Complexity of gap junctions between horizontal cells of the carp retina.

In the vertebrate retina, horizontal cells (HCs) reveal homologous coupling by gap junctions (gj), which are thought to consist of different connexins (Cx). However, recent studies in mouse, rabbit and zebrafish retina indicate that individual HCs express more than one connexin. To provide further insights into the composition of gj connecting HCs and to determine whether HCs express multiple connexins, we examined the molecular identity and distribution of gj between HCs of the carp retina. We have cloned four carp connexins designated Cx49.5, Cx55.5, Cx52.6 and Cx53.8 with a close relationship to connexins previously reported in HCs of mouse, rabbit and zebrafish, respectively. Using in situ hybridization, Cx49.5 expression was detected in different subpopulations of retinal neurons including HCs, whereas the Cx52.6 transcript was localized exclusively in HCs. Using specific antibodies, Cx55.5 and Cx53.8 were detected on dendrites of all four HC subtypes and axon terminals. Immunoelectron microscopy confirmed the presence of Cx55.5 and Cx53.8 in gap junctions between these processes and Cx55.5 was additionally observed in HC dendrites invaginating cone pedicles, suggesting its participation in the modulation of photoreceptor output in the carp retina. Furthermore, using single-cell RT-PCR, all four connexins were detected in different subtypes of HCs, suggesting overlapping expression patterns. Thus, the composition of gj mediating homologous coupling between subtypes of carp HCs appears to be more complex than expected. Moreover, BLAST searches of the preliminary carp genome, using novel sequences as query, suggest that most of the analyzed connexin genes are duplicated in carp.

[Effects of atypical employment on difficulties in falling asleep and maintaining sleep - gender differences in the lidA study]. / Ein- und Durchschlafstörungen in Abhängigkeit von atypischen Beschäftigungsformen - Geschlechterunterschiede in der lidA-Studie.

BACKGROUND AND AIM: Due to the increasing flexibilisation of the European labour market, new forms of atypical work organisation have been arising. Atypical employment may cause negative health effects similar to unemployment. Considering the health-promoting relevance of sleep for work productivity, we investigate if different forms of atypical employment are associated with difficulties falling and maintaining asleep among middle-aged male and female employees. METHODS: Data were retrieved from the 1(st) wave of the lidA study, a nation-wide survey among employees in Germany in 2011. According to the Integrated Employment Biography (IEB) of the Institute of Employment Research (IAB), participants were born in 1959 or 1965 and subject to mandatory social insurance contributions on 31.12.11. Our analysis is based on 4 544 participants. Using logistic regression models separately for men and women, difficulties falling and maintaining asleep were modelled to depend on years mostly spent in full-time, part-time, in marginal employment or in unemployment during the period from 1999-2010 as well as on years in the current position, fixed-term employment contract, organisational restructuring and dismissals at time of the survey in 2011. RESULTS: Women (9%) were more affected by difficulties falling and maintaining asleep than men (5%). Among women, past years mostly spent in full-time, part-time, marginal employment or in unemployment were not associated with sleep disturbances. Men who had mostly worked part-time or unemployment were more likely to report difficulties falling and maintaining asleep. Likewise, in men a fixed-term contract was linked with a higher risk of sleep disturbances. In women, witnessed dismissal in the working environment was a significant influencing factor. CONCLUSION: Atypical employment can be related to difficulties falling and maintaining asleep. In future research gender-specific reasons for atypical employment as well as adverse working conditions should be taken into account. Changes between different forms of atypical employment as well as cumulative measures of these employment exposures in employees' biographies should be included in future studies.

Characterization of immune system alterations following preoperative autologous blood donation for elective hip replacement surgery.

Preoperative autologous blood donation (PABD) has been shown to decrease natural killer (NK) cell function in cancer patients, raising concerns about an increased cancer recurrence risk owing to PABD. It is unclear whether PABD leads to other immunomodulatory effects that might affect more short-term risks like postoperative infectious complications in surgical patients. Lymphocyte subsets (CD4+ T cells, CD8+ T cells, B cells, NK cells) were determined in 86 consecutive patients donating 2 units of autologous whole blood prior to elective hip replacement surgery. In addition, cytokine secretion patterns of monocytes [tumour necrosis factor (TNF)] and lymphocytes [interferon-gamma, interleukin (IL)-2, IL-4, IL-10] upon stimulation were determined in a random subgroup of 58 patients. Analyses were performed immediately before the first donation and on the day prior to operation. Granulocytes increased during PABD by 4.6% (P < 0.01). Lymphocytes decreased by 8.8% (P < 0.01), accompanied by a relative rise in CD4+ T cells by 10.7% (P < 0.01) and in B cells by 10.3% (P < 0.01), and a fall of NK cells by 20.8% (P < 0.01). Stimulated TNF secretion of monocytes was suppressed (-12.3%, P < 0.01). The effect on the reactivity of lymphocytes and the T helper 1 (Th1)/Th2 balance were variable. The observed changes of innate and cellular immunity might influence the risk of perioperative infectious complications.

