Subclass Specificity Research Articles

Integrating Demographics and Imaging Features for Various Stages of Dementia Classification: Feed Forward Neural Network Multi-Class Approach.

MRI magnetization-prepared rapid acquisition (MPRAGE) is an easily available imaging modality for dementia diagnosis. Previous studies suggested that volumetric analysis plays a crucial role in various stages of dementia classification. In this study, volumetry, radiomics and demographics were integrated as inputs to develop an artificial intelligence model for various stages, including Alzheimer's disease (AD), mild cognitive decline (MCI) and cognitive normal (CN) dementia classifications. The Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset was separated into training and testing groups, and the Open Access Series of Imaging Studies (OASIS) dataset was used as the second testing group. The MRI MPRAGE image was reoriented via statistical parametric mapping (SPM12). Freesurfer was employed for brain segmentation, and 45 regional brain volumes were retrieved. The 3D Slicer software was employed for 107 radiomics feature extractions from within the whole brain. Data on patient demographics were collected from the datasets. The feed-forward neural network (FFNN) and the other most common artificial intelligence algorithms, including support vector machine (SVM), ensemble classifier (EC) and decision tree (DT), were used to build the models using various features. The integration of brain regional volumes, radiomics and patient demographics attained the highest overall accuracy at 76.57% and 73.14% in ADNI and OASIS testing, respectively. The subclass accuracies in MCI, AD and CN were 78.29%, 89.71% and 85.14%, respectively, in ADNI testing, as well as 74.86%, 88% and 83.43% in OASIS testing. Balanced sensitivity and specificity were obtained for all subclass classifications in MCI, AD and CN. The FFNN yielded good overall accuracy for MCI, AD and CN categorization, with balanced subclass accuracy, sensitivity and specificity. The proposed FFNN model is simple, and it may support the triage of patients for further confirmation of the diagnosis.

Deep Learning-Based Interpretable AI for Prostate T2W MRI Quality Evaluation

Prostate MRI quality is essential in guiding prostate biopsies. However, assessment of MRI quality is subjective with variation. Quality degradation sources exert varying impacts based on the sequence under consideration, such as T2W versus DWI. As a result, employing sequence-specific techniques for quality assessment could yield more advantageous outcomes. This study aims to develop an AI tool that offers a more consistent evaluation of T2W prostate MRI quality, efficiently identifying suboptimal scans while minimizing user bias. This retrospective study included 1046 patients from threecohorts (ProstateX [n=347], All-comer in-house [n=602], enriched bad-quality MRI in-house [n=97]) scanned between January 2011 and May 2022. An expert reader assigned T2W MRIs a quality score. A train-validation-test split of 70:15:15 was applied, ensuring equal distribution of MRI scanners and protocols across all partitions. T2W quality AI classification model was based on 3D DenseNet121 architecture using MONAI framework. In addition to multiclassification, binary classification was utilized (Classes 0/1 vs. 2). A score of 0 was given to scans considered non-diagnostic or unusable, a score of 1 was given to those with acceptable diagnostic quality with some usability but with some quality distortions present, and a score of 2 was given to those considered optimal diagnostic quality and usability. Partial occlusion sensitivity maps were generated for anatomical correlation. Three body radiologists assessed reproducibility within a subgroup of 60 test cases using weighted Cohen Kappa. The best validation multiclass accuracy of 77.1% (121/157) was achieved during training. In the test dataset, multiclassification accuracy was 73.9% (116/157), whereas binary accuracy was 84.7% (133/157). Sub-class sensitivity for binary quality distortion classification for class 0 was 100% (18/18), and sub-class specificity for T2W classification of absence/minimal quality distortions for class 2 was 90.5% (95/105). All three readers showed moderate to substantial agreement with ground truth (R1-R3 κ=0.588, κ=0.649, κ=0.487, respectively), moderate to substantial agreement with each other (R1-R2 κ=0.599, R1-R3 κ=0.612, R2-R3 κ=0.685), fair to moderate agreement with AI (R1-R3 κ=0.445, κ=0.410, κ=0.292, respectively). AI showed substantial agreement with ground truth (κ=0.704). 3D quality heatmap evaluation revealed that the most critical non-diagnostic quality imaging features from an AI perspective related to obscuration of the rectoprostatic space (94.4%, 17/18). The 3D AI model can assess T2W prostate MRI quality with moderate accuracy and translate whole sequence-level classification labels into 3D voxel-level quality heatmaps for interpretation. Image quality has a significant downstream impact on ruling out clinically significant cancers. AI may be able to help with reproducible identification of MRI sequences requiring re-acquisition with explainability.

