Amount Of Lysis Research Articles

Observing temporal variation in hemolysis through photoacoustics with a low cost LASER diode based system

Patients under hemolytic condition need continuous monitoring of lysis as depletion of Red Blood Cells (RBC) and the presence of antioxidant free hemoglobin (Hb) in excess amount due to hemolysis lead to severe deterioration of their health. Out of many modalities, Photoacoustics (PA) offers real time information noninvasively from deep lying blood vessels since Hb is the strongest chromophore in mammalian blood and the PA response of blood varies with the amount of Hb present. During hemolysis, total Hb content in blood however remains unchanged, thus, questions the use of PA in hemolysis detection. In this report, a hypothesis that the amplitude of the PA signal would not change with the amount of lysis is framed and tested by applying osmotic shock to the RBCs in hypotonic environment and the PA response is recorded over time using a low cost NIR based PA system. The experimental outcome indicates that PA amplitude falls off as lysis progresses in course of time consequently rejecting the hypothesis. The decaying PA response also carries the signature of RBC swelling during the early phase of lysis. The PA measurement can detect hemolysis as low as 1.7%. These findings further advocate transforming this NIR-PA system into a portable, noninvasive patient care device to monitor hemolysis in-vivo.

The Hypofibrinolytic Defect of Nephrotic Syndrome Is Directly Proportional to Disease Severity

IntroductionNephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury), hypoalbuminemia, and edema. Increasing evidence suggests that myriad abnormalities of the hemostatic system may be implicated in the pathogenesis of thrombosis during NS. Indeed, thromboelastometry studies by our group and others have suggested a resistance to fibrinolysis in animal models of NS. Research on NS-associated fibrinolytic effects in human patients is scarce however and complicated by conflicting results and limited mechanistic investigations. The aim of the present study was to delineate the relationship between disease severity (proteinuria and hypoalbuminemia) and fibrinolysis using two rodent models of NS and a cohort of human NS patients. We hypothesized that hypofibrinolysis is directly proportional to NS disease severity.MethodsUsing two well established rat models of NS, transgenic diphtheria toxin receptor (DTR) and puromycin aminonucleoside (PAN), we compared markers fibrinolysis to disease severity. A range of severity was induced by a single I.P. injection of diphtheria toxin (0-75 ng/kg IP) or PAN (0-150 mg/kg IV). On day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio (uPr:Cr). Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor and immediately analyzed with ROTEM before being spun down to platelet poor plasma (PPP). Samples were also collected from a local cohort of pediatric and adult NS patients (n=23), along with the corresponding clinical lab data for each patient. Plasma clot lysis assay (CLA) was performed using urokinase (50 IU). Plasma clot turbidity was measured by inducing clot formation with recalified PPP + 0.5 pM TF + 4uM phospholipids, and monitoring absorbance at 405nm. Fibrinolytic markers in plasma were measured by ELISA. Plasmin generation (PG) has not previously been studied in rats, however our initial results suggested that PG is substantially prolonged in rats compared to humans. Thus, through a series of experiments we first developed a PGA protocol optimized for use in rats. Final PG results were obtained over 12 hours using an MCA-fluorogenic substrate with addition of 10 nM tissue plasminogen activator (tPA).ResultsHypofibrinolysis: The amount of lysis at 60 min (LI60) on ROTEM was significantly correlated with proteinuria in both the DTR and PAN models (R2=0.167; P=0.015 & R2=0.740; P<0.001, respectively). The addition of tranexamic acid to the ROTEM assay (1mg/100ul), abolished lysis (increased the LI60 to 100%) in both healthy and NS rats (~8% & 3.5% abolishment, respectively), suggesting that the increased clot stability is indeed a fibrinolytic defect. These findings were confirmed with the CLA, such that plasma clot lysis was significantly negatively correlated with proteinuria (R2=0.196; P=0.007 & R2=0.214; P=0.010) and significantly positively correlated with hypoalbuminemia (R2=0.310; P<0.001 & R2=0.240; P=0.006), in the DTR & PAN models, respectively. Additionally, plasma clot lysis by CLA was decreased in NS patients with uPrCr ≥2 (n=16) vs. <2 mg/mg (n=7) (96.1 vs 55.2 %, respectively; P=0.041).Mechanisms of Hypofibrinolysis: In the DTR rats, disease severity was positively correlated with plasma concentrations of Fibrinogen, uPA, PAI-1, and plasma turbidity at baseline and following clot formation, suggesting a difference in clot structure related to disease state.Plasmin Generation: After optimizing the protocol for rats, we were able to obtain evaluable PG curves (Figure). PG increased with disease severity such that it was highly correlated with proteinuria and hypoalbuminemia in both rat models (P<0.001; Figure), as well as in our human samples (P=0.005).ConclusionsThese data suggest that NS thrombi are resistant to fibrinolysis in a manner that is proportional to disease severity. Paradoxically, increased plasmin generation implies that the fibrinolytic system is hyperactive during NS. Thus it appears that the nephrotic clots are inherently resistant to fibrinolysis, even in the face of the increased plasmin that is generated. We are currently examining fibrin clot structure in NS, hypothesizing that nephrotic plasma forms a denser fibrin network that is resistant to fibrinolysis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nephrotic Syndrome Clots Are Resistant to Fibrinolysis Despite Elevated Plasmin Generation

IntroductionNephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury), hypoalbuminemia, and edema. Importantly, NS is associated with a complex acquired hypercoagulopathy and a high prevalence (~25%) of life-threatening thrombotic complications. We have recently demonstrated that NS disease severity is directly proportional to hypercoagulability,in two well-established animal models of NS (puromycin aminonucleoside (PAN) and Adriamycin (ADR) rats). Furthermore, thromboelastometry studies by our group and others have suggested a resistance to fibrinolysis in both rats and humans with NS. Increasing evidence suggests that myriad abnormalities of the hemostatic system may be implicated in the pathogenesis of altered fibrin clot formation during NS; however the relationship between hypofibrinolysis and NS disease severity requires further validation and the mechanisms underlying the hypofibrinolytic defect remain unknown. Thus, the aim of the present study was to delineate the relationship between disease severity (proteinuria and hypoalbuminemia) and hypofibrinolysis using two rodent models of NS: a transgenic rat expressing the human diphtheria toxin receptor (hDTR) in a podocyte-specific manner and the well-established PAN rat model. We hypothesized that hypofibrinolysis is directly proportional to NS disease severity.MethodsUsing the hDTR rat, we compared markers of global hemostasis (ROTEM) and fibrinolysis to disease severity. A range of severity was induced by a single I.P. injection of diphtheria toxin (0, 25, 50 & 75 ng/kg; n= 7-8/group). On day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio. Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations] and immediately analyzed with ROTEM (whole blood; intem) before being spun down to platelet poor plasma (PPP). Plasma clot lysis assay (CLA) was performed using urokinase (50 IU). Plasmin generation was measured using a fluorogenic substrate in plasma with addition of 10 nM tissue plasminogen activator (tPA).ResultsHypercoagulopathy in the hDTR NS model: Disease severity (proteinuria and hypoalbuminemia) was significantly correlated with endogenous thrombin potential, peak thrombin, and hypercoagulopathic ROTEM parameters (clot formation time, intermediate firmness (amplitude at 10 & 20 min), and maximum clot firmness).Hypofibrinolysis: The amount of lysis at 60 min (LI60) on ROTEM was significantly correlated with proteinuria in both the DTR and PAN models (R2=0.167; P=0.015 & R2=0.740; P<0.001, respectively), suggesting that NS thrombi are resistant to fibrinolysis in a manner that is proportional to disease severity. These findings were confirmed with the CLA, such that plasma clot lysis was significantly negatively correlated with proteinuria (R2=0.196; P=0.007 & R2=0.214; P=0.010) and significantly positively correlated with hypoalbuminemia (R2=0.310; P<0.001 & R2=0.240; P=0.006), in the DTR & PAN models, respectively.Plasmin Generation Assay: Plasmin generation increased with disease severity, such that it was highly correlated with proteinuria and hypoalbuminemia in both models (P<0.001; Figure). Interestingly, there was no correlation between plasmin generation and plasma clot lysis. (P=0.101 & P=0.126 in DTR & PAN rats, respectively).ConclusionsNS disease severity is directly proportional to hypercoagulability in a podocyte-specific rodent model of NS; thus confirming our previously published findings in a podocyte-specific experimental model of NS. Moreover, this hypercoagulopathy leads to formation of a clot that is resistant to fibrinolysis. Paradoxically, the increased plasmin generation implies that the fibrinolytic system is also hyperactive in NS. Thus, it must be concluded that the clot is resistant, even to this increased plasmin that is generated. Future studies will explore the mechanisms underlying and in vivo significance of this fibrinolytic resistance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proteinuria Severity Is Directly Correlated with Markers of Hypofibrinolysis in a Podocyte Specific Experimental Model of Nephrotic Syndrome

IntroductionNephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury or dysfunction), hypoalbuminemia, and edema, and is associated with a complex acquired hypercoagulopathy and a high prevalence (~25%) of life-threatening thrombotic complications. However, anticoagulation is associated with a substantial risk for adverse bleeding events. Recently published epidemiology studies suggest that proteinuria severity is directly correlated with thrombotic risk. However, further validation of this candidate biomarker for thrombotic risk requires appropriate validation and adequate pathophysiologic explanation. We have recently demonstrated that proteinuria severity is directly proportional to hypercoagulability (as assessed by ex vivo and in vivo markers of thrombotic capacity),in two well-established animal models of NS (puromycin aminonucleoside (PAN) and Adriamycin (ADR) rats). Thromboelastometry studies also suggested a resistance to fibrinolysis during rat NS. Thus, the aim of the present study was to further delineate the relationship between proteinuria severity and hypofibrinolysis using a podocyte-specific rat model of NS. We hypothesized that hypofibrinolysis is directly proportional to severity of proteinuria.MethodsUsing a transgenic rat which expresses the human diphtheria toxin receptor (hDTR) on a podocyte specific promoter (podocin), we compared markers of global hemostasis (ROTEM) and fibrinolysis to proteinuria severity. A range of proteinuria severity was induced by a single I.P. injection of diphtheria toxin (0, 25, 50 & 75 ng/kg; n= 7-8/group). On Day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio. Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations] and immediately analyzed with ROTEM (whole blood; intem) before being spun down to platelet poor plasma (PPP). Plasma clot lysis assay (CLA) was performed using urokinase (50 IU). Thrombin and plasmin generation assays are currently being performed.ResultsThere were significant differences (P<0.005) between the highest proteinuria hDTR rat group and controls, in both hypercoagulopathic (clot formation time, maximum clot firmness, and clot size (amplitude at 10 & 20 min)), and hypofibrinolytic (amount of lysis at 60 min; LI60) ROTEM parameters. Importantly, there was a significant negative correlation between proteinuria severity and LI60 (R2 =0.362, P=0.02), suggesting that hypofibrinolysis is directly proportional to podocyte injury and therefore disease severity during NS. Preliminary results from the CLA (n=2 control & 2 high proteinuria) also suggest a marked impairment (~50% difference) in plasma clot lysis time in proteinuria rats.ConclusionsThese results demonstrate that proteinuria severity is directly proportional to both hypercoagulability and hypofibrinolytic capacity in a podocyte-specific rodent model of NS, thus confirming our recent findings in two other well-established animal models of NS in a third, more specific, experimental model of glomerular disease. Importantly, these data also strongly suggest a marked impairment in fibrinolysis during NS, which is directly correlated with proteinuria severity. Therefore it appears that severe proteinuria is associated with both hypercoagulopathic and hypofibrinolytic defects in the coagulation system. Future studies will delve into the molecular mechanisms involved in these defects. DisclosuresNo relevant conflicts of interest to declare.

