The Fgf8 subfamily (Fgf8, Fgf17 and Fgf18) is required for closure of the embryonic ventral body wall.

The closure of the embryonic ventral body wall in amniotes is an important morphogenetic event and is essential for life. Defects in human ventral wall closure are a major class of birth defect and a significant health burden. Despite this, very little is understood about how the ventral body wall is formed. Here, we show that fibroblast growth factor (FGF) ligands FGF8, FGF17 and FGF18 are essential for this process. Conditional mouse mutants for these genes display subtle migratory defects in the abdominal muscles of the ventral body wall and an enlarged umbilical ring, through which the internal organs are extruded. By refining where and when these genes are required using different Cre lines, we show that Fgf8 and Fgf17 are required in the presomitic mesoderm, whereas Fgf18 is required in the somites. This study identifies complex and multifactorial origins of ventral wall defects and has important implications for understanding their origins during embryonic development.

Generation and validation of novel conditional flox and inducible Cre alleles targeting fibroblast growth factor 18 (Fgf18).

BACKGROUND: Fibroblast growth factor 18 (FGF18) functions in the development of several tissues, including the lung, limb bud, palate, skeleton, central nervous system, and hair follicle. Mice containing a germline knockout of Fgf18 (Fgf18 -/- ) die shortly after birth. Postnatally, FGF18 is being evaluated for pathogenic roles in fibrosis and several types of cancer. The specific cell types that express FGF18 have been difficult to identify, and the function of FGF18 in postnatal development and tissue homeostasis has been hampered by the perinatal lethality of Fgf18 null mice. RESULTS: We engineered a floxed allele of Fgf18 (Fgf18 flox ) that allows conditional gene inactivation and a CreERT2 knockin allele (Fgf18 CreERT2 ) that allows the precise identification of cells that express Fgf18 and their lineage. We validated the Fgf18 flox allele by targeting it in mesenchymal tissue and primary mesoderm during embryonic development, resulting in similar phenotypes to those observed in Fgf18 null mice. We also use the Fgf18 CreERT2 allele, in combination with a conditional fluorescent reporter to confirm known and identify new sites of Fgf18 expression. CONCLUSION: These alleles will be useful to investigate FGF18 function during organogenesis and tissue homeostasis, and to target specific cell lineages at embryonic and postnatal time points.

Gastric inhibitory polypeptide immunoneutralization attenuates development of obesity in mice.

Previous reports have suggested that the abrogation of gastric inhibitory polypeptide (GIP) signaling could be exploited to prevent and treat obesity and obesity-related disorders in humans. This study was designed to determine whether immunoneutralization of GIP, using a newly developed specific monoclonal antibody (mAb), would prevent the development of obesity. Specific mAb directed against the carboxy terminus of mouse GIP was identified, and its effects on the insulin response to oral and to intraperitoneal (ip) glucose and on weight gain were evaluated. Administration of mAb (30 mg/kg body wt, BW) to mice attenuated the insulin response to oral glucose by 70% and completely eliminated the response to ip glucose coadministered with human GIP. Nine-week-old C57BL/6 mice injected with GIP mAbs (60 mg·kg BW(-1)·wk(-1)) for 17 wk gained 46.5% less weight than control mice fed an identical high-fat diet (P < 0.001). No significant differences in the quantity of food consumed were detected between the two treatment groups. Furthermore, magnetic resonance imaging demonstrated that subcutaneous, omental, and hepatic fat were 1.97-, 3.46-, and 2.15-fold, respectively, lower in mAb-treated animals than in controls. Moreover, serum insulin, leptin, total cholesterol (TC), low-density lipoprotein (LDL), and triglycerides were significantly reduced, whereas the high-density lipoprotein (HDL)/TC ratio was 1.25-fold higher in treated animals than in controls. These studies support the hypothesis that a reduction in GIP signaling using a GIP-neutralizing mAb might provide a useful method for the treatment and prevention of obesity and related disorders.

The functional diversity of essential genes required for mammalian cardiac development.