The Size of a Thyroid Nodule with ACR TI-RADS Does Not Provide a Better Prediction of the Nature of the Nodule: A Single-Center Retrospective Real-Life Observational Study.

We investigated if thyroid nodule size has a predictive value of malignancy on a par with composition, echogenicity, shape, margin, and echogenic foci, and what would be the consequence of observing the rule of the American College of Radiology (ACR) to perform a fine-needle aspiration biopsy (FNAB). We conducted a retrospective real-life observational study on 86 patients who underwent surgery after a standardized diagnostic protocol. We divided the TR3, TR4, and TR5 classes into sub-classes according to the size threshold indicating FNAB (a: up to the threshold for no FNAB; b: over the threshold for FNAB suggested). We computed sensitivity, specificity, and positive (PPV) and negative predictive value (NPV) for the different sub-classes and Youden's index (Y) for the different possible cutoffs. Each sub-class showed the following PPV (0.67, 0.68, 0.70, 0.78, 0.72), NPV (0.56, 0.54, 0.51, 0.52, 0.59), and Y (0.20, 0.20, 0.22, 0.31, 0.30). In this real-life series, we did not find a significant difference in prediction of malignancy between the sub-categories according to the size threshold. All nodules have a pre-evaluation likelihood of being malignant, and the impact and utility of size thresholds may be less clear than suggested by the ACR TIRADS guidelines in patients undergoing standardized thyroid work up.

Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (Solanum lycopersicum).

The post-translational import of nuclear-encoded chloroplast preproteins is critical for chloroplast biogenesis, and the Toc159 family of proteins is the receptor for this process. Our previous work identified and analyzed the Toc GTPase in tomato; however, the tomato-specific transport substrate for Toc159 is still unknown, which limits the study of the function of the TOC receptor in tomato. In this study, we expand the number of preprotein substrates of slToc159 receptor family members using slToc159-1 and slToc159-2 as bait via a split-ubiquitin yeast two-hybrid membrane system. Forty-one specific substrates were identified in tomato for the first time. Using slToc159-1GM and slToc159-2GM as bait, we compared the affinity of the two bait proteins, with and without the A domain, to the precursor protein, which suggested that the A domain endowed the proproteins with subclass specificity. The presence of the A domain enhanced the interaction intensity of slToc159-1 with the photosynthetic preprotein but decreased the interaction intensity of slToc159-2 with the photosynthetic preprotein. Similarly, the presence of the A domain also altered the affinity of slToc159 to non-photosynthetic preproteins. Bimolecular fluorescence complementation (BiFC) analysis showed that A domain had the ability to recognize the preprotein, and the interaction occurred in the chloroplast. Further, the localization of the A domain in Arabidopsis protoplasts showed that the A domain did not contain chloroplast membrane targeting signals. Our data demonstrate the importance of a highly non-conserved A domain, which endows the slToc159 receptor with specificity for different protein types. However, the domain containing the information on targeting the chloroplast needs further study.

B cells defined by immunoglobulin isotypes.

