Tooth agenesis in osteogenesis imperfecta related to mutations in the collagen type I genes.

BACKGROUND: Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, mainly caused by mutations in the collagen type I genes (COL1A1 and COL1A2). Tooth agenesis is a common feature of OI. We investigated the association between tooth agenesis and collagen type I mutations in individuals with OI. SUBJECTS AND METHODS: In this cohort study, 128 unrelated individuals with OI were included. Panoramic radiographs were analyzed regarding dentinogenesis imperfecta (DGI) and congenitally missing teeth. The collagen I genes were sequenced in all individuals, and in 25, multiplex ligation-dependent probe amplification was performed. RESULTS: Mutations in the COL1A1 and COL1A2 genes were found in 104 of 128 individuals. Tooth agenesis was diagnosed in 17% (hypodontia 11%, oligodontia 6%) and was more frequent in those with DGI (P = 0.016), and in those with OI type III, 47%, compared to those with OI types I, 12% (P = 0.003), and IV, 13% (P = 0.017). Seventy-five percent of the individuals with oligodontia (≥6 missing teeth) had qualitative mutations, but there was no association with OI type, gender, or presence of DGI. CONCLUSION: The prevalence of tooth agenesis is high (17%) in individuals with OI, and OI caused by a qualitative collagen I mutation is associated with oligodontia.

Maternally transmitted target antigen for unrestricted killing by NZB T lymphocytes.

A new target antigen for unrestricted killing was defined by NZB T lymphocytes which were immunized and restimulated with H-2-identical BALB/c spleen cells. These effector cells killed nearly all target cells tested, irrespective of their H-2 type, but did not kill NZB target cells. The response was shown to have three major components: unrestricted killing specific for Qed-1b, H-2d-restricted killing specific for minor histocompatibility antigens, and unrestricted killing specific for a new antigen, Mta. Mta is present on normal and mitogen-stimulated T and B lymphocytes and on several tumor lines. It was found on cells from 26 mouse strains tested, including two substrains of NZB, representing 9 different H-2 types and 14 different non-H-2 backgrounds. Analysis of the NX8 recombinant inbred lines (derived from Mta-NZB/Icr and Mta+C58/J parents) suggested that Mta is maternally transmitted. This was confirmed by typing of reciprocal F1 hybrids and backcrosses between positive and negative strains: Mta+ females bear Mta+ offspring and Mta- females Mta- offspring, irrespective of the phenotype of the males.

Genetic and cellular aspects of xenogeneic mixed leukocyte culture reaction.

The nature of the antigens stimulating xenogeneic lymphocytes was studied using "primed LD typing". Human lymphocytes were sensitized in vitro against mouse spleen cells and restimulated with spleen cells of mouse strains sharing non-H-2 antigens or various regions of H-2 with the initial stimulating strain. The largest thymidine uptake was caused by restimulation with cells from the specific primary stimulator or an H-2-identical strain. Species-specific antigens or strain-specific antigens carried in the C57BL/10 background account for less than 15% of the total stimulation; a non-H-2 antigen associated with the Mlsalpha genotype caused moderate restimulation, amounting to 25% of the average H-2 response. Within H-2, the strongest restimulation was caused by antigens controlled by the I-A subregion; the K and D regions caused moderate, the I-C and S regions very weak, and the I-B subregion no restimulation. Thus, the genetic control of antigens stimulating xenogeneic and allogeneic MLC responses requires T cells and adherent cells, but in the human-mouse MLC, both cell types must come from the human responder; the majority of the proliferating cells are T cells. It is suggested that allograft and xenograft reactions are fundamentally identical processes, and that the relative vigor of alloaggression may be explained by secondary potentiating mechanisms depending on species-specific interactions between aggressor and target cells.

Histocompatibility antigen-activated cytotoxic T lymphocytes. I. Estimates of the absolute frequency of killer cells generated in vitro.

A sensitive procedure employing limiting dilution of activated lymphocytes and 51Cr release from highly labeled target cells was used to derive minimal estimates of the absolute frequency of cytotoxic T lymphocytes (CTL) present in a population of mouse lymphocytes activated to alloantigens of the major histocompatibility complex in bulk mixed lymphocyte cultures. From this figure (0.7-1.2%), the maximal rate of target cell killing could be calculated to be approximately 4 targets/CTL/hour.

Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors.

Using limiting dilutions of responding cells in mouse mixed leukocyte cultures, we obtained direct estimates of the minimum frequency of precursors of cytotoxic T lymphocytes (CTL.P) for a variety of antigens. Depending on the strain combination, there were as many as 4-15 CTL.P reactive to DBA/2 among 10(4) lymph node cells. Taking into account that only 5-10% of peripheral T lymphocytes have the potential to develop into cytotoxic T lymphocytes (CTLs) (6), this implies that at least 1-2% of all CTL.P are responsive to any given H-2 haplotype difference. Precursors of cytotoxic cells thus have the same high frequency of cells reactive to alloantigens of the major histocompatibility complex as found among proliferating cells in graft-vs.-host reactions and mixed lymphocyte interactions. The frequencies of CTL.P reactive to xenoantigens (rat) or trinitrophenyl-modified self were less than half the frequency of alloreactive CTL.P. A minority of the CTL.P specific for one H-2 haplotype were also reactive to a third party H-2 haplotype, presumably on the basis of recognition of shared determinants. By dilution of sensitized cells from single microcultures, it was shown that a single CTL.P undergoes a minimum of three to four cell divisions and generates at least 8-16 CTLs after antigenic activation.

The generation of killer cells to trinitrophenyl-modified allogeneic targets by lymphocyte populations negatively selected to strong alloantigens.

Negatively selected mouse and rat lymphocyte populations, specifically deprived of alloreactivity to a particular major histocompatibility complex (MHC) haplotype, are nevertheless fully capable of responding to trinitrophenyl (TNP)-modified allogeneic stimulator cells and developing cytotoxic T-lymphocyte activity to TNP-altered allogeneic target cells. As for syngeneic systems, lytic expression of those responder killer cells also requires MHC identity between the target and stimulator cell populations. Such a finding argues strongly against two variations of the dual recognition hypothesis: like-like interactions and adaptive differentiation. Instead, these data favor either the altered self model or a third variation of the dual receptor model, where one of the relevent receptors is specific for the modifying antigen and the second is a low affinity receptor unable to be triggered in the absence of a modifying antigen.

Organization and evolution of D region class I genes in the mouse major histocompatibility complex.

Chromosome walking has been used to study the organization of the class I genes in the D and Qa regions of the MHC of the BALB/c mouse and in the D region of the AKR mouse. Five and eight class I genes are found in the D and Qa regions of the BALB/c mouse, respectively, while the AKR mouse contains only a single class I D region gene that has been identified by transfection as the Dk gene. Restriction map homologies and crosshybridization experiments suggest that the multiple class I genes in the D region of the BALB/c mouse have been generated by unequal crossing-over involving class I genes from the Qa region. The expanded D region of BALB/c and other H-2d haplotype mouse strains appears to be metastable, since evidence for gene contraction in the Dd region has been found in two mutant strains. Thus the D region and also the Qa region class I genes are in a dynamic state, evolving by gene expansion and contraction.

Mta, the maternally transmitted antigen, is determined jointly by the chromosomal Hmt and the extrachromosomal Mtf genes.

Mus spretus from four stocks, originating in Spain, Portugal, and Morocco, were tested for the maternally transmitted antigen, Mta. All expressed a variant form not found in other species of mice. Analysis of appropriate crosses with inbred mice showed that the spretus form of Mta is determined by a new allele, c, of the Hmt gene. The Hmtc allele has been isolated in coupling with four different H-2 haplotypes. It is possible to raise CTL specific for the spretus form of Mta. The maternally transmitted factor, Mtf alpha s, of spretus mice determines, in conjunction with the Hmta allele of C57BL/6, an Mta that is indistinguishable from the common form found in C57BL/6 and most other inbred mice. Our experiments show that the specificity of the cell surface antigen Mta is governed jointly by the cytoplasmic gene Mtf and the chromosomal gene Hmt. We propose that Hmt encodes a class I histocompatibility antigen that acts as a restricting element for the Mtf gene product, thus meeting the requirements of T killer cell recognition.

Generation of T cells with lytic specificity for atypical antigens. I. A mitochondrial antigen in the rat.

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.

Western pine beetle: specificity among enantiomers of male and female components of an attractant pheromone.

The flight response of both sexes of Dendroctonus brevicomis to the mixture of myrcene, racemic frontalin, and (1R,5S,7R)-(+)-exo-brevicomin and to the mixture of myrcene, (1S,5R)-(-)-frontalin and racemic exo-brevicomin was significantly greater than the response to the same mixtures in which the antipodes were substituted. The flight response to these two mixtures was also greater than the response to the ternary mixture of myrcene, racemic frontalin, and racemic exo-brevicomin (MFE). The walking response of both sexes to the mixture of myrcene, racemic frontalin, and (+)-exo-brevicomin was not different from the response to MFE. Substitution of the antipode lowered the response when compared to that of MFE. When evaporated with ponderosa pine turpentine, (-)-frontalin was active in the field while its antipode was not.

Lower-than-standard dose peg-IFN alfa-2a for chronic hepatitis C caused by genotype 2 and 3 is sufficient when given in combination with weight-based ribavirin.

Mono-therapy with pegylated interferon (peg-IFN) has shown that a lower-than-standard dose yields the same sustained viral response (SVR) rates as standard doses for chronic hepatitis C virus (HCV) infection caused by genotypes 2 or 3. Our aim was to see if a fixed, lower-than-standard dose of peg-IFN alfa-2a (135 microg weekly) in combination with ribavirin 11 mg/kg daily for 24 weeks yields sufficient SVR rates for genotypes 2 or 3. Hundred consecutive patients with a mean age of 44 years (range 20-69 years), 59 with genotype 3 and 41 with genotype 2, were studied. Rapid viral response (RVR) with HCV-RNA <15 IU/mL at treatment week 4 and SVR were calculated. RVR was achieved by 28/40 (70%) patients with genotype 2 and 41/58 (71%) with genotype 3. Significantly more genotype 2 patients with RVR achieved SVR 27/28 (96%) than genotype 2 patients who failed to achieve RVR, 8/12 (66%), P = 0.009. The corresponding figures for genotype 3 patients were 39/41 (95%) vs 11/17 (65%), respectively, P = 0.002. In total, SVR was achieved by 35/41 (85%) patients with genotype 2 and 51/59 (86%) patients with genotype 3, respectively. We found that 135 microg peg-IFN alfa-2a weekly was sufficient for treatment of genotype 2 and 3 chronic hepatitis C when combined with RBV dosed daily according to body weight. This combination yielded high SVR rates (85-86%) and may be cost-saving.

Minor histocompatibility antigens.

Histocompatibility antigens have been studied for over 50 years because they form a major obstacle to clinical transplantation. Human minor histocompatibility antigens remain ill-defined, but minor histocompatibility loci have been mapped on nearly every mouse chromosome. Recent molecular definition of several transplantation antigens suggests that they are by-products of an immune system poised to present viral antigens, and a mutation in any gene may give rise to a new minor histocompatibility antigen.

Antigen presentation by non-classical class I molecules.

Non-classical class I genes are no longer clearly distinguished from classical ones in mammals, and they are found also in fishes, frogs and chickens. They contribute to immune responses against pathogens. Given the number and diversity of class Ib products, their various tissue distribution patterns, and the wide range of peptides they bind, new functions are to be expected.

Decreased fracture rate, pharmacogenetics and BMD response in 79 Swedish children with osteogenesis imperfecta types I, III and IV treated with Pamidronate.

