Diabetes induces changes in ILK, PINCH and components of related pathways in the spinal cord of rats.

Recent work suggests that diabetes affects processing of peripheral, spinal and supraspinal signals in the spinal cord. However, there is little evidence for spinal cord lesions that would account for alterations in behavioral responses induced by experimental diabetes. Therefore, we assessed the expression of proteins that might affect neuronal cytoskeletal stability and thus promote dendritic and synaptic reorganization in diabetic rats. Expression of ILK, PINCH, PI3K, GSK-3beta, tau, MAP2, synaptophysin and drebrin in the lumbar spinal cord of non-diabetic and streptozotocin-diabetic rats was assessed by Western-blot analysis and immunocytochemistry after 8 and 20weeks of diabetes. The impact of diabetes on the proteins studied was duration-dependent with changes observed after 20 but not 8weeks of diabetes. ILK and PINCH proteins levels were significantly decreased and both colocalized to neurons and oligodendrocytes. PI3K protein levels were also significantly decreased, while GSK-3beta activity tended to be increased. Phosphorylation of tau and MAP2A/B protein expression were significantly increased, and expression of synaptophysin and drebrin were reduced in diabetic rats. Decreased ILK and PINCH as well as alterations of components of related signaling pathways are associated with tau hyperphosphorylation, MAP2 overexpression and reduction of synaptic proteins in the spinal cord of diabetic rats, suggesting that ILK and PINCH contribute to stabilization of axonal and dendritic structures. However, these changes are not likely the cause of altered behavioral responses in diabetic rats that occur after short-term diabetes, but may contribute to structural changes occurring in long-term diabetes.

Reduced frequency of HLA-DQ6 in individuals with a positive direct antiglobulin test.

BACKGROUND: The association between HLA class II antigens and the production of red cell autoantibodies was investigated in this study. STUDY DESIGN AND METHODS: Thirty-one individuals with a positive direct antiglobulin test (DAT) and 85. DAT-negative controls (cadaveric organ donors) were typed using a DNA-based method for class II HLA typing, that is, the polymerase chain reaction with sequence-specific primers. Amplified HLA alleles corresponding to the serologic specificities DR 1-18 and DQ 1-9 were identified. RESULTS: Analysis of DR and DQ frequencies showed that HLA-DQ6 was less frequent in DAT-positive individuals (19% vs. 53% in the control population), with a p value of 0.0014 and a corrected p value of 0.059, and relative risk of 0.23 (95% Cl = 0.09-0.58). The frequency of HLA-DQ6 was higher in asymptomatic DAT-positive blood donors (n = 8, 38% DQ6 positive, p = 0.48) than in DAT-positive hospital and clinic patients (n = 23, 13% DQ6 positive, corrected p value = 0.030, relative risk = 0.13, 95% Cl = 0.04-0.41), 96 percent of whom had evidence of clinical hemolysis. CONCLUSION: This study demonstrates that HLA-DQ6 may have a negative association with a positive DAT result in patients with evidence for hemolysis, and may be a resistance antigen for clinically relevant red cell autoantibodies.

Peyronie's disease is associated with an HLA class II antigen, HLA-DQ5, implying an autoimmune etiology.

PURPOSE: Previous studies using serological (protein based) HLA typing methods suggested an association between various HLA antigens and Peyronie's disease. We used a molecular (deoxyribonucleic acid based) HLA typing method to determine whether Peyronie's disease is associated with class II HLA antigens. MATERIALS AND METHODS: Class II HLA typing using the polymerase chain reaction-sequence specific primer method was performed in 31 patients with Peyronie's disease, 19 age matched urological patients with disorders other than Peyronie's disease and 75 major organ donor cadavers representative of the general population. RESULTS: The frequency of HLA-DQ5 was significantly increased in patients with Peyronie's disease (61% HLA-DQ5 positive) compared to age matched disease-free controls (11% HLA-DQ5 positive, corrected p = 0.003) and general population controls (25% HLA-DQ5 positive, corrected p = 0.005). The relative risk for Peyronie's disease associated with HLA-DQ5 was 12.7 and 4.6 compared to age matched and general population controls, respectively. CONCLUSIONS: This study shows a positive association for the HLA class II antigen HLA-DQ5 with Peyronie's disease, suggesting that HLA-DQ5 is a risk factor for Peyronie's disease and implying an autoimmune etiology for this disorder.

