Characterization of the HLA-A2.2 subtype: T cell evidence for further heterogeneity

Five blood donors were identified whose HLA-A2 is different from the common HLA-A2. Their A2 molecule (A2.2) had a more basic isoelectric point than normal A2 (A2.1). Cytotoxic T lymphocytes (CTL) restricted. by HLA-A2.1, specific for influenza A and Epstein-Barr viruses, failed to lyse virus-infected target cells with HLA-A2.2. Identical patterns were obtained with both viruses. CTL from four of the A2.2-positive donors recognized target cells prepared from others in the group that shared only the HLA-A2.2 antigen. The A2.2 antigen from one donor seemed to be different in that target cells were not recognized by CTL from donors with the normal A2.1 nor with basic A2.2. There seems, therefore, to be heterogeneity within the HLA-A2.2 subtype.     

Periodic catatonia: confirmation of linkage to chromosome 15 and further evidence for genetic heterogeneity

We earlier reported significant evidence for linkage on chromosome 15q15 in periodic catatonia, a sub-phenotype of schizophrenic psychoses. The disorder is characterized by qualitative hyperkinetic and akinetic psychomotor disturbances through acute psychotic episodes and debilitating symptoms in the long term, with psychomotor weakness, grimacing facial movements and apathy. Here, we confirm mapping of a major gene locus on chromosome 15q15 in a second genome scan in a new set of four multiplex families. Non-parametric multipoint linkage analyses identified a broad region with a maximum peak of Z(all) =3.91 ( P=0.006) and Z(lr) =3.04 at D15S1234 ( P=0.001), satisfying conventional criteria for confirmed linkage. Parametric affected-only analyses under an autosomal dominant model gave a maximum HLOD score of 1.65 (D15S1234) with an estimated 47% of families being linked. Analysis of individual families showed that one large family showed linkage, whereas two others could be clearly excluded, confirming genetic heterogeneity. No other locus reached suggestive levels of significance. Haplotype analysis on chromosome 15 in this and previously linked families placed the susceptibility region to a 11-cM interval between marker D15S1042 and D15S659. Periodic catatonia is the first sub-phenotype of schizophrenic psychoses with confirmed linkage despite the existence of considerable genetic heterogeneity.     

Properties of a goat anti-L antibody: further evidence for heterogeneity of the L antigen.

The preparation and properties of an antibody (anti-L) against low potassium type (LK) goat red cells raised in a high potassium type (HK) goat are described. This reagent stimulated active potassium transport, but showed only weak serological activity against low potassium type (LK) sheep and goat red cells. The results are discussed in relation to the hypothesis that anti-L antibody has two specificities--a sodium pump-stimulating activity (anti-Lp) and a serological activity (anti-L1y).     

Angiotensin-converting enzyme 2 autoantibodies: further evidence for a role of the renin-angiotensin system in inflammation

Traditionally viewed as important in the regulation of blood pressure, the renin-angiotensin system - and specifically the angiotensin-converting enzyme (ACE)-angiotensin (Ang) II-AT₁ receptor axis - may play a prominent role to promote inflammation and fibrosis. ACE2, a new component of the renin-angiotensin system, has emerged as a key enzyme that selectively degrades Ang II and generates Ang-(1-7), a bioactive peptide with anti-inflammatory and anti-fibrotic actions. Takahashi and colleagues demonstrate circulating titers of inhibitory autoantibodies against ACE2 in patients with systemic sclerosis. The current study reveals a potentially novel mechanism to attenuate the catalytic activity of ACE2, thereby promoting the actions of Ang II.     

Regulation of enzyme release from human polymorphonuclear leukocytes: further evidence for the independent regulation of granule subpopulations

Addition of 5-20 mM LiCl to purified human polymorphonuclear leukocytes led to the release of lysozyme, the specific granule constituent, but not the release of elastase which is in azurophilic granules. In contrast, 2.5-10 micrograms cytochalasin D/mL induced the release of both lysozyme and elastase. Addition of lipopolysaccharide to leukocytes did not induce enzyme release but primed cells for enhanced release induced by cytochalasin D. Lipopolysaccharide also primed cells for enhanced release of lysozyme by either N-formylmethionylleucylphenylalanine (fMLP) or Li+ but did not prime cells for elastase release by these stimuli. In contrast, fMLP + cytochalasin D interacted synergistically, leading to enhanced elastase release but not lysozyme release from the cells. Additional experiments with combinations of secretagogues and lipopolysaccharide yielded results consistent with the hypothesis that specific granules and subpopulations of azurophilic granules are under separate regulation and, thus, may be influenced by separate elements of intracellular second messenger systems.     

Cardiodynamic response to psychological and cold pressor stress: further evidence for stimulus response specificity and directional fractionation

In the present study 36 police officers were exposed to a psychological stressor (IQ quiz) and to cold pressor stress while several cardiovascular variables were monitored. Impedance cardiography was used to provide measures of heart rate, stroke volume, cardiac output, myocardial contractility, and total peripheral resistance. In addition, measures of systolic and diastolic blood pressure and peripheral skin temperature were obtained. A multivariate analysis of variance (MANOVA) indicated that significant increases in diastolic and systolic blood pressure during the cold pressor test were mediated by large increases in total peripheral resistance, whereas blood pressure elevation during the IQ quiz were accompanied by significant increases in heart rate and, to a lesser extent, cardiac output. Peripheral skin temperature decreased in response to each stressor. Additional analysis indicated a degree of stimulus specificity for several variables. For example, diastolic blood pressure showed greater increases to cold pressor than quiz, whereas systolic blood pressure increased more with the psychological than the physical stressor. Directional fractionation occurred for both myocardial contractility and cardiac output.     

HLA Haplotypes with C4B5; evidence for further allelic heterogeneity

Twenty-three individuals from various disease groups and normal controls were identified by immunofixation with anti-C4, C4-dependent lysis, determination of Rg (Rodgers) and Ch (Chido) phenotypes, and immunoblotting with C4-specific mouse monoclonal antibody. We found that one haplotype predominates with the C4B ^* 5 allele, HLA-A11, B22(55), Cw3, Bf ^* S, C4A ^* 4B ^* 5, which also carries the Ch ^{1,–2, 3} haplotype. The B5 allotype was also found with HLA-1360, HLA-1335 in Caucasoids, and HLA-B18 in non-Caucasoids; these carried the Ch ^{–1, –2, –3} haplotype. Our results are in accord with an earlier report of two B5 subtypes, B5Rg⁺ and B5Rg^– (Roos et al. 1984). The specificity of the mouse monoclonal antibodies IC4 and 21312 had been previously related to C4A and C4B, respectively, but our results suggest that they relate more closely to Rg and Ch determinants.     

Proliferation of chicken peripheral blood leukocytes in response to pokeweed mitogen is macrophage dependent

Chicken peripheral blood leukocytes (PBL) proliferate in vitro in response to a wide range of pokeweed mitogen (PWM) concentrations. The PWM response was not influenced by the major histocompatibility complex (MHC) haplotype nor by the intrinsic responsiveness of the PBL to concanavalin A (Con A). The results indicate that PWM under our conditions stimulates B-lineage cells, although a T-cell subset is also clearly induced to division. The PBL from B-cell-depleted animals gave substantially lower responses than those from normal controls. Pokeweed mitogen stimulation of PBL was adherent cell dependent. Thus the low PWM response of adherent cell-depleted PBL was reconstituted by the addition of irradiated unseparated PBL. Furthermore, purified irradiated adherent cells containing greater than 90% macrophages were also able to reconstitute PWM responses. Finally, we have shown that PWM was able to induce large numbers of B cells to produce cytoplasmic immunoglobulin. However, only a minor proportion of such cells were induced to immunoglobulin secretion.     

Inhibition of inflammation but not ankylosis by glucocorticoids in mice: further evidence for the entheseal stress hypothesis

Introduction

Studies in the spontaneous ankylosis model in aging male DBA/1 mice and in patients with ankylosing spondylitis provide evidence that inflammation and new tissue formation leading to joint or spine ankylosis are likely linked but largely uncoupled processes. We previously proposed the ''entheseal stress'' hypothesis that defines microdamage or cell stress in the enthesis as a trigger for these disease processes. Here, we further investigated the relationship between inflammation and ankylosis by focusing on the early phase of the spontaneous arthritis model.

