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CASPASE-12 (CASP12) has an anti-inflammatory function during infection, and is a risk factor for sepsis in African-Americans (AA). To determine if CASP12 could be protective for systemic lupus erythematosus (SLE) in AA, we genotyped AA SLE patients and controls. We found that, at best, there was a weak association between CASP12 genotype with the absence of anti-dsDNA autoantibodies in SLE patients. No effect was seen upon serum interleukin-1 beta levels, nor was any other protective effect noted for the CASP12 genotype, whether upon association with SLE, or any of the 11 American College of Rheumatology classification criteria. We concluded that CASP12 genotype thus does not influence the phenotype of SLE in AA. This raises the issue as to why this protein would not play a more significant role in a chronic inflammatory disease process.

In the vast majority of human populations, the gene encoding CASPASE-12 (CASP12) has a premature termination codon that precludes the production of protein. However, approximately 20% of persons of recent African descent have a single nucleotide polymorphism (#rs497116; A->G) that turns the stop codon into one encoding Arg. The subsequent functional allele is a risk factor for sepsis as it uniquely downregulates inflammatory cytokines in African-Americans (AA). To determine if CASP12 could be protective for systemic lupus erythematosus (SLE) in AA, we genotyped AA SLE patients and controls. There was a weak association between CASP12 genotype with the absence of anti-dsDNA autoantibodies in SLE patients. No effect was seen upon serum interleukin-1 beta levels, nor was any other protective effect noted for the CASP12 genotype, whether upon association with SLE, or any of the 11 American College of Rheumatology classification criteria. CASP12 genotype thus does not influence the phenotype of SLE in AA.

Summary Most humans lack a functional CASP12 gene, with the nonfunctional variant (CASP12p1), found in 100% of the Caucasian and east Asian population, and in approximately 80% of people of African descent. However, 20% of Africans carry an intact allele of CASP12, which produces a full-length pro-enzyme and increases the risk of sepsis. We examined CASP12 allele distribution in persons from central and southern Asia and found that CASP12 was significantly present in members of the Dravidian language group, particularly in persons from the Indian state of Tamil Nadu.

A series of indole ring containing compounds were designed based on the structure of the gp41 complex in the region of the hydrophobic pocket. These compounds were synthesized using a Suzuki Coupling reaction, and evaluated using a fluorescence binding assay and cell-cell fusion assay. The observed inhibition constant of compound 7 was 2.1microM, and the IC(50) for cell-cell fusion inhibition was 1.1microM. Assay data indicated that 7 is a promising lead compound for optimization into an effective low molecular weight fusion inhibitor.

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin-7 is cleaved by caspase-7, and we map two putative caspase-7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin-7 protein. Site-directed mutagenesis of these two caspase-7 cleavage sites in the polyQ-expanded form of ataxin-7 produces an ataxin-7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin-7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ length-dependent manner, expression of the non-cleavable D266N/D344N form of polyQ-expanded ataxin-7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase-7 truncation product of ataxin-7-69Q or -92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin-7, showed increased cellular toxicity. We also detected N-terminal polyQ-expanded ataxin-7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase-7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase-7 into the nucleus by polyQ-expanded ataxin-7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin-7 by caspase-7 may contribute to SCA7 disease pathogenesis.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.

The endoplasmic reticulum (ER) is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. It also regulates cellular responses to stress and intracellular Ca2+ levels. A variety of toxic insults can result in ER stress that ultimately leads to apoptosis. Apoptosis is initiated by the activation of members of the caspase family and serves as a central mechanism in the cell death process. The present study was carried out to determine the role of caspases in triggering ER stress-induced cell death. Treatment of cells with ER stress inducers such as brefeldin-A or thapsigargin induces the expression of caspase-12 protein and also leads to translocation of cytosolic caspase-7 to the ER surface. Caspase-12, like most other members of the caspase family, requires cleavage of the prodomain to activate its proapoptotic form. Caspase-7 associates with caspase-12 and cleaves the prodomain to generate active caspase-12, resulting in increased cell death. We propose that any cellular insult that causes prolonged ER stress may induce apoptosis through caspase-7-mediated caspase-12 activation. The data underscore the involvement of ER and caspases associated with it in the ER stress-induced apoptotic process.

