INAUGURAL ARTICLE by a Recently Elected Academy Member:Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A

Myostatin (MSTN) is a transforming growth factor-β (TGF-β) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-β family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Myostatin (MSTN) is a secreted signaling molecule that normally acts to limit skeletal muscle growth (for review, see ref. 1). Mice lacking MSTN exhibit dramatic increases in muscle mass throughout the body, with individual muscles growing to about twice the normal size (2). MSTN appears to play two distinct roles in regulating muscle size, one to regulate the number of muscle fibers that are formed during development and a second to regulate the growth of those fibers postnatally. The sequence of MSTN has been highly conserved through evolution, with the mature MSTN peptide being identical in species as divergent as humans and turkeys (3). The function of MSTN has also been conserved, and targeted or naturally occurring mutations in MSTN have been shown to cause increased muscling in numerous species, including cattle (3–5), sheep (6), dogs (7), rabbits (8), rats (9), swine (10), goats (11), and humans (12). Numerous pharmaceutical and biotechnology companies have developed biologic agents capable of blocking MSTN activity, and these have been tested in clinical trials for a wide range of indications, including Duchenne and facioscapulohumeral muscular dystrophy, inclusion body myositis, muscle atrophy following falls and hip fracture surgery, age-related sarcopenia, Charcot–Marie–Tooth disease, and cachexia due to chronic obstructive pulmonary disease, end-stage kidney disease, and cancer.The finding that certain inhibitors of MSTN signaling can increase muscle mass even in Mstn^−/− mice revealed that the function of MSTN as a negative regulator of muscle mass is partially redundant with at least one other TGF-β family member (13, 14), and subsequent studies have identified activin A as one of these cooperating ligands (15, 16). MSTN and activin A share many key regulatory and signaling components. For example, the activities of both MSTN and activin A can be modulated extracellularly by naturally occurring inhibitory binding proteins, including follistatin (17, 18) and the follistatin-related protein, FSTL-3 or FLRG (19, 20). Moreover, MSTN and activin A also appear to share receptor components. Based on in vitro studies, MSTN is capable of binding initially to the activin type II receptors, ACVR2 and ACVR2B (also called ActRIIA and ActRIIB) (18) followed by engagement of the type I receptors, ALK4 and ALK5 (21). In previous studies, we presented genetic evidence supporting a role for both ACVR2 and ACVR2B in mediating MSTN signaling and regulating muscle mass in vivo. Specifically, we showed that mice expressing a truncated, dominant-negative form of ACVR2B in skeletal muscle (18) or carrying deletion mutations in Acvr2 and/or Acvr2b (13) have significantly increased muscle mass. One limitation of the latter study, however, was that we could not examine the consequence of complete loss of both receptors using the deletion alleles, as double homozygous mutants die early during embryogenesis (22). Moreover, the roles that the two type I receptors, ALK4 and ALK5, play in regulating MSTN and activin A signaling in muscle in vivo have not yet been documented using genetic approaches. Here, we present the results of studies in which we used floxed alleles for each of the type II and type I receptor genes in order to target these receptors alone and in combination in muscle fibers. We show that these receptors are functionally redundant and that signaling through each of these receptors contributes to the overall control of muscle mass.     

Fixator-assisted nailing of tibial fractures: New surgical technique and presentation of first 30 cases

Background

Intramedullary nailing is considered a “gold standard” for treatment of tibial shaft fractures. However, some types of fractures are typically considered as “difficult for nailing”. This group includes the periarticular fractures, fractures of both bones at the same level, comminuted and segmental fractures of the tibia. Fixator-assisted nailing (FAN) is an effective method treatment of these types of fractures. The main requirements for the ideal reduction device are an ease of its installation and an ability of multiplanar fracture reduction. Fixator-assisted nailing (FAN) with the use of two perpendicular to each other monolateral tubular frames perfectly meets these requirements. In this study we present this new surgical technique and the analysis of first 30 cases.

