Discordance in HER2 Status in Gastro-esophageal Adenocarcinomas: A Systematic Review and Meta-analysis.

Trastuzumab combined with chemotherapy is standard of care for HER2 positive advanced gastro-esophageal cancers. The reported prevalence of HER2 discordance between primary tumors and corresponding metastases varies, hampering uniform patient selection for HER2 targeted therapy. This meta-analysis explores the influence of HER2 assessment methods on this discordance and investigates the prevalence of HER2 discordance in gastro-esophageal adenocarcinomas. PubMed, Embase and Cochrane databases were searched until January 2016. Differences in discordance rate between strict and broad(er) definitions of HER2 status were assessed using random-effect pair-wise meta-analysis. Random-effect single-arm meta-analyses were performed to assess HER2 discordance and the prevalence of positive and negative conversion. A significantly lower discordance rate in HER2 status between primary tumors and corresponding metastases was observed using a strict vs. broad definition of HER2 status (RR = 0.58, 95%CI 0.41-0.82), with a pooled discordance rate of 6.2% and 12.2%, respectively. Using the strict definition of HER2 assessment pooled overall discordance was 7% (95%CI 5-10%). The lowest discordance rates between primary tumors and corresponding metastasis are observed when using a strict method of HER2 positivity. Treatment outcomes of different studies will be better comparable if selection of eligible patients for HER2 targeted therapy is based on this strict definition.

Low pathogenic avian influenza A(H7N9) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies.

Infections with low pathogenic avian influenza (LPAI) A(H7N9) viruses have caused more than 100 hospitalized human cases of severe influenza in China since February 2013 with a case fatality rate exceeding 25%. Most of these human infections presented with severe viral pneumonia, while limited information is available currently on the occurrence of mild and subclinical cases. In the present study, a ferret model for this virus infection in humans is presented to evaluate the pathogenesis of the infection in a mammalian host, as ferrets have been shown to mimic the pathogenesis of human infection with influenza viruses most closely. Ferrets were inoculated intratracheally with increasing doses (>10 e5 TCID50) of H7N9 influenza virus A/Anhui/1/2013 and were monitored for clinical and virological parameters up to four days post infection. Virus replication was detected in the upper and lower respiratory tracts while animals developed fatal viral pneumonia. This study illustrates the high pathogenicity of LPAI-H7N9 virus for mammals. Furthermore, the intratracheal inoculation route in ferrets proofs to offer a solid model for LPAI-H7N9 virus induced pneumonia in humans. This model will facilitate the development and assessment of clinical intervention strategies for LPAI-H7N9 virus infection in humans, such as preventive vaccination and the use of antivirals.

Identification of helper T cell master regulator candidates using the polar score method.

The T helper paradigm is currently being revised from the Th1-Th2 dichotomy to a multi-state paradigm involving a number of different cell phenotypes. Transcriptional profiling using microarrays has been used to study the development of these phenotypes. There is however no clear consensus on how to approach the analysis of this data, especially in the context of cells that are triggered to expand rapidly, and massively change their gene expression pattern. We develop a method we call 'polar score' to identify genes that are related to T helper cell polarization. This method is designed to identify polarizing genes in a set where many genes change expression. To illustrate the use of this technique, we apply it to published T cell microarray data and compare it to conventional analysis methods. With the new method, we find evidence for the existence of IL9 producing T cells ('Th9 cells') that are induced by a combination of TGFß and IL4. We identify several candidate master regulator genes for this phenotype. Furthermore, treatment with TGFß and IL12 results in a Treg and Th17 hybrid cell phenotype.

Identification of a common HLA-DP4-restricted T-cell epitope in the conserved region of the respiratory syncytial virus G protein.