Antigen presentation by thymic epithelial cells (TEC) to T cells that undergo maturation is one of the major events in the selection of the T cell repertoire. We have already reported that medullary TEC lines (mTEC) established from newborn C57BL/6 (H-2b) mice are able to present a soluble antigen, ovalbumin (OVA), to OVA-specific, I-Ab restricted helper T cell lines but cortical TEC (cTEC) lines are not (Mizuochi, T. et al., J. Exp. Med. 1992. 175: 1601). In this report, to clarify the cause of this difference, we analyzed the biochemical nature as well as the distribution of both major histocompatibility complex (MHC) class II molecules and invariant chains (Ii) in both TEC by immunoprecipitation and laser confocal scanning microscopic analysis, as well as the expression of mRNA encoding H-2Ma or H-2Mb. Our results demonstrate that cTEC and mTEC are both able to present peptide antigens to peptide-specific, I-Ab-restricted helper T cell hybridoma and are able to present class II MHC alloantigens to an I-Ab-specific T cell line, that mRNA for H-2Ma and H-2Mb are expressed in both TEC, that cTEC and mTEC apparently incorporate tetramethylrhodamine isothiocyanate-labeled OVA in the same manner, and that the SDS-stable MHC class II molecules, onto which peptides were loaded, are formed in both cTEC and mTEC. However, these molecules were more rapidly degraded in mTEC than in cTEC. In addition, two Ii-derived polypeptides of approximately 21 kDa and 10 kDa were precipitated by the anti-class II monoclonal antibody Y3P; 10-kDa polypeptides were detected in the both TEC, while 21-kDa polypeptides were detected only in cTEC. Finally, beta chains of MHC class II with less sialylated oligosaccharides were precipitated from the cell surface of cTEC. Taken together, these results suggest that there are substantial differences in the antigen-presenting pathways of cTEC and mTEC, and these difference might be responsible for T cell selection events in the thymus.

In humans, a functional CASPASE-12 (CASP12) gene has been identified only in persons of African heritage and has been suggested to play a regulatory role in response to bacterial pathogens and in promoting and increased susceptibility to sepsis. The existence of a gene whose effect is deleterious, and which has been the subject of extensive negative selection in the rest of the human population, implies the simultaneous presence of some selective benefit for persons having CASP12. Given the importance of inflammatory immune responses in controlling the initial stages of infection, and the role that CASP12 plays in down-regulating inflammation, we hypothesize that pathogens which exploit the inflammatory response are restrained by an active CASP12 gene product. Several candidate pathogens are discussed.

A series of indole ring containing compounds were designed based on the structure of the gp41 complex in the region of the hydrophobic pocket. These compounds were synthesized using a Suzuki Coupling reaction, and evaluated using a fluorescence binding assay and cell-cell fusion assay. The observed inhibition constant of compound 7 was 2.1microM, and the IC(50) for cell-cell fusion inhibition was 1.1microM. Assay data indicated that 7 is a promising lead compound for optimization into an effective low molecular weight fusion inhibitor.

Molecular identification proved Mta, the maternally transmitted antigen of mice, to be a model minor histocompatibility (H) antigen. It consists of a peptide, MTF, that is presented on the cell surface by an H-2 class-I molecule, HMT. MTF is derived from ND1, a mitochondrially encoded protein, and the amino-terminal N-formyl-methionine is essential for binding to HMT; conservative substitutions at the sixth residue causes MTF to be a minor H antigen. HMT is encoded by the M3 gene at the telomeric end of the H-2 complex. The peptide-binding site of HMT is hydrophobic, and allelic forms of the mature protein differ by only three amino acids. Homologues and analogues of the mouse Mta system have recently been identified in rats.

