Influence of road salt thawing peaks on the inflow composition and activated sludge properties in municipal wastewater treatment.

Operational data over 2 years from three large Austrian wastewater treatment plants (WWTPs) with design capacities of 4 million, 950,000 and 110,000 population equivalent (PE) were examined. Salt peaks, due to thawing road salt were detected and quantified by electrical conductivity, temperature and chloride measurement in the inflow of the WWTPs. Daily NaCl inflow loads up to 1,147 t/d and PE-specific loads of 0.26-0.5 kg NaCl/(PE · y) were found. To mimic the plants' behaviour in a controlled environment, NaCl was dosed into the inflow of a laboratory-scale activated sludge plant. The influence of salt peaks on important activated sludge parameters such as sludge volume index, settling velocity and floc size were investigated. Influent and effluent were sampled extensively to calculate removal rates. Respiration measurements were performed to quantify activated sludge activity. Particle size distributions of the activated sludge floc sizes were measured using laser diffraction particle sizing and showed a decrease of the floc size by approximately two-thirds. The floc structure was examined and documented using light microscopy. At salt concentrations below 1 g/L, increased respiration was found for autotrophic biomass, and between 1 and 3 g NaCl/L respiration was inhibited by up to 30%.

Association of the length of time using computers and mobile devices with low back, neck and mid-back pains: findings from a birth cohort.

OBJECTIVES: Neck and low back pains are the leading causes of years lived with disability, and using computers or mobile devices in excess could be risk factors for back pain. Our aim was to evaluate the association of the length of time using computers and mobile devices with neck, mid-back and low back pains and the number of regions with pain. STUDY DESIGN: Cross-sectional study nested in the 1993 Pelotas birth cohort with young adults aged 22 years. METHODS: Outcomes analyzed were neck, mid-back and low back pains and the number of regions with pain. Exposures were the number of daily hours using computers and mobile devices. Crude and adjusted analyses were performed to estimate prevalence ratios using Poisson regression. RESULTS: Almost half of the sample reported having back pain, the low back pain being the most prevalent. Compared with individuals using mobile devices for less than one hour, the prevalence of neck pain was 1.41 and 1.81 times higher among individuals using mobile devices from three to seven hours and for seven or more hours per day, respectively. Neck pain prevalence was 1.47 times higher among individuals using computers for more than two hours than among those not using computers. Using mobile devices for seven hours or more was associated to 1.19 times higher prevalence of low back pain. CONCLUSION: Using mobile devices in excess was associated to neck and low back pains, while the use of computers in excess was associated only to neck pain. It is important that guidelines are developed to recommend the adequate length of time that computers and mobile devices should be used to prevent back pain.

HLA Class II Differentiates Between Thyroid and Polyglandular Autoimmunity.

The HLA class II genes are susceptibility genes for autoimmune endocrine diseases; however, scarce data are available pertaining to the determinants of genetic susceptibility to polyglandular autoimmunity (PGA). A total of 300 consecutive and unselected patients with either PGA or monoglandular autoimmune thyroid disease (AITD) and 100 healthy control subjects were genotyped for the HLA class II DRB1, -DQA1, and -DQB1 alleles. Compared to patients with AITD and controls, the HLA-DRB1*03 (pc =0.001), *04 (pc<0.001), -DQA1*03 (pc<0.001), and -DQB1*02 (pc =0.001) alleles were increased in patients with PGA. When dividing patients with Hashimoto's thyroiditis (HT) into those with PGA (PGA-HT) vs. those with HT as monoglandular disease, significant differences for the DRB1*03 (pc=0.001) and DQA1*03 (pc=0.001) alleles were observed. In contrast, the DQB1*02 allele was more prevalent in PGA patients with Graves' disease (PGA-GD) vs. those with monoglandular GD (pc=0.002). The HLA-DRB1*15 (pc =0.001), -DQA1*01 (pc =0.001), -DQB1*05 (pc =0.002) and -DQB1*06 (pc =0.002) alleles were significantly less present in PGA compared to monoglandular AITD and controls, thus indicating protective alleles. The HLA class II alleles differentiate between mono- and polyglandular autoimmunity in patients with autoimmune thyroid disease.

HLA class II haplotypes differentiate between the adult autoimmune polyglandular syndrome types II and III.

