Levels Of Isotypes Research Articles

Extracellular Acidity-mediated Expression of cPLA2γ Confers Resistance in Gastric Cancer Cells.

Extracellular acidity, a characteristic of solid tumors, has been proposed to be a critical factor for aggravating tumor malignancy and conferring resistance to therapeutics. Recently, acidity has been implicated in inflammatory responses, which are mediated through active lipid metabolites in various human tissues. In the present study, we investigated whether acidity can affect lipid-mediated signaling, and found that phospholipase A2 (PLA2) activity increased at acidic pH in SNU601 and AGS gastric carcinoma cell lines. To identify the PLA2 isoform that is responsible for the acidity-induced activity, we assessed mRNA levels of cPLA2 isotypes through real-time qPCR, and protein levels through immunoblot assay in cells cultured in acidic medium. It was found that acidic pH conditions markedly elevated the PLA2γ expression. A gene interference study using specific siRNA of cPLA2γ suggested that expression of cPLA2γ in acidic culture conditions may be associated with protection of cancer cells in acidic environment, as shown by cell viability and clonogenic assays. In addition, expression of cPLA2γ appeared to confer cell resistance to anticancer drugs under acidic pH conditions. Acidity-induced cPLA2γ expression may exert protective effects by imparting resistance to the gastric cancer cells under acidic environment.

High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 102 CoViD-19 and non-CoViD-19 patients. The SPRi assay simultaneously measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19. This new high throughput method can be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 and its mutants in vaccination programs.•Surface Plasmon Resonance imaging is an unprecedented technology for high throughput screening of antibody profiling of CoViD19 patients.•Fingerprinting of isotypes IgM, IgG and IgA can be performed for 384 patients in one run.•An affinity maturation effect was shown for patients recovering from CoViD19.

Relationship between natural and infection-induced antibodies in systemic autoimmune diseases (SAD): SLE, SSc and RA.

Infection or vaccine-induced T cell-dependent immune response and the subsequent high-affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease-specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine-induced and disease-related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme-linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti-CS) and F4 fragment (anti-F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n=92), rheumatoid arthritis (n=73) and systemic sclerosis (n=157) disease groups. Anti-measles IgG and anti-dsDNA IgG/IgM autoantibodies were measured using commercial and in-house indirect ELISA tests. In all SAD groups the anti-measles IgG-seropositive cases showed significantly higher anti-CS IgG titers (P=0·011). In anti-dsDNA IgG-positive SLE patients, we detected significantly higher levels of anti-CS and anti-F4 IgG nAAbs (P=0·001 and<0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti-CS and anti-F4 nAAbs in anti-dsDNA IgM-positive SLE patients (P=0·002 and 0·016, respectively). The association between IgG isotypes of pathogen- or autoimmune disease-related antibodies and the IgG nAAbs may underscore the immune response-inducible nature of the diseases investigated. The relationship between protective anti-dsDNA IgM and the IgM isotype of anti-F4 and anti-CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.

Neurolisteriosis in a previously asymptomatic patient with serum IgM deficiency: a case report

BackgroundListeria monocytogenes is an opportunistic pathogen of the central nervous system commonly associated with impaired cell-mediated immunity. We hereby present a case of adult neurolisteriosis where the only immunological feature persistently present was serum IgM deficiency, suggesting that non-specific humoral immunity may also play a central role in the control of neuroinvasion by Listeria monocytogenes.Case presentationA 62-year-old male who had never experienced severe infections presented with headache, nuchal rigidity and confusion. Neuroimaging was normal and lumbar puncture revealed pleiocytosis (760 leukocytes/mm3) and hypoglycorrhachia (34 mg/dL). The patient was treated empirically for bacterial meningitis. Indeed, further analysis of the CSF showed infection by Listeria monocytogenes, which was accompanied by reduced serum IgM levels that persisted well beyond the period of acute bacterial infection. Levels of IgG and IgA isotypes, along with peripheral blood counts of major leukocyte subsets, were at the same time largely preserved. Intriguingly, the absence of membrane-bound IgM on B cells was essentially complete in the acute post-infection period leading to a remarkable recovery after 12 months, suggesting that mechanisms other than defective membrane expression are underlying serum deficiency.ConclusionsAs far as we know, this is the first reported case of neurolisteriosis associated with IgM deficiency in an adult individual without a history of severe infections or other underlying conditions. A possible role of circulating IgM against invasive disease caused by Listeria monocytogenes, particularly in the early course of host-pathogen interaction, is discussed.

