Immunoglobulin Type Research Articles

Component-resolved diagnostics in the clinical and laboratory investigation of allergy.

The diagnosis and management of allergy is complex; the clinical symptoms associated with allergic reactions span a broad spectrum of severity, from mild hay fever-type symptoms through to life-threatening anaphylaxis. Obtaining an allergy-focused clinical history is therefore vital for identifying possible allergic triggers and directing testing. However, this focus could be changing as scientific and technological advances have paved the way for developments within in vitro testing for allergy. With knowledge of allergens at the molecular level expanding, there are now the facilities to characterize the sensitization profiles of allergy sufferers and determine the specific molecules (or components) against which the allergen-inducing immunoglobulin type E proteins have been produced. This technology is termed component-resolved diagnostics. We know that accurate identification of immunoglobulin type E specificity, the source of the causative allergen, and knowledge of potential allergic cross-reactivities are required for optimal clinical management of allergy patients. These factors can make allergy a diagnostic challenge outside of a specialist centre, and contribute to the difficulties associated with requesting and interpreting allergy tests. The incorporation of component-resolved diagnostics into current practice has provided a platform for patient-tailored risk stratification and improved the application of allergen-specific immunotherapy, revolutionizing specialist management of these patients. This review discusses the roles of each type of testing in allergy management and predictions for future pathways.

TOXOPLASMOSIS: SEROLOGIC PROFILE OF PREGNANT WOMEN AT THE BRAZZAVILLE UNIVERSITY CENTRE HOSPITAL (CHUB)

Introduction: Toxoplasmosis is a disease caused by an obligate intracellular coccidia Toxoplasma gondii, which is transmitted by cats. In pregnant women, it is a concern because of the severe complications to the foetus. The objective of this study is to determine the toxoplasma serologic profile in pregnant women at the Brazzaville University Centre Hospital (CHUB).
 Materials and Methods: This is a cross-sectional study from September 2015 to March 2016 (6 months) which includes sera from pregnant women received at the Parasitology Mycology laboratory of the CHUB for Toxoplasma serology. Immunoglobulins G and M searches were done by immuno-analysis (Biomerieux, Mini-Vidas technology). The data was analysed by the IBM SPSS version 20 software. The comparisons of proportion is done by the khi 2 test. The level of significance of statistical data were fixed at 5%.
 Results: The mean age of pregnant women included in our study was 27, 8+/- 6,84 with the extremes ages of 15 and 44 years.
 Toxoplasma seroprevalence in this study is 47,2% (68/144). The types of immunoglobulins (Ig) retrieved were IgG alone in 45,1% of cases (65/144), IgG associated to IgM in 2,8% of cases (4/144). Serological profiles were: no immunity (52,1%) immunised (41%), recent infection (1,4%) active infection (2,8%) equivocal result (2,8%).
 Conclusion: Toxoplasmosis is a zoonosis which represents a real public health issue in our environment, even when the level of immunised pregnant women seems high.

HISTOPATHOLOGICAL AND IMMUNOFLUORESCENT STUDIES IN CERTAIN SKIN DISORDERS

Background: This study was an attempt to evaluate the role of direct immunofluorescent technique to demonstrate the immunoglobulins in certain skin disorders, which are likely to have immunological mechanism in their pathogenesis.
 Methods: This study was carried out in department of Pathology, Sardar Patel Medical College & Associated group of Hospitals, Bikaner. This study was hospital based study on skin biopsy specimen recieved in the department of Pathology during the study period.
 Results: On immunofluorescent studies of these skin biopsies, 12 out of 16 cases of pemphigus were positive for fluorescence was intracellular area in epidermis. In lichen planus 5 out of 7 cases were positive for fluorescence and commonest site was dermo-epideremal junction. In dermatis-herpatiformis 2 out of 3cases were positive for fluorescence and commonest site was dermo-epideremal junction. IgG was commonest type of immunoglobulin’s demonstrate in 12 out of 16 cases of pemphigus followed by IgM (5 cases), IgA(1cases). In lichen planus IgM was commonest type of immunoglobulin’s demonstrate in 5 out of 7 cases of pemphigus followed by IgG (3 cases), IgA(1cases). In dermatitis herpatiformis IgA was commonest type of immunoglobulin’s demonstrate in 2 out of 3 cases of pemphigus followed by IgM (1 cases).
 Conclusion: We conclude that the demonstration of immunoglobulins in skin biopsies by direct fluorescent technique is a quite useful adjunct in diagnostic confirmation of pemphigus, lichen planus and dermatitis herpetiformis.
 Keywords: Direct immunofluorescence, Pemphigus, Histopathology.