BACKGROUND: Genetics of the adult autoimmune polyglandular syndrome (APS) is poorly understood. AIM: The aim of this study was to gain further insight into the genetics of the adult APS types. SITE: The study was conducted at a university referral center. METHODS: The human leukocyte antigen (HLA) class II alleles, haplotypes, and genotypes were determined in a large cohort of patients with APS, autoimmune thyroid disease (AITD), and type 1 diabetes and in healthy controls by the consistent application of high-resolution typing at a four-digit level. RESULTS: Comparison of the allele and haplotype frequencies significantly discriminated patients with APS vs AITD and controls. The HLA class II alleles DRB1*03:01 *04:01, DQA1*03:01, *05:01, DQB1*02:01, and *03:02 were observed more frequently (P<.001) in APS than in AITD and controls, whereas the alleles DRB1*15:01, DQB1*03:01, and *06:02 were underrepresented in APS vs AITD (Pc<.001) and controls (Pc<.01), respectively. The DRB1*03:01-DQA1*05:01-DQB1*02:01 (DR3-DQ2) and DRB1*04:01-DQA1*03:01:DQB1*03:02 (DRB1*04:01-DQ8) haplotypes were overrepresented in APS (Pc<.001). Combination of both haplotypes to a genotype was highly prevalent in APS vs AITD and controls (Pc<.001). Dividing the APS collective into those with Addison's disease (APS type II) and those without Addison's disease but including type 1 diabetes and AITD (APS type III) demonstrated DR3-DQ2/DRB1*04:01-DQ8 as a susceptibility genotype in APS III (Pc<.001), whereas the DR3-DQ2/DRB1*04:04-DQ8 genotype correlated with APS II (Pc<.001). The haplotypes DRB1*11:01-DQA1*05:05-DQB1*03:01 and DRB1*15:01-DQA1*01:02-DQB1*06:02 are protective in APS III but not in type II (Pc<.01). CONCLUSIONS: HLA class II haplotypes differentiate between the adult APS types II and III. Susceptible haplotypes favor the development of polyglandular autoimmunity in patients with AITD.

TRALI-new challenges for histocompatibility and immunogenetics in transfusion medicine.

Antibodies against human leukocyte antigens (HLAs) have long been associated with transfusion-related acute lung injury (TRALI). In contrast to febrile transfusion reactions and refractoriness to platelet transfusions in immunized patients, the causative antibodies in TRALI are present in the transfused blood component, i.e. they are formed by the blood donor and not by the recipient. Consequently, blood components with high plasma volume are particularly associated with TRALI. In addition to antibodies against HLAs, antibodies directed against human neutrophil antigens (HNAs) present in the plasma of predominantly multiparous female blood donors can induce severe TRALI reactions. Especially, antibodies to HLA class II and HNA-3a antigens can induce severe or even fatal ALI in critically ill patients. Over the last decade, the clinical importance of TRALI as major cause for severe transfusion-related morbidities has led to the establishment of new guidelines aimed at preventing this condition, including routine testing for HLA and -HNA antibodies for plasma donors with a history of allogeneic sensitization. This, in turn, poses new challenges for close collaboration between blood transfusion centers and histocompatibility and immunogenetics laboratories, for sensitive and specific detection of the relevant antibodies.

Detection of granulocyte antibodies using simultaneous analysis of specific granulocyte antibodies assay (SASGA).

OBJECTIVE: Granulocyte-associated antibodies can cause several clinical granulocytopenic disorders. The monoclonal-antibody-specific immobilization of granulocyte antigens (MAIGA) is currently used as the standard assay to specify these antibodies. Here we describe an assay for specific analysis of granulocyte antibodies (SASGA) which is able to simultaneously detect and specify granulocyte IgG- and IgM-antibodies using flow cytometry. METHODS: Bead populations with distinct fluorescence intensities were used as solid phase for immobilization of mAb. Typed granulocytes were incubated with human sera and a mix of three distinct mouse monoclonal antibodies against specific granulocyte antigens (for example CD16, CD11a, HLA class I). After cell lysis and incubation of lysate with beads, goat antibodies against human IgG and IgM antibodies were added. Seventy-one frozen sera of donors and patients previously implicated in transfusion reactions and various underlying disorders were analysed for specific granulocyte-binding antibodies using MAIGA and SASGA. RESULTS: The SASGA assay was able to simultaneously detect granulocyte-specific antibodies for different glycoproteins. Overall, the results of MAIGA and SASGA were concordant in 92·9%. 5 sera containing anti-HNA-1b (n=2) and -HLA class I (n=3) were not detected by MAIGA, but were recognized by the SASGA. In serial dilution tests with sera containing anti-HNA-1a, -1b, -2a and HLA class I, the SASGA assay detected the antibodies at higher dilutions than MAIGA. CONCLUSION: The SASGA assay permits reliable detection of specific granulocyte antibodies. Six distinct antibodies can be simultaneously determined. This method will potentially open the way to investigations on additional specific antibodies as it facilitates laboratory diagnosis.