Methods

Aging male DBA/1 mice from different litters were caged together at the age of ten weeks and studied for signs of arthritis. A group of DBA/1 mice were treated daily with dexamethasone (0.5 μg/g body weight). Severity of disease was assessed by histomorphology and by positron emission tomography (PET) using 2-[¹⁸F]fluoro-2-deoxy-D-glucose (¹⁸F-FDG) as a tracer. Bone loss in dexamethasone-treated or control mice was determined by in vivo dual-energy X-ray absorptiometry. Chemokine gene expression was studied ex vivo in dissected paws and in vitro in mesenchymal cells (periosteal and bone marrow stromal cells) by quantitative real-time PCR in the presence or absence of bone morphogenetic protein 2 (BMP2) and dexamethasone.

Results

Dexamethasone treatment did not affect incidence or severity of ankylosis, but led to an expected reduction in inflammation in the paws at week 15 as measured by PET tracer uptake. Treatment with dexamethasone negatively affected bone mineral density. Chemokines attracting neutrophils and lymphocytes were expressed in affected paws. In vitro, BMP2 stimulation upregulated chemokines in different mesenchymal joint-associated cell types, an effect that was inhibited by dexamethasone.

Conclusions

BMP signaling may be a trigger for both inflammation and ankylosis in the spontaneous model of ankylosing enthesitis. The lack of inhibition by glucocorticoids on new bone formation while causing systemic bone loss highlights the paradoxical simultaneous loss and gain of bone in patients with ankylosing spondylitis.     

Extreme heterogeneity of population response to climatic variation and the limits of prediction

Certain general facets of biotic response to climate change, such as shifts in phenology and geographic distribution, are well characterized; however, it is not clear whether the observed similarity of responses across taxa will extend to variation in other population‐level processes. We examined population response to climatic variation using long‐term incidence data (collected over 42 years) encompassing 149 butterfly species and considerable habitat diversity (10 sites along an elevational gradient from sea level to over 2,700 m in California). Population responses were characterized by extreme heterogeneity that was not attributable to differences in species composition among sites. These results indicate that habitat heterogeneity might be a buffer against climate change and highlight important questions about mechanisms maintaining interpopulation differences in responses to weather. Despite overall heterogeneity of response, population dynamics were accurately predicted by our model for many species at each site. However, the overall correlation between observed and predicted incidence in a cross validation analysis was moderate (Pearson's r = 0.23, SE 0.01), and 97% of observed data fell within the predicted 95% credible intervals. Prediction was most successful for more abundant species as well as for sites with lower annual turnover. Population‐level heterogeneity in response to climate variation and the limits of our predictive power highlight the challenges for a future of increasing climatic variability.     

A mutation in COL9A1 causes multiple epiphyseal dysplasia: further evidence for locus heterogeneity

Multiple epiphyseal dysplasia (MED) is an autosomal dominantly inherited chondrodysplasia. It is clinically highly heterogeneous, partially because of its complex genetic background. Mutations in four genes, COL9A2, COL9A3, COMP, and MATR3, all coding for cartilage extracellular matrix components (i.e., the alpha2 and alpha 3 chains of collagen IX, cartilage oligomeric matrix protein, and matrilin-3), have been identified in this disease so far, but no mutations have yet been reported in the third collagen IX gene, COL9A1, which codes for the alpha1(IX) chain. MED with apparently recessive inheritance has been reported in some families. A homozygous R279W mutation was recently found in the diastrophic dysplasia sulfate transporter gene, DTDST, in a patient with MED who had a club foot and double-layered patella. The series consisted of 41 probands with MED, 16 of whom were familial and on 4 of whom linkage analyses were performed. Recombination was observed between COL9A1, COL9A2, COL9A3, and COMP and the MED phenotype in two of the families, and between COL9A2, COL9A3, and COMP and the phenotype in the other two families. Screening of COL9A1 for mutations in the two probands from the families in which this gene was not involved in the recombinations failed to identify any disease-causing mutations. The remaining 37 probands were screened for mutations in all three collagen IX genes and in the COMP gene. The probands with talipes deformities or multipartite patella were also screened for the R279W mutation in DTDST. The analysis resulted in identification of three mutations in COMP and one in COL9A1, but none in the other two collagen IX genes. Two of the probands with a multipartite patella had the homozygous DTDST mutation. The results show that mutations in COL9A1 can cause MED, but they also suggest that mutations in COL9A1, COL9A2, COL9A3, COMP, and DTDST are not the major causes of MED and that there exists at least one additional locus.     

Autosomal dominant ataxia: Genetic evidence for locus heterogeneity from a cuban founder-effect population

The locus for autosomal dominant ataxia with a diagnosis of olivo-ponto-cerebellar atrophy at autopsy has been previously assigned to chromosome 6p. However, evidence for two alternative locations has been reported. We have recently described a large potential founder-effect population of such patients in the Holguin province of Cuba. With an estimated 1,000 patients available for analysis, this extensive cluster of families provides a unique opportunity for the definitive localization of the genetic mutation. Linkage analysis between the disease locus in this population and markers within and flanking the HLA region on chromosome 6 were undertaken in 12 families comprising over 100 affected individuals. Despite similarity in the clinical phenotype between those families where the disease locus has been reported to be linked to the HLA locus and the Cuban patients, no evidence of linkage to this region could be demonstrated in the latter. The disease locus was excluded from a 96-cM genetic interval of the short arm of chromosome 6, encompassing the F13A1-HLA-GLO1-MUT/D6S4 loci. These data strongly support the existence of genetic heterogeneity for the disease.     

The inhibition of lipopolysaccharide-induced macrophage inflammation by 4 compounds in Hypericum perforatum extract is partially dependent on the activation of SOCS3

Our previous studies found that 4 compounds, namely pseudohypericin, amentoflavone, quercetin, and chlorogenic acid, in Hypericum perforatum ethanol extract synergistically inhibited lipopolysaccharide (LPS)-induced macrophage production of prostaglandin E2 (PGE2). Microarray studies led us to hypothesize that these compounds inhibited PGE2 production by activating suppressor of cytokine signaling 3 (SOCS3). In the current study, siRNA was used to knockdown expression of SOCS3 in RAW 264.7 macrophages and investigated the impact of H. perforatum extract and the 4 compounds on inflammatory mediators and cytokines. It was found that the SOCS3 knockdown significantly compromised the inhibition of PGE2 and nitric oxide (NO) by the 4 compounds, but not by the extract. The 4 compounds, but not the extract, decreased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), while both lowered interleukine-1β. SOCS3 knockdown further decreased IL-6 and TNF-α. Pseudohypericin was the major contributor to the PGE2 and NO inhibition in cells treated with the 4 compounds, and its activity was lost with the SOCS3 knockdown. Cyclooxygenase-2 (COX-2) and inducible NO synthase protein expression were not altered by the treatments, while COX-2 activity was decreased by the extract and the 4 compounds and increased by SOCS3 knockdown. In summary, it was demonstrated that the 4 compounds inhibited LPS-induced PGE2 and NO through SOCS3 activation. The reduction of PGE2 can be partially attributed to COX-2 enzyme activity, which was significantly elevated with SOCS3 knockdown. At the same time, these results also suggest that constituents in H. perforatum extract were alleviating LPS-induced macrophage response through SOCS3 independent mechanisms.     

Phenotypic plasticity in response to environmental heterogeneity contributes to fluctuating asymmetry in plants: first empirical evidence

Fluctuating asymmetry (FA) is widely used to quantify developmental instability (DI) in ecological and evolutionary studies. It has long been recognized that FA may not exclusively originate from DI for sessile organisms such as plants, because phenotypic plasticity in response to heterogeneities in the environment might also produce FA. This study provides the first empirical evidence for this hypothesis. We reasoned that solar irradiance, which is greater on the southern side than on the northern side of plants growing in the temperate zone of the Northern Hemisphere, would cause systematic morphological differences and asymmetry associated with the orientation of plant parts. We used geometric morphometrics to characterize the size and shape of flower parts in Iris pumila grown in a common garden. The size of floral organs was not significantly affected by orientation. Shape and particularly its asymmetric component differed significantly according to orientation for three different floral parts. Orientation accounted for 10.4% of the total shape asymmetry within flowers in the falls, for 11.4% in the standards and for 2.2% in the style branches. This indicates that phenotypic plasticity in response to a directed environmental factor, most likely solar irradiance, contributes to FA of flowers under natural conditions. That FA partly results from phenotypic plasticity and not just from DI needs to be considered by studies of FA in plants and other sessile organisms.     

