MHC/Peptide-Specific Interaction of the Humoral Immune System: A New Category of Antibodies.

Abs bind to unprocessed Ags, whereas cytotoxic CD8(+) T cells recognize peptides derived from endogenously processed Ags presented in the context of class I MHC complexes. We screened, by ELISA, human sera for Abs reacting specifically with the influenza matrix protein (IMP)-derived peptide58-66 displayed by HLA-A*0201 complexes. Among 653 healthy volunteers, blood donors, and women on delivery, high-titered HLA-A*0201/IMP58-66 complex-specific IgG Abs were detected in 11 females with a history of pregnancies and in 1 male, all HLA-A*0201(-). These Abs had the same specificity as HLA-A*0201/IMP58-66-specific cytotoxic T cells and bound neither to HLA-A*0201 nor the peptide alone. No such Abs were detected in HLA-A*0201(+) volunteers. These Abs were not cross-reactive to other self-MHC class I alleles displaying IMP58-66, but bound to MHC class I complexes of an HLA nonidentical offspring. HLA-A*0201/IMP58-66 Abs were also detected in the cord blood of newborns, indicating that HLA-A*0201/IMP58-66 Abs are produced in HLA-A*0201(-) mothers and enter the fetal blood system. That Abs can bind to peptides derived from endogenous Ags presented by MHC complexes opens new perspectives on interactions between the cellular and humoral immune system.

Characterization and application of two RANK-specific antibodies with different biological activities.

Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools.

Gp120/CD4 blocking antibodies are frequently elicited in ART-naïve chronically HIV-1 infected individuals

Antibodies with the ability to block the interaction of HIV-1 envelope glycoprotein (Env) gp120 with CD4, including those overlapping the CD4 binding site (CD4bs antibodies), can protect from infection by HIV-1, and their elicitation may be an interesting goal for any vaccination strategy. To identify gp120/CD4 blocking antibodies in plasma samples from HIV-1 infected individuals we have developed a competitive flow cytometry-based functional assay. In a cohort of treatment-naïve chronically infected patients, we showed that gp120/ CD4 blocking antibodies were frequently elicited (detected in 97% plasma samples) and correlated with binding to trimeric HIV-1 envelope glycoproteins. However, no correlation was observed between functional CD4 binding blockade data and titer of CD4bs antibodies determined by ELISA using resurfaced gp120 proteins. Consistently, plasma samples lacking CD4bs antibodies were able to block the interaction between gp120 and its receptor, indicating that antibodies recognizing other epitopes, such as PGT126 and PG16, can also play the same role. Antibodies blocking CD4 binding increased over time and correlated positively with the capacity of plasma samples to neutralize the laboratory-adapted NL4.3 and BaL virus isolates, suggesting their potential contribution to the neutralizing workforce of plasma in vivo. Determining whether this response can be boosted to achieve broadly neutralizing antibodies may provide valuable information for the design of new strategies aimed to improve the anti-HIV-1 humoral response and to develop a successful HIV- 1 vaccine.

Isotype distribution of anticardiolipin antibodies in systemic lupus erythematosus prospective analysis of a series of 100 patients

A prospective study of IgG and IgM isotypes of anticardiolipin antibodies (aCL) in a series of 100 patients with systemic lupus erythematosus was carried out. To determine the normal range of both isotype titres a group of 100 normal control serum samples was studied and a log-normal distribution of IgG and IgM isotypes was found. The IgG anticardiolipin antibody serum was regarded as positive if a binding index greater than 2.85 (SD 3.77) was detected and a binding index greater than 4.07 (3.90) was defined as positive for IgM anticardiolipin antibody. Twenty four patients were positive for IgG aCL, 20 for IgM aCL, and 36 for IgG or IgM aCL, or both. IgG aCL were found to have a significant association with thrombosis and thrombocytopenia, and IgM aCL with haemolytic anaemia and neutropenia. Specificity and predictive value for these clinical manifestations increased at moderate and high anticardiolipin antibody titres. In addition, a significant association was found between aCL and the presence of lupus anticoagulant. Identification of these differences in the anticardiolipin antibody isotype associations may improve the clinical usefulness of these tests, and this study confirms the good specificity and predictive value of the anticardiolipin antibody titre for these clinical manifestations.

Distribution of collagen types I, III, and IV in gastric tissue of marmosets (Callithrix spp., Callitrichidae: Primates)

Extracellular matrix (ECM) components such as fibrillar collagens play a fundamental role in wound repair and have also been studied in association with the gastric ulcer healing process in gastroenterology. Nevertheless, there have been no studies in the literature to date regarding the description and characterization of ECM components, neither in normal nor in injured gastric tissue of primate species. The objective of this study was to investigate the expression of gastric collagen types I, III, and IV in marmosets (Callithrix sp.). Histological specimens from the stomach of 6 Callithrix jacchus, 12 C. kuhli, and 12 C. geoffroyi were evaluated. The specimens were immunostained with anti-types I and III collagen polyclonal antibodies and anti-type IV collagen monoclonal antibody. Collagen types I and III were detected in the submucosa and lamina propria between the mucosal glands while collagen type IV was detected in the muscularis mucosae, muscular layers, blood vessels, and gastric mucosa between the mucosal glands. It is hoped that these findings can contribute to future studies on the gastric extracellular matrix components in primates and to comparative studies in the area of gastroenterology.

