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Shore-based live holding is a required step for post-harvest economic success in the American lobster (Homarus americanus H. Milne Edwards, 1837) fishery. This study evaluated and quantified nutritional condition and gene expression of hepatopancreas during live storage of American lobsters harvested from inshore and offshore locations in Nova Scotia, Canada in early winter. Crude-fat percentage of lobster hepatopancreas and plasma biochemical indicators in haemolymph were used to evaluate the nutritional status of lobsters during live storage. Hepatopancreas crude-fat levels were significantly higher in lobsters from inshore in contrast to offshore locations. Similarly, inshore lobsters were easily distinguished from offshore lobsters because of higher haemolymph Brix and plasma triglyceride, cholesterol, and total protein concentrations. Elevated plasma aspartate aminotransferase activity in inshore lobsters when compared to offshore lobsters suggests that handling practices in the inshore fishery have more impact on harvested lobsters than offshore fishing practices. Inshore lobsters were nevertheless able to recover by four weeks in storage with no obvious mid- and long-term effects. Microarray analysis of hepatopancreas tissue resulted in gene expression profiles that routinely separated inshore from offshore lobsters at all storage time points. The biochemistry of haemolymph and the crude-fat and molecular gene expression analysis of hepatopancreas proved to be useful tools for differentiating lobster harvesting locations. Lobsters harvested inshore in January and stored at the wharf for one month in contrast to lobsters harvested fresh in February had similar gene expression and biochemical profiles; indicating that there is no disadvantage to the livestorage of lobsters caught early in the winter, compared to fresh caught lobsters a month later. [ABSTRACT FROM AUTHOR]

Ethanolic extracts of mycelia from Aspergillus niger (strain N402) grown in liquid media were observed to have haemolytic activity on bovine erythrocytes. This haemolytic activity decreased significantly during the time of growth (1-3 days). Moreover, when A. niger was grown on carbon-deprived medium, the efficiency of this haemolytic activity in the ethanolic extracts was much lower than when grown in carbon-enriched medium, and became almost undetectable after 3 days of growth in carbon-deprived medium. The lipid composition of these ethanolic extracts was analysed by liquid chromatography-electrospray ionisation tandem mass spectrometry. This haemolytic activity can be mainly linked to the relative levels of the molar ratios of the unsaturated fatty acids and lysophosphatidylcholines. [ABSTRACT FROM AUTHOR]

SUMMARY Objective To determine if column agglutination technology ( CAT) for titration of anti-D and anti-c concentrations produces comparable results to those obtained by continuous flow analyser ( CFA). Background Anti-D and anti-c are the two commonest antibodies that contribute to serious haemolytic disease of the foetus and neonate ( HDFN). Current practice in the UK is to monitor these antibodies by CFA quantification, which is considered more reproducible and less subjective than manual titration by tube IAT (indirect antiglobulin test). CAT is widely used in transfusion laboratory practice and provides a more objective endpoint than tube technique. Materials and methods Antenatal samples were (i) quantified using CFA and (ii) titrated using CAT with the reaction strength recorded by a card reader and expressed as a titre score ( TS). Results The TS rose in accordance with levels measured by quantification and was able to distinguish antibody levels above and below the threshold of clinical significance. Conclusion CAT titre scores provided a simple and reproducible method to monitor anti-D and anti-c levels. The method was sensitive to a wide range of antibody levels as determined by quantification. This technique may have the potential to replace CFA quantification by identifying those cases that require closer monitoring for potential HDFN. [ABSTRACT FROM AUTHOR]

The complete genome sequence of the bovine pathogen Mannheimia haemolytica A1 was analyzed byblast searches for the presence of two-component regulatory system proteins. Five complete sets of putative two-component systems were identified, and the NarQ/P system was further investigated. in silico analysis of the NarQ and NarP proteins showed features that are typical of the sensor and response regulator proteins. A narP knock-out mutant was constructed. The narP mutant has lost its ability to respond to NaNO3 in the media and fail to alter the expression of several proteins. One of the proteins that showed increased production in the parent strain in response to NaNO3 was analyzed by matrix-assisted laser desorption ionization time-of-flight MS. Unexpectedly, the protein was identified to be FbpA, a periplasmic component of the iron transporter system. Sequence analysis of the promoter region of fbpA identified motifs typical for NarP-regulated genes. The expression of the leukotoxin gene was also altered in the narP mutant as shown by Western immunoblot analysis and reverse transcription-PCR. [ABSTRACT FROM AUTHOR]

