Nina S. Levy

Haptoglobin: Basic and Clinical Aspects

Haptoglobin is an abundant hemoglobin-binding protein present in the plasma. The function of haptoglobin is primarily to determine the fate of hemoglobin released from red blood cells after either intravascular or extravascular hemolysis. There are two common alleles at the Hp genetic locus denoted 1 and 2. There are functional differences between the Hp 1 and Hp 2 protein products in protecting against hemoglobin-driven oxidative stress that appear to have important clinical significance. In particular, individuals with the Hp 2-2 genotype and diabetes mellitus appear to be at significantly higher risk of microvascular and macrovascular complications. A pharmacogenomic strategy of administering high dose antioxidants specifically to Hp 2-2 DM individuals may be clinically effective.

Vitamin E reduces cardiovascular disease in individuals with diabetes mellitus and the haptoglobin 2-2 genotype

AIMS Individuals with both diabetes mellitus (DM) and the Haptoglobin (Hp) 2-2 genotype are at increased risk of cardiovascular disease. As the antioxidant function of the Hp 2-2 protein is impaired, we sought to test the pharmacogenomic hypothesis that antioxidant vitamin E supplementation would provide cardiovascular protection to Hp 2-2 DM individuals. MATERIALS & METHODS We determined the Hp genotype on DM participants from two trials (HOPE and ICARE) and assessed the effect of vitamin E by Hp genotype on their common prespecified outcome, the composite of stroke, myocardial infarction and cardiovascular death. Data was analyzed with a fixed-effect model. These results were input into a simulation model, the Evidence Based Medicine Integrator, in order to estimate their long-term implications in a real-world population from Kaiser Permanente (CA, USA). RESULTS Meta-analysis of the two trials demonstrated a significant overall reduction in the composite end point in Hp 2-2 DM individuals with vitamin E (odds ratio: 0.58; 95% CI: 0.40-0.86; p = 0.006). There was a statistically significant interaction between the Hp genotype and vitamin E on the composite end point. In these trials, Hp typing of 69 DM individuals and treating those with the Hp 2-2 with vitamin E prevented one myocardial infarct, stroke or cardiovascular death. Lifelong administration of vitamin E to Hp 2-2 DM individuals in the Kaiser population would increase their life expectancy by 3 years. CONCLUSION A pharmacogenomic strategy of screening DM individuals for the Hp genotype and treating those with Hp 2-2 with vitamin E appears to be highly clinically effective.

Somatic Mutation in Anti-Phosphorylcholine Antibodies

A detailed analysis of the genes and proteins that participate in the murine immune response to PC has provided key insights at the structural level into the phenomenon of somatic mutation in B cells. Most anti-PC antibodies are encoded by 1 VH gene of the S107 subfamily, and 3 VK genes, VKT15 of the VK22 subfamily, VKM3 from the VK8 subfamily, and VK167 from the VK24 subfamily. No mutation was detected in these genes until the 2nd wk after immunization, indicating that mutation is under developmental control. The protein sequences of 73 heavy and light chains derived from the secondary response support the concept of developmental activation of mutation after antigen stimulation. No mutation was found in the IgM antibodies, whereas half of the IgG and IgA antibodies had mutation. Most of the mutated antibodies had higher affinity for antigen than their germline counterparts, which suggests that the major role of somatic mutation is to increase affinity rather than to create new specificities. Nucleotide sequencing established two hallmarks of mutation in immunoglobulin genes: mutations are targeted to a 1 kilobase region surrounding and including the rearranged variable gene, and they occur at an extraordinary frequency of 10(-2) nucleotide substitutions. Mutation is probably caused by DNA repair, and may occur during error-prone repair of nicked DNA around the variable gene or during mismatch repair of misaligned structural intermediates. The elucidation of this remarkable mechanism clearly requires studies of a more dynamic character. Two major questions that need to be answered are: what targets mutation to the variable gene, and what enzymes are involved?

Haptoglobin phenotype as a predictive factor of mortality in diabetic haemodialysis patients

Introduction: The mortality rate in diabetic dialysis patients (DDPs) is over 15% per year, with the cause of death most often attributed to cardiovascular disease (CVD) or bacterial infection (sepsis). Identification of genetic markers predictive of early mortality would be useful in the evaluation of therapies for the reduction of mortality rate in this population. Haptoglobin (Hp) is a polymorphic protein which appears to confer differential susceptibility to bacterial infection and CVD. We therefore proposed that Hp phenotype can predict mortality in DDPs. Methods: We tested this hypothesis prospectively in a longitudinal study of 392 dialysis patients from eight medical centres in Israel. Hp was determined by polyacrylamide gel electrophoresis. Patients were followed for all-cause mortality over a 3-year period. Results: We found that Hp phenotype was a significant predictor of mortality in DDPs stratified by age. In diabetic individuals over 60 years of age there was a decrease in mortality associated with the Hp 1-1 phenotype (P = 0.03). However, in younger DDPs the Hp 2-2 phenotype was associated with a decreased mortality rate (P = 0.003). Conclusion: Hp phenotype may be useful in the risk stratification algorithm and management of DDPs.

Restoration of blood flow by using continuous perimuscular infiltration of plasmid DNA encoding subterranean mole rat Spalax ehrenbergi VEGF

The optimal vector, regulatory sequences, and method of delivery of angiogenic gene therapy are of considerable interest. The Spalax ehrenbergi superspecies live in subterranean burrows at low oxygen tensions and its tissues are highly vascularized. We tested whether continuous perimuscular administration of Spalax vascular endothelial growth factor (VEGF) DNA could increase tissue perfusion in a murine hindlimb ischemia model. Placebo or VEGF ± internal ribosome entry site (IRES) was continuously administrated perimuscularly in the ischemic zone by using an infusion pump. None of the mice in the VEGF-treated group (>50 μg) developed visible necrosis vs. 33% of the placebo group. Microscopic necrosis was observed only in the placebo group. Spalax VEGF muscular infiltration resulted in a faster and more complete restoration of blood flow. The restoration of blood flow by VEGF was dose-dependent and more robust and rapid when using the VEGF–IRES elements. The flow restoration using continuous perimuscular infiltration was faster than single i.m. injections. Vessel density was higher in the VEGF and VEGF–IRES (−) groups compared with the placebo. Continuous perimuscular administration of angiogenic gene therapy offers a new approach to restore blood flow to an ischemic limb. Incorporation of an IRES element may assist in the expression of transgenes delivered to ischemic tissues. Further studies are needed to determine whether VEGF from the subterranean mole rat Spalax VEGF is superior to VEGF from other species. If so, 40 million years of Spalax evolution underground, including adaptive hypoxia tolerance, may prove important to human angiogenic gene therapy.

Generation of antibody diversity before and after immunization.

Immunoglobulin variable genes are a large, multigene family whose members rearrange at different times in development. The hallmark of the immune system is diversity, and it is intriguing to observe how B cells, which start out with unrearranged genes or zero diversity, rearrange germline genes to build a repertoire of greater than lo6 specificities. Which genes rearrange first? Is there a developmental program to rearrangement? How quickly does the repertoire diversify, and does it occur at a molecular level by randomly rearranging genes or at a cellular level after selection by antigen? Analysis of rearranged genes in B cells early in ontogeny has provided striking insights into the formation of the repertoire. Despite over 1,000 variable genes for the heavy chain (V,) in the germline, only a few, located near the joining (JH) genes, are rearranged.’” Although less is known about rearrangement of variable genes for the kappa light chain (VK), preliminary data indicate that genes from many different subfamilies are rearranged. Further analyses of rearranged VK genes may provide a molecular basis for the phenomenon of the programmed appearance of certain B cells during ontogeny.‘ Additional diversity is generated in immunoglobulin genes after antigen stimulation by somatic mutation. Much is known about the structural hallmarks of mutation in genes coding for antibodies from secondary responses. Nucleotide substitutions are clustered in a discrete region of DNA surrounding and including the rearranged V genesV6; they occur at a frequency of around 1%; and they are more common in IgG and IgA molecules than in I g h molecules.’ The elucidation of this unusual mechanism clearly requires studies of a more dynamic character. We have approached this problem by attempting to identify a population of B cells that is actively undergoing somatic mutation. The results indicate that mutation occurs early after B cells are stimulated with a primary injection of antigen. Mutated antibodies then undergo intense selection by antigen to produce those with high affinity. We propose that the major effect of somatic hypermutation is to increase affinity and not to create new specificities that would allow other gene products to participate in an immune response.”

Changing the face of diabetic care with haptoglobin genotype selection and vitamin e.

Research over the past 10 years in our laboratory has led to two major findings. The first is that haptoglobin (Hp) genotype can predict the risk of developing vascular complications in individuals with diabetes mellitus (DM), and the second, more far-reaching discovery, is that vitamin E treatment can significantly reduce vascular complications in individuals with DM and the Hp 2-2 genotype. The former finding has been well documented in numerous studies which included over 50,000 patients of diverse geographical and ethnic backgrounds. The latter discovery is more recent and less well accepted by the medical community due to confounding reports over the past 30 years regarding the efficacy of vitamin E treatment for vascular disease. We propose that the benefit of vitamin E treatment was not obvious in earlier studies due to the absence of any genetic basis for patient selection. Our studies dividing DM individuals into vitamin E treatment subgroups based on Hp genotype show a clear benefit for individuals of the Hp 2-2 genotype, while patients carrying the other two Hp genotypes are not affected or may be adversely affected by receiving vitamin E. These findings may explain the overall lack of benefit seen in previous vitamin E studies and emphasize the importance of carefully selecting which patients should receive vitamin E therapy. The pharmacogenomic paradigm discussed in this review potentially could result in a dramatic improvement in the health of millions of individuals worldwide using a treatment that is both accessible and affordable to all.