Presence Of Antibodies Research Articles

Antigen/HLA-agnostic strategies for Characterizing Tumor-responsive T cell receptors in PDAC patients via single-cell sequencing and autologous organoid application

Characterization of tumor-responsive T cell receptors (TCRs) is a critical step in personalized TCR-T cell therapy, and remains challenging for pancreatic ductal adenocarcinoma (PDAC). Here we report a proof-of-concept study to identify and validate antitumor TCRs in two representative PDAC patients using ultradeep single-cell TCR/RNA sequencing and autologous organoids, and reveal the phenotypic dynamics of TCR repertoire in different T cell expansions from the same patient. We first performed comparative sequencing on freshly harvested peripheral blood mononuclear cells (PBMCs) and uncultured tumor infiltrating lymphocytes (TILs), followed by reactivity tests of TIL-enriched TCRs with autologous organoids, in which two tumor-responsive TCRs were successfully characterized and the corresponding TILs were mostly tissue-resident memory-like T cells, and partially expressed both naïve and exhausted T cell markers. For the PDAC patient without high-quality TILs, PBMCs were cultured with neoantigen peptide (KRASG12D), organoids, or anti-CD3 antibody in presence, and experienced extensive clonal expansions within ten days. All derived PBMCs were sequenced in parallel (>82,000 cells), and TCRs enriched in both peptide- and organoid-experienced, but not anti-CD3-treated CD8 T cells, were assessed for their reactivity to antigen-presenting cells (APCs) and organoids, in which three neoantigen-reactive TCRs were identified as tumor-responsive, and the corresponding T cells were characterized by mixed transcriptional signatures including but not limited to typical exhausted T cell markers. Together, our study revealed that the combination of ultradeep single-cell sequencing and organoid techniques enabled rapid characterization of tumor-responsive TCRs for developing practical personalized TCR-T therapy in an antigen/human leukocyte antigen (HLA)-agnostic manner.

Serological profile and pleurisy lesions associated with Actinobacillus pleuropneumoniae in pig farms in North Macedonia

Actinobacillus pleuropneumoniae (App) is one of the most important swine respiratory pathogens that causes porcine pleuropneumonia and massive financial losses in pig industry. The objective of this study was to investigate App infection in five pig herds from North Macedonia experiencing clinical respiratory infections by serological testing and a slaughterhouse pleurisy evaluation system (SPES). In total, 250 blood samples were taken from pigs aged 6, 10, 14, 18, and 22 weeks. Ten animals per age category from each farm were sampled and analyzed for presence of antibodies against App. At the slaughterhouse, 50 lungs per herd from slaughtered age pigs were scored by the SPES for the presence of lesions associated with chronic pleurisy. The overall seroprevalence to App was 65.6%, ranging from 34% to 98% at the farm level. The highest seroprevalence was found in 6-week-old pigs in all farms, while significant differences were observed between farms in the 10-, 14-, 18-, and 22-week-old pig groups. Pleurisy associated with App was found in 26.4% of all examined lungs, with the mean SPES score being 0.75 (0.14 - 1.10). The percentage of SPES scores of 0, 1, 2, 3, and 4 in all lungs were 73.4%, 1.6%, 8.8%, 8.4%, and 7.6% respectively. A significant difference in mean SPES score was obtained between two farms. High seroprevalences of App detected on the tested farms were probably due to constant natural infection. The highest seroprevalences, measured in the youngest pigs, could be due to maternally-derived antibodies. Higher seroprevalence against App and lower SPES scores in some farms suggests immunity resulting from infection by corresponding field serotypes.

Seroprevalence of Toxoplasma gondii in people at an increased risk of HIV infection – a pilot study

<br><b>Introduction:</b> The observation of the epidemiology of toxoplasmosis may prevent the development of a severe form of the disease in HIV patients.</br> <br><b>Aim:</b> The aim of the study was to evaluate the seroprevalence of <i>Toxoplasma gondii</i> in the population of the Kuyavian-Pomeranian Voivodeship at high risk of contracting HIV.</br> <br><b>Material and methods:</b> Blood serum samples of 43 patients of the Consulting and Diagnostic AIDS Center were tested for the presence of anti-HIV-1/HIV-2 antibodies and p24 antigen, and for the presence of anti-toxoplasma IgM and IgG antibodies.</br> <br><b>Results:</b> Anti-toxoplasma IgG antibody prevalence of 53.5% (23/43) was found in the study population, while the examination of specific IgM antibodies was negative. A high IgG antibody avidity index was obtained in 18 (94.7%) seropositive samples. Thirty (69.77%) of the samples were female, and 13 (30.23%) were male. Among men, HIV was detected in 1 (7.69%), and IgG antibodies against <i>T. gondii</i> in 7 (53.85%) samples. IgG antibodies against <i>T. gondii</i> were found in 16 (53.33%) women. The HIV-positive individual was 24 years old. The presence of antibodies against <i>T. gondii</i> in the IgG class was found in people of different ages (in women aged 38.44 ±13.00 years old and in men aged 29.29 ±10.86 years old). The risky situation that could cause HIV infection is in most cases sexual contacts (79.07%).</br> <br><b>Conclusions:</b> High seroprevalence of <i>T. gondii</i> was found among the studied subjects at a high risk of HIV infection. Further research is required on a larger study group.</br>

Serum anti-malondialdehyde-acetaldehyde IgA antibody concentration improves prediction of coronary atherosclerosis beyond traditional risk factors in patients with rheumatoid arthritis

Patients with rheumatoid arthritis (RA) have increased atherosclerosis; oxidative stress may be a contributor. Oxidative stress produces immunogenic malondialdehyde-acetaldehyde (MAA) protein adducts and anti-MAA antibodies are detectable in human serum. We hypothesized that anti-MAA antibody concentrations are associated with coronary atherosclerosis in RA patients. Serum concentrations of anti-MAA antibodies (IgA, IgG, and IgM) were measured in 166 RA patients using ELISA cross-sectionally. Relationship between anti-MAA antibody concentrations and cardiovascular and metabolic measures and predictive accuracy of anti-MAA antibodies for presence of coronary artery calcium (CAC) and high CAC (≥ 300 Agatston units or ≥ 75th percentile) were assessed. Only serum IgA anti-MAA antibody concentration was associated with increased CAC, insulin resistance, and decreased high-density lipoprotein particle number. When added as an interaction term with ACC/AHA 10-year risk score plus high-sensitivity C-reactive protein, IgA anti-MAA antibody concentration improved the C-statistic for prediction of any CAC and high CAC compared to ACC/AHA 10-year risk score plus hs-CRP alone. IgA anti-MAA concentration is associated with multiple cardiovascular risk factors and modifies the relationship between ACC/AHA 10-year risk score and CAC in RA patients. IgA anti-MAA concentration could assist in prediction of atherosclerotic CVD and risk stratification when added to standard measures of cardiovascular risk.

Yaws elimination in Ecuador: Findings of a serological survey of children in Esmeraldas province to evaluate interruption of transmission.

The WHO roadmap for neglected tropical diseases includes yaws eradication requiring certification of elimination of transmission in all endemic and formerly endemic countries worldwide. A community-based programme for yaws control was considered to have achieved elimination of the infection in the endemic focus in Ecuador after 1993. We did a serosurvey of children in this focus to provide evidence for interruption of transmission. Survey of serum samples collected from children aged 2 to 15 years living in the formerly endemic and in geographically contiguous areas. A convenience sample of sera collected between 2005 were 2017 from non-yaws studies, were analyzed using immunochromatic rapid tests to screen (OnSite Syphilis Ab Combo Rapid Test) for Treponema pallidum-specific antibodies and confirm (DPP Syphilis Screen and Confirm) seroreactivity based on the presence antibodies to treponemal and non-treponemal antigens. Seroreactivity was confirmed in 6 (0.14%, 95% CI 0.06-0.30) of 4,432 sera analyzed and was similar in formerly endemic (0.11%, (95% CI 0.01-0.75) and non-endemic (0.14%, 95% CI 0.06-0.34) communities. All seroreactors were of Afro-Ecuadorian ethnicity and most were male (4/6) and aged 10 or more years (5/6), the latter possibly indicating venereal syphilis. Only 1 seroreactor lived in a community in the Rio Santiago, that was formerly hyperendemic for yaws. We observed very low levels of treponemal transmission in both formerly endemic and non-endemic communities which might be indicative of congenital or venereal syphilis and, if yaws, would likely be insufficient to maintain transmission of this endemic childhood infection. Additional surveys of children aged 1 to 5 years are planned in Rio Santiago communities to exclude yaws transmission.