Genes required for an organism to develop to maturity (for which no other gene can compensate) are considered essential. The continuing functional annotation of the mouse genome has enabled the identification of many essential genes required for specific developmental processes including cardiac development. Patterns are now emerging regarding the functional nature of genes required at specific points throughout gestation. Essential genes required for development beyond cardiac progenitor cell migration and induction include a small and functionally homogenous group encoding transcription factors, ligands and receptors. Actions of core cardiogenic transcription factors from the Gata, Nkx, Mef, Hand, and Tbx families trigger a marked expansion in the functional diversity of essential genes from midgestation onwards. As the embryo grows in size and complexity, genes required to maintain a functional heartbeat and to provide muscular strength and regulate blood flow are well represented. These essential genes regulate further specialization and polarization of cell types along with proliferative, migratory, adhesive, contractile, and structural processes. The identification of patterns regarding the functional nature of essential genes across numerous developmental systems may aid prediction of further essential genes and those important to development and/or progression of disease.

Obesity and the gastrointestinal tract: you are what you eat.

Obesity represents a complex multifactorial syndrome that develops from interactions among genetic and environmental factors and is a leading cause of illness and death. The prevalence of obesity in the United States has increased dramatically since 1975. Although often ignored, the gastrointestinal tract, and the gastrointestinal regulatory peptides in particular, constitutes an ideal starting point for defining and investigating obesity as it represents the route by which all nutrients are ingested, processed, and absorbed. Another important factor to consider when evaluating the etiology of obesity is the capacity for all animals to store nutrients. Insulin is the most potent anabolic hormone, and it appears to have evolved from the need to maximize energy efficiency, obviating the requirement to continuously forage for food. Organisms expressing this important peptide possessed a distinct survival advantage and flourished. During the course of evolution, insulin biosynthesis translocated from the intestine to pancreatic islets, which necessitated a messenger from the intestine to complete the "enteroinsular axis." The eventual development of glucose-dependent insulinotropic polypeptide (GIP) and other incretins fulfilled this requirement. GIP appears to offer an additional survival benefit by not only stimulating intestinal glucose transport and maximally releasing insulin to facilitate nutrient storage but also by its insulin-mimetic properties, including enhanced uptake of glucose by adipocytes. This physiological redundancy offered by insulin and GIP ensured the survival of organisms during times when food was scarce. As food is no longer scarce, at least in the West, this survival advantage appears to have contributed to the current obesity epidemic.

Glucose-dependent insulinotropic polypeptide monoclonal antibodies prevent and treat obesity in wild-type and hyperphagic mice.

OBJECTIVE: This study aimed to investigate whether a glucose-dependent insulinotropic polypeptide (GIP) monoclonal antibody (mAb) will promote weight loss in wild-type mice and to determine effects of this mAb in preventing weight gain in ob/ob mice. METHODS: Phosphate-buffered saline (PBS) or GIP mAb was injected intraperitoneally to wild-type mice fed a 60% high-fat diet (HFD). After 12 weeks, mice that received PBS were divided into two groups and were fed a 37% HFD for 5 weeks; one group received PBS, and one group received GIP mAb. In a separate study, PBS or GIP mAb was injected intraperitoneally to ob/ob mice fed normal mouse chow for 8 weeks. RESULTS: PBS-treated mice gained significantly more than those treated with GIP mAb, with no difference in food consumption detected. Obese mice fed a 37% HFD and PBS continued to gain weight (+2.1% ± 0.9%), whereas mice administered GIP mAb lost 4.1% ± 1.4% body weight (p < 0.01). Leptin-deficient mice consumed similar amounts of chow, and, after 8 weeks, the PBS- and GIP mAb-treated mice gained 250.4% ± 9.1% and 192.4% ± 7.3%, respectively (p < 0.01). CONCLUSIONS: These studies support the hypothesis that a reduction in GIP signaling appears to affect body weight without suppressing food intake and might provide a novel, useful method for the treatment and prevention of obesity.

Hepatic leukemia factor-expressing paraxial mesoderm cells contribute to the developing brain vasculature.

