Immune regulation by the ST6Gal sialyltransferase

The ST6Gal sialyltransferase controls production of the Siaα2-6Galβ1-4GlcNAc (Sia6LacNAc) trisaccharide, which is the ligand for the lectin CD22. Binding of CD22 to Sia6LacNAc is implicated in regulating lymphocyte adhesion and activation. We have investigated mice that lack ST6Gal and report that they are viable, yet exhibit hallmarks of severe immunosuppression unlike CD22-deficient mice. Notably, Sia6LacNAc-deficient mice display reduced serum IgM levels, impaired B cell proliferation in response to IgM and CD40 crosslinking, and attenuated antibody production to T-independent and T-dependent antigens. Deficiency of ST6Gal was further found to alter phosphotyrosine accumulation during signal transduction from the B lymphocyte antigen receptor. These studies reveal that the ST6Gal sialyltransferase and corresponding production of the Sia6LacNAc oligosaccharide are essential in promoting B lymphocyte activation and immune function.

Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma.

Nerve growth factor (NGF) serum levels were measured in 49 patients with asthma and/or rhinoconjunctivitis and/or urticaria-angioedema. Clinical and biochemical parameters, such as bronchial reactivity, total and specific serum IgE levels, and circulating eosinophil cationic protein levels, were evaluated in relation to NGF values in asthma patients. NGF was significantly increased in the 42 allergic (skin-test- or radioallergosorbent-test-positive) subjects (49.7 +/- 28.8 pg/ml) versus the 18 matched controls (3.8 +/- 1.7 pg/ml; P < 0.001). NGF levels in allergic patients with asthma, rhinoconjunctivitis, and urticaria-angioedema were 132.1 +/- 90.8, 17.6 +/- 6.1, and 7.6 +/- 1.8 pg/ml (P < 0.001, P < 0.002, and P < 0.05 versus controls), respectively. Patients with more than one allergic disease had higher NGF serum values than those with a single disease. When asthma patients were considered as a group, NGF serum values (87.6 +/- 59.8 pg/ml) were still significantly higher than those of control groups (P < 0.001), but allergic asthma patients had elevated NGF serum levels compared with nonallergic asthma patients (132.1 +/- 90.8 versus 4.9 +/- 2.9 pg/ml; P < 0.001). NGF serum levels correlate to total IgE serum values (rho = 0.43; P < 0.02). The highest NGF values were found in patients with severe allergic asthma, a high degree of bronchial hyperreactivity, and high total IgE and eosinophil cationic protein serum levels. This study represents the first observation (that we know of) that NGF is increased in human allergic inflammatory diseases and asthma.

Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene

The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the cyp19 gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the cyp19 gene. Male and female ArKO mice were born with the expected Mendelian frequency from F1 parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor α is deleted by targeted disruption.

Pancreatic β cells synthesize and secrete nerve growth factor

Differentiation and function of pancreatic β cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic β cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in β cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic β-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

Heme oxygenase 1 is required for mammalian iron reutilization

The majority of iron for essential mammalian biological activities such as erythropoiesis is thought to be reutilized from cellular hemoproteins. Here, we generated mice lacking functional heme oxygenase 1 (Hmox1; EC 1.14.99.3), which catabolizes heme to biliverdin, carbon monoxide, and free iron, to assess its participation in iron homeostasis. Hmox1-deficient adult mice developed an anemia associated with abnormally low serum iron levels, yet accumulated hepatic and renal iron that contributed to macromolecular oxidative damage, tissue injury, and chronic inflammation. Our results indicate that Hmox1 has an important recycling role by facilitating the release of iron from hepatic and renal cells, and describe a mouse model of human iron metabolic disorders.

Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome

Our preliminary family studies have suggested that some female first-degree relatives of women with polycystic ovary syndrome (PCOS) have hyperandrogenemia per se. It was our hypothesis that this may be a genetic trait and thus could represent a phenotype suitable for linkage analysis. To investigate this hypothesis, we examined 115 sisters of 80 probands with PCOS from unrelated families. PCOS was diagnosed by the combination of elevated serum androgen levels and ≤6 menses per year with the exclusion of secondary causes. The sisters were compared with 70 healthy age- and weight-comparable control women with regular menses, no clinical evidence of hyperandrogenemia, and normal glucose tolerance. Twenty-two percent of the sisters fulfilled diagnostic criteria for PCOS. In addition, 24% of the sisters had hyperandrogenemia and regular menstrual cycles. Circulating testosterone (T) and nonsex hormone-binding globulin-bound testosterone (uT) levels in both of these groups of sisters were significantly increased compared with unaffected sisters and control women (P < 0.0001 for both T and uT). Probands, sisters with PCOS, and hyperandrogenemic sisters had elevated serum luteinizing hormone levels compared with control women. We conclude that there is familial aggregation of hyperandrogenemia (with or without oligomenorrhea) in PCOS kindreds. In affected sisters, only one-half have oligomenorrhea and hyperandrogenemia characteristic of PCOS, whereas the remaining one-half have hyperandrogenemia per se. This familial aggregation of hyperandrogenemia in PCOS kindreds suggests that it is a genetic trait. We propose that hyperandrogenemia be used to assign affected status in linkage studies designed to identify PCOS genes.

Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin

We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis. Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund’s adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints. A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82%. The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins. The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect. Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon γ levels. Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio. These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response. This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.

Correction of obesity and diabetes in genetically obese mice by leptin gene therapy

The ob/ob mouse is genetically deficient in leptin and exhibits both an obese and a mild non-insulin-dependent diabetic phenotype. To test the hypothesis that correction of the obese phenotype by leptin gene therapy will lead to the spontaneous correction of the diabetic phenotype, the ob/ob mouse was treated with a recombinant adenovirus expressing the mouse leptin cDNA. Treatment resulted in dramatic reductions in both food intake and body weight, as well as the normalization of serum insulin levels and glucose tolerance. The subsequent diminishment in serum leptin levels resulted in the rapid resumption of food intake and a gradual gain of body weight, which correlated with the gradual return of hyperinsulinemia and insulin resistance. These results not only demonstrated that the obese and diabetic phenotypes in the adult ob/ob mice are corrected by leptin gene treatment but also provide confirming evidence that body weight control may be critical in the long-term management of non-insulin-dependent diabetes mellitus in obese patients.

Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor

Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin’s action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr986 within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr986 to Phe, which abrogates SHP-2 phosphorylation and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin’s weight-reducing effects in obese individuals.

The JC and BK human polyoma viruses appear to be recent introductions to some South American Indian tribes: There is no serological evidence of cross-reactivity with the simian polyoma virus SV40

In an effort to understand the unusual cytogenetic damage earlier encountered in the Yanomama Indians, plasma samples from 425 Amerindians representing 14 tribes have been tested for hemagglutination inhibition antibodies to the human JC polyoma virus and from 369 Amerinds from 13 tribes for hemagglutination inhibition antibodies to the human BK polyoma virus. There is for both viruses highly significant heterogeneity between tribes for the prevalence of serum antibody titers ≥1/40, the pattern of infection suggesting that these two viruses only relatively recently have been introduced into some of these tribes. Some of these samples, from populations with no known exposure to the simian polyoma virus SV40, also were tested for antibodies to this virus by using an immunospot assay. In contrast to the findings of Brown et al. (Brown, P., Tsai, T. & Gajdusek, D. C. (1975) Am. J. Epidemiol. 102, 331–340), none of the samples was found to possess antibodies to SV40. In addition, no significant titers to SV40 were found in a sample of 97 Japanese adults, many of whom had been found to exhibit elevated titers to the JC and BK viruses. This study thus suggests that these human sera contain significant antibody titers to the human polyoma viruses JC and BK but do not appear to contain either cross-reactive antibodies to SV40 or primary antibodies resulting from SV40 infection.

Tobacco smoke is a source of toxic reactive glycation products

Smokers have a significantly higher risk for developing coronary and cerebrovascular disease than nonsmokers. Advanced glycation end products (AGEs) are reactive, cross-linking moieties that form from the reaction of reducing sugars and the amino groups of proteins, lipids, and nucleic acids. AGEs circulate in high concentrations in the plasma of patients with diabetes or renal insufficiency and have been linked to the accelerated vasculopathy seen in patients with these diseases. Because the curing of tobacco takes place under conditions that could lead to the formation of glycation products, we examined whether tobacco and tobacco smoke could generate these reactive species that would increase AGE formation in vivo. Our findings show that reactive glycation products are present in aqueous extracts of tobacco and in tobacco smoke in a form that can rapidly react with proteins to form AGEs. This reaction can be inhibited by aminoguanidine, a known inhibitor of AGE formation. We have named these glycation products “glycotoxins.” Like other known reducing sugars and reactive glycation products, glycotoxins form smoke, react with protein, exhibit a specific fluorescence when cross-linked to proteins, and are mutagenic. Glycotoxins are transferred to the serum proteins of human smokers. AGE-apolipoprotein B and serum AGE levels in cigarette smokers were significantly higher than those in nonsmokers. These results suggest that increased glycotoxin exposure may contribute to the increased incidence of atherosclerosis and high prevalence of cancer in smokers.

Stimulation via CD40 can substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus

Reactivation of latent herpesviruses is a particular problem in immunocompromised individuals, such as AIDS patients, who lack effective CD4 T helper cell function. An important question is whether residual immune defenses can be mobilized to combat such opportunistic infections, in the absence of CD4 T cells. In the present study, we used a mouse model of opportunistic infection to determine whether stimulation via CD40 could substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus. Treatment with an agonistic antibody to CD40 was highly effective in preventing reactivation of latent murine gammaherpesvirus (MHV-68) in the lungs of CD4 T cell-deficient mice. CD8+ T cells were essential for this effect, whereas virus-specific serum antibody was undetectable and IFN-γ production was unchanged. This demonstration that immunostimulation via CD40 can replace CD4 T cell help in controlling latent virus in vivo has potential implications for the development of novel therapeutic agents to prevent viral reactivation in immunocompromised patients.

Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP

We have introduced a targeted mutation in SH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP− mice with lymphocyte choriomeningitis virus (LCMV) or Toxoplasma gondii was associated with increased T cell activation and IFN-γ production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP− mice produced increased IFN-γ and decreased IL-4, findings supported by decreased serum IgE levels in vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation of SH2D1A/SAP.

Genetic mapping of a murine locus controlling development of T helper 1/T helper 2 type responses.

Genetic background of the T cell can influence T helper (Th) phenotype development, with some murine strains (e.g., B10.D2) favoring Th1 development and others (e.g., BALB/c) favoring Th2 development. Recently we found that B10.D2 exhibit an intrinsically greater capacity to maintain interleukin 12 (IL-12) responsiveness under neutral conditions in vitro compared with BALB/c T cells, allowing for prolonged capacity to undergo IL-12-induced Th1 development. To begin identification of the loci controlling this genetic effect, we used a T-cell antigen receptor-transgenic system for in vitro analysis of intercrosses between BALB/c and B10.D2 mice and have identified a locus on murine chromosome 11 that controls the maintenance of IL-12 responsiveness, and therefore the subsequent Th1/Th2 response. This chromosomal region is syntenic with a locus on human chromosome 5q31.1 shown to be associated with elevated serum IgE levels, suggesting that genetic control of Th1/Th2 differentiation in mouse, and of atopy development in humans, may be expressed through similar mechanisms.

Long-term expression of erythropoietin in the systemic circulation of mice after intramuscular injection of a plasmid DNA vector.

Erythropoietin (Epo)-responsive anemia is a common and debilitating complication of chronic renal failure and human immunodeficiency virus infection. Current therapy for this condition involves repeated intravenous or subcutaneous injections of recombinant Epo. In this report, we describe the development of a novel muscle-based gene transfer approach that produces long-term expression of physiologically significant levels of Epo in the systemic circulation of mice. We have constructed a plasmid expression vector, pVRmEpo, that contains the murine Epo cDNA under the transcriptional control of the cytomegalovirus immediate early (CMV-IE) promoter, the CMV-IE 5' untranslated region, and intron A. A single intramuscular (i.m.) injection of as little as 10 micrograms of this plasmid into immunocompetent adult mice produced physiologically significant elevations in serum Epo levels and increased hematocrits from preinjection levels of 48 +/- 0.4% to levels of 64 +/- 3.3% 45 days after injection. Hematocrits in these animals remained elevated at greater than 60% for at least 90 days after a single i.m. injection of 10 micrograms of pVRmEpo. We observed a dose-response relationship between the amount of plasmid DNA injected and subsequent elevations in hematocrits. Mice injected once with 300 micrograms of pVRmEpo displayed 5-fold increased serum Epo levels and elevated hematocrits of 79 +/- 3.3% at 45 days after injection. The i.m. injected plasmid DNA remained localized to the site of injection as assayed by the PCR. We conclude that i.m. injection of plasmid DNA represents a viable nonviral gene transfer method for the treatment of acquired and inherited serum protein deficiencies.