Diagnostic pitfalls in neuromyelitis optica spectrum disorders

Neuromyelitis optica spectrum disorders are autoimmune disorders of the central nervous system marked by inflammatory demyelination, axonal loss and astrocytopathy, associated with lesions in the brain and in the spinal cord and with the presence of antibodies against aquaporin 4 (AQP4-IgG). Various studies on neuromyelitis optica spectrum disorders broaden the knowledge about their immunopathogenesis, clinical course, immunological assays, and magnetic resonance imaging findings. Nevertheless, in clinical practice differential diagnosis remains challenging, particularly in cases seronegative for AQP4-IgG. Essential clinical syndromes in neuromyelitis optica spectrum disorders, namely optic neuritis and acute myelitis, could also be observed in multiple sclerosis and other demyelinating diseases of the central nervous system. The early and accurate diagnosis is essential due to different prognosis and treatment strategies in these diseases. For example, therapies used in multiple sclerosis, including beta-interferons, natalizumab, fingolimod and alemtuzumab, may not only be ineffective in neuromyelitis optica spectrum disorders but even harmful and provoke relapses. Therefore, in a patient with a clinical neurological syndrome accompanied by demyelinating lesions in the central nervous system several investigative studies should be undertaken, including serological assays [AQP4-IgG, antibodies against myelin oligodendrocyte glycoprotein (MOG-Ab)], the cerebrospinal fluid examination and magnetic resonance imaging of the brain and spinal cord, and interpreted with caution. Due to clinical similarities and relatively common misdiagnoses, there is a need to summarise the typical and atypical clinical, laboratory and radiographic features in neuromyelitis optica spectrum disorders to avoid diagnostic pitfalls and inappropriate treatment.

Abstract LB070: Development of Claudin 18.2/CD3 bispecific antibody maintaining effector functions for better cancer therapy

Abstract Background: Tumors are heterogeneous in nature with highly suppressive microenvironment. The heterogeneity is distinguished by the presence of distinct cell populations, where many tumors are partial suppressed, thus lead to tumor relapse and develop drug-resistance after initial therapy. Most importantly, the heterogeneity in tumors raises considerable challenges to design effective treatment strategy with predictable outcomes. Paracellular permeability at tight junctions is largely determined by Claudins. Under aberrant condition, Claudins disrupt tight junction functions and promote tumor-initiation. Claudin 18.2 is significantly expressed in gastric and pancreatic cancers, while normal tissue expression is limited to the epithelium of the stomach, making it a promising target for cancer therapy. Although great efficacy has been observed for T-cell engagers, balance of safety-efficacy remains a major concern. Bispecific antibody (BsAb) with lower CD3-binding arm could have a huge potential to minimize unspecific T-cell activation while maintaining better-efficacy. Here, we designed Claudin 18.2/CD3 BsAb with weaker CD3-binding arm and maintain Fc-effector functions to broaden mechanisms of action including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Methods: Claudin 18.2 expressing and T-cells were used for Claudin 18.2 and CD3 binding analysis, respectively. T-cell activation was performed by co-culturing T-cells with Claudin 18.2+ and Chaudin 18.2- cells. Pancreatic cancer cell was co-cultured with human PBMCs for ADCC, T-cell activation and T-cell cytotoxicity studies. Co-culture of Jurkat and CD16+ NK cells were used for Fc-mediated T-cell activation study. Claudin 18.2+ and T-cell cells were used for CDC studies in presence of human complement serum. Results: While comparable Claudin 18.2 binding affinity was maintained, a significant reduction in CD3 binding was observed by Claudin 18.2/CD3 BsAb compared to a benchmark antibody. Although weaker CD3 binding, BsAb induced equivalent Claudin 18.2 specific T-cell activation in presence of Jurkat and/or human PBMCs compared to the benchmark. Moreover, it also induced ADCC of pancreatic cancer cell and T-cell activation in presence of PBMCs in dose-dependent manner. Claudin 18.2/CD3 BsAb induced CDC of Claudin 18.2+ cells but not T-cell cells in presence of human complement serum. While Claudin 18.2/CD3 BsAb maintained the Fc-mediated ADCC and CDC functions, it had no significant effect on Fc-mediated T-cell activation compared to the benchmark antibody in presence of CD16+ NK cells. Conclusion: We designed Claudin 18.2/CD3 BsAb with weaker CD3 binding to minimize unspecific T-cell activation while maintaining Fc-mediated-effector functions. These data suggest the development of Claudin 18.2/CD3 BsAb as a drug target for patients with gastric and pancreatic cancers. Citation Format: Amit K. Chaudhary, Mohd S. Zaman, Cai Huang, Jianbo Dong, Yue Liu. Development of Claudin 18.2/CD3 bispecific antibody maintaining effector functions for better cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB070.

Preparation of high concentration protein powder suspensions by milling of lyophilizates

Pharmaceutical formulations utilizing protein drugs as powders can be used as drug delivery systems in various ways. Besides powders for inhalation, another promising approach is their use as suspensions in non-aqueous liquids for subcutaneous administration providing high protein stability and good injectability. In this study protein powder suspensions were prepared using a swing-mill. Milling of lyophilizates containing a model monoclonal antibody in presence of the suspension vehicle was compared to cryogenic dry milling. Wet media milling led to injectable suspensions, but resulted in monomer loss and increase in protein aggregation. When the lyophilizates were cryogenic dry ball milled less aggregation and monomer loss were detected. Differences related to protein integrity were found for different process parameters, which were successfully optimized. If not cooled with liquid nitrogen, dry milling resulted in increased damage to the mAb. The type of polyol stabilizer, as well as the protein to stabilizer ratio, did not affect the preservation of protein integrity.As finding the right milling duration is time and resource intensive, a correlation between lyophilizate cake hardness and milling duration was established. Based on this approach high concentration lyophilizates were successfully micronized. Suspensions of cryogenic milled powders lead to clogging of 25G needles, which could be prevented by an additional sieving step. Depending on the suspension vehicle, low viscosity formulations (<10 mPa·s) even at high concentrations (≥100 mg/ml protein concentration) were obtained featuring good injectability.

Withdrawal Notice: Characterization of Glyoxal Modified LDL: Role in the Generation of Circulating Autoantibodies in Type 2 Diabetes Mellitus and Coronary Artery Disease

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Seroprevalence of Coxiella burnetii in cattle in the Belgrade epizootiological area

Q-fever is antropozoonosis which is caused by Coxiella burnetii, obligate intracellular pathogen. The most significant characteristics of this pathogen are resistance and stability in the environment, possibility of aerosol dissemination, and very low infective dose. C. burnetii can infect domestic and wild animals, rodents, birds and ticks. Q fever in animals is generally asymptomatic, although it can lead to reproductive disorders during pregnancy. The main route of infection in humans is inhalation of contaminated aerosol and dust. Serological studies have shown the presence of antibodies to C. burnetii in the serum samples of cattle in Belgrade epizootiological area. Seroprevalence of 18% was found in farm bred cattle, while it was only 1.5% in individual breeding. In farm bred cows that have suffered abortion prevalence was 49%, and only 1.9% in individual breeding. The overall results indicate that the circulation of this pathogen in cattle, in Belgrade epizootiological area, poses a health risk, not only to the cattle, but also to the humans, especially persons working with animals. Q fever control programs most often recommend serological research and vaccination of animals. Accordingly, it is necessary to define a strategy for the implementation of biosecurity measures and preventive measures against Q fever.

Abstract # 1768 [formula omitted]-Adrenoceptor dependent intracellular mechanisms of the learned immunosuppression in human CD4+ T cells

It is well documented that immune functions can be modulated by associative learning processes such as classical conditioning. Using the calcineurin inhibitor and immunosuppressant drug cyclosporine A as unconditioned stimulus, we have established a behavioral conditioning paradigm in humans and rats and have previously shown that the conditioned suppression of the IL-2 production in T cells is mediated via the sympathetic nervous system and β2-adrenoceptor (β2AR)-dependent mechanisms. The aim of the present study was to elucidate the intracellular mechanisms of the learned immunosuppression in human CD4+ T cells. We isolated CD4+ T cells from the peripheral blood by magnetic cell separation and stimulated the cells with anti-CD3/anti-CD28 antibodies in presence and absence of the selective β2AR agonist terbutaline (TERB). Subsequently, we analyzed the three main intracellular pathways known to be involved in the regulation of IL-2 expression: A nuclear factor ‘K-light-chain-enhancer’ of activated B cells (NFKB), a cAMP response element-binding protein (CREB) or a Calcineurin (CaN) dependent way. We are now investigating the link between the β2AR mediated pathway and the T cell receptor (TCR) mediated pathway and its influence on NFKB, CREB and CaN to find out which pathway is responsible for decreased production of IL-2 in human CD4+ T cells in the conditioning paradigm.

Homogeneous single-label tyrosine kinase activity assay for high throughput screening

Protein post-translational modifications (PTMs) are regulatory mechanisms carried out by different enzymes in a cell. Kinase catalyzed phosphorylation is one of the most important PTM affecting the protein activity and function. We have developed a single-label quenching resonance energy transfer (QRET) assay to monitor tyrosine phosphorylation in a homogeneous high throughput compatible format. Epidermal growth factor receptor (EGFR) induced phosphorylation was monitored using Eu3+-chelate labeled peptide and label-free phosphotyrosine specific antibody in presence of a soluble quencher molecule. In the QRET kinase assay, antibody binding to phosphorylated Eu3+-peptide protects the Eu3+-chelate from luminescence quenching, monitoring high time-resolved luminescence (TRL) signals. In the presence of specific kinase inhibitor, antibody recognition and Eu3+-chelate protection is prevented, allowing an efficient luminescence quenching. The assay functionality was demonstrated with a panel of EGFR inhibitors (AG-1478, compound 56, erlotinib, PD174265, and staurosporine). The monitored IC50 values ranged from 0.08 to 155.3 nM and were comparable to those found in the literature. EGFR activity and inhibition assays were performed using low nanomolar enzyme and antibody concentration in a 384-well plate format, demonstrating its compatibility for high throughput screening (HTS).