Detection of chimerism and early engraftment after allogeneic peripheral blood stem cell or bone marrow transplantation by short tandem repeats.

Chimerism can be monitored after HLA-matched allogeneic bone marrow transplantation (BMT) or allogeneic peripheral blood stem cell transplantation (PBSCT) by detecting polymorphisms in short tandem repeats (STR). The purpose of our study was to document early complete chimerism in BMT and PBSCT recipients using STR, and to determine whether the initial WBC recovery correlated with the days required to attain complete chimerism. A total of 5 patients (2 PBSCT and 3 BMT) were followed by STR after transplantation. Peripheral blood obtained prior to transplantation was used to determine the 2 most informative STR probes for each donor/recipient pair. STR were amplified by polymerase chain reaction (PCR) with 8 commercial probes, and PCR products were visualized with silver staining. Peripheral blood was evaluated daily post-transplantation for WBC counts and to identify the presence of mixed or full chimerism by STR. The sensitivity of the STR technique varied from 0.05 to 1%, depending on the probe. Full chimerism was documented between day 9 and 14 in PBSCT recipients and on day 14 and 16 in BMT recipients. The initial rise in WBC occurred within 3 days of the onset of full chimerism, indicating that full chimerism is a more sensitive indicator of early engraftment. Periodic recipient monitoring using STR after complete chimerism identifies those patients who revert to mixed chimeras. The STR method may be useful in future studies to determine the significance of early engraftment and the clinical implications of sustained complete chimerism or mixed chimerism.

Effect of crossmatching on outcome in organ transplantation.

The complement-dependent cytotoxicity (CDC) crossmatch and the flow cytometry crossmatch (FCXM) are both used prospectively in renal transplantation, and their use is under evaluation in other types of major organ transplantation. The FCXM is the more sensitive method and better predicts outcome in second and subsequent renal allografts. Improved survival has unmasked the detrimental effect of a positive crossmatch on outcome in liver transplantation. Because of the urgent need of liver transplant candidates, it is unrealistic to defer transplantation until a crossmatch-negative donor is found; however, additional therapeutic measures may be taken to improve outcome for crossmatch-positive liver recipients. Some reports suggest that prospective crossmatching may improve outcome for sensitized heart recipients, and, additionally, recent studies have demonstrated that HLA compatibility between donor and recipient is an independent variable affecting survival after heart transplantation, prompting a reassessment of the current practice of transplanting hearts without consideration of the HLA match.

A new LIM protein containing an autoepitope homologous to "senescent cell antigen".

Autoantibody eluted from aged human red blood cells was used to immunoscreen a human fetal liver expression library and led to the isolation of a cDNA encoding a novel 35.8 kDa protein with five LIM domains. An autoepitope homologous to the "senescent cell antigen" on the red blood cell membrane anion exchange protein identified by Kay et al. (P.N.A.S. 87:5734, 1990) was present in the first zinc finger of the third LIM domain. The gene for this novel protein is highly conserved in vertebrates, and its 4.6 kb mRNA is widely expressed in human tissues. Recombinant autoantigens such as the one reported here have potential applications in vitro for the purification, identification and quantitation of autoantibodies, and in vivo for the removal of autoantibodies, increasing red blood cell lifespan and reducing the need for transfusion.

Identification of a precursor genomic segment that provided a sequence unique to glycophorin B and E genes.