Benign familial neonatal convulsions: confirmation of genetic heterogeneity and further evidence for a second locus on chromosome 8q

The syndrome of benign familial neonatal convulsions (BFNC) is a rare, autosomal dominant form of epilepsy. It is characterized by spontanous seizures beginning within the first 6 months of life. In the majority of families linkage is to chromosome 20q markers. Based on the linkage results in one large BFNC kindred, genetic heterogeneity and existence of a second locus on chromosome 8 have been suggested. Here we report on a second BFNC family in which linkage to the EBN1 locus on chromosome 20q was excluded, confirming the genetic heterogeneity of this disorder. All affected family members experienced onset of seizures before the age of 2 months. Three BFNC subjects showed subsequent epileptic seizures after 12 months of age, showing that the risk of subsequent epilepsy is not restricted to the chromosome 20q linked BFNC families. A lod score of 0.99 was obtained with the marker D8S274, suggesting linkage to chromosome 8.     

The antitumor agent mitoxantrone binds cooperatively to DNA: evidence for heterogeneity in DNA conformation

The equilibrium binding of the antitumor compound DHAQ, or mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione], to various DNAs has been examined by optical titration and equilibrium dialysis methods. At low r (bound drug/DNA base pair) values, r less than 0.03, DHAQ binds, in a highly cooperative manner, to calf thymus and Micrococcus lysodeikticus DNAs. The binding isotherms for the interaction of DHAQ with Clostridium perfringens DNA and poly(dA-dT).poly(dA-dT) exhibit a small positive slope at low r values, suggestive of cooperative binding. In contrast, the binding of DHAQ to poly(dG-dC).poly(dG-dC) shows no evidence of cooperative binding even at very low r values. At higher r values (r greater than 0.05), the binding of DHAQ to all the DNAs studied is characterized by a neighbor-exclusion process. A model is proposed to account for the two modes of binding exhibited in the cooperative binding isotherms. The main feature of the proposed model is that local sequence and structural heterogeneity of the DNA give rise to sets of binding sites to which DHAQ binds in a highly cooperative manner, while the majority of the DNA sites bind DHAQ via a neighbor-exclusion process. This two-site model reproduces the observed binding isotherms and leads to the conclusion that DHAQ binds in clusters to selected regions of DNA. It is suggested that clustering may play a role in the physiological activity of drugs.     

The chemotactic response to PDGF-BB: evidence of a role for Ras

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase- mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, we find that Ras inhibition suppresses migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibited PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras failed to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, did migrate toward PDGF-BB. Cells expressing dominant-negative Ras were able to migrate toward soluble fibronectin demonstrating that these cells retained the ability to migrate. These results suggest that Ras is an intermediate in PDGF- stimulated chemotaxis but may not be required for fibronectin- stimulated cell motility.     

Immunocytochemical evidence suggesting heterogeneity in the population of sea urchin egg cortical granules

Unfertilized eggs of many species of animals contain cortical granules, which are specialized secretory granules that upon fertilization release their contents from the egg. The unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, contain cortical granules that all display an identical and elaborate internal morphology. It has been assumed that they all contain identical components. In this report we present immunocytochemical data which indicate that the cortical granule population of S. purpuratus eggs is heterogeneous. Two monoclonal antibodies are shown to react to the spiral lamellae region of approximately 20% of the cortical granules, implying that the contents of the reactive granules differ from the contents of the majority of the population. An egg protein of greater than 320 kDa is recognized by the antibody. These antibodies also stain a 130-kDa protein expressed on the surface of primary mesenchyme cells in later development. Both antibodies recognize a post-translational modification of this protein. This suggests that an antigenically similar epitope is present both on the 130-kDa primary mesenchyme cell-specific protein and in the cortical granules. To determine if the primary mesenchyme and cortical granule proteins are related, a fusion protein antibody specific for a region of the 130-kDa protein was used to stain unfertilized eggs. This antibody did not stain cortical granules. Thus, 20% of the cortical granules contain a molecule that has an epitope antigenically similar to the post-translational modification recognized in primary mesenchyme cells by the monoclonal antibodies.