F1 rats primed with normal parental strain lymphocyte populations and restimulated in culture with parental lymphoblasts generate potent cytotoxic T cell responses to unusual antigen systems. Here we describe in the Lewis (L)/DA anti-DA combination an antigen system most likely of mitochondrial origin with the following properties: it is transmitted maternally from DA strain females, inherited in an extra-chromosomal manner, restricted by class I RT1Aa major histocompatibility complex gene products, extinguished on target cells treated with chloramphenicol, and its pattern of expression in different rat strains correlates with restriction fragment-length polymorphisms of mitochondrial DNA. Sequence analysis of the rat ND1 gene indicates that the maternally transferred factor in the rat is not a homologue of the maternally transmitted factor responsible for the mitochondrial antigen in mice. In keeping with its inheritance from DA females, this antigen is present on target cells from (DA female x L male)F1 donors and all other F1 combinations derived from DA female parents, but absent from target cells from some F1 combinations (L/DA and Wistar-Furth [WF]/DA) derived from DA strain males. The presence of this antigen in other F1 combinations (Brown Norway [BN]/DA, August 2880 [AUG]/DA, and PVG/DA) indicates that this mitochondrial antigen system is shared by the DA, BN, and PVG strains, but not by the L and WF strains.

The RMA-S mutant T cell line is defective in H-2b restricted antigen presentation and has markedly reduced surface expression of Kb and Db. We examined RMA-S for the expression of the medial class I histocompatibility antigens Qa1b and Mta. While RMA-S targets varied in their susceptibility to lysis by cytotoxic T lymphocytes (CTL) specific for Qa1b, Mta levels were detectable but consistently low compared to the parent RMA cell line. Addition of synthetic ND1 alpha 1-26 or ND1 alpha 1-17 peptides that mimic MTF alpha (the ligand of Mta) increased killing of RMA-S by anti-Mta alpha CTL to levels comparable to or better than RMA, with 300 nM peptide being fully effective. None of the MTF peptides increased the killing of RMA-S by anti-H-2b or anti-Qa1b CTL, even at the highest (1 microM) peptide concentrations. RMA-S cells treated with 100 microM of either the ND1 alpha 4-26 or ND1 alpha 1-26 peptides showed a small increase in the fluorescent staining for beta 2-microglobulin but not for H-2Kb or H-2Db. These results show that Mta and Qa1b, although affected, are not obliterated by the defect in RMA-S cells; that the association of MTF peptides with HMT is exclusive; and that MTF enters the endoplasmic reticulum in the same fashion as other endogenous peptides.

MTF, a murine minor histocompatibility antigen, is maternally inherited and thought to be encoded by a mitochondrial gene. We sequenced the entire mitochondrial genomes from three strains that differ in MTF Mtf beta, Mtf gamma, and Mtf delta) and compared the sequences with the known, Mtf alpha, mitochondrial DNA sequence. We found only one site where all four genomes differed, affecting amino acid residue 6 of ND1, a subunit of NADH dehydrogenase. Incubation of non-Mtf alpha target cells with synthetic peptide ND1 alpha 1-17 (the first 17 amino acid of the ND1 protein of Mtf alpha mice) rendered them susceptible to lysis by MTF alpha-specific cytotoxic T cells (CTLs). Similarly, non-Mtf beta target cells were lysed by MTF beta-specific CTLs after incubation with the allelic form ND1 beta 1-17. Thus, Mtf is attributable to allelic variation at a single residue of the ND1 protein. Cells can therefore display peptides derived from mitochondrially encoded proteins, and such peptides can be histocompatibility antigens.

To evaluate the polymorphism and conservation of the major histocompatibility complex class Ib molecule Qa1 in wild mouse populations, we determined the nucleotide sequence of exons 1-3 of Qa1 of eight mouse haplotypes derived from wild mice, including Mus musculus domesticus, M. m. castaneus, M. m. bactrianus, and M. spretus, as well as two t haplotypes. Our data identify eight new alleles of Qa1. Taken together with previously published data on Qa1 among the common laboratory inbred strains, and in agreement with cytotoxic T-lymphocyte, serological, and biochemical data, these results further confirm the existence of two families of Qa1 molecules, Qa1(a)-like and Qa1(b)-like, and illuminate the extreme conservation of the peptide-binding region of these molecules, even across species.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.