Role of Stromal Cell-Mediated Notch Signaling in AML Resistance to Chemotherapy

Introduction: Our group has recently shown that bone marrow-mesenchymal stromal cell (BM-MSCs)-mediated Notch signaling may control survival and chemoresistance of B-acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia (CLL) cells. Conversely, the role of Notch signaling in acute myeloid leukemia (AML) remains controversial, as its contribution to the crosstalk between BM-MSCs and AML cells is still unknown. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML primary blast cells in co-culture with BM-MSCs.Methods: AML blast cells were obtained after informed consent from bone marrow samples (30) and peripheral blood (20) of AML patients, according to the Institutional guidelines. BM-MSCs were expanded from bone marrow of 12 healthy donors (BM-MSCs) and of 12 AML-patients (BM-MSCs*). PCR, FACS analysis and western immunoblotting were used to study the expression of Notch receptors and ligands, as well as Notch activation status, in AML cells and BM-MSCs. AML cells were co-cultured with BM-MSCs or BM-MSCs* at 10:1 (AML:BM-MSCs) ratio for 2 to 3 days in presence of Cytarabine, Etoposide, Idarubicin, as well as in presence or absence of anti-Notch-1, -2, -3, -4, anti-Jagged1, -2 and anti-DLL3 blocking antibodies or gamma secretase inhibitor-XII (GSI-XII). Cell viability was evaluated by Annexin-V/Propidium Iodide (PI) and MTT; proliferation and cell cycle were assessed through CFSE dilution and PI methods, respectively.Results: AML cells expressed Notch receptors and ligands, showing Notch-1, -2, Jagged-1, -2 and DLL-3 signature, while BM-MSCs/ BM-MSCs* showed expression of Notch-1, -2, -3, -4 and Jagged-1, -2 and DLL-1. We then analyzed Notch activation in different cell types by evaluating the expression of NICD-1, -2, -3 as well as Hes-1. We found that at least 50% of AML samples showed basal Notch activation while BM-MSCs/ BM-MSCs* showed slight Notch activation. The expression and activation pattern were modulated after 3 days of co-culture with either BM-MSCs or BM-MSCs*. The pan blockade of Notch signaling by GSI-XII were capable to inhibit AML cell proliferation as well as induce AML cell apoptosis in culture or in co-culture with BM-MSCs/ BM-MSCs*. The addition of chemotherapeutic agents decreased AML cell viability in culture, while a significant rescue from apoptosis was observed when cocultured with BM-MSCs or BM-MSCs*. Pan Notch signaling blockade by either GSI-XII or combination of Notch receptor-blocking antibodies in presence of chemotherapeutic agents significantly lowered the supportive role of BM-MSCs towards AML cell lines. The specific blockade of Notch-1, -2, -3 or Jagged-1, -2 rescued partially the chemosensibility, while blockade of Notch-4 or DLL-3 rescued totally the chemosensitivity of primary AML cells in co-culture with BM-MSCs.Conclusions: These results suggest that Notch signaling may represent a potential therapeutic strategy to overcome bone marrow stromal-mediated survival and chemoresistance of AML. Moreover Notch blocking antibodies were able to impair the survival benefit imparted by bone marrow stromal cells. Therefore blocking Notch antibodies could be a useful strategy to improve the efficiency of AML chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Seroprevalence of dogs to Leishmania infantum antigens in the territory of Crna Gora

Due to expansion of vector borne diseases and their importance for human health we have investigated the prevalence of antibodies to Leishmania infantum antigens in serum samples of dogs. Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. According to the World Health Organization the disease is a constant threat to 350 million people in 88 countries, provided that most Mediterranean countries belong to the area designated as endemic. Since Crna Gora is a country of Mediterranean basin, it is important to present the results of the investigation on the presence of antibodies on L. infantum antigens in blood serum of dogs, considering that dogs can be reservoirs and source of infection for humans. In the period from 2006. to 2010., there were tested 433 serum samples of dogs with clinical symptoms and 68 serum samples of dogs without symptoms of the disease. Those were dogs with visible symptoms of the disease such as alopecia, weight loss and lymphadenomegalia. In 66% of cases, there was known hematological profile with predominant hypochromic anemia and neutrophilic leukocytosis. As mentioned above, the investigation included also 68 asymptomatic dogs in good condition, out of which 45 were service dogs in use in military and police as well as 23 dogs from the asylum. By Indirect immunofluorescence test or Indirect Fluorescent Antibody Technique (IF-AT) and Direct Agglutination Test (DAT), there was determined the presence of specific antibodies to L. infantum antigens. Positive findings in the serum dilution 1:160 was deter?mined in 258 samples (59.58%) in the group of dogs with clinical symptoms, and in 5.9% in the tested asymptomatic dogs. A part of the territory of Crna Gora with its average annual air temperature of 14.6 - 17.8?C and mediterranean climate (long, warm and dry summer and mild, rainy winter) has all the prerequisites for the development and maintenance of the infection. The confirmed presence of five kinds of Phlebotomines on the territory of Crna Gora which are essential in the cycle of developing and maintaining of Leishmania species with dominant Phlebotomus neglectus species, additionally proves a great risk of occur?rence and maintenance of the disease. The obtained results of seroprevalence in dogs are in correlation with the results of 55 cases of visceral leishmaniasis recorded by the Institute for Public Health in people from 1992 to 2009. Taking into consideration that seronegative findings and absence of symptoms does not exclude the infection, as well as that seropositive finding does not necessarily confirm it, a high level of seroprevalence (of 59.58%) in dogs in urban regions of Crna Gora, recorded during 5 years period, is by itself an alarming epizootiological and epidemiological fact.

