Structural analysis of Fc/FcγR complexes: a blueprint for antibody design

The number of studies and the quality of the structural data of Fcγ receptors (FcγRs) has rapidly increased in the last few years. Upon critical examination of the literature, we have extracted general conclusions that could explain differences in affinity and selectivity of FcγRs for immunoglobulin G (IgG) based on structural considerations. FcγRs employ a little conserved asymmetric surface of domain D2 composed of two distinct subsites to recognize the well-conserved lower hinge region of IgG1-Fc. The extent of the contact interface with the antibody in subsite 1 of the receptor (but not in subsite 2), the geometrical complementarity between antibody and receptor, and the number of polar interactions contribute decisively toward strengthening the binding affinity of the antibody for the receptor. In addition, the uncertain role of the N-linked glycan of IgG for the binding and effector responses elicited by FcγRs is discussed. The available data suggest that not only the non-covalent interactions between IgG and FcγRs but also their dynamic features are essential for the immune response elicited through these receptors. We believe that the integration of structural, thermodynamic, and kinetic data will be critical for the design and validation of the next generation of therapeutic antibodies with enhanced effector capabilities.     

The association between CBS 844ins68 polymorphism and head and neck squamous cell carcinoma risk – a case-control analysis

Introduction

Susceptibility to head and neck squamous cell carcinoma may be modified by functional polymorphisms in genes involved in the folate pathway, such as cystathionine beta-synthase (CBS). The CBS 844ins68 polymorphism is associated with DNA methylation changes and cancer development.

Material and methods

A case-control retrospective study was conducted in 322 patients with head and neck squamous cell carcinoma and in 531 control subjects without cancer. The polymerase chain reaction-restriction fragment length polymorphism technique was used to genotype the polymorphism. For statistical analysis, χ² test was conducted to examine whether the genotypic frequency of CBS 844ins68 was in Hardy-Weinberg equilibrium and multiple logistic regression was used for comparisons between groups, and for interactions between the polymorphism and risk factors and clinical histopathological parameters.

Results

No significant difference in CBS 844ins68 genotypic distribution was observed between the groups. Age > 50 years, male gender and tobacco consumption were predictors of the disease with increased risk of 7.89 (95% CI: 5.56-11.21), 2.49 (95% CI: 1.72-3.62), 6.44 (95% CI: 4.63-8.96) and 2.29 times (95% CI: 1.71-3.06) respectively. There was no association between the distribution of the CBS 844ins68 genotype and risk factors for this disease. According to clinical histopathological parameters, CBS 884ins68 polymorphism presented high frequency in oral cavity (p < 0.05) and patients with the polymorphism presented less survival time (p < 0.05).

Conclusions

We concluded that the CBS 844ins68 polymorphism is not associated with HNSCC risk and there is increased risk of this disease in male gender individuals smokers aged over 50 years. In adittion, the polymorphism is more frequent in patients with oral cavity as primary site and in patients with less survival time.     

Epistatic interactions between Fc (GM) and FcγR genes and the host control of human immunodeficiency virus replication

Host genetic factors are thought to contribute to the interindividual differences in the control of human immunodeficiency virus (HIV) replication. The aim of the present investigation was to determine whether genes encoding GM and KM allotypes-genetic markers of immunoglobulin γ and κ chains, respectively-and those encoding Fcγ receptor (FcγR) IIa and IIIa are associated with the host control of HIV replication. A case-control design was employed among HIV-infected subjects, with a group that spontaneously controlled HIV replication ("controllers") as cases (n = 73) and those who did not control replication as controls (n = 100). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, direct DNA sequencing, and TaqMan genotyping assays. In Caucasian Americans, certain combinations of FcγR and GM genotypes were differentially distributed between controllers and noncontrollers. Among the noncarriers of the FcγRIIa arginine allele, GM21 noncarriers had over 7-fold greater odds of being controllers than the carriers of this allele (odds ratio [OR] = 7.47). These GM determinants also interacted with FcγRIIIa alleles. Among the carriers of the FcγRIIIa valine allele, GM21 noncarriers had over 3-fold greater odds of being controllers than the carriers of this allele (OR = 3.26). These results demonstrate epistatic interactions of genes on chromosomes 14 (GM) and 1 (FcγR) in influencing the control of HIV replication.     