Human glycophorin A, B, and E (GPA, GPB, and GPE) genes belong to a gene family located at the long arm of chromosome 4. These three genes are homologous from the 5'-flanking sequence to the Alu sequence, which is 1 kb downstream from the exon encoding the transmembrane domain. Analysis of the Alu sequence and flanking direct repeat sequences suggested that the GPA gene most closely resembles the ancestral gene, whereas the GPB and GPE genes arose by homologous recombination within the Alu sequence, acquiring 3' sequences from an unrelated precursor genomic segment. Here we describe the identification of this putative precursor genomic segment. A human genomic library was screened by using the sequence of the 3' region of the GPB gene as a probe. The genomic clones isolated were found to contain an Alu sequence that appeared to be involved in the recombination. Downstream from the Alu sequence, the nucleotide sequence of the precursor genomic segment is almost identical to that of the GPB or GPE gene. In contrast, the upstream sequence of the genomic segment differs entirely from that of the GPA, GPB, and GPE genes. Conservation of the direct repeats flanking the Alu sequence of the genomic segment strongly suggests that the sequence of this genomic segment has been maintained during evolution. This identified genomic segment was found to reside downstream from the GPA gene by both gene mapping and in situ chromosomal localization. The precursor genomic segment was also identified in the orangutan genome, which is known to lack GPB and GPE genes. These results indicate that one of the duplicated ancestral glycophorin genes acquired a unique 3' sequence by unequal crossing-over through its Alu sequence and the further downstream Alu sequence present in the duplicated gene. Further duplication and divergence of this gene yielded the GPB and GPE genes.

Immunogenicity and antigenicity of defined linear determinants of the MN sialoglycoprotein glycophorin A.

Many epitopes of the blood group MN sialoglycoprotein glycophorin A (GPA) are clinically important and have been implicated in alloimmunization and autoimmune hemolytic anemia. However, the factors that contribute to the immunogenicity and antigenicity of GPA epitopes are poorly understood. To study this question, linear determinants were defined to include all GPA exofacial regions that were free of linked carbohydrate (GPA 27-34, GPA 52-62, and GPA 65-70), and peptides corresponding to these amino acids were synthesized. All defined GPA determinants were shown to contain residues accessible to the immune system. Immunogenicity was measured by binding rabbit antisera, produced by using red cells (RBCs), RBC membrane, and purified GPA as immunogens, to peptide antigens in an enzyme-linked immunosorbent assay. GPA 27-34 and GPA 52-62 were shown to be immunogenic, but GPA 65-70 was not. Comparison of the binding of antisera to peptide and purified GPA antigens showed that the defined determinants were less immunogenic than other, nondefined GPA epitopes. Antigenicity algorithms (hydrophilicity, surface probability, flexibility, and antigenic index) predicted that GPA 27-34 and, to a lesser extent, GPA 52-62 would be antigenic, while GPA 65-70 would be nonantigenic. Antigenicity, measured by the binding of rabbit antipeptide sera to RBCs, RBC membrane, and purified GPA, was demonstrated for the GPA 52-62 determinant alone. Results differed among molecular forms of GPA, which indicated that the specific conformation assumed by GPA is important in determining the immunogenicity and antigenicity of its epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

Primate genes for glycophorins carrying MN blood group antigens.

Glycophorin A, B, and E genes were derived from a common ancestral gene and this gene family appeared during primate evolution, probably between orangutan and gorilla divergences. Based on the study of genomic structures of these human glycophorins and the genetic and immunological study of primate glycophorins, we hypothesize that chimpanzee and gorilla glycophorin B could possess a longer extracellular region and carry a stronger N blood group antigenicity compared with that of the human.

Glycophorin B and glycophorin E genes arose from the glycophorin A ancestral gene via two duplications during primate evolution.