Kinetics of hypotonic lysis of human erythrocytes

The curve of osmotic stability of erythrocytes is based on the amount of lysis as a function of salt concentration under fixed time incubation and represents an equilibrium situation after a sufficiently long time, although lysis is a rapid process. The curve is valid for the analysis of modulating agents that have influence on this equilibrium, but not for those that have influence on the lysis kinetics. This work has developed experimental conditions to study the hemolysis kinetics based on the interruption of lysis by hypertonicity at predetermined intervals of time. These conditions were used to evaluate the kinetics of hemolysis of 17 volunteers. The lysis curve as a function of time was statistically fitted to a hyperbola, using the analytical routine of the integrated kinetic model of Michaelis–Menten to determine the time required to promote lysis of half of the population of erythrocytes (t1/2) and the maximum absorbance (Amax) reached in the test. The results showed good variance among volunteers. The constant t1/2 was negatively correlated with total and LDL-cholesterol, and Amax, as it was expected, showed significant associations with hematological variables that are under the influence of hemoglobin levels. Stratification of the study population into two age groups (18–30 and 40–90 years old) showed that the t1/2 values were significantly lower in the older population. Although the study population has been small, the study showed that this kinetic approach of the erythrocyte lysis is very promising for analyzing the myriad of variables which have influence on the cell membrane.

In vitro microscopic imaging of rt-PA thrombolysis with 120-KHz ultrasound in a human clot model

Substantial enhancement of recombinant tissue plasminogen activator (rt-PA) thrombolysis can be achieved with diagnostic frequency ultrasound. Microscopic visualization of human whole-blood clots treated with human fresh frozen plasma (HFFP), rt-PA, and 120-KHz pulsed ultrasound offers a useful method to study the possible mechanisms. Whole human-blood clots were formed inside of glass micropipette (1.7-mm diameter) and placed in a water tank at 37&amp;lt;th&amp;gt;°C. The clot–plasma interface was imaged using an inverted optical microscope. Clots were exposed to HFFP (control), HFFP and rt-PA (0.0945 mg/ml), (sham), or HFFP, rt-PA (0.0945 mg/ml), and 120-KHz pulsed ultrasound (ultrasound treated) for 30 min. Thrombolysis at the clot surface was recorded with a CCD camera and the lytic front was analyzed as a function of time. Images of the clots were analyzed to quantify the overall amount of lysis and compared to a dynamic chemical model of enzymatic degradation in the presence of ultrasound-induced flow. Sham and ultrasound-treated clots showed significant lysis with the lytic rate of (1.33±0.02 microns/min; mean ± s.d.) and (5.17±0.10 microns/min), respectively. Ultrasound enhanced thrombolysis by a factor of 4. In addition, the lytic rate profile may offer a suggestion to the mechanism of enhancement. No thrombolysis was observed in control clots. [The authors of this study gratefully acknowledge the support of the Whitaker Foundation Grant RG-0128-01.]

Thrombus characterization with intravascular ultrasound: potential to predict successful thrombolysis.

To define analysis methods using radiofrequency (RF) data from intravascular ultrasound (IVUS) to characterize thrombus so that the success or failure of thrombolysis can be predicted. Experimentation was done in 2 phases: first, 40 clots created from platelet-rich (n=20) and platelet-poor (n=20) plasma were imaged with 20 and 30-MHz IVUS probes. The digitized RF signals were analyzed to determine the attenuation and slope of attenuation characteristics as a reference standard for the second part of the study. In this phase, 20 perfusion trials were performed with 10 platelet-rich and 10 platelet-poor samples in an in vitro arterial perfusion model; the composition of the samples was blinded during interrogation with IVUS. Alteplase (0.1 mg) was then infused directly into the thrombus for 1 hour; restoration of flow and amount of lysis were quantified. Significant differences were detected for the integrated attenuation value (p<0.001) between platelet-poor and platelet-rich clots interrogated with a 30-MHz probe, but no differences were noted using the 20-MHz catheter. However, both transducers were capable of differentiating platelet-rich and platelet-poor clots using a slope of attenuation analysis, which was successful in identifying all 20 (100%) of the blinded clots in the in vitro model. The speed and completeness of lysis were significantly higher (p<0.05) for platelet-rich (78%) than platelet-poor (21%) clots. IVUS preprocessed imaging is capable of differentiating various types of thrombus. This information can be used to predict the success or failure of pharmacological lysis techniques.