Genetic association between the HIF-1α P582S polymorphism and cervical cancer risk: a meta analysis

Background: Hypoxia-inducible factor-1 alpha (HIF-1α) P582S polymorphism has been reported to increase transactivation capacity of HIF-1α, which is prone to tumorigenesis. Several published case-control studies on the association between P582S polymorphism and cervical cancer have shown mixed results. In this study, we chose to perform a meta-analysis to assess the association. Methodology/Principal findings: We conducted a meta-analysis consisting of four studies with a total of 846 cases and 991 controls. All data were collected and overall comparison was performed among all subjects. Using the fi xed effects model, the homozygous and the recessive models showed a significant increase in the risk of cervical cancer (the pooled OR=6.32, 95% CI=2.28-17.55, Phet=0.348; the pooled OR=5.86, 95% CI=2.13-16.11, Phet=0.394 respectively). Publication bias was not significantly indicated in this analysis. Conclusions: This meta-analysis demonstrates that HIF-1α P582S polymorphism may be associated with the risk of cervical cancer.     

FcγRIIB: a modulator of cell activation and humoral tolerance

An immune response needs to be tightly regulated to prevent excessive inflammation, which may result in the destruction of healthy tissues. At the molecular level, the strength of an immune response is determined by the integration of a multitude of positive and negative signals. This review will focus on IgG-dependent immune responses and discuss how the inhibitory receptor FcγRIIB may be involved in regulating both the afferent and efferent phases of such a response. Furthermore, we will discuss recent evidence suggesting that FcγRIIB may have important functions beyond the negative regulation of signals transduced by the B-cell receptor or activating FcγRs and could be responsible for the activity of agonistic antibodies in vivo.     

Influence of FcγRIIIb polymorphism on its ability to cooperate with FcγRIIa and CR3 in mediating the oxidative burst of human neutrophils

Considering that human neutrophil FcγRIIa and FcγRIIIb receptors interact synergistically with CR3 in triggering neutrophil functional responses, allelic polymorphisms in these receptors might influence such interactions. We assessed whether FcγRIIIb polymorphisms affect FcγR/CR cooperation in mediating the neutrophil oxidative burst (OB), in particular the FcγRIIIb/CR3 cooperation that occurs via lectin-saccharide-like interactions. The OB of human neutrophil antigen (HNA)-1a-, HNA-1b-, and HNA-1a/-1b-neutrophils stimulated with immune complexes, opsonized or not with serum complement, was measured by the luminol-enhanced chemiluminescence assay. Compared with HNA-1a-neutrophils, HNA-1b-neutrophils exhibited reduced FcγR-stimulated OB, but increased FcγR/CR-stimulated OB. It suggests that (i) FcγR and CR cooperate more effectively in HNA-1b-neutrophils, and (ii) the HNA-1b allotype influences the FcγRIIIb cooperation with FcγRIIa, but not with CR3. HNA-1a- and HNA-1b-neutrophils exhibited similar OB responses elicited via CR3 alone or via FcγR/CR-independent pathways. In addition, the level of FcγRIIIb, FcγRIIa, and CR3 expression did not differ significantly among the neutrophil groups studied. Together, these results demonstrate that the HNA-1b allotype influences the functional cooperation between FcγRIIIb and FcγRIIa, and suggest that the difference in the glycosylation pattern between HNA-1a and HNA-1b does not affect the FcγRIIIb cooperation with CR3.     

FcγRIIB: a modulator of cell activation and humoral tolerance

An immune response needs to be tightly regulated to prevent excessive inflammation, which may result in the destruction of healthy tissues. At the molecular level, the strength of an immune response is determined by the integration of a multitude of positive and negative signals. This review will focus on IgG-dependent immune responses and discuss how the inhibitory receptor FcγRIIB may be involved in regulating both the afferent and efferent phases of such a response. Furthermore, we will discuss recent evidence suggesting that FcγRIIB may have important functions beyond the negative regulation of signals transduced by the B-cell receptor or activating FcγRs and could be responsible for the activity of agonistic antibodies in vivo.     

Is there a relation between Chlamydia infection and primary biliary cirrhosis?