Modulation of the Systemic Immune Response in Suckling Rats by Breast Milk TGF-β2, EGF and FGF21 Supplementation.

Breast milk is a rich fluid containing bioactive compounds such as specific growth factors (GF) that contribute to maturation of the immune system in early life. The aim of this study was to determine whether transforming growth factor-β2 (TGF-β2), epidermal growth factor (EGF) and fibroblast growth factor 21 (FGF21), compounds present in breast milk, could promote systemic immune maturation. For this purpose, newborn Wistar rats were daily supplemented with these GF by oral gavage during the suckling period (21 days of life). At day 14 and 21 of life, plasma for immunoglobulin (Ig) quantification was obtained and spleen lymphocytes were isolated, immunophenotyped and cultured to evaluate their ability to proliferate and release cytokines. The main result was obtained at day 14, when supplementation with EGF increased B cell proportion to reach levels observed at day 21. At the end of the suckling period, all GF increased the plasma levels of IgG1 and IgG2a isotypes, FGF21 balanced the Th1/Th2 cytokine response and both EGF and FGF21 modified splenic lymphocyte composition. These results suggested that the studied milk bioactive factors, mainly EGF and FGF21, may have modulatory roles in the systemic immune responses in early life, although their physiological roles remain to be established.

High TUBB2A expression in childhood T‐ALL is correlated with the clinical outcome

AbstractIntroductionMicrotubules are polymers that perform functions such as mitosis, intracellular transport, cell morphology, and ciliary and flagellar motility. Since microtubules are taking active part in cell division, they are among direct targets of several antimitotic drugs.MethodsExpression levels of tubulin isotypes were analyzed in microarray data of childhood diagnostic T‐ALL samples (n = 31) and healthy thymocytes (n = 7). Findings were validated with qPCR in separate T‐ALL cohort (n = 48), and clinical correlation analyses were performed. TUBB2A's effects were tested with siRNA‐mediated knockdown in MOLT4 cell line, and apoptosis assay was carried out at 24, 48, and 72 hours time points.ResultsIn microarray data, TUBB2A was found to be the only differentially expressed tubulin isotype (adj. P value = .01), which was validated by qPCR (P = .02). Samples representing differentiation stages of T cell showed an increasing trend of TUBB2A toward mature T‐cell stage. TUBB2A expression was significantly higher in high‐risk group patients (P = .026) and in a group with WBC counts &gt;100 (×109cells/L) (P = .029). High TUBB2A was also found to be a predictor of shorter OS (P = .029) and RFS (P = .042).ConclusionAberrant expression of TUBB isotypes can affect the balance of microtubules or microtubule‐associated proteins, which might lead to drug resistance/relapse. Contribution of cytoskeleton proteins to drug resistance needs further investigation, and understanding aberrant expression and mode of action of microtubules will improve therapy strategies.