Human glycophorin A, B, and E genes are homologous from the 5'-flanking region to 1 kilobase downstream from the exon encoding the transmembrane region. Analysis of human Alu sequences at the transition site from the homologous to nonhomologous region suggested that the GPA gene most closely resembles the ancestral gene, whereas GPB and GPE genes arose by homologous recombination within the Alu repetitive sequence, and acquired 3' sequences from an unrelated gene (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623; Kudo, S., and Fukuda, M. (1990) J. Biol. Chem. 265, 1102-1110). To understand glycophorin gene evolution in primate phylogeny, transmembrane and Alu regions of several primate genomes were amplified by the polymerase chain reaction and their sequences were analyzed. These studies revealed that the GPA gene was present in all primates studied, and the GPB gene was present in pygmy chimpanzee, chimpanzee, and gorilla, but absent from orangutan and gibbon. GPE gene was present in all species with a GPB gene, but was detected in only 7 out of 16 gorillas. The 24-base pair insertion sequence found in the transmembrane exon of the human GPE gene was shown to be derived from the ancestral GPB gene and was inserted into the ancestral GPE gene prior to gorilla divergence. The recombination site in the GPA gene was confirmed to be within an Alu repetitive sequence. We conclude that GPB and GPE genes arose from an ancestral GPA gene via two gene duplications occurring during primate evolution, prior to gorilla divergence.

Propranolol treatment affects parameters of human immunity.

Elevated sympathetic activity can modulate parameters of immunity. We investigated the role of the low sympathetic activity in resting healthy volunteers by treating them with the beta-adrenergic antagonist propranolol (3 x 40 mg/d for 7 days). Propranolol treatment increased the number of circulating T cells but not that of other white blood cells. Similarly, Con A-stimulated lymphocyte proliferation and IL-2 formation were enhanced. Although the number of circulating NK cells did not change, NK-cell activity was reduced markedly after propranolol treatment. These alterations are not a mirror image of the changes observed under conditions of elevated sympathetic activity but demonstrate that withdrawal of the endogenous sympathetic tone by drugs such as propranolol can modulate parameters of human immunity.

Molecular analysis of a hybrid gene encoding human glycophorin variant Miltenberger V-like molecule.

The genomic structure of a human glycophorin variant, Miltenberger class V-like molecule (MiV*), was examined. Southern blot analysis of total genomic DNA revealed that the 5' half of the MiV* gene derived from glycophorin A (GPA) gene whereas the 3' half derived from glycophorin B (GPB) gene. This structure is reciprocal to another glycophorin variant, Sta, which has a GPB-GPA hybrid structure. The genomic sequences around the crossing-over point were amplified by polymerase chain reaction, and the sequences were determined. Comparison of the nucleotide sequences of the GPA, GPB, and MiV* genes indicates that the crossing-over point is located in the region around the 3' end of intron 3 of the GPA gene. This place is different from the crossing-over point for Sta, which was found to be highly homologous to that for haptoglobin-related genes. However, the nucleotide sequences within the presumptive crossing-over point for the MiV* gene were found to be homologous in a reverse orientation to the crossing-over point proposed for haptoglobin-related genes. These results suggest strongly that homologous recombination through unequal crossing over can be facilitated by specific genomic elements such as those in common for formation of MiV*, Sta, and haptoglobin-related genes. The present study also localized the gene of the third glycophorin, GPE, at chromosome 4, q31.1 band, the same locus as for the GPA and GPB genes. The results indicate that GPE was not involved in generating MiV* or Sta hybrid gene despite the fact that it is localized adjacent to the GPA and GPB genes.

Identification of the crossing-over point of a hybrid gene encoding human glycophorin variant Sta. Similarity to the crossing-over point in haptoglobin-related genes.

One of the human glycophorin variants, Stones (Sta), has been shown to be the product of a hybrid gene of which the 5'-half derived from the glycophorin B (GPB) gene whereas the 3'-half derived from the glycophorin A (GPA) gene. The present study reveals the crossing-over point of this hybrid gene from the analysis of polymerase chain reaction products. The genomic sequences encompassing the region corresponding to exon 3 to exon 4 of GPA were amplified by polymerase chain reaction with oligonucleotide primers synthesized according to GPA and GPB genomic sequences (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623). After subcloning the products, the nucleotide sequences derived from GPA, GPB, and putative Sta genes were determined. Comparison of the nucleotide sequences of GPA, GPB, and Sta genes indicate that the crossing-over took place 200 base pairs upstream from the first nucleotide of exon 4. Intriguingly, the nucleotide sequence surrounding the putative crossing-over point is homologous to the crossing-over point proposed for haptoglobin genes (Maeda, N., McEvoy, S.M., Harris, H.F., Huisman, T.H.J., and Smithies, O. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7395-7399). These results suggest strongly that homologous recombination through unequal crossing-over can be facilitated by specific genomic elements, such as those in common in these two crossing-over events. The present study also revealed that this Sta individual has a variant GPA gene; substitution of adenine for guanine at the nucleotide for codon 39 results in substitution of lysine for arginine at amino acid 39, and loss of an SstI restriction site.