The ability of B cells to generate antibodies and provide long-lived protective immunity is the cornerstone of vaccination and has contributed to the success of modern medicine. The nine different antibody subclasses produced by humans have effector functions that differ according to antigen type and route of exposure. Expression of the appropriate isotype is critical for effective humoral immunity, and it is becoming clear that subclass specificity is to some extent reflected at the cellular level. Understanding the mechanisms that govern the induction, expansion, and maintenance of B cells expressing different antibody subclasses informs the strategic manipulation of responses to benefit human health. This article provides an overview of the mechanisms by which the different human antibody subclasses regulate immunity, presents an update on how antibody subclass expression is regulated at the cellular level and highlights key areas for future research.

Insufficient Anthrax Lethal Toxin Neutralization Is Associated with Antibody Subclass and Domain Specificity in the Plasma of Anthrax-Vaccinated Individuals.

Anthrax vaccine adsorbed (AVA) is a significant line of defense against bioterrorist attack from Bacillus anthracis spores. However, in a subset of individuals, this vaccine may produce a suboptimal quantity of anti-protective antigen (PA), antibodies that are poorly neutralizing, and/or antibody titers that wane over time, necessitating annual boosters. To study individuals with such poor responses, we examine the properties of anti-PA in a subset of vaccinated individuals that make significant quantities of antibody but are still unable to neutralize toxin. In this cohort, characterized by poorly neutralizing antibody, we find that increased IgG4 to IgG1 subclass ratios, low antibody avidity, and insufficient antibody targeting domain 4 associate with improper neutralization. Thus, future vaccines and vaccination schedules should be formulated to improve these deficiencies.

An integrated multi-objective whale optimized support vector machine and local texture feature model for severity prediction in subjects with cardiovascular disorder.

The left ventricle (LV) myocardium undergoes deterioration with the reduction in ejection fraction (EF). The analysis of its texture pattern plays a major role in diagnosis of heart muscle disease severity. Hence, a classification framework with co-occurrence of local ternary pattern feature (COALTP) and whale optimization algorithm has been attempted to improve the prediction accuracy of disease severity level. This analysis is carried out on 600 slices of 76 participants from Kaggle challenge that include subjects with normal and reduced EF. The myocardium of LV is segmented using optimized edge-based local Gaussian distribution energy (LGE)-based level set, and end-diastolic and end-systolic volumes were calculated. COALTP is extracted for two distance levels (d = 1 and 2). The t-test has been performed between the features of individual binary classes. The features are ranked using feature ranking methods. The experiments have been performed to analyze the performance of various percentages of features in each combination of bin for fivefold cross-validation. An integrated whale optimized feature selection and multi-classification framework is developed to classify the normal and pathological subjects using CMR images, and DeLong test has been performed to compare the ROCs. The optimized edge embedded to level set has produced better segmented myocardium that correlates with R = 0.98 with gold standard volume. The t-test shows that texture features extracted from severe subjects with distance level "1" are more statistically significant with a p value (< 0.00004) compared to other pathologies. This approach has produced an overall multi-class accuracy of 75% [confidence interval (CI) 63.74-84.23%] and effective subclass specificity of 70% (CI 55.90-81.22%). The obtained results show that the multi-objective whale optimized multi-class support vector machine framework can effectively discriminate the healthy and patients with reduced ejection fraction and potentially support the treatment process.

Evaluation of anti-gal enzyme-linked immunosorbent assay for the diagnosis of Indian post-kala-azar dermal leishmaniasis.