Heavy/light chain assay in the monitoring of multiple myeloma

Serum protein (SPE) and immunofixation electrophoresis (IFE) have been extensively validated for the routine use of identifying, characterising and quantifying monoclonal proteins. However, accurate quantitation of IgA monoclonal proteins can be difficult when they migrate in to the β fraction, due to co-migration with transferrin and complement components. The heavy/light chain (HLC) immunoassay is an additional tool for measuring intact immunoglobulin monoclonal proteins. Therefore, we aimed to examine the clinical utility of the HLC assay for the disease monitoring of IgG and IgA multiple myeloma (MM) patients. A total of 177 samples from 30 MM patients (21 IgG and 9 IgA) were analysed retrospectively with median number of six follow up samples per patient (range 3-13). Serum free light chains (sFLC) and HLC were quantified using Freelite and Hevylite immunoassays. Details of M-protein concentration, β-globulin levels, total immunoglobulin levels and disease treatment response were obtained from the laboratory and patient information system. Passing-Bablok regression analysis was performed to compare (i) M-protein quantification with involved HLC (iHLC) and (ii) total immunoglobulin with summated HLC pairs for each immunoglobulin type (e.g., IgGκ+IgGλ). For 127 IgG MM samples, IgG iHLC levels showed a good correlation with SPE quantification (iHLC y=0.96x+4.9; r=0.917) and summated HLC showed a good correlation with total IgG concentration (summated HLC y=0.94x+5.74; r=0.91). In total, 95/127 (75%) IgG MM follow-up samples had an abnormal HLC ratio and 122/127 (96%) had a positive SPE, probably due to the lower sensitivity of HLC assay in detecting clonality in patients with IgG MM. Consistent with this, one patient assigned a very good partial response by International Myeloma Working Group criteria would be assigned a complete response based on HLC measurements. For 50 IgA MM samples, 42/50 (84%) had an abnormal HLC ratio. Conversely, 50/50 (100%) of M-proteins showed β fraction migration and were difficult to accurately quantify by SPE. Therefore, M-protein concentration and iHLC did not correlate as well in IgA MM (y=1.9x-8.4; r=0.8) compared to IgG MM. However, there was good correlation between total IgA and summated IgA HLC (IgAκ+IgAλ y=1.35x-0.33; r=0.95). Of the 8/50 (16%) IgA samples with a normal HLC ratio, 6/8 (75%) were consistent with the disease status being in complete remission. Interestingly, in one IgA MM patient, SPE and IFE were negative, but the serum FLC ratio and involved FLC were highly abnormal, consistent with the presence of light chain escape. Our data suggest HLC measurements could add value to the current disease monitoring of MM patients. In IgG MM patients, the M-protein level correlated well with HLC values. The HLC assay complements the serum FLC assay and is especially useful for monitoring of IgA MM patients who display M-proteins migrating in the β region on SPE.

Effect of herbal immunomodulators on immune status, haematological and serum biochemical parameters of Japanese quails (Coutrinix japanica) during summer stress

As the body temperature of birds rise, growth and production parameters tend to decline. In order to address the problem of heat stress, an experiment was conducted to investigate the effect of different herbal immunomodulators on the haemobiochemical and immune status of Japanese quails. Japanese quails (Coutrinix japanica) (120; day-old) were randomly divided into 4 treatment groups, viz. T1 (control diet), T2 (Stressroak liquid), T3 (Withania somnifera and Phyllanthus emblica liquid), T4 (Glycrrhiza glabra, Tribulus terrestris and Asparagas racemosus liquid) with 6 replicates of 5 birds each. The haematological data revealed that total erythrocyte count, packed cell volume, haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin were significantly high in all test groups (T2, T3 and T4) compared to control. Total leukocyte count, mean corpuscular haemoglobin concentration, total serum protein, albumin, globulin, A/G ratio, triglycerides, cholesterol and blood glucose were not affected by herbal immunomodulators at 5th week of age. Immune response as revealed by delayed type of hypersensitivity (measurement of skin thickness) and serum immunoglobulin were significantly high in all the test groups compared to control. It can be concluded that herbal immune modulators improve haemotological and immune status of Japanese quails without affecting serum biochemical parameters.

QIP-MS: A specific, sensitive, accurate, and quantitative alternative to electrophoresis that can identify endogenous m-proteins and distinguish them from therapeutic monoclonal antibodies in patients being treated for multiple myeloma

Serum protein (SPE) and immunofixation electrophoresis (IFE) have been extensively validated for the routine use of identifying, characterising and quantifying monoclonal proteins. However, accurate quantitation of IgA monoclonal proteins can be difficult when they migrate in to the β fraction, due to co-migration with transferrin and complement components. The heavy/light chain (HLC) immunoassay is an additional tool for measuring intact immunoglobulin monoclonal proteins. Therefore, we aimed to examine the clinical utility of the HLC assay for the disease monitoring of IgG and IgA multiple myeloma (MM) patients.A total of 177 samples from 30 MM patients (21 IgG and 9 IgA) were analysed retrospectively with median number of six follow up samples per patient (range 3–13). Serum free light chains (sFLC) and HLC were quantified using Freelite and Hevylite immunoassays. Details of M-protein concentration, β-globulin levels, total immunoglobulin levels and disease treatment response were obtained from the laboratory and patient information system. Passing–Bablok regression analysis was performed to compare (i) M-protein quantification with involved HLC (iHLC) and (ii) total immunoglobulin with summated HLC pairs for each immunoglobulin type (e.g., IgGκ+IgGλ).For 127 IgG MM samples, IgG iHLC levels showed a good correlation with SPE quantification (iHLC y=0.96x+4.9; r=0.917) and summated HLC showed a good correlation with total IgG concentration (summated HLC y=0.94x+5.74; r=0.91). In total, 95/127 (75%) IgG MM follow-up samples had an abnormal HLC ratio and 122/127 (96%) had a positive SPE, probably due to the lower sensitivity of HLC assay in detecting clonality in patients with IgG MM. Consistent with this, one patient assigned a very good partial response by International Myeloma Working Group criteria would be assigned a complete response based on HLC measurements. For 50 IgA MM samples, 42/50 (84%) had an abnormal HLC ratio. Conversely, 50/50 (100%) of M-proteins showed β fraction migration and were difficult to accurately quantify by SPE. Therefore, M-protein concentration and iHLC did not correlate as well in IgA MM (y=1.9x–8.4; r=0.8) compared to IgG MM. However, there was good correlation between total IgA and summated IgA HLC (IgAκ+IgAλ y=1.35x–0.33; r=0.95). Of the 8/50 (16%) IgA samples with a normal HLC ratio, 6/8 (75%) were consistent with the disease status being in complete remission. Interestingly, in one IgA MM patient, SPE and IFE were negative, but the serum FLC ratio and involved FLC were highly abnormal, consistent with the presence of light chain escape.Our data suggest HLC measurements could add value to the current disease monitoring of MM patients. In IgG MM patients, the M-protein level correlated well with HLC values. The HLC assay complements the serum FLC assay and is especially useful for monitoring of IgA MM patients who display M-proteins migrating in the β region on SPE.

Features of humoral answer in experimental animal tularemia with different sensitivity to infection

Tularemia is an anthropozoonotic infection caused by Francisella tularensis. In clinical and sanitary-epidemiological practice, traditional diagnostics methods in tularemia are based on serological assays for detecting specific antibodies, allowing to diagnose it and estimate durability of patients’ immunity after vaccination. Previously, it was shown that specific serum antibodies in patients recovered after tularemia, unlike to those vaccinated with the live tularemia vaccine F. tularensis 15 NIIEG, can interact with specific epitopes on lipopolysaccharides isolated from strains of various subspecies — F. tularensis (Ft) and F. novicida (LPS Fn), while LPS Fn-specific immunoglobulins are lacked in the blood of vaccinated individuals. A set of experiments on identifying antibodies with similar specificity in laboratory animals of various species — mice, guinea pigs and rats with differed sensitivity to tularemia, administered with live tularemia vaccine strain as well as virulent F. tularensis strains to simulate vaccine-mediated and infectious processes, respectively was conducted. A methodical approach has been developed that allows to analyze humoral response in modelled infectious process in animals highly sensitive to tularemia such as BALB/c mice and guinea pigs that consisted of preliminary immunization with live tularemia vaccine followed by infection with virulent F. tularensis strains. It was shown that induction of specific anti-LPS Ft antibodies occured in these animal species, both after vaccination and infection with virulent strains. It was noted that, unlike guinea pigs and rats, mice both during vaccination and infection were characterized by significantly lower titers of LPS Ft-specific antibodies. However, no specific interaction between mouse serum and LPS Fn might be detected. Moreover, two types of immunoglobulins with different antigen specificities to the LPS Ft and LPS Fn epitopes were detected by dot-blot analysis in guinea pigs immunized with live tularemia vaccine, followed by infection with a virulent strain. In addition, antibodies to LPS Fn were also detected in the serum of rats infected with virulent, but not vaccine-based, F. tularensis strains. Thus, previous experimental data on the production of immunoglobulins with different antigenic specificity were confirmed in an experimental tularemia modelled in rats and guinea pigs that demonstrated a diagnostic significance and feasibility of using LPS Fn to confirm tularemia infection in humans.