Platelets do not adsorb HLA class I molecules during storage of pooled platelet concentrates.

The origin of HLA class I molecules on platelets is still under discussion. Adsorption of HLA molecules on platelets using specific experimental conditions has been described. The study presented investigates whether there is a significant elution and adsorption of HLA class I molecules on platelets during storage of pooled random platelet concentrates (PRPC) under routine conditions. Platelet concentrates (PCs) from whole blood were prepared from HLA-A2-positive and HLA-A2-negative donors, pooled and stored under routine conditions. In addition, platelets from HLA-A2-negative donors were pelleted and resuspended in cell-free plasma from HLA-A2-positive donors. HLA-A2-positive PCs (positive control), HLA-A2-negative PCs (negative control) and HLA-A2-negative platelets in plasma from HLA-A2-negative donors were stored simultaneously. Binding of FITC-conjugated monoclonal murine antihuman HLA-A2 antibodies (anti-HLA-A2-mab) was measured during 5-day storage by flow cytometry. An increased binding of anti-HLA-A2-mab during storage was found on HLA-A2-negative platelets (P < 0.005) independently whether they were incubated with cell-free plasma or platelets from HLA-A2-positive donors or autologous HLA-A2-negative cell-free plasma. However, non-specific binding of IgG controls increased equally, whereas anti-HLA-A2-mab binding to platelets from HLA-A2-positive donors did not decrease during storage. This study suggests that there is no significant elution and adsorption of HLA class I antigens of platelets in pooled PCs during storage under the usual conditions for platelet storage. Increased anti-HLA-A2-mab signal was due to non-specific binding. Therefore, HLA class I compatible platelets should maintain their compatibility for an immunized patient when stored in a pool with HLA incompatible platelets and shortened survival after transfusion should not be expected.

Perspectives of paid whole and plasma donation.

Although payment of blood donors is rejected by the WHO, the FDA, the EU, and the Red Cross (RC), in Germany, monetary compensation of expenses is permitted not only for plasmapheresis but also for whole blood donation. The structure and organisation of the institutions ensuring the blood supply in Germany and the pertaining aspects of blood safety will be discussed. Data reported to the health authorities show that the frequency of transfusion-transmitted infection markers in the German donor population is low and that only very few infections have been transmitted through blood. This is underlined by a detailed analysis of the paid donor population of a small university blood service (UBS). The analysis documents a very stable and reliable cohort of predominantly repeat donors. Unpaid RC donors of blood units transfused to patients at the university hospital of Marburg showed a sixteen-times higher sero-conversion rate than those of the UBS (p < 0.0001). However, in a survey, 77% of the paid donors denied continuation of blood donation in the event of payment being stopped. Therefore non-remuneration would result in acute blood supply shortages. Since increased blood shortages are to be expected anyway in the near future, all measures improving the supply of safe blood, including monetary compensation, should be objectively discussed without prejudice.

Does hemodilution enhance coagulability?

UNLABELLED: Recent publications reported enhanced coagulability in hemodilution determined by TEG. In contrast, earlier reports have shown prolongation of in-vivo bleeding time in anemia. In order to take a closer look at this discrepancy undiluted and diluted anticoagulated blood samples (20 % with saline solution, hydroxyl-ethyl starch 6 % (HES), autologous platelet poor plasma (PPP)) were investigated by TEG (n = 10), ball (n = 10), and hook coagulometer (n = 15) as well as tests simulating primary hemostasis ex vivo (Platelet Function Analyzer PFA-100, n = 10). RESULTS: Dilution with plasma changed TEG parameters in a way, when started by recalcification of the blood sample, which is characteristic of enhanced coagulability (r decreased in all and k in 8 of 10 samples, maximal amplitude increased in 9 out of 10). With HES, changes in TEG parameters mainly indicated reduced coagulability (k increased in 7 out of 10, MA decreased in 10 out of 10). When the coagulation was additionally activated by PTT reagent (InTEG) the TEG parameters also mainly showed hypocoagulation with the three dilution solutions. Coagulation times with ball and hook coagulometers were significantly prolonged by dilution especially with saline (+ 25 % and + 17 %, p < 0.001). Dilution always significantly (often abnormally) prolonged closure time in PFA-100 (saline + 41 +/- 18 %, PPP + 37 +/- 20 %, HES + 69 +/- 24 %) demonstrating disturbance of primary hemostasis, particularly with HES. CONCLUSIONS: From the results obtained it can be concluded that the changes in the classical TEG (without addition of PTT-reagent), suggesting an enhanced coagulability, may be caused methodically as they are also found with autologous PPP. On the other hand, a disturbance of the primary hemostasis in hemodilution has to be taken into account from the results seen with the PFA-100 and a number of published data.

