Rare blood donors: a personal approach.

The National Blood Group Reference Laboratory (NBGRL) in Israel was established in Jerusalem in 1971 and transferred to Magen David Adom (MDA), National Blood Services in 1995. This laboratory was the inspiration of the first author of this article for over 30 years. The realization of this vision was made possible by the cooperation of colleagues and laboratory workers in blood transfusion services throughout the country. The aim of the service was to provide diagnostic help in resolving immunohematologic problems found in the blood banks and clinics in Israel. In the beginning, only a part-time technician performed the work and testing was done using very limited reagents. The service was expanded by personal visits to all of the 22 blood banks in Israel to explain the aim of this new service and to educate them about the importance of resolving each and every case. One major issue was the cost involved in referring problems but it was decided at the outset that these would be covered by the government to ensure that a workup would be performed for all referred cases. The expansion of the service could not have been achieved without the help of the SCARF program. This voluntary service enabled us to identify the first rare donors in Israel, resolve complex cases, and find compatible blood for our patients. To illustrate the importance of the NBGRL in Israel and the rapid resolution of cases referred, several individual stories are described. The purpose of this review is to show the importance of the NBGRL in identifying rare blood groups and in providing and coordinating services and the importance of keeping in close contact with the rare donors to encourage and promote their donations, which may save lives.

Persistent anti-Dra in two pregnancies.

The Drori (Dr(a)) antigen is one of the ten high-prevalence antigens of the Cromer blood system, which are carried on decayaccelerating factor (DAF, CD55). The Dr(a-) phenotype was first described in a 48-year-old Jewish woman from Bukhara. Her serum contained an antibody to a high-prevalence antigen named anti-Dra. Most known individuals with the Dr(a-) phenotype are Jews from the geographic area of Bukhara, but individuals from Japan have also been described. Antibodies in the Cromer blood group system, including anti-Dra,have never been reported to cause HDN. In most of the cases with anti-Dra examined in Israel, the antibodies have been subtyped as IgG2 and IgG4. This report is of a woman with Dr(a-) phenotype and an anti-Dr(a) titer of 256 to 512 in her serum, observed during two successive pregnancies. At birth, the RBCs of the first- and second-born child were negative and positive in the DAT, respectively, and neither manifested clinical signs of HDN. The disappearance of Cromer system antibodies, including anti-Dra in midpregnancy, has been described in a previous study. In that study, it was theorized that the antibodies in the serum of the women were adsorbed onto placental DAF. The finding of a high anti-Dra titer in two successive pregnancies in this patient, with a positive DAT for the RBCs of one of the two babies at term, differs from published reports, suggesting that a different mechanism might be involved.

Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

Regulation of acetylcholine receptor gene expression in human myasthenia gravis muscles. Evidences for a compensatory mechanism triggered by receptor loss.

Myasthenia gravis (MG) is a neuromuscular disorder mediated by antibodies directed against the acetylcholine receptor (nAChR) resulting in a functional nAChR loss. To analyze the molecular mechanisms involved at the muscular target site, we studied the expression of nAChR subunits in muscle biopsy specimens from MG patients. By using quantitative PCR with an internal standard for each subunit, we found that the levels of beta-, delta-, and epsilon-subunit mRNA coding for the adult nAChR were increased in severely affected MG patients, matching our previous data on the alpha-subunit. Messenger levels were highly variable in MG patients but not in controls, pointing to individual factors involved in the regulation of nAChR genes. The fetal subunit (gamma-chain) transcripts were almost undetectable in the extrajunctional region of MG muscle, suggesting that gene regulation in MG differs from that in the denervation model, in which nAChR gamma-subunit mRNA is reexpressed. Nicotinic AChR loss mediated by monoclonal anti-nAChR antibodies in both the TE671 muscle cell line and cultured normal human myotubes induces a similar increase in beta- alphand delta-subunit mRNA levels, suggesting the existence of a new muscular signaling pathway system coupled to nAChR internalization and independent of muscle electrical activity. These data demonstrate the existence of a compensatory mechanism regulating the expression of the genes coding for the adult nAChR in patients with MG.

The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor.

Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR alpha subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR alpha subunit (H alpha1-210), and to portions of it that encompass either the MIR (H alpha1-121) or the ligand binding site of AChR (H alpha122-210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments H alpha1-121 and H alpha1-210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments H alpha1-121 and H alpha1-210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment H alpha122-210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia.

Acetylcholine receptor alpha-subunit and myogenin mRNAs in thymus and thymomas.

Myasthenia gravis is an autoimmune disorder characterized in most cases by serological antibody against the acetylcholine receptor (AChR). Evidence for intrathymic localization of AChR suggests that the thymus has an important role in the pathogenesis of this disorder. Using reverse transcription followed by the polymerase chain reaction, we have demonstrated AChR alpha-subunit mRNA in thymuses and thymomas from patients with and without myasthenia gravis. We have also studied the expression of myogenin which is known to be involved in the regulation of AChR expression. By using the reverse transcription polymerase chain reaction, we found myogenin mRNAs in all of the thymuses and thymomas. Thus, both AChR alpha-subunit and myogenin mRNA are present in all of these specimens. By immunohistochemistry myoid cells (desmin and myoglobin positive) were present in all (four of four) thymuses studied and in two of five thymomas. Thus, in thymomas, nonmyoid cells might express both AChR and myogenin. These results indicate that cells within the thymus and thymoma express AChR and its regulatory protein myogenin and that such cells, under certain conditions, might play a role in the triggering of myasthenia gravis.

Acetylcholine receptor and myogenic factor gene expression in Torpedo embryonic development.

The mRNA levels of acetylcholine receptor (AChR) and myogenic factors were followed during embryonic development of Torpedo skeletal muscle and its homologue, the electric organ. A different developmental pattern of AChR gene expression was found in these two tissues: a slight decrease in the muscle, and a marked increase, concomitant with synapse formation, in the electric organ. However, the developmental pattern of MyoD and MRF4 mRNA levels was similar in both tissues, with no significant changes during development. This is in contrast with the sharp increase in the expression of AChR in the electric organ and may suggest that the burst in the expression of AChR during the differentiation of myotubes into electrocytes is not regulated by changes in the myogenic factor mRNA levels.

Increased gene expression of acetylcholine receptor and myogenic factors in passively transferred experimental autoimmune myasthenia gravis.

The passive transfer of myasthenia gravis by injection of mAb against muscle acetylcholine receptor (AChR) alpha-subunit, results in increased expression of AChR subunit genes, mainly at synaptic regions. The gene expression of AChR and of other muscle-specific proteins is regulated in a similar manner in passively transferred experimental autoimmune myasthenia gravis (EAMG) and in AChR-induced EAMG. Administration of AChR-specific mAb leads to a significant reduction in muscle AChR content and to an elevation in the mRNA levels corresponding to the adult, synaptic type of the receptor, as shown by Northern blot and in situ hybridization analyses. The mRNA levels of the myogenic factors myogenin and MRF4 are also increased moderately, whereas MyoD transcript levels remain unchanged. Thus, passive transfer of EAMG by mAb directed to defined epitopes of AChR alpha-subunit provides a suitable model for analyzing and following the cascade of molecular events triggered by anti-AChR immunopathologic antibodies and may shed light on the regulatory mechanisms underlying the human disease as well.

Changes in the expression of mRNAs for myogenic factors and other muscle-specific proteins in experimental autoimmune myasthenia gravis.

The regulation of genes for acetylcholine receptor (AChR), myogenic factors and other muscle-specific proteins has been analyzed in experimental autoimmune myasthenia gravis (EAMG) and following denervation. The levels of the transcripts for the myogenic factors, MyoD1, myogenin and MRF4, were measured using Northern blot analysis. Myogenin and MRF4 transcript levels were observed to be 3.1- and 2.6-fold higher in muscle of rats with EAMG than in controls, respectively. MyoD1 levels, however, remained unchanged. The increases in AChR, myogenin and MRF4 mRNAs were one order of magnitude higher in 2-week denervated muscle than in the myasthenic muscle. The levels of muscle creatine kinase (MCK), alpha-actin and muscle dystrophin transcripts were also analyzed. Dystrophin levels were found to be 1.7- and 4.7-fold higher in EAMG and denervated muscle, respectively, than in controls; in contrast, MCK and alpha-actin levels remained unchanged.

Regulation of acetylcholine receptor gene expression in rats treated with alpha-bungarotoxin.

Regulation of acetylcholine receptor (AChR) gene expression was analyzed in alpha-bungarotoxin (alpha-BTX) treated rats. A reduction in available 125I-alpha-BTX binding sites was accompanied by an increase in the various AChR transcripts. The increase in the AChR alpha-, beta- epsilon- and delta-subunit mRNAs was similar to that observed in rats with experimental autoimmune myasthenia gravis (EAMG). Unlike in EAMG, the gamma-subunit transcripts reappeared following alpha-BTX treatment. The quantitative differences in the levels of AChR transcripts between alpha-BTX treatment and EAMG on one hand and denervation on the other hand, support the notion that the regulation of AChR gene expression is controlled by muscle activity and by neuronal factors as well. We also demonstrate in this report that myogenin transcripts increase following alpha-BTX treatment as well as following denervation, whereas MyoD1 transcripts remain stable.

Acetylcholine receptor gene expression in experimental autoimmune myasthenia gravis.