Incidence Analysis of Monoclonal Gammopathy of Undetermined Significance in People over 40 Years of Age

To study the distribution of monoclonal gammopathy of undetermined significance(MGUS) in different age, sex and ethnic people over 40 years old. Five hundred and ninety-six people(over 40 years old) examened in the Health Examination center of the First Affiliated Hospital of Xinjiang Medical University from July 2017 to September 2017 were selected. Among 596 people, male 310, female 286, Han people 488, and Uygur ethnic people 108. According to age, 596 people were divided into 3 groups, (40-59 years old group, 60-79 years old group, over 80 years old group). First, all samples were screened by capillary serum protein electrophoresis. If the suspected monoclonal bands were found in the electrophoretogram, and then the specific protein types were determined by serum immunofixation electrophoresis. The total incidence of MGUS in 596 screened population was 4.027%. The incidence of MGUS in 40-59 years old group, 60-69 years old group and over 80 years old group were 1.762%, 2.929% and 10% respectively, and the differences among the groups were statistically significant(P<0.05). The incidence of MGUS in male (5.806%) was significantly higher than that in female (2.097%)(χ2=5.177，P<0.05). Binary Logistic regression analysis showed that over 80 years old and male were independent risk factors for MGUS(P=0.001, OR=4.188, 95%CI: 1.814-9.673, P=0.048, OR=2.605, 95%CI: 1.009-6.725). The types of immunoglobulin in patients with MGUS were mostly IgG, IgG(66.7%) was significantly more than IgA (29.2%)(χ2=21.375，P<0.05)，and there was no significant difference in the incidence of MGUS between people with in Kappa and Lambda. The age increase and male may increase the incidence of MGUS, the IgG is the most common type of immunoglobcdin in pathogenesis of MGUS, so the early screening should be done.

Intralaboratory Reliability and Variability for Allergen-Specific Immunoglobulin Type E Serology Testing.

Atopic dermatitis is a very common condition affecting dogs and often managed with allergen-specific immunotherapy, which requires accurate identification of causative allergens. Serology testing is used commonly. Serum was collected from 35 atopic dogs and separated into three samples each (1, 2, and 3). Samples 1 and 2 were sent to IDEXX Laboratories the same day; sample 3 was stored at -80°C and submitted ∼30 days later. Specific immunoglobulin type E reactivity to various allergens were determined using monoclonal anti-canine enzyme-linked immunosorbent assay (ELISA) and expressed as ELISA absorbance units. Percent difference ranged from 14.30 to 127.34% for samples 1 and 2. These values increased when comparing samples a month apart (21.78 to 129.65%). Between samples 1 and 2, for each allergen there were differences in interpretation 15.18% of the time; 32 of 35 dogs (91.4%) had at least one allergen with a different interpretation. Comparing sample 3 and the average of samples 1 and 2, differences in interpretation increased to 22.32%; all dogs had at least one allergen that was interpreted differently. These differences in interpretation can alter immunotherapy. Overall, results show the need for better reliability for allergen-specific immunoglobulin type E serology testing using monoclonal anti-canine ELISA.

Allergen immunotherapy improves defective follicular regulatory T cells in patients with allergic rhinitis

The function of follicular regulatory T (TFR) cells, especially in regulating IgE production in patients with allergic diseases, is poorly understood. We sought to investigate the phenotype, function, and clinical relevance of TFR cells in patients with allergic rhinitis (AR). The phenotype and frequency of tonsillar and circulating TFR cells were characterized by using flow cytometry. TFR cell function was examined in an assay by coculturing with follicular helper T cells and B cells. The associations between TFR cells and the clinical features in patients with AR before and after allergen immunotherapy (AIT) were analyzed. TFR cells were detected in germinal centers of tonsils, but compared with subjects without AR, the frequencies decreased in patients with AR who were allergic to house dust mites. Circulating TFR cells in blood were phenotypically and numerically correlated with tonsillar TFR cells, and a reduction of circulating TFR cells but not total or CXCR5- regulatory T cells was noted in patients with AR compared with healthy control subjects. Moreover, circulating TFR cells in patients with AR showed a specific defect in suppressing IgE production but were capable of suppressing production of other immunoglobulin types. We identified negative associations of circulating TFR cell frequencies and function with antigen-specific IgE levels or disease severity in patients with AR. After AIT, the frequencies and function of circulating TFR cells were improved, which positively associated with disease remission. Impairment in TFR cells might contribute to aberrant IgE production in patients with AR, and AIT improves defective TFR cell function. TFR cells might serve as a potential biomarker to monitor clinical response to AIT.