Recent genetic lineage tracing studies reveal heterogeneous origins of vascular endothelial cells and pericytes in the developing brain vasculature, despite classical experimental evidence for a mesodermal origin. Here we provide evidence through a genetic lineage tracing experiment that cephalic paraxial mesodermal cells give rise to endothelial cells and pericytes in the developing mouse brain. We show that Hepatic leukemia factor (Hlf) is transiently expressed by cephalic paraxial mesenchyme at embryonic day (E) 8.0-9.0 and the genetically marked E8.0 Hlf-expressing cells mainly contribute to the developing brain vasculature. Interestingly, the genetically marked E10.5 Hlf-expressing cells, which have been previously reported to contain embryonic hematopoietic stem cells, fail to contribute to the vascular cells. Combined, our genetic lineage tracing data demonstrate that a transient expression of Hlf marks a cephalic paraxial mesenchyme contributing to the developing brain vasculature. This article has an associated First Person interview with the first author of the paper.

Effects of gastric inhibitory polypeptide (GIP) immunoneutralization on mouse motor coordination and memory.

Gastric inhibitory polypeptide (GIP) is a regulatory peptide expressed in the mammalian upper small intestine, and both GIP and its receptor (GIPR) are expressed in the cortex and hippocampus regions of the brain as well. While learning and memory deficits have been observed in GIPR-/- mice, the effects of peripheral GIP immunoneutralization on motor-coordination, learning, and memory have not been examined. In the present study, adult GIPR-/- mice (KO) and age-matched wild-type C57BL/6 J mice (WT) received weekly vehicle PBS injections for 12 weeks, while a third group of wild-type mice were injected weekly for 12 weeks with 30 mg/kg body weight humanized GIP-mAb (AB) to assess the possibility of long-term effects of peripheral GIP antagonism on rodent memory and behavior. All mice groups then underwent a battery of tests that evaluated motor behavior, body coordination, and memory. Performance deficits in several memory studies after 12 weeks of treatment were demonstrated in KO, but not in AB or WT mice. Body coordination performance showed no significant differences among the 3 groups. A similar short-term study (3 injections over 9 days) was also conducted and the results were similar to those from the long-term study. Thus, short-term and long-term peripheral GIP antagonism by GIP-mAb did not appear to affect learning and memory in mice, consistent with the notion that the GIP-mAb does not cross the blood brain barrier. Furthermore, our studies indicate that GIP signaling in the brain appears to involve local neurocrine pathways.

Fatty acid transport protein-2 regulates glycemic control and diabetic kidney disease progression.

Kidney disease is one of the most devastating complications of diabetes, and tubular atrophy predicts diabetic kidney disease (DKD) progression to end-stage renal disease. We have proposed that fatty acids bound to albumin contribute to tubular atrophy by inducing lipotoxicity, after filtration across damaged glomeruli, and subsequent proximal tubule reabsorption by a fatty acid transport protein-2-dependent (FATP2-dependent) mechanism. To address this possibility, genetic (Leprdb/db eNOS-/-) and induced (high-fat diet plus low-dose streptozotocin) mouse models of obesity and DKD were bred with global FATP2 gene-deleted mice (Slc27a2) and then phenotyped. DKD-prone mice with the Slc27a2-/- genotype demonstrated normalization of glomerular filtration rate, reduced albuminuria, improved kidney histopathology, and longer life span compared with diabetic Slc27a2+/+ mice. Genetic and induced DKD-prone Slc27a2-/- mice also exhibited markedly reduced fasting plasma glucose, with mean values approaching euglycemia, despite increased obesity and decreased physical activity. Glucose lowering in DKD-prone Slc27a2-/- mice was accompanied by ß cell hyperplasia and sustained insulin secretion. Together, our data indicate that FATP2 regulates DKD pathogenesis by a combined lipotoxicity and glucotoxicity (glucolipotoxicity) mechanism.

GATA-4 upregulates glucose-dependent insulinotropic polypeptide expression in cells of pancreatic and intestinal lineage.