Evolution of the glycophorin gene family in the hominoid primates.

Analysis of nucleotide sequences of the human glycophorin A (GPA) and glycophorin B (GPB) genes has indicated that the GPA gene most closely resembles the ancestral gene, whereas the GPB gene likely arose from the GPA gene by homologous recombination. To study the evolution of the glycophorin gene family in the hominoid primates, restricted DNA on Southern blots from man, pygmy chimpanzee, common chimpanzee, gorilla, orangutan, and gibbon was probed with cDNA fragments encoding the human GPA and GPB coding and 3'-untranslated regions. This showed the presence in all of the hominoid primates of at least one GPA-like gene. In addition, at least one GPB-like gene was detected in man, both chimpanzee species, and gorilla, strongly suggesting that the event that produced the GPB gene occurred in the common ancestor of man-chimpanzee-gorilla. An unexpected finding in this study was the conservation of EcoRI restriction sites relative to those of the other four enzymes used; the significance of this observation is unclear, but raises the question of nonrandomness of EcoRI restriction sites in noncoding regions. Further analysis of the evolution of this multigene family, including nucleotide sequence analysis, will be useful in clarification of the evolutionary relationships of the hominoid primates, in correlation with the structure and function of the glycophorin molecules, and in assessment of the role of evolution in the autogenicity of glycophorin determinants.

Multiple restriction fragment length polymorphisms associated with the Vc determinant of the MN blood group-related chimpanzee V-A-B-D system.

Twelve restriction fragment length polymorphisms (RFLPs) were detected in common chimpanzee using two restriction enzymes (HindIII and MspI) and four DNA probes to the coding regions of the human glycophorin A (GPA) and glycophorin B (GPB) genes and their 3'-untranslated regions. Seven RFLPs correlated with red cell expression of the Vc determinant of the MN blood group-related V-A-B-D system and five RFLPs correlated with nonexpression of this antigen. Animals heterozygous for the V allele that encodes the Vc determinant had all 12 polymorphic restriction fragments and appeared to show reduced intensity of probe hybridization to these fragments, consistent with the presence of a V and a non-V allele. No RFLPs were detected with EcoRI, SstI, or BamHI, in spite of the relatively large segment of DNA (at least 20 kb) involved in the polymorphisms. The RFLPs were chimpanzee specific and were not found in man, gorilla, orangutan, or gibbon. Multiple RFLPs distinguishing primate species are rare and may be useful markers for molecular evolution.

Adrenergic control of circulating lymphocyte subpopulations. Effects of congestive heart failure, dynamic exercise, and terbutaline treatment.

The current studies were undertaken to explore the relationship between enhanced sympathetic nervous activity and lymphocyte subset distribution in three settings: congestive heart failure, dynamic exercise, and beta-adrenergic agonist treatment. We compared the number and subset distribution of circulating lymphocytes in 36 patients with congestive heart failure and 31 age-matched control subjects. The number of circulating lymphocytes was lower in heart failure than in control. This was due to a reduction in Tsuppressor/cytotoxic and natural killer cells without significant alteration of Thelper cells. The extent of the alteration was similar in patients with idiopathic and ischemic heart failure, but the reduction was more pronounced in patients with New York Heart Association class III-IV than in class I-II. The plasma catecholamine elevation in heart failure was also independent of etiology but more pronounced in the more severely ill patients. We also assessed lymphocyte subsets after acute stimulation of sympathetic activity by dynamic exercise and after treatment with the beta-adrenergic agonist terbutaline. Dynamic exercise until exhaustion increased the number of circulating lymphocytes in healthy controls and heart failure patients in a subset-selective manner. By contrast, a 7-d treatment with terbutaline caused a reduction in the circulating number of lymphocytes in some subsets that was identical to that seen in heart failure patients. We conclude that prolonged sympathetic activity reduces the number of circulating lymphocytes by a beta-adrenergic mechanism. Such alterations might be involved in the pathophysiology of heart failure and other disease states involving increased activity of the sympathetic nervous system.

