Lysis Of Sheep Red Blood Cells Research Articles

The Species Composition of Microbiota and Biological Properties of Dominant Bacterial Species in the Lacrimal Fluid in Patients with Endogenous Uveites

The purpose: to study the species composition of the lacrimal fluid microbiota in patients with endogenous uveitis and the biological properties of dominant species of bacteria.Patients and Methods. A bacteriological study of the lacrimal fluid of 107 patients (114 eyes) with endogenous uveitis and 28 practically healthy persons (control) was carried out. The research was performed using the conventional methods. The isolated microorganisms were identified up to the species. We studied the main biological properties of bacteria — the presence of persistence and virulence factors of isolated dominant species.The results. 43 strains of various types of bacteria were isolated from the lacrimal fluid of patients with uveitis, bacteria of the genus Staphylococcus prevailed, S. aureus was isolated in 23.3 % of cases, and the remaining strains were representatives of coagulase-negative staphylococci. The species composition of the bacteria isolated from practically healthy individuals did not differ in essence. Isolated strains of S. aureus had a significant set of virulence factors. All isolates lysed sheep red blood cells, 80 % of the cultures had lecithovitellase and 60 % — DNA-se activity. Representatives of coagulase-negative staphylococci were characterized only by hemolytic activity. In the study of persistence factors, it was found that all the studied strains of S. aureus and the vast majority of representatives of coagulase-negative staphylococci isolated from tears were resistant to lysozyme. Biofilm formation was more typical for S. aureus strains 90 % versus 34.4 % for representatives of coagulase-negative staphylococci (t = 2.194, p = 0.034). Similar patterns were found in relation to complement resistance, 90 % of S. aureus cultures and 37.5 % of coagulase-negative staphylococci (t = 2.055; p = 0.046).Conclusion. Among the microorganisms isolated from the lacrimal fluid, Staphylococcus bacteria prevailed, from which S. cohnii was most often isolated. S. aureus to a greater extent than coagulase-negative staphylococci had a certain set of virulence and persistence factors. The microbiocenosis of the lacrimal fluid of patients with endogenous uveitis did not differ from practically healthy individuals. The presence of S. aureus in lacrimal fluid is a risk factor for the development of endogenous uveitis in cases of chronic oral and ENT organ diseases.

Incomplete Complement Inhibition in Patients with PNH on Eculizumab - 5 Year Experience from the National PNH Service Leeds

Background Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired disorder characterised by intravascular hemolysis and thrombosis. Patients with symptomatic PNH are commenced on the complement inhibitor eculizumab (600mg weekly for 5 weeks then 900mg 2 weekly). This monoclonal antibody targets C5 in the complement cascade, halting terminal complement activation thus inhibiting intravascular hemolysis. In some patients intravascular hemolysis is not adequately controlled on the standard regimen. Patient symptoms, transfusion requirements and raised Lactate dehydrogenase (LDH) levels are indicators for suboptimal control of PNH and review of eculizumab dosing. The 50% hemolytic complement (CH50) test is a functional assay assessing capability of serum complement components of the classical pathway to lyse sheep red blood cells pre-coated with rabbit anti-sheep red blood cell antibody. Patients with complement inhibited PNH should demonstrate absent lysis. As the test is expensive and difficult to organise, we tested if incomplete complete blockade as determined by CH50 activity would be better to confirm under-dosing than LDH value. Methods The Leeds (UK) PNH National Service reviewed patients who underwent CH50 assay between January 2015 and March 2020. All patients were on eculizumab with clinical concerns regarding suboptimal control of PNH. Patients receive eculizumab infusions intravenously every 14 days and routine follow up from the PNH Service. Serum samples were obtained 24 hours prior to infusions for CH50 assay; LDH values were routinely collected. Complete complement blockade was defined by <10% CH50 activity; intravascular hemolysis was indicated by LDH value >1.5x upper limit of normal (ULN). Confidence intervals were set at 95% and significance set at p<0.05. Results In the study period, 327 tests (median 2, range 1 - 8) were carried out in 146 patients (median age 54 years, range 16 - 89; 74 female). 81% (265) were successful; 19% (62) were unsuccessful due to processing errors. Of the successful tests, 74% (197 in 127 patients) indicated complete complement blockade and 26% (68 in 38 patients) indicated incomplete blockade. Of the patients with incomplete blockade, 68% (26) demonstrated complete blockade on repeat testing and 32% (12) had their eculizumab dose increased. Clinical symptoms of under-dosing in the 12 patients requiring a dose increase included increased transfusion requirements and/or breakthrough hemolysis (7), pregnancy (2; both returned to 900mg post pregnancy) and significant lethargy (3). Of the patients requiring a dose increase, 3 were on 1200mg before 2015; their dose was increased to 1500mg. Repeat testing was carried out in 10/12 patients after dose increase; 8 indicated complete blockade; 2 patients were incompletely blocked at 1200mg and received a further dose increase to 1500mg. Further testing indicated complete blockade in 1 patient; 1 required a 3rd dose increase to 1800mg due to incomplete blockade and ongoing transfusion requirement. Corresponding LDH values were analysed; median LDH for the complete blockade group was 1.16xULN (range 0.54 - 2.16) and 1.28xULN (range 0.76 - 2.38) for the incomplete blockade group. LDH values were not significantly higher in the incomplete blockade group compared to the complete blockade group, p=0.08. There was no significant difference in LDH values pre- and post-dose increase, p=0.38 (Figure 1); median pre-dose increase LDH 1.14xULN; median post-dose increase LDH 1.13xULN. Correlation coefficient shows that CH50 activity was positively correlated with LDH value, r(123)=0.18, p=0.04. Conclusion We report the effective utilisation of CH50 analysis where there is clinical concern of suboptimal control of PNH. All patients demonstrating hemolytic activity on CH50 assays indicated subsequent complement blockade following increase of eculizumab dose. Increasing eculizumab is costly requiring robust evidence of suboptimal complement inhibition; a positive correlation between CH50 activity and LDH values was shown however this is not sufficient to guide clinical decisions. LDH values of the incomplete blockade group were not significantly higher than those with complete blockade, suggesting the use of LDH values as an assessment of complement inhibition in patients with ongoing symptoms or transfusion requirements is not sufficient to guide eculizumab dose increases. Disclosures Munir: Alexion: Honoraria; F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Pike:Apellis: Research Funding. Arnold:Alexion Pharmaceuticals: Honoraria. Hillmen:AstraZeneca: Consultancy, Speakers Bureau; Alexion: Consultancy, Research Funding, Speakers Bureau; Acerta: Other: Financial or material support; Roche: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; Pharmacyclics: Other: Financial or material support, Research Funding; Janssen: Consultancy, Other: Financial or material support, Research Funding, Speakers Bureau; Apellis: Consultancy, Research Funding, Speakers Bureau; Gilead: Other: Financial or material support, Research Funding. Griffin:Alexion Pharmaceuticals: Honoraria, Other: Conference Support; Biocryst: Membership on an entity's Board of Directors or advisory committees.

MON-279 Correlation Between Haemolitic Complement Activity (CH50) and Growth Hormone Response to Stimulation in Adult Growth Hormone Deficiency: Preliminary Data

It is known that GH, directly or via IGF-1 activity, influences many aspects of immune response; both humoral and cellular branches are modulated, but no data are available about complement function. CH50 is a screening assay for the activation of the classical complement pathway and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). On the other hand, a low inflammation is present in GH deficiency (GHD) and it is related to cardiovascular risk of such situation, also in partial forms.Therefore, we performed a cohort study in a group of adult GHD patients evaluating CH50 levels to further explore the pattern of inflammatory markers in this condition.We included 17 patients with total GHD (GH peak after stimulation with GHRH+arginine <9 ng/ml with BMI<30 or <4 ng/ml with BMI≥30 Kg/m2; n=15) or partial GHD (peak between 9 and 16 ng/ml with BMI<30 Kg/m2 n=2) with mean±SEM BMI 27.7±2.6 kg/m2 and mean±SEM age 52.5±2.6 ys. The etiology of GHD, assessed by MRI, was: primary empty sella (n=7); post-surgical (n=1) pituitary adenoma (n=4); idiopathic (n=4), pineal cyst (n=1); CH50 has been assayed by turbidimetric method. Mean±SEM CH50 of entire cohort was 50.75±2.5 U/l (normal range 12.5-100 U/ml); however, a significant inverse correlation was observed between GH AUC and CH50 levels (Spearman r=-0.49; p=0.04).This datum joint to our previous observations about increased free light chains of immunoglobulins in GHD, substantiating the hypothesis of chronic low-grade inflammation affecting this condition, which in turn could negatively influence GH secretion. These data could be of interest especially in patients recognized as “idiopathic” GHD, where no pituitary morphologic alterations or anamnestic information (previous surgery, trauma or irradiation) were found. However, these data cannot be conclusive and further studies are needed to establish the causal relationships between these parameters, also in a greater cohort of patients.

Structure determination of CAMP factor of Mobiluncus curtisii and insights into structural dynamics

Bacterial vaginosis (BV) is a common type of vaginal inflammation caused by a proliferation of pathogenic bacteria, among which Mobiluncus curtisii. In our previous studies on M. curtisii genome, we identified the presence of a genomic fragment encoding a 25 kDa pore-forming toxin, the CAMP factor, which is known to be involved in the synergistic lysis of erythrocytes namely CAMP reaction. However, whether this hypothetical gene product has hemolytic activity is unknown. Moreover, its relative structure and function are not yet solved. Here we found that the M. curtisii CAMP factor is a monomer at pH 4.4 and oligomer at pH > 4.6. Hemolysis assays showed that M. curtisii CAMP factor could lyse sheep red blood cells efficiently in pH 5.4–7.4. Negative staining electron microscope analysis of the CAMP factor revealed ring-like structures at pH above 4.6. Additionally, the crystal structure of M. curtisii CAMP factor, determineded at 1.85 Å resolution, reveals a 5 + 3 helix motif. Further functional analysis suggested that the structural rearrangement of the N-terminal domain might be required for protein function. In conclusion, this structure-function relationship study of CAMP factor provides a new perspective of the M. curtisii role in BV development.

DETEKSI HEMAGLUTININ, HEMOLISIN DAN KOAGULASE SECARA FENOTIPIK DAN GENOTIPIK PADA Staphylococcus aureus ISOLAT ASAL BROILER

Staphylococcus aureus is a bacterium causing diseases in animals and human. Staphylococcus aureus in broilers cause septicemia, tendosinovitis, dermatitis, endocarditis, wound infections, arthritis and bumblefoot. In this research, 24 isolates of Staphylococcus aureus from broiler were characterized of its virulent factors including the presence of haemagglutinin, ability to agulate the plasma in tubular coagulase test as well presipitate formation in clumping factor test, and haemolysis types. By polymerase chain reaction (PCR) were genotypically detected genes coa, clf, hlaA, and hlaB. All of the isolates (100%) had haemagglutinin, capable to agglutinate and precipitae of rabbit plasma. All isolates could lyse sheep red blood cells with the type of α-hemolysis (45.8%), β-hemolysis (50.0%) and γ-haemolysis (4.2%). Genotypically, all isolates (100%) had coa and clf genes, hlaA gene 7(0.8%) and hlaB gene (29.2%).

Measuring the 50% Haemolytic Complement (CH&lt;sub&gt;50&lt;/sub&gt;) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540 nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Characterization of L‐cysteine desulfhydrase from Prevotella intermedia

Hydrogen sulfide is responsible for lysis of red blood cells and is a major compound for oral malodor. To clarify the production mechanism of hydrogen sulfide in Prevotella intermedia, we found an L-cysteine desulfhydrase gene (lcs) homologue on the genome database of P. intermedia ATCC25611 and characterized its gene product. The lcs gene homologue cloned into pGEX6p-1 vector was expressed in Escherichia coli and purified. Lcs activity was assayed by detection of the reaction products (hydrogen sulfide and pyruvate) or its derivatives from L-cysteine. Site-directed mutagenesis was used to convert an amino acid of the Lcs molecule. The purified lcs gene product catalysed the degradation of L-cysteine to pyruvate, ammonia, and hydrogen sulfide, indicating that the protein is L-cysteine desulfhydrase. The enzyme required pyridoxal 5'-phosphate as a cofactor, and it was highly active at pH 7.0 and completely inhibited by ZnCl(2). The K(m) and V(max) of the enzyme were 0.7 mm and 4.2 micromol/min/mg, respectively. Replacement of Tyr-59, Tyr-118, Asp-198, and Lys-233 with any of the amino acids resulted in the complete disappearance of Lcs activity, implying that these amino acids are essential for enzyme activity. In addition, hydrogen sulfide produced by this enzyme lysed sheep red blood cells and modified hemoglobin. These results show the enzymatic properties of L-cysteine desulfhydrase from P. intermedia ATCC25611 and also suggest that the Lcs enzyme, which produces hydrogen sulfide from L-cysteine, is closely associated with the pathogenesis of P. intermedia.

A novel cationic-peptide coating for the prevention of microbial colonization on contact lenses

To develop an antimicrobial peptide with broad spectrum activity against bacteria implicated in biomaterial infection of low toxicity to mammalian cells and retaining its antimicrobial activity when covalently bound to a biomaterial surface. A synthetic peptide (melimine) was produced by combining portions of the antimicrobial cationic peptides mellitin and protamine. In contrast to the parent peptide melittin which lysed sheep red blood cells at >10 microg ml(-1), melimine lysed sheep red blood cells only at concentrations >2500 microg ml(-1), well above bactericidal concentrations. Additionally, melimine was found to be stable to heat sterilization. Evaluation by electron microscopy showed that exposure of both Pseudomonas aeruginosa and Staphylococcus aureus to melimine at the minimal inhibitory concentration (MIC) produced changes in the structure of the bacterial membranes. Further, repeated passage of these bacteria in sub-MIC concentrations of melimine did not result in an increase in the MIC. Melimine was tested for its ability to reduce bacterial adhesion to contact lenses when adsorbed or covalently attached. Approximately 80% reduction in viable bacteria was seen against both P. aeruginosa and S. aureus for 500 microg per lens adsorbed melimine. Covalently linked melimine (18 +/- 4 microg per lens) showed >70% reduction of these bacteria to the lens. We have designed and tested a synthetic peptide melimine incorporating active regions of protamine and mellitin which may represent a good candidate for development as an antimicrobial coating for biomaterials. Infection associated with the use of biomaterials remains a major barrier to the long-term use of medical devices. The antimicrobial peptide melimine is an excellent candidate for development as an antimicrobial coating for such devices.

Survey for potentially necrotizing spider venoms, with special emphasis on Cheiracanthium mildei

It has proven difficult to identify those spiders which cause necrotic lesions. In an effort to design a simple, inexpensive screening method for identifying spiders with necrotizing venoms, we have examined the venom gland homogenates of a variety of spider species for their ability to cause red blood cell lysis. Those venoms which were positive were further examined for the presence of sphingomyelinase D, and their ability to evoke necrotic lesions in the skin of rabbits. Sphingomyelinase D is known to be the causative agent of necrosis and red blood cell lysis in the venom of the brown recluse spider ( Loxosceles reclusa), and our assumption was that this would be the same agent in other spider venoms as well. This did not prove to be the case. Of 45 species examined, only the venom of L. reclusa and Cheiracanthium mildei lysed sheep red blood cells. Unlike L. reclusa venom, however, C. mildei venom did not possess sphingomyelinase D nor did it cause necrotic lesions in the skin of rabbits. We present evidence suggesting that a phospholipase A 2 is the hemolytic agent in C. mildei venom.

CH50 and Its Use in the Allergist–Clinical Immunologist's Practice

The CH50 reflects the ability of serum complement to lyse sheep red blood cells (RBCs); its value is the reciprocal of the dilution of serum to lyse 50% of antibody coated sheep RBCs. Reduction of the CH50 occurs when individual complement component(s) are deficient or consumed. The CH50 may be useful for a variety of medical specialties and is most helpful diagnostically when laboratory results are interpreted in the context of the history and physical exam. The primary utility of the CH50 in the practice of an allergist-immunologist is to screen for complement-deficiency associated immunodeficiency (primarily classic or terminal complement component deficiencies) and some vasculitides (usually immune-complex induced and/or small vessel vasculitis). Absent or significantly reduced individual complement components may result in recurrent sinopulmonary bacterial infections, Neisserial meningitis, or sepsis. A reduced CH50 in this situation warrants quantification and functional assays of individual complement components The sensitivity and specificity of a reduced CH50 limit the value of using this test for vasculitis screening unless painful, persistent urticarial lesions or purpura is observed. Thus, the CH50 has definite limitations but serves as a costeffective, screening test with the appropriate clinical presentation.

Susceptibility of beta-hemolytic streptococci to nine antimicrobial agents among four medical centers in Salt Lake City, Utah, USA

A multicenter study was performed to evaluate the susceptibility of beta-hemolytic streptococci to nine antimicrobial agents. MICs were performed in cation-supplemented Mueller-Hinton broth with 3.5% lysed sheep red blood cells according to NCCLS guidelines. A total of 646 isolates were tested: 300 (46%) group A; 170 (26%) group B; 38 (6%) group C, 35 (5%) group F; 83 (17%) group G; and 20 (3%) nongroupable. Six percent of the total isolates were resistant to one or more of the antibiotics tested. Approximately 7% of 387 strains from the University of Utah Hospital and Clinics were resistant to erythromycin. Four isolates were resistant to clindamycin. Six strains (3%) from Primary Children's Medical Center (207 tested) were resistant to one or more of the macrolides. Resistance was rare at the LDS Hospital and the Salt Lake Veteran's Affairs Hospital. Overall, resistance among beta-memolytic streptococci in this geographic location does not seem to be a significant problem, except at the tertiary care university hospital.

Complement Lytic Activity Has No Role in the Pathogenesis of Autoimmune Diabetes in NOD Mice

The NOD mouse is widely used as a model of organ-specific autoimmunity because it develops specific autoimmune destruction of pancreatic beta-cells. Although it is clear that T-cells and monocytes are necessary for beta-cell destruction, humoral factors, such as antibodies and complement, may also contribute to tissue damage. Attempts to cure diabetes in experimental models by immunoisolation of transplanted islets has raised the need to protect the islets from the relatively small components of the complement cascade. In this study, we report that NOD mice have no complement lytic activity and that the exclusion of complement is unnecessary in this model. Sera from young NOD mice were unable to lyse sheep red blood cells coated with rabbit antibody. Lytic activity of NOD sera was reconstituted by mixing with C4-deficient CBA sera, but not C5-deficient DBA/2 sera, indicating the presence of C4, but the absence of C5 activity in NOD sera. Lytic activity of NOD sera could be reconstituted with human C5 electrofocused in polyacrylamide gel. The polymerase chain reaction was used to amplify fragments from genomic DNA corresponding to the region of Hc (the gene encoding C5) in DBA/2 mice, which carries a 2-base pair deletion responsible for the lack of C5 protein expression in these mice. DBA/2 and NOD mice from several colonies produced a fragment 2 bases shorter than that generated from the wild-type allele in BALB/c mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic activity of adenylate cyclase toxin from Bordetella pertussis is not required for hemolysis

Adenylate cyclase (AC) toxin from Bardetella pertussis enters cells to cause supraphysiologic increases in cAMP, AC toxin is also hemolytic. Substitution of Lys-58 with a methionine residue by site-directed mutagenesis of the structural gene for AC toxin, cyaA, and introduction of this mutation onto the B. pertussis chromosome results in an organism that synthesizes an enzyme-deficient AC toxin molecule. This mutant toxin molecule exhibits 1000-fold reduction in enzymatic activity relative to wild-type and has no toxin activity in J774 cells. The enzyme-deficient toxin molecule is not, however, impaired in its ability to lyse sheep red blood cells. In order to ascertain the importance of these two separate activities of AC toxin in vivo the enzyme-deficient organisms were used to infect infant mice. The hemolytic, enzyme-deficient mutant organisms are reduced in virulence relative to wild-type organisms after intranasal challenge indicating that, although the enzymatic activity of AC toxin does not contribute to hemolysis, it is this property of the toxin which is important for virulence of B. pertussis.

Identification of a lymphokine that stimulates eosinophil differentiation in vitro. Its relationship to interleukin 3, and functional properties of eosinophils produced in cultures.

Factors stimulating eosinophil differentiation in vitro have been studied by means of a liquid bone marrow culture system in which the number of eosinophils is estimated directly by morphology or indirectly by assay for eosinophil peroxidase. The results show that eosinophil colonies are not formed in agar, emphasizing the importance of the liquid culture system. Three types of evidence identify a novel lymphokine, eosinophil-differentiating factor (EDF). (a) Coordinate analysis of lymphokine activity in media conditioned by a panel of parasite antigen and another panel of alloantigen-reactive T cell clones indicates that EDF is distinct from interleukin 2 (IL-2), IL-3, and bone marrow proliferation activity (BMPA). (b) A T hybrid (NIMP-TH1) produces EDF but no IL-2, IL-3, interferon, or colony-stimulating factor. (c) Gel filtration of conditioned media (CM) indicates that NIMP-TH1 and a T clone (NIMP-T2) produce EDF (Mr 46,000). NIMP-T2 also produced IL-3 (Mr 26,000) but this was easily separated from EDF. IL-3 is also shown to have eosinophil differentiation activity (EDA) but this represents a very small proportion of the EDA in T2-CM. Fractionation of WEHI-3-CM indicates that EDA from this source has a similar elution profile to IL-3 (Mr 35-36,000). Furthermore, a comparison of the relative activities in purified IL-3 and WEHI-3-CM indicates that all the EDA can be attributed to the IL-3 in the latter. EDF is shown to stimulate production of eosinophils in long-term bone marrow cultures; the kinetics of eosinophil production suggests that EDF is acting on committed precursors in the bone marrow. The transient nature of eosinophil production suggests that precursors from multipotential stem cells are not produced. The eosinophils produced in these cultures are morphologically normal and functional in that they lysed sheep red blood cells coated with IgG1, IgG2a, and IgG2b, but not with IgM, IgA, or IgE. In addition, they were capable of adhering to and killing Schistosoma mansoni schistosomula.

Inside-out vesicles prepared from complement lysed sheep red blood cells.

Antibody sensitized sheep red blood cells were lysed to 68% by diluted human serum. The ghosts were shown to undergo vesiculation induced by low ionic strength buffer and gentle shearing forces like osmotically lysed ghosts. About 45% of the vesicles derived from complement lysed ghosts (as referred to vesicle protein) remained floating on top of a linear 3-18% dextran T110 gradient during ultracentrifugation. Using antibodies as probes for sideness about 90% of the floating vesicles displayed an inside-out orientation. Floating inside-out vesicles bear a slightly reduced but significant number of C9 molecules on their inverted extracellular side of the membrane as compared to the (leaky or collapsed) vesicles banding in the gradient. This finding suggests that the floating is not due to a lack of C5b-9 (under the assumption that all C9 was bound to C5b-9). It is concluded that lysis with diluted human serum may result partly in ineffective C5b-9 complexes and/or in lesions with limited permeability which do not impair vesicle floating on top of polymer gradients.

The study of antibodies and antigens dissociated from the immune complexes extracted from ovarian carcinoma ascitic fluid.

In previous studies in our laboratory, heteroantisera were produced in rabbits by immunization with extracts of ascitic fluids from ovarian epithelial carcinomas. Reverse passive hemagglutination (RPHA) assay of these sera was used for the immunodiagnosis of ovarian cancer. Antibodies attached to lysed sheep red blood cells were able to detect tumor antigens in 63% of ovarian carcinoma sera. In the present study we have shown that the antigens detected in these assays are in fact contained in circulating immune complexes and are recoverable by disassociation with 8 M urea and separation of antibody and antigen by ion exchange chromatography. The antibodies thus obtained from immune complexes (IC) were assayed by indirect immunofluorescence against a variety of paraffin-embedded tissue sections. Poorly differentiated ovarian epithelial carcinomas and papillary serous adenocarcinomas, as well as the tumors grown by implantation of the latter in nude mice, were all positively stained, while the Krukenberg tumor metastatic to the ovary, the benign ovarian cyst, the normal ovary, and the normal fallopian tube were all negative. Complete blocking of immunofluorescence staining by prior absorption of antibody with the dissociated antigen was achieved. The present study demonstrates that the antibodies and antigens dissociated from the immune complexes isolated from the ascitic fluids provide useful reagents for immunodiagnosis.

Characterization of the effects of asparaginase from escherichia coli and a glutaminase‐free asparaginase from vibrio succinogenes on specific cell‐mediated cytotoxicity

AbstractAsparaginases isolated from Escherichia coli and Erwinia carotovora are effective in the treatment of acute lymphoblastic leukemia in man. During treatment with either of these enzymes, patients frequently experience pronounced toxicity, including liver and pancreatic dysfunction and immunosuppression. The capabilities of these enzymes to hydrolyze L‐glutamine, an important amino acid in mammalian intermediary metabolism, have led investigators to suggest that the glutaminase activity may be responsible for the observed toxicities. Unlike other asparaginases, an enzyme isolated in our laboratory from Vibrio succinogenes, has been shown to be a potent antilymphoma agent and to be highly specific for L‐asparagine. Our previous work has established that the glutaminase‐free asparaginase from V. succinogenes does not suppress the in vivo humoral or cell‐mediated immune responses of mice to sheep red blood cells, whereas the E. coli enzyme abolishes these responses. In this study, we describe the mechanisms by which E. coli asparaginase suppresses specific antibody‐dependent cell‐mediated cytotoxicity. An analysis of the kinetics of appearance of the specific cell‐mediated cytotoxic response of spleen cells after immunization with sheep red blood cells revealed that it paralleled the IgG plaque‐forming cell response. In addition, 18‐h culture supernatants from immune spleen cells conferred upon non‐immune cells the ability to specifically lyse sheep red blood cells. Pretreatment of immune supernatants with protein A‐Sepharose 4B absorbed out the soluble arming factor. An analysis of E. coli asparaginase‐induced suppression of specific antibody‐dependent cell‐mediated cytotoxicity revealed that a decreased synthesis of specific IgG was responsible for the observed reduction in cytotoxic reactivity. When culture supernatants from spleen cells of immunized animals not treated with asparaginase were added to spleen cells from E. coli asparaginase‐treated mice, however, there was an increased cytotoxic response. This suggested that there was an increase in the number or ratio of effector cells for the specific antibody‐dependent cell‐mediated cytotoxic response in spleens of enzyme‐treated animals. Through the characterization of the immunosuppressive effects of two asparaginases—one having the catalytic capability to hydrolyze L‐glutamine and the other lacking this catalytic activity—we now have evidence that the immunosuppressive effects of E. coli asparaginase on specific antibody‐dependent cell‐mediated cytotoxicity cannot be the result of asparagine depletion alone. Our data demonstrate that the glutaminase‐free asparaginase from V. succinogenes is not immunosuppressive and suggest that an asparaginase with high substrate specificity for L‐asparagine may be a safer treatment for leukemia patients.

Isolation of human T lymphocytes: Comparison between nylon wool filtration and rosetting with neuraminidase (VCN) and 2-aminoethylisothiouronium bromide (AET)-treated sheep red blood cells (SRBC)

Isolation of human T lymphocytes by rosetting with SRBC, centrifugation on Ficoll-Hypaque gradients and lysis of SRBC, which reportedly affects the functional activities of lymphocytes, was compared to isolation of T lymphocytes by filtration through a nylon wool column. The two methods were similar in yields and purity of T lymphocytes. No selective loss of T cell subpopulations was found in T lymphocytes isolated by filtration through a nylon wool column as judged by the percentage of Tγ and Tμ and the suppresor and helper activity in functional assays. Therefore, filtration through a nylon wool column is a useful method to purify T lymphocytes and has the advantage of being comparatively simple and quick to perform and of avoiding manipulations which may affect the functional activity of T lymphocytes.

Tolerance to heterologous erythrocytes.

Injection of a water-soluble nonantigenic fraction obtained from lysed sheep red blood cells virtually abolishes the subsequent immune response to the red cells. The suppression is systemic and appears to be serum mediated.

The Detection of Autoantibody-Forming Cells

Haemolysis in gel was used to demonstrate the release of antibody to the basic protein of myelin from frozen sections of lymph nodes and central nervous system (CNS) of guinea pigs with experimental autoimmune encephalomyelitis (EAE). The antibody, which lysed sheep red blood cells coated with basic protein, appeared to be released from thawed lymphoid cells in the tissues, rather than to have diffused from serum, since no haemolytic foci were found in control sections from other vascular tissue, namely spleen. The haemolysis was due to the release of specific anti-basic protein antibody, since lysis was inhibited by free basic protein, and sheep red blood cells coated with an antigen unrelated to basic protein were not lysed. Anti-basic protein antibody produced locally by cells in the CNS may be an important determinant in the pathogenesis of EAE. The application of this technique of haemolysis in gel to frozen sections of brain from patients with multiple sclerosis should indicate whether the large amount of γ-globulin found, and presumably produced locally, in the CNS in multiple sclerosis has any antibody specificity for the basic protein of myelin.