A thorough examination of glucose-dependent insulinotropic polypeptide (GIP) expression has been hampered by difficulty in isolating widely dispersed, GIP-producing enteroendocrine K-cells. To elucidate the molecular mechanisms governing the regulation of GIP expression, 14 intestinal and pancreatic cell lines were assessed for their suitability for studies examining GIP expression. Both STC-1 cells and the pancreatic cell line betaTC-3 were found to express GIP mRNA and secrete biologically active GIP. However, levels of GIP mRNA and bioactive peptide and the activity of transfected GIP reporter constructs were significantly lower in betaTC-3 than STC-1 cells. When betaTC-3 cells were analyzed for transcription factors known to be important for GIP expression, PDX-1 and ISL-1, but not GATA-4, were detected. Double staining for GIP-1 and GATA-4 in mouse duodenum demonstrated GATA-4 expression in intestinal K-cells. Exogenous expression of GATA-4 in betaTC-3 cells led to marked increases in both GIP transcription and secretion. Lastly suppression of GATA-4 via RNA interference, in GTC-1 cells, a subpopulation of STC-1 cells with high endogenous GIP expression resulted in a marked an attenuation of GIP promoter activity. Our data support the hypothesis that GATA-4 may function to augment or enhance GIP expression rather than act as an initiator of GIP transcription.

Role of hepatic cytochrome p450s in the pharmacokinetics and toxicity of cyclophosphamide: studies with the hepatic cytochrome p450 reductase null mouse.

Cyclophosphamide (CPA) is an anticancer prodrug that is dependent on cytochrome P450 (CYP) metabolism for its therapeutic effectiveness. In spite of the use of CPA in the clinic for over 50 years, little is known about the relationship between its toxicokinetics and therapeutic response. We have employed a powerful new model, the Hepatic Cytochrome P450 Reductase Null (HRN) mouse, which has almost no hepatic cytochrome P450 activity, to study the toxicokinetics of CPA and to establish in vivo the role of hepatic P450 metabolism in its pharmacokinetics. In HRN mice the in vitro metabolism and intrinsic clearance of CPA was over 6-fold lower than in wild-type animals. This change in CPA metabolism was also reflected in vivo, with a profound difference in the pharmacokinetics of both CPA and its metabolites. At a CPA dose of 100 mg/kg, the Cmax, plasma area under the curve (AUC) and half-life were increased by 2.6-, 6.2-, and 3.2-fold, respectively, in the HRN mice. Similar changes were also observed at a dose of 300 mg/kg. These data confirm that hepatic metabolism is the major route of CPA elimination and disposition. The primary metabolites of CPA, 4-hydroxycyclophosphamide (4-OH-CPA) and 3-dechloroethylcyclophosphamide, were still formed, but at altered rates in the HRN mice. At 100 mg/kg the t1/2 for 4-OH-CPA was increased 1.8-fold, the Cmax reduced 1.7-fold, and the AUC remained unchanged. This latter finding shows that P450-mediated oxidative metabolism is essential for the clearance of this compound. Toxicokinetic analysis of CPA-induced myelosuppression and granulocytopenia showed that at high doses (> or =100 mg/kg) there was no difference in myelotoxicity between the wild-type and HRN mice. However, at lower doses (< or =70 mg/kg) a significant difference was observed, with little toxicity seen in HRN mice but at least a 45% reduction in the bone marrow granulocyte population in wild-type mice. Meta-analysis of the toxicity experiments showed the myelotoxicity of CPA was found to be closely correlated with the Cmax of 4-OH-CPA (r2= 0.80, P = 0.002). As the therapeutic effectiveness of CPA has been linked to the AUC for 4-OH-CPA, the finding that 4-OH-CPA Cmax may determine its level of myelotoxicity indicates that the therapeutic index could be altered by changing the method of CPA administration. Furthermore, monitoring 4-OH-CPA Cmax may identify individuals at most risk of CPA side effects.

Cell-specific expression of glucose-dependent-insulinotropic polypeptide is regulated by the transcription factor PDX-1.