Alterations in Ca2+ homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose-regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress-induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c-mediated caspase activation in a cell-free system, and expression of GRP78 blocks both caspase activation and caspase-mediated cell death. GRP78 forms a complex with caspase-7 and -12 and prevents release of caspase-12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm to set in motion the cytosolic component of the ER stress-induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death.

Received: 15 May 1998 / Revised: 23 June 1998The nucleotide sequence data reported in this paper have beensubmitted to the GenBank nucleotide sequence database andhave been assigned the number AF059493H. R. Gaskins (Y)University of Illinois at Urbana-Champaign,1207 W. Gregory Drive, Urbana, IL 61801, USAT. Chun 7 H. R. GaskinsDepartment of Animal Sciences, University of Illinois atUrbana-Champaign, Urbana, IL 61801, USAC. J. Aldrich 7 E. HermelEvansville Center, Indiana University School of Medicine,Evansville, IN 47712, USA

Class I histocompatibility antigen display is defective in the RMA-S mutant cell line due to a mutation in the Tap-2 gene, which encodes a peptide transporter. Incubation of RMA-S cells with oligomycin, an inhibitor of mitochondrial ATPase, strongly increased lysis by cytotoxic T lymphocytes (CTL) specific for the class Ib antigen H2-M3, and lysis by Qa-1 b -specific CTL was restored. Oligomycin did not affect normal class I display on RMA cells. Treatment of RMA-S cells with other inhibitors of mitochondrial function failed to increase lysis by anti-H2-M3 or Qa-1 b CTL. Lysis by allogenic CTL specific for H-2 b antigens was either not enhanced or only weakly increased, depending upon the H-2 haplotype of the alloreactive effector cells used.

CASPASE-12 (CASP12) modulates inflammation due to its ability to down-regulate inflammatory cytokines, particularly interleukin-1. Most humans possess the inactive pseudogene allele (CASP12p1) but a functional, non-truncated CASP12 allele [single nucleotide polymorphism #rs497116; A->G; ter124R] is found in 20% of persons of African descent. CASP12 is a risk factor for sepsis in persons of African descent. Rheumatoid arthritis (RA) is a serious and complex autoimmune disease that involves significant joint inflammation and destruction, and interleukin-1 contributes to the pathogenesis of RA. To determine if CASP12 may be protective against RA due to its anti-inflammatory functions, we genotyped 742 patients with rheumatoid arthritis and 342 controls from the Consortium for Longitudinal Evaluation of African-Americans with Early Rheumatoid Arthritis and the Veterans Affairs Rheumatoid Arthritis registries. Of RA patients, 17 (2.29%) were homozygous for CASP12, 146 (19.7%) were heterozygous, and 579 (78%) were homozygous for CASP12p1. These numbers did not vary significantly from controls. No significant differences were observed for CASP12 genotype on age of disease onset, seropositivity for rheumatoid factor or anti-CCP, baseline joint erosion, joint space narrowing scores, overall disease scores, C-reactive protein levels or erythrocyte sedimentation rates. Thus, CASP12 does not protect against RA in African-American patients, nor mitigates disease parameters.

CASPASE-12 (CASP12) modulates inflammation by down-regulating inflammatory cytokines. CASP12 is a risk factor for sepsis in persons of African ancestry, as the functional CASP12 allele is present in about 20% of persons of African descent. Nearly all other human populations lack a functional CASP12 gene. Systemic lupus erythematosus (SLE) is a serious and complex autoimmune disease affecting multiple organ systems and is most common in African-American (AA) women. To determine if CASP12 may be protective against SLE, 596 AA SLE patients and 296 healthy controls from the Lupus Family Registry and Repository were genotyped for CASP12. Serum from 30 patients and controls was assayed by antibody arrays for 23 inflammatory cytokines and chemokines. There was a significant association between CASP12 and protection against serositis. CASP12 homozygosity was seen only in controls, and CASP12 was only in female, but not male patients without serositis compared to those with serositis. In male patients, CASP12 was protective against the development of arthritis. Expression of the Mig chemokine was statistically different between patients and controls, independent of genotype. No influence of CASP12 genotype was seen upon serum cytokine levels in patients vs controls. The results indicate that CASP12 is protective against lupus serositis and arthritis in AA patients, with possible gender influences for both. In addition, chemokines may contribute to the pathogenesis of SLE.