Expression and Clinical Significance of C/EBPα Gene in Elderly Multiple Myeloma

To investigate the expression of C/EBPα gene in elderly patients with multiple myeloma （MM） and its prognostic significance. Sixty-nine olderly patients with multiple myeloma (MM) treated in our hospital from February 2015 to October 2017 were selected and enrolled in the MM group, 38 healthy persons received physical examination were selected and enrolled in the control group. The bone marrow of 2 groups was collected and the mononuclear cells were isolated.The mRNA expression level of C/EBPα gene in mononuclear cells was determined by RT-PCR, the Western blot was used to detect the protin expression level of PBMNC C/EBPα, and the protein level of C/EBPα in bone marrow was detected by immunohistochemistry. The correlations of C/EBPα gene expression with the clinical characteristics and survival time in MM patients were analyzed. The expression level of mRNA and protein of C/EBPα in MM patients was significantly lower than that in the control group (P<0.05). The expression level of C/EBPα gene significantly correlated with the ISS stage, CRP, Calcium, β2-MG, LDH and the percentage of myeloma cells in MM patients (P<0.05). The expression of C/EBPα gene was not correlate with sex, age, immunoglobulin typing, Hb in MM patients (P>0.05).Immunohistochemical staining showed that the bone marrow samples of the control group were stained more deeply, and the staining intensity in bone marrow samples of MM patients with CR, PR and relapse was successively descended. The protein level of C/EBPα in CR patients with MM was significantly higher than that in PR and relapsed patients by Western blot (P<0.05). Kaplan-Meier survival analysis showed that OS and DFS in the patients with high expression of C/EBPα gene were higher than those in low expression group (P<0.05). Multivariate Cox regression analysis showed that CRP,ratio of myeloma cells and C/EBPα gene were independent factors affecting OS and PFS (P<0.05). The expression level of C/EBPα gene in MM patients is low that may stimulate the genesis of MM, and the expression of C/EBPα gene closely relates with the development of MM disease.

Is complement C1q a potential marker for tumor burden and immunodeficiency in multiple myeloma?

Multiple myeloma (MM) patients are immunodeficient. Complement C1q is an important cofactor of both nonspecific and humoral immunity, and it participates in the immunomodulation of multiple tumors. Thus, this study aimed to determine the risk factors and clinical significance of C1q expression in MM patients. In total, 193 MM patients were examined. The mean value of C1q in the patient group (130.46 ± 36.17 mg/L), was lower than that in the control group (anemia and nonmalignant hematologic disease). C1q dynamically changed with different MM stages and was recovered to normal levels when the disease was in remission; however, it decreased again after disease progression. The risk factors of C1q reduction included Durie-Salmon stage and the immunoglobulin type of the disease. In conclusion, C1q appears to be a useful biomarker of tumor burden and a prognostic factor of disease relapse. C1q is a potential marker of the immunodeficiency status in MM patients.

Pollen/Fruit Syndrome: Clinical Relevance of the Cypress Pollen Allergenic Gibberellin-Regulated Protein.

A pollen/food-associated syndrome (PFAS) has been described between peach and cypress pollen. Cross-reactive allergens were characterized which belong to the Gibberellin-regulated protein (GRP) family, BP14 in cypress pollen and Pru p 7 in peach. GRP are small cationic protein with anti-microbial properties. A patient suffering from a peach/cypress syndrome was explored clinically and biologically using 2 types of immunoglobulin E (IgE) multiarray microchip, immunoblots and a basophil activation test to assess the clinical relevance of various extracts and purified allergens from fruits or cypress pollen. In addition to PR10 sensitization, the patient showed specific IgE to Pru p 7, BP14 and allergen from pomegranate. These last 3 allergens and allergenic sources are able to induce ex vivo basophil activation characterized by the monitoring of the expression of CD63 and CD203c, both cell surface markers correlated with a basophil mediator release. Up to 100% of cells expressed CD203c at 50 ng/mL of BP14 protein. In contrast, snakin-1, a GRP from potato sharing 82% sequence identity with Pru p 7 did not activate patient's basophils. These results strongly suggest that, like Pru p 7, BP14 is a clinically relevant allergenic GRP from pollen. Allergen members of this newly described protein family are good candidates for PFAS where no cross-reactive allergens have been characterized.

Facilities and services of postexposure prophylaxis in anti-rabies clinics: A national assessment in India.

The rabies postexposure prophylaxis (PEP) is provided through anti-rabies clinics in the country. It was considered important to assess their facilities under a nationwide multi-centric survey. The objective of this study is to assess the facilities available for PEP at the anti-rabies clinics and to ascertain the PEP provided at the anti-rabies clinics. The cross-sectional assessment was made from May 2017 to January 2018 in selected seven states of India. Thirty-five anti-rabies clinics from both Government and private; urban and rural areas from the states were assessed by an expert team using a pretested checklist for facilities and services available for PEP. On an average, 10 new animal bite cases were attended at each anti-rabies clinic per day. The cold chain facilities for rabies biologicals were satisfactory. The facilities for wound washing (54.3%) and the use of antiseptics to animal bite wounds were not adequate. Rabies vaccines were administered by intramuscular in 54.3% and by intradermal route in 45.7% of the cases. The vaccine stock-outs were reported only in the government sector (18.5%). The type of rabies immunoglobulin (RIG) used was equine (63.2%) and human (36.8%); given free of cost in 40% of Anti rabies clinics. The local wound infiltration of RIG was in practice at 58.6% of anti-rabies clinics. The stock-out of RIG was more in private (50%) hospitals than in Government (40.7%) hospitals. The facilities available for PEP at the anti-rabies clinics were inadequate and have to be improved across the country.

Advances in Intestinal Mucosal Immunoglobulins of Teleost Fish (Review)

Immunoglobulins are globulins with antibody activity or antibody-like structures, and fish were the first vertebrates to develop immunoglobulins. There are three major types of immunoglobulins (IgM, IgD and IgZ) in teleost fish, which play an important role in resisting pathogen infection. For fish, the intestinal tract is not only the place for nutrient digestion but also the largest endocrine organ. The intestinal mucosal barrier system can block pathogenic microorganisms and toxins and is important for immunologic defense. Therefore, the study of the molecular mechanism of fish immunoglobulin in intestinal mucosa is of great significance to the selection of immune prevention strategies for fish diseases. At present, information on the immune system of intestinal mucosa in fish is scarce, and there are still many problems in the function of newly identified antibodies, which need further research. In this study, we have summarized current knowledge about the types, gene structure, biological function, and immune response of immunoglobulins in teleost fish, and intestinal mucosal immune tissue of fish. The scientific questions in this field were also presented according to the progress from the latest research in fish immunology. The aim of present study is to provide a theoretical basis for the study of fish immunology.

Advances in Intestinal Mucosal Immunoglobulins of Teleost Fish (A Review)

Immunoglobulins are globulins with antibody activity or antibody-like structures, and fish were the first vertebrates to develop immunoglobulins. There are three major types of immunoglobulins (IgM, IgD and IgZ) in teleost fish, which play an important role in resisting pathogen infection. For fish, the intestinal tract is not only the place for nutrient digestion but also the largest endocrine organ. The intestinal mucosal barrier system can block pathogenic microorganisms and toxins and is important for immunologic defense. Therefore, the study of the molecular mechanism of fish immunoglobulin in intestinal mucosa is of great significance to the selection of immune prevention strategies for fish diseases. At present, information on the immune system of intestinal mucosa in fish is scarce, and there are still many problems in the function of newly identified antibodies, which need further research. In this study, we have summarized current knowledge about the types, gene structure, biological function, and immune response of immunoglobulins in teleost fish, and intestinal mucosal immune tissue of fish. The scientific questions in this field were also presented according to the progress from the latest research in fish immunology. The aim of present study is to provide a theoretical basis for the study of fish immunology.

Modulation of Immune Tolerance via Siglec-Sialic Acid Interactions.

One of the key features of the immune system is its extraordinary capacity to discriminate between self and non-self and to respond accordingly. Several molecular interactions allow the induction of acquired immune responses when a foreign antigen is recognized, while others regulate the resolution of inflammation, or the induction of tolerance to self-antigens. Post-translational signatures, such as glycans that are part of proteins (glycoproteins) and lipids (glycolipids) of host cells or pathogens, are increasingly appreciated as key molecules in regulating immunity vs. tolerance. Glycans are sensed by glycan binding receptors expressed on immune cells, such as C-type lectin receptors (CLRs) and Sialic acid binding immunoglobulin type lectins (Siglecs), that respond to specific glycan signatures by triggering tolerogenic or immunogenic signaling pathways. Glycan signatures present on healthy tissue, inflamed and malignant tissue or pathogens provide signals for “self” or “non-self” recognition. In this review we will focus on sialic acids that serve as “self” molecular pattern ligands for Siglecs. We will emphasize on the function of Siglec-expressing mononuclear phagocytes as sensors for sialic acids in tissue homeostasis and describe how the sialic acid-Siglec axis is exploited by tumors and pathogens for the induction of immune tolerance. Furthermore, we highlight how the sialic acid-Siglec axis can be utilized for clinical applications to induce or inhibit immune tolerance.