Dialysis membranes in convective treatments.

substitution fluid. No dialysis fluid is present. Thistechnique offers the advantage of excellent clearancesHaemodialysis is basically divided into two categories: for large molecules. The treatment, however, has onlylow-flux dialysis and high-flux dialysis. Low-flux dia- been applied in Europe because of the need for largelysis includes the standard haemodialysis technique in quantities of substitution fluid in commercially pre-which dialysers with low hydraulic permeability are pared bags. (ii) Haemodiafiltration is a form of therapyutilized. Blood and dialysate flow rates are at 250 and in which diffusion and convection are conveniently500 ml/min, respectively and the average duration is combined. Dialysate is circulated countercurrent to4 h. When the dialyser surface area is increased, and blood but the amount of ultrafiltration exceeds theflow rates of blood and dialysate are increased up to programmed patient weight loss and therefore the final400 and 800 ml/min, respectively, the treatment is fluid balance is achieved by the infusion in the venousdefined as high efficiency haemodialysis [1]. In this line of appropriate amounts of substitution fluid [4–6].treatment the clearance of small molecules is remark- Due to the high costs related to replacement solutions,ably increased and the treatment time generally can be new approaches have been proposed in Europe forshortened, but the clearance for middle to large molec- performing haemofiltration and haemodiafiltration [7].ules is still very low due to the permeability character- Part of the fresh dialysate is diverted from its line toistics of the membrane. the dialyser, it is filtered and it finally is used as aClassic cellulosic membranes such as cuprophan are replacement solution. These on-line treatments havetypically low-flux membranes i.e. permeability to water the advantage of reduced costs, unlimited productionis in the range of 5–6 ml/h/mmHg/m2, they are strongly of substitution fluid and no need for tons of commer-hydrophilic and remarkably thin to permit an optimal cially produced fluid in bags. On the other hand, theutilization in diffusive treatments such as microbiological and chemical purity of the on-linehaemodialysis. prepared fluid may represent a concern since no on-lineOn the other hand, classic high-flux synthetic mem- controls can be made. (iii) High-flux dialysis is abranes originally were fully hydrophobic, strongly technique in which highly permeable dialysers areasymmetric and with a thick wall. They were employed utilized in conjunction with an accurate ultrafiltrationprincipally in convective therapies such as haemofiltra- control of the dialysis machine. Because of the naturetion due to their high hydraulic permeability, i.e. of the hollow fibre dialyser, the correct water balance30–40 ml/h/mmHg/m2, and their high sieving coeffi- is achieved thanks to an internal filtration in thecients [2]. proximal part and a backfiltration in the distal part ofOnly recently has it been possible to design synthetic the filter [8]. As a consequence, some of the advantagesmembranes with reduced wall thickness and a mixed of removing larger molecules are maintained becausehydrophobic–hydrophilic structure. This has permitted of the internal convection, while the need for replace-their use in new techniques such as high-flux haemodia- ment solution is avoided by a significant amount oflysis and haemodiafiltration in which diffusion and backfiltration of fresh dialysate. In this way, someconvection are conveniently combined [3]. of the advantages of haemodiafiltration are maintainedWhen high-flux membranes are utilized, three differ- with a simpler and easier layout of the technique [8].ent approaches can be chosen: (i) haemofiltration is a Again, the dialysate purity is a key issue in thisfully convective treatment in which large amounts of treatment where bacterial products could be back-ultrafiltrate are produced and replaced by a sterile transported into the blood together with the reverseflux of water. This may affect the entire concept ofbiocompatibility of the system. Concerning this aspect,

Apoptosis and other effects of radiation in normal human urothelial cells.

In this paper, an attempt is made to identify endpoints that might be of potential use in the quantification of radiation effects in human tissues. Irradiated cultures of cells that are not selected for clonogenic survival but are left in situ to grow after irradiation show a wide variety of morphological and biochemical abnormalities. These include nuclear fragmentation and other evidence of programmed cell death, but they also include a considerable amount of lysis, necrosis, and persistent abnormal growth and function, which are expressed in the progeny of irradiated cells. Induction of proteins associated with stress or shock responses, growth and cell cycle control, and control of apoptosis are also seen and may persist. The dose dependence of these various responses is documented, because it probably determines to a large extent the outcome of radiation exposure in terms of whether a cell dies, divides normally, or develops genomic instability, mutation, and ultimate carcinogenic progression of the progeny. Clearly, a cell that dies presents no further threat to the organism, nor does a fully repaired cell. Therefore, a major challenge facing radiation protection research is to define the population at risk of surviving with damage. The results show that there is a variation in response to radiation between different patient cultures that is detectable in an explant culture system of primary normal human urothelium. The growth pattern and protein expression postirradiation is consistent with apoptosis being a major determinant of low dose response to radiation. This form of death appears to be suppressed at higher doses and, in the majority of subjects, results in the presence of a highly abnormal population of cells, even though the population size is the same whether their progenitors were irradiated or not.