Glucose-dependent insulinotropic polypeptide (GIP) is a potent stimulator of insulin secretion and comprises an important component of the enteroinsular axis. GIP is synthesized in enteroendocrine K-cells located principally in the upper small intestine. The homeobox-containing gene PDX-1 is also expressed in the small intestine and plays a critical role in pancreatic development and in the expression of pancreatic-specific genes. Previous studies determined that the transcription factors GATA-4 and ISL-1 are important for GIP expression. In this study, we demonstrate that PDX-1 is also involved in regulating GIP expression in K-cells. Using immunohistochemistry, we verified the expression of PDX-1 protein in the nucleus of GIP-expressing mouse K-cells and evaluated the expression of PDX-1, serotonin, and GIP in wild-type and PDX-1(-/-) mice at 18.5 d after conception. Although we demonstrated a 97.8% reduction in the number of GIP-expressing cells in PDX-1(-/-) mice; there was no statistical difference in the number of serotonin-positive cells. Additionally, PDX-1 transcripts and protein were detected in a GIP-expressing neuroendocrine cell line, STC-1. Electromobility shift assays using STC-1 nuclear extracts demonstrated the specific binding of PDX-1 protein to a specific regulatory region in the GIP promoter. Using chromatin immunoprecipitation analysis, we demonstrated binding of PDX-1 to this same region of the GIP promoter in intact cells. Lastly, overexpression of PDX-1 in transient transfection assays led to a specific increase in the activity of GIP/Luc reporter constructs. The results of these studies indicate that the transcription factor PDX-1 plays a critical role in the cell-specific expression of the GIP gene.

The Drosophila insulin receptor independently modulates lifespan and locomotor senescence.

The Insulin/IGF-like signalling (IIS) pathway plays an evolutionarily conserved role in ageing. In model organisms reduced IIS extends lifespan and ameliorates some forms of functional senescence. However, little is known about IIS in nervous system ageing and behavioural senescence. To investigate this role in Drosophila melanogaster, we measured the effect of reduced IIS on senescence of two locomotor behaviours, negative geotaxis and exploratory walking. Two long-lived fly models with systemic IIS reductions (daGAL4/UAS-InRDN (ubiquitous expression of a dominant negative insulin receptor) and d2GAL/UAS-rpr (ablation of insulin-like peptide producing cells)) showed an amelioration of negative geotaxis senescence similar to that previously reported for the long-lived IIS mutant chico. In contrast, exploratory walking in daGAL4/UAS-InRDN and d2GAL/UAS-rpr flies declined with age similarly to controls. To determine the contribution of IIS in the nervous system to these altered senescence patterns and lifespan, the InRDN was targeted to neurons (elavGAL4/UAS-InRDN), which resulted in extension of lifespan in females, normal negative geotaxis senescence in males and females, and detrimental effects on age-specific exploratory walking behaviour in males and females. These data indicate that the Drosophila insulin receptor independently modulates lifespan and age-specific function of different types of locomotor behaviour. The data suggest that ameliorated negative geotaxis senescence of long-lived flies with systemic IIS reductions is due to ageing related effects of reduced IIS outside the nervous system. The lifespan extension and coincident detrimental or neutral effects on locomotor function with a neuron specific reduction (elavGAL4/UAS-InRDN) indicates that reduced IIS is not beneficial to the neural circuitry underlying the behaviours despite increasing lifespan.

Cell-specific expression of the glucose-dependent insulinotropic polypeptide gene functions through a GATA and an ISL-1 motif in a mouse neuroendocrine tumor cell line.

BACKGROUND/AIMS: Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that, in the presence of glucose, stimulates insulin secretion from beta-cells. GIP is expressed in gastrointestinal K-cells. Prior analysis of the GIP promoter demonstrated that 193 bases of the promoter are required to direct cell specific expression. Here we sought to identify and characterize the transcription factors involved. RESULTS: By mutational analysis of the GIP promoter in a neuroendocrine cell line (STC-1), we identified two regions located between bases -193 and -182 and bases -156 and -151 that, when independently altered, were responsible for a 90% and 85% reduction in transcription, respectively. When we compared these two regions with known motifs from transcription factor databases, we identified the cis elements as potential GATA and ISL-1 binding sites. With subsequent electrophoretic mobility shift analysis (EMSA) using STC-1 nuclear extracts, we demonstrated the ability of these regions to form specific DNA protein complexes. Furthermore, we utilized antisera to confirm the specific binding of GATA-4 to the upstream site and ISL-1 to the downstream element. CONCLUSION: These findings provide evidence for the involvement of the transcription factors GATA-4 and ISL-1 in the cell-specific expression of the GIP gene.