Safety and efficacy of unmodified whole blood vs. buffy coat-depleted red cell concentrates in autologous transfusion of elective orthopaedic surgery patients.

Storing autologous blood as whole blood (WB) has been proposed for increasing the cost-effectiveness of preoperative autologous blood donation programmes. However, experimental data suggest that autologous leucocytes might lead to immunomodulation similar to the effect attributed to allogeneic leucocytes. In a retrospective analysis, the postoperative outcome of 120 patients undergoing elective orthopaedic surgery and having donated up to two units of autologous WB (AWB) was compared with that of a control group of 52 patients, whose autologous donation had been processed into buffy coat-depleted red cell concentrates (RCC). At least one autologous unit, but no allogeneic units, had been transfused in all analysed patients. Donation schemes were equally efficacious in both groups. There was no significant difference in postoperative infection rates between the two groups. Overall rates were 7.7% in the RCC group and 8.3% in the WB group. Surgical, thromboembolic and other recorded complications, length of postoperative hospital stay and days of the use of antibiotics were also not significantly different between the two groups. The results of this study suggest that transfusion of up to two units of unmodified AWB is as efficacious as the transfusion of autologous RCC and does not negatively influence the postoperative outcome in elective orthopaedic surgery.

Singlet oxygen (1O(2)) disrupts platelet aggregates.

INTRODUCTION: Important mediators of activated polymorphonuclear leukocytes (PMN) are the oxidants HOCl and chloramine, which generate the nonradical photon-emitting oxidant singlet oxygen (1O(2)). Since 1O(2) inhibits platelet aggregation, we became interested in a possible oxidant mediated reversibility of platelet aggregation. METHODS: Chloramine T (CT) is a stable 1O(2) generator that mimics the natural chloramine N-chloro-taurine. Platelet-rich plasma (PRP) was incubated with CT 0-8 min after addition of the aggregation agonist (10 microM adenosine-5'-diphosphate, ADP, or 5 microg/ml collagen) and the aggregation was monitored. Platelet function was also analyzed by the platelet function analyzer, PFA-100. Fifty microliters of 200 micromol/l ADP was added to 400 microl PRP. After 1 min at 37 degrees C, 50 microl of 0 or 30 mmol/l CT was added, and after an incubation for 3 min at 37 degrees C, 50 microl of 25% glutaraldehyde was added. The samples were analyzed in a transmission microscope at x3000 and x7000 magnification. RESULTS: Chloramines inhibit platelet function in PRP: about 1 mM CT suppresses 50% of the aggregatory capacity of thrombocytes in normal PRP (effective dose 50%, ED(50)=1 mM chloramine), which is identical to the ED(50) for CT in whole blood. The ADP- or collagen-induced platelet aggregation can be reversed by addition of CT: up to 2 min after the addition of ADP as the aggregation inducer, the aggregation is reversible to more than 70% by addition of a 1O(2) release-inducer (3 mM CT). In contrast, addition of CT 8 min after the addition of ADP results only in about 50% reversal of platelet aggregation. The electron microscopic images of platelets before ADP, after incubation for 4 min at 20 micromol/l ADP, after incubation for 1 min at 20 micromol/l ADP, and a further incubation for 3 min at 3 mmol/l CT demonstrate an ADP-dependent formation of platelet aggregates, which are disrupted by 1O(2) into the single platelets; a phenomenon comparable to the decomposition of a puzzle or the continental drift of the major earth plates. The morphology of oxidized and unoxidized platelets is similar. CONCLUSION: This study demonstrates that 1O(2) inhibits and reverses platelet aggregation. The physiologic signal action and the direct anticoagulant action of 1O(2) might be a new principle for pharmacologic intervention in atherothrombosis.

Arginine inhibits hemostasis activation.

BACKGROUND: The diagnosis and the therapy of in vivo hemostasis activation is of great clinical importance. Artefactual changes of the hemostasis (i.e., coagulation or fibrinolysis) in vitro have to be prevented. Usual in vitro anticoagulation by sodium citrate does not fully inhibit coagulation--or fibrinolysis--activation. Therefore, there is need for a simple physiologic inhibitor of hemostasis activation both in diagnosis and therapy of hemostasis activation. METHODS: Whole blood clotting time (WBCT), prothrombin time (PT), activated partial thromboplastin time (APTT), in vitro bleeding test closure time (IVBT-CT), and whole blood aggregometry (WBA) were determined in normal human blood or plasma, supplemented with increasing concentrations of L-arginine or guanidine. RESULTS: Arginine in concentrations of 5-100 mM inhibited the WBCT, PT, APTT, IVBT-CT, and WBA. Arginine (50 mM) resulted in a two-fold prolongation of WBCT, PT, or IVBT-CT (the anti-epinephrine action is superior to the anti-ADP action), a four-fold prolongation of APTT or a 60% inhibition of WBA. CONCLUSION: L-Arginine (or guanidine) inhibited the activation of hemostasis. Arginine might be used as hemostasis stabilizer both in the diagnosis and therapy of hemostasis activation. The usage of arginine as an in vitro hemostasis inhibitor might be indicated in the diagnosis of hemostasis activation, as occurring in pharmacological thrombolysis or disseminated intravascular coagulation (DIC). The storage of blood or blood products might be improved by arginine stabilization. The amino acid (and nitric oxide precursor) L-arginine could be an interesting new pharmacologic agent to inhibit a pathologic hemostasis activation.

Singlet oxygen inhibits agonist-induced P-selectin expression and formation of platelet aggregates.

Major mediators of activated polymorphonuclear leukocytes (PMN) are the oxidants HOCl and chloramine, which are a source for the nonradical photon-emitting oxidant singlet oxygen (1O2). We were interested in a possible platelet-modulating activity of 1O2. As a stable 1O2 source we chose the mild oxidant chloramine T (CT), which mimics the natural chloramine N-chloro-taurine. Freshly drawn native whole blood from donors (n = 5) was incubated at 0 to 3 mM CT for 1 minute at 37 degrees C. Then saline. 10 microM adenosine diphosphate (ADP), 5 microg/mL collagen, or 6.25 microM thrombin receptor activator peptide (TRAP) were added and the mixtures were allowed to incubate for 3 minutes at 37 degrees C. Aliquots of activated blood were fixed in 1% para-formaldehyde. After removal of the fixative, platelets were labeled with anti-CD61-FITC and anti-CD62P-PE antibodies and analyzed by flow cytometry. An oxidant concentration-dependent decrease in the expression of P-selectin appeared (at 3 mM CT to 39, 23, and 20% of the 100% saline control level for ADP, collagen, and TRAP, respectively). There was also an oxidant concentration-dependent decrease in the formation of platelet aggregates (at 3 mM CT to 8, 12, and 13% of the 100% saline control level for ADP, collagen, and TRAP, respectively; the 50% effective dose was 1.0 to 1.5 mM chloramine). In ADP- and TRAP-stimulated platelets, an oxidant-mediated increase in platelet fragments appeared (at 3 mM CT: three- to fourfold of the initial value). The addition to the blood of 30 mM of the oxyradical scavenger mannitol in contrast to excess methionine did not antagonize these oxidative modulations of platelet activation. The results were confirmed using equimolar concentrations of NaOCI and N-chloro-taurine. This study shows that 1O2 inhibits platelets, decreasing the expression of CD62P and the formation of platelet aggregates. Activated PMN might modulate hemostasis, shifting it into an antithrombotic state. The physiologic signal action and the direct anticoagulant action of 1O2 (released by chloramines such as vancomycin) might be a new principle for pharmacologic intervention in atherothrombosis.

Thrombin converts singlet oxygen (1O2)-oxidized fibrinogen into a soluble t-PA cofactor. A new method for preparing a stimulator for functional t-PA assays.