Association between E-selectin expression and histopathological modification of glomerular lesions by non-nephritogenic IgM antibodies in experimental lupus nephritis

Objective. To examine the role played by E-selectin in bystander IgM-mediated modification of glomerular lesions in experimental lupus nephritis.Methods. Experimental lupus SCID mice were induced by an intraperitoneal injection of clone 7B6.8, which was derived from a MRL/lpr mouse and shown to induce wire-loop type glomerular lesions. Mice were subsequently administered clone Sp6, a non-nephritogenic IgM antibody- producing hybridoma. E-selectin expression was then evaluated in glomeruli showing histopathological conversion of lesions from wire-loop-like to a cell-proliferative form. We also investigated the effects of a circulating soluble form of E-selectin (sE-selectin) on the modification of glomerular lesions in this lupus model.Results. In experimental lupus mice, glomerular E-selectin expression significantly increased during the conversion from wire-loop-like glomerular lesions to a cell-proliferative type mediated by a non-nephritogenic bystander IgM antibody in presence of a nephritogenic antibody. Intraglomerular infiltration of CD68 + macrophages correlated significantly with the glomerular level of E-selectin expression. In addition, overexpression of circulating sE-selectin significantly suppressed conversion to cell-proliferative glomerular lesions and glomerular macrophage infiltration in these lupus model mice.Conclusions. The histopathological modification of lupus nephritis by non-nephritogenic bystander IgM antibodies is associated in part with glomerular E-selectin expression.

Autoimmune limbic encephalitis and anesthesia

Keywords Limbic encephalitis AnesthesiaTo the Editor:We report the anesthetic management of a patient withautoimmune limbic encephalitis scheduled for endovascu-lar aortic aneurysm repair surgery. This rare disorder isusually paraneoplastic and is characterized by memoryimpairment, seizures, psychiatric symptoms, autonomicderangement, involuntary movements, and cognitive dys-function [1]. It is also usually characterized by the presenceof antibodies against various antigens, including thoseagainst N-methyl-D-aspartate (NMDA) receptors [1, 2].Therapy consists of immunosuppressants in combinationwith the management of seizures and psychiatric symptoms.Our patient was a 66-year-old man, American Society ofAnesthesiologists stage III. His medications included pred-nisolone, valproate, bromazepam and mirtazapine. Generalanesthesia was induced with midazolam, propofol, fentanyland rocuronium and was maintained with propofol and rem-ifentanilinfusions.BloodpressureandBispectralIndexlevelsshowed significant intraoperative variability, with a relativeresistance to propofol infusion (Electronic SupplementaryMaterial1).Sugammadex2 mg kg

AB0142 Immunosuppressive effects of glucocorticoids and regulatory t cells on cd28null t cells in vitro

BackgroundCD28null T cells are terminally differentiated T cells lacking CD28 co-receptor. These cells display properties of proinflammatory killer cells1, 2 and are suggested to be resistant to apoptosis in vivo3....

Autoimmune polyglandular syndrome, type 2 associated with myasthenia gravis

Autoimmune polyglandular syndrome type 2 is defined as adrenal insufficiency associated with autoimmune primary hypothyroidism and/or with autoimmune type 1 diabetes mellitus, but very rare vith myasthenia gravis. We presented a case of an autoimmune polyglandular syndrome, type 2 associated with myasthenia gravis. A 49-year-old female with symptoms of muscle weakness and low serum levels of cortisol and aldosterone was already diagnosed with primary adrenal insufficiency. Primary hypothyroidism was identified with low values of free thyroxine 4 (FT4) and raised values of thyroidstumulating hormone (TSH). The immune system as a cause of hypothyroidism was confirmed by the presence of thyroid antibodies to peroxidase and TSH receptors. Myasthenia gravis was diagnosed on the basis of a typical clinical feature, positive diagnostic tests and an increased titre of antibodies against the acetylcholine receptors. It was not possible to confirm the immune nature of adrenal insufficiency by the presence of antibodies to 21-hydroxylase. The normal morphological finding of the adrenal glands was an indirect confirmation of the condition as well as the absence of other diseases that might have led to adrenal insufficiency and low levels of both serum cortisol and aldosterone. Hormone replacement therapy, anticholinergic therapy and corticosteroid therapy for myasthenia gravis improved the patient's general state of health and muscle weakness. This case report indicates a need to examine each patient with an autoimmune disease carefully as this condition may be associated with another autoimmune diseases.

Autoimmune pancreatitis

Autoimmune pancreatitis is a form of chronic pancreatitis of presumed autoimmune aetiology. The disease is characterised with clinical, serological, histomorphological and imaging features. Autoimmune pancreatitis is recognised as a T-cell-mediated specific disease with lymphoplasmatic infiltration of pancreatic tissue and pancreatic parenchyma fibrosis. Serum immunoglobulin IgG or IgG4 and antibodies (rheumatoid factor, lactoferrin antibodies, carbonic anhydrase II, etc) are usually increased. But the lack of specific biochemical markers is a major drawback in the diagnosis of autoimmune pancreatitis. The Japan Pancreas Society proposed diagnostic criteria for autoimmune pancreatitis as the presence antibodies, pancreas enlargement and pancreatic duct narrowing, lymphoplasmatic infiltration, response to corticosteroid therapy, and association with other autoimmune diseases such as autoimmune hepatitis, sclerosing cholangitis, primary biliary cirrhosis, sialoadenitis, inflammatory bowel disease and Sjögren syndrome. New criteria (HISORt Criteria) incorporate imaging changes, organ involvement, specific elevation of IgG4 subclass and histopathological markers. Autoimmune pancreatitis could be associated with diabetes mellitus and exocrine pancreatic dysfunction. Clinically, autoimmune pancreatitis is a disease with mild symptoms; severe attacks of abdominal pain are not typical. Typically, pancreatic calcifications and pseudocyst are absent; on the other hand jaundice and/or pancreatic mass are frequent signs, and both make differential diagnosis with pancreatic cancer difficult. From a practical point of view, in an elderly male presenting with obstructive jaundice and pancreatic mass, autoimmune pancreatitis is one of the differential diagnoses to avoid unnecessary surgical therapy.

Development of enzyme-linked immunosorbent assay for prostaglandin D 2 using the stable isosteric analogue as a hapten mimic and its application

For the determination of prostaglandin (PG) D 2 produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD 2 is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD 2. In an attempt to get a specific antibody for PGD 2, we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD 2, a novel, chemically stable, isosteric analogue of PGD 2. We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD 2. To develop the enzyme-linked immunosorbent assay (ELISA) for PGD 2, the immobilized antigen using the stable PGD 2 derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD 2. The optimization of the assay provided a sensitive calibration curve for PGD 2 from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD 2 was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD 2 in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD 2 in the maturation medium of adipocytes at 37 °C caused the chemical conversion into PGJ 2 derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD 2 before the conversion starts to occur.