Modeling of red blood cell life-spans in hematologically normal populations

Despite the impact of red blood cell (RBC) Life-spans in some disease areas such as diabetes or anemia of chronic kidney disease, there is no consensus on how to quantitatively best describe the process. Several models have been proposed to explain the elimination process of RBCs: random destruction process, homogeneous life-span model, or a series of 4-transit compartment model. The aim of this work was to explore the different models that have been proposed in literature, and modifications to those. The impact of choosing the right model on future outcomes prediction--in the above mentioned areas--was also investigated. Both data from indirect (clinical data) and direct life-span measurement (biotin-labeled data) methods were analyzed using non-linear mixed effects models. Analysis showed that: (1) predictions from non-steady state data will depend on the RBC model chosen; (2) the transit compartment model, which considers variation in life-span in the RBC population, better describes RBC survival data than the random destruction or homogenous life-span models; and (3) the additional incorporation of random destruction patterns, although improving the description of the RBC survival data, does not appear to provide a marked improvement when describing clinical data.

Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes

The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.

Pharmacokinetics and clinical efficacy of cyclosporine treatment of dogs with steroid-refractory inflammatory bowel disease

The usual treatment of dogs with inflammatory bowel disease (IBD) consists of administration of immunosuppressive doses of steroids. However, some dogs are refractory to steroid treatment and pose a significant challenge to the veterinarian. Because cyclosporine A (cyA) has been shown to be effective in steroid-resistant IBD in humans, the purpose of this study was to investigate the pharmacokinetics and clinical efficacy of PO cyA treatment in dogs with steroid-refractory IBD (n = 14). All dogs were treated with cyA 5 mg/kg PO q24h for a period of 10 weeks. A clinical activity score was assigned to assess severity of clinical signs before and after treatment. The total number of infiltrating lymphocytes and T cells in duodenal biopsies were assessed before and after treatment in 9 dogs. In addition, serum concentration of cyA was measured in 8 dogs over a 24-hour period. Pharmacokinetic profiles in dogs with IBD were similar to those of healthy dogs. Improvement of clinical signs was observed in 12 of 14 dogs with IBD. Median clinical activity score after treatment with cyA was significantly reduced from a median score of 9 to a median score of 5 (P = 0.001). T cell numbers in duodenal biopsies were significantly decreased after treatment from a median +/- 95% range in the villous region of 28 (19-30) cells/10,000 microm2 before versus 7 (0-10)/10,000 microm2 after treatment, P = 0.01; and from a median +/- 95% range number in the crypt region of 15 (6-23) cells/10,000 microm2 before versus 4 (0-9)/10,000 microm2 after treatment, P = 0.02, implying T cell lysis as a possible mechanism of action. In conclusion, based on this small study, cyA appears to be an effective alternative drug in dogs with IBD that are refractory to immunosuppressive doses of steroids.

Metastatic disease to the parotid gland

OBJECTIVES: The purpose of this study was to review the clinico-pathologic findings and outcome of patients with metastasis to the parotid lymph nodes and gland. METHODS: Retrospective study. Thirty-four out of 520 patients with a parotid mass treated at our institution met the criteria for this study. Age, gender, clinical findings, histopathology, treatment. and outcome were analyzed. RESULTS: Twenty-three patients had metastases to parotid lymph nodes from a squamous cell carcinoma of the skin, seven from a cutaneous malignant melanoma after a mean interval of 15 months, two from a ductal breast carcinoma, one with a metastatic disease from a rhabdomyosarcoma of the lower limb, and one from a Merkel cell carcinoma. The 5-year cause-specific and absolute survival for squamous cell carcinoma was 64% and 48% respectively and 71% and 57% for malignant melanomas. CONCLUSIONS: Metastatic disease to the parotid gland is mostly caused by squamous cell carcinoma. Despite combined treatment modalities long-term survival remains poor.

