Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification

A considerable number of deletions of variable size and position that involve the beta-globin gene complex on chromosome 11 are associated with the clinical entities of hereditary persistence of fetal hemoglobin (HPFH) and delta beta thalassemia. Specific deletions appear to be associated with consistent phenotypes and some are known to be recurrent. To facilitate the molecular diagnosis of uncharacterized patients with HPFH and delta beta thalassemia, oligonucleotide primers have been designed to enzymatically amplify deletion-specific products for nine known deletions, which include those responsible for HPFH-1, HPFH-2, HPFH-3, Spanish (delta beta)zero thalassemia, hemoglobin (Hb) Lepore, Sicilian (delta beta)zero thalassemia, Chinese G gamma(A gamma delta beta)zero thalassemia, Asian-Indian inversion-deletion G gamma(A gamma delta beta)zero thalassemia, and Turkish inversion-deletion (delta beta)zero thalassemia. Using this approach, we have successfully characterized the molecular basis for delta beta thalassemia in 23 individuals from 16 families of diverse ethnic origins. Thirteen individuals from this group were shown to be heterozygous for the 13.4- kb Sicilian deletion, two were heterozygous for the 100-kb Chinese G gamma(A gamma delta beta)zero deletion, four were heterozygous for the Turkish form of inversion-deletion delta beta thalassemia, and three were heterozygous for the Asian-Indian form of inversion-deletion G gamma(A gamma delta beta)zero thalassemia. One Vietnamese subject was heterozygous for a 12.6-kb deletion, which we have fully characterized at the molecular level. Sequence analysis of the breakpoint regions of the Chinese deletion and the Turkish rearrangement indicates that, in each case, the mutation is likely to have arisen from a single origin. This hypothesis is supported by the evident geographical clustering of the various deletions described here.     

Residual amounts of glycoprotein Ib concomitant with near-absence of glycoprotein IX in platelets of Bernard-Soulier patients

A study of the Bernard-Soulier syndrome in two unrelated families using different polyclonal antibodies in a sensitive immunoblot assay showed residual amounts of platelet membrane glycoprotein (GP) lb in the eight homozygotes, as well as the near-absence of GPlb beta and GPIX. The eight heterozygotes studied showed a double band pattern for GPlb and about half the normal level of GPlb beta and GPIX. Therefore, we conclude that the Bernard-Soulier syndrome is heterogeneous and is probably not due to gene deletions.     

Augmentation of neutrophilic granulocyte progenitors in the bone marrow of mice with tumor-induced neutrophilia: cytochemical study of in vitro colonies

Transplantation of CE mammary carcinoma into mice has been shown to produce marked neutrophilia. Previous studies in vivo indicated a significant increase in marrow neutrophil production in these mice, but regulatory mechanisms of this neutrophilia have not been well understood. In order to obtain information about neutrophil production mechanisms at the progenitor cell level, the profile of marrow granulocyte-macrophage progenitors in mice with neutrophilia induced by this tumor was quantitatively analyzed by cytochemical staining of in vitro colonies to distinguish colonies of neutrophils (N-colony), macrophages (M-colony), and mixed cells (NM-colony). Cell cycle kinetics of progenitors were studied by in vivo administration of cytocidal drugs. The absolute number of N-colonies in a femur increased significantly and reached three times normal three to four weeks after tumor implantation. The number of NM-colonies also increased significantly by the fourth week, but the number of M-colonies was unchanged. The number of N-colonies in a femur related directly to the degree of neutrophilia. The increased number of N-colonies from the marrow of tumor-bearing mice was not attributed to a different time course of colony growth nor to a different sensitivity to CSA; instead, a significantly larger fraction of neutrophilic progenitors from the tumor-bearing mice were in active cell cycle than were those of normal mice. The day 14 tumor-bearing mouse serum demonstrated N-colony stimulating activity while the sera of normal mice and day 7 tumor- bearing mice were inhibitory for in vitro colony growth. These studies demonstrated an increase in the numbers and turnover rate of marrow neutrophilic progenitors in CE tumor-induced neutrophilia, suggesting that this tumor stimulates proliferation of these progenitors in vivo.     

Bone marrow ablation followed by allogeneic marrow grafting during first complete remission of acute nonlymphocytic leukemia

Of 33 patients who had undergone allogeneic bone marrow transplantation during first complete remission of acute nonlymphocytic leukemia, 21 patients have now been followed in continued complete remission for 6- 64 mo (median greater than 18 mo) without maintenance chemotherapy. The median age of the surviving patients is 27 yr. Transplant-related complications occurring throughout the first year after marrow grafting were fatal in 7 patients, and leukemic recurrence led to the death of 5 patients. The actuarial long-term disease-free survival is 60% and the actuarial remission rate is 79%.     