Risk factors associated with the frequency of antibodies against Babesia bovis and Babesia bigemina in cattle in southern Mozambique

The study aimed to evaluate the risk factors associated with the frequency of IgG antibodies against Babesia bovis and B. bigemina in cattle in southern Mozambique. Eight hundred and nine serum samples were collected from cattle in three provinces namely Maputo, Gaza and Inhambane, and tested by indirect enzyme-linked immunosorbent assay (i-ELISA) to assess the humoral immune response towards B. bovis and B. bigemina. The chi-square test at 5% significance was used to determine whether there was an association between gender, age and geographic origin of seropositive animals. The overall prevalence was 78.8% (548/695) for B. bovis and 76.0% (528/695) for B. bigemina. The origin of the animals showed a significant association (p<0.05) with seropositivity to both agents, while gender and age was not associated (p>0.05). Maputo province had the highest rate of positive animals, with 93.7% (118/126) for B. bovis and 97.6% (123/126) for B. bigemina. In Gaza province 77.3% (321/415) of the animals were positive for B. bovis and 67.5% (280/415) for B. bigemina, while in the province of Inhambane the levels of seropositivity were 70.8% (109/154) and 81.2% (125/154) for B. bovis and B. bigemina respectively. In the present study, the frequency of cattle positive for B. bovis and B. bigemina was shown to increase among older age groups, suggesting that infection and re-infection persisted even after the primary infection. Thus, this region is considered to be in a state of enzootic stability with regards to B. bovis and B. bigemina.

Cardiac Troponin Specific Autoantibodies: Analytical Tools for Exploring Their Impact on Cardiac Troponin I Testing

Cardiac troponin (cTn) I and T are the recommended biomarkers for the diagnosis and risk stratification of patients with suspected acute coronary syndrome (ACS), a major cause of cardiovascular death and disability worldwide. It has recently been demonstrated that cTn-specific autoantibodies (cTnAAb) can negatively interfere with cTnI detection by immunoassays to the extent that cTnAAb-positive patients may be falsely designated as cTnI-negative. The aim of this thesis was to develop and optimize immunoassays for the detection of both cTnI and cTnAAb, which would eventually enable exploring the clinical impact of these autoantibodies on cTnI testing and subsequent patient management. The extent of cTnAAb interference in different cTnI assay configurations and the molecular characteristics of cTnAAbs were investigated in publications I and II, respectively. The findings showed that cTnI midfragment targeting immunoassays used predominantly in clinical practice are affected by cTnAAb interference which can be circumvented by using a novel 3+1-type assay design with three capture antibodies against the N-terminus, midfragment and C-terminus and one tracer antibody against the C-terminus. The use of this assay configuration was further supported by the epitope specificity study, which showed that although the midfragment is most commonly targeted by cTnAAbs, the interference basically encompasses the whole molecule, and there may be remarkable individual variation at the affected sites. In publications III and IV, all the data obtained in previous studies were utilized to develop an improved version of an existing cTnAAb assay and a sensitive cTnI assay free of this specific analytical interference. The results of the thesis showed that approximately one in 10 patients with suspected ACS have detectable amounts of cTnAAbs in their circulation and that cTnAAbs can inhibit cTnI determination when targeted against the binding sites of assay antibodies used in its immunological detection. In the light of these observations, the risk of clinical misclassification caused by the presence of cTnAAbs remains a valid and reasonable concern. Because the titers, affinities and epitope specificities of cTnAAbs and the concentration of endogenous cTnI determine the final effect of circulating cTnAAbs, appropriately sized studies on their clinical significance are warranted. The new cTnI and cTnAAb assays could serve as analytical tools for establishing the impact of cTnAAbs on cTnI testing and also for unraveling the etiology of cTn-related autoimmune responses.

Clearance-inducing antibodies are responsible for protection against the acute phase of Trypanosoma cruzi infection in mice

A study was conducted on mice infected with strains Y and CL of Trypanosoma cruzi. The ability of anti-Y and anti-CL sera to induce complement-mediated lysis, immune clearance and protection against the acute phase of the infection was studied using homologous anti-Y or anti-CL serum tested with the Y or CL strain, or heterologous anti-Y serum tested with the CL strain or anti-CL serum tested with the Y strain. Complement-mediated lysis was induced by both homologous and heterologous antisera but protection was afforded only by homologous antisera. Immune clearance was induced by homologous but not by heterologous antisera. Antisera with high clearance ability were able to confer protection whereas antisera with high lytic ability were not. These results show a high correlation between the antibody ability to induce clearance and to confer protection and suggest that clearance rather than lysis is responsible for protection against the acute phase of the infection. The mechanism of antibody protection against the acute phase of the infection is discussed.

