Gene Therapy for Hemophagocytic Lymphohistiocytosis (HLH): Fixing a Criticial ‘Circuit Breaker’ in the Immune System.

Abstract 3158 In addition to its well-known role in host defense, perforin-dependent cytotoxicity also has a critical immune regulatory function. Genetic defects of perforin underlie many cases of hemophagocytic lymphohistiocytosis (HLH), a fatal disorder of extreme immune activation. While the initial treatment of HLH is largely focused on suppressing dangerous inflammation, hematopoietic cell transplantation (HCT) is required for long-term correction of the underlying immune regulatory defect. Unfortunately, mortality after HCT in patients with HLH is unusually high for a non-malignant disorder, ranging from 20 to 50%. We developed a model of HLH in which perforin deficient (prf-/-) mice develop all of the features of HLH after challenge with lymphocytic choriomeningitis virus (LCMV). Recent studies from our group have demonstrated that transplantation of wild type (WT) bone marrow into prf-/- mice rescues them from the subsequent development of HLH after challenge with LCMV. In this model, perforin-dependent immune regulation appears to function in trans, with a critical threshold of 10% WT chimerism required to protect animals from HLH. This low threshold is consistent with clinical reports of very limited numbers of patients with long term mixed chimerism after HCT who appear to be protected from HLH recurrence. These data have also provided a clear target for gene therapy efforts. We have begun to test the feasibility of autologous gene correction using lentiviral vectors in this model system. Hematopoietic stem and progenitor cells (HSC) from prf-/- mice were transduced with lentivirus vectors expressing perforin under the control of ubiquitous or tissue-specific promoters. When transduced HSC were transplanted into lethally irradiated prf-/- mice, we have observed levels of in vivo gene correction ranging from 10–80%, depending on the promoter and transduction protocols utilized. We find that the transduced CD8 T cell and NK cell progeny display significant correction of their cytotoxic defects. Challenge of gene corrected prf-/- mice with LCMV has revealed significant correction of the underlying immune regulatory defect and protection from the development of HLH. However, reestablishing normal perforin-dependent immune regulation with gene corrected prf-/- HSC's appears to be somewhat less efficient than with mixed WT:prf-/- chimerism. Thus, further optimization of perforin gene control by lentiviral vectors appears to be warranted. Results of ongoing studies related to vector optimization and reestablishing physiologic perforin-mediated immune regulation will be presented. Because normal perforin expression in only a fraction of lymphocytes is sufficient to rescue individuals from HLH, this disorder is an attractive target for gene therapy approaches. Demonstration of long term gene correction and rescue from HLH in our animal model will pave a path for translational efforts testing gene therapy for HLH in clinical trials. Disclosures: No relevant conflicts of interest to declare.

Transepithelial HCO3− absorption is defective in renal thick ascending limbs from Na+/H+ exchanger NHE1 null mutant mice

In the medullary thick ascending limb (MTAL) of rat kidney, inhibiting basolateral Na+/H+ exchange with either amiloride or nerve growth factor (NGF) results secondarily in inhibition of apical Na+/H+ exchange, thereby decreasing transepithelial HCO3− absorption. To assess the possible role of the Na+/H+ exchanger NHE1 in this regulatory process, MTALs from wild-type and NHE1 knockout (NHE1−/−) mice were studied using in vitro microperfusion. The rate of HCO3− absorption was decreased 60% in NHE1−/− MTALs (15.4 ± 0.5 pmol·min−1·mm−1 wild-type vs. 6.0 ± 0.5 pmol·min−1·mm−1 NHE1−/−). Transepithelial voltage, an index of the NaCl absorption rate, did not differ in wild-type and NHE1−/− MTALs. Basolateral addition of 10 μM amiloride or 0.7 nM NGF decreased HCO3− absorption by 45–49% in wild-type MTALs but had no effect on HCO3− absorption in NHE1−/− MTALs. Inhibition of HCO3− absorption by vasopressin and stimulation by hyposmolality, both of which regulate MTAL HCO3− absorption through primary effects on apical Na+/H+ exchange, were similar in wild-type and NHE1−/− MTALs. Thus the regulatory defect in NHE1−/− MTALs is specific for factors (bath amiloride and NGF) shown previously to inhibit HCO3− absorption through primary effects on basolateral Na+/H+ exchange. These findings demonstrate a novel role for NHE1 in transepithelial HCO3− absorption in the MTAL, in which basolateral NHE1 controls the activity of apical NHE3. Paradoxically, a reduction in NHE1-mediated H+ extrusion across the basolateral membrane leads to a decrease in apical Na+/H+ exchange activity that reduces HCO3− absorption.