Elimination of kala azar from India is challenging as there are potential reservoirs of Leishmania donovani in patients with post-kala-azar dermal leishmaniasis (PKDL). The vast repertoire of carbohydrate moieties on L. donovani is known to elicit specific and strong humoral responses in patients with kala azar. The present study was undertaken to evaluate the diagnostic performances of anti-gal antibodies using enzyme-linked immunosorbent assay for successful serological diagnosis of PKDL in Indian patients and to differentiate cases of past cured visceral leishmaniasis infections. We developed Gal enzyme-linked immunosorbent assay to measure specific anti-gal IgG isotype in the sera of 71 Indian patients with PKDL. The diagnostic efficacy of the newly developed assay was evaluated for precision, sensitivity and accuracy. Gal2 enzyme-linked immunosorbent assay revealed three-fold increased anti-gal titers in 71 patients with active PKDL compared to controls. Subclass enzyme-linked immunosorbent assay analysis further revealed enhanced IgG2 and IgG3 anti-gal titers in patients with PKDL compared to control subjects. The rank order for specificity and sensitivity for IgG subclasses was IgG3>IgG2>IgG4>IgG1. The area under the curve values of 0.98 and 0.99 were obtained for IgG and IgG3 Gal2 enzyme-linked immunosorbent assays respectively. Overall sensitivity and specificity were 95.7% (95% CI: 88.1-99.1) and 98.1% (95% confidence interval: 90.1-99.9), and 98.5% (95% CI: 92.4-99.9) and 98.1% (95% CI: 90.1-99.9), respectively. Intra-assay coefficient of variation was 1.5% and inter-assay coefficient of variation was 11.7%. The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. Taken together, anti-gal titers detected through Gal2 enzyme-linked immunosorbent assay can serve as an effective diagnostic tool in disease elimination setting and help in better case management in endemic districts.

Genome-Wide Association Study on Immunoglobulin G Glycosylation Patterns.

Immunoglobulin G (IgG), a glycoprotein secreted by plasma B-cells, plays a major role in the human adaptive immune response and are associated with a wide range of diseases. Glycosylation of the Fc binding region of IgGs, responsible for the antibody’s effector function, is essential for prompting a proper immune response. This study focuses on the general genetic impact on IgG glycosylation as well as corresponding subclass specificities. To identify genetic loci involved in IgG glycosylation, we performed a genome-wide association study (GWAS) on liquid chromatography electrospray mass spectrometry (LC–ESI-MS)—measured IgG glycopeptides of 1,823 individuals in the Cooperative Health Research in the Augsburg Region (KORA F4) study cohort. In addition, we performed GWAS on subclass-specific ratios of IgG glycans to gain power in identifying genetic factors underlying single enzymatic steps in the glycosylation pathways. We replicated our findings in 1,836 individuals from the Leiden Longevity Study (LLS). We were able to show subclass-specific genetic influences on single IgG glycan structures. The replicated results indicate that, in addition to genes encoding for glycosyltransferases (i.e., ST6GAL1, B4GALT1, FUT8, and MGAT3), other genetic loci have strong influences on the IgG glycosylation patterns. A novel locus on chromosome 1, harboring RUNX3, which encodes for a transcription factor of the runt domain-containing family, is associated with decreased galactosylation. Interestingly, members of the RUNX family are cross-regulated, and RUNX3 is involved in both IgA class switching and B-cell maturation as well as T-cell differentiation and apoptosis. Besides the involvement of glycosyltransferases in IgG glycosylation, we suggest that, due to the impact of variants within RUNX3, potentially mechanisms involved in B-cell activation and T-cell differentiation during the immune response as well as cell migration and invasion involve IgG glycosylation.

IgG Subclass Specificity Discriminates Restricted IgM Rheumatoid Factor Responses From More Mature Anti-Citrullinated Protein Antibody-Associated or Isotype-Switched IgA Responses.