Dominant IgM synthesis against the soluble form of the prevailing variant surface glycoprotein from TeAp-N/D1 Trypanosoma equiperdum throughout the experimental acute infections of horses with non-tsetse transmitted Trypanozoon parasites

ABSTRACT Two horses were infected with distinct non-tsetse transmitted Trypanozoon Venezuelan stocks, namely TeAp-N/D1 Trypanosoma equiperdum and TeAp-El Frio01 Trypanosoma evansi. Preceding reports have revealed that a 64-kDa antigenic glycopolypeptide (p64), which is the soluble form of the predominant variant surface glycoprotein from TeAp-N/D1 T. equiperdum, can be used as a good antigen for immunodiagnosis of animal trypanosomosis. Here, the course of the experimental acute infection in both horses was monitored by evaluating total anti-p64 IgG and particular anti-p64 γ-specific IgG and μ-specific IgM isotypes in sera using indirect enzyme-linked immunosorbent assays. Both equines showed a maximum of whole anti-p64 antibody generation, which dropped to readings below the maximum but always above the positive cutoff point. Levels of specific IgG and IgM isotypes oscillated throughout the course of the experiments. Essentially, the γ-specific IgG response remained very close to the cutoff point, whereas the μ-specific IgM response displayed values that were mostly above the positive cutoff point, showing a major peak that coincided with the maximum of complete anti-p64 IgG production. These results showed that horses infected with non-tsetse transmitted Trypanozoon parasites developed an immune reaction characterized by a dominant IgM generation against the p64 antigen.

Association Between Centromere- and Topoisomerase-specific Immune Responses and the Degree of Microangiopathy in Systemic Sclerosis.

Autoreactive antibody responses, including the use of several isotypes of autoantibodies, have been shown to be associated with clinical outcome in several rheumatic autoimmune diseases. The goals of this study were to evaluate whether (1) anticentromere antibody (ACA)- and antitopoisomerase antibody (ATA)-specific isotype expression, and (2) organ involvement are associated with the degree of microangiopathy in systemic sclerosis (SSc). ACA and ATA IgG, IgM, and IgA levels were measured in baseline serum samples of ACA IgG-positive (+) and ATA IgG+ patients with SSc. The degree of microangiopathy was determined based on nailfold videocapillaroscopy (NVC) images collected at the same point in time. Logistic regression analyses with autoantibodies, clinical characteristics, isotype expression, and ACA and ATA IgG, IgM, and IgA levels as independent variables, and NVC pattern as the dependent variable were performed. In 164 patients, isotype levels and degree of microangiopathy were evaluated. Logistic regression confirmed the association of the degree of microangiopathy with the presence of digital ulcers (OR 3.07, 95% CI 1.43-6.60), interstitial lung disease (OR 3.41, 95% CI 1.11-10.61), and pulmonary arterial hypertension (OR 5.58, 95% CI 2.05-17.81). ATA positivity was associated with more severe microangiopathy (OR 2.09, 95% CI 1.05-4.13). Patients who expressed solely ACA IgG showed a trend towards less severe microangiopathy compared to patients also expressing ACA IgM and/or IgA. Levels of ACA IgG and ATA IgM were found to be associated with microangiopathy severity. We observed an association between ACA and ATA responses and the degree of microangiopathy in SSc. These findings might indicate that the breadth of the autoimmune response, as reflected by autoantibody production and microvascular damage, interacts in the pathophysiology of SSc.

Toll-like receptor signaling directly modulates B cell antibody responses

Abstract Toll-like receptor (TLR) signaling alone or in concert with other cellular or molecular signals helps shape responses by different cell types. Murine B lymphocytes express cell surface-bound TLRs 1, 2, 4, 6 and endosomal TLRs 3, 7, 9. This study aims to delineate the effects of these TLRs on B cell responses including proliferation, activation and antibody class-switch recombination (CSR). We stimulated mature, naïve B cells in vitro with individual TLR agonists alone or together with T cell help (via ligation of CD40) and the TH2 cytokine interleukin-4 (IL-4). We measured proliferation by CellTrace Violet assay, cell activation by surface expression of B cell activation markers, and analyzed antibody class switching by surface expression and secreted levels of antibody isotypes by flow cytometry. We found that in the presence of a minimal level of anti-CD40, stimulation with TLRs 1/2, 2/6, 4, and 9 agonists induced cell proliferation and up-regulation of activation markers including CD69 and CD80. While both types of TLR agonists down-regulated class switching to IgG1, stimulation of the surface-bound TLRs (1/2, 2/6 and 4) increased class switching to IgG3 and IgG2b. In contrast, stimulation of endosomal TLR9 inhibited switching to all isotypes. The addition of IL-4 induced robust cell proliferation and class switching to IgG1, and TLR 1/2, 2/6, 4, and 9 agonists further enhanced these IL-4-mediated B cell responses. Altogether, our data demonstrate differential effects of surface bound and endosomal TLRs on B cell responses. Activation of surface-bound TLRs, but not endosomal TLR9, triggers CSR to TH1-associated antibody isotypes, while both groups of TLRs synergize the effect of IL-4 on driving CSR to IgG1.