This project was conducted to investigate the comparative efficiency of competitive ELISA (cELISA), single radial haemolysis test (SRH) and countercurrent immunoelectroosmophoresis (CIEOP) for the diagnosis of Peste des Petits Ruminants (PPR) in sheep and goats. Serum samples from 198 sheep and 82 goats were collected from three different government livestock farms and all the samples were run simultaneously with three serological tests. The samples found positive for PPR antibodies through cELISA, SRH and CIEOP were 96 (34.2%), 55 (19.6%) and 67 (23.9%), respectively. Kappa statistics was used to evaluate the concordance between the laboratory-based test (cELISA) and field-based tests (SRH and CIEOP). Kappa statistics scores for cELISA vs SRH and CIEOP were 0.5851 (95% confidence interval 0.4848-0.6854) and 0.6668 (95% confidence interval 0.5733-0.7603), respectively, which indicate a "moderate" agreement between cELISA and SRH and "substantial" agreement between cELISA and CIEOP. SRH and CIEOP revealed a relative diagnostic sensitivity with cELISA of 54.1% and 64.5% and relative diagnostic specificity of 98.3% and 97.2%, respectively. The data suggested that for mass screening and control of PPR, these serological tests proved practical in the absence of cELISA since they have a high relative diagnostic specificity and a satisfactory relative diagnostic sensitivity. [ABSTRACT FROM AUTHOR]

Abstract: The in vitro effects of polycyclic aromatic hydrocarbons (PAHs) on two plasmatic immune parameters, lysozyme concentration and haemolytic alternative complement activity, of the European sea bass, Dicentrarchus labrax, were tested using field (10−7 and 10−9 mgmL−1) and high concentrations (10−3 and 10−5 mgmL−1) observed during oil spills. Peripheral blood from 105 fish was collected, centrifuged at 1200g, for 10min, at 4°C and three plasma pools, each of 35 fish, were constituted. Two oils (heavy fuel oil and light cycle oil) and 16 pure PAHs, selected on the basis of the American Environmental Protection Agency list (US EPA), were tested in vitro on the two humoral immune parameters. Only three pure PAHs (anthracene, chrysene and dibenz[a,h]anthracene) modulated lysozyme concentration. Acenaphthene, acenaphthylene, anthracene, benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, pyrene and light cycle oil modified the haemolytic alternative complement activity after 4h of incubation. This study investigates the direct effects of several PAHs on fish humoral immune functions and describes the haemolytic complement activity of fish as suitable biomarkers of oil pollution. [Copyright &y& Elsevier]

During a decade-long, high endemic situation with severe group A streptococcal disease in western Norway, a cluster of 16 patients with invasive streptococcal disease was hospitalized during a period of 11 weeks. A study including clinical characteristics and molecular epidemiology of the outbreak was initiated. Relevant clinical information was obtained from the medical records of the patients. Nine of the 16 patients had soft tissue infection, and seven of these had streptococcal toxic shock syndrome (STSS). Mortality, both overall and among those with STSS, was 25%. Streptococcal isolates from these patients were characterized by serogrouping and emm sequence typing. The emm amplicons were further characterized by sequence analysis and restriction fragment length polymorphism ( emm RFLP) analysis. The streptococci were identified as group A streptococcus (GAS) in 11 patients and group G streptococcus (GGS) in four patients. The patients with GGS infection were older than the patients with GAS infection, and all patients infected with GGS had predisposing comorbidities. Isolates from 13 patients were available for emm gene analysis and found to belong to nine different emm types. Similar differentiation was obtained with emm RFLP in GAS. Hence, the outbreak was polyclonal. Results suggestive of horizontal gene transfer and recombination between the emm genes of GAS, group C streptococcus and GGS were found in the isolates from seven patients. Such genetic recombination events suggest a possible role in pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: After we could recently demonstrate a beneficial effect of environmental enrichment on AD-like brain pathology in female TgCRND8 mice [Ambrée O, Leimer U, Herring A, Görtz N, Sachser N, Heneka MT, et al. Reduction of amyloid angiopathy and Abeta plaque burden after enriched housing in TgCRND8 mice: involvement of multiple pathways. Am J Pathol 2006;169:544–52] the present study focuses on the behavioural effects of environmental enrichment with special emphasis on learning and memory performance in this AD model. In the first experiment spontaneous exploration, locomotor activity and anxiety-related behaviour were assessed as the performance in learning tasks can be biased substantially by exploratory behavioural traits. In the second experiment spatial memory in the Barnes maze test and object recognition memory were examined. Regarding exploratory behaviour transgenic mice from standard housing condition were statistically indistinguishable from wild-type controls. Enrichment had comparable effects in both genotypes indicated by higher levels of exploration and locomotor activity. In transgenic mice the elevated plus-maze revealed less anxiety-related behaviour due to enrichment in contrast to wild-type mice that statistically did not differ in anxiety-related behaviour. Concerning learning and memory performance, cognitive deficits of standard housed transgenic mice could be demonstrated in both learning tasks. Surprisingly, in both housing conditions a significantly higher number of transgenic mice refused to explore any objects compared to wild-type mice. Furthermore, the Barnes maze test revealed deficits of the transgenic mice in spatial memory compared to wild-type mice whereas no effect of environmental enrichment was detectable. Thus environmental enrichment increased exploratory behaviour and decreased anxiety-related behaviour but could not clearly ameliorate deficits in learning and memory performance of TgCRND8 mice. [Copyright &y& Elsevier]