Gastrin stimulates receptor-mediated proliferation of human esophageal adenocarcinoma cells.

The prevalence of esophageal adenocarcinoma in the setting of Barrett's metaplasia continues to increase in Western nations at a rate greater than any other cancer. The trophic properties of gastrin have been documented in gastric, pancreatic and colon cancer cell lines, suggesting a potential role for this regulatory peptide in the growth of these malignancies. The aims of these studies were to identify and characterize the presence of functional cholecystokinin type-2 (gastrin) receptors on the membranes of human esophageal adenocarcinoma cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated the presence of cholecystokinin type-2 receptor transcripts in human esophageal adenocarcinoma cell lines. Competitive binding assays revealed specific binding of gastrin in SEG-1 cells (IC50 of 2.4 x 10(-8) M). This finding was confirmed by laser scanning confocal microscopy through internalization of rhodamine green labeled gastrin heptapeptide in SEG-1 cells. Gastrin caused a dose-dependent increase in proliferation of SEG-1 cells when compared to controls. This effect was abolished by co-incubation with L365,260, a CCK-2-specific receptor antagonist. Gastrin-induced phosphorylation of the p44 and p42 mitogen-activated protein kinases was demonstrated by Western blot analysis. In conclusion, the studied human esophageal adenocarcinoma cell lines possess cholecystokinin type-2 (gastrin) receptors. Receptors bind gastrin, resulting in increased proliferation in SEG-1 cells.

Diversity and professional excellence.

There are compelling moral and practical reasons why the memberships in professions should mirror the populations that they serve. In order to address this general issue more exactly, this essay will confine itself to a particular case as an emblem for the general point. The particular case is the profession of orthopedic surgeons in the United States. From an examination of this specific case, it is hoped that more general issues of racial, ethnic, and gender fairness within the professions will also be addressed.

Interactions of apoptotic cells with macrophages in radiation-induced bystander signaling.

Nontargeted effects that result in ongoing cellular and tissue damage show genotype-dependency in murine models with CBA/Ca, but not C57BL/6, exhibiting sensitivity to induced genomic instability. In vivo, radiation exposure is associated with genotype-dependent macrophage activation, and these cells are a source of bystander signaling involving cytokines and reactive oxygen and nitrogen species. The mechanisms responsible for macrophage activation and production of damaging bystander signals after irradiation are unclear. Macrophages from CBA/Ca exhibit an M1 (proinflammatory) phenotype compared to the M2 (anti-inflammatory) phenotype of C57BL/6 macrophages. Using the murine RAW264.7 macrophage-like cell line, we show that the ability of macrophages to interact with apoptotic cells and their responses to interaction varies significantly according to macrophage phenotype. Nonstimulated and M2 macrophages induce anti-inflammatory markers arginase and TGFß after engulfment of apoptotic cells. In contrast, M1 macrophages do not induce anti-inflammatory responses, but express the proinflammatory markers NOS2, IL-6, TNFα, superoxide and NO, able to contribute to a damaging microenvironment. Macrophages stimulated with both inflammatory and anti-inflammatory agents prior to exposure to apoptotic cells induce a mixed response. The results indicate a complex cross-talk between macrophages and apoptotic cells and demonstrate that phagocytic clearance of apoptotic cells induced by genotoxic stress can produce microenvironmental responses consistent with the induction of a chromosomal instability phenotype in sensitive CBA/Ca mice with M1 macrophage activation, but not in resistant C57BL/6 mice with M2 macrophage activation. Modulation of macrophage phenotypes may represent a novel approach for reducing the nontargeted effects of radiation.

Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study.

The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL) can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h) with fludarabine (n=7) or chlorambucil (n=3). The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15) than in vitro (σ2=1.33) and the results failed to correlate (r(2)=0.18, p=0.22). p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro) responses associated with changes in white cell count (p=0.026). Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses.