[The critical hemoglobin value in newborn infants, infants and children].

The optimum and critical hemoglobin concentrations are determined by the oxygen demand of the tissues and several oxygen transport parameters (i.e., blood flow, arterial oxygen saturation, oxygen affinity of hemoglobin, and the critical venous oxygen pressure). Most of the oxygen transport parameters change markedly during the first weeks after birth. Oxygen consumption and cardiac output in neonates are three times those of adults on a body weight basis. Due to the high oxygen affinity of fetal hemoglobin, the oxygen unloading capacity of hemoglobin in neonates is about 50% less than in adults. From oxygen transport parameters and oxygen consumption we have calculated the optimum and the critical hemoglobin concentrations for preterm and full-term neonates during the first weeks after birth. A hemoglobin concentration of 15 g/dl appears optimal for preterm and full-term infants at birth as well as for adults. The calculated minimum acceptable hemoglobin concentration is 6 g/dl for children and adults, 12 g/dl for preterm infants and 11 g/dl for full-term neonates at birth. Due to the postnatal decrease in oxygen affinity, the minimum acceptable hemoglobin concentration decreases by approximately 1 g/dl/week for the first 5-6 weeks until the minimum value of 6 g/dl for children and adults is reached. The minimum hemoglobin concentration should be 2 g/dl higher in patients who require increased oxygen or suffer from other serious disorders. A minimum hemoglobin concentration of 10 g/dl is recommended in children with leukemia or other oncological disease. In infants and children with chronic hypoxemia (cyanotic congenital heart disease) the minimum hemoglobin concentration should be increased by the percentage of arterial oxygen desaturation.


[Perioperative blood coagulation problems in children].

During childhood preoperative coagulation diagnosis is performed to prove or to rule out an inborn coagulation disorder or an acquired von Willebrand disease. The coagulation system of the newborn differs considerably from that of the adults as well as the time in which the single parameters reach adult values. Reducing the coagulation screening to the determination of aPTT and Quick test neglects severe hemostaseological disorders easily, such as von Willebrand disease which is often observed in childhood and often combined with normal aPTT values. Sometimes children affected with hypertrophy of the adenoids have temporary aPTT prolongations combined with normal values for the other coagulation parameters possibly due to lupus inhibitors. When children with deficiencies of coagulation factors need high doses of coagulation concentrates, the number of laboratory controls may be reduced by determination of recovery and the half-life period some time before.


[Diagnostic strategies for detection of the von Willebrand syndrome].

The von Willebrand disease (vWD) is the most severe coagulopathy. Because of the complex biochemical structure of the von Willebrand factor (vWF), a great number of types and subtypes of the vWD were found. A screening of vWD can only be done by examining the bleeding time, the ristocetin cofactor activity (risto) and by an immunological determination of the vWF concentration. Examinations of 200 patients with a bleeding tendency showed that the ratio vWF/risto < 0.7 indicates a high probability for an abnormal multimeric structure of vWF. The exact determination of the vWD subtype then has to be done by a SDS-agarose gel electrophoresis. In 16.8% of our patients we found a decreased vWF concentration in the platelets. These patients showed normal plasmatic coagulation factors, but a bleeding tendency and a prolonged bleeding time. For diagnosis of vWD the bleeding time, immunological determination of the vWF and the risto should be done first. If a ratio vWF/risto < 0.7 or a prolonged bleeding time with a bleeding tendency is found, the separation of the vWF multimers into plasma and platelets and the determination of the vWF concentration in platelets should be carried out for an exact diagnosis of vWD.


[Autologous blood donation and erythropoiesis].

Autologous blood donation before elective surgery has been widely endorsed as good transfusion practice. Although only 6% of patients undergoing elective orthopedic surgery are unable to donate 3 autologous units, 40% cannot donate 4 units because they become anemic. According to the results of our study this must be due to different problems of bone marrow dynamics, which cannot be overcome by unselected use of human recombinant erythropoietin.


[Preoperative autologous blood donation in heart surgery patients: laboratory parameters with oral iron substitution].

In this study we analyzed some hematologic parameters of 228 autologous blood donors at a cardiosurgic clinic. The standard schedule of drawing autologous blood at our clinic is 4 units whole blood (450 ml) and 1 unit plasma (750 ml) for a time of 5 weeks under iron substitution (equivalent to 240 mg Fe). In younger women this kind of drawing blood leads to an unacceptable decrease of hemoglobin concentration. The preoperative autologous blood donation results in an increase of erythropoiesis on the day of clinical admittance. The best parameters to supervise the hematologic situation of the outpatient autologous blood donors are the hemoglobin concentration and the hematocrit.


[Hemoglobin follow-up of orthopedic autologous blood transfusion patients].

Patients undergoing autologous donation prior to elective orthopedic surgery show better Hb values during the perioperative period compared to those without autologous donation, all patients in both groups receiving no homologous transfusion. The widely assumed improvement of Hb regeneration during the postoperative period according to an erythropoiesis stimulated by preoperative autologous donation is not supported by our data.


[Combination of a simple hollow fiber system with leukocyte filter for production of leukocyte depleted erythrocyte concentrates and plasma].

Leukocyte-depleted red cell concentrate (RCC) and plasma were separated by a hollow fiber filter system combined with a leukocyte filter without any additional devices. The RCC with 100 ml additive solution had a weight of 329 g; hematocrit was 0.55, free hemoglobin 16 mg/dl; leukocytes were (0.6 +/- 0.6) x 10(9)/l. The plasma (268 g) contained 5.4 g/dl of total protein, and only a few blood cells; clotting factor VIII activity 75%, all satisfying the guidelines.


[Cryopreservation of erythrocytes with hydroxyethyl starch. In vitro results leading to an autologous retransfusion model in the dog].

Although the effectiveness of hydroxyethyl starch (HES) as a cryoprotectant for human red blood cells (HRBC) is well known, no clinical application has evolved so far. In contrast to glycerol HES has the advantage of causing no hemolysis per se. This offers the opportunity of a one-step procedure without a time consuming postthaw washing procedure prior to transfusion. In this study the in vitro results obtained with red blood cells from 8 dogs (DRBC) are reported and compared to HRBC (n = 5). It turned out that DRBC had a similar 2,3-DPG and a lower ATP content. Postthaw survival in terms of saline stability differed markedly (67 +/- 6 and 86 +/- 2%, respectively). DRBC were more susceptible to hypotonic stress than HRBC. Nevertheless, after cryopreservation 91% (HRBC) and 92% (DRBC) of the original 2,3-DPG were found in the thawed RBC concentrates.


Cryopreserved autologous platelet transfusions in alloimmunized patients with acute leukemia.

Autologous platelets of 5 alloimmunized patients with acute leukemia in remission were cryopreserved with 5% dimethylsulfoxide in liquid nitrogen and retransfused in the following therapy-induced thrombocytopenic phase. The mean platelet recovery after freezing, thawing and washing was 85 +/- 6%. The mean corrected 1-hour increment in platelet counts was 11 (4-27) x 10(9)/l, i.e. 61% in comparison with fresh platelet transfusions in other patients. In vivo function of frozen platelets was documented by improvement of the posttransfusion bleeding time. Cryopreserved autologous platelets function hemostatically and can be used even for completely refractory patients and thus permit curative antileukemic therapy.


[Rheologic studies before and after therapeutic cytapheresis and plasma exchange treatment].

Changes in rheological properties of human blood before and after platelet pheresis were measured in patients with myeloproliferative diseases as well as in a control group of platelet donors. Although the subjective feeling of the patients during measurement did improve, a better rheological situation could not be demonstrated by the rheological parameters used. A reduction of platelets does not influence the viscosity of whole blood or of plasma. On the other hand, the viscosities of whole blood and plasma differed significantly before and after plasma-pheresis in patients with monoclonal gammopathies. The control group comprised patients without hyperviscosity syndrome and did not show any change in rheological parameters.


Two years serial lymphocytapheresis of progressive hyperleukocytosis in a patient with a non-Hodgkin's lymphoma.

A patient with non-Hodgkin's lymphoma was treated by ambulant lymphocytapheresis once a month over a period of 2 years and 8 months. The cytoreductive effect resulted in a decrease of white blood cells from 410/nl to 130/nl. The patient tolerated the procedure, which lasted 3-4 h, without any side effects. He could be kept in good condition by monthly apheresis. So lymphocytapheresis is a means to reduce hyperleukocytosis in patients with non-Hodgkin's lymphoma.


[Therapeutic thrombocytapheresis in patients with myeloproliferative diseases with the cell separators Fresenius AS 104 and Cobe Spectra: biocompatibility and safety].

We studied biocompatibility, safety and efficiency of the two cell separators AS 104 (Fresenius) and Spectra (Cobe) during therapeutic thrombocytaphereses. Although some patients have very high platelet levels and coagulation as well as circulatory equilibrium is easily disturbed, no important activation of coagulation or complement was observed. In respect to patient's safety both cell separators performed very well.


[Hemorheologic findings in hyperviscosity syndrome and their modification by plasmapheresis].

A patient suffering from refractory immunocytoma was treated with therapeutical plasmapheresis. To estimate the hemorheological risk during and at the end of the therapy, hemorheological measurements were performed. Hematocrit, aggregation index, filtration index, plasma viscosity, colloid osmotic pressure and protein concentrations were analyzed. The efficiency of the plasmapheresis treatment is demonstrated by the reduction of the plasma viscosity from 5.5 to 2.3 mPa (57% reduction) and of the filtration index from 43.9 to 29.7 (32% reduction). The colloid osmotic pressure decreased from 3.4 to 2.5 kPa (26% reduction), plasma protein concentration from 106 to 86 g/l (19% reduction) and IgM concentration from 100 to 82 g/l (18% reduction), while the albumin concentration remained constant.


Selection of unrelated bone marrow donors: does the current procedure warrant complete MHC class II identity?

Bone marrow transplantation from unrelated donors is being used increasingly for the treatment of patients with leukemia and several other hematologic disorders. Selection of unrelated bone marrow donors currently relies on serological HLA identity and negative mixed lymphocyte reactions between donor/recipient pairs. As serological HLA-DP typing is not feasible, we used the HLA-DPB1 oligonucleotide typing method to investigate whether the current selection procedure can guarantee complete MHC class II identity. In 40 consecutive patients, one third (62/193) serologically HLA-A, -B, -C, -DR and -DQ identical donors were found to be MLC-negative with a relative response below 5%. HLA-DPB1 oligonucleotide typing of these MLC-negative donors revealed that again only one third (20/62) was also identical for DP with their presumptive recipients. In the majority of pairs a disparity in graft-versus-host direction or in host-versus-graft direction of at least one allele was seen. These data indicate that, in spite of the strict MLC criteria used, the current procedure did not warrant complete MHC class II identity. This implies that oligotyping for DPB1 can improve matching and should be introduced for typing of volunteers.


[11th International Histocompatibility Workshop 1991: Personal data for the Multiple Sclerosis Study].

In our multiple sclerosis (MS) study as a part of the 11th IHWS we HLA-typed 6 MS families with 9 patients and defined the complement polymorphisms (BF, C2, C4) of these families. The aims of the study were the definition of the MS susceptibility gene and the investigation of the involvement of other factors in the etiopathogenesis of MS. The MS study of the IHWS demonstrated a strong association with HLA-DRw15 and -DWw6 in a Caucasian population. The heterozygous C2 deficiency in our family PD1 linked with the haplotype A25 B18 DR2 BFS C4A4 C4B2 confirmed by complement titration may express the participation of complement factors in the etiopathogenesis of MS resulting in immunogenetic heterogeneity of MS. Analysis of the 3 MS pairs of sisters shows the linkage of HLA with the assumed MS susceptibility gene. This could not be confirmed in the whole MS family study of the 11th IHWS.


[Attempts in developing a standardized test system of immunologic reactivity].

Stimulation of lymphocytes with mitogens and antigens is an established model for in vitro testing of immunoreactivity. Due to the great variability of blastogenic response the interpretation of these results is difficult. Our results suggest that multivariate experiments and statistics improve the interpretation.


[Strong and weak histocompatibility antigens and immune response].

In blood samples of 88 healthy donors various red cell, HLA-A, -B, -Cw and -Dr antigens were determined by standard methods. Lymphocyte reactivity was measured by 3H-thymidine uptake into DNA of lymphoblastic cells stimulated by 14 different mitogens and antigens. Cluster analysis confirmed by t test and F test defined 3 distinct clusters of lymphocyte reactivity. Cluster 1 in 70% [relative risk (r.r.) 109, p < 0.001] and cluster 3 in 92% (r.r. 33, p < 0.01) showed significantly different cumulations of 3 or more increased characteristic red cell and HLA antigens. Our data suggest that the patterns of both red cell and HLA antigens play a major role in defining immunoreactivity.


Abbott HCV EIA 2nd generation: a new screening assay.

An enzyme immunoassay for the detection of antibodies against structural and nonstructural domains of the hepatitis C virus (EIA-2) was compared to a first generation test (EIA-1) that can only detect antibodies against one nonstructural antigen (c100). EIA-2 revealed elevated detection rates as assessed in high risk and patient populations as well as lower repeat reactive rates in volunteer blood donors. Research test systems proved an increase in apparent specificity.


[Gene amplification with PCR and sequence specific HLA oligonucleotide typing].

Genetic polymorphism in the HLA class II region has been identified by the analysis of the polymerase chain reaction (PCR) products using sequence-specific oligonucleotide (SSO). The PCR-SSO method permits precise and direct analysis of allelic variations with as little as 1 microgram of genomic DNA. The standardized, uniform hybridization and critical wash protocol of the Eurotransplant typing kit enables HLA typing independent of gene expression and quality of lymphocytes. One of the advantages of this technique is that the definition of splits is much better than typing by serology.


One year's experience with two different image analysis systems for automated reading of the contrast fluorescence test.

We have tested two different personal-computer-based color image analysis systems for automated reading of the microlymphocytotoxicity test (LCT) for HLA-A,B,C typing and screening. Over 17,000 single LCT reactions were prepared using the simultaneous double fluorescent variant of the LCT (contrast fluorescence test, CFT). All tests were read visually by experienced laboratory staff members. For digital image analysis, an automated scanning system was used. The reactions were first recorded on a videotape recorder using a color (CCD) videocamera und subsequently analyzed with the two different image analysis systems by specifically developed programs. Good correlation (r = 0.89) of the score values assigned by digital image analysis with the visual tray reading was obtained. Since also the other main performance characteristics of the prototype system were acceptable for routine application, we may conclude that digital image analysis is a feasible and very interesting new technique for automated evaluation of the LCT.


[Automated measurement of cell destruction with the Patimed System in comparison with conventional HLA typing].

HLA molecules serve as identity markers for immunocompetent cells which respond to pathogens and transplant tissue antigens to distinguish self from non-self. The continuous discovery of new HLA polymorphisms which are associated with various diseases or are involved in graft rejection continuously increases the number of HLA antigens which need to be identified for each individual. We defined the HLA class I and HLA class II antigens with the NIH standard microcytotoxicity system and compared the results with those obtained using the commercially available automated Patimed system. Our results show that serological identification of HLA antigens with the Patimed system is reliable. It can be recommended for routine as well as for research purposes.


ABO-incompatible bone marrow transplantation.

In the past 10 years 201 HLA-identical bone marrow transplantations (BMT) were performed with major ABO incompatibility in 41 (20%) and minor ABO incompatibility in 35 (18%) patients. ABO compatibility between donor and recipient showed no influence on granulocyte and platelet recovery after BMT. Erythrocyte reconstitution was significantly (p < 0.01) delayed for about 1 week in major ABO-incompatible BMT. In addition, a pure red cell aplasia lasting for 2-5 months occurred in 6 out of 21 blood group 0 patients who received transplants of group A. The rate of graft rejection, incidence of graft-versus-host disease as well as the leukemic relapse rate were similar in ABO-compatible and ABO-incompatible BMT. The probability of a 10-year survival after BMT is independent of ABO compatibility between donor and recipient.


[Isoagglutinins after bone marrow transplantation with minor ABO incompatibility].

Sera of 22 patients after BMT with blood group change from A to 0 were investigated. Early after BMT 59% of the patients developed a temporary anti-A whereas late after BMT none of the patients tested showed isoagglutinins against A even though the red blood cells were of type 0 and anti-B was present. The onset of these anti-A isoagglutinins correlated with production of leukocytes by the donor BM and is regarded as a sign of GvHR. Late after BMT a permanent immunological tolerance to the A antigen of the recipients' old blood group seems to exist.


[Acquired autoimmune hemolytic anemia after liver transplantation by "auto"-anti-A and "auto"-anti-P1].

A 47-year-old man (group A) received a cadaver liver from a group 0 donor. On day 8 after the transplant the recipient developed severe immune hemolysis and showed a positive direct antiglobulin test due to two antibodies (anti-A and anti-P1) of transplant donor origin. The possible mechanisms involved in "autoantibody" formation in the recipient are discussed.


Immune-haemolytic anaemia (IHA) after solid organ transplantation due to rhesus antibodies of donor origin: report of 5 cases.

During a 3-year period we observed 5 patients who developed IHA after transplantation of solid organs (4 kidneys, 1 liver) due to irregular anti-erythrocyte antibodies of donor origin (4 times anti-D, once anti-D+C). The onset of acute haemolysis was usually within 2-3 weeks after transplantation; however, 1 patient developed acute haemolysis as late as day 116 after renal transplantation. The course of the clinical and serological signs (DAT positive, 'auto'-antibodies in serum and eluate) was typical and self-limited in all cases, the function of the transplanted organ was usually not affected. All patients could be managed by transfusion of antibody-compatible blood. The administration of azathioprine (1 case) did not seem to have any beneficial effect. The causative antibodies could be demonstrated retrospectively in the serum of 1 donor. Another donor had no detectable irregular anti-erythrocyte antibodies at the time of transplantation, suggesting a secondary immune response of transplanted lymphatic tissue in this case. Both recipients of a kidney from this donor developed IHA. Serum samples of the 2 other donors were not available for investigation. We suggest that a sensitive screening for irregular anti-erythrocyte antibodies in the donor's serum should be performed before any transplantation of solid organs. If such antibodies are found, organs should only be used after irradiation. However, as our experiences show, this cannot prevent IHA in each case. Once haemolysis develops, the correct serological diagnosis and the transfusion of antibody-compatible red blood cells are most important.


[Comparative study of antibody identification in the gel centrifugation test (ID Microtyping System), solid phase antiglobulin test (Solidscreen Capture R, Ready ID) and tube test].

The gel centrifugation test (ID Microtyping System)--a new method for antibody screening--was compared with solid phase systems (Solidscreen, Capture R, Ready ID) and the conventional tube test. 141 different antibodies were tested and the results were compared. The ID Microtyping System identified 138 (98%) of all antibodies, the tube test 110 (78%), Capture R 86 (61%), Ready ID 79 (56%) and Solidscreen 75 (53%). The results in identification of all antibodies except cold agglutinins (n = 107) were: in the ID Microtyping System 98% (105), tube test 76% (82), Capture R 70% (75), Ready ID 68% (73) and Solidscreen 61% (65).


[Superiority of the gel centrifugation method (ID System) in detection of erythrocyte antibodies].

We report on the direct comparison of gel centrifugation technique and tube testing for antibody (AB) screening (ABS) under controlled routine conditions. 3,000 blood samples were screened for AB by gel centrifugation (ID-System, bromelin 37 degrees C and room temperature, indirect antiglobulin test with LISS) and a sensitive tube test (T; bromelin two-phase test 37 degrees C/room temperature and indirect antiglobulin test with 22% bovine albumin) in parallel. By ID significantly more relevant and potentially hemolytic AB (51 vs. 35 AB/1.7 vs. 1.2%) could be detected: anti-E 4, anti-C 1, anti-D 4, anti-CW 2, anti-C 2, anti-Jk (a) 2, anti-Jk (b) 1. 11 of these even remained negative in tube test when retested with increased sensitivity and taking additional (homozygous) test cells. In addition, naturally occurring but rarely hemolytic AB (35 vs. 23 AB/1.2 vs. 0.8%) were also more frequently detectable by ID: anti-Le (a) 6, anti-Le (b) 2, anti-P1 6. In contrast, only two AB were only positive in T: anti-Le (a) 1, anti-Le (ab) 1. The main disadvantage of ID was its frequent positivity (7.7 vs. 4.3%) due to irrelevant cold AB (anti-I, anti-HI, anti-H) and unspecific factors. This can be partly reduced by omission of the bromelin test at room temperature (ID 3.0%, T 1.5%) as the detection of relevant AB is not affected. The frequency of naturally occurring AB was still the same as in T (0.7%) when bromelin at room temperature was omitted in both techniques. Further advantages of ID are simplicity, small volumes of sera and reagents and easy evaluability.


[Positive direct antiglobulin test (DAGT) using the gel system].

In the gel test system the direct antiglobulin test (DAGT) shows a considerably higher sensitivity than in the tube test. Some 40% of gel test positive DAGTs prove to be negative when tested in the tube system. Most of the (auto-)antibodies detected by gel test DAGT appear to be bound integrally to the patient's own red cells and therefore are of questionable importance in view of future blood transfusions. The high sensitivity of the gel test DAGT certainly is of interest but implicates unwanted additional work for the immune hematological laboratory.


[Examination of a Berlin blood donation branch for antibodies to hepatitis C virus with the anti-HCV test and for circulating HCV-RNA using polymerase chain reaction].

Hepatitis C virus (HCV) is responsible for the majority of cases of transfusion-related hepatitis. We performed a first-generation anti-HCV EIA in 665 repeat and 168 first-time blood donors from Berlin. 4.7 and 4.2%, respectively, showed at least one indeterminate or positive result. We further looked for HCV genome in the plasma of 20 donors with reactive anti-HCV-EIA doing a polymerase chain reaction (nested PCR). The control group consisted of 20 patients with chronic hepatitis C. The PCR was negative in all examined blood donors, but was positive in 17 of 20 controls. These findings raise the question, if a positive anti-HCV test correlates with infectiosity.


Results of the DGTI workshop on the evaluation of the reactivity of monoclonal anti-D.

All D+ samples were detected by all monoclonal antibodies (mabs) without any problem. Very few of the 3,000 D- samples showed a +/- to ++ reaction with some of the IgG type mabs using the Coombs technique. It is still open whether these are specific reactions with weakly positive samples, which were negative with polyclonal sera. IgM type mabs detected up to 100% of the Du samples dependent on the Rh phenotype and the technique used (CCDuee > CcDuee > ccDuEe). Most of the CCDuee samples reacted very strongly and should no longer be regarded as Du.


Human monoclonal antibodies for the differentiation of RhD categories.

With the introduction of human monoclonal antibodies against antigens of the Rh system it was possible to solve a series of problems as, for instance, the permanent availability of reagents. Another problem, the classification of D categories with a panel of specific monoclonal antibodies, also might be solved by the production of such antibodies. Especially the classification of cat. VI cells is of great importance. Here we are presenting a panel of 5 human monoclonal antibodies useful for the definition of Rh-D categories. The antibodies are designated as BS221, BS227, BS228, BS230 and BS231. By positive or negative reactions with expected category cells it is possible to determine the corresponding category. All cat. VI cells of a defined panel could be identified. Of particular interest is the antibody BS221 due to its reactivity with cat. VI cells. The fact, however, that BS221 misses the detection of some cat. VI cells indicates a further subdivision of cat. VI.


[Anti-M after transfusion as an indication of a genetic variant of the MN locus].

We report a case of an anti-M antibody (titer 128) and MN red blood cells (RBC) in a 76-year-old female German patient. In our case, however, RBC showed weak reactions to human anti-M compared with strong reactions using rabbit anti-M. Papain treatment destroyed the RBC reactivity to human anti-M whereas the strong reactivity to rabbit anti-M was unchanged. This pattern was also demonstrable in the patient's son and grandson. Our results indicate the existence of a rare allele at the MN locus in this family.


[Liberation of the T-antigen in bacteremia].

Many microorganisms are able to produce neuraminidase, which can uncover the T antigen on red blood cells and cause hemolysis. We studied T activation in 224 patients with positive blood cultures. Only those patients were included who had real bacteremia according to clinical parameters and microbiological results. None of our patients showed T transformation. We conclude that T activation is a rare or a very passing phenomenon which has less importance in routine diagnosis of sepsis.


[Detection of platelet-specific alloantigens in 400 unselected blood donors].

Antibodies against platelet specific alloantigens cause neonatal alloimmune thrombocytopenia, posttransfusion purpura, and they are sometimes found in polytransfused patients. In the last few years, new alloantigens were discovered in addition to the Zw-, Bak-, and Ko-alloantigens. In order to obtain representative data for the frequency of all platelet alloantigens in the European population, we typed 400 blood donors of our institution. No significant differences between our findings and data already published were found for the antigens of the HPA-1, -2, -3, and -5 systems; however, no HPA-4b (Yuka)-positive and no Naka-negative individual was found among the 400 blood donors tested. The Siba and HPA-2b antigens proved to be identical. The 'low-frequency' alloantigen Sra was not identified among the 400 individuals tested.


[Clinical and serologic studies in 34 patients with post-transfusion purpura].

Posttransfusion purpura (PTP) is a rare transfusion reaction characterized by a severe bleeding tendency and thrombocytopenia which occurs approximately 1 week after transfusion of platelet-containing blood components in patients previously immunized against platelet alloantigens. 34 cases of PTP were studied. Our patients were all female with a mean age of 60.8 years (35-78 years, n = 32). The inciting blood components were whole blood (4) or red cell concentrates (28). The interval between transfusion and onset of purpura ranged from 2 to 14 days with a clear maximum at 7 and 8 days. In 11 of 13 patients (85%) transfusion was accompanied by febrile, nonhemolytic transfusion reactions. Hemorrhagic symptoms lasted 9.4 days (3-37 days, n = 16). The mean minimal platelet count was 7.1 x 10(3)/microliters [(0-28) x 10(3)/microliters, n = 29]. The platelet count rose to over 50 x 10(3)/microliters after 13.9 days (2-61 days, n = 26), over 100 x 10(3)/microliters after 17.0 days (3-75 days, n = 22). In 1 patient, PTP led to death due to intracranial hemorrhage. 22 patients were treated with corticosteroids, 20 patients with intravenous immunoglobulins (IVIG), 17 of these patients received both. Therapy with IVIG was successful in 14 of 19 patients, whereas platelet transfusions (n = 18) were not able to evaluate the platelet count. Serological analysis showed that antibodies against the HPA-1a antigen either alone (18), in combination with HLA antibodies (12) or with anti-HPA-5b (1) were responsible in 91.2%, while antibodies against other platelet antigens were rarely implicated. In elution experiments HPA-1a antibodies could be eluted from the autologous HPA-1a-negative platelets. We suppose that these antibodies had a pseudo-specificity and were involved in the destruction of the patients' own platelets.


[Heparin-associated thrombocytopenia: successful therapy of patients after prospective selection of a compatible heparinoid with the heparin-induced platelet activation test].

Diagnosis of HAT type II and treatment of thromboembolic complications in these patients are difficult. Recently we have developed the heparin-induced platelet activation (HIPA) assay which allows a rapid confirmation of the tentative diagnosis of HAT type II. In vitro studies with sera of 25 patients revealed cross-reactivity to the LMW heparins Fragmin, Fraxiparin and Clexane whereas a LMW heparinoid, Org 10172 (Orgaran), did not. In a prospective study this heparinoid was selected for 10 HAT patients, for whom further parenteral anticoagulation was required. In 7 of these patients who received LMW heparins prior to laboratory investigations low platelet counts persisted under treatment with LMW heparins and 2 patients developed additional thromboembolic complications. Upon treatment with Org 10172 platelet counts normalized in 9 patients, in 1 patient thrombocytopenia was unrelated to parenteral anticoagulation, in 1 patient platelet count normalized after discontinuation of Org 10172. We conclude that the HIPA assay allows the laboratory diagnosis of HAT type II and the selection of a compatible heparin or heparinoid for further parenteral anticoagulation.


Case report: acute renal failure, thrombocytopenia and nonhemolytic icterus probably caused by mefenamic acid (Parkemed)-dependent antibodies.

A 65-year-old, previously healthy man developed acute renal failure, severe thrombocytopenia and hepatic icterus after a small dose of mefenamic acid (Parkemed). Drug-dependent antibodies reacting against platelets could be identified as the most probable cause for this acute and rapidly reversible disorder. A concomitant hemolytic reaction was not observed and accordingly no drug-dependent red cell antibodies could be demonstrated. The drug-specific antibodies were found only during the acute phase using the platelet immunofluorescence test and a solid-phase immunoassay but not with the monoclonal antibody specific immobilization of platelet antigens assay. After discontinuation of the drug the patient steadily improved and fully recovered until day 22 after admission and drug removal. The clinical course strongly suggests that drug-dependent antibodies against mefenamic acid and/or its metabolites reacting by immune complex mechanism were responsible not only for the thrombocytopenia but also for the renal and hepatic failure.


[Development of HLA antibodies in thrombocyte substitution with cell separator products].

In a retrospective study we investigated the development of HLA antibodies in patients who received platelet concentrates from cell separators. 118 hematological/oncological patients from the Frankfurt University Clinics were investigated. They received between 4 and 66 platelet concentrates for the duration of 30 months. All patients had a negative antibody screening on admission. 31% developed either transient (15%) or permanent (16%) lymphocytotoxic antibodies. The increasing number of platelet transfusions did not correlate with the development of HLA antibodies, but the appearance of these antibodies seemed to be dependent on the disease. Permanent antibodies appeared in 8% of patients with acute leukemia, whereas 38% of patients suffering from CL, lymphoma, MDS and myeloma produced antibodies. Some patients (18) received granulocyte transfusions as well. It is striking that 11% of these patients developed permanent and 28% transient HLA antibodies. There exist no data about recent transfusions or previous pregnancies. To lower the rate of sensitization in patients with diseases such as CL, lymphoma, MDS and myeloma, it should be discussed whether leukocyte-depleted platelet concentrates should be given to these patients.


Detection of antibodies to HCV: comparison of a c100-3 EIA and the 2nd generation EIA.

The reactivity of 558 serum specimens in Abbott 1st and 2nd generation EIAs was compared. A significant increase in HCV seropositivity was especially found in high risk groups such as hemophiliacs (57.7 vs. 73.1%), IVDA (54.5 vs. 61.8%), HIV positives (21.8 vs. 25.5%) or patients on dialysis (15.7 vs. 16.9%). Reactivity of all sera which only reacted in the 2nd generation EIA was 'confirmed' by using supplemental research assays. In contrast, sera which were highly reactive in the c100-3 assay but could not be confirmed by Neutralization Assay were weakly positive or negative in the 2nd generation test.


[Clinical importance of granulocyte-specific antibodies].

Clinical and laboratory data of 184 patients with immune neutropenia were evaluated. They suffered from autoimmune neutropenia (n = 165), alloimmune neonatal neutropenia (n = 18) and from transfusion-associated lung injury (n = 1). Autoimmune neutropenia was predominantly found in patients below 3 years. Patients were usually affected by benign bacterial infections. The peripheral blood count showed normal or diminished leukocyte counts with median absolute neutrophil counts of 285 cells/microliters. Bone marrow examination revealed in 60% of the cases a hypercellular marrow with a shift to the left. In 36% the bone marrow was normal and in 4% a hypocellular marrow was found. Spontaneous remission occurred in all newborns and, so far, in 4 patients with autoimmune neutropenia. Symptomatic treatment of the infections was sufficient in most of the patients.


[Clinical aspects of prenatal prevention of rhesus incompatibility].

The postnatal treatment with anti-D immunoglobulin to prevent rhesus sensitization is successful in about 90% of all rhesus-negative mothers at risk. Failures derive mostly from large fetomaternal hemorrhages during the last months of pregnancy. Studies from Canada, Great Britain and Sweden have shown that the injection of an additional dosage of anti-D during the 28th to 34th week of pregnancy results in a further 90% reduction of the failure rate. Although there is only a limited number of cases of hemolytic diseases in the newborn, the cost-effect ratio of this prophylactic treatment calculated for the Federal Republic of Germany shows not only a medical but also an economic benefit.


[New possibilities in diagnosing hemolytic disease of newborn infants].

The diagnosis and management of HDN relies on measurement of maternal anti-D, amniotic fluid analysis and fetal blood sampling by chordiocentesis. However, amniocentesis and chordiocentesis have substantial risks of fetomaternal hemorrhage and subsequent increase in maternal anti-D. In addition to quantitation the functional activity of maternal anti-D has been determined by measuring the interaction of red blood cells sensitized by maternal serum in monocyte-monolayer assays. We assessed the functional activity of anti-D by titration of the sensitized red blood cells using selected sera with rheumatoid factor (RF) as human anti-IgG. First experiments using monoclonal anti-D showed a good correlation between erythrophagocytosis and RF titers. The bilirubin-protein ratio in amniotic fluid may be of great value in predicting the severity of HDN, as shown in 94 cases with severe and 39 cases with moderate disease. Amniotic fluid analysis is complicated by the presence of hemoglobin; we developed a computer program to solve this problem. To improve the serological diagnosis of ABO incompatibility, we measured IgG-anti-A, B in 1,392 maternal and newborn sera applying a sensitive gel test with Coombs serum. Furthermore, we determined the hemolytic activity of anti-A, B by microscopic observation of the morphological changes of red blood cells.


SLP assay: a rapid assay for detection of red blood cell antibodies in the serology of pregnancies.

A rapid manual test for the detection of red cell antibodies called SLP assay has been developed and compared with the sensitivity of the antiglobulin assay. Acid-soluble proteins (SLP) from human leukocytes cause aggregation of human red blood cells. SLP represents a group of proteins consisting of 5 fractions of different positively charged macromolecules, which are able to reduce the negative charge of the red cells. Reduction of the negative charge results in a nonspecific hemagglutination of different strengths, depending on the SLP fractions used. This hemagglutination can be reversed by neutralizing the SLP with heparin. In the case of blood group-antibody-mediated aggregation the hemagglutination is nonreversible despite neutralization with heparin and remains stable for several hours. Because of the high sensitivity of the SLP assay all blood group antibodies from the IgM type as well as from the IgG type are detectable even in low concentrations. The sensitivity of the SLP assay is comparable to the antiglobulin assay.


[Specificity and incidence of erythrocyte antibodies in pregnant patients with intrauterine transfusions for fetal erythroblastosis].

The specificity and frequency of irregular erythrocyte alloantibodies in serum obtained from 85 pregnant women managed by a total of 480 intrauterine transfusions for treatment of fetal erythroblastosis was examined over a 4-year observation period. 138 alloantibodies reactive in the indirect antiglobulin test were detected. Their specificities were widespread. The frequency of non-anti-D alloantibodies primarily responsible for fetal immunohemolysis confirmed by elution from fetal red cells increased to 8% compared with studies performed in the 70s. 16 (19%) patients developed additional alloantibodies after onset of intrauterine transfusion therapy. Regarding the fact of the high incidence of secondarily induced alloantibodies, the high prevalence of antibody mixtures and the occurrence of rare alloantibodies against blood group antigens with weak immunogenic potency, we concluded that many of the patients were 'high responders'. Therefore the role of fetomaternal transplacental hemorrhage induced by invasive intrauterine examination methods and transfusions is discussed here. It obviously has to be considered as the main cause of the immunohematologic complications.


[Incidence of the detection of erythrocyte antibodies in relation to screening test cells].

A variety of antibody screening tests are available to detect immunization in patients' sera. However, antibodies can be observed only if corresponding antigens are present on test cells used by an antibody screen. By comparing available test cell kits of different manufacturers we revealed various specificities of antigens present or absent on these cells. Using by an antibody screen Wr(a+), Co(b+) and Kp(a+) test cells additionally to available test cells, we detected in 1000 sera of patients 13 anti-Wr(a) and 1 anti-Co(b) besides 17 antibodies with different specificities.


[Granulocyte-specific and HLA antibodies in pregnancy: incidence and clinical value].

Postpartum sera of 1,016 unselected women were examined for granulocyte-specific and HLA antibodies. A total of 11 out of 1,016 sera (1.1%) were only reactive with neutrophils. Cytotoxic HLA antibodies were detected in 24%, noncytotoxic HLA antibodies in 4.8% of the sera. All antibodies belonged to the IgG 1 and IgG 3 subclasses. NA1 and NB1 specificity were each determined in one serum, two sera contained NA2-specific antibodies. After 1 year all antibodies were no more detectable. As none of the newborns from immunized mothers developed neutropenia, the incidence of alloimmune neonatal neutropenia seems to be lower than 0.1%.


[Neonatal isoimmune thrombocytopenia by anti-GP IIb/IIIa].

Neonatal alloimmune thrombocytopenic purpura is caused by maternal platelet-specific alloantibodies which react on fetal platelets with the corresponding antigen. We describe a 30-year-old female patient with type I Glanzmann thrombasthenia who developed an isoantibody against the platelet GP IIb/IIIa complex before or during pregnancy. The platelet count in the male newborn was 35 x 10(9)/l, but there were no signs of cerebral hemorrhage. Following infusion of 2 g/day i.v. IgG on 3 consecutive days, platelet counts rose to normal values. An isoantibody reacting with the GP IIb/IIIa complex of all platelet donors tested was found in the maternal serum using the MAIPA assay. Thus, immune-mediated neonatal thrombocytopenia may be caused by platelet isoantibodies against GP IIb/IIIa as well as by platelet-specific alloantibodies and by maternal autoantibodies.


[Effects of an infusion pump with peristaltic mechanism of action on various blood components in vitro].

Apart from a slight hemolysis at very low flow rates, the peristaltic infusion pump IVAC 560 has no harmful effects on blood components such as packed red cells, whole blood, platelet concentrates and fresh frozen plasma. So it may be a convenient option to perform volume processed transfusions.


[Decreasing the risk of post-transfusion non-A, non-B hepatitis by anti-HCV screening].

In May 1990 a specific enzyme immunoassay (EIA) for NANBH was developed by recombinant DNA technology which detects antibodies to a virus called hepatitis C virus (HCV). The anti-HCV EIA was manufactured by Ortho Diagnostic Systems with recombinant antigens from Chiron Corp. based on extraction from high infectious titer chimpanzee plasma RNA after transcription into cDNA. We tested the anti-HCV prevalence of blood donors and hemodialysis patients. The anti-HCV prevalence with the first generation test was 0.52% (Ortho), 0.87% (Abbott) in blood donors and 4.16% in hemodialysis patients. The second generation anti-HCV test (Abbott) with improved sensitivity and specificity comprises 0.25% repeated anti-HCV-positive blood donors and 8.2% anti-HCV-positive hemodialysis patients.


Biocompatibility of synthetic oxygen carriers and fluorosurfactants.

A system for testing the biocompatibility of synthetic oxygen carriers is described including tests of hemolysis, complement activation and proliferation of cell lines or lymphocytes. Of 17 surfactants tested in this system, 6 were compatible in all tests while the other compounds showed individually differing patterns of incompatibility. We conclude that, in order to conduct a meaningful screening, a series of different assays has to be applied. Addition to our system of further assays covering cytokine induction and phagocytosis should be attempted.


[Secondary hemochromatosis in polytransfused patients with myelodysplastic syndromes].

In a retrospective analysis we assessed the data of 46 patients with myelodysplastic syndromes (MDS), who had received more than 50 blood transfusions during the course of disease. The number of units given ranged from 50 to 155 (mean 79). 20 patients (RA n = 4, RARS n = 12, RAEB n = 1, RAEB/T n = 2, CMML n = 1), followed up between 8 and 108 months (mean survival time 39.4 months), developed a secondary hemochromatosis. More than 40% of the patients showed signs of heart failure, in some cases accompanied by cardiac arrhythmias. 11 patients also suffered from hepatopathy and 5 developed diabetes mellitus. Secondary hemochromatosis was particularly common in patients with RARS. Refractory congestive heart failure secondary to hemochromatosis was the cause of death in 14 patients, whereas none died from hepatic insufficiency. We conclude that the risk of secondary hemochromatosis should not be neglected in polytransfused patients with MDS. In some cases, particularly those with favorable prognostic features of MDS, it may shorten life expectancy. The availability of a new oral iron chelator (1,2-dimethyl-3-hydroxypyrid-4-one or L1) offers a promising and practicable approach to prevent this complication.


[Prevalence of lupus anticoagulant, autoimmune hemolysis, thrombocytopenia and disorders of platelet function in unselected patients with SLE].