BACKGROUND: Osteogenesis imperfecta (OI) is an inherited heterogeneous bone fragility disorder, usually caused by collagen I mutations. It is well established that bisphosphonate treatment increases lumbar spine (LS) bone mineral density (BMD), as well as improves vertebral geometry in severe OI; however, fracture reduction has been difficult to prove, pharmacogenetic studies are scarce, and it is not known at which age, or severity of disease, treatment should be initiated. MATERIALS AND METHODS: COL1A1 and COL1A2 were analyzed in 79 children with OI (type I n=33, type III n=25 and type IV n=21) treated with Pamidronate. Data on LS BMD, height, and radiologically confirmed non-vertebral and vertebral fractures were collected prior to, and at several time points during treatment. RESULTS: An increase in LS BMD Z-score was observed for all types of OI, and a negative correlation to Δ LS BMD was observed for both age and LS BMD Z-score at treatment initiation. Supine height Z-scores were not affected by Pamidronate treatment, The fracture rate was reduced for all OI types at all time points during treatment (overall p<0.0003, <0.0001 and 0.0003 for all OI types I, III and IV respectively). The reduced fracture rate was maintained for types I and IV, while an additional decrease was observed over time for type III. The fracture rate was reduced also in individuals with continued low BMD after >4yrs Pamidronate. Twice as many boys as girls with OI type I were treated with Pamidronate, and the fracture rate the year prior treatment was 2.2 times higher for boys (p=0.0236). Greater Δ LS BMD, but smaller Δ fracture numbers were observed on Pamidronate for helical glycine mutations in COL1A1 vs. COL1A2. Vertebral compression fractures did not progress in any individual during treatment; however, they did not improve in 9%, and these individuals were all >11years of age at treatment initiation (p<0.0001). CONCLUSION: Pamidronate treatment in children with all types of OI increased LS BMD, decreased fracture rate, and improved vertebral compression fractures. Fracture reduction was prompt and maintained during treatment, irrespective of age at treatment initiation and collagen I mutation type.

Do nonclassical, class Ib MHC molecules present bacterial antigens to T cells?

Immune responses to bacterial antigens that appear unrestricted by the MHC may involve oligomorphic MHC class Ib molecules. One example is H-2M3, which binds N-formylated peptides and presents a Listeria peptide to cytotoxic T cells from infected mice. Lack of polymorphism makes these molecules a promising target for peptide vaccines.

Cytoplasmic inheritance of a cell surface antigen in the mouse.

Mta is a cell surface antigen of the mouse and serves as a target for specific T killer lymphocytes. Using a killer cell assay, the antigen has been found in 72 strains of laboratory mice and, with one exception, in all tested samples of mice caught in the wild or bred from such, including Mus molossinus, Mus castaneus and Mus spretus. Five strains of rats, non-inbred NMRI mice, most substrains of NZB mice and the closely related strain NZO are negative for Mta. In reciprocal F1 crosses between several Mta+ and two Mta- strains, the antigen is maternally transmitted; that is, Mta+ females bear only positive offspring, whereas Mta- females bear only negative offspring, regardless of the genotype of the male. Since 34 foster-nursed mice had the Mta type of their genetic mothers, the factor that determines expression of Mta must be transmitted before birth and not via the milk. The cytoplasmic genes of Mta+ strains have been combined with the chromosomal genes of Mta- strains, and vice versa, by repeated backcrossing. All progeny retained the Mta type of their maternal lines. Thus, the Mta type is determined solely by maternal inheritance and is not influenced by chromosomal genes. We found no evidence of incompatibility between the cytoplasmic factors and nuclear genes of Mta- and Mta+ strains.

MtDNA-encoded histocompatibility antigens.

Mitochondrially encoded H antigens are by-products of a system that has evolved in vertebrates to present peptides from intracellular pathogens on the cell surface for detection by CTLs, which can lyse the infected cell. CTL lines and clones with defined specificity against mitochondrial H antigens, which can be maintained in culture for long periods, offer a unique tool in mitochondrial genetics. Expression of polymorphic mitochondrial H antigens depends on both the presence and the activity of the corresponding mitochondrial genome, and CTLs can provide strong selection against cells displaying their cognate antigen.

