Iron metabolism markers and haptoglobin phenotypes in susceptibility to HSV-1 or/and HSV-2 lesion relapses.

Different haptoglobin (Hp) phenotypes play a role in several pathologic processes including infectious diseases. In order to evaluate the role of iron storage and metabolism in susceptibility to herpetic manifestations, we studied the frequency of the Hp phenotypes and iron metabolism in patients affected by H. Simplex virus 1 or 2 (HSV-1 or HSV-2), compared with controls. Hp phenotype and iron metabolism were determined in 100 patients with recurrent HSV-1 or HSV-2 manifestations during the relapses, and in 110 healthy subjects. The frequencies of the three Hp phenotypes in the patient group compared to the control group were 18% versus 14.5% p = NS for Hp 1.1, 25% versus 40% p = 0.03 for Hp 2.2 and 57% versus 45.5% p = NS for Hp 2.1. All iron metabolism parameters tested showed significant differences between patients and controls; haemoglobin (Hb), ferritin, and serum iron were lower, while transferrin was higher in the patients than in controls. Reductions in iron availability may be a risk factor for relapsing lesions of HSV-1 or HSV-2. Hp 2.2 phenotype may offer some protection against the recurrence of Herpes labialis or genitalis manifestations.

A unique tetrameric structure of deer plasma haptoglobin--an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure.

Similar to blood types, human plasma haptoglobin (Hp) is classified into three phenotypes: Hp 1-1, 2-1 and 2-2. They are genetically inherited from two alleles Hp 1 and Hp 2 (represented in bold), but only the Hp 1-1 phenotype is found in almost all animal species. The Hp 2-2 protein consists of complicated large polymers cross-linked by alpha2-beta subunits or (alpha2-beta)n (where n>or=3, up to 12 or more), and is associated with the risk of the development of diabetic, cardiovascular and inflammatory diseases. In the present study, we found that deer plasma Hp mimics human Hp 2, containing a tandem repeat over the alpha-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, i.e. (alpha-beta)4, although the locations of -SH groups (responsible for the formation of polymers) are exactly identical to that of human. Denaturation of deer Hp using 6 m urea under reducing conditions (143 mmbeta-mercaptoethanol), followed by renaturation, sustained the formation of (alpha-beta)4, suggesting that the Hp tetramers are not randomly assembled. Interestingly, an alpha-chain monoclonal antibody (W1), known to recognize both human and deer alpha-chains, only binds to intact human Hp polymers, but not to deer Hp tetramers. This implies that the epitope of the deer alpha-chain is no longer exposed on the surface when Hp tetramers are formed. We propose that steric hindrance plays a major role in determining the polymeric formation in human and deer polymers. Phylogenetic and immunochemical analyses revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage.

Haptoglobin phenotype and risk of cervical neoplasia: a case-control study.

BACKGROUND: Haptoglobin is an acute-phase glycoprotein that influences host response to infections and tumours. The haptoglobin locus is polymorphic with 2 classes of alleles (Hp(1) and Hp(2)) yielding 3 phenotypes: Hp1-1, Hp2-2, and Hp2-1 with structurally and functionally distinct protein products, suggesting that haptoglobin polymorphism may influence susceptibility to infections and cancers. METHODS: We examined the relation between haptoglobin phenotype and high-grade cervical intraepithelial neoplasia (CIN) in a hospital-based case-control study. Cases (n = 307) were women with biopsy-confirmed CIN-2 or CIN-3. Controls (n = 358) were a random sample of women with normal cytology. The PGMY polymerase chain reaction and reverse line blot methods were used for HPV detection and genotyping. Haptoglobin phenotype was determined by polyacrylamide gel electrophoresis. RESULTS: Among controls, phenotype distribution corresponded to allele frequencies of 0.39 for Hp(1) and 0.61 for Hp(2) with no significant deviation from the Hardy-Weinberg equilibrium (p=0.66). With all women included in the analysis, the Hp1-1 phenotype was associated with increased risk of CIN (OR contrasting Hp1-1 vs. Hp2-2 = 1.0; 95% CI: 0.6-1.5). However, in analyses restricted to HPV-positive participants, the Hp1-1 phenotype was associated with 2.7-fold (95% CI: 1.0-7.2) higher risk of CIN. CONCLUSIONS: If confirmed, these findings indicate an increased risk of CIN among women with the Hp1-1 phenotype.

Quantitative variations of the isoforms in haptoglobin 1-2 and 2-2 individual phenotypes.