Origin and pathogenesis of antiphospholipid antibodies

Antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies that are detected in the serum of patients with a variety of conditions, including autoimmune (systemic lupus erythematosus), infectious (syphilis, AIDS) and lymphoproliferative disorders (paraproteinemia, myeloma, lymphocytic leukemias). Thrombosis, thrombocytopenia, recurrent fetal loss and other clinical complications are currently associated with a subgroup of aPL designating the antiphospholipid syndrome. In contrast, aPL from patients with infectious disorders are not associated with any clinical manifestation. These findings led to increased interest in the origin and pathogenesis of aPL. Here we present the clinical features of the antiphospholipid syndrome and review the origin of aPL, the characteristics of experimentally induced aPL and their historical background. Within this context, we discuss the most probable pathogenic mechanisms induced by these antibodies.

Detection of specific IgE antibodies in parasite diseases

Activation of Th1 or Th2 cells is associated with production of specific immunoglobulin isotypes, offering the opportunity to use antibody measurement for evaluation of T cell function. Schistosomiasis and visceral leishmaniasis are diseases associated with Th2 activation. However, an IgE response is not always detected in these patients. In the present study we evaluated specific IgE antibodies to S. mansoni and L. chagasi antigens by ELISA after depletion of serum IgG with protein G immobilized on Sepharose beads or RF-absorbent (purified sheep IgG antibodies anti-human IgG). In schistosomiasis patients, specific IgE to SWAP antigen was demonstrable in only 10 of 21 patients (48%) (mean absorbance ± SD = 0.102 ± 0.195) when unabsorbed serum was used. Depletion of IgG with protein G increased the number of specific IgE-positive tests to 13 (62%) and the use of RF-absorbent increased the number of positive results to 20 (95%) (mean absorbances ± SD = 0.303 ± 0.455 and 0.374 ± 0.477, respectively). Specific IgE anti-L. chagasi antibodies were not detected in unabsorbed serum from visceral leishmaniasis patients. When IgG was depleted with protein G, IgE antibodies were detected in only 3 (11%) of 27 patients, and the use of RF-absorbent permitted the detection of this isotype in all 27 visceral leishmaniasis sera tested (mean absorbance ± SD = 0.104 ± 0.03). These data show that the presence of IgG antibodies may prevent the detection of a specific IgE response in these parasite diseases. RF-absorbent, a reagent that blocks IgG-binding sites and also removes rheumatoid factor, was more efficient than protein G for the demonstration of specific IgE antibodies.

How are antibodies involved in the protective mechanism of susceptible mice infected with T. cruzi?

Host resistance to Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the acute phase of the infection the presence of IFN-g, TNF-a, IL-12 and GM-CSF has been closely associated with resistance, whereas TGF-ß and IL-10 have been associated with susceptibility. Several investigators have demonstrated that antibodies are responsible for the survival of susceptible animals in the initial phase of infection and for the maintenance of low levels of parasitemia in the chronic phase. However, how this occurs is not yet understood. Our results and other data in the literature support the hypothesis that the protective role of antibodies in the acute phase of infection is dependent mostly on their ability to induce removal of bloodstream trypomastigotes from the circulation in addition to other concomitant cell-mediated events.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 ± 70 vs 1451 ± 235 × 10² neutrophils/mg; P<0.01) and in the kidneys (526 ± 89 vs 852 ± 73 × 10² neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Production and characterization of monoclonal antibodies to a Brazilian bovine herpesvirus type 5

Antigens of a bovine herpesvirus type 5 (BHV-5), isolated from a cow with a neurological infection in Rio Grande do Sul State, Brazil, were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs). Eleven hybridomas secreting mAbs directed at BHV-5 antigens were obtained after two fusions and screening of 356 hypoxanthine-aminopterin-thymidine-resistant clones. The mAbs reacted at dilutions up to 1:500 (hybridoma culture supernatant) and up to >1:10,000 (ascitic fluid) in an indirect fluorescent antibody assay (IFA) and in immunoperoxidase staining of BHV-5-infected cells. Four mAbs (1D12, 2E2, 2G10 and 4E4) showed virus-neutralizing activity against the parental BHV-5 isolate. Five mAbs (1F3, 2A6, 2F9, 2G10 and HB24L) reacted in Western immunoblotting with a protein of approximately 90 kDa. Three other mAbs (2E2, 3D6 and 4E4) reacted in IFA with antigens of a BHV-1 mutant glycoprotein C- negative strain, demonstrating that they are directed at a viral antigen other than glycoprotein C. The eleven mAbs tested reacted with 20 BHV-5 field isolates and nine mAbs reacted with 10 BHV-1 isolates. Two mAbs (1F3 and 2F9) failed to react with BHV-1 field isolates, although they displayed a weak and nonreproducible reaction with the BHV-1 reference strain Los Angeles. These mAbs may be very useful in distinguishing between BHV-1 and BHV-5 infections since most of the traditional reagents and techniques are unable to do so. One mAb (2F9) was shown to bind to viral antigens by immunohistochemistry of histological sections of the brain of a BHV-5-infected calf. These results demonstrate that the mAbs produced here are suitable for use in a variety of immunological techniques and therefore may be useful for diagnostic and research purposes.