Expression and purification of catalytic domain of botulinum neurotoxin serotype ‘F’: immunological characterization and its application in detection

ABSTRACTBotulinum neurotoxin (BoNT) is the most toxic biomolecule known to the world (⁓100 times toxic than cyanide) and has been listed as category ‘A’ biowarfare agent. In the present investigation, we cloned the catalytic domain of BoNT type ‘F’. Maximum recombinant BoNT/F protein expression was achieved at 21°C, 0.75 mM IPTG at 6 h post induction. The rBoNT/F protein was purified and confirmed subsequently by MS-MS analysis as well as Western blot. High titer polyclonal antibodies were generated and elevated level of IgG1 isotypes was observed. Plate ELISA was developed for the detection of BoNT/F with the sensitivity of 3.9 ng/mL. Limits of Detection (LOD) was established for different fruit juices through spiking studies using rBoNT/F Lc protein as antigen and achieved a detection limit of 15.62 ng/mL for apple juice followed by 31 ng/mL for litchi and 62 ng/mL for grape, orange, and mango juices. The developed system has the potential for the detection of BoNT/F in food as well as environmental samples.

Immune responses to Mycobacterium tuberculosis membrane-associated antigens including alpha crystallin can potentially discriminate between latent infection and active tuberculosis disease.

Changes in expression of membrane antigens may accompany the transition of Mycobacterium tuberculosis (Mtb) from 'dormant' to 'active' states. We have determined whether antibody and T cell responses to Mtb membrane (MtM)-associated antigens, especially the latency-induced protein alpha crystallin (Acr), can discriminate between latent tuberculosis infection (LTBI) and active TB (ATB) disease. Study subjects comprised a previously described cohort of healthcare workers (HCWs, n = 43) and smear-positive ATB patients (n = 10). HCWs were further categorized as occupational contacts (OC, n = 30), household contacts of TB (HC, n = 8) and cured TB (CTB, n = 5). Levels (ΔOD) of serum antibody isotypes (IgG, IgA and IgM) were determined by ELISA and blood T cell proliferative responses were determined by flow cytometry using Ki67 protein as marker for DNA synthesis. Antibodies to MtM and Acr were predominantly IgG and their levels in HCWs and ATB did not differ significantly. However, HCWs showed a significantly higher level of anti-MtM IgM and a significantly lower level of anti-Acr IgA antibodies than the ATB patients. Also, a larger proportion of HCWs showed a high (>1) ΔODAcr/ΔODMtM ratio for IgG. HCWs also showed a higher, though not significantly different from ATB, avidity of anti-MtM (IgG) antibodies. A higher proportion of HCWs (35% of OC, 62.5% of HC and 20% of CTB), compared with ATB (10%) showed a positive T cell response to Acr along with significant difference (P <0.05) between HC and ATB. A significant correlation (r = 0.60, P <0.0001) was noted between T cell responses of HCWs towards Acr and MtM (reported earlier by us) and both responses tended to decline with rising exposure to the infection. Even so, positive responses to Acr (38.5%) were significantly lower than to MtM (92%). Neither antibody nor T cell responses to either antigen appeared affected by BCG vaccination or reactivity to tuberculin. Results of the study suggest that the levels of IgM antibodies to MtM, IgA antibodies to Acr and proliferative T cell responses to both the antigens can potentially discriminate between LTBI and active TB disease. They also underscore the necessity of SOPs for antibody assays.

