Reverse mutations – spontaneous amelioration or cure of inherited disorders?

Some recent publications indicate that inherited disorders can ameliorate or possibly disappear if mutations responsible for the disease revert to normal. This review tries to summarize our current knowledge about reverse mutations as this information may be of special interest for attempts at somatic gene therapy. Received: 14 July 1997 / Accepted: 2 March 1998     

Enhancement of chemotactic factor-stimulated neutrophil oxidative metabolism by leukotriene B4

Leukotriene B4 (LTB4) is a potent primary stimulator of neutrophil chemotaxis, aggregation, and degranulation and induces superoxide production at higher concentrations. In order to determine whether LTB4 modulates neutrophil responses to oxidative stimuli, human neutrophils (PMNs) were incubated with LTB4 prior to stimulation with f-Met-Leu-Phe (fMLP, 10(-7) mol/L), opsonized zymosan (OZ, 250 micrograms/mL), or phorbol myristate acetate (PMA, 32 nmol/L). Superoxide (O2-) production by stimulated PMNs was assessed by the superoxide dismutase-inhibitable reduction of cytochrome c. LTB4 alone did not stimulate O2- production in concentrations below 10(-7) mol/L and had no effect on the O2- assay. In the concentration range of 10(-12) to 10(-8) mol/L, LTB4 did not alter O2- release induced by OZ or PMA. In contrast, LTB4-treated cells demonstrated enhanced O2- production following exposure to fMLP, and in the presence of 10 nmol/LLTB4, generated 180% +/- 41% of O-2 quantities produced by control cells (n = 23). Enhancement was LTB4 dose-dependent, was maximal in the range of 1 to 10 nmol/L LTB4, was not reversed by removal of the lipid from the medium prior to fMLP stimulation, and was not dependent on the presence of Ca++ or Mg++ in the suspending medium. Chemiluminescence of fMLP-stimulated neutrophils was increased to 323% of controls in neutrophils preincubated with 10 nmol/L LTB4. Unlike augmentation of oxidative responses to fMLP seen with other degranulating stimuli, enhancement by LTB4 was not correlated with an increase in 3H-fMLP receptor binding. These results indicate that, in addition to its primary effects on neutrophil function, LTB4 modulates PMN oxidative responses to the chemotactic peptide and, thus, may amplify the release of oxygen metabolites at inflammatory foci.     

Physical and functional interactions between Stat5 and the tyrosine- phosphorylated receptors for erythropoietin and interleukin-3

Erythropoietin (Epo) and interleukin-3 (IL-3) stimulate activation of the Jak2 tyrosine kinase and induce tyrosine phosphorylation and activation of Stat5. In the present study, we have shown that Epo or IL- 3 stimulation induces binding of Stat5 to the tyrosine-phosphorylated Epo receptor (EpoR) or IL-3 receptor beta subunit (betaIL3), respectively, in IL-3-dependent 32D cells expressing the EpoR. The binding of Stat5 to these cytokine receptors was shown to be rapid and transient, occurring within 1 minute of stimulation of cells and significantly decreasing after 5 minutes of cell treatment. In vivo binding experiments in COS cells showed that binding of Stat5 to the EpoR was mediated through the Stat5 Src homology 2 (SH2) domain. In vitro binding studies further showed that Stat5, but not other Stats examined, bound specifically to tyrosine-phosphorylated recombinant EpoR fusion proteins. In these in vivo and in vitro binding studies, Stat5 bound, albeit to a lesser degree, to truncated EpoR mutants in which all the intracellular tyrosines except Y-343 were removed. Furthermore, EpoR-derived synthetic phosphotyrosine peptides corresponding to Y-343, Y-401, Y-431, and Y-479 inhibited the in vitro binding of Stat5. When expressed in 32D cells, a mutant EpoR in which all the intracellular tyrosines were removed by carboxy-terminal truncation showed a significantly impaired ability to induce tyrosine phosphorylation of Stat5, particularly at low concentrations of Epo, but exhibited an increased sensitivity to Epo for growth signaling as compared with the wild-type EpoR. These results indicate that Stat5 specifically and transiently binds to the EpoR through the interaction between the Stat5 SH2 domain and specific phosphorylated tyrosines, including Y-343, in the EpoR cytoplasmic domain. It was implied that betaIL3 may also have similar Stat5 docking sites. The Stat5 docking sites in the EpoR were shown to facilitate specific activation of Stat5, which, however, may not be required for the EpoR-mediated growth signaling.     