20 Years of Transplant Program in Multiple Myeloma - a Single Centre Experience

Aims: The aim is to present the results of 20 years of autologous stem cell transplant (ASCT) program in multiple myeloma (MM) at our site.Patients and methods: Since 1997 till 2017, a cohort of 348 patients underwent ASCT at the Department of Hemato-Oncology of the University Hospital Olomouc. Altogether 274 were 1st ASCT, 62 were 2nd transplants, the rest were tandem or third ASCT. The patients had standard baseline characteristics, including age, gender, immunoglobulin and light chain type, Durie-Salmon (DS) and International Staging System (ISS).The induction regimens for 1st ASCT included VAD (vincristin, adriamycine, dexamethasone) in 34%, THAL in 15%, BTZ based regimens in 34%, the rest (17%) were combined polychemotherapeutic regimens. The re-induction regimens before 2nd ASCT included CAD (cyclophosphamide, adriamycine, dexamethasone) in 27%, BTZ in 31% and THAL based regimens in 18%, other patients (24%) had LEN based regimens, polychemotherapy or no re-induction. 163 patients (47%) had maintenance, mostly interferon based (25%), conventional chemotherapy based (9%) or THAL based (7%), and 53% of patients had no maintenance.We assessed response rates, ie the rate of complete remission (CR), very good partial remission (VGPR), partial remission (PR), minimal response (MR), stable disease (SD), progressive disease (PG) and overall response rate (ORR) based on International Myeloma Working Group (IMWG) criteria. The survival measures of individual treatment lines were assessed by means of progression free survival (PFS). We assessed the efficacy of ASCT with respect to treatment line, pre-transplant and post transplant response achieved (day+100), and with respect to DS and ISS as well as to treatment modality used for induction and maintenance.For statistical estimation we used Kaplan-Meier curves, Log rank test (Mantel-Cox), Post-hoc tests according to Dunn, Pearson ChiSquare test, Fisher´s Exact Test, Kruskal-Wallis test, and McNemar-Bowker Test.Results: The response rates after 1st ASCT were following: CR 32,1%, VGPR 26,5%. PR 34,8%, MR and SD 3% and PG 3,4%. Median PFS after 1st ASCT was 35 months and it corresponded to the depth of treatment response: CR 48 months, VGPR 36 months, PR 33 months, MR and SD 13 months, PG 9 months, p<0,0001. Pre-transplant responses showed a trend towards better outcomes with deeper responses but beyond statistical significance (p = 0,077). Advanced DS stage correlated with worse PFS (stage I 58 months, stage II 42 months, stage III 31 months). Patients under age 59 years tended to have slightly better PFS (37 vs 34 months, p = 0,078).Both pre and posttransplant responses were significantly better after novel drugs than conventional chemotherapy, still, there was no statistically significant difference in PFS. None of the maintenance (chemotherapy, interferon, THAL-based) lead to a better PFS. There were very few patients treated with BTZ, LEN or ixazomib maintenance precluding valid statistical analysis. There were no differences in either responses or PFS with regard to treatment line but there were only 19 patients not having ASCT as their first regimen.The response rates after 2nd ASCT were as follows: CR 26,7%, VGPR 13,3%, PR 38,3%, MR+SD 8,3% and PG 13,3%. Median PFS after 2nd ASCT was 20 months, decreasing with inferior responses (CR 34,8 months, VGPR 22,5 months, PR 33,1 months, MR and SD 18,8 months, PG 6,6 months p<0,0001). There were no differences in PFS with respect to either ISS, DS stage or re-induction regimen. Patients transplanted in their first relapse tended to have better PFS than in later relapses.Conclusion: The use of ASCT is still the gold standard in MM. Regardless of treatment line, it achieves significant outcomes. Novel drugs induce deeper pre-transplant responses, still, the major advantage against conventional chemotherapy is in the speed of response and absence of severe adverse events, enabling more patients to reach ASCT. Maintenance therapy accounts for better survival, however, this is true only for novel drugs (such as bortezomib, lenalidomide) whereas older modalities including interferone, post-transplant chemotherapy, steroids or thalidomide do not possess such activity. The second ASCT is comparable to to current treatment modalities in relapsed MM with fair post-transplant outcomes as well as PFS, with high rate of complete responses.Supported by the grant IGA-LF-2018-004 andMH CR - RVO (FNOl, 00098892). DisclosuresNo relevant conflicts of interest to declare.