Possible Involvement of Calmodulin in the Maintenance of Plasma Membrane Integrity in Oat Protoplasts both under Isotonic Conditions and after Hypotonic Shock

Summary Protoplasts from Avena sativa were incubated with several calmodulin inhibitors including chlorpromazine (CPZ), trifluoperazine (TFP), tetracaine and R24571, and the effect on plasma membrane stability investigated under isotonic conditions as well as after hypotonic shock. Isotonic lysis was obtained if the drug concentration exceded 5 µM for R24571, 50 µM for TFP, 100 µM for CPZ and 400 µM for tetracaine, respectively. At lower drug concentrations protoplasts were stable in isotonic solution but the amount of lysis after hypotonic shock was strongly enhanced. Using tetracaine, lysis was additionally dependent on the external p H. The results suggest that calmodulin is important for maintaining plasma membrane integrity both under isotonic conditions and after osmotic shock.

Effect of ionophore A23187 on plasma membrane integrity in isolated protoplasts of Avena sativa

It is argued that modulation of the cytoplasmic Ca 2+ of plant cell protoplasts should be expected to decrease their ability to incorporate membrane material and thus to resist osmotic shock. Protoplasts from Avena sativa were incubated with ionophore A23187 under various conditions, and the extent of protoplast lysis was determined by the use of fluorescein diacetate. The amount of lysis was strongly dependent on both the extracellular Ca 2+ concentration and the extracellular pH. The influence of the ionophore concentration itself and the incubation time were also investigated. The results suggest that a certain level of intracellular Ca 2+ is important for maintaining plasma membrane integrity, even under isotonic conditions.

Basics and practice in evaluating plasminogen.

There exist different ways of assays of plasminogen which give information about different properties of this proenzyme. The concentration of plasminogen can be determined by its antigenicity. Since the normal concentration of plasminogen in plasma is between 15 and 25 mg/dl the test can be carried out by simple methods such as radial immunodiffusion on Partigen plates. The possibility of errors is small and there is no need of special apparatus. The disadvantages are the lapse of 24 h until the result is available and the fact that the knowledge of the concentration does not give any information about the activity. The activity can be measured by different coagulation tests. A typical assay would involve activation of plasminogen to plasmin, addition of plasminogen-free thrombin and measuring of the lysis time. The result is however, dependent on more than one variable. Plasmin is rapidly inhibited by alpha-2-antiplasmin (APL) and there is also a dependence of the lysis time on the amount of clottable fibrinogen in the test system. Better results can be obtained by the use of diluted test plasma and addition of a constant amount of plasminogen-free fibrinogen. A different way would be the use of the euglobulin fraction instead of plasma. This has however, the possible disadvantage of incomplete precipitation of plasminogen. Instead of coagulation tests the activity can also be determined when diluted activated plasma is placed on plasminogen-free fibrin plates and the amount of lysis in the plate is recorded. All assays of this group also depend on the method of activation of plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysis of Plasmodium falciparum by ferriprotoporphyrin IX and a chloroquine-ferriprotoporphyrin IX complex.

Ferriprotoporphyrin IX (FP) and a chloroquine-FP complex lysed isolated Plasmodium falciparum parasites as judged by decreases in the turbidity of parasite suspensions and by ultrastructural changes. Exposure of parasite suspensions to 50 microM FP or to a complex formed from 50 microM FP and 20 MicroM chloroquine reduced the number of identifiable parasites and caused swelling and loss of internal detail in those that were identifiable. The amount of lysis was dose-dependent over the range of 10 to 50 microM FP. Formation of a chloroquine-FP complex reduced, but did not eliminate, the toxicity of FP. Since there is evidence indicating that a chloroquine-FP complex forms when chloroquine-susceptible parasites are exposed to chloroquine, we suggest that accumulation of this complex may account for the chemotherapeutic effect of chloroquine against P. falciparum.