To investigate the presence and patterns of specific IgG subclass recognition by IgM rheumatoid factor (IgM-RF) and IgA-RF with a newly developed enzyme-linked immunosorbent assay (ELISA), which can discriminate between polyspecific and restricted RF responses. Polyspecific and restricted RF responses were determined with our ELISA, which uses individually coated recombinant IgG subclasses instead of polyclonal IgG as target antibodies. Fine specificity was determined using target antibodies with single amino acid mutations in the Fc region. In a screening panel of 93 sera that were previously found to be IgM-RF positive in a conventional RF assay, we were able to discriminate between sera with polyspecific IgM-RF responses (i.e., RF responses directed against all 4 IgG subclasses) and those with restricted IgM-RF responses, with low or absent relative reactivity against IgG2, IgG3, or IgG4. We found the largest variation for anti-IgG3 reactivity. Samples without detectable anti-IgG4 reactivity formed an independent group from the other restricted RF responses and the polyspecific RF responses. The specificity of these anti-IgG4-negative sera could be pinpointed to single amino acid differences between IgG1 and IgG4. Polyspecific RF responses more often showed signs of RF response maturation, with more isotype switching to IgA-RF, as compared to restricted RF responses. In a cohort of IgM-RF+ and/or anti-citrullinated protein antibody (ACPA)-positive arthralgia patients, we found restricted RF responses in 35% (49 of 140) of RF+/ACPA- patients, while RF+/ACPA+ patients, who have a much higher risk of developing rheumatoid arthritis, virtually always (123 of 128 [96%]) showed a polyspecific RF response. IgG subclass-specific RF distinguishes between immature restricted RF responses and potentially more pathogenic, ACPA-associated polyspecific responses.

IgG subclass specificity to C1q determined by surface plasmon resonance using Protein L capture technique

Recombinant monoclonal antibodies (mAbs) have become an important category of biological therapeutics. mAbs share the same structures and biological functions as endogenous IgG molecules. One function is complement-dependent cytotoxicity (CDC) initiation by binding of C1q. Traditionally, ELISA methods have been utilized to measure C1q binding. A new robust capture method was established in this study to measure the binding affinity of C1q to antibodies by surface plasmon resonance (SPR). The utility of this method was demonstrated by determination of the difference in IgG subclass specificity of C1q binding.

Abstract 4245: A comparative analysis of PI3K inhibitors in human tumor primary culture microspheroids

Abstract Altered phosphoinosotide metabolism is a hallmark of many human malignances. The phosphoinsotide kinases (PI3K) of which there are several sub-classes, have been explored as targets for cancer therapy. Beyond the first generation pan-PI3K inhibitors Wortmannin &amp; LY294002, new, clinically active agents with subclass specificity and/or dual inhibitory functions (PI3K &amp; mTOR) have been developed, including BEZ235 (PI3K/mTOR), GDC0941(αβδ), BYL719(α) &amp; Idelalisib (δ). To examine the activity of these agents we applied Ex Vivo Analysis of Programmed Cell Death (EVA/PCD) (Nagourney, R et al Anticancer Res., 2012)to human tumor microspheroids, isolated directly from surgical specimens. Lethal concentrations (LC50's) interpolated from 5-point dose response curves were compared for activity and for correlation by Pearson Moment. Results: Micro-molar activity favored BEZ &amp;gt; BYL &amp;gt; LY. Correlation coefficients reveal BYL vs. BEZ (N=15, R =0.69, P&amp;lt; 0.01); BYL vs. LY (N=7, R=0.74, P=0.1); BEZ vs. LY (N=61, R=0.52, P&amp;lt;0.001). A comparison of activity for hematologic (Heme) vs. Solid tumors (solid) for LY: Heme &amp;gt; Solid; BEZ: Solid &amp;gt; Heme; BYL: Heme &amp;gt; Solid with preliminary evidence for Idelalisib showing Heme &amp;gt; Solid. Conclusions: The PI3K inhibitors are active in both Heme and Solid tumors, with relative concordance between agents, but some evidence of disease specificity. Additional comparisons with GDC 0941 and drug combination studies are being conducted to further characterize the clinical activity and combinatorial potential of this class of agents. EVA/PCD analyses, conducted in human tumor primary cultures, have the capacity to evaluate drug activity, synergy and sequence dependence offering clinically relevant insights into novel drug applications. Citation Format: Robert Alan Nagourney, Paula J. Bernard, Federico Francisco, Steven S. Evans. A comparative analysis of PI3K inhibitors in human tumor primary culture microspheroids. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4245. doi:10.1158/1538-7445.AM2014-4245

A split-ubiquitin yeast two-hybrid screen to examine the substrate specificity of atToc159 and atToc132, two Arabidopsis chloroplast preprotein import receptors.