CD26-Related Serum Biomarkers: sCD26 Protein, DPP4 Activity, and Anti-CD26 Isotype Levels in a Colorectal Cancer-Screening Context.

Current screening trials are showing reduction in colorectal cancer incidence and mortality. However, participation rates are often low, and blood-based tests could complement existing screening strategies. CD26 protein (sCD26) and its dipeptidyl peptidase IV (DPP4) enzymatic activity in circulation have been proposed as biomarkers for colorectal cancer and other diseases. However, changes in sCD26 and DPP4 levels show complex degrees of correlation, and their physiological or pathophysiological role is unclear. The aim of this study was to analyse if anti-CD26 autoantibodies are related to sCD26 and DPP4 and to determine their relevance in a context of colorectal cancer screening for complementing the value of sCD26 and DPP4 as biomarkers. These biomarkers were measured in a large prospective cohort (n = 497, except the anti-CD26 antibodies, evaluated in 125 samples) that included a subgroup of individuals that were positive for the faecal immunological occult blood test (FIT) (n = 86) and underwent a colonoscopy (n = 47). We confirmed for the first time higher DPP4 activity in men compared to women (Student's t test, p = 0.002), though this difference between sexes was not seen for serum sCD26 protein. These biomarkers correlated (R = 0.246, p = 0.003) only in women. Correlations were found between anti-CD26 isotypes but not with DPP4 activity or sCD26 concentration, except for a negative correlation only in men between anti-CD26 IgA isotype and sCD26 (R = −0.232, p = 0.044), and an almost significant negative correlation between anti-CD26 IgG and sCD26 limited to FIT-positive men. Interestingly, patients with advanced adenomas displayed the most elevated mean levels of anti-CD26 IgA, IgM, and particularly IgG (Mann-Whitney U test, p = 0.030) in comparison with the other FIT positives without adenomas, and these levels did not correlate with sCD26 or its DPP4 activity. Our preliminary results suggest that the combination of these measures using sex as confounder could perhaps be used as biomarkers for colorectal disease. It also suggests that events affecting the gut influence the levels of anti-CD26 antibodies, which show little or no effect in antigen clearance. These findings should be confirmed in a larger cohort of individuals with colonoscopy. The physiological origin of the sex differences observed should be further addressed.

Extract of Ascaris suum induces TGF-β and early production of IgG1 in experimental autoimmune hepatitis.

In experimental autoimmune hepatitis (EAH) of Th1 profile, an extract of adult Ascaris suum worms (ASC) was previously found to deviate the immune response to a Th2/IL-10 pattern. Here, the effects of treatment with ASC on production of TGF-β and the anti-Ascaris isotypes IgG1 and IgG2a in EAH were evaluated. EAH was induced in BALB/c mice, intravenously with concanavalin A. Two hours later, these animals received ASC (EAH+ASC group) or PBS vehicle (EAH group). IgG1 and IgG2a were evaluated 8 h, 24 h and 7 d after induction. TGF-β was measured in a splenocyte culture at this last time. The isotype levels in the EAH group were low throughout the kinetics. In the EAH+ASC group, there was significant production of IgG1 at 24 h and 7 d, but of IgG2a only at 7 d. There was statistically greater production of TGF-β in the EAH+ASC group. The higher levels of IgG1 and TGF-β in this group suggest that an additional Th1 response control route exists in EAH, which needs to be investigated.