Haptoglobin is a hemoglobin-binding protein presenting in humans three distinct phenotypes (Hpt 1-1, Hpt 1-2, or Hpt 2-2). The Hpt 1-2 and Hpt 2-2 phenotypes are in turn represented by populations of isoforms. The relative amounts of the major isoforms of Hpt 1-2 and Hpt 2-2 were found to differ not only in different individuals, but also in the same individual before and after a physical effort. Exercise-dependent changes in the plasma concentrations of ascorbate, urate, alpha-tocopherol, retinol, and glutathione were also observed, but correlations between such changes and those of the amount for any isoform were not found. Samples of Hpt 1-2 or Hpt 2-2 were challenged with oxidants (H(2)O(2) with ferrous ions, spermine-NO, KO(2), and 3-morpholinosydnonimine), but the isoform levels were not altered. Hpt 2-2 isoforms were present in Hpt 1-2, as minor species. Furthermore, different isoforms exhibited different hemoglobin binding abilities. Thus, these parameters should also be taken into consideration in studies correlating Hpt phenotypes prevalence with pathologies or functional differences.

A new method for fast haptoglobin phenotyping and hemoglobin binding capacity calculation based on capillary zone electrophoresis.

A capillary zone electrophoresis method was developed for haptoglobin (Hp) phenotyping in hemoglobin (Hb) supplemented serum. The method allows a complete resolution of the major haptoglobin phenotypes Hp 1-1, Hp 2-1, and Hp 2-2 based on the difference in charge-to-mass ratio of their Hb-Hp complexes. Identification of these phenotypes was achieved by their significant differences in migration times and their marked difference in electrophoretic pattern. Our method showed full agreement with starch gel electrophoresis. Furthermore, following neuraminidase treatment of the serum, the Hp subtypes Hp1-1FF, FS and SS could be resolved, based on the same criteria as the phenotyping. The new electrophoretic method allowed typing of the rare phenotypes Hp 2-1 modified (Hp 2-1M) and Hp Johnson. The calculated hemoglobin binding capacity of serum correlates well with the nephelometrically determined haptoglobin concentration. The new method for typing haptoglobin gives prospectives for fast haptoglobin typing and Hp 1-1 subtyping.

Distribution of haptoglobin phenotypes in a Korean population, using the semi-automated PhastSystem.

We have established a new phenotyping method for haptoglobin, based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis using the PhastSystem (Pharmacia Biotech, Uppsala, Sweden), followed by immunoblotting for detection. We measured haptoglobin concentrations and determined the haptoglobin phenotypes of 316 healthy Koreans using this method: 31 (9.8%) were of Hp 1-1 type, 140 (44.3%) of Hp 2-1 type and 145 (45.9%) of Hp 2-2 type. The haptoglobin allele frequencies were calculated to be 0.32 for Hp1 and 0.68 for Hp2. We were able to visualize up to 12 bands from the human Hp 2-2 polymeric series, with molecular weights in the range 171.9 x 10(3) to 802.2 x 10(3). The reference range of serum haptoglobin concentrations obtained by the IFCC (International Federation of Clinical Chemistry) standard method was 0.27-2.14 g/L. The serum haptoglobin concentration in Koreans was similar to that of Caucasians, but the Hp1 allele frequency was lower in Koreans. Our method could be used in clinical laboratories as a simple and practical method of haptoglobin phenotyping. In addition, the Hp 2-2 polymeric series could be used as high molecular weight standards.

Haptoglobin polymorphism, iron metabolism and mortality in HIV infection.

BACKGROUND: Three phenotypes of the antioxidant protein haptoglobin are known: Hp 1-1, Hp 2-1 and Hp 2-2. OBJECTIVES: To investigate the outcome of HIV infection according to haptoglobin type. DESIGN AND METHODS: Haptoglobin phenotypes were determined using starch gel electrophoresis in serum obtained from 653 HIV-infected Caucasians in the AIDS reference centers of Gent (n = 184), Antwerp (n = 309), and Luxembourg (n = 160). Survival was compared between haptoglobin types using Kaplan-Meier curves. Plasma HIV-1 RNA was quantified by reverse transcriptase PCR. Serum iron, transferrin saturation, ferritin, and vitamin C were assayed to evaluate iron-driven oxidative stress in 184 HIV-infected patients and 204 controls. RESULTS: The haptoglobin type distribution amongst the patients (17.6% Hp 1-1, 49.9% Hp 2-1, 32.5% Hp 2-2) corresponded to that of the controls. Kaplan-Meier curves showed a higher mortality for the Hp 2-2 group (P = 0.0001; adjusted mortality risk ratio, 1.78; 95% confidence interval, 1.25-2.54). Median survival time was 11.0 years (Hp 1-1 and Hp 2-1) versus 7.33 years (Hp 2-2). Plasma HIV-1 RNA levels prior to antiviral therapy and their increase over 1 year were highest in Hp 2-2 patients (P = 0.03 and 0.003, respectively). The Hp 2-2 type was associated with higher serum iron, transferrin saturation, and ferritin levels and with low vitamin C concentrations. Furthermore, ferritin concentrations were higher in HIV-infected patients than in controls (P < 0.0001). CONCLUSION: HIV-infected patients carrying the Hp 2-2 phenotype show a worse prognosis, which is reflected by a more rapid rate of viral replication (in the absence of antiviral treatment). They also accumulate more iron and oxidize more vitamin C, suggesting that less efficient protection against haemoglobin/iron-driven oxidative stress may be a direct mechanism for stimulating viral replication.

[Haptoglobin (HPA)-subtypes in Swiss populations]. / Haptoglobin (HPA)-Subtypen in schweizerischen Populationen.

A subtyping of the haptoglobins of thirteen small Swiss populations, who live in mountainous regions of the Alps, and four larger Swiss populations has been undertaken. The impact of population and sample size on allele frequencies is discussed.

Haptoglobin phenotyping by newly developed monoclonal antibodies. Demonstration of haptoglobin uptake into peripheral blood neutrophils and monocytes.

We have generated two IgG murine mAbs that recognize native human haptoglobin (Hp). These mAbs, 3A8 and 4B2, efficiently bind to the Hp complex regardless of the serum donor's phenotype. The specificity of mAb 3A8 was confirmed by immunoaffinity purification of 3A8-binding material from human serum and subsequent N-terminal amino acid sequencing of the invariant 40-kDa chain. mAb 3A8 and 4B2 were also reactive with cell-associated Hp when studied by immunocytochemistry. When human peripheral blood leukocytes were tested, 90% of the granulocytes and a lesser (and variable) fraction of monocytes displayed an intense intracytoplasmatic granular staining. This was confirmed by flow cytometric analysis of permeabilized leukocytes and by demonstrating the presence of Hp (of the expected Hp serum phenotype) in extracts of washed granulocytes by immunoblotting. Leukocytes obtained from a Hp phenotype 2-2 donor, incubated in culture medium supplemented with 10% serum from a donor possessing the Hp 1-1 phenotype, contained both Hp phenotypes when analyzed by immunoblotting after a 6-h incubation period. In addition, Hp was actively exocytosed by granulocytes following their exposure to Candida albicans. These observations suggest that exogenous Hp is concentrated within granulocytes and not synthesized de novo and is, in turn, exocytosed following neutrophil activation. Northern blotting analysis is consistent with the lack of haptoglobin gene transcription in granulocytes. These findings together with the earlier observations that Hp modulates granulocyte activity suggest that Hp levels may be enhanced locally at sites of inflammation to modulate granulocyte activity.

The effects of putrefaction on haptoglobin phenotyping from tooth fragments.

Tooth fragments freshly extracted from humans were stored for various times and temperatures under both dry and moist conditions. The fragments were reduced to powder and proteins eluted. Purified haptoglobin fractions were separated, detected and phenotyped using the Phast Gel electrophoresis system using gradient gels. Haptoglobin phenotypes were demonstrable but became less detectable as time increased.

Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamide gels rehydrated in presence of 2-mercaptoethanol.

A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase-treated plasma samples by using dry miniaturized (interelectrode distance: 55 mm) polyacrylamide gel, rehydrated in presence of 2-mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4-6.5 and pH 6-8) followed by immunoblotting. The presence of 2-mercaptoethanol in the gel prevented refolding of the Hp alpha and Hp beta chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp alpha allelic products (1S, 1F, 2FS, 2SS and 2FF). A population study carried out with 250 unrelated individuals living in Central Spain is also presented.

[The haptoglobin-streptococcus relationship]. / Die Haptoglobin-Streptokokken-Relation.

In many experiments beginning in 1977 Prof. O. Prokop and Prof. W. Köhler discovered that the human haptoglobins are able to agglutinate group G-streptococci, but the reaction depends on the haptoglobin type. Haptoglobins of the types Hp 2-2 and Hp 2-1 are complete agglutinins and haptoglobin of the Hp 1-1 type is incomplete ("blocking") antibody. Later it was found that streptococci with the T4--antigen (from Lancefield types A, C and G) are suitable for the test.