Cyclin D1 in non-small cell lung cancer: a key driver of malignant transformation

PURPOSE: To review the evidence implicating the deregulation of cyclin D1 in the pathogenesis of non-small cell lung cancer (NSCLC), and to discuss the opportunities for targeted clinical intervention. METHODS: Data published until June 2006 are summarized, and previously unpublished results from our own research are included. RESULTS: In normal cells, cyclin D1 complexes with and activates cyclin-dependent kinases (CDK) and acts as a transcriptional regulator. The protein is frequently overexpressed in a wide range of cancers, sometimes coincident with CCND1 (cyclin D1) gene amplification (5-20% of tumours). A low level of somatic mutations have been seen in certain tumours. CCND1 is amplified in NSCLC and cyclin D1 is frequently overexpressed in tumours and pre-invasive bronchial lesions, generally from one parental allele. Mutation analyses revealed a frequent CCND1 gene polymorphism (A870G) that modulates alternative splicing and allows expression of an alternative cyclin D1 transcript (transcript cyclin D1b). The encoded cyclin D1b protein lacks a specific phosphorylation site required for nuclear export. Genotype has been correlated with the risk and/or severity of disease or drug response across a range of malignancies, including lung cancer. Together, these findings suggest a strong pathological role for cyclin D1 deregulation in bronchial neoplasia. CONCLUSION: Current data indicate that cyclin D1 overexpression is not a consequence of, but rather a pivotal element in the process of malignant transformation in the lung and other tissues. This understanding may open new avenues for lung cancer diagnosis, treatment and prevention.

Cyclin D1 (CCND1) A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer (NSCLC) patients

PURPOSE: The cyclin D1 (CCND1) A870G gene polymorphism is linked to the outcome in patients with resectable non-small cell lung cancer (NSCLC). Here, we investigated the impact of this polymorphism on smoking-induced cancer risk and clinical outcome in patients with NSCLC stages I-IV. METHODS: CCND1 A870G genotype was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis (RFLP) of DNA extracted from blood. The study included 244 NSCLC patients and 187 healthy control subjects. RESULTS: Patient characteristics were: 70% male, 77% smokers, 43% adenocarcinoma, and 27% squamous cell carcinoma. Eighty-one percent of the patients had stages III-IV disease. Median age at diagnosis was 60 years and median survival was 13 months. Genotype frequencies of patients and controls both conformed to the Hardy Weinberg equilibrium. The GG genotype significantly correlated with a history of heavy smoking (>or=40 py, P=0.02), and patients with this genotype had a significantly higher cigarette consumption than patients with AA/AG genotypes (P=0.007). The GG genotype also significantly correlated with tumor response or stabilization after a platinum-based first-line chemotherapy (P=0.04). Survival analysis revealed no significant differences among the genotypes. CONCLUSION: Evidence was obtained that the CCND1 A870G gene polymorphism modulates smoking-induced lung cancer risk. Further studies are required to explore the underlying molecular mechanisms and to test the value of this gene polymorphism as a predictor for platinum-sensitivity in NSCLC patients.

Prospective study of fetal DNA in serum and disease activity during pregnancy in women with inflammatory arthritis

OBJECTIVE: Rheumatoid arthritis (RA) usually improves during pregnancy and recurs postpartum. Fetal cells and cell-free DNA reach the maternal circulation during normal pregnancy. The present study investigated dynamic changes in levels of fetal DNA in serum from women with RA and inflammatory arthritis during and after pregnancy to test the hypothesis that the levels of circulating fetal DNA correlate with arthritis improvement. METHODS: Twenty-five pregnant patients were prospectively studied. A real-time quantitative polymerase chain reaction panel targeting unshared, paternally transmitted HLA sequences, a Y chromosome-specific sequence, or an insertion sequence within the glutathione S-transferase M1 gene was used to measure cell-free fetal DNA. Results were expressed as fetal genomic equivalents per milliliter (gE/ml) of maternal serum. Physical examinations were conducted during and after pregnancy. RESULTS: Levels of fetal DNA in women with improvement in or remission of arthritis were higher than those in women with active disease, especially in the third trimester. Overall, an inverse relationship between serum fetal DNA levels and disease activity was observed (P < 0.001). Serum fetal DNA increased with advancing gestation, reaching median levels of 24 gE/ml (range 0-334), 61 gE/ml (range 0-689), and 199 gE/ml (range 0-2,576) in the first, second, and third trimesters, respectively, with fetal DNA clearance observed postpartum. Arthritis improvement was initially noted in the first trimester for most patients, increased further or was sustained with advancing gestation, and was active postpartum. CONCLUSION: Changes in serum fetal DNA levels correlated with arthritis improvement during pregnancy and recurrence postpartum. Immunologic mechanisms by which pregnancy might modulate RA activity are described.