Prevalence of lupus anticoagulant (LA) in patients with systemic lupus erythematosus (SLE) and clinical manifestations vary widely between different clinical series. We investigated the relation between LA, autoimmune hemolytic anemia (AIHA), thrombocytopenia and platelet dysfunction in 80 unselected patients with SLE. AIHA was found in 6 patients (7.5%) and thrombocytopenia in 10 patients (12.5%), which was not related to platelet aggregation abnormalities. Compared to controls, patients with SLE showed significantly prolonged aPTT and kaolin clotting time (KCT), but platelet aggregation induced by both collagen and thrombin was not impaired. LA activity as defined by Rosner et al. (index for LA/ICA) was found in 15 patients (18.9%). Only 7 of these patients showed a positive platelet neutralization test (Triplett) and 9a positive tissue thromboplastin inhibition test (Schleider). In our SLE patients 23.7% have suffered from at least one thrombotic complication. In patients with LA activity thromboembolic complications were increased (p < 0.05). Thrombocytopenia was found in 6% of LA-negative but in 20% of LA-positive patients.


[Electronic data processing-assisted serial automation of current methods in blood group serology].

The introduction of special centrifugable racks with a transparent bottom into the conventional typing of blood groups in glass tubes facilitates the simultaneous work on and reading of a maximum of 32 complete ABO, Rhesus and Kell typings in one series. As a result of the facts that it is unnecessary to label the individual tubes and that the pipetting of serum and erythrocyte suspensions is done automatically and through the unmistakable classification of the samples by means of bar-coding, the manual work is reduced to about 50%. This modified typing method combines the advantages of the conventional typing method in glass tubes with the advantages of an efficient microplate technique. There is no qualitative loss in the implementation and reading of the analysis.


Detection of antibodies to hepatitis C virus in blood donors and their relationship to surrogate markers.

Stored serum samples from 7,179 nonselected blood donors were tested for anti-HCV using Ortho EIA first generation. Results were compared to data acquired by anti-HBc testing and ALT levels found in routine testing. 24 donors (0.33%) were repeatedly reactive with Ortho HCV EIA, 230 (3.20%) were anti-HBc-positive and 138 (1.92%) had raised ALT levels > or = 36 IU/l. A low correlation was found between HCV antibody screening with EIA and surrogate testing. When tested in addition with the Abbott HCV EIA, 20 of the 24 Ortho EIA-positive subjects showed a positive reaction. In the Abbott neutralization test 13 of these 20 (65%) were reactive. 8 (33.33%) of the 24 Ortho-EIA-positive donors were positive in the two-antigen-RIBA (first generation), 8 were indeterminate and 8 were nonreactive. The neutralization test and the RIBA can be used as supplementary tests fo further analyze HCV-EIA-positive specimens.


[Sex and age specific GPT limit values in blood donors].

Excluding blood donations with elevated serum ALT from transfusion is only justified under the assumption that these more frequently transmit unrecognized hepatitis infections. The distribution of such infections in the donor population (with respect to age and sex) should be similar to that of hepatitis B and C, respectively. The prevalence of the latter two infections among our blood donors is the same in both sexes. The exclusion rate, however, is 3-4 times higher for men if equal ALT limits are applied. We therefore determined which ALT limits would give equal exclusion rates for donations of male and female blood donors in order to balance the risk of unrecognized hepatitis in blood donations from the two sex groups. In a second step, we also took each donor's age group into account and determined individual ALT limits for different age and sex groups.


[Distribution of the incidence of SGPT activity in blood donors].

Predonation SGPT (ALT) testing was performed in 5,706 blood donors (3,944 male, 1,762 female) with the Reflotron system. The values of SGPT activities (IU/l; 25 degrees C) are not normally distributed, whereas the log10 SGPT distribution is largely Gaussian (the arithmetic mean log10 +/- SD for female donors is 0.98 +/- 0.14 and for male donors 1.08 +/- 0.16, the antilog of the mean log10 9.5 and 12.1 IU/l, respectively). In the following the impact of SGPT testing at various exclusion levels on the percentage of deferred donors is shown: (1) female: cutoff level > 18.0 IU/l (mean log10 +2.0 SD) 2.8%, > 19.5 IU/l (+2.25 SD) 2.1%, > 21.1 IU/l (+2.5 SD) 1.8% and > 24.8 IU/l (+3.0 SD) 1.2% deferment. (2) male: cutoff level > 25.2 IU/l (mean log10 +2.0 SD) 2.7%, > 27.6 IU/l (+2.25 SD) 2.3%, > 30.3 IU/l (+2.5 SD) 1.4% and > 36.4 IU/l (+3.0 SD) 0.8% deferment. Using the current cutoff value of > 45 IU/l (according to German regulations, 1987) only 0.2% female (with SGPT > mean log10 +4.5 SD) and 0.3% male (with SGPT > mean log10 +3.5 SD) donors would be excluded. These findings demonstrate that different SGPT exclusion levels for men and women are necessary and that the current cutoff value of > 45 IU/l does not exclude donors who should better be rejected.


[Etiology of increased SGPT activity in blood donors].

From all blood donors screened within a given period of 6 months for SGPT (ALT) immediately before donation, 166 (141 male, 25 female) were found to have SGPT activities above our cutoff value of > 28 IU/l (25 degrees C). In order to define the characteristics of these blood donors they were asked for detailed medical history followed by a thorough physical examination and further serologic and biochemical testing. Factors associated with elevated SGPT activity included daily alcohol consumption (82 donors), infections associated with abnormal DCs (23), therapy with hepatotoxic drugs (8), infections that had been treated with antibiotics (6), strenuous physical exercises (5), body building supported by anabolics (2), acute HBV infection (1), acute HCV infection (1), alcohol/drug abuse and positive test for anti-HCV antibodies (1), anti-CMV positivity with absolute and relative lymphocytosis (1). In 36 cases factors that could have been associated with an increased SGPT activity were not verified. A critical valuation of the presently accepted cutoff value of 45 IU/l (25 degrees C) is given.


[High precision, reproducibility and practical application of modified alanine aminotransferase determination with the microtiter plate].

In order to improve the alanine aminotransferase (ALT) assay by using microtiter plates, a new 96-channel multiple syringe of high precision and incubation chambers with precise regulation of the temperature operated by computer were developed. In accordance with the recommendation of the German Society of Clinical Chemistry for the optimized standard method, the ALT assay was modified for the procedure at 25 degrees C. The modification was comparable with the best conventional methods which, however, are very time-consuming. The ALT assay has the advantages of the microtiter plate, e.g., secure sample identification, on-line documentation and reduction of costs and time for the analysis.


[Kinetic measurement of GPT with the microtiter plate].

Experiences with a kinetic method for measurement of SGPT in microtiter plate using an automatic sample processing device and Medusa software (Biotest) were evaluated. The correlation coefficient in parallel assessment of samples in a clinical routine laboratory was found to be 0.935 (p < 0.0001). The correlation coefficient comparing the internal standard dilution with actual recordings was found to be 0.999 (p < 0.0001). Automated screening of SGPT in microtiter plates seems thus to be reliable as well as feasible in blood bank routine.


[Diagnosis of the presence of SGPT in blood donors].

Presampling determination for hemoglobin has been pursued for a long time, but now it is becoming possible to examine further parameters such as SGPT (ALT). The SGPT predonation testing is best performed with the Reflotron system (Fa. Boehringer Mannheim) since whole blood samples drawn into capillary tube can be used. Between 1985 and 1988 1.35-1.75% of an average of 22,000 whole blood donations were found to have SGPT levels above the accepted cutoff (> 30 IU/l, 25 degrees C). The cost per test using the UV kinetics and the overall expenses of unusable collections were compared with the cost of SGPT predonation testing. An average of DM 20,000.-per year has been saved. Therefore prescreening of SGPT in blood banking is a highly cost-effective approach.


[ALAT (GPT) screening with the microtitration plate].

Sera of donors with values above the controls have been retested by the optimized standard method at 37 degrees C. 103 of 420 sera which had to be retested also showed elevated ALT in the optimized standard method. Therefore we conclude that the ALT microplate test used is suitable as screening test in blood donors.


Recombinant antigens in viral diagnosis.

DNA fragments coding for a variety of different viral antigens have been cloned and expressed in Escherichia coli. Selected purified recombinant antigens were used for detection of specific antibodies by means of the ELISA technique. This approach has been used for the development of four different ELISAs for the detection of HIV- and EBV-specific antibodies.


[Follow-up of blood donors with indeterminate reaction to anti-HIV 1/2 EIA or isolated detection of antibodies to HIV-1 core antigens (anti-p24)].

Over a period of 20 months we identified 10 voluntary blood donors with indeterminate reactions in anti-HIV 1/2 EIA tests. Western blot (WB) patterns of 6 of these 10 donors showed isolated core antibodies (anti-p24), in 4 cases WB patterns were negative. After a median follow-up period of 58 weeks (range 12-93) we have not seen a case of typical seroconversion.


Treponema pallidum membrane protein A ELISA: a new test for screening and diagnosis of syphilis.

A new ELISA test system for screening antibodies directed against Treponema pallidum is presented. The ELISA is based on a recombinant DNA-derived T. pallidum membrane protein A (TmpA). It makes the production of the kit easier as it abandons the necessity to culture T. pallidum in living animals. The specificity and sensitivity of the ELISA are comparable with those of the TPHA and FTA-ABS. The test gives only a positive result when there are antibodies against the TmpA antigen and, therefore, it gives in screening (e.g. blood donors) no false-positive reaction in persons who, due to an old well-treated infection, have a so-called serological scar. As these donors are noninfective, they should be concerned as negative in syphilis screening.


[Prevalence of Borrelia burgdorferi antibodies in Hamburg blood donors].

One thousand regular blood donors of the Department of Transfusion Medicine at the University Hospital in Hamburg were screened for antibodies against the Lyme disease spirochete, B. burgdorferi. 7.2% were initially reactive in the enzyme immunoassay, 37.5% of which were confirmed by immunoblot. The seroprevalence of anti-B. burgdorferi antibodies thus is 2.7% in Hamburg blood donors. 25 of 27 positive donors received a physical exam, which did not reveal any symptoms of acute or chronic Lyme disease. 24 of these 25 donors were tested for B. burgdorferi-specific DNA in urine by polymerase chain reaction, which came out negative in all cases. Introduction of B. burgdorferi antibody screening is not regarded an effective means to prevent transfusion-transmitted Lyme disease.