Skin graft rejection caused by the maternally transmitted antigen Mta.

Mta is a medial histocompatibility antigen of the mouse. It does not stimulate a primary mixed lymphocyte response and stimulates only a very weak secondary response. Primary skin grafts are rejected with a mean survival time of 59 days by Mta- NZB recipients, and 39 days by recipients of the C57BL/6 background. Rejection is accelerated in recipients primed against Mta with a skin graft or cells, especially when these differ by multiple minor histocompatibility antigens. Mta is determined by a maternally transmitted, extrachromosomal genetic element, so backcross mice reject skin from their inbred, homozygous paternal strain. Mta, therefore, constitutes a new exception to the classic laws of transplantation.

Bone marrow cell transplants involving donors and hosts with haplotypes derived from spretus mice.

Intra-H2 recombinant inbred mice derived from matings between B10 (H2b) and B10.SP2 (H2sp2) mice, with an H2 haplotype derived from Mus spretus, have been used to map genes at H2. Recombinants 10115, 10484, R40, 9347, and 9950 were used as donors or hosts in bone marrow cell (BMC) transplants in irradiated mice. From previous studies of Mus musculus mice, the antigens (Ag) on BMC appear to be inherited recessively. The mechanisms offered include codominant inheritance of transacting genes that regulate expression of BMC Ag (Hh hypothesis) and codominant inheritance of class I Ag motifs capable of sending "negative signals" to effector natural killer (NK) cells (missing self hypothesis). Our results indicate that stem cell donors that express the same class I Ag, but differ at genes between Bat2 and Tnfa in the H2-S/D interval, can differ in immunogenicity of transplanted stem cells. The structural gene for the H2sp2 Ag appears to map telomeric of Bat2 and is codominantly inherited. An H2b gene capable of inhibiting expression of the H2sp2 Ag (or contributing to class I motifs capable of inhibiting NK cell mediated lysis of H2sp2 BMC) maps in the Bat2/Tnfa gene segment, but requires homozygosity for this function and may require the H2-Db gene as well. Although H2sp2 hosts reject H2b BMC, hosts (10115, 10484, R40, and 9347 strain) that are H2b in the centromeric, and H2sp2 in the telomeric, portion of H2 accept H2b BMC grafts. These two observations have not been made with haplotypes entirely of Mus musculus origin. The data do not support the Hh hypothesis, and are consistent with the missing self hypothesis only if the gene (requiring homozygozity for function) in the Bat2/Tnfa region codes for a particular protein or peptide that associates with Db to generate a "protective motif."

Decreased physical performance of congenic mice with mismatch between the nuclear and the mitochondrial genome.

Maternal transmission of mitochondrial DNA (mtDNA) allows us to generate mtDNA congenic strain by repeating backcrosses of female mice to male mice of an inbred strain, which carries different mtDNA haplotype from that of the female progenitor. Since genetic backgrounds of inbred strains commonly used (e.g., C57BL/6J [B6] and BALB/c) are mainly derived from an European subspecies of Mus musculus domesticus, congenic strains, in which mtDNA originated from an Asian subspecies M. musculus musculus or an European species M. spretus, give in vivo condition that mismatch occurs between the mitochondrial and the nuclear genome. So far, little has been known how the mismatch condition affects the physiological phenotype of the mice. To address this question, we established two mtDNA congenic strains, C57BL/6J(B6)-mtSPR and BALB/c-mtSHH, which carry M. spretus- and M. m. musculus-derived mtDNAs, representing the conditions of interspecific and intersubspecific mitochondrial-nuclear genome mismatch, respectively. Using these congenic strains, we examined their physical performance by measuring their running time on a treadmill belt until exhaustion. The result clearly showed that the mtDNA congenic strains manifested a significant decrease in the level of physical performance, when compared with their progenitor strains. It also appeared that the congenic mice manifested growth rate. Thus, all results indicated that mismatch between the mitochondrial and the nuclear genome causes phenotypic changes in individuals of mice.