Hematopoietic and endothelial progenitor cell trafficking in patients with myeloproliferative diseases

BACKGROUND AND OBJECTIVES. The presence of circulating hematopoietic progenitor cells in patients with myeloproliferative diseases (MPD) has been described. However, the exact nature of such progenitor cells has not been specified until now. The aim of this work was to investigate the presence of endothelial precursor cells in the blood of patients with MPD and to assess the role of the endothelial cell lineage in the pathophysiology of this disease. DESIGN AND METHODS. Endothelial progenitor cell marker expression (CD34, prominin (CD133), kinase insert domain receptor (KDR) or vascular endothelial growth factor receptor 2 (VEGFR2), and von Willebrand factor) was assessed in the blood of 53 patients with MPD by quantitative polymerase chain reaction. Clonogenic stem cell assays were performed with progenitor cells and monocytes to assess differentiation towards the endothelial cell lineage. The patients' were divided according to whether they had essential thrombocythemia (ET, n=17), polycythemia vera (PV, n=21) or chronic idiopathic myelofibrosis (CIMF, n=15) and their data compared with data from normal controls (n=16) and patients with secondary thrombo- or erythrocytosis (n=17). RESULTS. Trafficking of CD34-positive cells was increased above the physiological level in 4/17 patients with ET, 5/21 patients with PV and 13/15 patients with CIMF. A subset of patients with CIMF co-expressed the markers CD34, prominin (CD133) and KDR, suggesting the presence of endothelial precursors among the circulating progenitor cells. Clonogenic stem cell assays confirmed differentiation towards both the hematopoietic and the endothelial cell lineage in 5/10 patients with CIMF. Furthermore, the molecular markers trisomy 8 and JAK2 V617F were found in the grown endothelial cells of patients positive for trisomy 8 or JAK2 V617F in the peripheral blood, confirming the common clonal origin of both hematopoietic and endothelial cell lineages. INTERPRETATION AND CONCLUSIONS. Endothelial precursor cells are increased in the blood of a subset of patients with CIMF, and peripheral endothelial cells bear the same molecular markers as hematopoietic cells, suggesting a primary role of pathological endothelial cells in this disease.

Galectin-3 modulates T cell activity and is reduced in the inflamed intestinal epithelium in IBD

BACKGROUND: Galectins are involved at different stages in inflammation. Galectin-3, although mostly described as proinflammatory, can also act as an immunomodulator by inducing apoptosis in T cells. The present study aims to determine galectin-3 expression in the normal and inflamed intestinal mucosa and to define its role in T cell activity. MATERIALS AND METHODS: Galectin-3 was detected by quantitative polymerase chain reaction with total RNA from endoscopic biopsies and by immunohistochemistry. Biopsies and peripheral blood mononuclear cells (PBMC) were stimulated in vitro and were used to assess the functional consequences of inhibition or exogenous addition of galectin-3. RESULTS: Galectin-3 is expressed at comparable levels in controls and inflammatory bowel disease (IBD) patients in remission. In the normal mucosa, galectin-3 protein was mainly observed in differentiated enterocytes, preferentially at the basolateral side. However, galectin-3 was significantly downregulated in inflamed biopsies from IBD patients. Ex vivo stimulation of uninflamed biopsies with tumor necrosis factor led to similar galectin-3 messenger RNA downregulation as in vivo. When peripheral blood mononuclear cells (PBMC) were analyzed, galectin-3 was mainly produced by monocytes. Upon mitogen stimulation, we observed increased proliferation and decreased activation-induced cell death of peripheral blood T cells in the presence of galectin-3-specific small interfering RNA. In contrast, exogenous addition of recombinant galectin-3 led to reduced proliferation of mitogen-stimulated peripheral blood T cells. CONCLUSIONS: Our results suggest that downregulation of epithelial galectin-3 in the inflamed mucosa reflects a normal immunological consequence, whereas under noninflammatory conditions, its constitutive expression may help to prevent inappropriate immune responses against commensal bacteria or food compounds. Therefore, galectin-3 may prove valuable for manipulating disease activity.

Cartilage tissue engineering for degenerative joint disease

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.