Post-translational import of nucleus-encoded chloroplast pre-proteins is critical for chloroplast biogenesis, and the Toc159 family of proteins serve as receptors for the process. Toc159 shares with other members of the family (e.g. Toc132), homologous GTPase (G−) and Membrane (M−) domains, but a highly dissimilar N-terminal acidic (A−) domain. Although there is good evidence that atToc159 and atToc132 from Arabidopsis mediate the initial sorting step, preferentially recognizing photosynthetic and non-photosynthetic preproteins, respectively, relatively few chloroplast preproteins have been assigned as substrates for particular members of the Toc159 family, which has limited the proof for the hypothesis. The current study expands the number of known preprotein substrates for members of the Arabidopsis Toc159 receptor family using a split-ubiquitin membrane-based yeast two-hybrid system using the atToc159 G-domain (Toc159G), atToc132 G-domain (Toc132G) and atToc132 A- plus G-domains (Toc132AG) as baits. cDNA library screening with all three baits followed by pairwise interaction assays involving the 81 chloroplast preproteins identified show that although G-domains of the Toc159 family are sufficient for preprotein recognition, they alone do not confer specificity for preprotein subclasses. The presence of the A-domain fused to atToc132G (Toc132AG) not only positively influences its specificity for non-photosynthetic preproteins, but also negatively regulates the ability of this receptor to interact with a subset of photosynthetic preproteins. Our study not only substantiates the fact that atToc132 can serve as a receptor by directly binding to chloroplast preproteins but also proposes the existence of subsets of preproteins with different but overlapping affinities for more than one member of the Toc159 receptor family.

Identification, Biochemical and Structural Evaluation of Species-Specific Inhibitors against Type I Methionine Aminopeptidases

Methionine aminopeptidases (MetAPs) are essential enzymes that make them good drug targets in cancer and microbial infections. MetAPs remove the initiator methionine from newly synthesized peptides in every living cell. MetAPs are broadly divided into type I and type II classes. Both prokaryotes and eukaryotes contain type I MetAPs, while eukaryotes have additional type II MetAP enzyme. Although several inhibitors have been reported against type I enzymes, subclass specificity is scarce. Here, using the fine differences in the entrance of the active sites of MetAPs from Mycobacterium tuberculosis , Enterococcus faecalis , and human, three hotspots have been identified and pyridinylpyrimidine-based molecules were selected from a commercial source to target these hotspots. In the biochemical evaluation, many of the 38 compounds displayed differential behavior against these three enzymes. Crystal structures of four selected inhibitors in complex with human MetAP1b and molecular modeling studies provided the basis for the binding specificity.

Differential distribution of phospholipase C beta isoforms and diaglycerol kinase-beta in rodents cerebella corroborates the division of unipolar brush cells into two major subtypes

Sublineage diversification of specific neural cell classes occurs in complex as well as simply organized regions of the central and peripheral nervous systems; the significance of the phenomenon, however, remains insufficiently understood. The unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons that occur at high density in vestibulocerebellum. As they are classified into subsets that differ in chemical phenotypes, intrinsic properties, and lobular distribution, they represent a valuable neuronal model to study subclass diversification. In this study, we show that cerebellar UBCs of adult rats and mice form two subclasses-type I and type II UBCs-defined by somatodendritic expression of calretinin (CR), mGluR1α, phospholipases PLCβ1 and PLCβ4, and diacylglycerol kinase-beta (DGKβ). We demonstrate that PLCβ1 is associated only with the CR(+) type I UBCs, while PLCβ4 and DGKβ are exclusively present in mGluR1α(+) type II UBCs. Notably, all PLCβ4(+) UBCs, representing about 2/3 of entire UBC population, also express mGluR1α. Furthermore, our data show that the sum of CR(+) type I UBCs and mGluR1α(+) type II UBCs accounts for the entire UBC class identified with Tbr2 immunolabeling. The two UBC subtypes also show a very different albeit somehow overlapping topographical distribution as illustrated by detailed cerebellar maps in this study. Our data not only complement and extend the previous knowledge on the diversity and subclass specificity of the chemical phenotypes within the UBC population, but also provide a new angle to the understanding of the signaling networks in type I and type II UBCs.

Development of the Corticothalamic Projections

In this review we discuss recent advances in the understanding of corticothalamic axon guidance; patterning of the early telencephalon, the sequence and choreography of the development of projections from subplate, layers 5 and 6. These cortical subpopulations display different axonal outgrowth kinetics and innervate distinct thalamic nuclei in a temporal pattern determined by cortical layer identity and subclass specificity. Guidance by molecular cues, structural cues, and activity-dependent mechanisms contribute to this development. There is a substantial rearrangement of the corticofugal connectivity outside the thalamus at the border of and within the reticular thalamic nucleus, a region that shares some of the characteristics of the cortical subplate during development. The early transient circuits are not well understood, nor the extent to which this developmental pattern may be driven by peripheral sensory activity. We hypothesize that transient circuits during embryonic and early postnatal development are critical in the matching of the cortical and thalamic representations and forming the cortical circuits in the mature brain.

Distinct patterns of blood-stage parasite antigens detected by plasma IgG subclasses from individuals with different level of exposure to Plasmodium falciparum infections

BackgroundIn endemic regions naturally acquired immunity against Plasmodium falciparum develops as a function of age and exposure to parasite infections and is known to be mediated by IgG. The targets of protective antibodies remain to be fully defined. Several immunoepidemiological studies have indicated an association of cytophilic anti-parasite IgG with protection against malaria. It has been hypothesized that the initial antibody responses against parasite antigens upon first few Plasmodium falciparum infections is dominated by non-protective IgG2/IgG4 and IgM antibodies, which then gradually develop into protective response dominated by cytophilic IgG1 and IgG3 antibodies.MethodsNaturally occurring IgG antibodies against P. falciparum blood-stage antigens were analysed from plasma samples collected from four groups of individuals differing in age and level of exposure to P. falciparum infections. Western Blot profiling of blood-stage parasite antigens displaying reactivity with individual plasma samples in terms of their subclass specificities was conducted. Parasite antigens detected by IgG were grouped based on their apparent molecular sizes resolved by SDS-PAGE as high molecular weight (≥ 70 kDa) or low molecular weight (< 70 kDa). The number of discernable low molecular weight parasite antigens detected by different IgG subclass antibodies from each plasma sample was recorded. Using Wilcoxons rank sum test these reactivities were compared amongst groups of individuals with different levels of exposure to P. falciparum infections.ResultsIgG4 and IgM antibodies in plasma samples from all groups detected very few parasite antigens. IgG2 antibodies from all groups detected a common pattern of high molecular weight parasite antigens. Cytophilic IgG subclasses in plasma samples from individuals with higher levels of exposure to P. falciparum infections distinctly detected higher numbers of low molecular weight parasite antigens.ConclusionsIn the present study, there was no evidence for switching of antibody responses from non-cytophilic to cytophilic subclasses against blood-stage parasite antigens as a likely mechanism for induction of protective immunity against malaria.

