An in vitro model for toxin-mediated vascular leak syndrome: ricin toxin A chain increases the permeability of human endothelial cell monolayers.

Vascular leak syndrome (VLS) is the dose-limiting toxicity observed in clinical trials of immunotoxins containing ricin toxin A chain (RTA). RTA itself is thought to cause VLS by damaging vascular endothelial cells, but the exact mechanism remains unclear. This is partially due to the paucity of appropriate models. To study VLS, we developed an in vitro model in which human umbilical vein-derived endothelial cells were first grown to confluence on microporous supports and then cultured under low pressure in the presence or absence of RTA. Endothelial cell barrier function was assessed by measuring the volume of fluid that passed through each monolayer per unit time. We found that RTA significantly increased monolayer permeability at times and concentrations consistent with the onset of VLS in patients treated with RTA-based immunotoxins. Scanning electron microscopy showed that intercellular gaps formed in endothelial monolayers exposed to RTA. Intercellular gap formation followed endothelial cell death caused by the enzymatic activity of RTA. We conclude that RTA is directly toxic to endothelial cells in vitro and speculate that this contributes to VLS in vivo.

HLA-DQB1-associated susceptibility that distinguishes Hashimoto's thyroiditis from Graves' disease in type I diabetic patients.

Insulin-dependent diabetes (IDDM) is frequently associated with autoimmune thyroid disease (ATD) within families. In these families, HLA polymorphism may modulate the susceptibility to each disease. Families with IDDM were further categorized as to the presence of ATD. IDDM-affected subjects from families without ATD were compared with subjects with ATD or with IDDM and ATD from IDDM/ATD families and with a control group. IDDM susceptibility in IDDM/ATD families was negatively associated with the presence of DQB1*0602 [relative risk (RR) = 0.038; P = 0.0001; corrected P (Pc) = 0.0005] and *0301 (RR = 0.3; P = 0.002; Pc = 0.01) and positively associated with the presence of DQB1*0201 (RR = 3.4; P = 0.0007; Pc = 0.0035) and *0302 (RR = 5; P = 0.0001; Pc = 0.0005), regardless of ATD. Compared with the IDDM-only group, the ATD-only group had an increased frequency of subjects with DQB1*0602 (RR = 0.14; P = 0.031), suggesting that the known IDDM-protective effect of this allele may be independent of susceptibility to ATD; however, this difference was not significant when the P value was correlated for the number of alleles tested. In these families, susceptibility to ATD was only associated with DQB1*0201 (RR = 5.71; P = 0.0043; Pc = 0.021). Among subjects with DQB1*0201, there was a weak negative association between the presence of DQB1*0302 on the second haplotype and Hashimoto's thyroiditis (RR = 0.237; P = 0.026; Pc > 0.05). We conclude that in IDDM/ATD families, IDDM-affected subjects are at risk for ATD, especially those carrying DQB1*0201. This risk may be influenced by the alleles carried on the second haplotype, with DQB1*0302 (or a closely linked gene) protecting from Hashimoto's thyroiditis and favoring Graves' disease.

Frequent HLA class I and DP sequence mismatches in serologically (HLA-A, HLA-B, HLA-DR) and molecularly (HLA-DRB1, HLA-DQA1, HLA-DQB1) HLA-identical unrelated bone marrow transplant pairs.