Over the past two decades, a number of studies have failed to provide direct evidence of specific microbial chronic infection in primary biliary cirrhosis (PBC). However, a recent report suggests that there is a specific association of Chlamydia pneumoniae in patients with PBC and that C. pneumoniae or similar antigens might play a role in the pathogenesis of disease. To determine if Chlamydia infection is associated with PBC, we applied a combination of immunological and molecular approaches to investigate (a) the serological reactivity against two common Chlamydia human pathogens, C. pneumoniae and C. trachomatis, by immunoblotting, (b) the presence of Chlamydia in liver samples of patients with PBC and controls by PCR amplification of Chlamydia specific 16S rRNA and (c) the presence of Chlamydia proteins in liver samples of patients with PBC and controls by immunohistochemical staining. By immunoblotting, C. trachomatis and C. pneumoniae specific serological antibodies were found in 52/57 (91.2%) AMA positive PBC, 7/33 (21/2%) of AMA negative PBC, 1/25 (4%) PSC, 0/15 (0%) Sjorgen's syndrome and 0/20 (0%) systemic lupus erythematosus patients and 0/20 (0%) healthy volunteers at 1:200 sera dilution. PBC sera reacted to Chlamydia and E. coli lysates in western blots up to a maximum of 10(-4) dilution. However, PCR amplification of the Chlamydia specific 16S rRNA gene was negative in 25/25 PBC livers but positive in 1/4 PSC liver, 3/6 in other liver disease controls and 1/4 normal liver samples. While two commercially available specific monoclonal antibodies stained positive controls (Chlamydia infected HEp-2 cells) they failed to detect Chlamydia antigens in PBC livers. The detection of Chlamydia specific antibodies but not Chlamydia rRNA gene and Chlamydia antigens in PBC suggests that Chlamydia infection is not involved in PBC.     

Discrepancy in Dengue Virus Neutralizing Antibody Titers between Plaque Reduction Neutralizing Tests with Fcγ Receptor (FcγR)-Negative and FcγR-Expressing BHK-21 Cells

Protective immunity against dengue virus (DENV) is best reflected by the presence of neutralizing antibodies. The conventional plaque reduction neutralizing test (PRNT) is performed using Fcγ receptor (FcγR)-negative cells. Because FcγR plays a key role in antibody-dependent enhancement, we examined neutralizing antibody titers of mouse monoclonal antibodies and human serum samples in PRNTs using FcγRIIA-negative and FcγRIIA-expressing BHK cells. There was a discrepancy in dengue virus neutralizing antibody titers between PRNTs using FcγRIIA-negative versus FcγRIIA-expressing BHK cells. Neutralizing antibody titers to DENV-1 and DENV-2 tested with monoclonal antibodies, and with most of the human serum samples, were higher in assays using BHK cells than those using FcγRIIA-expressing BHK cells. The results suggest that neutralizing antibody titers determined using FcγRIIA-expressing cells may better reflect the protective capacity of anti-DENV antibodies, as the major target cells of DENV infection are FcγR-positive cells.Dengue virus (DENV), a member of the family Flaviviridae, represents a major health problem in tropical and subtropical regions of the world. There are four serotypes, dengue virus types 1 to 4 (DENV-1 to DENV-4). DENV causes a wide range of symptoms, from mild febrile illness known as dengue fever (DF) to severe life-threatening illness, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Infection with one serotype induces life-long protection against homologous serotypes, but protection against other serotypes is short-lived. In secondary infection, cross-reactive, nonneutralizing antibodies bind to DENV. DENV-antibody complexes are taken up more efficiently by Fcγ receptor (FcγR)-expressing cells, and higher levels of viremia develop (5, 7, 10, 12, 15, 16). This phenomenon, known as antibody-dependent enhancement (ADE), is considered to be a risk factor for DHF and DSS.Protective immunity against DENV is best reflected by the presence of neutralizing antibody. High neutralizing antibody levels induced by primary infection are considered central in offering life-long protective immunity against the homologous serotypes. Thus, a vaccine against DENV infection is expected to induce high levels of neutralizing antibodies against all four serotypes. The plaque reduction neutralizing test (PRNT) is a widely accepted approach to measure the neutralizing activities of antibodies (14). PRNTs, which employ Vero, LLC-MK2, or BHK-21 cells (11, 14) are, however, limited to measuring neutralizing activities of viral infectivity in the absence of FcγR (1). It is possible that neutralizing antibody titers of anti-DENV antibodies induced by natural infection or by vaccines may differ when assayed in the presence of enhancing activity. The neutralizing antibody titers determined using FcγR-expressing BHK-21 cells may better reflect protective immunity, because the principal target cells of DENV are FcγR-expressing cells, such as monocytes (6). In the present study, we sought to determine if neutralizing antibody titers were at the same or different levels when BHK-21 cells and cell lines expressing FcγR were used as the assay cells.     

Might there be a link between mannose-binding lectin polymorphism and dental caries?

The aim of this study is to analyze two polymorphisms in exon 1 of Mannose-binding lectin (MBL) gene in children with carious teeth and children caries-free in order to determine the frequencies of these polymorphisms and to investigate possible association between MBL polymorphisms and dental caries. Fourty-two children with carious teeth and 40 children caries-free participated in the study. Two-hundred microliters of peripheral blood samples were taken in EDTA tubes and genomic DNA was isolated. PCR-RFLP method was used with BanI and MboII digestion enzymes. The overall distribution of genotypes did not significantly differ between two groups and there was also no significant difference in the allele frequency of codon 54 wild type (allele A) (p=0.884, p=0.585). It has been concluded that further investigations may be required to show possible association between MBL and dental caries in which high number healthy children are participated.     

Genetic dissection of a major haplotype associated with arthritis reveal FcγR2b and FcγR3 to act additively

A haplotype with tightly linked Fc gamma receptor (FcγR) genes is known as a major locus controlling immune responses and autoimmune diseases, including arthritis. Here, we split a congenic fragment derived from the NOD mouse (Cia9) to study its effect on immune response and arthritis in mice. We found that arthritis susceptibility was indeed controlled by the FcγR gene cluster and a recombination between the Fc γ R2b and Fc γ R3 loci gave us the opportunity to separately study their impact. We identified the NOD-derived Fc γ R2b and Fc γ R3 alleles as disease-promoting for arthritis development without impact on antibody secretion. We further found that macrophage-mediated phagocytosis was directly correlated to Fc γ R3 expression in the congenic mice. In conclusion, we positioned Fc γ R2b and Fc γ R3 alleles as disease regulatory and showed that their genetic polymorphisms independently and additively control innate immune cell activation and arthritis.     

Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance?

Two carp tumor necrosis factor alpha (TNFalpha) genes have been cloned and sequenced. Both TNF1 and TNF2 sequences have several polymorphisms in the 3' UTR and TNF2 has a polymorphism in the coding sequence. Lipopolysaccharide and the protozoan blood flagellate Trypanoplasma borreli induced expression of TNFalpha in carp head kidney phagocytes when added in vitro. Differential expression was observed, with TNF2 being higher expressed than TNF1. We used the TNFalpha-specific inhibitor pentoxifylline to demonstrate the involvement of carp TNFalpha in the induction of nitric oxide and in the stimulation of cell proliferation. In addition, two carp lines differing in their resistance to T. borreli were typed for the TNF2 polymorphism and association between one isoform and resistance was found.     

FcγRIIa polymorphism and anti-malaria-specific IgG and IgG subclass responses in populations differing in susceptibility to malaria in Burkina Faso

FcγRIIa is known to be polymorphic; and certain variants are associated with different susceptibilities to malaria. Studies involving the Fulani ethnic group reported an ethnic difference in FcγRIIa-R131H genotype frequencies between the Fulani and other sympatric groups. No previous studies have addressed these questions in Burkina Faso. This study aimed to assess the influence of FcγRIIa-R131H polymorphism on anti-falciparum malaria IgG and IgG subclass responses in the Fulani and the Mossi ethnic groups living in Burkina Faso. Healthy adults more than 20 years old belonging to the Mossi or the Fulani ethnic groups were enrolled for the assessment of selected parasitological, immunological and genetic variables in relation to their susceptibility to malaria. The prevalence of the Plasmodium falciparum infection frequency was relatively low in the Fulani ethnic group compared to the Mossi ethnic group. For all tested antigens, the Fulani had higher antibody levels than the Mossi group. In both ethnic groups, a similar distribution of FcγRIIa R131H polymorphism was found. Individuals with the R allele of FcγRIIa had higher antibody levels than those with the H allele. This study confirmed that malaria infection affected less the Fulani group than the Mossi group. FcγRIIa-R131H allele distribution is similar in both ethnic groups, and higher antibody levels are associated with the FcγRIIa R allele compared to the H allele.     

No association between atopic asthma and a coding variant of FcεR1β in a Japanese population

Susceptibility to atopic diseases is known to involve genetic factors. The Gly237 allele of a polymor-phism (Glu237Gly) of the FcεR1β gene is reportedly associated with atopic asthma in Japanese. To confirm this association, we conducted transmission disequilibrium tests in 76 families identified through atopic asthmatics. A case-control study was also carried out in atopic asth-matic subjects and non-atopic controls. The Gly237 allele was not preferentially transmitted to atopic asthma-affected offspring. Neither the Gly237 allele nor the Gly237/Gly237 + Glu237/Gly237 genotypes were significantly more prevalent in the atopic asthmatics than in the controls. This study failed to confirm a substantial role of the Gly237Glu polymorphism of the FcεR1β gene in the genetic predisposition for atopic asthma in this Japanese population. Received: February 22, 1999 / Accepted: April 16, 1999     

A novel FCGR3A intragenic haplotype is associated with increased FcγRIIIa/CD16a cell surface density and population differences

FcγRIIIa (CD16a) cell surface density affects immune complex binding and initiation of effector mechanisms. Investigations into the clinical relevance of variable FcγRIIIa surface density require baseline data from healthy individuals. In this study, proportions of FcγRIIIa-positive leukocyte subsets and corresponding FcγRIIIa cell surface densities were determined in whole blood from 53 healthy individuals (22 Black individuals, 31 Caucasians). Compared to Caucasians, Black individuals had significantly lower proportions of FcγRIIIa-positive natural killer (NK) cells (95.2% vs. 96.9%) and CD8⁺ T lymphocytes (9.6% vs. 11.7%), whereas the opposite was true for monocytes (24.2% vs. 16.3%). However, Black individuals had significantly lower FcγRIIIa surface densities on monocytes and NK cells compared to Caucasians (P < 0.001). We investigated FCGR3A gene copy number and novel polymorphisms, obtained from full gene sequencing, in relation to FcγRIIIa expression levels on NK cells. The broad range of FcγRIIIa surface densities was not attributed to variable FCGR3A gene copy number (all individuals had 2 gene copies except for 2/53 (3.8%) with one extra copy). However, a novel 3-SNP/1-indel FCGR3A intragenic haplotype may account for the significantly increased FcγRIIIa surface densities (P < 0.0001) and may explain the population differences. This genetic determinant may serve as predictive marker for a high-expressing FcγRIIIa phenotype.     

High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

PurposeHost genetic variants in activating natural killer (NK) cell receptors may contribute to differences in severity of COVID-19. NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) responses play, however, a controversial role in SARS-CoV-2 infections. It is unclear whether proinflammatory and cytotoxic SARS-CoV-2-specific ADCC responses limit disease severity or rather contribute to the immunopathogenesis of severe COVID-19.MethodsUsing a genetic association approach and subsequent in vitro antibody-dependent NK cell activation experiments, we investigated whether genetic variants in the FcγRIIIa-encoding FCGR3A gene, resulting in expression of either a low-affinity or high-affinity variant, and individual SARS-CoV-2-specific ADCC response contribute to COVID-19 severity.ResultsIn our study, we showed that the high-affinity variant of the FcγRIIIa receptor, 158-V/V, is significantly over-represented in hospitalized and deceased patients with COVID-19, whereas the low-affinity FcγRIIIa-158-F/F variant occurs more frequently in patients with mild COVID-19 (P < .0001). Furthermore, functional SARS-CoV-2 antibody-specific NK cell-mediated ADCC assays revealed that significantly higher proinflammatory ADCC responses occur in hospitalized patients with COVID-19, and are especially observed in NK cells expressing the FcγRIIIa-158-V/V variant (P < .0001).ConclusionOur study provides evidence that pronounced SARS-CoV-2-specific NK cell-mediated ADCC responses are influenced by NK cell FcγRIIIa genetic variants and are a hallmark of severe COVID-19.     

Detecting chronotype differences associated to latitude: a comparison between Horne--Östberg and Munich Chronotype questionnaires

Background: Chronotype, phase preference to perform activities during a 24-hour day, represents distinct circadian temporal organization of living organisms. Morning and evening types can be identified by questionnaires such as Horne and Östberg (HO) and Munich Chronotype Questionnaire (MCTQ). Environmental factors, such as different light–dark cycles experienced at different latitudes, interact with the organisms’ circadian timekeeping system. Therefore, chronotype is expected to vary as a result of different geographical locations.

Aim: To identify differences in chronotype distribution in populations of two Brazilian cities, Natal and Sao Paulo, located at different latitudes.

Subjects and methods: Two specific questionnaires, the Horne and Östberg Questionnaire (HO) and the Munich Chronotype Questionnaire (MCTQ), were used to identify chronotypes of undergraduate students from São Paulo and Natal.

Results: The comparison of the curve distributions of HO and MCTQ scores between both cities allowed one to observe that, while HO curves of São Paulo and Natal overlapped, MCTQ curves showed a clear shift towards eveningness in São Paulo.

Conclusion: This experiment confirmed results from previous studies that the farther away from the equator, the longer the delay of the sleep phase. It was also concluded that MCTQ is better at detecting this phenomenon.