Activated phagocytes, particularly polymorphonuclear leukocytes (neutrophils), by means of oxidative photonic burst, i.e., the combined activation of NADPH-oxidase and myeloperoxidase, generate large amounts of oxidants of the hypochlorite/chloramine type that are an important physiologic source for the nonradical, photon-emitting oxidant singlet oxygen (1O2), which (in the dark blood stream) is both a signal and an agent of defense against bacteria or fibrin. 1O2-oxidized fibrinogen or oxidized fibrin monomer has previously been shown to be unpolymerizable, and methionine to methionine sulfoxide-oxidized fibrinogen occurs in circulating blood. The present study demonstrates that thrombin converts oxidized fibrinogen into a soluble stimulator of tissue-type plasminogen activator (t-PA). After addition of 0.1 IU thrombin to 25 microl oxidized normal human plasma and an incubation time of 10 min (room temperature), t-PA activity increases about 20-fold when compared with oxidized plasma without the addition of thrombin. Thus, since oxidized fibrin monomer is a t-PA cofactor, thrombin-degraded oxidized fibrinogen can be used as a stimulator in functional t-PA assays.

Flow cytometric determination of RBC survival in autoimmune hemolytic anemia.

BACKGROUND: In cases of warm autoimmune hemolytic anemia (WAIHA), crossmatch incompatible RBCs are most often used for transfusion. The determination of the in vivo survival of transfused and autologous RBCs in WAIHA is helpful in the assessment of the efficacy of transfusion and other therapeutic interventions. CASE REPORT: A 38-year-old man presented with acute WAIHA, thrombocytopenia, and neutropenia. Steroids and IVIG therapy were ineffective, and the patient received RBCS: Because of increasing hemolysis and persisting thrombocytopenia, splenectomy was performed, resulting in partial remission. Further improvement was achieved by immunosuppressive therapy. MATERIALS AND METHODS AND RESULTS: Survival of transfused and autologous RBCs was determined, using a flow cytometric method based on the determination of different blood group antigens of patient and donor RBCS: The survival of autologous and transfused RBCs before splenectomy was determined on two consecutive days. The life span of autologous RBCs remained rather stable at 69 and 64 hours on Days 10 and 11, respectively, whereas the life span of transfused RBCs decreased from 186 hours to 25 hours. After splenectomy, the life span of transfused RBCs almost normalized: 43 days at postsplenectomy Day 3 and 87 days at postsplenectomy Day 69. CONCLUSION: Flow cytometry was successfully used to determine changing hemolytic activity during the clinical course of WAIHA. Additionally, the survival of transfused RBCs could be measured, which may be helpful to judge for the compatibility of allogeneic RBCS: Thus, we were able to show the therapeutic inefficacy of steroids and immunoglobulins, and quick improvement after splenectomy.

Influence of ABO blood groups on primary hemostasis.

BACKGROUND: Recently, the in vitro bleeding time test (IVBT) was proved to be a very sensitive screening method for the detection of vWD, showing rather good correlation between the closure time and the level of vWF. The vWF levels have been found to be significantly lower in healthy humans who are group O than in those who belong to the other ABO blood groups (non-group O). The aim of this study was to detect whether these differences in vWF levels in normal persons correspond to differences in nonvascular primary hemostasis when investigated by the IVBT. MATERIAL AND METHODS: Healthy blood donors (n = 162) without evidence of hemostatic disorders, without ingestion of drugs for at least 2 weeks, and with normal in vivo bleeding time endpoints, normal factor VIII clotting activity levels, normal structure of vWF multimers, and normal ristocetin-induced platelet aggregation were examined by IVBT. IVBT was performed with two automated systems (Thrombostat 4000, VDG [TST]; and a platelet function analyzer (PFA-100, Dade Behring [PFA]). CaCl2 and ADP were used as aggregants for the two TST tests (TST-CaCl2 and TST-ADP), and ADP- or epinephrine (Epi)-coated membranes were used with the two PFA tests (PFA-ADP and PFA-Epi). RESULTS: Closure time in the IVBT significantly correlated with the blood groups, but in reverse order (as did blood volume; data not shown): TST-ADP (mean +/- SD): group O, 89 +/- 14.6 seconds versus non-group O, 82 +/- 13 seconds (p<0.01); TST-CaCl(2): group O, 154 +/- 28.9 seconds versus non-group O, 140 +/- 31.3 seconds (p<0.01); PFA-ADP: group O, 91 +/- 13.4 seconds versus non-group O, 86 +/- 11.9 seconds (p<0.05); PFA-Epi: group O, 112 +/- 15.4 seconds versus non-group O, 104 +/- 16.7 seconds (p<0.05). Donors with vWF < or =77.5 % had longer closure time than those with vWF >77.5 % (p<0.05). CONCLUSION: Significant ABO-group-specific differences in nonvascular primary hemostasis could be found by IVBT. The differences are small, however, and lie within the normal range. Whether these differences have any biologic relevance can only be speculated.