Role of ppGpp in rpoS Stationary-Phase Regulation in Escherichia coli

ABSTRACT The bacterial sigma factor RpoS is strongly induced under a variety of stress conditions and during growth into stationary phase. Here, we used rpoS-lac fusions in Escherichia coli to investigate control acting at the level of RpoS synthesis, which is especially evident when cells approach stationary phase in rich medium. Previous work has shown that the small molecule ppGpp is required for normal levels of RpoS in stationary phase. Despite the attraction of a model in which the ppGpp level controls stationary-phase induction of RpoS, careful measurement of rpoS-lac expression in a mutant lacking ppGpp showed similar effects during both exponential growth and stationary phase; the main effect of ppGpp was on basal expression. In addition, a modest regulatory defect was associated with the mutant lacking ppGpp, delaying the time at which full expression was achieved by 2 to 3 h. Deletion analysis showed that the defect in basal expression was distributed over several sequence elements, while the regulatory defect mapped to the region upstream of the rpoS ribosome-binding site (RBS) that contains a cis-acting antisense element. A number of other genes that have been suggested as regulators of rpoS were tested, including dksA, dsrA, barA, ppkx, and hfq. With the exception of the dksA mutant, which had a modest defect in Luria-Bertani medium, none of these mutants was defective for rpoS stationary-phase induction. Even a short rpoS segment starting at 24 nucleotides upstream of the AUG initiation codon was sufficient to confer substantial stationary-phase regulation, which was mainly posttranscriptional. The effect of RBS-proximal sequence was independent of all known trans-acting factors, including ppGpp.

Identification of a Gene in Staphylococcus xylosus Encoding a Novel Glucose Uptake Protein

ABSTRACT By transposon Tn917 mutagenesis, two mutants ofStaphylococcus xylosus were isolated that showed higher levels of β-galactosidase activity in the presence of glucose than the wild type. Both transposons integrated in a gene, designatedglcU, encoding a protein involved in glucose uptake inS. xylosus, which is followed by a glucose dehydrogenase gene (gdh). Glucose-mediated repression of β-galactosidase, α-glucosidase, and β-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type. In addition to the pleiotropic regulatory effect, integration of the transposons into glcU reduced glucose dehydrogenase activity, suggesting cotranscription of glcU and gdh. Insertional inactivation of the gdh gene and deletion of the glcU gene without affecting gdh expression showed that loss of GlcU function is exclusively responsible for the regulatory defect. Reduced glucose repression is most likely the consequence of impaired glucose uptake in the glcU mutant strains. With cloned glcU, an Escherichia colimutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present. Therefore, glucose is internalized by glcU in nonphosphorylated form. A gene from Bacillus subtilis,ycxE, that is homologous to glcU, could substitute for glcU in the E. coli glucose growth experiments and restored glucose repression in the S. xylosus glcU mutants. Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases. It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes. GlcU and YcxE are the first examples whose specificity, glucose, has been determined.

A Mutation in a Purported Regulatory Gene Affects Control of Sterol Uptake in Saccharomyces cerevisiae

ABSTRACT Aerobically growing wild-type strains of Saccharomyces cerevisiae are unable to take exogenously supplied sterols from media. This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis. Mutants which can take up sterols aerobically in heme-competent cells have been selected. One of these mutations, designated upc2-1, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements. During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that upc2-1was hypersensitive to Ca2+. Using resistance to Ca2+ as a screening vehicle, we cloned UPC2 and showed that it is YDR213W, an open reading frame on chromosome IV. This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. The single guanine-to-adenine transition in upc2-1 gives a predicted amino acid change from glycine to aspartic acid. The regulatory defect explains the semidominance and pleiotropic effects of upc2-1.

Regulation of Cl- permeability in normal and cystic fibrosis sweat duct cells

Reabsorptive cells of the human sweat gland normally exhibit a high basal Cl- permeability but are markedly impermeable to Cl- in cystic fibrosis (CF). We examined the possibility that the reduced basal Cl- permeability of CF sweat duct cells in primary culture is due to a defective regulation of plasma membrane Cl- permeability by prostaglandin E2 (PGE2), which is endogenously produced by cultured sweat duct cells. The macroscopic Cl- permeabilities of normal and CF sweat duct cells were assessed using a halide-specific fluorescent dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, in combination with fluorescence digital-imaging microscopy. The Cl- and Br- permeabilities of normal sweat duct cells were markedly reduced by inhibiting endogenous PGE2 production with indomethacin. This inhibition of Cl- permeability by indomethacin was largely reversed by the addition of PGE2 (10 nM to 1 microM), but not forskolin. Conversely, PGE2 failed to stimulate the low Cl- permeabilities of sweat duct cells cultured from CF subjects. Our results support the following conclusions: 1) a defective regulation of Cl- permeability in CF is a feature of reabsorptive as well as secretory epithelial cells, and 2) the nature of this regulatory defect extends beyond altered Cl- permeability regulation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

The Inab phenotype: characterization of the membrane protein and complement regulatory defect

Recent demonstration that Cromer-related human blood group antigens reside on decay-accelerating factor (DAF) has led to identification of an apparent null phenotype (Inab) for erythrocyte DAF. This study examined expression of other phosphatidylinositol (PI)-anchored proteins by Inab erythrocytes and showed that the PI-linked membrane proteins acetylcholinesterase (AchE) and lymphocyte function-associated antigen-3 (LFA-3) are normally expressed by these cells. Furthermore, studies of the complement sensitivity of Inab RBCs demonstrated them to be abnormally complement sensitive, with an apparent defect in downregulation of C3 convertase activity. Thus, the Inab phenotype appears to represent an instance of hereditary erythrocyte DAF deficiency whose mechanism differs from that of paroxysmal nocturnal hemoglobinuria (PNH) and which is unassociated with clinically evident hemolytic disease.