Systemic use of non‐biologic corticosteroids in orofacial diseases

Systemic non‐biologic agents have long been in clinical use in medicine – often with considerable efficacy, albeit with some adverse effects – as with all medications. With the advent of biologic agents, all of which currently are restricted to systemic use, there is a growing need to ensure which agents have the better therapeutic ratio. The non‐biologic agents (NBAs) include a range of agents, most especially the corticosteroids (corticosteroids). This study reviews the corticosteroids in systemic use in management of orofacial mucocutaneous diseases; subsequent studies discuss corticosteroid‐sparing agents used in the management of orofacial diseases, such as calcineurin inhibitors used to produce immunosuppression; purine synthetase inhibitors; and cytotoxic and other immunomodulatory agents.     

Detection of Hepatitis B Virus DNA Sequences in Liver in HBsAg Seronegative Patients with Liver Disease with and without Anti-HBc Antibodies

Excluding studies from Brechot and co-workers, little supporthas been found for a role of the hepatitis B virus in the pathogenesisof HBsAg seronegative patients with predominantly chronic liverdiseases, including primary liver cancer. In this study liverDNA from 59 predominantly British patients (four cases withpaired biopsies, 6–12 months apart) with different, mostlychronic, liver diseases was analysed by molecular hybridization.All were seronegative for HBsAg and serum hepatitis B virusDNA (dot blot hybridization) and their liver diseases were believedto be unrelated to hepatitis B virus infection. Hepatitis Bvirus DNA was detected in liver of 11 (18.6 per cent) patients;nine had episomal(3.2 Kb) DNA and eight had higher molecularweight bands suggesting integrated forms. Six patients werealso seronegative for anti-HBc. Patients of UK and non-UK originwere equally represented. Hepatitis B virus DNA was detectedin serum of six of nine patients tested using the polymerasechain reaction. The detection of hepatitis B virus DNA in liverand in serum by this assay in a significant proportion of patientswith chronic liver disease, hitherto unsuspected of being hepatitisB virus-related, suggests a possible role for this virus inlow- as well as high-prevalence countries.     

Percutaneous umbilical blood sampling and umbilical vein transfusions. Rapid serologic differentiation of fetal blood from maternal blood

Percutaneous umbilical blood samples (PUBS), obtained under ultrasound guidance, are used for prenatal diagnosis and management of hemolytic disease of the newborn (HDN) and other fetal disorders. Rapid testing at the time of sampling is vital to distinguish fetal from maternal blood. Blood typing was performed by slide technique in the treatment room during 38 procedures on 25 patients. Anti-I was used to test 50 presumed PUBS; venous I-positive maternal blood was tested in parallel. Because anti-I cannot detect fetal blood after umbilical vein transfusion (UVT) of I-positive donor blood, ABO and Rh blood typing reagents were used to test 29 samples when maternal and fetal or donor blood groups differed. Monoclonal reagents were used for optimal detection of weak AB antigens in fetal blood. Avid, chemically modified anti-D was used for Rh typing. Blood typing showed 27 (34%) of 79 samples to be maternal blood. Fetal blood was obtained in 8 of 10 cases investigated for fetal disorder and in 16 cases of potential HDN (anti-D, 5; -CD, 5; -cE, 2; -K, 2; -c; -E). The absence of HDN (antigen-negative fetus) was determined in 4 cases. UVT afforded live birth of 9 of 10 infants with HDN and was not indicated in two cases.     

Electronic verification of donor-recipient compatibility: the computer crossmatch