DEC-205 receptor-mediated long-circling nanoliposome as an antigen and Eucommia ulmoides polysaccharide delivery system enhances the immune response via facilitating dendritic cells maturation

DEC-205 receptor-mediated dendritic cells (DC) targeting nanoliposomes is a promising delivery system in eliciting an immune response against pathogens. When this delivery system carries both antigen and immunomodulator, it can effectively regulate the DC function as well as the initial T cell response. To maximize the desired therapeutic effects of Eucommia ulmoides Oliv. polysaccharides (EUPS), and induce an efficient humoral and cellular immune response against an antigen, we encapsulated the OVA and EUPS in long-circling nanoliposomes and conjugated it with anti-DEC-205 receptor antibody to obtain a DEC-205-targeted nanoliposomes (anti-DEC-205-EUPS-OVA-LPSM). The physicochemical properties and immune-modulating effects were investigated in vitro and in vivo by a series of the experiment to evaluate the targeting efficiency of anti-DEC-205-EUPS-OVA-LPSM. In vitro, anti-DEC-205-EUPS-OVA-LPSM (160 μg mL−1) could enhance DCs proliferation and increase their phagocytic efficiency. In vivo anti-DEC-205-EUPS-OVA-LPSM remarkably promoted the OVA-specific IgG and IgG isotypes levels, enhanced the splenocyte proliferation, and induced the NK cell and CTL cytotoxicity. Besides, the anti-DEC-205-EUPS-OVA-LPSM enhanced the maturation of DCs. These findings suggest that the DEC-205 receptor antibody-conjugated EUPS nanoliposome can act as an efficient antigen delivery system to enhance the cellular and humoral immune response by promoting DC maturation. This indicates that the anti-DEC-205-EUPS-OVA-LPSM has significant potential as an immune-enhancing agent and antigen delivery system.

Myocardial protection in cardiac surgery: Del Nido versus blood cardioplegia.

Objectives:del Nido cardioplegia which was traditionally used for myocardial protection in pediatric congenital heart surgery is now being extensively utilized in adult cardiac surgery. The aim of this study was to compare the safety and efficacy of del Nido cardioplegia (DNC) with blood cardioplegia (BC).Materials and Methods:This is a historical cohort study using secondary data. Two hundred and eighty six patients who underwent coronary artery bypass graft (CABG) or valve surgery were included. They were divided into 2 matched cohorts of which 143 patients received BC and 143 patients received DNC.Results:There was no difference in cardiopulmonary bypass time (P = 0.516) and clamp time (P = 0.650) between the groups. The redosing of cardioplegia was significantly less for DNC (1.13 vs. 2.35, P = <0.001). The post bypass hemoglobin was higher for DNC (9.1 vs. 8.7, P = 0.011). The intraoperative and postoperative blood transfusion was comparable (P = 0.344) (P = 0.40). The incidence of clamp release ventricular fibrillation (P = 0.207) was similar. The creatine kinase-MB isotype levels for the CABG patients were comparable on all 3 days (P = 0.104), (P = 0.106), and (P = 0.158). The postoperative left ventricle ejection fraction was lesser but within normal range in the DNC group (53.4 vs. 56.0, P = <0.001). The duration of ventilation (P = 0.186), ICU days (P = 0.931), and postoperative complications (P = 0.354) were comparable. There was no 30-day mortality or postoperative myocardial infarction in both the groups.Conclusion:DNC provides equivalent myocardial protection, efficacy, and surgical workflow and had comparable clinical outcomes to that of BC. This study shows that DNC is a safe alternate to BC in CABG and valve surgeries.

Anticardiolipin antibody and anti-β2 glycoprotein I antibody are potential risk markers of ischaemic stroke in Chinese adults.