Human mononuclear cell antibody-dependent cell-mediated cytotoxicity toward red blood cells of various Rh genotypes.

Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human peripheral blood mononuclear cells (85 % lymphocytes and 15 % monocytes) or lymphocyte-enriched (98 % lymphocytes) effector cells against human red blood cell (HRBC) targets of various Rh genotypes, with or without papain treatment, was tested in an 18-hour 51Cr release assay. The HRBC were sensitized with Rh0GAM, an anti-Rh0 (D) IgG fraction. Mononuclear cell effectors were found to lyse papain treated Rh0 (D) homozygous cells to a greater degree than Rh0 (D) heterozygotes. The genotypes of the HRBC targets, in order of the amount of lysis from highest to lowest was R1R1, R2R2 or R1R2, R2 r, R1r, R0r, rr. When the target cells were not papain treated and mononuclear cell effectors were used, the amount of lysis was low and the % specific lysis for Rh0 (D) homozygote and heterozygote HRBC was equivalent. Lymphocyteenriched effector cells did not lyse the homozygous target cells to a greater degree than the heterozygotes, although the amount of lysis for both types of HRBC was greater than that noted using mononuclear cells as effectors.

Immunological studies in chronic lymphocytic leukemia—II. Natural killer- and antibody-dependent cellular cytotoxicity potentials of malignant and non-malignant lymphocyte subsets and the effect of α-interferon

Malignant and non-malignant lymphocyte subsets from untreated B- CLL patients were isolated by sequential rosette sedimentation procedures and tested, together with unfractionated lymphocytes, in the natural killer ( NK) and antibody dependent cellular cytotoxicity (ADCC) assays. Though addition of α-interferon (α- IFN) enhanced the NK potential in all patients, unfractionated lymphocytes alone exhibited grossly defective effector capacities in both assays when compared to normal donors. Whereas the malignant mouse rosette-forming cells were devoid of both NK and ADCC function, the non-malignant T-cells, though by themselves deficient in NK function, could be boosted to subnormal levels by addition of α- IFN. Likewise, a negatively selected Null cell fraction was also shown to exhibit considerable NK and ADCC function. Addition of α- IFN to ADCC cultures had no effect on the amount of lysis. NK experiments with a panel of target cells with varying NK sensitivity showed that B- CLL patients after the addition of α- IFN were able to lyse even the resistant DAUDI cell line. These findings, which indicate a strong regulatory role of α- IFN on the NK function of non-malignant lymphocytes in CLL, are discussed in relation to our present knowledge concerning NK cells in normal donors.

Early Steps in Specific Tumor Cell Lysis by Sensitized Mouse T Lymphocytes

Abstract It has previously been shown by several investigators that Mg++ in the absence of appreciable Ca++ is sufficient to support the formation of specific adhesions between cytolytic T lymphocytes (CTL) and tumor target cells but that programming of the targets for lysis is strongly Ca++ dependent. However, we recently found that Ca++, in the presence of suboptimal concentrations of Mg++, is capable of synergizing with Mg++ in supporting adhesion formation. The latter observation raised the possibility that the structure of the adhesion in the absence of Ca++ might be unphysiologic and incompatible with programming for lysis. Thus it was conceivable that the role of Ca++ in programming for lysis might be limited to its demonstrated role in adhesion. In the present report we have examined the abilities of other alkaline earth metals to substitute for calcium in supporting adhesion formation and programming for lysis. In the presence of Mg++ at a concentration optimal to support adhesion, Sr++ could replace Ca++ in programming for lysis. The concentration of Sr++ required for maximum cytolysis was 5- to 20-fold greater than the optimum Ca++ concentration; nevertheless when Sr++ was present at an optimum concentration, the amount of lysis was identical to that observed in the presence of an optimum concentration of Ca++. In contrast to its ability to replace Ca++ in programming for lysis, Sr++ could not substitute for Ca++ in potentiating adhesion formation in the presence of suboptimal Mg++. Ba++ could not substitute for Ca++ in either adhesion formation or programming for lysis but inhibited both in the presence of Ca++. Likewise, high concentrations of Mg++ in the absence of Ca++ did not enhance programming for lysis. The ability of Sr++ to replace Ca++ in cytolysis but not in adhesion formation indicates that the role of Ca++ in programming for lysis is distinct from its demonstrated role in adhesion and the calcium is required only at the former site. It is possible that the role of Ca++ in programming for lysis is to act as a co-factor essential to the activity of a CTL surface protein involved in the lytic mechanism. Alternatively, Ca++ influxes into CTL may trigger activation of the lytic mechanism in a manner analogous to the stimulus-secretion coupling seen in many secretory phenomena. The observation that the order of efficacy of alkaline earth metals is supporting programming for lysis is Ca++ τ Sr++ τ Ba++ or Mg++ is compatible with either possibility.

Capacity of human oral leucocytes to mediate antibody-dependent cell-mediated cytotoxicity

The ability of human oral leucocytes to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) was studied using a 51chromium ( 51Cr)-release assay. Oral leucocyte or peripheral blood leucocyte effector cells were reacted with antiserum-treated 51Cr-labelled murine lymphoma target cells and 51Cr release was measured. Morphologically-comparable, neutrophil-rich leucocyte populations from either peripheral blood or the mouth were capable of mediating ADCC, the difference in capacity of the 2 groups being reflected by the amount of lysis seen at different effector cell:target cell ratios. The ADCC mediating property of leucocytes may help to explain immune injury in oral tissue.

Editorial: News on thromboembolism from London.

The British Journal of Surgery for March 1974 contained two noteworthy reports on thromboembolism. Gordon-Smith et al 1 have put to clinical use a test for fibrinolytic activity that measures the amount of isotope liberated in three hours from a clot that they tagged by first injecting the patient with 125 I-fibrinogen. This method gives a more accurate figure for fibrin digested than tests measuring the time necessary to achieve a given amount of lysis. It has importance in postoperative measurement since the higher levels of fibrinogen normally present at that time can obscure the other types of tests. The authors found agreement with the observation of others of increased activity during operation, but did not confirm a substantial decrease in fibrinolytic activity in the subsequent early postoperative days that could be appreciated by conventional statistical methods. When they looked more closely at the first postoperative day by special statistical

Mechanisms of Increased Susceptibility to C Lysis of Abnormal Human Red Blood Cells

Abstract The cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) are abnormally susceptible to lysis by C and cold-reactive antibodies, but for different reasons. For PNH cells, the fixation of antibody, C1 and C4 is the same as for normal cells. However, the amount of C3 fixed/C4 is increased 3- to 7-fold. The amount of C3 fixed for a given degree of lysis is about one-third that of normal cells. In a less sensitive variant of PNH cells, the amount of C3 fixed/C4 was about the same as for the markedly abnormal cells, but the amount of C3 fixed for a given amount of lysis was the same as for normal cells. Normal cells treated with sulfhydryl-reactive agents resembled PNH cells in these reactions. HEMPAS cells fix 2 to 3 times as much of some, but not other, examples of anti-I. The amount of C1 fixed/Ab is normal.

Detection of Bound C3 by a New Immunochemical Method

Abstract C3 bound to sheep erythrocytes (EC3) reacts with anti-C3 antibody. The C3-anti-C3 complex fixes complement and can be detected quantitatively by either 1) the C1 fixation and transfer test or 2) the amount of lysis which occurs when whole complement is added to the erythrocyte-C3-anti-C3 complex. The hemolytic activity of anti-C3 antibody is inhibited by fluid phase C3 or by C3 bound to immune complexes. We have devised a quantitative test for C3 antigen based on the inhibition of anti-C3 antibody by C3. The C3 to be measured (in either bound or fluid phase form) is added to a standard amount of anti-C3 antibody. Residual antibody activity is then determined by adding the absorbed antiserum to sheep EC3 indicator cells and measuring lysis of the cells which occurs after addition of whole guinea pig complement. By comparing the observed lysis with an inhibition-of-lysis curve obtained with known C3 antigen, the amount of test antigen can be estimated. The method is simple, rapid and reproducible and can detect nanogram amounts of bound C3.