The incidence of both acute kidney injury (AKI, previously referred to as acute renal failure) and chronic kidney disease (CKD) is reaching epidemic proportions. In both situations, early intervention can significantly improve the prognosis. However, the paucity of early, predictive, non-invasive biomarkers has impaired our ability to institute potentially effective therapies for these common clinical conditions in a timely manner. The current status of one of the most promising novel biomarkers, namely neutrophil gelatinase-associated lipocalin (NGAL), is presented in this review. The evidence for the role of NGAL measurements in a variety of clinical situations leading to AKI (cardiac surgery, kidney transplantation, contrast nephropathy, haemolytic uraemic syndrome and in the intensive care setting) or to CKD (lupus nephritis, glomerulonephritides, obstruction, dysplasia, polycystic kidney disease, IgA nephropathy) is explored. The emerging utility of standardized clinical platforms for reliable measurement of NGAL in plasma (Triage® NGAL Device; Biosite Incorporated) and urine (ARCHITECT® analyzer; Abbott Diagnostics) is also discussed. It will be important in future studies to validate the sensitivity and specificity of NGAL concentration measurements in clinical samples from large cohorts and from multiple clinical situations. Such studies will be facilitated by the anticipated widespread availability of standardized commercial tools in the near future. [ABSTRACT FROM AUTHOR]

Phenylhydrazine (PHZ) and its derivatives were first given a medical application at the end of the 19th century but with with very little benefit. However, this compound seems to be very useful in models studying mechanisms of haemolytic anaemia. Phenylhydrazine induces a reactive oxygen species formation, peroxidation of lipids and oxidative degradation of spectrin in the membrane skeleton. PHZinduced haemolytic injury seems to be derived from oxidative alternations to red blood cell proteins. This compound can modulate immune reactions. [ABSTRACT FROM AUTHOR]

A calf thymus extract was prepared and fractionated into lipid and non-lipid fractions. The non-lipid fraction was isolated from the calf thymus extract using the Folch method. The components isolated from the non-lipid fraction were characterized by IR, NMR, biuret and HPLC method. The results of the analyses indicated the presence of peptides. The lipid fraction contained phospholipids, glycolipids and neutral lipids. The biological activity of both the isolated lipid and peptide fractions was determined by the in vivo hemolytic plaques method in Wistar rats with an involuted thymus. The peptide and phospholipid fractions of the thymus extract showed a significant increase of hemolytic plaques. The glycolipid and neutral lipid fraction failed to express a significant immunological response. [ABSTRACT FROM AUTHOR]

This study characterized larvicidal activity against the bean bug, Riptortus clavatus (Hem., Alydidae), associated with a strain of Bacillus thuringiensis serovar morrisoni (H8ab). Purified crystals and solubilized crystal proteins exhibited only low-level activities, while the supernatant of broth culture contained rapid and strong larvicidal activity. Heating at 100°C for 10 min destroyed the activity. Two extracellular vegetative proteins, with molecular masses of 40 and 45 kDa, were obtained by diethylaminoethyl (DEAE)-cellulose column chromatography from the bacterial culture fluid. Both proteins were related to the known haemolysins of Bacillus cereus, showing strong cytolytic activity against sheep erythrocytes. The bean bug-killing activity was not associated with individual proteins; however, strong activity was induced when two proteins were combined. The combined proteins were toxic to larvae in the early stage of first instar but not against larvae of later instars and adults. Larvae of the diamondbackmoth, Plutella xylostella, were not killed by these proteins. [ABSTRACT FROM AUTHOR]