[Infections with Borrelia burgdorferi in Würzburg blood donors: antibody prevalence, clinical aspects and pathogen detection in antibody positive donors].

The prevalence of antibodies against Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, was determined in a group of blood donors from the Würzburg area (Southern Germany). 26 of 472 donors (5.5%) tested positive in a hemagglutination test. When performing immunoblots only 13 donors (2.7%) gave rise to B. burgdorferi-specific antibodies. 9 of them were examined in more detail by anamnesis, physical examination, determination of inflammation parameters of the blood and polymerase chain reaction (PCR) analysis of urine. All persons were asymptomatic for Lyme borreliosis. One of 4, who remembered a tick bite, actually had suffered from an erythema migrans 5 years ago. Another one had been affected by fever, headaches and pains in the limbs, arthralgia and motoric disorder in both hands 6 months before examination. Analysis of the blood did not provide any evidence of an acute infection. In the urine of 2 donors we detected B. burgdorferi-specific DNA by PCR. No seroconversion due to blood transfusion could be observed, when 9 recipients of blood products provided by the 13 seropositive donors were serologically reexamined. PCR analysis of urine samples of 5 recipients was also negative.


Effect of a phorbol ester on basic surface properties of trichomonads.

The effect of nanomolar concentrations of 12-O-tetradecanoilphorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus was evaluated by means of measurements of the parasites' surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites' net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.


Physicochemical characterization of Escherichia coli. A comparison with gram-positive bacteria.

Eight Escherichia coli strains were characterized by determining their adhesion to xylene, surface free energy, zeta potential, relative surface charge, and their chemical composition. The latter was done by applying X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR). No relationship between the adhesion to xylene and the water contact angles of these strains was found. Three strains had significantly lower surface free energies than the other strains. Surface free energies were either obtained from polar and dispersion parts or from Lifshitz-van der Waals and acid/base parts of the surface free energy. A correlation (r = 0.97) between the polar parts and the electron-donor contributions to the acid/base part of the surface free energy was found. The zeta potentials of all strains, measured as a function of pH (2-11), were negative. Depending on the zeta potential as a function of pH, three groups were recognized among the strains tested. A relationship (r = 0.84) was found between the acid/base component of the surface free energy and the zeta potential measured at pH = 7.4. There was no correlation between results of XPS and IR studies. Data from the literature of XPS and IR studies of the gram-positive staphylococci and streptococci were compared with data from the gram-negative E. coli used in this study. It appeared that in these three groups of bacteria, the polysaccharide content detected by IR corresponded well with the oxygen-to-carbon ratio detected by XPS.


Evidence for lymphocyte chemotaxis toward monocytes during PHA-induced aggregation in vitro.

An important, early phenomenon during the development of immune cell interactions in vitro is the formation of multicellular aggregates. We have developed a quantitative assay to determine the kinetics of multicellular aggregate formation within a heterotypic population of cells on a flat surface. This assay follows the time rate of change in the value of an aggregation index for cells in undisturbed culture. For an initial, well-separated population of cells, the index is a minimum and remains at this value if the cells do not move and interact. By contrast, for conditions that promote active cell movement followed by interaction, the index value increases with time. The index, which reflects cells' relative spatial distributions, is an "indirect enumeration" of the number of cells within aggregates as a function of time. We used this index to follow the aggregative behavior of a population of freshly isolated human peripheral lymphocytes and monocytes. Previous studies have shown that monocytes are centrally located within aggregates and that lymphocytes move to surround monocytes. In order to test if lymphocyte movements are random or directed prior to interactions with monocytes, we formulated a simple model to describe changes in the expected number of cells in an "idealized aggregate" as a function of time. A comparison of the model curves with curves generated from the changes in the aggregation index shows that the best fit derives from a model that involves directed movement of lymphocytes toward monocytes. These results suggest that monocytes produce a chemoattracting agent for lymphocytes for these experimental conditions.


Characterization of eukaryotic cell surfaces prior to and after serum protein adsorption by X-ray photoelectron spectroscopy. Fibroblasts, HELA epithelial, and smooth muscle cells.

Elemental surface concentration ratios N/C,O/C, and P/C of fibroblasts, HELA epithelial cells, and smooth muscle cells, prior to and after washing in the absence or presence of serum proteins, were determined by X-ray photoelectron spectroscopy. Cell surfaces appeared to adsorb hardly any serum proteins, and the relatively high P/C, as compared to N/C and O/C, elemental surface concentration ratio indicated that the cell surfaces consisted mainly of the phospholipid bilayer, with little or no proteins present. The lack of adsorption of serum proteins to the cell surfaces seems at odds with the common notion that cells require adhesive proteins in order to adhere and spread. However, the adsorption behavior of cellularly produced proteins may be completely different, particularly since they seem to be able to displace adsorbed serum proteins from biomaterials surfaces. Interestingly, only HELA epithelial cells (a tumor cell line) appeared to adsorb a very small amount of proteins.


Surface charge and hydrophobicity of wild and mutant Crithidia fasciculata.

Surface charge of wild-type Crithidia fasciculata and three drug-resistant mutants (TR3, TFRR1, and FUR11) was studied by direct zeta-potential determination and ultrastructural cytochemistry. Surface tension was also investigated by measurements of the advancing contact angle formed by the protozoa monolayers with drops of liquids of different polarities. The individual zeta potential varies markedly among the C. fasciculata cells. The wild and FUR11 mutant strains displayed lower negative surface charge (-12.5 and -9.5 mV, respectively) as compared with the TR3 (-14.8 mV) and TFRR1 (-14.7 mV) mutant strains. Binding of cationized ferritin (CF) was observed at the cell surface of wild and mutant strains of C. fasciculata. Neuraminidase treatment reduced the negative surface charge in the TFRR1 and TR3 mutants in about 37 and 29%, respectively, whereas no significant change was observed with the wild and FUR11 mutant strains. These findings suggest that sialic acid residues are the major anionogenic groups on the surface of C. fasciculata. The density of sialic acid residues per cell in wild and mutant strains of C. fasciculata falls in a range of 1.4 x 10(4) to 3.6 x 10(4). Marked differences of hydrophobicity were also observed. For example, the TFRR1, FUR11, and TR3 drug-resistant mutant strains showed higher contact angle values (55.4, 54.2, and 49.3, respectively) than the wild-type (35.6), as assessed by alpha-bromonaphtalene.


Ion flow through biomembranes. Physical theory explains its high sensitivity.

Numerous biomembranes exhibit a sensitivity to changes in electrical potential greater than predicted as possible from the classical application of the Boltzmann relation, a phenomenon which has long defied explanation, the actual sensitivity of some Na+ channels being many times greater than the classical limit. This paper explains, using a minimum of mathematics, how the very rapid gating effect of adsorbed Ca2+ (or other impermeable divalent cations) can directly affect the conductance of channels, and thus interact with the electric field within the channel to produce a change in the potential across the channel's gate much greater than the change in the membrane potential, with a corresponding change in the fraction of open conformational gates and change in conductance. These results are not in conflict with the Boltzmann relation, the necessary energy being made available from the total potential difference across the membrane by a long unrecognized stochastic process; the full mathematical theory is given in cited references.


Depth profiling of the elemental surface composition of the oral microorganism S. salivarius HB and fibrillar mutants by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy (XPS) on microbial cell surfaces requires freeze-drying of cells, and as a result, the cell surface appendages flatten out on the cell surface and form a collapsed fibrillar mass. At present, it is unclear how the density, length and composition of these fibrils influence the elemental surface composition as probed by XPS. The sampling depth of XPS can be varied by changing the electron take-off angle. In this article, we made a depth profiling of the collapsed fibrillar mass of Streptococcus salivarius HB and fibril-deficient mutants by angle-dependent XPS. Methylamine tungstate negative staining and ruthenium red staining followed by sectioning revealed distinct classes of fibrils with various lengths on each of the strains. Interpretation of the angle dependence of the oxygen/carbon (O/C) and phosphorus/carbon (P/C) surface concentration ratios of these strains was difficult. However, the angle dependence of the nitrogen/carbon (N/C) surface concentration ratio could be fully interpreted: N/C did not vary with sampling depth on a bald strain, S. salivarius HBC12 and on S. salivarius HB7, a strain with a dense array of fibrils of uniform length. N/C decreased with sampling depth in case of a sparsely fibrillated strain, S. salivarius HBV51 and eventually reached the value observed for the bald strain, HBC12. A high N/C at small sampling depth was observed for S. salivarius HB with protruding, protein rich fibrils. We conclude that elemental depth profiling of microbial cell surfaces by XPS can be interpreted to coincide with structural and biochemical information on the cell surface as obtained by electron microscopy and can therefore be considered as a useful technique to study structural features of cell surfaces in combination with electron microscopy.


Coordinate loss of growth regulatory factors following castration of rats carrying the Dunning R3327 G prostatic tumor.

Hormonal manipulation of prostate cancer is an effective therapy for metastatic disease. Unfortunately, following an initial response tumors reestablish themselves as hormone independent variants and progress. This study was designed to assess the interrelationship of cytokeratin P (Cyto P), vimentin, epidermal growth factor receptor (rEGF) and tissue testosterone following androgen deprivation therapy. Animals bearing the hormone dependent Dunning R3327 G subline prostatic adenocarcinoma were surgically castrated and progressing tumors from both hormone intact and castrated groups were quantitatively assayed for immunohistologic reactivity against the described markers. The results demonstrate a significant (p < 0.05) decrease in cytokeratin (Cyto P), rEGF and testosterone levels following castration. When the expression of both rEGF and Cyto P are related to the tissue testosterone content, it is observed that the ratio between rEGF and testosterone remains essentially unchanged (0.65 +/- 0.21 to 0.65 +/- 0.41), suggesting that in the Dunning R3327 G subline, rEGF expression is coordinately under androgen control. At least some cytokeratin expression also appears to be particularly sensitive to androgen levels, since the ratio between Cyto P and testosterone decreased from 0.92 +/- 0.39 to 0.35 +/- 0.41 following castration. In contrast, following castration, the expression of vimentin was unaffected.


Studies on the quantification of proteoglycans by the dimethylmethylene blue dye-binding method. Specificity, quantitation in synovial lavage fluid, and automation.

The dimethylmethylene blue (DMMB) dye-binding technique is widely used for the quantification of sulfated glycosaminoglycans (sGAG) and proteoglycans. We conducted further studies on this technique in our laboratory and found that concentrations of DNA and RNA in excess of 20 micrograms/ml interfered negatively with the detection of sGAGs; interference was eliminated by using DNase and RNase. Hyaluronan at 40 micrograms per ml did not interfere with the detection of sGAG. However, because of the higher concentrations of hyaluronan in synovial lavage fluid, it was necessary to treat this fluid with Streptomyces hyaluronidase in order to quantify sGAG. The DMMB assay was automated with a laboratory work station and compared to the standard method.


Evaluation of combined assays of serum ferritin, alpha-1-antitrypsin and alpha-fetoprotein in liver cancer.

Serum ferritin (SF), alpha-1-antitrypsin (AAT) and alpha-fetoprotein (AFP) were examined preoperatively in 66 patients with intrahepatic space-occupied lesions revealed by B-real time ultrasonography. Elevated SF levels (> 300 micrograms/L in males and > 180 micrograms/L in females), AAT levels (4.2 g/L), and AFP levels (> 20 micrograms/L) were shown in 84%, 71% and 66% respectively of 55 patients with liver cancer. Combined analysis indicates that if all the three tests are negative, liver cancer can be essentially excluded; and positive AFP can rule out hepatic hemangioma. So combined assays of SF, AAT and AFP are valuable in the diagnosis and differential diagnosis of liver cancer.


Rapid immunostaining of live nerve for identification of sensory and motor fasciculi.

Six New Zealand white rabbits were anesthetized with pentobarbital, and sciatic nerves were exposed and cut at the mid thigh. Both proximal and distal ends were incubated directly with Blue-SAb in a micropipe of 5 mm diameter covering the nerve trunk ends for 30 minutes at room temperature. After removal of the micropipe, the nerve ends were washed with physiological saline. Blue-stained fasciculi, i.e., sensory fasciculi were seen among unstained ones under the operating microscope. This method requires no histological sections. Neural cells of the spinal cord and ganglion were cultured in RPM11640 medium containing Bright Blue. The growth and metabolism of the neural cells were tested by MTT assay and their morphology was observed. Statistical difference between the experiment and control groups was determined, indicating that Bright Blue had no effect on the neural cells and their repairing power. This rapid immunostaining technique offers a good approach for the identification and accurate coaptation of sensory fasciculi in peripheral nerve repair.


[An immunohistochemical study of occult micrometastases in regional lymph nodes of patients with stage I non-small cell lung carcinoma].

739 regional lymph nodes from 94 patients with stage I non-small cell lung carcinoma (NSCLC) were studied by immunohistochemistry. These lymph nodes, contained no metastasis as assessed by conventional histopathology, were recut. A series consecutive sections from the original blocks were immunostained with polyclonal and monoclonal antibodies to keratins, carcinoembryonic antigen (CEA) and human milk fat globulin membrane antigen (HMFG-2). Single tumor cells or small clusters of tumor cells, not visible on routine examination, were readily detected. The actual number of lymph nodes that contained occult tumor cells was 123 (16.6%) from 53 patients (56.4%). The majority of 102 immunostaining positive nodes were distributed in the hilar (29%) and peribronchial (25%) regions. Our data indicate that: 1. a series consecutive sections and immunohistochemistry may greatly increase the diagnostic yield of occult micrometastases in lymph nodes. 2. the high incidence of occult metastases in NSCLC may be of importance in relation to their rapid dissemination and high death rate. 3. the high frequency of occult nodal metastases in NSCLC raises questions in regard to our presently used criteria for staging, prognosis and treatment of ostensibly stage I disease. 4. perhaps resections of hilar and peribronchial lymph nodes will have an important clinical significance in prevention of wide dissemination of tumor cells.


[The production and identification of partial serogroup--specific monoclonal antibodies against leptospires hebdomadis serogroup].

Hebdomadis is one of the major serogroups found in China. In Sichuan, this serogroup appears to have a close relationship with local outbreak of leptospirosis. BALB/c mice were immunized intrasplenically with outer envelope of serogroup Hendomadis serovar hebdomadis strain 245. Spleen cells were fused with SP2/0 myeloma cells, two monoclonal antibodies Af2 and Bb2 were produced by hybridoma technique. McAb Af2 and McAb Bb2 were identified to be IgG1 and IgG3 by immunodiffusion respectively. Specificities of these two McAbs were determined by MAT; both reacted to 8 serovars (hebdomadis, nona, kambale, kremastos, worsfoldi, jules, maruborincana) of Hebdomadis serogroup. The agglutination titres of McAb Af2 and McAb Bb2 were 1:640-1:2,500,000 and 1:320-1:2,500,000, respectively. The two McAbs did not agglutinate with serovar kabura of Hebdomadis serogroup, they did not agglutinate with 11 serovars of Sejroe serogroup, 4 serovars of Mini serogroup, 18 representive serovars of L. interrogans in 18 serogroups, L. biflexa strain Patoc I and Leptonema illini. So it was found that McAb Af2 and McAb Bb2 showed partial serogroup specificity for Hebdomadis by agglutination.


[Radioimmunoassay of serum and CSF myelin basic protein and its application to patients with acute cerebrovascular accident].

Myelin basic protein (MBP) was measured in the serum and CSF of patients with acute cerebrovascular disease (CVD, 34 cases), demyelinating disorders (DMD, 30 cases) and other neurological diseases (OND, 26 cases) by using a double antibody radioimmunoassay (RIA). Patients with acute CVD had a mean serum MBP concentration and positivity rate much higher than those with DMD and OND. The differences were significant (P < 0.05). In CSF, MBP levels in patients with acute CVD and patients with DMD were significantly greater than those in OND patients (P < 0.05). The results also show that there was a linear relationship between the CSF MBP levels and the serum MBP levels in patients with acute CVD (r = 0.72, P < 0.01), but no such relationship in patients with DMD and OND. The amount of serum MBP was also significantly correlated to the severity of acute CVD, to the level of consciousness disorder and limb paralysis, and to the extent and site of the cerebral lesion at CT-scan (P < 0.05). This study shows that the measurement of brain specific MBP in serum as a marker of cerebral damage may have clinical value in the diagnosis and prognosis of patients with CVD.


[Alpha 2 megaloglobulin inhibits the activation of rabbit platelet by Chinese Agkistrodon acutus venom].

An in vitro study of alpha 2-mengaloglobulin (alpha 2MG) in the inhibition of the activation of washed rabbit platelet by Chinese Agkistrodon acutus venom (CAAV) was reported. Results showed that CAAV induced aggregation rate of washed rabbit platelet was positively related to the CAAV concentration, and the maximal aggregation rate was 43.8 +/- 9.9% at the concentration of 100 micrograms/ml. Meanwhile the release of serotonin (5-HT) and platelet factor 4(PF4) from the platelet reached maximum at the CAAV concentrations of 0.81 +/- 0.07 microgram/ml and 39.08 +/- 2.78%, respectively. Platelet aggregation and the release of 5-HT and PF4 induced by CAAV could be inhibited by the addition of 0.25% alpha 2 MG into the platelets both 1 minute before and 1 minute after the addition of CAAV. When alpha 2 MG was added to the platelets 1 minute prior to the addition of CAAV, the platelet aggregation and the ultrastructural changes which could be induced by CAAV were not observed, and the morphology of the rabbit platelets was the same as that of the normal counterparts.


The mossy fiber terminals in the hippocampal formation of Callithrix jacchus: comparative and evolutionary considerations.

The distribution of mossy fiber terminals in the regio inferior of the hippocampus of Callithrix jacchus was studied by means of Timm's method. The topographic distribution of Timm-positive zones in the hilus, in the suprapyramidal and intrapyramidal areas of the CA3 subfield is described. A Timm-positive reaction in intragranular strips and supragranular zones, the presence of Timm-negative zones in the infragranular border of the fascia dentata were found in this species. A comparison between mossy fiber distribution in Callithrix jacchus and that in human was carried out in an attempt to identify interspecies differences in the mossy fiber system in the hippocampus of primates. The hypothesis of a possible functional relevance of the supra- and intrapyramidal mossy fiber terminals on the control of hippocampal pyramidal neurons is expressed.


Study of neovascularization of the jejunal graft--a new animal model.

The development of a new rabbit model for study of neovascularization of the jejunal graft is described. The relationship between stripping of the serosal membrane and the speed of the neovascularization process was analyzed. The results demonstrate that the visceral peritoneum acts like a physical barrier, retarding the formation of the new nutrient blood supply from recipient bed to the jejunal graft.


Functional abnormalities of circulating natural killer cell subpopulations in patients with dilated cardiomyopathy.

We investigated abnormalities in natural killer (NK) cells in the myocardium and circulating blood of 38 patients with idiopathic dilated cardiomyopathy (DCM), 18 patients with hypertrophic cardiomyopathy, 8 patients with primary amyloidosis, and 12 age-matched normal control subjects. Immunohistochemical staining of myocardial biopsies revealed a significantly greater number of CD57-positive NK cells in patients with DCM than that in controls (3.7 +/- 2.7 v.s. 1.9 +/- 1.6, p < 0.05). The New York Heart Association functional class, left ventricular ejection fraction, myocardial fiber diameter, and interstitial fibrosis volume fraction did not differ significantly between the DCM patients who died within five years of diagnosis and the 31 surviving DCM patients. However, there were significantly fewer CD57-positive NK cells in patients who died than in surviving patients (p < 0.05). There were no significant differences in the peripheral NK cell activity or the number of NK subset cells between the 16 patients with DCM (n = 16) and the 12 age-matched normal controls. In normal controls, the number of some NK cell subpopulations (CD16+, CD57+, CD16+ CD57+, and CD8+ CD57+ cells) were positively correlated with NK cell activity. In patients with DCM, there was no correlation between the number of NK cell subpopulations and NK cell activity. Our findings indicate that functional abnormalities exist in NK cell subpopulations in patients with DCM, and that these abnormalities may be related to the pathogenesis of DCM.


Impact of serum concentration of the medium and fasting on the imprintability of the insulin receptors of Chang liver cells.

When the cells of the Chang cell line came into interaction with a hormone (insulin) an imprinting-like phenomenon took place. The binding capacity of the receptors strengthened and this feature was transmitted to the descendant generations. The quality of the nutrient medium influenced the development of imprinting, when the cells were maintained in a medium containing 2% serum it was more difficult to evoke imprinting than in case the cells were kept in a medium containing 10% serum. If the cells were cultured kept in Tyrode (physiological) solution for 24 hours the possibility to evoke imprinting was lost. Difference could be observed between the behaviour of receptors in nuclear membrane and that of receptors in the plasma membrane; i.e. changes were more dynamic in the plasma membrane.


[The influence of paraquat on cell cycle--analysis of cell kinetics using DNA/BrdU staining].

We examined the in vitro and in vivo effects of paraquat on the cell cycle. After we exposed paraquat to the cultured cell (MDCK), we examined cell kinetics by flow cytometry with BrdU-PI double staining. And we administered paraquat intravenously to the rats, we collected and separated rat's liver and pulmonary cells, and we examined the cell kinetics by same method. Consequently our study found that the cells in the S-phase were noted to be abundant, and we suggested that paraquat apparently arrested the cell cycle at S time, and that DNA damage was importance in the mechanism of paraquat toxicity.


Intravenous urography pre-prostatectomy: an evaluation of its use.

The pre-operative intravenous urograms of 120 consecutive patients who had prostatectomy for benign prostatic hypertrophy (BPH) were studied. Of these, seventy-eight patients (65%) had normal intravenous urograms (IVU) while 42 patients had abnormal IVU. In this study serum creatinine above 2.0mg% and Blood urea above 35mg% proved valuable indices for possible selection of patients with BPH likely to show significant obstructive disease on IVU. This is not only cost saving, but also reduces unnecessary radiation to the patients.


Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Presence and significance of some antibodies/autoantibodies in patients with acute myocardial infarction and idiopathic dilated cardiomyopathy.

By using ELISA technique we determined the anti-myosin, actin, collagen type I and IV, cholesterol and phosphotyrosine proteins antibodies in sera from patients with acute myocardial infarction and idiopathic dilated cardiomyopathy. In idiopathic dilated cardiomyopathy we found a significant increase in five out of six types of antibodies tested. The patients with acute myocardial infarction reveal high levels of anti-myosin antibodies only. Our results suggest that some of the autoantibodies studied by us have a prognostic value and could be involved in maintaining cardiac dysfunctions.


Substance P like-immunoreactivity release from enterochromaffin cells of rat caecum mucosa. Inhibition by serotonin and calcium-free medium.

Location, endogenous contents, and release of Substance P like-immunoreactivity were investigated in the rat caecum, using Immunohistochemistry and RadioImmunoAssay. Our immunohistochemical results indicate that Substance P was present both in the neuromuscular and mucosal compartments of this intestinal structure. However, detection of the peptide in the enterochromaffin cells of the mucosa remained very difficult. That may be explained by the very low endogenous contents of Substance P detected in the mucosa, using RIA. As we have already described a serotonin release from rat caecum mucosa, we show, now, that Substance P like-immunoreactivity may be released from the same structure. This release was stable, calcium-dependent, inhibited by serotonin, and not influenced by the chemical depolarization. Our data demonstrate an active release of Substance P like-immunoreactivity from intestinal mucosa, in the rat caecum. It seems that the endogenous pool of Substance P like-immunoreactivity is involved as a functional pool. The mechanisms responsible of this release seem to be different than that observed for the serotonin release. Substance P like-immunoreactivity may be released in precise physiological conditions, or even, in pathological conditions.


[The regeneration of hematopoietic and connective tissues].

The data are summarized on comparative investigations by the author and co-workers concerning regenerative process in hemopoietic and connective tissues of representatives of molluscs, cyclostomes, fishes and mammals. In gastropods, a regeneration stimulus strikingly increases the volume of amebocyte reproduction foci in the pericardium (primitive organ of hemopoiesis), accelerates the amebocyte yield into the peripheral blood and induces the appearance of DNA-synthesizing cells in the latter. The stimulation of precursor cell reproduction in hemopoietic organs and the acceleration of their maturation and yield into the peripheral blood are common processes in the regeneration response for hemopoietic systems of mammals, fishes and cyclostomes. In contrast to fishes and cyclostomes, adult mammals show the absence of dividing (DNA-synthesizing) cells and mature segmentonuclear granulocytes demonstrate neither RNA nor protein synthesis. In all forms studied, the stromal type cells of hemopoietic nature (fibroblast-like, fixed macrophages, etc.) are a constant and continuously renewed component of connective tissue and play a considerable role in its regeneration.


Comparison of solution structures of mutant bovine pancreatic trypsin inhibitor proteins using two-dimensional nuclear magnetic resonance.

Structural perturbations due to a series of mutations at the 30-51 disulfide bond of bovine pancreatic trypsin inhibitor have been explored using NMR. The mutants replaced cysteines at positions 30 and 51 by alanine at position 51 and alanine, threonine, or valine at position 30. Chemical shift changes occur in residues proximate to the site of mutation. NOE assignments were made using an automated procedure, NASIGN, which used information from the wild-type crystal structure. Intensity information was utilized by a distance geometry algorithm, VEMBED, to generate a series of structures for each protein. Statistical analyses of these structures indicated larger averaged structural perturbations than would be expected from crystallographic and other information. Constrained molecular dynamics refinement using AMBER at 900 K was useful in eliminating structural movements that were not a necessary consequence of the NMR data. In most cases, statistically significant movements are shown to be those greater than approximately 1 A. Such movements do not appear to occur between wild type and A30A51, a result confirmed by crystallography (Eigenbrot, C., Randal, M., & Kossiakoff, A.A., 1990, Protein Eng. 3, 591-598). Structural alterations in the T30A51 or V30A51 mutant proteins near the limits of detection occur in the beta-loop (residues 25-28) or C-terminal alpha-helix, respectively.


Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel.

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.


Antigen F1 from Yersinia pestis forms aqueous channels in lipid bilayer membranes.

Antigen F1 is a protein of 17 kDa produced by Yersinia pestis when it is cultured at 37 degrees C. When incorporated into planar lipid bilayer membranes this protein induces fluctuations on membrane conductance typical of the formation of ionic channels. These fluctuations reveal two distinct unitary conductance sizes, one in the range of 800 to 1400 pS and the other in the range of 140 to 600 pS. Zero current potential measurements in the presence of a salt gradient show that the channel is not significantly ion selective. The reversal potential measured in the presence of 0.5 M KCl on the cis side and 0.1 M KCl on the trans side was 3.58 +/- 3.98 mV (N = 7). The non-selectivity of the channel, in addition to its large conductance, suggests that it forms large aqueous pores. The present results, taken together with other data showing that antigen F1 inhibits the activity of phagocytic cells, suggest that antigen F1 acts by forming aqueous pores in the membrane of these target cells.


Some pharmacological properties of prolonged administration of Ukrain in rodents.

Some pharmacological properties of Ukrain administered intraperitoneally (i.p.) once daily for three months in mice and rats of both sexes were studied. A three month treatment with Ukrain significantly depressed the spontaneous locomotor activity and did not affect the motor coordination of mice and rats of both sexes. Ukrain did not affect the body weight gain as well as the mass of internal organs; the exception was an increase in the mass of the spleen in rats. Biochemical studies indicated that a three month treatment with Ukrain depressed the whole brain dopamine (DA) concentrations and did not affect the noradrenaline (NA) concentrations. Long-term administration of Ukrain showed no characteristic changes in the concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the whole brain. The observed changes after three months' treatment with Ukrain are similar in mice and rats of both sexes.


A cDNA clone representative of a novel splicing pattern of the D. melanogaster dunce gene.

The D. melanogaster dunce gene is involved in both the learning and memory processes of the fly. The gene encodes for a cAMP-specific phosphodiesterase, a function playing a central role in the regulation of the intracellular cAMP level. Molecular cloning of dunce has so far not been completely achieved, although it is known that the gene encodes a large set of RNAs and has a complex organization, extending for more than 140 kilobases and containing several genes within its introns. Here we report the isolation and the characterization of 21/7, a cDNA clone representative of a novel dunce splicing pattern. The nucleotide sequence of this clone led to the identification of a dunce exon included in at least one transcript so far uncharacterized.


[Molecular biological analysis of cystic fibrosis--a model example for the strategy of "reverse genetics"].

The elucidation of the basic defect causing cystic fibrosis (CF) is a paradigm for the application of "reverse genetics" to the analysis of human genetic disease. Following this strategy, linkage analysis localized the responsible gene for CF on chromosome 7. Chromosome mediated gene transfer and chromosome walking and jumping led to the isolation of the CFTR-gene and its cDNA. A major 3 bp deletion mutation (DeltaF508) and more than 100 other mutations of this gene have been identified as molecular basis of cystic fibrosis. The CFTR-amino acid sequence, obtained by conversion of the cDNA-sequence, indicates that CFTR belongs to a group of integral membrane transport proteins (ABC-proteins). The normal cAMP-stimulated chloride-transport, lacking in CF-cells is restored by transfer and expression of CFTR-cDNA-recombinants in these cells. CFTR is most likely itself a chloride channel. The molecular identification of this gene has already led to substantial advances in diagnosis and prevention of this disease. New therapeutic approaches by pharmacological means or gene therapy are expected from the further molecular and functional analysis of the CFTR-gene.


Development of an African-American role model intervention to increase breast self-examination and mammography.

Minorities and indigent populations have low participation rates in breast cancer education and screening programs, and suffer from higher morbidity and mortality. Attitudes, norms, and values of such populations are best addressed by breast cancer patients of the same race and cultural background who serve as role models. This article describes the development and pilot study of an intervention program using role models as part of a "Witness" presentation. Programs were held in participants' local African-American churches and community centers. The organization of the program was based on an educational model (4MAT) that identified learning styles and brain hemisphere dominance. Preliminary results with 78 African-American women indicate that the program design is effective in reaching low-income, less-educated African-American women who did not believe themselves to be at high risk for breast cancer. Three-month follow-up demonstrated a significant increase in the practice of BSE and 19% had a mammogram.