The products of the class II-like H2-M genes of the major histocompatibility complex are required for class II antigen processing. We sequenced H2-Ma and Mb from several mouse strains to determine whether these genes are polymorphic like the classical H2-A and E genes, or are oligomorphic, like H2-O. Both Mb loci appear to be transcribed and are distinct from each other. Mb1 and Mb2 differ by about 11% at the nucleotide level and are most dissimilar in their second exons (corresponding to the beta 1 domain). Relative to the published Mb1d haplotype sequence, the products of the b, g7, f, and k2 alleles of Mb1 from Mus musculus domesticus and the separate mouse species Mus spretus differ by only one to four amino acids. The majority of the changes occurred in the second exon of Mb1, in contrast to HLA-DMB, the human orthologue. Little polymorphism was seen for Mb2, and Ma was invariant in all strains tested. The similarity of the g7 allele to those from other haplotypes makes it unlikely that the M class II genes play a role in the autoimmune diabetes of NOD strain mice. The M genes are regulated in a manner similar to classical class II genes, in that they are upregulated by IFN-gamma in macrophages, and to a lesser extent by IL4 in B cells. When modeled on the crystal structure of the HLA-DR1 class II molecule, nearly all of the differences between M beta 1 and M beta 2 affect residues facing away from the putative peptide binding groove.

In humans, a functional CASPASE-12 (CASP12) gene has been identified only in persons of African and Southern Indian heritage. CASP12 has been suggested to play a regulatory role in response to bacterial pathogens and in promoting and increased susceptibility to sepsis by down-regulating the production of inflammatory cytokines such as interleukin-1 (IL1). The existence of a gene whose effect is deleterious, and which has been the subject of extensive negative selection in the rest of the human population, implies the simultaneous presence of some selective benefit for persons having CASP12. Given the importance of inflammatory immune responses in controlling the initial stages of infection, and the role that CASP12 plays in down-regulating inflammation, we hypothesize that pathogens which exploit the inflammatory response are restrained by an active CASP12 gene product. A number of candidate pathogens can be ruled out on the basis that IL1 is required for their clearance, while others may select for CASP12 by how they utilize the IL1 response as a part of their pathogenesis.

Mouse beta 2-microglobulin (beta 2m) is polymorphic. Sequences of five allelic wild mouse B2m genes have been determined from the large exons of genomic DNA using the polymerase chain reaction. Relative to the standard B2m(a) allele, the products of four alleles of Mus musculus origin (w2, w3, w4, and w5), differ by only one or two amino acids. w5 has a single nucleotide change, Asp85--Val, and is identical to the c allele. w3 has two changes, Val(-13)--Ile and Lys44--Glu. w2 differs at Arg81--Thr and w4 at His34--Gln, and they share the Asp85--Val change with B2mc and B2mw5. w5 and c cells are lysed by S19.8, a monoclonal antibody specific for beta 2mb (Ala85), in a complement-mediated cytotoxicity assay, whereas w4 cells are not. Thus, distant changes appear to introduce subtle conformational effects on beta 2m structure. Five independent isolates of Mus spretus (w1) differ the most from B2m(a), with 12 amino acid changes and only one silent substitution. Replacements predicted from the nucleotide sequence occur in loops of the molecule facing away from the class I heavy chain and not in regions where beta 2m associates with class I alpha 3 domains. Concordantly, the w1-5 allelic forms of beta 2m associate well with H-2 heavy chains. The many amino acid changes in the spretus sequence and the paucity of silent substitutions suggest that B2m has been subject to positive selection.