The reverse hemolytic plaque assay has been used for several years to study hormone release from various endocrine cell types. The basic method utilizes a monolayer (consisting of indicator erythrocytes and the cells under study) that is fixed to the floor of an incubation chamber. Antibody directed against a peptide or protein is added to the chamber. Peptides released from the cells under study complex with the antibody and bind to protein-A on the surface of the indicator erythrocytes. The addition of complement causes the indicator cells to lyse, forming a “plaque” or zone of hemolysis surrounding the secreting cells. The size or rate of formation of these plaques can be used as indices to monitor peptide or protein release. In addition to this standard procedure, the plaque assay can be modified by using loose or unattached indicator cells and is termed the loose plaque assay (LPA). The LPA for a particular peptide can be used alone, sequentially with an assay directed toward another peptide, or repeatedly on the same cells to monitor release over time. In light of the fact that plaque assays do not compromise the function of living cells, it is possible to combine these plaque assays with other procedures such as immunocytochemistry, in situ hybridization, fluorescent microscopy, electrophysiology, and electron microscopy to explore other facets of the secretory process in conjunction with release. When taken together, the plaque assay has been quite useful in the study of endocrine cell secretion. Moreover, with the many adaptations possible, it should be particularly valuable in the future for the study of peptide release in other cell types such as neurons. [Copyright &y& Elsevier]

The authors used the reverse hemolytic plaque assay to investigate whether single retinal and optic lobe cells of juvenile and adult crayfish secrete crustacean hyperglycemic hormone (CHH) and whether the secretion rate depends on extracellular serotonin (5-HT) concentration. Nearly 25% of individual retinal and optic lobe cells of juvenile and adult organisms secrete CHH in response to KCl depolarization. In this condition, CHH secretion increased as a function of 5-HT concentration. In both cases, the dose–response curve indicates two different populations of CHH-secreting cells. Juveniles showed a higher CHH secretion index than did adult organisms, demonstrating a developmental interstage variation of CHH secretion. The authors conclude that (1) retinal CHH-secreting cells correspond to a population of retinal tapetal cells and (2) optic lobe CHH-secreting cells correspond to two subpopulations of CHH of medulla terminalis–X organ. [Copyright &y& Elsevier]

Primary Sjogren's Syndrome (SS) is an autoimmune disease characterized by dry eyes and dry mouth due to lymphocytic infiltration of lacrimal and salivary glands. Biopsies of their salivary glands provided an opportunity to characterize the phenotypic and functional properties of inflammatory site lymphocytes. We found that the salivary gland lymphocytes (SGL) of SS patients differed from the peripheral blood lymphocytes of the same patients because: a) SGL lacked lymphocytes reactive with anti-Leu-7 and anti-Leu-11 monoclonal antibodies; b) SGL lacked natural killer (NK) activity; and c) SGL lacked the ability to suppress polyclonal B cell responses in the presence of complement fragment C3a, a function that requires the presence of Leu-7+ cells. These studies also showed that the SGL of SS patients differed from tonsillar lymph node (LN) lymphocytes of immunologically normal individuals because tonsillar LN contained Leu-7+ T cells, and tonsillar LN could suppress polyclonal B cell responses in the presence of the complement fragment C3a. The absence of this regulatory subset in the salivary glands of SS patients may contribute to pathogenesis, because these cells may be important in the suppression of polyclonal antibody synthesis and in the elimination of neoplastic or viral infected cells.

Vaccination and administration of monoclonal antibody cocktails are effective tools to control the progression of infectious diseases and to terminate pandemics such as COVID-19. However, the emergence of SARS-CoV-2 mutants with enhanced transmissibility and altered antigenicity requires broad-spectrum therapies. Here we developed a panel of SARS-CoV-2 specific mouse monoclonal antibodies (mAbs), and characterized them based on ELISA, Western immunoblot, isotyping, and virus neutralization. Six neutralizing mAbs that exhibited high-affinity binding to SARS-CoV-2 spike protein were identified, and their amino acid sequences were determined by mass spectrometry. Functional assays confirmed that three mAbs, F461G11, F461G15, and F461G16 neutralized four variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta) These mAbs are promising candidates for COVID-19 therapy, and understanding their interactions with virus spike protein should support further vaccine and antibody development., (Copyright © 2021. Published by Elsevier B.V.)

The prospect of utilizing the cichlid teleost tilapia ( Oreochromis niloticus ) as an alternative experimental model to mammals for preliminary chemical immunotoxicity risk assessment is being evaluated by examining the National Toxicology Program's standard battery of rodent immunotoxicity assays in chemicaltreated tilapia. The present report examines the hemolytic plaque forming cell assay (PFC), a quantitative indicator of antibody production, in tilapia exposed to the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P). Reduced antibody production against sheep red blood cell (SRBC) antigen in response to B\[a]P was observed using the PFC assay, via reduction in plaque number. Under specific immunization circumstances, increased plaque formation was observed in chemical-exposed fish, an effect also reported in rodents. Although the normal teleost immune responsiveness was weaker than seen with mice under comparable conditions (presumably due to differences in antibody structure of teleosts), tilapia werefound toexhibit well-defined primary and secondary humoral responses to SRBC, and an immunotoxic response to B[a]P comparable to the rodent model. [ABSTRACT FROM AUTHOR]

The reverse haemolytic plaque assay was developed to measure antigen (AgE or rye grass I)-specific IgE-plaque-forming cells from the peripheral-blood lymphocytes of ragweed- and grass-allergic patients. The anti-IgE-developing antisera was shown to be isotype-specific, and the response of the assay was inhibited by 52% by the addition of 10 pg of antigen. In addition, the assay was shown to have a reproducibility (s.d.) of 15%. The blood lymphocytes from all fifteen atopic (grass and ragweed) patients were shown to form antigen-specific IgE-plaque-forming cells during the pollen season (mean value 115 cells) and up to 6 months after the season (mean value 56 cells). Cycloheximide appears to block the formation of the plaque-forming cells. This method appears to be sensitive and reproducible enough to study in vitro IgE antibody synthesis of peripheral-blood lymphocytes from atopics. [ABSTRACT FROM AUTHOR]

In 50 old donors antibody synthesis has been detected using a protein A haemolytic plaque assay. Data provide evidence that the plaque forming cell (PFC) capacity of aged peripheral mononuclear cells (PBMC) is significantly depressed (0.01 > P > 0.001) in comparison to controls. Additionally, suppression is mediated by adherent cells, since monocyte depleted lymphocytes regain the ability of generating spontaneous plaques. The inhibitory effect of monocytes seems to be prostaglandin-dependent, since indomethacin pre-treated PBMC give rise to a normal number of plaques when compared to young mononuclear cells. [ABSTRACT FROM AUTHOR]

Interactions between heparin and protamine in normal human serum previously were found to activate the primary complement pathway and deplete the early-acting complement components. In the present investigation, the role of heparin-protamine complexes, immunoglobulins, C1 q and C1-1NH in this interaction was studied. Heparin and protamine, like antibody and antigen, were found to combine in multiple proportions; those proportions which resulted in optimal precipitation also induced maximal C consumption. Complexes formed in serum contained C1 q and IgG, but the amount of C1 q was insufficient to account for the total depletion of C1 haemolytic activity, and immunoglobulins were not required for activation of complement to occur: maxima! consumption was observed in immunoabsorbed hypogammaglobulinaemic sera in which IgG, IgM and IgA were undetectable (<5, <10 and <7 7mu;g/mI, respectively). Purified Cl q aggregated heparin-protamine complexes formed or forming in the presence or absence of serum. The ability to induce maximal C consumption was rapidly lost as heparin and protamine interacted, and complexes preformed either in serum or buffer, or supernates thereof, were relatively ineffective in complement consumption. Thus, the consumption of complement seems to occur via transient reactivity with Cl, probably at the level of C1 q. It seems to be limited by the ability of free heparin to potentiate the activity of Cl-INH, and perhaps also by its direct effect upon the early acting C components. These experiments support the concept that complement activation by interactions between polyelectrolytes such as heparin and protamine, like interactions between antibodies and antigens, may have a role in the initiation of inflammatory reactions by direct activation of the complement system. [ABSTRACT FROM AUTHOR]

Cultures of spleen cells from Swiss, C57B1 or DBA/2 mice stimulated with 2,4,6-trinitrophenyl (TNP) conjugated sheep erythrocytes (SRBC) were used for studying the in vitro responses to TNP and native SRBC antigens and the frequency of occurrence of cells responding to both antigens. The response was revealed by plating the cultured cells with both native SRBC and TNP-conjugated pigeon erythrocytes (TNP-PRBC). Specific responses were obtained in all the cultures. Bispecific haemolytic plaque-forming cells (PFC) were detected in almost all cultures of individual Swiss mice cells with a frequency of 2.5-14 PFC/106 cells recovered. In DBA/ 2 cell cultures bispecific PFC were found in half the cultures (2.5-8.3/106 cells) and in C57B1 cell cultures in 30 per cent of the cultures (7-21/106 cells) When cells from individual Swiss mice immunized in vivo with TNP-SRBC were mixed as an allogeneic culture from the 2nd day after immunization in the presence of TNP-SRBC, the frequency of bispecific PFC increased from 8 to 30/106 cells. Mixed allogeneic cultures of normal C57B1 and DBA/2 cells yielded high specific responses with regular occurrence of bispecific. PFC only when the numbers of cells cultured together was small. However, when allogeneic cells were mixed 24 hours after starting the cultures, all responses were stimulated and bispecific PFC were found in considerable numbers (4-33/106 cells). Cross-reactivity between TNP-PRBC and native SRBC antigens was studied by culturing cells with each of the antigens and plating the cells with both, or by immunizing in vivo with SRBC or PRBC and culturing the cells with both antigens from the 2nd to the 5th day after immunization with both antigens. In no instance did bispecific PFC exceed background levels (0.1-0.6/106 cells) in these control experiments.

