Lung transplantation is often complicated by acute and/or chronic rejection leading to graft-function loss. In addition to the HLA donor-specific antibodies (HLA-DSA), a few autoantibodies are ...correlated with the occurrence of these complications. Recently, antibodies directed against non-classical HLA molecules, HLA-G, -E, and -F have been detected in autoimmune diseases, like systemic lupus erythematosus. Non-classical HLA molecules are crucial in the immunological acceptance of the lung graft, and some of their isoforms, like HLA-G*01:04 and -G*01:06, are associated with a negative clinical outcome. The aim of this study is to determine the frequency of detection of HLA-G antibodies in lung transplant recipients (LTRs) and their impact on the occurrence of clinical complications. After incubating the cell lines SPI-801, with and without three different HLA-G isoform expression, with sera from 90 healthy blood donors and 35 LTRs (before and after transplantation), HLA-G reactivity was revealed using reagents from commercial monoclonal antibody immobilization of platelet antigen assay (MAIPA ApDIA®). Only one serum from one blood donor had specific reactivity against the HLA-G transduced lines. Non-specific reactivity in many sera from LTRs was observed with transduced- and wild-type cell lines, which may suggest recognition of an autoantigen expressed by the SPI-801 cell line. In conclusion, this study allowed the development of a specific detection tool for non-denatured HLA-G antibodies. These antibodies seem uncommon, both in healthy subjects and in complicated LTRs. This study should be extended to patients suffering from autoimmune diseases as well as kidney and heart transplant recipients.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Immunohematology laboratories are regularly facing transfusion issues due to serological weaknesses. Altered (partial) RH antigens account for most of them. In some situations, RHCE variant alleles ...are involved. Herein we present our three-step molecular exploration, with allele frequencies, that has efficiently untangled RH2 phenotype weaknesses and discrepancies in our 2017–2021 cohort. In the last 5 years, the PACA Corse EFS molecular platform received 265 samples from healthy blood donors or patients with C and C/e typing difficulties. The first-intention technique (DNA array and real time PCR for RHCE*CeRN research) detected RHCE variant alleles in 143 cases (54%). The RHCE alleles classically found in African populations were the most frequent, with RHCE*CeRN allele in 40 cases (15%) and (C)ces haplotype type 1 and 2 in 26 cases (10%). A “CE” effect haplotype was suspected in 56 cases, due to the uncommon DCE haplotype that may explain the low C expression. When there were no RHCE*Ce or RHCE*CE alleles, we then searched for RHD polymorphisms by DNA array. We detected the RHD*DAU5 and RHD*DIVa in 18 and 7 cases respectively, suggesting that C ambiguity is related to the presence of these alleles which has never been described with DAU5. If no variant RHCE and RHD alleles were detected, we finally sequenced the 10 exons of both RHCE and RHD genes according to the clinical context and found seven new RHCE alleles. Thus, this molecular strategy would improve the knowledge of RHCE variants’ expression and, thus, optimize the transfusion management.
BACKGROUND: Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1α and HIF-1β/aryl hydrocarbon ...receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known. METHODS: Using real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1α and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1α splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival. RESULTS: HIF-1α isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1αTAG) and HIF-1α736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1αTAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1αTAG (P = 0.048), and total HIF-1α (P = 0.048) mRNA levels. HIF-1αTAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1αTAG mRNA levels correlated with shortened metastasis free survival (P = 0.01). CONCLUSIONS: Our results show for the first time that mRNA expression of a HIF-1αTAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.See commentary: http://www.biomedcentral.com/1741-7015/8/45
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) Chikungunya (CHIKV), O'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group is investigating the natural history, ...epidemiology and medical management of infection by these viruses, to identify knowledge gaps and to propose recommendations for direct future investigations and rectification measures. Here, we present the first report dedicated to diagnostic aspects of CHIKV, ONNV and MAYV. Regarding diagnosis of the disease at the acute phase, molecular assays previously described for the three viruses require further evaluation, standardized protocols and the availability of international standards representing the genetic diversity of the viruses. Detection of specific IgM would benefit from further investigations to clarify the extent of cross-reactivity among the three viruses, the sensitivity of the assays, and the possible interfering role of cryoglobulinaemia. Implementation of reference panels and external quality assessments for both molecular and serological assays is necessary. Regarding sero-epidemiological studies, there is no reported high-throughput assay that can distinguish among these different viruses in areas of potential co-circulation. New specific tools and/or improved standardized protocols are needed to enable large-scale epidemiological studies of public health relevance to be performed. Considering the high risk of future CHIKV, MAYV and ONNV outbreaks, the Working Group recommends that a major investigation should be initiated to fill the existing diagnostic gaps.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Standardization efforts of real time quantitative PCR (RQ-PCR), such as the Europe Against Cancer (EAC) protocol, are actually essential. However, all the stages of the preanalytical phase (blood ...collection, conservation and procedures of cellular separation) which influence the ex vivo expression of many genes are not controlled. Various kits for stabilizing the RNA at the time of blood collection were developed: PAXgene Blood kit (Qiagen) and the new Tempus Blood RNA kit (Applied Biosystems). PAXgene Blood was already validated to monitor minimal residual disease (MRD) in onco-hematologic pathologies. In order to evaluate the Tempus Blood RNA kit, it was compared to unstabilized EDTA blood/Ficoll/TRIzol protocol and PAXgene Blood kit. The RNAs extracted by the different methods were assessed for quantity, quality and detection of control genes (
GUS and
ABL) and fusion gene transcripts
BCR-ABL and
TEL-AML1 by RQ-PCR.
Our study shows that using the EAC protocol, the new Tempus Blood RNA kit allows the detection of fusion gene transcript with the same sensitivity as the two other protocols. Altogether, our data suggest the possible use of such a technology for MRD follow-up in myeloproliferative diseases and acute leukemias. So, further multicentric studies on leukemic patients should be performed to validate this technique in these applications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background
The RH system is one of the most polymorphic blood group systems with numerous allele variants affecting Rh polypeptides expression. This complexity is at the origin of difficulties for ...transfusion of African patients especially sickle cell disease patients requiring chronic transfusion therapy with high risk of immunization. As a complete survey of RH variants is lacking in African populations, we performed red blood cell genotyping to determine the type and frequency of RHD and RHCE alleles in sub‐Saharan African populations.
Study Design and Methods
A total of 347 blood samples were collected from individuals of six nonpygmoid and three pygmoid populations. RH typing was performed using two single‐tube multiplex polymerase chain reaction amplifications (BioArray Solutions, Immucor).
Results
All six sub‐Saharan nonpygmoid populations exhibited constant variety in both type and frequency of aberrant RHD and RHCE alleles. Predicted partial RH1 (1.8%) and RH5 (0.9%) phenotypes were less than expected. Conversely, predicted partial phenotype RH2 (5.5%) was frequent. Data confirmed the high frequency of samples positive for the non–clinically significant RH10/RH20 antigens (39.5%) and revealed a high frequency of RH54 (DAK, 8.1%). The pygmoid groups showed higher percentages of predicted partial RH antigens and greater heterogeneity reflecting wide genetic differentiation.
Conclusion
Our data show that frequencies of aberrant RHD and RHCE alleles were similar, irrespective of location and ethnicity. In view of the predicted frequencies and relative clinical significance of both private antigens and high‐prevalence antigens absent, the most relevant assays for individuals of African descent in a transfusion setting are for 1) partial RH2 in the patient and 2) RH54 (DAK) in the donor.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
BACKGROUND
Knowledge of RH variants in African populations is critical to improving transfusion safety in countries with populations of African ancestry and to providing valuable information and ...direction for future development of transfusion in Africa. The purpose of this report is to describe RH diversity in individuals from Mali.
STUDY DESIGN AND METHODS
Blood samples collected from 147 individuals self‐identified as Dogon and Fulani were analyzed for Rh antigens and alleles.
RESULTS
The most common RHD allele variant was RHD*DAU0. Five predicted partial‐D phenotypes were attributed to RHD*DAU3 or RHD*DIVa. Neither RHD*DAR nor RHD*DIIIa was found. Investigation of RHCE revealed three predicted partial‐e antigens encoded by RHCE*ce(254G) in trans to RHCE*cE. Regarding C antigen, 28 Fulani typed as C+ and 16 of 28 harbored at least one RHCE*Ce‐D(4)‐ce, two being homozygous and predicted to show a rare RH:32,−46 phenotype. A new RHCE*ceTI with replacement of Exon 2 by RHD (RHCE*ceTI(D2)) was identified in Dogon and was identified by inheritance study to be in cis to RHD*DIVa. These samples typed C– with anti‐C polyclonal antibody and monoclonal antibodies (MoAbs) MS24, P3X2551368+MS24, and MS273, but positive with anti‐RhCe MoAb‐BS58. The same pattern was observed in sample with RHD*DIVa/RHCE*ceTI.
CONCLUSION
Our survey indicated an uneven distribution of RH variant alleles between Dogon and Fulani, suggesting that study in well‐documented cohorts is warranted. A high incidence of predicted partial‐C phenotype encoded by RHCE*Ce‐D(4)‐ce was found in Fulani. Further study will also be needed to clarify the clinical significance of the new DIVa/ceTI(D2) haplotype encoding partial D and variant ce antigens.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
BACKGROUND
RhD phenotypes that express a significantly reduced amount of RhD antigen per red blood cell may be mistyped as RhD‐negative by standard serologic methods. The molecular identification of ...weak D Type 1, 2, or 3 carriers allows managing them as RhD‐positive and, thus, rationalizes the use of RhD‐negative stock units and the administration of Rh‐immunoglobulin prophylaxis, avoiding unnecessary costs and possible side effects.
STUDY DESIGN AND METHODS
One sample was investigated for confirming a D−C−E+c+e− phenotype. Rh phenotyping was performed with the microplate direct hemagglutination test. DNA array analysis was performed using the BeadChip wRhD kit, and the RHD gene was explored by sequencing to determine the molecular background associated with RhD‐negative phenotype.
RESULTS
Molecular investigations showed a lack of amplification of Exons 3 through 7 and c.1154G>T transversion in Exon 9, suggesting an RHD‐CE‐D composite on a weak D Type 2 background. We attempted to precisely identify the two recombination sites generating this hybrid allele. The 5′ and 3′ breakpoints were located in Introns 2 and 7, which showed concentration of mobile Alu sequences most likely involved in the RHD‐cE(3‐7)‐weak D Type 2 allele.
CONCLUSION
Altogether, we identified the first example of an RHD‐CE‐D large hybrid allele on a weak D Type 2 background associated with an RhD‐negative phenotype. By investigating the RHCE‐D breakpoint zones, we suggest a mobile element‐mediated recombination.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK