Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomatous growths in many organs. Sixty to seventy percent of cases are sporadic and appear to represent new mutations. TSC exhibits locus heterogeneity: the TSC2 gene is located at 16p13.3 whilst the TSC1 gene, predicted to encode a novel protein termed hamartin, has recently been cloned from 9q34. With the exception of a contiguous gene deletion syndrome involving TSC2 and PKD1 , TSC1 and TSC2 phenotypes have been considered identical. We have now comprehensively defined the TSC1 mutational spectrum in 171 sequentially ascertained, unrelated TSC patients by single strand conformation polymorphism and heteroduplex analysis of all 21 coding exons, and by assaying a restriction fragment spanning the whole locus. Mutations were identified in 9/24 familial cases, but in only 13/147 sporadic cases. In contrast, a limited screen revealed TSC2 mutations in two of the familial cases and in 45 of the sporadic cases. Thus TSC1 mutations were significantly under-represented among sporadic cases (Fisher's exact p -value = 3.12 x 10(-4)). Both large deletions and missense mutations were common at the TSC2 locus whereas most TSC1 mutations were small truncating lesions. Mental retardation was significantly less frequent among carriers of TSC1 than TSC2 mutations (odds ratio 5.54 for sporadic cases only, 6.78 +/- 1.54 when a single randomly selected patient per multigeneration family was also included). No correlation between mental retardation and the type of mutation was found. We conclude that there is a reduced risk of mental retardation in TSC1 as opposed to TSC2 disease and that consequent ascertainment bias, at least in part, explains the relative paucity of TSC1 mutations in sporadic TSC.      

Functional analysis and intracellular localization of the human menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1)

The Menkes protein (MNK or ATP7A) is an important component of the mammalian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure and function of this protein and to elucidate its role in cellular copper homeostasis, a cDNA construct encoding the full-length MNK protein was cloned into a mammalian expression vector under the control of the CMV promoter. Transfection of this plasmid construct into CHO-K1 cells yielded clones that expressed MNK at varying levels. Detailed characterization of four clones showed that an increase in MNK protein expression led to a corresponding increase in the level of copper resistance of the cells. Subcellular localization studies showed that in the parental CHO-K1 and the transfected cell lines, MNK was located in a post-Golgi compartment which, based on immunogold electron microscopic analyses, most likely represented the trans -Golgi network (TGN). When the extracellular copper concentration was increased, MNK in the clones as well as in CHO-K1 cells was redistributed to the cytoplasm and plasma membrane, but returned to the TGN under basal, low copper conditions. This report presents the first ultrastructural evidence for the association of MNK with vesicles within the cell and with the TGN and plasma membrane. It also demonstrates the stable expression of a functional MNK protein from a cDNA construct in mammalian cells, as well as the copper-induced redistribution of MNK in a cell line (CHO-K1) that was not selected for copper resistance or overexpression of MNK.      

Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone

In contrast to the many detailed studies of Graafian follicles, the biology of small follicles in the human ovary is poorly understood and the trigger for follicular growth initiation remains unknown. No practical model exists to study preantral follicle growth in the human because of their slow growth rate and lack of an effective culture system. We therefore tested ovarian xenografts as a new strategy to study the early stages of ovarian follicular growth in vivo. Mice homozygous for severe combined immunodeficiency (SCID) and hypogonadism (hpg) received human ovarian xenografts under their kidney capsules. Follicle growth was assessed by morphology and proliferating cell nuclear antigen (PCNA) immunostaining. The grafts were recovered after 11 (short-term) and 17 weeks (long-term), and serially sectioned. During the last 6 weeks of long-term grafting, mice were randomized to receive either placebo or 1 IU of purified follicle stimulating hormone (FSH) s.c. on alternating days. After 11 weeks of grafting, the most advanced follicles had a maximum of two granulosa cell layers. In the absence of FSH administration, follicles did not progress beyond the two-layer stage even after 17 weeks of grafting, and the oestradiol levels remained undetectable. In the FSH-treated long-term grafts, follicles had grown to antral stages and resulted in oestradiol levels as high as 2070 pmol/l. Growth initiation indices did not differ between control and FSH-treated grafts. This study demonstrates that follicles can survive and grow in human ovarian tissue grafted under the renal capsules of immunodeficient mice for at least 17 weeks, and indicate that xenograft models are potentially useful for studying human follicle development. Using this physiological model, we showed that FSH is required for follicle growth beyond the two-layer stage, although growth initiation is independent of gonadotrophin stimulation.      

Transplantation in reproductive medicine: previous experience, present knowledge and future prospects

The use of transplantation in reproductive medicine has beenconsidered by physicians and scientists alike for many years.Despite being side-tracked into futile pursuits of rejuvenationin the early days, the possibility of usefulness remains, particularlyfor preserving fertility in patients undergoing ablative chemo-or radiotherapy. These aims have been enhanced by advances intissue cryopreservation. When isolated primordial folliclesare transferred in the mouse, or ovarian tissue slices are graftedinto sheep, it is possible to obtain follicular survival withsubsequent maturation and oestrogen secretion and even restorefertility to sterilized hosts. For preservation of fertility,autografts avoid both the immunological problems of allograftsand the ethical dilemmas when using donor tissue. In the male,the concept of spermatogonial cell transfer after isolationand frozen storage of cells recovered from a testicular biopsyis most attractive, since it may provide another option forrescuing fertility in cancer patients, and provide a much neededone in children. Recent results demonstrate that gonocytes fromimmature mice injected into the tubules of sterilized hostsrestore spermatogenesis and produce fertile spermatozoa. Furthermore,the gonocytes can be stored frozen prior to transfer and stillproduce fertile tubules. This review presents a broad historyof transplantation in the male and female genital tracts aswell as attempts to anticipate possible future developments.     

Cytokine-specific regulation of urokinase receptor (CD87) expression by U937 mononuclear phagocytes

Mononuclear phagocytes concentrate urokinase-type plasminogen activator (uPA) at the cell surface by expressing membrane uPA receptors (uPAR). This study examines the ability of exogenous cytokines to alter expression of membrane-associated uPA and uPAR in U937 mononuclear phagocytes. Cells were stimulated with recombinant interferon gamma (IFN gamma) or tumor necrosis factor alpha (TNF alpha), followed by immunolabeling for uPA or uPAR and flow cytometry. IFN gamma increased surface uPA 2.2-fold relative to unstimulated controls (P < .001), whereas TNF alpha had no significant effect. Likewise, maximal uPA binding capacity was increased 2.8-fold by IFN gamma (P < .02), but was not affected by TNF alpha. In unstimulated cells, 50% of receptors were occupied by endogenously generated uPA, and this proportion was not affected by either cytokine. IFN gamma upregulated uPAR 2.1-fold relative to unstimulated controls (P < .001), whereas TNF alpha had no effect. In contrast to effects on surface protein, TNF alpha induced a substantial increase in uPAR mRNA, equaling the effect of IFN gamma. In addition, both cytokines doubled the intracellular uPAR pool (P < .01). By contrast, TNF alpha induced a 2.5-fold increase in the level of uPAR protein released into conditioned medium (compared with unstimulated cells), whereas IFN gamma had no effect. These results indicate that uPAR expression is regulated in a cytokine-specific fashion. Some stimuli, such as TNF alpha, may increase uPAR synthetic activity without a corresponding change in membrane expression, because of enhanced release of uPAR from the cell. Cytokine-specific modulation of uPAR may be important in regulating the function of mononuclear phagocytes in inflammation and tissue repair.     

Characterization of colony-stimulating activity produced by human monocytes and phytohemagglutinin-stimulated lymphocytes

Human colony-stimulating activity (CSA) may support the proliferation of both human and murine granulocyte-macrophage progenitor cells (CFU- C) or, in the case of human urinary CSA, may only stimulate murine bone marrow CFU-C. CSA produced in the culture media of monocytes and macrophages and phytohemagglutinin-stimulated lymphocytes from human peripheral blood was characterized for both human and mouse marrow CFU- C stimulating activities. During the initial phase of a long-term cultures of monocytes, both human- and mouse-active CSA (MnCM-HM) were produced. In later phases of culture, however, only mouse-active CSA (MnCM-M) was produced. Fractionation on Sephadex G-150 revealed two functionally distinct species from MnCM-HM and lymphocytes conditioned medium, a high molecular weight factor (MW greater than 150,000) which stimulated mouse but not human colony formation, and a low molecular weight species (MW 25,000-35,000) which was active against both mouse and human target cells. However, MnCM-M revealed only one high molecular weight species (greater than 150,000), active only on mouse marrow. The possible biologic significance of such an activity is discussed.     

Early Changes in Bone Density,Microarchitecture, Bone Resorption,and Inflammation Predict the Clinical Outcome 12 Weeks After Conservatively Treated Distal Radius Fractures: An Exploratory Study

Fracture healing is an active process with early changes in bone and inflammation. We performed an exploratory study evaluating the association between early changes in densitometric, structural, biomechanical, and biochemical bone parameters during the first weeks of fracture healing and wrist‐specific pain and disability at 12 weeks in postmenopausal women with a conservatively treated distal radius fracture. Eighteen patients (aged 64 ± 8 years) were evaluated at 1 to 2 and 3 to 4 weeks postfracture, using high‐resolution peripheral quantitative computed tomography (HR‐pQCT), micro‐finite element analysis, serum procollagen type‐I N‐terminal propeptide (P1NP), carboxy‐terminal telopeptide of type I collagen (ICTP), and high‐sensitive C‐reactive protein (hsCRP). After 12 weeks, patients rated their pain and disability using Patient Rated Wrist Evaluation (PRWE) questionnaire. Additionally, Quick Disability of the Arm Shoulder and Hand (QuickDASH) questionnaire and active wrist range of motion was evaluated. Linear regression models were used to study the relationship between changes in bone parameters and in hsCRP from visit 1 to 2 and PRWE score after 12 weeks. A lower PRWE outcome, indicating better outcome, was significantly related to an early increase in trabecular bone mineral density (BMD) (β ?0.96 [95% CI ?1.75 to ?0.16], R² = 0.37), in torsional stiffness (?0.14 [?0.28 to ?0.004], R² = 0.31), and to an early decrease in trabecular separation (209 [15 to 402], R² = 0.33) and in ICTP (12.1 [0.0 to 24.1], R² = 0.34). Similar results were found for QuickDASH. Higher total dorsal and palmar flexion range of motion was significantly related to early increase in hsCRP (9.62 [3.90 to 15.34], R² = 0.52). This exploratory study indicates that the assessment of early changes in trabecular BMD, trabecular separation, calculated torsional stiffness, bone resorption marker ICTP, and hsCRP after a distal radius fracture provides valuable information regarding the 12‐week clinical outcome in terms of pain, disability, and range of motion and validates its use in studies on the process of early fracture healing. © 2014 American Society for Bone and Mineral Research.     

Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates

We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG- CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of progenitor cells that can be collected by leukapheresis than does rhG-CSF alone, that blood cells mobilized by low-dose rhSCF plus rhG-CSF contain marrow repopulating cells, and finally that using a single 2-hour leukapheresis to collect cells, the blood cells mobilized by low-dose rhSCF plus rhG-CSF engraft lethally irradiated recipients more rapidly than do blood cells mobilized by rhG- CSF alone.