Evidence for gene recombination in FCGR3 gene variants.

BACKGROUND AND OBJECTIVES: The genes encoding the Fcgamma receptors (FcgammaR) IIIa and IIIb (FCGR3A and FCGR3B) are clustered on chromosome 1 band q23-24 and exhibit allelic polymorphism. We investigated the molecular basis of additional new FCGR3 genomic variation. MATERIALS AND METHODS: A segment shared by FCGR3A and FCGR3B containing the polymorphic nucleotide positions 141, 147, 227, 266, and 277 in exon 3 was cloned and sequenced from genomic DNA of 30 donors and 3 bacterial artificial chromosome (BAC) clones. A mixture consisting of isolated FCGR3B*2- and FCGR3A- plasmids was cloned and sequenced as well. Additionally, nucleotide databases were screened for clones with variant FCGR3 sequences. RESULTS: A total of 12 FCGR3 variants defined by the polymorphic positions were detected in whole blood genomic DNA from 23 of 24 FCGR3B*2 and/or FCGR3B*3 positive donors, the DNA from two of three BAC clones and in the DNA mixture of isolated FCGR3B*2- and FCGR3A- plasmids. CONCLUSION: Nucleotide exchanges of the variants were non-random and resulted from two alternative nucleotides present in one of the polymorphic position of the basic FCGR3 forms. Polymerase chain reaction (PCR) artefacts cannot be excluded as origin of new variants, but there is strong evidence that at least two variants are the result of a somatic recombination.

Recommendations of the ISBT Working Party on Granulocyte Immunobiology for leucocyte antibody screening in the investigation and prevention of antibody-mediated transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is currently one of the most common causes of transfusion-related major morbidity and death. Among the many TRALI mediators, leucocyte antibodies have been identified as important triggers of severe TRALI. STUDY DESIGN AND METHODS: These recommendations were compiled by experts of the ISBT Working Party on Granulocyte Immunobiology, based on the results obtained in eight international granulocyte immunology workshops, their personal experiences and on published study results. RESULTS: Leucocyte antibody screening has to include the detection of human leucocyte antigen (HLA) class I, class II and human neutrophil alloantigen antibodies using established and validated techniques. HLA class I antibody detection should be restricted to antibodies clinically relevant for TRALI. To avoid unnecessary workload, TRALI diagnosis should be assessed by consultation with the reporting clinician and thorough exclusion of transfusion-associated circulatory overload/cardiac insufficiency. In patients diagnosed with TRALI having donors with detectable leucocyte antibodies, evidence of leucocyte incompatibility should be provided by either cross-matching or typing of patient for cognate antigen. CONCLUSION: Leucocyte antibody screening for the immunological clarification of TRALI cases as well as for identification of potentially alloimmunized blood donors is feasible and can be performed in a reasonable and quality assured manner. This practice can contribute to the prevention of antibody-mediated TRALI.

The FCGR2A--Arg131 variant is no major mortality factor in the elderly--evidence from a German centenarian study.