Acetylcholine receptor (AChR) gene expression was analyzed in experimental autoimmune myasthenia gravis (EAMG) in rabbits, rats and mice. An increase in AChR transcripts was demonstrated to be exclusively associated with myasthenic symptoms and with a severe loss in membrane AChR. An increase of alpha-, beta-, epsilon-, and delta-subunit specific mRNAs (5.2-, 1.6-, 3.2- and 3.7-fold, respectively), which code for the adult type of AChR (alpha 2 beta epsilon delta) was observed in EAMG in rats. The gamma-subunit transcript was not detectable in myasthenic or healthy rats. It appears that the regulatory control of AChR gene expression in EAMG is different from that observed upon denervation.

Pausing, switching and speed fluctuation of the bacterial flagellar motor and their relation to motility and chemotaxis.

Wild-type Escherichia coli and Salmonella typhimurium cells, tethered to glass by their flagella, rotate with brief intermittent pauses, the prevalence of which is decreased by attractants and increased by repellents. By attaching latex beads to filaments of a S. typhimurium mutant having straight rather than helical flagella, it was established that the flagella on free cells also pause intermittently. Pausing is therefore an intrinsic feature of the motor and not an artifact associated with tethering. In tethered cells of wild-type strains and non-chemotactic mutants defective in transducers, chemotaxis proteins, or the flagellar switch, both the classical response to chemotactic stimuli (change in direction of rotation from counterclockwise to clockwise or vice versa), and the pausing response to such stimuli, were linked together. No separate signal for pausing was found. In comparing different strains under different stimulation conditions, it was found that cells that never reversed seldom if ever paused, while cells that reversed frequently paused frequently. It is suggested that pausing is the result of futile switching events. A modified description of tumbling and chemotaxis is provided in which pausing, as well as reversal, has a role. Suppression of reversals and pauses by attractant stimuli commonly resulted in an increase in the speed of counterclockwise rotation; this may be because of suppression of pauses or reversals that are too brief to be detected. The clockwise rotation rate of unstimulated cells, which commonly was faster than their counterclockwise rate, was not further increased by repellent stimuli. The rotation rate of any given cell under any given condition was found to fluctuate on all time-scales measured. The study also revealed that some of the common repellents of E. coli and S. typhimurium slow down or stop the motor; these effects are not mediated by the chemotaxis machinery or intracellular pH.

Increased levels of acetylcholine receptor alpha-subunit mRNA in experimental autoimmune myasthenia gravis.

To gain insight into the regulatory mechanisms underlying the blockade and loss of acetylcholine receptor (AChR) in myasthenia, we have followed AChR alpha-subunit mRNA levels in leg muscles of myasthenic and normal rabbits and rats. Northern blots of RNA preparations from normal and myasthenic animals were hybridized with a mouse AChR alpha-subunit cDNA probe. Our experiments indicate a specific increase (4-7-fold) in the levels of alpha-subunit mRNA in animals with experimental autoimmune myasthenia gravis (EAMG), in comparison with control animals. Actin mRNA levels were essentially unchanged. Our results thus suggest that EAMG is accompanied by an increased level of AChR gene transcription.

An evaluation of the SCM test for the diagnosis of cancer of the breast.

The SCM test (structuredness of the cytoplasmic matrix) consists of measuring the fluorescence polarization of fluorescein which is introduced into a particular sub-group of peripheral lymphocytes. The test has a non-specific part for the general detection of cancer and a specificity procedure which is based on the use of specific cancer extracts. In this article we deal only with the latter in relation to breast cancer. Blood samples from 94 patients have been tested; six of these had mastectomy performed previously; 83 underwent consecutively a surgical procedure and histology was obtained; five were only clinically examined. In 45/49 (92%) patients, correlation between a positive specificity test and tissue malignancy was found. Out of 35 patients with non-malignant proliferative lesions (as found by histology), 25 reacted positively in the test. Two out of five patients with non-malignant, non-proliferative lesions reacted positively in the test. Five patients who were defined as normals by clinical examination reacted negatively in the test. These results indicate the potential of the SCM test for detecting breast malignancy. The clinical implications of the test for cancer diagnosis are discussed.

Distribution and modulation of surface charges of cells from human leukemia-lymphoma lines at various stages of differentiation.

Untreated and retinoic acid (RA) treated human leukemia-lymphoma cell lines reflecting hematopoietic cells at various stages of differentiation, were examined electron microscopically for their surface negative charge distribution using cationized ferritin (CF), an electron dense label of anionic sites. The results indicate that there is a correlation between the CF labeling density/distribution and the stage of lymphoid cell differentiation. Viable unfixed null cell lines show a low CF labeling density with few and small CF patches. A gradual increase in CF labeling density and increase in size and number of CF patches correlates with the stage of differentiation on cell lines of both T or B origin. Treatment of viable unfixed cells with 10(-5) MRA for 10 days seems to prevent the CF-induced formation of CF patches, resulting in a continuous and even distribution of the CF label, similar to that observed on the surface of cells fixed before CF labeling. Some correlation between the distribution of surface anionic sites and the malignant potential of the human leukemic lines could be detected.