CD7-positive acute leukemia lacking T cell receptor gene rearrangements and terminal deoxynucleotidyl transferase expression suggesting pre-T cell origin.

In this report, we describe a case of acute leukemia possessing a unique immunophenotype. Leukemic cells isolated from peripheral blood were analyzed by automated fluorescence-activated flow cytometry. They demonstrated the following antigen pattern: CD7+, CD38+, transferrin receptor+, HLA-DR+, common ALL antigen (CALLA)-, and terminal deoxynucleotidyl transferase (TdT)-. No expression of B cell or myeloid antigens was observed. The observed antigen pattern suggests a pre-T cell origin. In addition, the tumor cells contained germline T cell receptor beta (T beta) and gamma (T gamma) chain genes, and a germline immunoglobulin heavy chain (JH) gene. These findings support the concept that pre-T cell leukemias may demonstrate germline T beta and T gamma genes as well as lack TdT expression. In addition, our results suggest that CD7 expression may be one of the earliest events in T cell differentiation, occurring prior to both T beta an T gamma gene rearrangements, and TdT expression.

A new method for isolation of human lymphocyte subsets reveals differential regulation of beta-adrenergic receptors by terbutaline treatment.

We adapted a technique for isolation of mononuclear leukocyte (MNL) subsets with immunomagnetic beads to the study of beta-adrenergic receptors. Mixed MNL cells were sequentially incubated with monoclonal antibodies specific for certain MNL subsets. Sheep antimouse antibodies coupled to magnetic beads were then added, and the desired MNL subset was pulled out with a magnet. This method yielded subsets with high purity and did not alter beta-receptor density or function. Healthy volunteers were treated for 7 days with the beta 2-selective agonist terbutaline (5 mg t.i.d.). Terbutaline treatment decreased beta-receptor number and isoproterenol-stimulated cyclic adenosine monophosphate (cAMP) generation in natural killer cells, helper T cells, and suppressor/cytotoxic T cells but not in B cells. The decrease was greatest in suppressor/cytotoxic T cells and least in helper T cells. Thus beta-adrenergic receptor regulation by agonists appears to differ among MNL subsets.

Hybrid sialoglycoprotein content of St(a+) red cells.

St(a+) red cells contain a ficin-resistant hybrid sialoglycoprotein (SGP) consisting of the amino terminus of Ss sialoglycoprotein (Ss SGP) and the carboxyl terminus of MN sialoglycoprotein (MN SGP). Ficin-modified St(a+) but not St(a-) red cells were agglutinated by monoclonal antibodies NN5 (anti-N) and 31 (detecting a sialic acid-dependent determinant on the SGPs). SDS-polyacrylamide gel electrophoresis showed three periodic acid-Schiff-staining protein bands in ficin-modified St(a+) but not St(a-) red cell-membranes; these bands bound monoclonal antibodies NN5 and 31 on Western blots. Using Scatchard analysis of 125I-NN5 IgG binding to M+N- red cells, estimates were obtained of 25,000 Ss SGP molecules per red cell per s allele, 33,000 Ss SGP molecules per red cell per S allele, and 75,000 hybrid SGP molecules per red cell per hybrid allele. These results are consistent with the expression of the hybrid SGP gene at a level intermediate between the MN SGP gene and the Ss SGP gene. Monoclonal antibodies NN5 and 31 may be useful in screening ficin-treated red cells for hybrid SGP variants.