Describes a reverse hemolytic plaque assay to measure the release of atrial natriuretic peptide (ANP) from individual rat atrial cardiocytes. Determination of proportion of cardiomycytes secreting ANP; Determination of proportion of ANP-synthesizing and secreting cells; Time course analysis; Effects of dexamethasone exposure.

The effect of ultraviolet-inactivated feline leukaemia virus (UV-FeLV) on the development of feline immunoglobulin-secreting cells (ISC) was investigated using a reverse haemolytic plaque assay. Low concentrations of UV-FeLV at 2× 10-4 to 2.0 μg/ml stimulated the production of ISC. By contrast, the same concentration of UV-FcLV suppressed the development of pokeweed mitogen (PWM)-drivcn ISC. Maximum suppression of ISC occurred al 50/J g/ml of UV-FeLV. The generation of an interferon resistant to acid, heat and sodium dodecyt sulphate (SDS) in the media of lymphocyte cultures incubated with PWM was also significantly suppressed in the presence of 0'2 /ig/ ml of UV-FeLV. These findings suggest that non-infectious viral particles appear to modify feline immunoglobulin and interferon-secreting systems. [ABSTRACT FROM AUTHOR]

Investigates effects of lifelong dietary restriction (DR) on growth hormone (GH) immunoreactivity and secretory function of somatotropes. Lifelong DR modulates somatotropes response to GH-releasing hormone (GHRH) using reverse hemolytic plaque assay (RHPA); Hypothalamic release and synthesis of GHRH; Major action of DR on somatotropes.

The effects of D-penicillamine (D-Pen) on the production of immunoglobulins (Ig) and anti-microbial antibodies (Ab) by human mononuclear cells (MNC) cultured in vitro were analyzed by hemolytic plaque forming cell (PFC) assays. Polyclonal Ig and Ab production, induced by pokeweed mitogen (PWM), was not affected by D-Pen in pharmacologically relevant concentrations, unless Cu2+ was added. Likewise, D-Pen + Cu2+, but not D-Pen alone, affected Polyclonal Ig production induced by Epstein-Barr virus. The production of interleukin-2 and B cell growth factor by phytohaemagglutinin-stimulated T cells was not inhibited by D-Pen; again D-Pen + Cu2+ markedly reduced the production of these cytokines. By contrast, D-Pen significantly reduced the antigen-induced antibody production without requirement of additional Cu2+. Addition of cytokine-containing supernatant to MNC treated with D-Pen + Cu2+ tended to increase the PWM-induced Ig responses, but had no effect on the antigen-induced Ab production of MNC cultured with D-Pen. Thus, the mechanisms by which D-Pen suppresses Polyclonal and antigen-induced B lymphocyte responses are different. [ABSTRACT FROM AUTHOR]

The influence of sodium aurotbiomalate on the secretion of immunoglobulins by normal human lymphocytes in intro was investigated by means of a reverse hemolytic plaque forming cell (PFC) assay, .Aurothiomalate inhibited the PFC response induced by pokeweed mitogen (PWM) and by Epstein-Barr virus (EBV) in a dose dependent manner. The inhibition was irreversible, as pre-incubation for 2 h with the drug followed by extensive washing and further culture in gold salt-free medium still caused an inhibition of the PFC response to PWM and to EBV. Cell proliferation was not significantly affected, suggesting that the inhibition of PFC formation was not due to cytotoxicity. Pre-incubation of monocytes/macrophages (Møs). T lymphocytes and B lymphocytes with the gold compound prior to culture with PWM showed that Mø's and B cells were highly sensitive, whereas T lymphocytes were resistant to the drug. The findings indicate that aurothiomalate inhibits the polyclonally induced PFC response by interfering with accessory Mø function and by affecting the B lymphocyte itself. [ABSTRACT FROM AUTHOR]

We investigated the susceptibility to human complement (C) of xeno-erythrocytes into which phosphatidylinositol (Pl)-anchored human C regulatory protein, decay-accelerating factor (DAF) or CD59 had been incorporated. Erythrocytes of sheep (Esh), swine (Esw), dog (Edg), and guinea pig (Egp), unsensitized with human natural antibody (Ab), were used as xeno-target. C-mediated lysis of erythrocytes (E) was induced in both classical and alternative pathways in parallel with the density of the sensitized Ab, except for Egp. The efficacy of DAF/CD59-mediated protection of the xeno E from human C, however, differed among these E species. In both classical and alternative pathways, Esh or Esw, which are non-activator surfaces, were protected by the incorporated DAF or CD59, DAF being more effective than CD59. On the other hand, CD59 was more effective than DAF in both pathways in protection of Egp, which is an alternative pathway activator. To elucidate this different behaviour of DAF and CD59, C3 step inhibition by the incorporated DAF or CD59 was measured. DAF was effective in the suppression of classical pathway-mediated C3 deposition in Esh, Esw and Egp, but not in Edg, while CD59 exhibited negligible effects in this regard. Next, inhibition of the lysis by CD59 was tested by haemolytic assay. CD59 did not block the C5b-8- mediated lysis in any xeno E. It also barely blocked C5b-9-mediated lysis, except in the case of Egp, in which CD59 partly blocked C9 attack. Membrane constituents on targets other than the incorporated complement inhibitors may be a crucial factor in the induction of cytolysis and, presumably, in hyperacute rejection. [ABSTRACT FROM AUTHOR]

A sensitive reverse haemolytic plaque assay was used on freshly isolated blood cells from normal human subjects to show that T lymphocytes and monocytes were both necessary for immunoglobulin production by unstimulated B cells cultured only for the time necessary to form plaques. When lymphocyte preparations were fully depleted of T cells by E-rosette formation overnight on ice followed by Ficoll-Hypaque centrifugation, the number of plaque-forming cells was reduced by up to 94% this reduction was reversed by the replacement of T cells, although excess T cells suppressed plaque formation. Moreover, when T-cell function was blocked by 10–1000 ng of two monoclonal anti-T-cell antibodies, OKT3 or UCHT1. this significantly reduced or abolished spontaneous IgG plaque, and higher concentrations of either OKT3 or UCHT1 reduced the numbers of IgA and IgM plaques formed by B cells. The role of monocytes in spontaneous plaque formation was investigated. The removal of plastic-adherent cells from mononuclear cell preparations did not consistently result in a reduction in the numbers of plaques, but complement-mediated lysis of monocytes with either of two monoclonal antibodies with specificity for rnonocytes, OKM1 and FMC1T, reduced by 50% the number of IgG, IgA and IgM plaques. This effect was reversed by addition of as few as 1% plastic-adherent cells. Decreased plaque formation by B cells, resulting from either blocking of T-cell function with monoclonal antibody or complement-mediated lysis of monocytes, or both, was fully reversed by soluble factors present in cell-free conditioned medium from lectin-activated T cells. Thus spontaneous plaque formation by human peripheral blood B cells requires T cells and a small number of monocytes, and the major function of these cells is to help B cells by the production of soluble factors. [ABSTRACT FROM AUTHOR]

In a haemolytic plaque assay staphylococcal strain Cowan 1 was shown to induce polyclonal antibody secretion In human blood lymphocytes, whereas Haemophilus influenzac and Escherichia coli gave low responses. Diplococcus pneumoniae and haemoiytic streptococci generally did not activate blood cells. All five bacteria could activate spleen, tonsil and adenoid cells both to polyclonal Ig secretion and increased DNA synthesis. Thus blood cell reactivity does not necessarily reflect the response pattern in other lymphatic organs. The adenoid was shown to contain lymphocytes more responsive to bacteria normally residing in nasopharynx than cells residing in other lymphatic organs. On the other hand, spleen and mesenteric lymph node contain a subpopulation of cells highly responsive to bacteria such as Escherichia coli normally residing in the bowel. Therefore, we conclude that there exists a functional compartmentalization of lymphocytes in distinct secondary lymphoid organs. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 70-year-old woman with autoimmune haemolytic anaemia. She was successfully treated with intravenous rituximab and she was presented with bilateral and symmetrically distributed well- defined erythematous plaques. The article discusses the Erythema annulare centrifugum, presenting with erythematous macules and papules with central clearing.

The article offers information on the thrombotic thrombocytopenic purpura (TTP) as a life-threatening syndrome with microangiopathic haemolytic anaemia (MAHA), thrombocytopenia and renal impairment symptoms. It mentions that immunosuppression is used as daily therapeutic plasma exchange (TPE) to decrease autoantibody production .

Total haemolytic complement was reduced in blister fluids of four pemphigus vulgaris patients when compared to serum complement levels and other serum and blister fluid proteins. Complement levels in most control blister fluids, on the other hand, more closely approached their corresponding serum levels. Haemolytic C1, C4, C2, C3 and C5, measured in two pemphigus sera and blister fluids, were not measurable in one blister fluid and were extremely low in the second patient. C3 proactivator (C3PA) was absent from both of these blister fluids. Three of the blister fluids exhibited anti-complementary activity when tested with normal human serum. By adding one blister fluid to normal human serum, inhibition of haemolytic C1, C2. C3 and C5 with conversion of C3 and C3PA occurred. Activation of complement locally in pemphigus blister fluids would suggest a pathogenetic role for complement in this disease. [ABSTRACT FROM AUTHOR]