The cellular immune response to respiratory syncytial virus (RSV) is important in both protection and immunopathogenesis. In contrast to HLA class I, HLA class II-restricted RSV-specific T-cell epitopes have not been identified. Here, we describe the generation and characterization of two human RSV-specific CD4(+)-T-cell clones (TCCs) associated with type 0-like cytokine profiles. TCC 1 was specific for the matrix protein and restricted over HLA-DPB1*1601, while TCC 2 was specific for the attachment protein G and restricted over either HLA-DPB1*0401 or -0402. Interestingly, the latter epitope is conserved in both RSV type A and B viruses. Given the high allele frequencies of HLA-DPB1*0401 and -0402 worldwide, this epitope could be widely recognized and boosted by recurrent RSV infections. Indeed, peptide stimulation of peripheral blood mononuclear cells from healthy adults resulted in the detection of specific responses in 8 of 13 donors. Additional G-specific TCCs were generated from three of these cultures, which recognized the identical (n = 2) or almost identical (n = 1) HLA-DP4-restricted epitope as TCC 2. No significant differences were found between the capacities of cell lines obtained from infants with severe (n = 41) or mild (n = 46) RSV lower respiratory tract infections to function as antigen-presenting cells to the G-specific TCCs, suggesting that the severity of RSV disease is not linked to the allelic frequency of HLA-DP4. In conclusion, we have identified an RSV G-specific human T helper cell epitope restricted by the widely expressed HLA class II alleles DPB1*0401 and -0402. Its putative role in protection and/or immunopathogenesis remains to be determined.

Moderate local and systemic respiratory syncytial virus-specific T-cell responses upon mild or subclinical RSV infection.

Respiratory syncytial virus (RSV) infections are a major cause of severe respiratory disease in infants. It has been shown that there is an increased frequency of childhood wheezing in ex-bronchiolitic preteen children. This was postulated to be mediated by a vigorous virus-specific Th2 response influencing the further development of the immune system. Little is known about the possible role of the immune response to clinically mild RSV infections in this respect. We have studied the RSV-specific cellular immune response in infants with a laboratory-confirmed RSV upper respiratory tract infection (URTI; n = 13, mean age 12 months, range 2-22 months) in comparison with infants with non-RSV mediated URTI (n = 9, mean age 9.3 months, range 4-18 months) or infants with severe RSV bronchiolitis (n = 11, mean age 2.3 months, range 1-6 months). RSV-specific cytokine-producing cells were enumerated using the ELISPOT method in peripheral blood mononuclear cells and nasal brush T-cells, collected during the acute and convalescent phase of the infection. Mixed Th1 (IFN-gamma) and Th2 (IL-4 and IL-13) responses were detected in all three groups. Frequencies of RSV-specific T-cells were lower in both URTI groups than in the RSV bronchiolitis group, and not significantly different between the RSV URTI and the non-RSV URTI group. The absence of vigorous virus-specific Th2 responses upon mild RSV infection does not support the hypothesis that these infections influence the development of the immune system and that they predispose for the development of atopic disease.

HLA class I-restricted cytotoxic T-cell epitopes of the respiratory syncytial virus fusion protein.

Virus-specific cytotoxic T lymphocytes (CTL) play a major role in the clearance of respiratory syncytial virus (RSV) infection. We have generated cytotoxic T-cell clones (TCC) from two infants who had just recovered from severe RSV infection. These TCC were functionally characterized and used to identify HLA class I (B57 and C12)-restricted CTL epitopes of RSV.

An alternative conditioning regimen for induction of specific skin graft tolerance across full major histocompatibility complex barriers.

Previously, we developed a well-tolerated single-day protocol for induction of stable multilineage chimerism and permanent donor-specific tolerance across major histocompatibility complex (MHC) barriers, with preservation of the host's normal immune responses. In our murine model, recipient mice were treated with a single dose of anti-CD3, anti-CD4, low dose total body irradiation (TBI; 3-6 Gy) and allogeneic bone marrow cells. An alternative cytoreductive strategy that is well-recognized in bone marrow transplantation, but has not been evaluated extensively in organ allograft recipients, involves the use of a combined chemotherapeutic drug treatment. The present data show that conditioning with low dose TBI, in a MHC-disparate donor-recipient combination, can be successfully substituted by a combined single low-dose dimethyl myleran (DMM)/cyclophosphamide (CY) therapy, resulting in both stable, mixed chimerism and specific skin graft tolerance.

Long-term detection of microchimaerism in peripheral blood after pretransplantation blood transfusion.

Renal allograft survival is prolonged after pretransplantation blood transfusion. The aim of this study was to test retrospectively the development and persistence of microchimaerism after pretransplantation blood transfusion and to assess whether the type of blood transfusion (partially matched [= sharing of at least one HLA-B and one HLA-DR antigen between blood donor and recipient] versus mismatched) influences the (continued) presence of donor-type cells. A sensitive nested PCR technique based on HLA-DRB1 allele-specific amplification using sequence-specific primers (detection level: one donor cell among 10(5) recipient cells) for detection of donor cells was implemented in our laboratory. We studied 21 patients for microchimaerism in the peripheral blood compartment, following blood transfusion. Our preliminary data show that microchimaerism was detectable up to 8 weeks after blood transfusion. In all patients receiving a partially matched blood transfusion, donor-type cells were detected in the first week after transfusion, in 7/8 patients 2-4 weeks after transfusion, and in some patients up to 8 weeks after transfusion. After mismatched transfusion a tendency to shorter duration of microchimaerism was observed.

Specific tolerance induction and organ transplantation.

Induction of tolerance to histocompatibility antigens of an organ donor would eliminate the need for long-term administration of nonspecific immunosuppressive drugs associated with an increased risk of infection and malignancies. Recently, we established a murine model in which recipient mice were treated with a single dose of anti-CD3, anti-CD4, low dose of total body irradiation (TBI) and allogeneic bone marrow cells. Our results clearly demonstrate that stable multilineage mixed chimerism, immunocompetence and permanent donor-specific skin graft tolerance across full major histocompatibility (MHC) barriers can be successfully achieved in this way. The observations that the preparative regimen and skin transplantation can be performed on the same day, and that a significant reduction in irradiation dose is sufficient in haploidentical donor-recipient combinations (MHC-sharing effect), bring our protocol closer to clinical use.

Molecular heterogeneity of second and fourth components of complement and their genes in systemic sclerosis and association of HLA alleles A1, B8 and DR3 with limited and DR5 with diffuse systemic sclerosis.

Deficiency of the complement component C4 at the functional, protein and gene level and deficiency of complement component C2 at the functional level were investigated and HLA analysis was performed on patients with limited and diffuse systemic sclerosis (SSc). One of the patients with limited SSc (n = 15) had subnormal C4, 1 subnormal C2 and 1 subnormal C4 and C2 activities; the latter patient had HLA alleles A11;B35;Dw1 associated with type II C2 deficiency and therefore most likely had a defect at the C2 locus. One of the patients with diffuse SSC (n = 12) had subnormal C4 and 1 subnormal C4 and C2 activities. C2 deficiencies in patients other than the one with the haplotype associated with C2 deficiency appeared not to be determined by the gene at the C2 locus. The incidence of partial C2 deficiency in a normal Caucasian population is reported to be 16 in 10,000, and that of partial C4 deficiency also appears to be very low. The percentages of C4A*Q0 and C4B*Q0 alleles in normal controls (n = 45) were within the reported range. Seven patients with limited SSc (n = 14) had one or two C4A*Q0 alleles and 2 with diffuse SSc (n = 13) had one C4A*Q0 allele. Thus, the incidence of C4A*Q0 was higher than normal in limited SSc and within the normal range in diffuse SSc. The two-sided Fisher's exact test applied on these data revealed that the association of C4A*Q0 with limited SSc did not reach a significant level (p = 0.10). Two of the 3 patients with limited SSc, who had two C4A*Q0 alleles, carried a heterozygous C4A-21-hydroxylase A (OHA) gene segment deletion as detected by Southern blotting. There was no correlation between the subnormal activity of C4 and the occurrence of one or two C4A*Q0 (and C4A-21-OHA segment deletion). HLA alleles A1, B8 and DR3 (p = 0.002) were associated with limited SSc (n = 23) and DR5(w11) (p = 0.018) with diffuse SSc (n = 17).

The human prostate-specific transglutaminase gene (TGM4): genomic organization, tissue-specific expression, and promoter characterization.

Human prostate-specific transglutaminase (hTGP) is a cross-linking enzyme secreted by the prostate. In this study, we performed dot blot analysis of 50 normal human tissues to demonstrate unambiguously the prostate-specific expression of hTGP. Furthermore, we elucidated the genomic organization of the TGM4 gene, the gene encoding hTGP. The structure of this gene displays striking similarity to that of other transglutaminase (TGase) genes. The TGM4 gene spans approximately 35 kb of genomic DNA and consists of 13 exons and 12 introns. The main transcription initiation site is located 52 bp upstream of the translational start codon. A hTGP splice variant of intron 1 was detected. This splice variant contains an in-frame antisense Alu element insertion. The TGM4 promoter was analyzed by sequencing and transfection experiments. At positions -1276 to -563, the promoter harbors a cyclophilin pseudogene with 94% similarity to the cyclophilin A cDNA. Deletion mapping of the TGM4 promoter in the transiently transfected human prostate cancer cell line PC346C showed comparable activity of 2.1-, 1.5-, and 0.5-kb promoter fragments.

Donor-specific immunological non-responsiveness after liver transplantation.

Since the rate of immunological losses of liver allograft after the immediate posttransplant period is much lower than in other organs, we studied the immune responses against donor HLA antigens in 18 patients with a good long-term outcome to determine whether the development of a state of immunological non-responsiveness to donor antigens might account for this favorable outcome. The reactivity against donor spleen cells was measured before and 2 years after transplantation. The reactivity in mixed lymphocyte culture (MLC) and the frequencies of cytotoxic T cell precursors (CTLp) were determined. Responses against third-party spleen cells were determined concurrently to exclude a generalized reduction of immunocompetence due to chronic immunosuppressive treatment. Before orthotopic liver transplantation, the majority of patients had normal T cell responses against donor antigens that were comparable to those against third-party antigens. Two years after transplantation, donor-specific MLC non-reactivity had developed in 10 of the 18 (56%) patients. In addition, 15 of 18 (83%) patients had developed donor-specific cytotoxic T cell (CTL) non-responsiveness; 2 had reduced numbers of CTLp against both donor and third-party cells, while the remaining patient had maintained reactivity against donor antigens. In conclusion, donor-specific non-responsiveness is present in the majority of patients 2 years after successful liver transplantation, but occurs predominantly at the CTL level.

Haematopoietic stem cells can induce specific skin graft acceptance across full MHC barriers.

Previously, we and others have demonstrated in several animal models that the establishment of stable haematopoietic chimerism through allogeneic bone marrow transfusion provides an effective means for the development of specific transplantation tolerance. However, a major limitation to the clinical application of allogeneic bone marrow transfusion in immunosuppressed recipients for induction of tolerance to solid grafts, is the risk of graft-versus-host disease (GVHD). Therefore, it is important to identify the cell population needed for the induction of mixed chimerism and tolerance. Haematopoietic stem cells have the capacity of self-renewal and multilineage differentiation, and have been shown to reduce the risk of GVHD. We studied transfusion of two rich sources of stem cells, namely allogeneic fetal liver cells and a subset of purified bone marrow-derived progenitor cells (c-kit+) into anti-T cell monoclonal antibody-treated, low-dose irradiated recipient mice. Our data revealed that stable multilineage mixed chimerism and permanent donor-specific tolerance for skin, even when transplanted directly following conditioning, can be successfully achieved in this way, with no signs of GVHD.

A 3-basepair deletion in repeat 1 of the LDL receptor promoter reduces transcriptional activity in a South African Pedi.

We have examined a naturally occurring mutation in the promoter region of the low density lipoprotein receptor (LDLR) gene of a South African Black patient with a clinical diagnosis of familial hypercholesterolemia (FH). The mutation constitutes a 3-bp deletion at nucleotide position -92 (FH Pedi-2) in the distal Sp1 binding site in repeat 1 of the LDLR promoter. The patient carries a second mutant LDLR allele containing a 1-bp deletion in exon 2 (FH Pedi-1) that gives rise to a frameshift mutation. Consistent with low receptor activity previously observed in cultured fibroblasts from the patient (5-15%), the rate of LDL receptor synthesis was markedly reduced to less than 20% of normal. DNase I footprint analysis indicated that the -92 mutation abolished binding of Sp1 to repeat 1 in the LDLR promoter. Transcription studies in transfected cells using normal and mutant promoter fragments linked to a luciferase reporter gene demonstrated that the promoter fragment containing the -92 mutation had approximately 10% of normal promoter activity. These findings indicate that the distal Sp1 binding site is essential for maximal activity of the normal intact LDLR promoter.

HLA-DRB4 gene encoded HLA-DR53 specificity segregating with the HLA-DR7, -DQ9 haplotype: unusual association.

HLA phenotyping of a leukemia patient of Caucasoid origin revealed the presence of the serological HLA-DR53 specificity. Comprehensive pedigree analysis demonstrated that the HLA-DR53 specificity segregated with the HLA-DR7, -DQ3 haplotype. High resolution PCR- SSP genotyping of the HLA class II genes revealed the presence of the HLA-DRB4*0101101 allele segregating together with the HLA-DRB1*0701, -DQA1*0201 and DQB1*03032 alleles. This finding is in contrast to known linkages in that thus far, the HLA-DR7, -DQ9 haplotype has only been described in association with the non-expressed HLA-DRB4*0103102N allele. The existence of this "novel" haplotype may be explained by a homologous recombinational event that occurred between the HLA-DR7, -DR53, -DQ2 and the HLA-DR7, -DQ9 haplotypes.

Influence of HLA-DRB1* incompatibility on the occurrence of rejection episodes and graft survival in serologically HLA-DR-matched renal transplant combinations.

BACKGROUND: The aim of the present study was to analyze the effect of HLA-DRB1* mismatches on graft function and graft survival in 92 patients who received serologically HLA-DR split antigen-matched cadaveric renal transplants. METHODS: The polymorphic second exon of the HLA-DRB1 alleles was typed using the sequence-specific oligonucleotides technique. RESULTS: The results show that in 26 of the 92 analyzed combinations, one or more HLA-DRB1* mismatches were found (28%). The analysis of the occurrence of treatable rejection episodes during the first 3 months after transplantation demonstrated a significantly higher incidence of rejection episodes in the HLA-DRB1*-mismatched group: 18 of 26 (69%) in the HLA-DRB1*-mismatched group against 23 of 66 (35%) in the HLA-DRB1*-matched group (P(uncorr)=0.0033). However, no effect of HLA-DRB1* mismatches on graft survival was found, although in general graft survival in the whole patient group was negatively influenced by the occurrence of rejection episodes during the first 3 months after transplantation (P(uncorr)=0.0008). In contrast, in the HLA-DR4-matched donor-recipient combinations (n=28), the effect of mismatching for the HLA-DRB1*04 alleles seemed to have a pronounced effect not only on the occurrence of rejection episodes but also in the form of diminished graft survival. CONCLUSIONS: Thus, this study indicates that the existence of HLA-DRB1* allele mismatches in renal transplant recipients, matched for the serologically defined HLA-DR split antigens, is not harmful for the transplant. The exception is the HLA-DRB1*04 mismatch, which seems to be deleterious for the grafted organ.

Specific tolerance induction and transplantation: a single-day protocol.

Bone marrow transfusion is a well-established method for induction of mixed hematopoietic chimerism and donor-specific tolerance in animal models. This procedure, however, is inapplicable in clinical transplantation using cadaveric donors due to the interval (1 week to 7 months) between tolerance induction and organ transplantation. For clinical use, it is essential that allografts be placed at the time of bone marrow transfusion. In the present study, we performed skin transplantation within 1 hour after a nonlethal conditioning regimen. Recipient mice were treated with anti-CD3, anti-CD4, low-dose total body irradiation (3 to 6 Gy TBI) and fully mismatched or haploidentical donor bone marrow cells. Stable multilineage chimerism and specific T-cell nonresponsiveness developed. Donor skin grafts were permanently accepted. These results suggest that this single day protocol has clear potential for application in both cadaveric and living-related organ transplantation.

5' regulatory nucleotide sequence of an HLA-A*0101null allele.

We have previously demonstrated an HLA-A*0101null allele segregating in a family with the HLA-B8, -Cw7, -DR3, -DR52, -DQ2 haplotype. In the present study the regulatory elements with known transcription enhancement activity of the silenced HLA-A*0101 allele were analyzed. In the enhancer B element, a T was substituted for a C at position - 106, whereas no other alterations were found in the adjacent 5' section of the HLA-A*0101null allele. This substitution was not seen in the enhancer B elements of the corresponding genes involved in normal HLA-A*0101 membrane expression. Comparison of enhancer B element sequences of classical functional major histocompatibility complex (MHC) class I alleles demonstrated a high degree of conservation. In contrast, many MHC class I pseudogenes showed mutation in their enhancer B boxes. These results may indicate that the single mutation detected in the enhancer B element plays a pivotal role in the abolishment of membrane expression of the HLA-A*0101null allele.