The murine major histocompatibility complex (MHC) contains two genes (Ham-1 and Ham-2) that encode members of a super-family of ATP-dependent transport proteins. These genes are believed to mediate the transport of peptide antigen from the cytoplasm into the lumen of the endoplasmic reticulum for binding by MHC class I molecules. Evidence for such a function has come from the rescue of class I surface expression by a cloned copy of the human homologue of Ham-1, PSF-1, in a human cell line that is defective in antigen processing. A mutant murine cell line, RMA-S, has an identical antigen-processing-defective phenotype. Here we show that expression of a cloned copy of the Ham-2 gene in RMA-S cells results in recovery of the ability to process and present class I-restricted antigens to cytotoxic T lymphocytes, and in partial recovery of class I surface expression. Processing defects for classical (H-2 K and D) and non-classical (Qa1 and HMT) class I molecules are corrected by Ham-2. These data indicate that both MHC-linked transporter genes are probably required for class I antigen processing, and that the functional transporter in this pathway may consist of a Ham-1/Ham-2 heterodimer.

In rodents, caspase-12 (casp12) is a negative regulator of interleukin-1 production. Most humans lack a functional CASP12 gene, with a non-functional variant (CASP12p1), found in 100% of the Caucasian and East Asian population, and in approximately 80% of people of African descent. However, 20% of sub-Saharan Africans carry an intact allele of CASP12, which produces a full-length non-catalytic proenzyme and is associated with an increased risk of sepsis. Using PCR-based single nucleotide polymorphism analysis and DNA sequencing, we examined CASP12 allele distribution in individuals from Central and Southern Asia and found that CASP12 was significantly present in members of the Dravidian language group (the predominant ethnic and language group of southern India), particularly persons from Tamil Nadu. The CASP12 allele also occurs at a higher frequency in individuals with the Australoid morphotype that predominates in southern India, as compared to those with the Caucasoid morphotype prevalent in central and northern India. The data suggest that there is a north-to-south gradient of increasing prevalence for the CASP12 allele. As the CASP12p1 allele has been rigorously selected for in populations outside of Africa and southern Asia, we hypothesize that a pathogen that exploits the inflammatory immune response in Africa and southern India may be exerting positive selective pressure on CASP12.

A number of cytotoxic T cell responses in rats and mice are restricted by medial or non- classical class I antigens of the major histocompatibility complex (MHC). The class I heavy chain encoded by the H-2M3 gene presents MTF, an N-formylated peptide derived from the amino terminus of the mitochondrially-encoded ND1 protein, which is polymorphic in the sixth residue. H-2M3 shows minimal polymorphism in mice, and it is more similar to its rat ortholog, RT1.M3, than to any other H-2 class I gene. In rats, a minor antigen is presented by an RT1.C-encoded restriction element. Other examples of antigen presentation by medial class I antigens are reviewed. Whereas the majority of medial class I genes have ill-defined, if any, function, we propose that some may serve to present special antigens, and that these neo-classical class I genes are highly conserved between species and may be localized at the distal end of the MHC.

Casp12 encodes the protein caspase 12 (casp12), which has a downregulatory effect upon IL-1 activation and subsequent inflammatory immune reactions. Casp12 is primarily a pseudogene (Casp12p1) in most humans, but ~20% of people of African ancestry have a functional, intact form of Casp12. This allele is increased in African-American individuals with severe sepsis. We examined Casp12 allele distribution in populations of Indians and Central Asians and in a pilot screen found that a small number of Tamils (2 of 10; 20%) possess the intact Casp12 allele. Gujratis, Punjabis and other Central Asians (Baluchi, Iranian, Pakistani or Afghani) were all homozygous for Casp12p1, as were nearly all other Indians (18/19; 95%). Thus, small but distinct populations outside of Africa still harbor Casp12. To examine the biochemical properties of the protein, we expressed recombinant human casp12. By fractionation techniques, casp12 was found exclusively in the cytosol. In order to better characterize the role of casp12 in the inflammatory process, we generated a rabbit polyclonal antiserum to casp12 and used it to determine if the protein interacted directly with components of the inflammasome. Immunoprecipitation of casp12 revealed that the protein did not interact with ASC, Cardinal, or Caspase 5. Casp12 may thus exert its downregulatory effects by direct interaction with IL1.