Presence Of Red Cells Research Articles

A study of clinicopathological features and selected laboratory parameters in a cohort of Sri Lankan patients with lupus nephritis

Introduction: Lupus nephritis (LN) is a complication of systemic lupus erythematosus that results in significant morbidity and mortality. Several studies have shown an association between certain pathological lesions and laboratory parameters in LN.Objective: The objectives were to describe the clinicopathological features and laboratory parameters in a cohort of Sri Lankan patients with biopsy-proven LN and determine the associations between the pathological features and laboratory parameters.Method: This was a retrospective, cross-sectional study of all renal biopsies of LN reported at the Department of Pathology, Faculty of Medicine, University of Colombo from January 2017 to December 2021.Results: Eighty cases were identified. The majority were females (95%, 76/80). The mean age at biopsy was 27 ± 10.71 years. Subnephrotic range proteinuria (43.8%, 35/80) was the commonest presentation. The most frequent histological type was class IV (66.3%, 53/80) followed by class II (15%, 12/80) and class III (10%, 8/80). Serum creatinine showed a significant association with both activity and chronicity indices. Glomerulosclerosis showed an association with serum creatinine levels while cellular/fibrocellular crescents were associated with both serum creatinine levels and red cells in urine.Conclusion: In this cohort of Sri Lankan patients with LN, the serum creatinine level showed an association with activity/chronicity indices and the presence of glomerulosclerosis and crescents in the renal biopsy. The presence of red cells in urine showed an association with crescents.

Immunohistochemical Analysis of Endothelial Cells in Vascular Transformation of Lymph Node Sinuses: Vascular or Lymphatic Differentiation?

Vascular transformation of sinuses (VTS) is an uncommon and benign lesion, defined by conversion of lymph node sinuses into complex, anatomizing and endothelial-lined channels. Despite the name of VTS, which implies a change in differentiation from lymphatic to vascular endothelium, very few studies have systematically examined VTS with modern immunohistochemical markers commonly used in clinical laboratories. It is unclear whether endothelial cells in VTS display pure vascular or lymphatic differentiation, or both. A total of 11 cases with a diagnosis of VTS (identified in the tissue archives of the Cleveland Clinic between 1992 and 2015) were reviewed and confirmed. Twenty cases of benign lymph nodes without specific diagnoses were used as control tissues. Immunohistochemical stains were performed on formalin-fixed, paraffin-embedded lymph node tissue using an automated immunohistochemistry platform with antibodies against CD31, CD34, D2-40, and ERG. Positivity in the VTS lesions was defined as distinct expression in the appropriate cell compartment in ≥20% of cells. In control cases, staining was evaluated in both vascular and lymphatic channels-vascular structures were identified by presence of red cells or well-formed vascular walls and lymphatics by anatomic location and absence of vascular features. In the VTS lesions, D2-40 expression was absent in the lesional endothelial cells of 5/11 (45%) cases. In the cases lacking D240 expression, uninvolved lymphatic endothelium maintained expression. CD34 expression was also seen in 6/11 (54%), CD31 was seen in 10/11 (90%), and ERG expression was seen in all cases. In all the control cases, D240 expression was exclusively seen in lymphatic endothelial cells and not seen in vascular endothelial cells (eg, vascular channels in the hilum). CD34 was weakly positive in the lymphatic endothelium of only 7/20 (35%) control cases, but expressed in 20/20 (100%) control cases in the vascular endothelium. 20/20 (100%) of control cases showed expression of CD31 and ERG in both vascular and lymphatic endothelium. VTS lesional endothelial cells demonstrate patterns of vascular markers that show mixed blood vascular and lymphatic features. There appears to be a degree of alignment toward endothelial differentiation with decreased expression of D2-40 in some cases.