Autocrine stimulation by erythropoietin (Epo) requires Epo secretion

Erythropoietin (Epo) autocrine stimulation has been implicated in erythroblastic leukemia. To examine whether this stimulation could occur intracellularly, we developed Epo autocrine models of stimulation in the human pluripotent UT-7 cell line. Retroviral expression of Epo totally abolished the growth factor requirement of UT-7 cells. Autonomous proliferation was not cell density-dependent and occurred at a unicellular level, showing a genuine autocrine mode of stimulation. Total blockage of Epo secretion induced by the endoplasmic reticulum- retention amino acids Lys-Asp-Glu-Leu (KDEL) signals in 11 lines prevented autonomous proliferation, whereas a leaky retention system, observed in 3 other lines, resulted in limited autocrine stimulation without true long-term autonomous proliferation. Production of Epo, in contrast to KDEL-modified Epo, induced reductions in Epo binding, Epo receptor (EpoR) mRNA, and phosphorylation levels similar to those induced by the addition of exogenous Epo to the parental cell line. In addition, autonomous growth and survival were inhibited by the addition of Epo-neutralizing antibodies, affording evidence that autocrine stimulation through EpoR activation takes place on the cell surface. Finally, phenotypic analysis of the virus-infected clones indicated that Epo production did not change the differentiative capacities of UT- 7 cells. All these data show that Epo autocrine stimulation is dependent on Epo secretion and takes place on the cell surface. From all analyzed parameters, the effects of Epo autocrine stimulation and those of exogenously added Epo appear to be identical.     

Histocompatible unrelated volunteer donors compared with HLA nonidentical family donors in marrow transplantation for aplastic anemia and leukemia

We treated 14 patients by transplantation of marrow from unrelated volunteer donors. Eight patients had severe aplastic anemia, 3 had chronic granulocytic leukemia, and 3 had Fanconi's anemia. The results are compared with those of a group of 14 similar patients transplanted concurrently from human leukocyte antigen (HLA)-mismatched family members: Sustained engraftment was achieved in 8 of 14 patients in both groups; one additional patient survived with autologous marrow reconstitution following an unrelated donor transplant. In the unrelated donor group, 6 of 9 evaluable patients developed grade III through IV acute graft-v-host disease, as compared with 4 of 9 patients after family-mismatched transplants. Overall survival was similar in the two groups. In the unrelated donor group 4 of 14 (29%) patients survived (median survival 1,299 days) as compared with 5 of 14 (36%) in the mismatched-family donor group (median survival 808 days). In both groups, patients with HLA phenotypically matched donors fared better than those with donors who were mismatched for one or more HLA antigen. Of the patients transplanted from HLA phenotypically matched donors 6 of 12 patients (50%) survived, as compared with 3 of 16 patients (19%) transplanted from HLA-mismatched donors. We conclude that unrelated donor bone marrow transplantation (BMT) should be considered in those cases of leukemia or bone marrow failure in which the chance of cure using conventional therapy is remote and a HLA genotypically or phenotypically matched family donor is not available.     

Management of pain associated with debridement of leg ulcers: a randomized,multicentre, pilot study comparing nitrous oxide–oxygen mixture inhalation and lidocaïne–prilocaïne cream

Background Mechanical debridement of fibrin and/or necrosis promotes healing of arterial and venous leg ulcers but is limited by pain associated with the procedure. Objective The main objective of this study was to compare the respective analgesic effect of nitrous oxide oxygen mixture (NOOM) inhalation and lidocaïne–prilocaïne cream (LPC) application during the mechanical repeated debridement of chronic arterial and venous leg ulcers. Methods In this randomized, multicentre, open‐label study, pain was evaluated before and after each care and debridement session using a Visual Analog Scale (VAS) and a Verbal Rating Scale (VRS), in the context of usual debridement and wound care process. The Quality of debridement and tolerability of the treatments were also assessed. Results Forty‐one patients were randomized: 20 received NOOM and 21 LPC. Pain assessed by VAS and VRS was more intense in the NOOM group than in the LPC group (5.29 vs. 3.68 and 2.87 vs. 1.71, P < 0.001, for the two scales respectively). No differences were found concerning quality of debridement, safety or tolerability between the two groups. Conclusion This pilot study demonstrates the superiority of the LPC over NOOM for pain control during the mechanical debridement of chronic leg ulcers.     

Plasma applications in medicine with a special focus on dermatology

The recent tremendous progress in understanding physical plasma phenomenon, together with the development of new plasma sources has put growing focus on the application of plasmas in health care. Active plasma components, such as molecules, atoms, ions, electrons and photons, reactive species, ultraviolet radiation, optical and infrared emission and heat have the ability of activating, controlling and catalysing reactions and complex biochemical procedures. Thermal and non‐thermal (i.e. cold) plasmas – both already widely established in medicine – are used for various therapeutic applications. Particularly in dermatology, plasma applications hold big potential, for example, in wound healing, such as efficient disinfection or sterilization, therapy of various skin infections or tissue regeneration. This review gives an overview on potential plasma applications in medicine – including the recent research on skin diseases – and summarizes possible interactions between plasmas and living tissue.     

Microsatellite markers developed through pyrosequencing allow clonal discrimination in the invasive alga Caulerpa taxifolia

Polymorphic microsatellites were developed for the invasive green algae Caulerpa taxifolia using next generation DNA sequencing. Results showed a limited rate of microsatellites for the amount of sequences, possibly explaining failure of previous attempts for microsatellite development through classical methods. Eight polymorphic loci were selected that exhibited polymorphism and a null or negligible rate of amplification failure. The number of alleles per locus ranged from two to seven. The reconstruction of Multi Locus Genotypes and the heterozygosity and departure from Hardy–Weinberg equilibrium confirmed the influence of clonal reproduction and showed the usefulness of this set of markers to successfully discriminate clonal lineages and analyze the clonal and genetic composition of algal beds. These markers will be used to further investigate the clonal composition and genetic structure in populations of C. taxifolia and to attempt retracing the origin of and pathways followed by invasive clonal lineages.