Monoclonal antibodies against human haptoglobin.

Three monoclonal antibodies: 2.36.71.41, 7.60.66.55, and 18.4.40. 80 to human haptoglobin 2-1 were produced, purified and characterized. The affinity constants ranged within 0.3-2.4 x 10(8) M-1. The monoclonal antibodies 7.60.66.55 and 18.4.40.80 reacted with beta subunit of haptoglobin, showed similar epitope affinities and epitope densities on main haptoglobin types. However, the epitope on the haptoglobin molecule for the monoclonal antibody 18.4.40.80 occupied somewhat more surface than that for the antibody 7.60.66.55. The monoclonal antibody 2.36.71.41 was able to bind both alpha and beta chains of haptoglobin. In ELISA affinity reactions this antibody achieved with haptoglobin 2-2 the plateau phase at absorbance values 15% higher than with haptoglobin 2-1, and 60% higher than with haptoglobin 1-1. End-point titration of the monoclonal antibody 2.36.71.41 against three haptoglobin types showed differences in titer, indicating distinct epitope densities.

Monoclonal antibody to SER immune suppressor detects polymeric forms of haptoglobin.

A series of monoclonal antibodies has been developed which is directed to a serum immunosuppressive factor, known as suppressive E-receptor factor (SER). SER, purified from the body fluids of cancer patients, is a polymeric form of haptoglobin, which is 100-1,000 times more potent an immunosuppressor than normal plasma haptoglobin and is immunochemically analogous to the neonatal form. Unlike the neonatal haptoglobin found in cord blood, SER, however, does not contain bound-hemoglobin. One group of monoclonal antibodies described in this study detects the polymeric forms of haptoglobin (SER) under non-denaturing conditions, but fails to recognize SER under the denaturing conditions of SDS-PAGE. A second group of monoclonal antibodies reacts only with the alpha subunit of haptoglobin but not with the beta subunit; in contrast, the commercially prepared polyclonal antisera to haptoglobin react with both the alpha and beta subunit. The average level of SER in normal human plasma (n = 19) was 1.0 micrograms/ml, regardless of age or sex. Since macrophages appear to secrete SER but do not synthesize haptoglobin, SER may represent an oxidized form of plasma haptoglobin generated from macrophages activated during an inflammatory response. These studies suggest that SER may be a negative feed-back regulator of immune response produced by activated macrophages.

Typing and subtyping of haptoglobin from native serum using disc gel electrophoresis in alkaline buffer: application to routine screening.

A method with which the six common phenotypes of human haptoglobin can be identified using unseparated serum is described. In contrast to other reported methods, both typing and subtyping of haptoglobin can be performed by polyacrylamide gel electrophoresis in alkaline buffer using 0.1-4.0 microliter of native serum with hemoglobin added. Haptoglobin-hemoglobin complexes are visualized by their peroxidase activity using benzidine and barium peroxide. This relatively inexpensive and fast method seems particularly well suited for the typing and subtyping of haptoglobin from minute amounts in large series of sera and other body fluids and thus may be useful in medical genetics and forensic medicine.

Haptoglobulin phenotypes in leprosy.

Haptoglobulin phenotyping was carried out in fifty controls and in thirty five leprosy patients. In controls the incidence Hp phenotypes 2-2, 2-1 and 2-1 (Mod) is 76%, 16% and 8% respectively. In leprosy patients, the incidence of phenotypes 2-2, 2-1, 1-1 and 0-0 is 77%, 11%, 3% and 9% respectively. The incidence of phenotype 2-2, 1-1 and 0-0 is more in leprosy patients than in controls and is significant (p less than 0.05). In none of the leprosy patients phenotype 2-1 (Mod) was recorded.

[Haptoglobins, streptococcus agglutination and streptococcal infections]. / Haptoglobine, Streptokokkenagglutination und Streptokokkeninfektionen.

The haptoglobins in man and in mammals react with streptococci bearing the T4-antigen. Hp 2-2 and Hp 2-1 react with high titres like complete agglutinins while Hp 1-1 acts like a blocking antibody. It is surprising to observe the similarity of the behaviour of the haptoglobins and of IgG and IgM. Streptococci carrying Hp 1-1 yield a positive Coombs-test. The Coombs-serum however, is an anti-human-haptoglobin-body from sheep. By means of supplements it has been possible to increase partly considerably the titres of Hp 2-1 and Hp 2-2 against the streptococci.