The rates of graft-versus-host disease (GVHD) and rejection are significantly higher among recipients of unrelated donor marrow (BM) than in recipients of marrow from HLA-identical siblings, even when donors and recipients are mixed lymphocyte culture (MLC) compatible and serologically and Dw identical. It has been hypothesized that phenotypically silent HLA class I and DP sequence mismatches might be associated with these differences, but little is known about their incidence. We have sequenced the HLA-A, HLA-B, HLA-C, HLA-DPA1, and HLA-DPB1 genes expressed by 12 unrelated marrow transplant pairs, 11 of whom were molecularly matched at DRB, DQA1, and DQB1 loci. Nine of these pairs were also HLA-A and HLA-B matched by serology. Six of these nine "HLA-identical" pairs were HLA-A (2 of 6), HLA-B (1 of 6), and HLA-C (6 of 6) mismatched at the sequence level. The mismatched class I alleles of all these pairs had strikingly different sequence motifs in the six specificity pockets of their antigen recognition site, and in five pairs they also had sequence differences at positions implicated in T-cell receptor (TCR) binding. Two of the three pairs who were serologically mismatched for one HLA-A or HLA-B antigen were also sequence mismatched at HLA-C. Finally, 10 of 11 pairs tested expressed different DP sequences. These data indicate that HLA class I, especially HLA-C, and DP sequence mismatches are frequent among unrelated subjects defined as HLA identical by current typing methods. We speculate that these sequence differences may explain, at least in part, the higher incidence of acute GVHD and rejection in unrelated BM transplantation as opposed to transplantation between HLA-identical siblings. Because of their high frequency, the role of HLA-A, HLA-B, HLA-C, and HLA-DP mismatches in transplantation outcome is now amenable to direct study.

HLA class I sequence-based typing.

HLA oligogenotyping has been used successfully to characterize most phenotypically undetectable variants of class II genes. Limitations inherent to the class I system have, however, complicated the application of this and other molecular approaches to HLA class I typing. We have previously shown that HLA class II polymorphism can be analyzed by a SBT approach. Here we present a class I-SBT strategy that provides complete sequence information for the two most polymorphic exons of the HLA-A, -B, and -C alleles. HLA class I SBT is based on direct sequencing of PCR-amplified HLA-A, -B, and -C cDNAs and requires a total of six cDNA -PCR-sequencing reactions (two per locus) and 13 different oligonucleotides. Each combination of oligonucleotides per reaction results in locus-specific sequence ladders and allows identification of both alleles in heterozygotes. Application of HLA-A, HLA-B, and HLA-C SBT to 26 homozygous and 32 serologically heterozygous samples has resulted in the identification of 24 novel class I nucleotide sequences encoding 17 new major histocompatibility complex class I products. An unexpected high degree of heterogeneity was found at the HLA-C locus with 14 novel sequences. Although there was a good correlation between the serologic phenotypes and SBT results, HLA-C SBT of most HLA-C serologically homozygous samples (heterozygous for HLA-A and/or -B) revealed heterozygozity (six of eight). SBT, the first molecular typing approach that has been generalized to both class I and class II genes, may be of special interest in applications demanding high sensitivity and specificity, such as in paternity testing or in the evaluation of the effects of sequence allelism in the outcome of unrelated bone marrow transplantation.

HLA class II "typing": direct sequencing of DRB, DQB, and DQA genes.

Routine clinical HLA class II typing is based largely on serological and cellular methods. These methods have many drawbacks that have led to the evaluation of molecular approaches to typing, including restriction fragment length polymorphism studies and oligotyping. We present here an alternative molecular approach, sequence-based typing (SBT), that allows direct determination of the sequences of all HLA class II polymorphic genes, thus providing the most detailed information currently possible in this regard. The data presented here using SBT are based on direct sequencing of polymerase chain reaction (PCR)-amplified DRB, DQB, and DQA cDNAs using a limited number of oligonucleotides. The oligonucleotides are designed to allow simultaneous determination of allelic sequences in any heterozygote as well as characterization of DRB isotypic complexity. Two types of amplification oligonucleotides (nonconserved and/or conserved) are used for DRB typing, which involves a maximum of four simultaneous cDNA/PCR/sequencing reactions. The first of these reactions only uses conserved oligonucleotides and is designed to detect all the different DRB transcripts present in any given heterozygote; the other three reactions use nonconserved oligonucleotides and are designed to ensure the unambiguous interpretation of the most complex DRB heterozygote combinations. Characterization of DQA1 and DQB1 sequences can be performed by using conserved oligonucleotides and only involves one reaction per locus. We have applied SBT to 43 homozygous cell lines and to 38 different heterozygote combinations that had previously been serologically typed. In all cases we were able to determine the allelic composition at DRB1, DRB3/4/5 and/or DQB1, and DQA1 loci of these cell lines and subjects; our results, analyzed by blind protocol, were consistent with the serological phenotypes. SBT can be extended to class I and class III genes and is automatable. We believe that this strategy deserves further evaluation as a possible HLA typing method.

Alloreactive T cells can distinguish between the same human class II MHC products on different B cell lines.

Certain allele-specific alloreactive T cell clones do not recognize the products expressed by some B cell lines that, according to typing methods other than sequencing, carry the allelic molecules recognized by these clones. In order to characterize the naturally occurring sequence polymorphisms putatively responsible for the differential allorecognition of these class II molecules, we have determined the third and/or second exon nucleotide sequences of HLA-DRB1, -DRB3/4/5, -DQB1, and -DQA1 genes from 35 representative lymphoblastoid cell lines. In some cases, the lack of recognition correlates with the presence of single amino acid substitutions in either the second or third hypervariable region (HVR) of the first domain of these molecules. In other cases, the differentially allorecognized class II molecules have identical second and/or first domain amino acid sequences. These findings indicate that a) class II MHC-alloreactive T cell clones can distinguish between molecules with identical amino acid sequences expressed by B cell lines established from unrelated individuals; b) allorecognition of class II molecules is sensitive to naturally occurring single amino acid substitutions in either the second HVR of class II molecules, which is unavailable to interact with TCR residues, or the third HVR. Our results also suggest that 1) in different B cell lines, identical class II molecules may present different endogenous peptides, which may behave as histocompatibility Ag; 2) the peptide-binding specificity of a class II molecule may be affected by amino acid substitutions in its second HVR (Ag-binding site); and 3) human class II allorecognition may be restricted by epitopes contributed by residues of their third HVR.

Detection of novel sequence heterogeneity and haplotypic diversity of HLA class II genes.

Nucleic acid sequences of the second exons of HLA-DRB1, -DRB3/4/5, -DQB1, and -DQA1 genes were determined from 43 homozygous cell lines, representing each of the known class II haplotypes, and from 30 unrelated Caucasian subjects, comprising 60 haplotypes. This systematic sequence analysis was undertaken in order to a) determine the existence of sequence microheterogeneity among cell lines which type as identical by methods other than sequencing; b) determine whether direct sequencing of class II genes will identify the presence of more extensive sequence polymorphism at the population level than that identified with other typing methods; c) accurately determine the molecular composition of the known class II haplotypes; and d) study their evolutionary relatedness by maximum parsimony analysis. The identification of seven previously unidentified haplotypes carrying five new allelic amino acid sequences suggests that sequence microheterogeneity at the population level may be more frequent than previously thought. Maximum parsimony analysis of these haplotypes allowed their evolutionary classification and indicates that the higher mutation rate at DRB1 compared to DQB1 loci in most haplotypic groups is inversed in specific haplotype lineages. Furthermore, the extent and localization of gene conversions and point mutations at class II loci in the evolution of these haplotypes is significantly different at each locus. Identification of additional HLA class II molecular microheterogeneity suggests that direct sequence analysis of class II HLA genes can uncover new allelic sequences in the population and may represent a useful alternative to current typing methodologies to study the effects of sequence allelism in organ transplantation.

Liquid crystals as a potential ointment vehicle.

A lecithin-water lyotropic liquid crystal was used as an ointment vehicle for a hydrocortisone formulation. The hydrocortisone was soluble in the liquid crystalline phase up to 5% by weight. The diffusion coefficient determined for the hydrocortisone in the liquid crystalline phase was 5.5 X 10(-9) cm X s-1, which is four magnitudes higher than the corresponding value for skin.