Guidelines for reporting experiments involving animals: the ARRIVE guidelines

British Journal of Pharmacology (BJP) is pleased to publish a new set of guidelines for reporting research involving animals, simultaneously with several other journals; the ‘ARRIVE’ guidelines (Animals in Research: Reporting In Vivo Experiments). This editorial summarizes the background to the guidelines, gives our view of their significance, considers aspects of specific relevance to pharmacology, re-states BJP''s guidelines for authors on animal experiments and indicates our commitment to carrying on discussion of this important topic. We also invite feedback via the British Pharmacological Society website.     

Large normal and reduced penetrance alleles in Huntington disease: instability in families and frequency at the laboratory,at the clinic and in the population

Sequeiros J, Ramos EM, Cerqueira J, Costa MC, Sousa A, Pinto‐Basto J, Alonso I. Large normal and reduced penetrance alleles in Huntington disease: instability in families and frequency at the laboratory, at the clinic and in the population. Large normal (‘intermediate’) alleles may produce de novo expansions in Huntington disease; nevertheless, there is very little evidence about their population prevalence and impact in daily practice, and there are conflicting reports about the extent of their instability. We estimated the frequency of large normal alleles (27–35 CAGs) and of reduced penetrance alleles (36–39 CAGs), as well as the frequency of genotypes carrying them, in (i) a diagnostic laboratory, (ii) a genetic counselling clinic and (iii) the general population. Large normal alleles were present in 6% of a large control sample, 7% of consultands who took pre‐symptomatic testing and 7% of samples in the laboratory. Reduced penetrance alleles were found in 1 of 1772 control chromosomes (0.1% of individuals), 5% of 146 pre‐symptomatic testees and over 2% of 1214 diagnostic samples (350 families). All 16 alleles sized 27–32 CAGs seemed to be transmitted stably; alleles ≥ 36 repeats were unstable in five families. Seven small full penetrance alleles contracted into the reduced penetrance range, but none into the large normal range. Evidence showed that large normal alleles are relatively frequent and that those with reduced penetrance are not a rare event, either at the laboratory or the clinic. This reinforces the need to understand the genomic context of repeat instability in each family and population.     

Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases

Loss of a whole chromosome 7 or a deletion of the long arm, del(7q), are recurring abnormalities in malignant myeloid diseases. To determine the location of genes on 7q that are likely to play a role in leukemogenesis, we examined the deleted chromosome 7 homologs in a series of 81 patients with therapy-related or de novo myelodysplastic syndrome or acute myeloid leukemia. Our analysis showed that the deletions were interstitial and that there were two distinct deleted segments of 7q. The majority of patients (65 of 81 [80%]) had proximal breakpoints in bands q11-22 and distal breakpoints in q31-36; the smallest overlapping deleted segment was within q22. The remaining 16 patients had deletions involving the distal q arm with a commonly deleted segment of q32-33. To define the proximal deleted segment at 7q22 at a molecular level, we used fluorescence in situ hybridization with a panel of mapped yeast artificial chromosome (YAC) clones from 7q to examine 15 patients with deletion breakpoints in 7q22. We determined that the smallest overlapping deleted segment is contained in a well- defined YAC contig that spans 2 to 3 Mb. These studies delineate the region of 7q that must be searched to isolate a putative myeloid leukemia suppressor gene, and provide the necessary cloned DNA for more detailed physical mapping and gene isolation.     

Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing human megakaryocytes

The localization of three known alpha-granule proteins, thrombospondin (TSP), von Willebrand factor (vWF), and fibrinogen (Fg) has been studied in human megakaryocytes (MK) by immunofluorescence and immunoelectron microscopy. For this study, highly purified populations of MK were prepared from human bone marrow either by counterflow centrifugal elutriation or by cell culture from normal subjects and from two patients with megakaryoblastic leukemia. In normal bone marrow immature MK, TSP, and vWF were observed in the Golgi-associated vesicles and in small immature alpha-granules; in mature MK, they were found in the matrix of the mature large alpha-granules. Surprisingly, Fg was detected neither in the Golgi area, nor in the small precursors of alpha-granules; it was only found in the mature alpha-granules but this labeling was generally weaker than in blood platelets. In order to confirm these differences between the expression of Fg and vWF or TSP additional studies were performed on cultured maturing MK: immunofluorescent and ultrastructural immunogold labeling confirmed that vWF appeared early in the maturation while the same immature MK were negative for Fg. In the late maturation stage, the three proteins were detected in the alpha-granules. In order to know whether Fg was lately synthesized or endocytosed from the outside medium, normal MK were grown in the presence of either normal or afibrinogenemic plasma, and normal serum. Fg was detected only in the alpha-granules of MK grown in normal plasma. Similar results were observed with malignant MK, whose maturation was independent of the culture conditions. In conclusion, this study brings immunocytochemical evidence that vWF and TSP are synthesized by immature MK, whereas Fg appears later in the MK alpha-granules and its expression is dependent of the presence of an exogenous Fg source.     

Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interaction with IL-1, IL-3, IL-6, and GMCSF

Recombinant human interleukin-5 (rhIL-5), in either liquid or semi- solid cultures, selectively induced eosinophil production from normal human bone marrow, with no activity on other cell lineages. The time course of eosinophil production induced by murine IL-5, rhIL-3, and rh granulocyte-macrophage colony stimulating factor (GMCSF) was similar to rhIL-5. The rate of eosinophil maturation in vitro was independent of the stimulating cytokine, mature eosinophils being produced after 4 to 5 weeks in liquid culture with each of these cytokines. The eosinophils produced in response to each cytokine were morphologically indistinguishable, and had the ultrastructural features of maturity except that the electron-dense material in the granules had not formed into crystalline cores. Neither rhIL-1 nor rhIL-6 alone, or in combination with rhIL-5 or rhIL-3, induced eosinophil differentiation or proliferation under the conditions used. rhIL-3 and rhGMCSF induced more eosinophil colonies than rhIL-5, rhIL-5 had an additive, not synergistic, effect on eosinophil colony production when combined with either rhIL-3 or rhGMCSF, suggesting that rhIL-5 stimulates a smaller and possibly different population of eosinophil progenitors. However, rhIL-5 induced the greatest eosinophil production in liquid cultures, suggesting that although it may act on a smaller population of precursors, it is able to stimulate more proliferative steps than either rhIL-3 or rhGMCSF.     

Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1

CD9 (p24) and PECAM1 (CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and PECAM-1 were found lining the membrane of the same granules that contained fibrinogen and vWF in their matrix. CD9 and PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (ADP) or induce granule secretion (thrombin). After treatment with ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and gold particles were still found decorating the periphery of the centralized alpha- granules. After thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha- granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule- specific receptors such as P-selectin) or by endocytosis from the plasma membrane (for proteins distributed in the two compartments).     

Carrier detection in hemophilia A: a cooperative international study. I. The carrier phenotype

Eight laboratories in six countries cooperated to clarify several issues concerning the phenotypes of heterozygous carriers of hemophilia "A." Plasma levels of factor VIII (F.VIII:C, formerly VIII:C) and von Willebrand factor (VWF:Ag, formerly VIIIR:Ag) of carriers and normal women were determined by various "in-house" methods; a single lyophilized plasma standard was used for all assays. Analysis of the collated data from 336 carriers (296 obligatory carriers and 40 sporadic carriers) and 137 normal women showed that there was no difference in the F.VIII:C levels of "paternal" carriers (women who had obtained the abnormal gene from their fathers) and "maternal" carriers. Neither was there a difference in the VWF:Ag levels of normal women and either type of carrier. Age was found to have a significant effect on both F.VIII:C and VWF:Ag, values being higher at very young and very old ages, the minima occurring in the 25- to 30-year range. ABO blood type had a striking effect. Women of types A, B, and AB (designated non- O in the study), both normals and carriers, had significantly higher levels of both factors than did women of type O. Analysis by laboratories showed that differences in mean levels of both factors between laboratories were highly significant. It was concluded that age, ABO blood type, and laboratory variation should be taken into account in carrier detection.