aCL and anti-β2 glycoprotein I antibody (aβ2GPI) are autoantibodies associated with thromboembolic diseases. Here we investigated whether they are correlated with ischaemic cardiovascular disease in a Chinese population. Serum total aCL and aβ2GPI isotypes (IgA, IgG or IgM, separately) were measured in 11 015 Chinese adults. Differences of antibody level between disease and non-disease groups were examined by t-test. The correlation between antibody and ischaemic cardiovascular disease was determined by logistic regression analysis. Performance of risk prediction models employed aCL or aβ2GPI isotypes was evaluated by C statistic, net reclassification improvement index and integrated discrimination improvement. Total aCL and aβ2GPI isotypes maintained low levels and increased with increasing age except total aCL and aβ2GPI IgG in participants older than 70 years. When distinguishing ischaemic cardiovascular disease by coronary heart disease (CHD) and ischaemic stroke, the stroke group had higher levels of aCL and aβ2GPI isotypes than the non-stroke group, while the CHD group only had a slightly higher aβ2GPI IgG than non-CHD groups. aCL and aβ2GPI were positively correlated with stroke but not with CHD, and improved the performance of conventional risk factors for stroke risk prediction, with C statistic from 0.769 (95% CI 0.744, 0.793) to 0.777 (95% CI 0.754, 0.800) (aβ2GPI IgG, P = 0.0091), and 0.778 (95% CI 0.754, 0.801) (aβ2GPI IgA, P = 0.0793). Stroke risk could be better reclassified by aCL and aβ2GPI, in association with both net reclassification improvement and integrated discrimination improvement statistics (P < 0.05). aCL and aβ2GPI are associated with ischaemic stroke and have added value for stroke risk prediction.

Fabrication of Microarrays for the Analysis of Serological Antibody Isotypes against Food Antigens.

Food intolerance is delayed adverse food reactions which follow consumption of specific foods. The underlying mechanisms are not well understood, but food intolerance is often considered as a type 2 hypersensitivity reaction mediated by immunoglobulin G (IgG) antibody. To understand the causes of food intolerance, it is important to investigate sensitization patterns of food-specific IgGs (sIgG) in relation to dietary patterns and physical conditions. Conventional approaches to measure serological IgGs often require large volumes of serum, thus are not suitable for highly multiplexed assays. To overcome this impracticality, we developed a highly sensitive method to screen the sIgGs and other antibody isotypes against 66 antigens with minimal amount of serums. We prepared a microarray by immobilizing food antigens on activated glass slides. Human sera and their dietary information were obtained from 30 subjects. Aliquots (200 nl) of sera were analyzed against 66 food antigens in parallel. sIgG levels were determined and analyzed in relation to subjects’ dietary patterns. The levels of antibody isotypes were also examined to understand the relationship between allergy and food intolerance. The developed microarray showed exceptional performances in antibody screening and demonstrated the potential to be used as an automated assay system.

Renal tubular P-glycoprotein expression is reduced in plasma cell disorders.

BackgroundP-glycoprotein (P-gp) transports many chemicals that vary greatly in their structure and function. It is normally expressed in renal proximal tubular cells. We hypothesized that P-gp expression influences light chain excretion. Therefore, we investigated whether renal tubular P-gp expression is altered in patients with plasma cell disorders.MethodsWe evaluated renal biopsy specimens from patients with plasma cell disorders (n = 16) and primary focal segmental glomerulosclerosis (the control group, n = 17). Biopsies were stained with an anti-P-gp antibody. Loss of P-gp expression was determined semi-quantitatively. Groups were compared for loss of P-gp expression, and clinical variables.ResultsP-gp expression loss was more severe in patients with plasma cell disorders than it was in those with glomerulonephritis (P = 0.021). In contrast, clinical and histological parameters including serum creatinine, level of urinary protein excretion, and interstitial fibrosis/tubular atrophy grade were not significantly different between the groups. P-gp expression loss increased with age in patients with plasma cell disorders (P = 0.071). This expression loss was not associated with serum creatinine, the level of urinary protein excretion or the interstitial fibrosis/tubular atrophy grade. There was no significant association between the severity of P-gp expression loss with the types and serum levels of light chains, isotypes and serum immunoglobulin levels.ConclusionRenal tubular P-gp expression is significantly down-regulated in patients with plasma cell disorders characterized by nephrotic range proteinuria. Additional studies are needed to determine whether reintroduction of renal tubular P-gp expression would mitigate the proximal tubular injury that is caused by free-light chains.