The functional single nucleotide polymorphism rs1801274 in the FCGR2A gene (His131Arg) influences the efficiency of hIgG2 binding, the main isotype produced in response to encapsulated bacteria like Streptococcus pneumoniae and Haemophilus influenzae. In contrast to the receptor with the His131 allele, FcgammaRIIa-Arg131 binds hIgG2 poorly and carriers of this variant have been shown to be much more susceptible to succumb to bacterial pneumonia or meningitis. As bacteraemic pneumonia is one of the leading causes of death in elderly individuals, we hypothesized that the Arg131 variant could be a major mortality factor in the old. We analysed the FCGR2A-His131Arg polymorphism in a group of 408 German centenarians and two samples of younger Germans aged 60-75 and 18-49 years, respectively. No statistically significant differences were observed between the three age groups, neither at the allele nor at the genotype level. Apparently, the ability to reach old age is largely unaffected by the genetically determined efficacy of the FCGR2A-based immune response. However, the severely reduced ability of FCGR2A-131Arg carriers to eliminate encapsulated bacteria must apparently be compensated by an alternative mechanism, possibly involving other genetic survival factors.

Post-transfusion purpura in a patient with HPA-1a and GPIa/IIa antibodies.

Post-transfusion purpura is a rare bleeding disorder characterized by severe and sudden thrombocytopenia within 3-12 days after blood transfusion. Typically, preformed antibodies directed against human platelet antigens, especially HPA-1a, are associated with the clinical symptoms. A 46-year-old female presenting to the hospital with acute progressive kidney insufficiency and anaemia received two units of packed red blood cells (RBC) within 2 days. On day 7, platelet count felt from 414 to 189 x 10(9) L(-1) and 1 day later dropped to 4 x 10(9) L(-1). Four platelet concentrates were applied without success. After serological confirmation of an HPA-1a antibody, the patient was treated with intravenous gamma immunoglobulin (ivIgG), and the platelet count increased to normal values on day 17. In addition to the persisting HPA-1a alloantibody, an antibody reactive with GPIa/IIa of HPA-5a- and HPA-5b-positive platelets was detected during the acute phase of thrombocytopenia. After complete remission, the patient was transfused with four units of packed RBC from HPA-1a-negative donors, and platelet counts remained normal.

Fcgamma receptor-mediated immune phagocytosis depends on the class of FcgammaR and on the immunoglobulin-coated target cell.

BACKGROUND AND OBJECTIVES: Three human Fcgamma receptors (FcgammaR) are known to mediate immune phagocytosis. A variety of different phagocytic assays have been described, but their comparability is complicated by the use of different effector cells and different antibody-coated target cells. The aim of this study was to determine the influence of these variable components on the FcgammaR-mediated phagocytosis. MATERIALS AND METHODS: We sensitized human red blood cells (RBC) with polyclonal human anti-D (huaD), or with human monoclonal anti-D of the isotypes IgG1 (huIgG1) or IgG3 (huIgG3). Sheep RBC coated with rabbit immunoglobulin (RBC-RAS) were also used. Monocytes or polymorphonuclear neutrophils (PMN) were incubated with different FcgammaR-specific antibodies or their F(ab')2 fragments to determine the contribution of the different FcgammaRs on these effector cells in the phagocytic process of different antibody-coated target cells. RESULTS: huaD-RBC and huIgG1-RBC were preferentially ingested via the FcgammaRI on monocytes and, to a minor extent, also by the FcgammaRII. PMN ingested these target cells only after induction of the FcgammaRI by interferon-gamma (IFN-gamma). huIgG3-RBC extensively formed rosettes with monocytes but were seldom ingested. RAS-RBC phagocytosis was induced primarily via the FcgammaRI on monocytes and was mediated by the FcgammaRII on PMN. CONCLUSION: When performing phagocytosis assays with different effector and target cells, one has to take into account that phagocytosis is mediated by different FcgammaR, making comparability of these assays more difficult.

HLA-antibody testing: the immune phagocytosis inhibition test is superior to the PRA-STAT and NIH lymphocytotoxic test with respect to specificity.

We compared the specificity and sensitivity of four different methods for the detection of antibodies specific for HLA antigens. The NIH version of the complement-dependent cytotoxic test (CDC) was used as the gold standard to which we compared two Fcgamma receptor (FcgammaR)-dependent immune phagocytosis inhibition tests (IPI) and one commercial enzyme-labelled immunosorbent assay (ELISA) with soluble HLA class I-antigen preparations bound to the plate (PRA-STAT). Both IPI tests are based on the fact that HLA-antibodies specifically bind to antigens on the monocyte surface via their Fab portion, and in so doing block a neighbouring FcgammaR with their Fc region. This blockade prevents phagocytosis of IgG-coated red blood cells (RBCs), which can be measured either microscopically (IPIm) or photometrically (IPIp). The four assays were used in blind tests on 20 human alloantisera or monoclonal antibodies with known HLA-antigen reactivities. Additionally, two monoclonal antibodies and one human serum were titrated to elucidate the sensitivity of each test. After all tests were completed, the identities of the samples were disclosed. Both IPI methods detected and identified all clinically relevant HLA class I and class II specific antibodies. In contrast, the CDC was not able to detect noncytotoxic HLA-antibodies and HLA class II specific antibodies; however, it detected clinically insignificant IgM lymphocytotoxins. The PRA-STAT assay enabled identification of all cytotoxic and noncytotoxic IgG antibodies with specificity for HLA-class I antigens. With respect to sensitivity, the CDC and the IPI methods were superior to the PRA-STAT. These facts demonstrate the advantage of IPI methods in the detection of clinically relevant HLA-antibodies.

NA1/NA2 antisera inhibit FcgammaRI- but not FcgammaRII- mediated phagocytosis.

BACKGROUND AND OBJECTIVES: Alloantibodies against the granulocyte-specific NA antigens play an important role in alloimmune neonatal neutropenia. As the NA system is located on the FcgammaRIIIb, the influence of NA-specific antibodies on granulocyte function is of special interest. MATERIALS AND METHODS: We tested alloantisera specific for NA1 and NA2 for their ability to influence FcgammaR-mediated phagocytosis of polymorphonuclear neutrophils by use of different FcgammaR-specific targets. Red blood cells coated with human IgG anti-D served as specific targets for FcgammaRI-mediated phagocytosis while mouse IgG1 anti-glycophorin A was used to coat red blood cells (RBCs) to obtain FcgammaRII specific targets. To test for a hypothetical induction of phagocytosis by FcgammaRIIIb we used D-- RBCs coated with human monoclonal anti-D as target cells for unprimed neutrophils. RESULTS: Granulocyte phagocytosis was directly induced by FcgammaRI and FcgammaRII but not by FcgammaRIIIb. NA1 alloantisera significantly inhibited FcgammaRI-mediated phagocytosis of IFN-gamma-stimulated neutrophils if the corresponding antigen was expressed. Conversely, NA2 alloantisera inhibited FcgammaRI-mediated phagocytosis in NA2-positive individuals. There was no effect of NA1- and NA2-specific alloantibodies on FcgammaRII-mediated phagocytosis. CONCLUSION: NA-specific alloantisera inhibit the FcgammaRI-induced phagocytosis in primed neutrophils, but they do not significantly inhibit their FcgammaRIIa-specific phagocytosis of mIgG1-coated RBCs.

The NA"null" phenotype of a young man is caused by an Fc gammaRIIIB gene deficiency while the products of the neighboring Fc gammaRIIA and Fc gammaRIIIA genes are present.

A 27-year-old man with an allergy to house dust mites was found to lack the Fc gammaRIIIb on his neutrophils. Cell surface marker and PCR techniques were used to investigate possible reasons for this deficiency. Agglutination and immunofluorescence assays using the man's neutrophils together with NA1- and NA2-specific antibodies were negative, and there was no reaction with the Fc gammaRIII-specific mAb 3G8. Indirect immunofluorescence demonstrated the presence of the CD24 molecule, which, like the Fc gammaRIIIb, is anchored to the cell membrane by glycosylphosphatidylinositol. Thus a lack of the Fc gammaRIIIb cell membrane anchor was excluded. PCR analysis confirmed the absence of the NA1 and NA2 alleles. The individual was therefore typed as NA"null". The products of those genes located together with the Fc gammaRIIIB gene within a complex on chromosome 1 (q23-24) were examined. Fc gammaRII was demonstrated on monocytes and B cells with the use of Fc gammaRII-specific monoclonal antibodies. About 5% of the individual's peripheral blood monocytes were positive with the 3G8 antibody, indicating the presence of Fc gammaRIIIa. From these data we concluded that the Fc gammaRIIIb deficiency on the neutrophil cell surface of this individual is due to a lack of the Fc gammaRIIIB gene while excluding a lack of the Fc gammaRIIA and the Fc gammaRIIIA genes.

Rapid typing of the human Fc gamma receptor IIA polymorphism by polymerase chain reaction amplification with allele-specific primers.

BACKGROUND: The human Fc gamma receptor IIa (Fc gamma RIIa) is expressed in two polymorphic forms, Fc gamma RIIa-H131 and Fc gamma RIIa-R131, that differ by the replacement of histidine by arginine at position 131. This replacement is caused by a single-nucleotide exchange of A-->G. The resulting receptor forms differ in their binding to human IgG2 and mouse IgG1, which may lead to a different immunologic defense to bacterial polysaccharides and encapsulated bacteria. STUDY DESIGN AND METHODS: A rapid and easy polymerase chain reaction(PCR) method of genotyping the Fc gamma RIIa was developed. Allele-specific primers discriminate between the Fc gamma RIIa-H131 and the Fc gamma RIIa-R131 forms of the receptor. The results were compared with those obtained by another DNA-based genotyping method, in which PCR-amplified DNA was hybridized with allele-specific oligonucleotides, and with a functional phagocytosis assay using mouse IgG1-coated red cells as target antigens. RESULTS: The genotypes deduced from the PCR with allele-specific primers were in complete accordance with those obtained by the data from the hybridization of PCR-amplified DNA with allele-specific oligonucleotides. Furthermore, the Fc gamma RIIa genotypes of 28 individuals in all cases corresponded to the functional phenotypes. CONCLUSION: The use of PCR with allele-specific primers provides a rapid and easily performed method for the determination the Fc gamma RIIa polymorphism.

Inhibition of monocyte and polymorphonuclear granulocyte immune phagocytosis by monoclonal antibodies specific for Fc gamma RI, II and III.

We tested two Fc gamma receptor I (Fc gamma RI); six Fc gamma RII; and six Fc gamma RIII-specific monoclonal antibodies (mAb) for their capacity to inhibit monocyte and polymorphonuclear granulocyte (PMN) immune phagocytosis which is mediated by Fc gamma R. We used human red blood cells (rbc) coated with hIgG1 or mIgG1 as Fc gamma RI- and Fc gamma RII-specific target cells, respectively. The Fc gamma RI-specific mAbs 22.2 and 32.2 did not inhibit Fc gamma RI- or Fc gamma RII-specific monocyte immune phagocytosis. The Fc gamma RII-specific mAbs IV.3, CIKM5, FLI8.2, FLI 8.26, 2E1, and 41H16 inhibited Fc gamma RII-specific monocyte immune phagocytosis in all Fc gamma RIIa high-responder (HR) individuals but did not inhibit Fc gamma RI-specific phagocytosis. Using PMN, FL18.2 and 2E1 only partially inhibited phagocytosis in HR individuals, but the Fc gamma RIII-specific mAbs 3G8, DJ130c. MFM-154. B88-9 and MG38 completely inhibited Fc gamma RII-specific phagocytosis if the corresponding antigen was available on the cell surface. In these cases phagocytosis inhibition may be explained by cross-linking of Fc gamma RII and Fc gamma RIII via one antibody molecule, with the Fab portion binding to Fc gamma RIII and the Fc portion binding to Fc gamma RII.

Linkage disequilibria between Duffy blood groups, Fc gamma IIa and Fc gamma IIIb allotypes.

Fc gamma receptors (Fc gamma R) on white blood cells and the Duffy blood group antigens on red blood cells are coded for on the long arm of chromosome 1. They appear in different allotypic forms: the high responder/low responder (HR/LR) forms of Fc gamma RIIa, the alloantigens NA1 and NA2 of Fc gamma RIIIb and the Duffy blood group antigens Fya and Fyb. The aim of this study was to analyze possible linkage disequilibria between these allotypic immunomodulatory receptors, and thus provide evidence for the existence of a hypothetical gene complex. The Duffy phenotype was determined by the indirect antiglobulin test, NA1/NA2 phenotypes by the granulocyte agglutination test and the HR/LR polymorphism by an immunophagocytosis assay. Two haplotypes were found to be in linkage disequilibrium. For the haplotype NA2, Fyb we calculated a delta-value of -0.07 and for the haplotype NA1, HR we obtained a delta-value of -0.02.