Effect of metal in Schiff bases of chitosan adsorbed on glassy carbon electrode in the inhibition of sphingomyelinase C toxin

This study was conducted to assess the catalytic electrode surface adsorption and capture properties of different metal chitosan derivatives in aqueous phosphate buffer solution (pH = 7.3). Early, recent work showed that the response of Iron chitosan complex with R = -CH3 on the periphery, over blood red cells in presence of sphingomyelinase C was protected. The effect of others substituent (R = -Br, -Cl, -F, NO2, -OCH3, -H) on the periphery of the Schiff base ligand did not show correlation with the oxidation of sphingomyelinase C and its biological response. For this reason, various adsorbed metal (M = Fe of recent work, Cu, Ni and Co) complexes of chitosan and Schiff bases on glassy carbon electrode for the oxidation of sphingomyelinase C were investigated and compared, each one with -CH3 group on the periphery of the Schiff base. UV–Vis and IR-TF spectroscopies, electrochemistry and microscopy assay were performed; then, the metal effect underlying. For the Schiff base, cobalt and copper complexes did not proved to be a remarkable cellular protector in presence of the enzyme, but the nickel complex showed to be a cellular protector at short time, this conclusion help to proposal a reaction mechanism for the electrochemical and biological studies.

Factors Involved in Maintaining Haemostasis in Patients with Myelodysplastic Syndrome.

Etiopathogenesis of myelodysplastic syndrome (MDS) might cause per se an anomalous haemostasis that can be even more deteriorated by thrombocytopaenia. So, evaluation of haemostasis in patients with MDS rises as a necessity.This work aimed to characterize haemostasis in non-bleeder MDS patients with a platelet count similar to healthy controls to establish differences between the two groups not related to thrombocytopaenia.Thromboelastometry in samples from MDS patients suggested the existence of at least two antagonistic processes: one of them giving a hypocoagulable pattern (prolonged clotting time and lower α angle) and another conferring a procoagulant profile (decreased fibrinolysis). Hypocoagulable state might be due to a decreased ability of platelets to be stimulated and to the presence in plasma of a factor/s that prolonged the time to initiate thrombin generation. This factor/s might be antibodies as this effect was observed in samples from MDS patients with an associated autoimmune-inflammatory condition.Otherwise, hypercoagulable state seemed to rely on an increased presence of red cell- and monocyte-derived microparticles and to the increased exposure of phosphatidylserine that served as scaffold for binding of coagulation factors.We concluded that haemostasis in MDS patients is a complex process influenced by more factors than platelet count.

Male with rhinosporidiosis of urethra: A case report

A 45-year old male presented with a six months history of a polypoidal lesion at external urethral meatus. There was a history of dysuria with bloody discharge from the lesion. The past and personal history was insignificant except for occasional bathing in a water pond. General physical examination and examination of nose, oral cavity and eyes was unremarkable. On local examination, a small red, fleshy, sessile lesion was seen at the external urethral orifice measuring 0.7 x 0.6 x 0.2 cm, clinically resembling a urethral caruncle. Urine routine examination showed presence of red cells. The lesion was completely resected under spinal anaesthesia. Histopathology showed stratified squamous lined tissue which reveals many sporangia containing endospore. Subepithelial region shows granulation tissue and is infiltrated with chronic inflammatory cells suggestive ofrhinosporidiosis. No other treatment was given. Patient was well after 3 months with no evidence of recurrence during follow up.

Retrospective Comparison of Clinical Characteristics and In-Hospital Outcomes among Diabetic and Non-Diabetic Adults with Acute Pyelonephritis.

Acute Pyelonephritis (APN) is a common infection in community. Diabetes Mellitus (DM) may have different effect on clinical characteristics and outcomes of APN compared to non-diabetic individuals. To compare clinical characteristics and assess outcomes of APN patients with and without DM. A retrospective analysis of 122 patients with DM (n=61) and without DM (n=61) was conducted at a single, private, urban set-up from Gujarat, India. Clinical symptoms, laboratory investigations, antibiotics treatment and outcomes in terms of mortality and prolonged hospitalization (10 days and above) were compared in two groups. Mean age was significantly higher in diabetics than non-diabetics (55.2±12.5 vs 41.5±17.3, p<0.0001) and females were proportionally higher in both groups (65.6% Vs 62.3%, p=0.706). Fever was most frequent symptom (83.6% Vs 90.2%, p=0.283) followed by nausea/vomiting (50.8% Vs 63.9%, p=0.143), dysuria (66.7% Vs 74.4%, p=0.433) and flank pain (8.2 Vs 13.1, p=0.379). Backache/back pain (47.5% Vs 29.5%, p=0.041) and Chronic Kidney Disease (CKD) (63.9% Vs 45.9%, p=0.045) were significantly higher in diabetics than non-diabetics. Mean hospital stay did not vary significantly in two groups (7.0±3.2 Vs 6.50±2.9, p=0.346) but proportion of patients with longer hospital stay was higher in DM (16.4% Vs 8.2%). Elevated white cell count, erythrocyte sedimentation rate, C-reactive protein, serum creatinine and presence of red cell in urine (> 5/ high power field [hpf]) did not vary significantly in two groups. Cephalosporin-beta-lactamase inhibitor (Cefaperazone-Sulbactam/Cefepime-Tazobactam) was the most prescribed antibiotic in both the groups. No deaths were observed in any group during this evaluation period. Only raised ESR (>30 mm/hr) {Odds Ratio (OR): 1.58, 95% Confidence Interval (CI) 1.36-1.82, p=0.004} and presence of CKD (OR: 1.71, 95% CI 1.30-2.25, p=0.008) were found to be the significant predictors of prolonged hospitalization in overall population. APN in diabetic and non-diabetic patients has similar clinical characteristics. Impact of diabetes on clinical outcomes of mortality and prolonged hospitalization warrants further investigation in a large, randomized, prospective trial.

Cross-Sectional Distributions of Platelet-Sized Particles in Blood Flow through Microchannels

In blood flow through microvessels, platelets are known to have enhanced concentrations near the vessel wall, which is the so-called “near-wall excess”. In the present study, the spatial distribution of platelet-sized polystyrene particles in red cell suspension flowing through microchannels with a square cross-section of 50 μm width was measured by a confocal laser scanning microscope. The measurements were performed at the flow rate of 1.0 μL/min for various particle diameters (d = 1.0 - 4.8 μm) and various volume fractions of red cells (hematocrit; Hct = 0 - 40 %). In the absence of red cells, the particles were uniformly distributed in the channel cross-section. For all cases studied, their distributions were also uniform at the inlet of the channel. At downstream cross-sections, high concentrations of particles near the channel wall were obtained in the presence of red cells. This tendency was enhanced for higher hematocrits and larger particles. The cross-sectional distribution of the particles revealed enhanced particle probability near the channel corners rather than near the center of the channel faces, suggesting that the particles are focused mainly close to the corners due to the square cross-section of the channel.

Hémorragie intra-alvéolaire associée à une dengue et à une leptospirose

The intra-alveolar hemorrhage syndrome is defined by the presence of red cells in the alveolar lumen and can lead to acute respiratory failure. Among the infectious etiologies of this syndrome, leptospirosis is a common cause, whereas in dengue, the intra-alveolar hemorrhage is exceptional. We report a patient aged 46 years, with no particular history, who presented a clinical picture involving acute respiratory failure, hemoptysis, bilateral alveolar images and anemia. The intra-alveolar hemorrhage has been authenticated by bronchoalveolar lavage. The etiological showed infection by both dengue and leptospirosis.

Clopidogrel anti-platelet effect: An evaluation by optical aggregometry, impedance aggregometry, and the Platelet Function Analyzer (PFA-100™)

Platelet aggregation inhibition by clopidogrel may be suboptimal in 4–30% of patients. Traditionally, optical aggregometry is used to assess clopidogrel's anti-platelet effects by inhibition of ADP-induced aggregation in platelet rich plasma. Red blood cells are an important source of ADP and, thus, are known to modulate platelet function. Because the whole blood aggregation by impedance method assesses platelet function in a physiological milieu, we compared clopidogrel response by this method with the optical method in platelet rich plasma (PRP) and the Platelet Function Analyzer (PFA-100™). Platelet function studies were performed in 17 healthy subjects at baseline and after 10 days of clopidogrel intake (75 mg/day). Optical and impedance aggregometry were performed after addition of ADP (10 and 20 µM) and collagen (1 and 2 µg/mL). For PFA-100™ analysis, whole blood closure time was measured in collagen-coated cartridges with ADP and epinephrine. All subjects except one showed a decrease in ADP-induced aggregation using both aggregation methods. However, ADP-induced platelet aggregation was significantly inhibited when assessed in whole blood as compared to the optical method (71 ± 34% vs. 34.2 ± 23%, p = 0.0002); this suggests that whole blood aggregometry is more sensitive in the detection of clopidogrel effect in the presence of red cells, which are known to modulate platelet function. The PFA-100™ ADP closure time was slightly prolonged above the reference interval in only 5/17 (29%) subjects, suggesting that this instrument is not able to detect clopidogrel effect. We conclude that whole blood aggregation appears to be more sensitive in detecting clopidogrel effect compared with the platelet rich plasma method; the PFA-100™ was unable to detect clopidogrel effect in the majority of the subjects.

Autosomal-dominant familial hematuria with retinal arteriolar tortuosity and contractures: A novel syndrome

Autosomal-dominant forms of hematuria have been mostly related to mutations in the COL4A3/COL4A4 genes. Patients with thin basement membrane (BM) disease do not have extrarenal manifestations, while those with Alport syndrome often present with hearing loss, anterior lenticonus, and dot-and-fleck retinopathy. We performed a phenotypic study and a candidate gene approach in a four-generation family presenting with autosomal-dominant hematuria associated with extrarenal manifestations. Renal biopsy was analyzed for determination of BM thickness and expression of chains of type IV collagen. Linkage to 18 candidate genes/loci was investigated using polymorphic microsatellite markers. In all affected patients, hematuria without proteinuria was associated with muscular contractures and retinal arterial tortuosities responsible for retinal hemorrhages. Cardiac arrhythmia, Raynaud phenomena, and brain MRI abnormalities were also observed. Despite the presence of red cells in tubule sections, no glomerular abnormalities were found by electron microscopy. Expression of type IV collagen chains and glomerular BM thickness was normal. We searched for a molecular defect affecting either BM or angiogenesis. Linkage analyses of genes encoding BM components (COL4A3/COL4A4, COL6A1, COL6A2, COL6A3, FBLN1), and angiogenic factors or their receptors (VHL, ANPT1, ANPT2, TIE, TEK, NOTCH2, NOTCH3, NOTCH4, DLL4, JAG1, JAG2) and of the facio-sapulo-humeral dystrophy and 3q21 loci failed to show segregation of the disease with those gene loci. We have identified a new inherited hematuria syndrome associated with retinal vessel tortuosities and contractures. We recommend performing a fundus examination in patients with familial hematuria and episodes of visual impairment, as well as a urinary analysis in patients with retinal arterial tortuosity or congenital muscular contractures.

Protein and platelet interactions with thermally denatured fibrinogen and cross-linked fibrin coated surfaces

In this work the hypothesis that a mature, cross-linked fibrin clot, pre-formed on a biomaterial, may be relatively nonthrombogenic was investigated. A cross-linked fibrin layer was formed on polyethylene which had been precoated with thermally denatured fibrinogen. Plasma protein adsorption and platelet interactions with the cross-linked fibrin and denatured fibrinogen surfaces were investigated. The adsorption of albumin, fibrinogen, and fibronectin from plasma was measured. For all three proteins, the cross-linked fibrin surface exhibited much higher levels of adsorption than either the thermally denatured fibrinogen or the polyethylene surface. Vroman peaks were observed for fibrinogen and fibronectin on polyethylene but not on the cross-linked fibrin and thermally denatured fibrinogen materials. In dilute plasma the thermally denatured fibrinogen surface showed considerable resistance to protein adsorption. However, at plasma concentrations greater than about 5% normal, this protein resistance was apparently lost. Platelet interactions (adhesion and release of granule constituents from adherent platelets) using suspensions of washed platelets in the presence of red cells were investigated at shear rates of 50, 300, and 525 s -1 using a cone and plate apparatus. The levels of platelet adhesion on the different surfaces were in the order: adsorbed fibrinogen>cross-linked fibrin>thermally denatured fibrinogen=polyethylene. Platelets on the cross-linked fibrin surface also showed high levels of release indicating significant platelet activation. Scanning electron microscopic observations were in agreement with the platelet adhesion and release data, showing only a few (but well-spread) adherent platelets on the cross-linked fibrin surface.

Factors Affecting Virus Photoinactivation by a Series of Phenothiazine Dyes

A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions. One of the dyes, 1,9-dimethyl-3-dimethylamino-7-dimethylaminophenothiazine (1,9-dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties. In order to understand why the virucidal specificity of 1,9-dimethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9-dimethyl-3-diethylamino-7-dibutylaminophenothiazine [compound 4-140], 1,9-dimethyl-3-dimethylamino-7-diethylaminophenothiazine [compound 6-136]). All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (> 0.5), but 1,9-dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB. In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2-octanol : water) ranging from 0.11 for MB to 3560 for compound 4-140. The dyes had the following affinities for DNA: 1,9-dimethylmethylene blue > compound 6-136 > MB approximately compound 4-140. This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds. Results with the most hydrophobic compound, 4-140, contrasted with those obtained with 1,9-dimethylmethylene blue. Compound 4-140 had a high affinity for protein and a low affinity for DNA. Although compound 4-140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer. However, unlike results with 1,9-dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4-140 was completely inhibited by the presence of red cells and plasma. Thus, the high affinity of 1,9-dimethylmethylene blue for DNA and the dye's efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizer's ability to inactivate viruses without adversely affecting anucleate red cells.

Inhibition of red cell aggregation prevents spontaneous echocardiographic contrast formation in human blood.

Spontaneous echocardiographic contrast (SEC) is a pattern of blood echogenicity that has been attributed to ultrasonic backscatter from blood cell aggregates that form under low shear conditions. Patients with left atrial SEC have an increased thromboembolic risk. This study examined the role of red cell and platelet aggregates in the pathogenesis of SEC in human blood and the effects on SEC of antithrombotic therapy and red cell disaggregatory agents. Blood echogenicity was examined with the use of quantitative videodensitometry over a controlled range of flow velocities in an in vitro model characterized by nonlaminar flow conditions. One hundred ninety study samples were prepared from single fresh blood donations (40 to 120 mL) from 24 healthy volunteers and 11 patients. Whole blood echogenicity was unaltered by depletion of platelets, stimulation of platelet aggregation with adenosine diphosphate, or inhibition of platelet aggregation with aspirin. Low flow-related echogenicity increased with increasing hematocrit (P<.001) but was abolished when red cells were lysed selectively with saponin (P<.001). In the presence of red cells, low flow-related echogenicity increased with increasing fibrinogen concentration (P<.001) and with plasma paraproteins. Low flow-related echogenicity in whole blood was unaltered by heparin and warfarin but was reduced in a dose-dependent manner by dextran 40 (40 mg/mL, 70% reduction, P<.001) and poloxamer 188 (8 mg/mL, 47% reduction, P<.001), which inhibited red cell aggregation. These results support protein-mediated red cell aggregation as the mechanism of SEC in human blood. Inhibition of red cell aggregation, indexed by resolution of SEC, may provide an alternative to anticoagulant and antiplatelet therapy to reduce cardiac thromboembolic risk.

In vitro addition of glutathione to blood from zinc-deficient rats corrects platelet defects: Impaired aggregation and calcium uptake

Zinc deficiency in rats impairs platelet aggregation, and this defect is associated with the failure of platelets to take up external calcium. Recent data show that the red cell plasma membrane from zinc-deficient rats contains a lower than normal concentration of protein sulfhydryl groups, a defect that can be corrected readily in vivo by zinc repletion. The purpose of this study was to determine the effect of zinc deficiency on sulfhydryl concentration of platelet membrane proteins and to determine if impaired platelet function can be corrected in vitro by treatment of blood with glutathione (GSH), a physiological reducing agent. Immature male rats were fed low zinc diets based on egg white or EDTA-treated soy protein (<1 mg/kg Zn) and the same diets supplemented with 100 mg/kg Zn (control). For membrane analysis, rats were fed soy-based diets for 3 weeks; for reversal experiments they were fed egg white-based diets for 2 weeks. Compared with controls, rats fed the low zinc diets had approximately 15% lower concentration of membrane protein sulfhydryls and their platelets exhibited impaired aggregation coupled with decreased calcium uptake when stimulated with ADP. Treatment of whole blood from deficient rats with 0.2 mM GSH restored aggregation and calcium uptake of washed platelets to control levels. The presence of red cells was essential for the reversal process in as much as treatment of platelet-rich-plasma or washed platelets with GSH had no effect. Treatment of control blood with GSH did not affect platelet aggregation, but decreased calcium uptake. The lower calcium uptake by controls suggests that a feedback mechanism in deficient platelets was not corrected by GSH treatment. The results show that selective sulfhydryl groups in platelet membranes are lost during zinc deficiency and that platelet function can be restored to normal by treatment of blood with GSH. There seems to be an association of oxidation-susceptible thiol groups with a calcium channel and that these groups are protected by normal physiological levels of zinc.

A cone-and-plate device for the investigation of platelet biomaterial interactions.

A device based on the cone-and-plate flow geometry commonly employed for viscometry was developed for the investigation of cell-surface interactions. The cone-and-plate geometry is capable of generating uniform, constant shear-rate flow fields, and control of cone rotational speed allows for easy variation of fluid shear rate. The current design is adapted for use with any material that is available in the form of a flat plate (film or coating). It also allows for replicate samples (the same or different surfaces) to be evaluated simultaneously. The device was tested under varying flow conditions for its ability to measure platelet adhesion from suspensions of washed platelets containing red cells. Collagen- and albumin-coated polymer materials were used as "standard" surfaces of known platelet reactivity (high and low, respectively). Adhesion to the collagen-coated surface was measured over a range of shear rate from 0 to 300 s(-1) and times up to 15 min. Platelet adhesion was observed to increase with increasing shear rate and time. Adhesion was significantly higher in the presence of red cells as has been observed by others. Effective platelet diffusion coefficients, calculated from the data on adhesion to the collagen surface, increased with increasing shear rate. Very little platelet adhesion to the albumin-coated surface, known to be unreactive to platelets, was observed when measured over a 15 min time period at 300 s(-1) shear rate, indicating that the device itself does not stimulate the platelets in the flow field. The data generated provide validation for this device as a simple means of measuring cell adhesion under controlled flow conditions to any smooth surface available in flat plate form.

23] Inhibition of platelet adhesion as functional test for vitamin E status

Publisher Summary This chapter describes inhibition of platelet adhesion as functional test for Vitamin E status. Implantation of substances in the form of rings of material into blood vessels provides a method of measuring the adhesion of platelets and subsequent thrombus formation in a test commonly known as the Gott ring test. Whole blood is more suitable for evaluating solely the quantitative aspect of platelet adhesion. The rate of transport of platelets to the flow chamber wall is greatly enhanced by the presence of red cells, roughly a hundred times the transport rates expected on the basis of platelet diffusivity assessed from their molecular weight. Various methods are available to quantify adherent platelets. Computer-aided analyses of the individual photomicrograph frames resulting from each experimental run are employed in the chapter. When whole blood is the perfusate, qualitative investigations are generally not possible; only quantitative measurements at given points in time can be performed.

Rescue of GATA-1-Deficient Embryonic Stem Cells by Heterologous GATA-Binding Proteins

Totipotent murine embryonic stem (ES) cells can be differentiated in vitro to form embryoid bodies (EBs) containing hematopoietic cells of multiple lineages, including erythroid cells. In vitro erythroid development parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. With the EB in vitro differentiation assay, constructs expressing heterologous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results presented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA-1), as determined by size and morphology of EBs, presence of red cells, and globin gene expression. Furthermore, GATA-3 and GATA-4, which are normally expressed in different tissues, and a protein consisting of the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 transcription activation domain were able to compensate for the GATA-1 defect. Chimeric molecules in which both zinc fingers of mGATA-1 were replaced with the zinc fingers of human GATA-3 or with the single finger of the fungal GATA factor areA, as well as a construct bearing the zinc finger region alone, displayed rescue activity. These results suggest that neither the transcription activation domains of mGATA-1 nor its zinc fingers impart erythroid cell specificity for its action in vivo. Rather, it appears that specificity is mediated through the cis-acting control regions which determine spatial and temporal expression of the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.

In vitro leukocyte adhesion to modified polyurethane surfaces: III. Effect of flow, fluid medium, and platelets on PMN adhesion

The operation of filters used to remove leukocytes from red cell concentrates may depend on the adhesion and mechanical trapping of leukocytes. If adhesion is a major component of filtration then filter materials which augment leukocyte adhesion will be useful. In previous leukocyte adhesion studies, done without flow, poly(ethyleneimene) (PEI) modified polyurethane (PU) films were shown to have greater adhesion when compared with unmodified PU. Since filtration is done under flow conditions, it was decided to study PMN adhesion at a number of flow rates using an established parallel plate flow cell. The influence of divalent cations, plasma and platelets were investigated in the presence of red cells, 40% Hematocrit. The number of adherent PMNs to the PEI modified films was always substantially higher than that for the unmodified ones when the shear rate was set at 30 s-1. When using Tyrode's solution containing albumin, with or without divalent cations, a maximum in PMN adhesion was found between the shear rates of 10 and 100 s-1. With Tyrode's solution containing albumin and with 10% (v/v) plasma in saline, the addition of platelets increased PMN adhesion when divalent cations were absent. Adhesion levels with 10% (v/v) plasma in saline were reduced when compared to Tyrode's solution containing albumin without divalent cations. These results support the use of filtration conditions where the concentration of plasma is reduced and the concentration of divalent cations is increased. Detailed evaluation of filter function with flow rate is also recommended. A cell adhesion promoting polymer coating, such as PEI, may be useful in improving filter efficiency.

A New Method For Continuous Measurement of Platelet Adhesion Under Flow Conditions

Platelet adhesion to the subendothelial tissue or biomaterial surface is an initial step in the formation of the hemostatic plug or thrombus. To clarify the mechanism of platelet adhesion in vivo, a simple and reproducible method for quantitation of platelet adhesion under flow conditions is required. In this study, a mechanical device for continuous recording of platelet adhesion under flow conditions using a quartz crystal microbalance was developed, an extremely sensitive sensor capable of measuring mass changes in the nanogram range. The relationship between surface mass change (delta W) and resonant frequency change (delta f) for pie-zoelectric crystals is given by the Sauerbrey equation: delta f = -fo2 delta W/NA pi (fo; fundamental frequency (5MHz) A; the area of quartz plate undergoing oscillation N; frequency constant for AT-cut quartz crystal pi; density of quartz crystal). Although the perfusion of buffer solution alone or addition of washed red cell suspension (Ht 30%) and formaldehyde-fixed platelets have no effect on delta f, addition of washed platelets in the presence of red cells caused an increase in delta f. To confirm that the increase in delta f results from platelet adhesion to the material surface, adherent and subsequently aggregated platelets were visualized by fluorescence microscopy using fluorescein-isothiocyanate labeled antiplatelet membrane glycoprotein Ib monoclonal antibody. There was a good correlation between the increase in delta f and the extent of platelet adhesion and subsequent aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of N-hydroxyphenetidine in phenacetin-induced hemolytic anemia

Phenacetin is well known to cause hemolytic anemia and methemoglobinemia in humans. Early mechanistic studies clearly established a causal role for active/reactive drug metabolites in the process but did not unequivocally identify these metabolite(s) or resolve the question of whether these two hemotoxicities are mechanistically linked. As part of ongoing studies on the mechanism underlying arylamine-induced hemotoxicities, we have recently shown that the arylhydroxylamine metabolites of aniline and dapsone mediate the hemolytic activity of aniline and dapsone, respectively. The present study was undertaken to determine if N-hydroxyphenetidine (PNOH), the known arylhydroxylamine metabolite of phenacetin, is responsible for phenacetin-induced hemolytic anemia. As measured by decreased survival of 51Cr-labeled erythrocytes in rats, phenacetin, p-phenetidine, and PNOH were all hemolytic in vivo, with PNOH being significantly the most potent of the three. In vitro exposure of 51Cr-tagged erythrocytes to PNOH, followed by transfusion into isologous rats, resulted in a concentration-dependent reduction in erythrocyte survival, indicating that PNOH is a directacting hemolytic agent. Phenacetin and p-phenetidine were inactive. Phenacetin, p-phenetidine, and PNOH all produced dose-dependent methemoglobinemia in rats. In parallel in vitro studies, PNOH elevated methemoglobin levels, p-phenetidine and phenacetin did not. However, attempts to identify PNOH in the blood of phenacetin- and p-phenetidine-treated rats were unsuccessful, despite the use of a highly sensitive analytical method. Hemotoxic concentrations of PNOH were found to be highly unstable in the presence of red cells, though relatively stable in the buffer vehicle alone. Inhibitors of acetylation ( p-aminobenzoic acid [PABA]) and deacetylation (bis-[ p-nitrophenyl]phosphate [BNPP]), used to alter the cyclic interconversion of phenacetin and p-phenetidine, caused changes in phenacetin hemotoxicity that indicated the hemotoxin was a deacetylated metabolite distal to p-phenetidine. These data are consistent with the hypothesis that PNOH, formed during the metabolic clearance of phenacetin, mediates phenacetin-induced hemolytic anemia and methemoglobinemia through direct toxic actions in the erythrocyte.