Donor Cell Myeloma: A Unique Case

Leukemia relapse occurring in donor cells, so called donor cell leukemia (DCL) after allogeneic hematopoietic stem cell transplantation has been previously reported in the literature. Some authors have suggested that the development of DCL is perhaps a more common occurrence than traditionally thought. Donor cell myeloma (DCM) seems to be less frequent than DCL. This 46-year old male when first seen in 2000 was diagnosed with stage IIIa multiple myeloma. A monoclonal IgA kappa spike was recorded at diagnosis. Treatment with melphalan and prednisone was delivered every four to six weeks for a total of 22 courses. Fourty months after the initial diagnosis, an M2 acute myelogenous leukemia was identified. Treatment with chemotherapy resulted in complete remission. Matched UCB cells were localized at the London Cord Blood Bank. The UCB belonged to a male product of a white western European mother and a black Nigerian father who was a carrier of hemoglobin S. Hemoglobins A, F and S were detected in the UCB, consonant with sickle cell trait. The patient was allografted employing the “Mexican” NST conditioning regimen, granulocyte count recovered to more than 0.5 x 109/L on day 14, with the platelet count never dropping below 20 x 109/L. On day +40, the polymorphic microsatellite markers revealed mixed chimerism. The hemoglobin S gene was identified on day +20 and on day +60, full chimerism was shown. Cyclosporine A was stopped on day +350. The patient returned 170 months after the transplant with low back pain and the bone marrow aspiration disclosed 80% abnormal plasma cells, an IgA kappa monoclonal spike of 3.1 gr/dl, and complete chimerism. Malignant plasma cells were sorted by means of flow cytometry before genetic fingerprinting; cells were stained with an admixture of fluorescent monoclonal antibodies and cells co-expressing dim CD45, bright CD38 and CD56 were sorted out to ≥99% purity. Sorted cells were shown to have donor origin (Figure 1). The patient was treated with thalidomide, dexamethasone and bortezomib and the monoclonal spike disappeared; an autologous stem cell transplant is planned. Most people consider that the development of a malignancy in the cells of the donor is a rare event and very few prospective studies have analyzed the real prevalence of this phenomenon. Prospectively, we have found that 7% (95% CI 2.9 to 13.6%) of patients with leukemic activity after an allogeneic graft do have a donor cell-derived leukemia; this figure contrasts with those described elsewhere in non-prospective studies. A major problem in the analysis of donor cell derived malignancies is that demonstration of the donor cell origin of malignant activity. In this case, the demonstration of DNA of the donor in the fluorescence-activated sorted malignant plasma cells is indicative of the origin of the myeloma cells. Interestingly, the immunoglobulin type produced by the initial myeloma cells is the same as that of the donor-cell myeloma; Despite being two myelomas producing the same immunoglobulin subtype, both should be considered as de novomalignancies and as such, treated; we have previously shown that donor cell leukemias do have a response when treated as de novo, non-secondary leukemias. To our best knowledge, this is the second report of DCM following allogeneic HSCT. Prior to this case, Kim et al reported a DCM after an allogeneic transplant in a patient with refractory anemia with ringed sideroblasts. Previously, two cases have been reported of donor-origin MM, but they occurred in patients who underwent solid organ transplantation of the kidney and heart-lung. Kumar et alreported a case of DCM developing after unrelated allogeneic HSCT in the both donor and recipient but they did not conducted a comprehensive molecular cytogenetic study. In the case published by Maestas et al, an abnormal proliferation of plasma cells was identified in the donor, thus making possible that a malignant plasma cell clone was already present in the donor stem cells. In summary, we have clearly shown that this patient has had three different malignancies: 1) De novomultiple myeloma, 2) Secondary acute myelogenous leukemia and 3) De novodonor cell-derived multiple myeloma. The mechanisms involved in these episodes could be useful to better understand tumorigenesis. DisclosuresNo relevant conflicts of interest to declare.

Changes of blood coagulation indicators in patients with lymphoplasmacytic lymphoma and their clinical significances

Objective To observe the alteration and clinical significances of blood coagulation indicators in patients with lymphoplasmacytic lymphoma (LPL). Methods Twenty patients who were newly diagnosed LPL in the First People's Hospital of Changzhou from January 2008 to October 2017 and twenty healthy controls were studied. The patients were treated by chemotherapy, plasma exchange, supplement of coagulation factor or other supportive therapy. The parameters of prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT), D-dimer (D-D), and platelet count (Plt) were detected in LPL group and healthy controls. Results The levels of PT and APTT in LPL group were dramatically higher than those in control group [(12.9±1.2) s vs. (11.6±0.9) s, (41.7±9.8) s vs. (24.7±2.9) s], and the level of Plt in LPL group was lower than that in control group [112×109/L (3×109/L-379×109/L) vs. 210×109/L (170×109/L-271×109/L)], and the differences were statistically significant (all P 0.05). There were no statistical differences in PT, APTT, FIB, TT, D-D and Plt levels among LPL patients with different types of immunoglobins (all P > 0.05). After treatment, all the coagulation abnormalities got relieved and no patient died of hemorrhage or thrombosis. Conclusions The LPL patients have coagulation disorders and hypercoagulability, and this is independent of the type of immunoglobulin. Clinical attention should be paid to monitoring coagulation indicators to prevent the occurrence of adverse reactions. Key words: Lymphoplasmacytic lymphoma; Blood coagulation factors; Platelets