Duration of frequent or severe respiratory tract infection in adults before diagnosis of IgG subclass deficiency.

Many adults with IgG subclass deficiency (IgGSD) experience long intervals of frequent/severe respiratory tract infection before IgGSD diagnosis, but reasons for delays in IgGSD diagnoses are incompletely understood. We performed a retrospective study of 300 white adults (ages ≥18 y) with IgGSD including frequency analyses of age at IgGSD diagnosis, duration of frequent/severe respiratory tract infection before IgGSD diagnosis, and age at onset of frequent/severe infection (calculated). We performed multivariable regressions on age at diagnosis, infection duration, and age at infection onset using these variables, as appropriate: sex; age at diagnosis; diabetes; autoimmune condition(s); atopy; allergy; corticosteroid use; body mass index; serum immunoglobulin isotype levels; blood lymphocyte subsets; three IgGSD-associated human leukocyte antigen-A and -B haplotypes; and referring physician specialties. Mean age at diagnosis was 50 ± 12 (standard deviation) y (median 50 y (range 19–79)). There were 247 women (82.3%). Mean infection duration at IgGSD diagnosis was 12 ± 13 y (median 7 y (range 1–66)). Mean age at infection onset was 38 ± 16 y (median 38 y (range 4, 76)). Age at infection onset was ≥18 y in 95.7% of subjects. Regressions on age at diagnosis and infection duration revealed no significant associations. Regression on age at infection onset revealed one positive association: age at diagnosis (p <0.0001). We conclude that the median duration of frequent/severe respiratory tract infection in adults before IgGSD diagnosis was 7 y. Older adults may be diagnosed to have IgGSD after longer intervals of infection than younger adults. Duration of frequent/severe respiratory tract infection before IgGSD diagnosis was not significantly associated with routine clinical and laboratory variables, including referring physician specialties.

Delineating the interaction of combretastatin A-4 with αβ tubulin isotypes present in drug resistant human lung carcinoma using a molecular modeling approach

Tubulin isotypes are known to regulate microtubule dynamic instability and contribute to the development of drug resistance in certain types of cancers. Combretastatin-A4 (CA-4) has a potent anti-mitotic, vascular disrupting and anti-angiogenic activity. It binds at the interface of αβ tubulin heterodimers and inhibits microtubules assembly. Interestingly, the CA-4 resistant human lung carcinoma shows alteration of βI and βIII isotype levels, a higher expression of βI tubulin isotype and a decreased expression of βIII tubulin isotypes has been reported in drug resistant cell lines. However, the origin of CA-4 resistance in lung carcinoma is not well understood. Here, we investigate the interaction and binding affinities of αβI, αβIIb, αβIII and αβIVa tubulin isotypes with CA-4, employing molecular modeling approaches. Sequence analysis shows that variations in residue composition at the CA-4 binding pocket of βI, βIII and βIVa tubulin isotypes when compared to template βIIb isotype. Molecular docking result shows that the CA-4 prefers ‘cis’ conformation in all αβ-tubulin isotypes. Molecular dynamics simulation reveal role of H7 helix, T7 loop and H8 helix of β-tubulin in lower binding affinity of αβI and αβIII isotypes for CA-4. The order of binding energy for CA-4 is αβIIb > αβIVa > αβI > αβIII. This suggest that drug resistance is induced in human lung carcinoma cells by altering the expression of β-tubulin isotypes namely βI and βIII which show lowest binding affinities. Our present study can help in designing potential CA-4 analogs against drug-resistant cancer cells showing altered expression of tubulin isotypes.Abbreviations:CA-4combretastatin-A4MDmolecular dynamicsRMSDroot mean square deviationDSSPdictionary of secondary structure of proteinsVMDvisual molecular dynamicsCommunicated by Ramaswamy H. Sarma