Cloning and characterization of ovine immunoglobulin G Fc receptor III (FcγRIII)

Receptors for the Fc regions of immunoglobin G (IgG) play a critical role in immunoregulation and immune defenses against pathogens. In this study, we describe the cloning, eukaryotic expression and IgG subclass specificity of ovine Fc gamma receptor III (FcγRIII). The newly cloned ovine FcγRIII cDNA contains a 940 bp open-reading frame (ORF), and is predicted to encode a 250 amino acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a short cytoplasmic tail. The overall identity of the ovine FcγRIII amino acid sequence to its cattle, pig and human counterparts was 83.2%, 62.0%, 60.7%, respectively. Overlapping PCR was performed with the extracellular domain of ovine FcγRIII and the transmembrane and intracellular region of ovine Fc gamma chain to construct a chimeric receptor. Rosetting analysis showed that transfected COS-7 cells required Fc receptor gamma chain for the expression of FcγRIII on the surface. COS-7 cells expressing FcγRIII were able to bind chicken erythrocytes sensitized with ovine IgG1, but not IgG2. Identification of ovine FcγRIII will further our understanding of the ovine immune system.

Genetic characterization and ligand specificity of the ovine Fc gamma receptor I (ovFcγRІ)

Receptors for the Fc region of IgG (FcγRs) play a critical role in the immune system and host protection against infection. In this study, we describe the cloning, mRNA expression and IgG subclass specificity of ovine Fc gamma receptor I (ovFcγRI). The ovFcγRI cDNA contains a 1047 bp open-reading frame, and is predicted to encode a 349 amino acid trans-membrane glycoprotein composed of three immunoglobulin-like domains, a trans-membrane region and a short cytoplasmic tail. The overall identity of the ovine FcγRI to its cattle, human and mouse counterparts at the level of the amino acid sequence was 92%, 61% and 54%, respectively. Rosetting analysis shows that COS-7 cells were transfected with an expressional vector carrying the cDNA open-reading frame of ovFcγRI and expressed this receptor on the surface. Identification of ovine FcγRI will aid in the understanding of molecular basis of the ovine immune system and further studies of the receptor function.

Autoantibody responses in autoimmune ovarian insufficiency and in Addison's disease are IgG1 dominated and suggest a predominant, but not exclusive, Th1 type of response

Steroid-producing cell autoantibodies (SCAs) directed against 21-hydroxylase autoantibodies (21OHAbs), 17alpha-hydroxylase autoantibodies (17OHAb), and cholesterol side-chain cleavage enzyme (side-chain cleavage autoantibodies, P450sccAb) characterize autoimmune primary ovarian insufficiency (SCA-POI). The aim of the study was to analyze IgG subclass specificity of autoantibodies related to adrenal and ovarian autoimmunity. We studied 29 women with SCA-POI, 30 women with autoimmune Addison's disease (AAD) without POI, and 14 patients with autoimmune polyendocrine syndrome type 1 (APS1). 21OHAb isotypes were also analyzed in 14 subjects with preclinical AAD. Samples from 30 healthy women served as control group to determine the upper level of normality in the isotype assays. Immunoradiometric assays with IgG subclass-specific secondary antibodies. In 21OHAb-positive sera, IgG1 isotype was detected in 90% SCA-POI and non-POI AAD sera and 67% APS1 patients. IgG1 isotype was found in 69% 17OHAb-positive SCA-POI and 100% 17OHAb-positive APS1 sera, and in 60% P450sccAb-positive SCA-POI and 80% P450sccAb-positive APS1 sera. For 21OHAb, IgG4 isotype was detected in 17% SCA-POI, 7% non-POI AAD, and 8% APS1 sera. None of the 17OHAb-positive sera was positive for IgG4. In P450sccAb-positive sera, 15% POI and 20% APS1 sera were positive for IgG4. Two 21OHAb-positive SCA-POI (7%), one 21OHAb-positive AAD (3%), three P450sccAb-positive SCA-POI (15%), and two P450sccAb-positive APS1 (20%) sera were positive for IgG4, in the absence of IgG1. All preclinical AAD sera resulted as positive for IgG1-21OHAb, but not for IgG4-21OHAb. The autoantibody responses in POI and AAD are IgG1 dominated, which suggests a predominant Th1 response. Selective IgG4 isotype specificity identified a small subset of patients with Th2-oriented response.