High-dose etoposide and cyclophosphamide without bone marrow transplantation for resistant hematologic malignancy

Seventy-five patients with resistant acute leukemia or lymphoma received high-dose cyclophosphamide and etoposide to explore the activity of this combination in resistant hematologic malignancies, and to determine the maximum doses of these drugs that can be combined without bone marrow transplantation. Etoposide was administered over 29 to 69 hours by continuous infusion corresponding to total doses of 1.8 g/m2 to 4.8 g/m2. Cyclophosphamide, 50 mg/kg/d, was administered on 3 or 4 consecutive days total 150 to 200 mg/kg ideal body weight). At all dose levels myelosuppression was severe but reversible. Mucosal toxicity was dose-limiting with the maximum tolerated dose level combining etoposide 4.2 g/m2 with cyclophosphamide 200 mg/kg. Continuous etoposide infusion produced stable plasma levels that were lower than would be achieved after administration by short intravenous infusion, and this could explain our ability to escalate etoposide above the previously reported maximum tolerated dose. There were 28 complete (35%) and 12 partial (16%) responses. Median duration of complete response (CR) was 3.5 months (range 1.1 to 20+). Seventeen of 40 patients (42%) with acute myelogenous leukemia (AML) achieved CR, including 6 of 20 (30%) with high-dose cytosine arabinoside resistance. We conclude that bone marrow transplantation is not required after maximum tolerated doses of etoposide and cyclophosphamide. This regimen is active in resistant hematologic neoplasms, and the occurrence of CR in patients with high-dose cytosine arabinoside-resistant AML indicates a lack of complete cross-resistance between these regimens.     

Heterozygous Mutations in Natriuretic Peptide Receptor‐B (NPR2) Gene as a Cause of Short Stature

Based on the observation of reduced stature in relatives of patients with acromesomelic dysplasia, Maroteaux type (AMDM), caused by homozygous or compound heterozygous mutations in natriuretic peptide receptor‐B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some cases of idiopathic short stature (ISS). We enrolled 192 unrelated patients with short stature and 192 controls of normal height and identified seven heterozygous NPR2 missense or splice site mutations all in the short stature patients, including one de novo splice site variant. Three of the six inherited variants segregated with short stature in the family. Nine additional rare nonsynonymous NPR2 variants were found in three additional cohorts. Functional studies identified eight loss‐of‐function mutations in short individuals and one gain‐of‐function mutation in tall individuals. With these data, we were able to rigorously verify that NPR2 functional haploinsufficiency contributes to short stature. We estimate a prevalence of NPR2 haploinsufficiency of between 0 and 1/26 in people with ISS. We suggest that NPR2 gain of function may be a more common cause of tall stature than previously recognized.     

Adenosine: an endogenous inhibitor of neutrophil-mediated injury to endothelial cells.

Since adenosine and its analogue 2-chloroadenosine prevent neutrophils from generating superoxide anion in response to chemoattractants, we sought to determine whether these agents could inhibit neutrophil-mediated injury of endothelial cells. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM) enhanced the adherence of neutrophils to endothelial cells twofold (18 +/- 2% vs. 39 +/- 3% adherence, P less than 0.001) and caused substantial neutrophil-mediated injury to endothelial cells (2 +/- 2% vs. 39 +/- 4% cytotoxicity, P less than 0.001). 2-Chloroadenosine (10 microM) not only inhibited the adherence of stimulated neutrophils by 60% (24 +/- 2% adherence, P less than 0.001) but also diminished the cytotoxicity by 51% (20 +/- 4% cytotoxicity, P less than 0.002). Furthermore, depletion of endogenously released adenosine from the medium by adenosine deaminase-enhanced injury to endothelial cells by stimulated neutrophils (from 39 +/- 4% to 69 +/- 3% cytotoxicity, P less than 0.001). Indeed, in the presence of adenosine deaminase, even unstimulated neutrophils injured endothelial cells (19 +/- 4% vs. 2 +/- 2% cytotoxicity, P less than 0.001). These data indicate that engagement of adenosine receptors prevents both the adhesion of neutrophils and the injury they cause to endothelial cells. Adenosine inhibits injury provoked not only by cells that have been stimulated by chemoattractants but also by unstimulated cells. Based on this model of acute vascular damage we suggest that adenosine is not only a potent vasodilator, but plays the additional role of protecting vascular endothelium from damage by neutrophils.