The erythroid defect in Diamond Blackfan anemia (DBA) is known to be intrinsic to the stem cell, but its molecular pathophysiology remains obscure. Using a 2-phase liquid erythroid culture system, we have demonstrated a consistent defect in DBA, regardless of clinical severity, including 3 first-degree relatives with normal hemoglobin levels but increased erythrocyte adenosine deaminase activity. DBA cultures were indistinguishable from controls until the end of erythropoietin (Epo)-free phase 1, but failed to demonstrate the normal synchronized wave of erythroid expansion and terminal differentiation on exposure to Epo. Dexamethasone increased Epo sensitivity of erythroid progenitor cells, and enhanced erythroid expansion in phase 2 in both normal and DBA cultures. In DBA cultures treated with dexamethasone, Epo sensitivity was comparable to normal, but erythroid expansion remained subnormal. In clonogenic phase 2 cultures, the number of colonies did not significantly differ between normal cultures and DBA, in the presence or absence of dexamethasone, and at both low and high Epo concentrations. However, colonies were markedly smaller in DBA under all conditions. This suggests that the Epo-triggered onset of terminal maturation is intact in DBA IntroductionDiamond Blackfan anemia (DBA) is a rare congenital red cell aplasia, which classically presents with profound aregenerative anemia in early infancy, often in association with physical anomalies and growth retardation. [1][2][3] The anemia responds to steroid therapy in up to 70% of patients, but eventually around 40% of affected individuals are dependent on long-term transfusion programs. [1][2][3][4] Spontaneous remissions may occur, even in patients who have never previously achieved transfusion independence. [1][2][3][4][5] Sustained remissions have occasionally been reported in response to interleukin-3 (IL-3). 6,7 Recently, clinical responses to prolactin or metoclopramide have been described, but only after prolonged administration. 8 Remissions, whether spontaneous or treatment induced, are usually associated with a residual erythropoietic defect shown by persistent mild anemia and macrocytosis, 1-3 with increased erythrocyte adenosine deaminase (eADA) activity. [9][10][11] The pathophysiologic basis for this characteristic pattern of erythroid failure and remission in DBA remains elusive, even after the identification of the gene encoding ribosomal protein S19 (RPS19) as the first DBA gene, 12 mutated in up to 25% of affected individuals. 13 RPS19 was not an obvious candidate gene for this disorder, being ubiquitously expressed in its normal ribosomal role. Gene transfection studies support the association between RPS19 mutations and impaired erythroid maturation, 14 but the specific contribution of RPS19 to normal and abnormal erythropoiesis has yet to be defined.The demonstration of impaired erythroid differentiation in vitro of enriched erythroid progenitor cells 15 and of purified CD34 ϩ cells 16 from patients with DBA provides strong evidence fo...

Expression of the virB operon, encoding the type IV secretion system required for Brucella suis virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against B. suis VirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism Agrobacterium tumefaciens, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and B. suis from which chvE was deleted was fully virulent in a mouse model. Comparative analyses of various Brucella species revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canis and B. suis, as well as the Brucella abortusand Brucella melitensis-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental B. abortus and B. melitensis strains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virB expression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the virB operon are not uniformly regulated in different Brucella species. Since VirB proteins were shown to modulate Brucella phagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.Bacteria belonging to the genus Brucella are gram-negative facultative intracellular pathogens of various wild and domestic mammals, and they also cause severe zoonotic infections in humans. Traditionally, three major species are distinguished by their preferences for certain animal hosts; Brucella abortus has a preference for cattle, Brucella melitensis has a preference for caprines, and Brucella suis has a preference for hogs. Whereas B. abortus is the livestock pathogen with the greatest economic impact, B. melitensis and B. suis account for most clinical cases in humans (15,42).In an attempt to unravel Brucella virulence factors by transposon mutagenesis, the crucial role of an operon similar to the virB operon of Agrobacterium tumefaciens encoding a type IV secretion system (T4SS) was revealed (35). The importance of the virB operon for Brucella virulence was further confirmed by signature-tagged mutagenesis both in vitro in a human macrophage infection model (24) and in vivo with mice (26). Further studies indicated that a complete Brucella virB operon was required for wild-type virulence in mice (47) or in macrophagelike cells (52,53). In nonphagocytic HeLa cells, the absence of some functional VirB proteins (B2, B4, and B9) did not...

Diamond Blackfan Anemia (DBA) is a rare congenital red cell aplasia, presenting in early infancy. The anemia is characteristically steroid responsive in the majority of cases, but eventually 40% of affected individuals are dependent on long-term transfusion and chelation programmes, underscoring the need for alternative therapies. A report by Abkowitz et al (Blood2002;100:2687) suggests a potential benefit of prolactin, induced indirectly by metoclopramide treatment, in a proportion of patients with steroid refractory DBA. We have therefore investigated the in vitro effect of prolactin on erythropoiesis in DBA, using a 2-phase liquid erythroid culture system in which we have previously demonstrated a severe consistent erythroid defect in DBA, and an enhancing effect of added steroids in both normal and DBA cultures. Peripheral blood mononuclear cells were cultured in serum-free medium containing 50ng/ml IL-3, 100ng/ml SCF, 1μg/ml cyclosporin A in the absence of erythropoietin (EPO) for 7 days (phase I), before transfer of non-adherent cells to phase II culture, with medium as for phase I plus 2U/ml EPO. Erythroid output was expressed as the total number of hemoglobinised cells generated after 7 days in phase II culture per cell transferred from phase I. In the absence of steroids, the addition of prolactin (PRL) 20–200ng/ml to both phases had no effect on erythroid output in normal (n=10) or DBA (n=9) cultures (table) normal (n=10) DBA (n=9) Interaction between prolactin and dexamethasone on erythroid output in normal and DBA cultures (mean±SEM) no PRL PRL 50ng/ml no PRL PRL 50ng/ml no dex 5.31±1.38 4.87±1.39 0.24±0.1 0.18±0.07 dex 10−7M 10.30±1.43 10.44±1.40 1.85±0.84 1.86±0.87 We then studied the potential interaction between PRL and steroids, given their known synergy in lactogenesis. While PRL 50ng/ml had no overall effect on mean erythroid output in the presence of 10−7M dex (table), there was striking variation between cultures. Notably, in 3/10 normal cultures there was an apparent prolactin-induced inhibition of the normal steroid stimulatory effect. A similar phenomenon was observed in DBA cultures, with apparent synergy between 50ng/ml PRL and 10−7M dex in 3/9 (erythroid output in PRL+dex 139%, 141% and 180% of the erythroid output with dex alone), but inhibition in 3/9 (erythroid output in PRL+dex 26%, 62%, 87% of output with dex alone). Interestingly, the inhibitory effect of prolactin in DBA cultures appeared to be both more common and more pronounced at the lower PRL concentration of 20ng/ml, equivalent to the top end of the physiological range. Conversely, the stimulatory effect appeared to be more pronounced at lower concentrations of steroid (10−8M). These observations would be consistent with a dose-dependent enhancement by prolactin of steroid sensitivity, causing a left shift of the bell-shaped steroid dose response curve. The complex dose-dependence between steroids and prolactin may be of relevance to the potential therapeutic effect of metoclopramide, which increases endogenous levels of both cortisol and prolactin. Until the interactions between prolactin and steroids are more completely understood, we would advise caution in using metoclopramide in other than transfusion-dependent and steroid refractory DBA, as our results predict the risk of inhibition of steroid responsiveness in vivo, with exacerbation of anemia.