course of both studies FGF23 has proven to be a strong and independent predictor of mortality, even in serum phosphate ranges which are presently considered relatively safe, at least according to KDOQI target levels for stage CKD 5D (<5.5 mg/dl/<1.78 mmol/l). The apparent link between serum phosphate and FGF23 in this dialysis population raises the question of whether FGF23 may reflect more appropriately the biological harm of individual phosphate levels than the actual phosphate levels themselves (illustrated in Figure 1)? In favour of this theory is the observation by Jean et al. that FGF23 was also apparently related to vascular calcification. Gutierrez et al. just published a link between FGF23 levels and left ventricular hypertrophy in CKD patients [6]—a phenomenon that could potentially be caused by arterial stiffness and increased pulse wave velocity. Alternatively, we should possibly examine whether such enormously elevated FGF23 concentrations, as those present in dialysis patients, are capable of inflicting damage at any tissue sites other than the kidney (such as the myocardium), perhaps by actions on other FGF receptors (FGFR) than on the prototypical dimeric FGFR:klotho receptor. There are two observations in the study by Jean et al. that deserve close attention as hypothesis-generating features: