Increased risk of strontium ranelate-related SJS/TEN is associated with HLA.

UNLABELLED: Severe adverse drug reactions (ADR) of Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) in some patients receiving strontium ranelate have been reported, but the risk factors are unclear. We show that HLA-A*33:03 and B*58:01 are significantly associated with patients who developed SJS/TEN; and provide the first evidence that genetic risk factors are involved in strontium ranelate-associated SJS/TEN. INTRODUCTION: In this study, HLA as a genetic risk factor was assessed among osteoporotic patients prescribed with strontium ranelate that developed severe cutaneous adverse drug reactions (SCARs) compared with those who were tolerant. METHODS: Genomic DNA isolated from peripheral blood mononuclear cells (PBMCs) of patients was HLA typed using sequencing-based typing method to determine their HLA profiles. RESULTS: Osteoporotic patients who are currently on strontium ranelate were enrolled in the study (n = 76). Tolerant controls were defined as patients who received strontium ranelate for a minimum of 3 months (range 3 months to 8 years) with no reports of any cutaneous reactions as these reactions usually occur within the first 12 weeks after starting treatment. Retrospective cases of SJS/TEN were also identified (n = 5). The majority of the accrued samples were of Han Chinese descent: controls (n = 72) and cases (n = 4). All cases and controls were genotyped at four HLA genes, namely HLA-A, HLA-B, HLA-C, and HLA-DRB1. In comparing the samples of Han Chinese descent (72 controls and 4 cases), we found significant associations with HLA-A*33:03 (p = 0.002) and HLA-B*58:01 (p = 0.023). There was no significant association with any HLA-C or HLA-DRB1 alleles. CONCLUSIONS: This study reveals that the occurrence of SJS/TEN in Han Chinese patients receiving strontium ranelate is HLA associated. This has important clinical implications for understanding the underlying mechanisms for this ADR as well as evaluating the potential role of genetic pre-screening for osteoporotic patients who may be prescribed strontium ranelate.

Building pharmacogenetics into a pharmacovigilance program in Singapore: using serious skin rash as a pilot study.

To study the possible genetic associations with adverse drug reactions (ADR), the Singapore Health Sciences Authority (HSA) has piloted a program to collect DNA and phenotype data of ADR cases as part of its pharmacovigilance program. Between 2009 and 2012, HSA screened 158 cases of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). To assess the association between HLA-B*1502 and carbamazepine (CBZ)-induced SJS/TEN, 13 cases and 26 drug-tolerant controls were analyzed. All 13 CBZ-SJS/TEN cases and 3/26 controls were HLA-B*1502 positive (odds ratio 181, 95% confidence interval: 8.7-3785, P=6.9 × 10(-8)). Discussions of the finding with the Ministry of Health and an expert panel led to the decision to make HLA-B*1502 testing the standard of care prior to first use of CBZ in Asians and to subsidize the genotyping test at public hospitals. This program illustrates the role of a regulatory authority in advancing the use of pharmacogenetics for drug safety.

Reduced ADP-ribosylation by PARP1 natural polymorphism V762A and by PARP1 inhibitors enhance Hepatitis B virus replication.

HepG2 and Huh7 cell lines are frequently used as models to study viral hepatitis and hepatocellular carcinoma. However, they exhibit significantly different capacities in their ability to support hepatitis B virus (HBV) replication. To investigate the basis for this, transcription factor-binding motifs at the HBV core promoter (HBVCP) were tested in luciferase reporter constructs to identify the possible role of host factors. Among the transcription factors screened: PARP1, SP1, HNF4α, HNF3, hB1F and HNF1, deletion of the PARP1 binding motif abrogated transcriptional activity at the HBVCP in HepG2 but not Huh7 cells. Sequencing of the PARP1 gene revealed that HepG2 cells carried an Ala762 allele which has low ADP-ribosylation activity, which was shown to have increased PARP1 binding affinity to its cognate motif thus resulting in higher transcriptional activity. PARP1 inhibitors that are being developed as broad cancer therapeutics also target PARP1 ADP-ribosylation enzymatic function. Four PARP1 inhibitors: PJ-34, ABT888, AZD2281 and AG014699 were tested for their effect on HBV replication. All four small molecules effectively enhanced HBV replication in vitro, confirming the role of PARP1 in HBV replication and that alteration of ADP-ribosylation by mutation or drugs can affect HBV replication. Our data demonstrate that natural polymorphisms in the host affecting proteins such as PARP1 can have a significant effect on HBV replication. Hence, patients who are infected with HBV and are on clinical trials involving PARP1 inhibitors may be at risk from unintended side-effects such as exacerbation of HBV replication.

Population-specific recombination sites within the human MHC region.

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.

B*13:50, a novel HLA-B*13 allele, identified by sequence-based typing.

We report a novel HLA-B*13 allele, B*13:50, found using high-resolution sequence-based typing in a Chinese donor. B*13:50 differs from B*13:01:01 by a single-nucleotide substitution (A→T) at position 482, in exon 3.

Identification of a novel HLA-C allele, HLA-C*03:85, in a Singaporean Chinese.

HLA-C*03:85 differs from C*03:03:01 by a single nucleotide substitution at position 276, in exon 2.

Inhibition of lymphocyte proliferation by sera from patients with hepatocellular carcinoma: lack of correlation with serum alpha-fetoprotein levels.

Sera from 23 hepatocellular carcinoma (HCC) patients were studied for their effects on lymphocyte proliferation during polyclonal mitogen stimulation and one-way mixed lymphocyte culture (MLC). The effect of 25% HCC serum on tritiated thymidine uptake of cultures varied from slight augmentation to 95% inhibition; however, no relationship was found between the potency of inhibition and serum alpha-fetoprotein (AFP) concentration. Purified AFP from the ascitic fluid of an HCC patient had no effect on MLC or cultures containing pokeweed mitogen; but at a concentration of 200 micro g/ml, AFP inhibited up to 30% of the proliferative responses in cultures containing phytohemagglutinin or concanavalin A. Results strongly suggest that the AFP in HCC sera is not inhibitory in itself.

An integrated approach in the discovery and characterization of a novel nuclear protein over-expressed in liver and pancreatic tumors.

An integrated approach in protein discovery through the use of multidisciplinary tools was reported. A novel protein, Hcc-1, was identified by analysis of the hepatocellular carcinoma (HCC)-M cell proteome. The assembled EST sequence of the 210 amino acid novel protein was subsequently confirmed by rapid amplification of cDNA ends (RACE). A total of 687 bp at the 5' untranslated region of Hcc-1 was identified. Promoter activity and several upstream open reading frames (uORFs) were demonstrated at this region. Bioinformatics prediction showed that the first 42 amino acids of the protein is a SAP domain with sequence matches to hnRNP from various vertebrate species. The Hcc-1 protein was localized to the cell nucleus while the gene was localized to chromosome 7q22.1. Hcc-1 cDNA level was increased in pancreatic adenocarcinoma. The level was also increased in well-differentiated hepatocellular carcinoma but decreases as the carcinoma progressed to a poorly differentiated stage.

Macrophage depletion using Cytodex.

Cytodex (microcarrier) beads were adapted for use as an adhering surface to deplete macrophages from a mouse spleen cell population. Non-adherent cells recovered from the incubation of one mouse spleen with 50 mg of Cytodex showed good viability and had markedly reduced proliferative response to the T cell mitogens phytohemagglutinin and concanavalin A. Response to lipopolysaccharide was, however, less clearly affected. Peritoneal exudate cells, and adherent cells recovered from the Cytodex by trypsinization, were able to restore the mitogenic response of non-adherent cells.

Towards the MHC-peptide combinatorics.

The exponentially increased sequence information on major histocompatibility complex (MHC) alleles points to the existence of a high degree of polymorphism within them. To understand the functional consequences of MHC alleles, 36 nonredundant MHC-peptide complexes in the protein data bank (PDB) were examined. Induced fit molecular recognition patterns such as those in MHC-peptide complexes are governed by numerous rules. The 36 complexes were clustered into 19 subgroups based on allele specificity and peptide length. The subgroups were further analyzed for identifying common features in MHC-peptide binding pattern. The four major observations made during the investigation were: (1) the positional preference of peptide residues defined by percentage burial upon complex formation is shown for all the 19 subgroups and the burial profiles within entries in a given subgroup are found to be similar; (2) in class I specific 8- and 9-mer peptides, the fourth residue is consistently solvent exposed, however this observation is not consistent in class I specific 10-mer peptides; (3) an anchor-shift in positional preference is observed towards the C terminal as the peptide length increases in class II specific peptides; and (4) peptide backbone atoms are proportionately dominant at the MHC-peptide interface.

A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.

Effect of recombinant gamma interferon on hepatocellular carcinoma cell lines.

The effects of recombinant gamma interferon (rIFN-gamma) were tested on 4 different hepatocellular carcinoma (HCC) cell lines PLC/PRF/5, HepG2, Hep3B and SK-Hep1. Exposure of these HCC cells to 100/ml of rIFN-gamma induced high levels of major histocompatibility complex (MHC) class I expression on their membrane, but not class II antigens. The enhancement of class I antigen synthesis occurs without a corresponding increase in synthesis of HBsAg by HBV genome positive PLC/PRF/5 and Hep3B cells. A 5 log dose of rIFN-gamma also did not trigger the integrated HBV genome into active replication as indicated by a lack of HBcAg and HBeAg synthesis. These results point to a possible role for the use of rIFN-gamma to enhance tumour cell recognition by the host immune system via the increased MHC class I expression.

Elevated blood levels of soluble tumor necrosis factor receptors in nasopharyngeal carcinoma: correlation with humoral immune response to lytic replication of Epstein-Barr virus.

Nasopharyngeal carcinoma (NPC) is tightly associated with Epstein-Barr virus (EBV) infection and a heavy infiltration of lymphoid cells in the tumor tissue. Although various lines of evidence have shown that the immune systems of NPC patients have the potential to attack the tumor cells, it is not yet understood how this potential is blocked. In this study we determined the circulatory soluble tumor necrosis factor receptors (sTNFRI and sTNFRII), which are proven to be inhibitory to the anti-tumor effects of tumor necrosis factor-alpha (TNF-alpha), in NPC patients. The serum concentration of both sTNFRI and sTNFRII was determined with an ELISA method, and shown to be significantly higher in 28 NPC patients than in matched healthy controls. This elevation was found to be positively correlated with the serum titers of IgA against EBV early antigens and viral capsid antigens in NPC patients, suggesting that the increased serum concentration of sTNFRI and sTNFRII is possibly due to the EBV infection in NPC tumor cells. This is partly supported by FACS analysis of the circulatory T cells. Phenotypical expression of activation markers such as CD25, CD38, CD69 and CD71 in blood T cells was not significantly different between the NPC and control individuals, indicating the elevation of the sTNFRs is indeed derived from the local immune response in the tumor area. Based on these results, it seems that the increased sTNFRs may act as an inhibitor to decrease the host immune response towards tumor cells in NPC patients.

Histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors: report from The World Marrow Donor Association. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. The ability to identify unrelated stem cell donors in one country for patients in another country requires cooperation and standardization in many areas. The adoption of guidelines for histocompatibility testing, such as those summarized in this report, will facilitate these opportunities and rapidly provide accurately typed donors for patients in need.

Inhibition of staphylococcal enterotoxin-driven lymphocyte proliferation by anti-MHC class II monoclonal antibody.

Staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules and the V beta region of T cell receptors (TCR) and subsequently induces T cell proliferation. This mitogenicity is the basis of pathological effects seen in food poisoning and toxic shock syndrome. Toxin-specific monoclonal antibodies have previously been shown to be effective in blocking toxin stimulated T cell responses. In this study, a monoclonal antibody, 52BL1, was found to be a potent inhibitor of SEA-, SEB-, SEC1-, SED-, and SEE-induced lymphocyte proliferation assays, which indicates that a single anti-HLA (human leukocyte antigen) class II antibody is effective in blocking the biological effects of these toxins. These results demonstrate the possibility of using anti-HLA class II antibodies in a clinical setting as an antagonist to staphylococcal enterotoxinmediated pathogenesis.

Cellular and molecular approaches to developing human monoclonal antibodies as drugs.

Attempts to bring murine monoclonal antibodies into clinical therapeutic use have always been thwarted by the body's reaction to foreign antigens. Although there has been some success with the use of murine monoclonal antibodies in the control of renal transplant rejection, its potential will remain limited. The solution then is to produce human mAbs, which are much less immunogenic in the patient and therefore, allow repeated administration. However, for the past decade, efforts to produce human monoclonal antibodies were focused on using the hybridoma fusion technique and Epstein-Barr virus transformation to generate lymphoblastoid cell lines. It is now certain that these two methods in their present form will yield neither the variety nor the quantity of human monoclonal antibodies sufficient for therapeutic use. A compromise approach which is to "humanise" murine antibodies by substituting up to 90-95% of their amino acids with human sequences will most likely become the method of choice in human monoclonal antibody production. These "chimeric" monoclonal antibodies have been shown to be much less immunogenic in patients and is a sign that monoclonal antibodies will soon be an important mode of immunotherapy.

Hepatitis B virus and hepatocellular carcinoma.

Hepatitis B virus (HBV) has been implicated as a causative factor for the development of hepatocellular carcinoma (HCC). However, no oncogenes have been identified within the HBV genome and furthermore, the virus is frequently fragmented after it integrates into the hepatocyte genome. Therefore it has been difficult to understand how HBV brings about genetic damage to result in HCC. Recent data from our laboratory show that despite fragmentation, the HBx gene is consistently retained in a functional form and therefore suggest that this gene is important to HCC development. In 4 out of 6 HCC cell lines studied, the DR2 integration sequence was destroyed during integration and points to a possible preferred target integration site. The growing importance of HBx in HCC development is supported by data from other laboratories showing that transgenic mice carrying the HBx gene alone frequently develop HCC. This review discusses the role of HBV and other factors involved in liver carcinogenesis.

Immunotoxins: the potential in cancer treatment.

Since its development in 1975, monoclonal antibody (mAb) technology has greatly enhanced our ability to analyse complex antigenic systems as well as improve the sensitivity and speed of many diagnostic tests. In particular, the study of tumour associated antigens using mAbs have revealed that many transformed cell phenotypes have useful markers on their plasma membrane, cytoplasm, or as secreted forms which can be used in developing diagnostic assays. Therapeutic application of these anti-tumour mAbs has however, been slow relative to the research and diagnostic applications. This article will discuss how the therapeutic effectiveness of anti-tumour mAbs can be enhanced by coupling them to drugs, toxins or radionucleids; and review the current advances and problems related to the application of these mAb conjugates.

Subtype analysis of the hepatitis B surface antigen using monoclonal antibodies.

A series of monoclonal antibodies (mAb) were produced against the hepatitis B surface antigen (HBsAg) and their specificity tested against 2 separate panels of known HBsAg subtypes. Using an enzymeimmunoassay, mAbs that specifically bind to the 'a', 'd' and 'w' epitopes were identified. There were also a number of mAbs that expressed binding patterns that do not correspond to the standard serological classification--which is not unusual since the mAbs were of murine origin. These mAbs are at present being used to replace the conventional goat or guinea-pig derived HBsAg subtyping antisera which are available in limited quantities and often vary in titer.