Background: This article describes standard operating procedures (SOPs) for a computer crossmatch to replace the immediate-spin crossmatch for ABO incompatibility between patient blood samples submitted for pretransfusion testing and the blood component selected for transfusion. These SOPs were developed following recent changes to the Standards for Blood Banks and Transfusion Services of the American Association of Blood Banks (AABB). Study Design and Methods: SOPs were developed, utilizing currently available software, for pretransfusion testing. The SOP for donor unit processing entails bar code entry of the unit number, component name, and ABO/Rh type; computer entry and interpretation of serologic reactions; warning of discrepancies between bar code-entered blood type and result interpretation; and quarantine of the donor unit in such instances. The SOP for patient sample testing requires bar code entry of specimen accession number, which accesses patient demographics; computer entry and interpretation of ABO/Rh tests; repeat blood typing at the time of crossmatch if only one patient blood type is on record; and warning if there are nonconcordant current and historical blood types. The computer crossmatch SOP requires bar code entry of specimen accession and donor unit numbers; release of group O red cells pending resolution of discrepancies; and immediate-spin crossmatch during computer downtime. Tables validated on- site prompt warning messages and prevent both computer crossmatch and release if blood components of the wrong ABO type are selected. Results: These SOPs meet the requirements of the 15th edition of the AABB Standards. Projected annual time savings at this institution are > 100,000 workload recording units. Further benefits include reduced patient sample volume requirements, less handling of biohazardous material, and elimination of unwanted positive or negative reactions associated with the immediate-spin crossmatch. Release of incompatible blood components when the wrong patient blood type is on record is addressed by requiring the use of group O red cells in the absence of two concordant blood types, one of which must be from a current sample. Conclusion: A combination of existing computer programs and carefully developed SOPs can provide a safe and efficient means of detecting donor-recipient incompatibility without performance of serologic crossmatch.     

Discrepancies in reverse ABO typing due to prozone

Three group O sera manifesting prozone in reverse ABO tests are reported. All were implicated in erroneous blood typing results. One sample failed to react with A1 red cells (RBCs) in immediate-spin (IS) tests, had anti-A and -B titers of 8192 and 2048, respectively, by indirect antiglobulin technique (IAT), and was from a diabetic patient; the parenteral administration of A substance present in porcine insulin is a possible cause of hyperimmunity in this case. The second sample was from the recipient of a single unit of group B fresh-frozen plasma; the serum anti-A and -B titers were 10,240 by IAT, but only weak reactions with A1 and B RBCs were noted in routine IS reverse typing tests; the hyperimmunity in the patient concerned was likely due to crossreacting anti-A, B stimulated by B-active glycoproteins and/or glycolipids in the transfused plasma. The third serum also had anti-A and anti-B IAT titers of 10,240 but did not react with A1 and B RBCs by IS; the hyperimmunity in this case may be related to sepsis from intestinal flora carrying A- and/or B-like antigens. These antibodies lysed A1 and/or B RBCs in tests incubated at room temperature (RT) and strongly agglutinated those RBCs by IS when diluted 10-fold with saline. The absence of the prozone phenomenon in tests with RBCs suspended in diluents containing EDTA is consistent with the previously published mechanism for anti-A prozone: namely, the steric hindrance of agglutination by the C1 component of human complement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Can the reading for serologic reactivity following 37 degrees C incubation be omitted?

The need to detect antibodies that agglutinate and/or hemolyze red cells (RBCs) directly at 37 degrees C, but do not react in subsequently performed indirect antiglobulin tests (IATs), is of concern relative to the streamlining and automation of antibody detection methods. To determine incidence and significance of such reactions, data from 87,480 tests, which used low-ionic-strength saline, 10-minute incubation at 37 degrees C, and anti-IgG, were analyzed for unexpected antibodies. There were 3590 positive tests, of which 475 showed reactions at 37 degrees C but not in subsequently performed IATs (37 + IAT-). Of these, 196 reactions were due to autoantibodies or other factors usually considered insignificant with respect to the survival of transfused incompatible RBCs, 176 were due to alloantibodies of questionable clinical significance (M, Lea, P1, etc.), and 103 were associated with alloantibodies of potential clinical significance (63 E, 27 K, 5 Jka, 4 D, 3 cE, and 1 C). This latter reaction was seen in 72 patients, with two 37 + IAT-antibodies occurring in each of 3 patients. Of the 75 potentially significant 37 + IAT-antibodies, 57 were seen in patients recently exposed to homologous RBCs, 13 in patients with a history of transfusion and/or pregnancy, and 5 in patients with no known exposure to homologous RBCs. IAT reactivity was observed in subsequent samples with 27 of these antibodies. The predictive value of a 37 + IAT-test was 21.7 percent for a potentially significant antibody. The incidence was 0.12 percent of all tests for unexpected antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)