[Follow-up in a boy with Leydig cell tumor after selective surgery]

A 8(6/12) year-old-boy presented with precocious puberty and a slightly enlarged left testis. After a detailed examination a Leydig cell tumour was diagnosed. Surgical exploration revealed an encapsulated tumour, 2.7 cm in length, which was selectively removed without orchidectomy. Within one year the clinical signs of pubertal precocity disappeared, the bone age did not further advance and height velocity declined from 8.2 cm / year (+3.9 SDS) to 4.1 cm/year (-1.0 SDS). Physiologically, he entered puberty at the chronological age of twelve years, presenting at that age, in comparison to his peer group, a slightly decreased pubertal growth spurt. However, bearing in mind that being precocious in puberty he started in fact his pubertal growth spurt at a far earlier age, therefore, this acceleration of height before operation has to be added to the centimetres gained during pubertal development thereafter resuiting consequently in an absolute normal pubertal growth spurt. This underlines the fact that the individual growth spurt and, therefore, the total amount of centimetres gained is very much robust. Ten years later, the patient ended up well within his familial target height and remained free of disease. We report on a long-term follow-up of a prepubertal boy after testis-sparing surgery for Leydig-cell-tumour.

CD40 ligation protects bronchial epithelium against oxidant-induced caspase-independent cell death

CD40 and its ligand regulate pleiotropic biological responses, including cell proliferation, differentiation, and apoptosis. In many inflammatory lung diseases, tissue damage by environmental or endogenous oxidants plays a major role in disease pathogenesis. As the epithelial barrier is a major target for these oxidants, we postulated that CD40, the expression of which is increased in asthma, plays a role in the regulation of apoptosis of bronchial epithelial cells exposed to oxidants. Using 16HBE 14o- cells exposed to oxidant stress, we found that ligation of CD40 (induced by G28-5 monoclonal antibodies) enhanced cell survival and increased the number of cells in G2/M (interphase between DNA synthesis and mitosis) of the cell cycle. This was associated with NF-kappaB and activator protein-1 activation and increased expression of the inhibitor of apoptosis, c-IAP1. However, oxidant stress-induced apoptosis was found to be caspase- and calpain-independent implicating CD40 ligation as a regulator of caspase-independent cell death. This was confirmed by the demonstration that CD40 ligation prevented mitochondrial release and nuclear translocation of apoptosis inducing factor. In conclusion, we demonstrate a novel role for CD40 as a regulator of epithelial cell survival against oxidant stress. Furthermore, we have identified, for the first time, an endogenous inhibitory pathway of caspase-independent cell death.

Expression of HB-EGF by retinal pigment epithelial cells in vitreoretinal proliferative disease

The heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated in wound-healing processes of various tissues. However, it is not known whether HB-EGF may represent a factor implicated in overstimulated wound-healing processes of the retina during proliferative retinopathies. Therefore, we investigated whether human retinal pigment epithelial (RPE) cells, which are crucially involved in proliferative retinopathies, express and respond to HB-EGF. RPE cells express mRNAs for various members of the EGF-related growth factor family, among them for HB-EGF, as well as for the EGF receptors ErbB1, -2, -3, and -4. The gene expression of HB-EGF is stimulated in the presence of transforming and basic fibroblast growth factors and by oxidative stress and is suppressed during chemical hypoxia. Exogenous HB-EGF stimulates proliferation and migration of RPE cells and the gene and protein expression of the vascular endothelial growth factor (VEGF). HB-EGF activates at least three signal transduction pathways in RPE cells including the extracellular signal-regulated kinases (involved in the proliferation-stimulating action of HB-EGF), p38 (mediates the effects on chemotaxis and secretion of VEGF), and the phosphatidylinositol-3 kinase (necessary for the stimulation of chemotaxis). In epiretinal membranes of patients with proliferative retinopathies, HB-EGF immunoreactivity was partially colocalized with the RPE cell marker, cytokeratins; this observation suggests that RPE cell-derived HB-EGF may represent one factor that drives the uncontrolled wound-healing process of the retina. The stimulating effect on the secretion of VEGF may suggest that HB-EGF is also implicated in the pathological angiogenesis of the retina.

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.

Immune cell migration across the blood–brain barrier: molecular mechanisms and therapeutic targeting

The endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier protect the CNS from the constantly changing milieu within the bloodstream. The BBB strictly controls immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS, such as viral or bacterial infections, or during inflammatory diseases, such as multiple sclerosis, immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of the available information on immune cell entry into the CNS is derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Consequently, our current knowledge on traffic signals mediating immune cell entry across the BBB during immunosurveillance and disease results mainly from experimental data in the EAE model. Therefore, a large part of this review summarizes these findings. Similarly, the potential benefits and risks associated with therapeutic targeting of immune cell trafficking across the BBB will be discussed in the context of multiple sclerosis, since elucidation of the molecular mechanisms relevant to this disease have largely relied on the use of its in vivo model, EAE.