Incorporation of rabbit anti-μ serum into agarose gels inhibited direct anti-SRBC PFCs using chicken complement, but developed equal numbers of indirect PFCs binding guinea-pig complement. The majority of attempts to demonstrate direct PFCs against HSA were unsuccessful, although low numbers of barely discernible PFCs were obtained by using HSA-SRBC sensitized with carbodiimide at room temperature; indirect PFCs were developed, however, by the anti-p serum. During the primary response, developed anti-HSA PFCs could not be assigned to IgM or IgG classes using reduction and alkylation. Concanavalin A completely inhibited direct anti-SRBC and Sill PFCs, although developed γM PFCs were only inhibited partially. The identity of direct and anti-: developed PFCs against Sill was demonstrated by the replica technique. None of the approaches applied provided any support for the existence of structural heterogeneity of IgM within the PFC population. [ABSTRACT FROM AUTHOR]

The concept that antigen has a continuous role in the recruitment and differentiation of immune progenitor cells was tested with optimum and suboptimum doses of heterologous erythrocytes in mice. These studies further evaluated an immune cell maturation scheme in which continuous antigenic stimulation is required for both the recruitment of ‘antigen-sensitive units’ and the expansive proliferation of a distinct sensitized cell compartment, which undergoes irreversible differentiation to functional antibody-forming cells. Haemolytic plaque-forming cell capacity during both the primary and secondary immune reactions were studied, both in the intact animal and with the spleen cell transfer technique. This in vivo culture technique was used to measure the sensitized cell compartment in the absence of existing antibody regulatory mechanisms. The results clearly demonstrate a higher detectable secondary immune capacity in the suboptimum antigen dose group than in the optimum antigen dose group. This was demonstrated for both the 19S and 7S cellular responses, as well as with humoral antibody levels measured in the spleen cell recipient mice. It can be concluded that in the presence of a suboptimum dose of antigen, which rapidly diminishes during the early intervals of the primary response, there is adequate recruitment with subsequent preservation or rescue from antigen-mediated depletion of the sensitized cell compartment, at the expense of the detectable primary response. [ABSTRACT FROM AUTHOR]

The open carboxymethylcellulose (CMC) haemolytic plaque technique was used to study the antibody-forming capacity of daughter ceils arising from single plaque-forming ceils (PFC) after in vitro mitosis. Spleen cells from mice immunized 2–5 days previously with sheep red blood cells (SRBC) were used. The number of in vitro mitoses was increased by giving mice colcemid 2 hours before killing. The experimental protocol involved allowing cells to form haemolytic plaques in a liquid medium; selecting the largest available PFC; transferring them to wash droplets to await colcemid-escape and completion of mitosis; separating the two daughter ceils from each other by micromanipulation; transfer of each daughter cell pair to a CMC plaque-revealing monolayer; and continuous assessment of plaque formation rate by daughter cell pair members. The whole procedure was carried out with the micromanipulation set up at 37°. Altogether, seventeen experiments on IgM (direct)-PFC and two on IgG (enhanced)-PFC were performed. Of eighty-seven daughter cell pairs transferred, eighty-six behaved symmetrically with both daughter ceils forming antibody (seventy-four cases) or not forming antibody (twelve cases). Arguments are presented to suggest that this failure rate of 14 per cent is due to technical factors inherent in the micromanipulation steps. In the great majority of cases, daughter cells were symmetrical in size and plaque formation rate. Plaques from daughter ceils were always of similar morphology, and when mixed sheep and goat RBC monolayers were used to reveal plaques, both cells of a pair always behaved similarly in that either both formed a clear plaque or both formed a turbid plaque. The results are discussed in the light of the hypothesis of Tannenberg and Malaviya (1968) for asymmetric divisions amongst PFC, which they do not favour. It is stressed, however, that the possibility of asymmetric divisions amongst antibody- forming cell precursors is not eliminated. [ABSTRACT FROM AUTHOR]

The production of complement was investigated using a modification of Jerne's haemolytic plaque technique. Guinea-pig spleen and bone marrow cells were suspended in layers of agarose containing, as indicator cells, sensitized sheep erythrocytes (EA) or intermediate products consisting of EA and some of the components of complement. R reagents or serum, in which certain components of the C′3 complex had been inactivated, were added after incubation. No plaque formation due to release of complement components was observed. Bone marrow and spleen cells produced plaques when incubated with sensitized or non-sensitized erythrocytes and a reagent containing cobra venom. A method was developed for the preparation of thin films of agarose in which tissue cells and erythrocytes were suspended in a single cell layer. Single cells and clumps of cells present in the peritoneal exudate of normal guinea-pigs produced plaques when incubated with EA or EAC′4 and R1. Occasionally, similar clumps of cells also produced plaques with EA or EAC′1 and R4. The plaque-forming cells appeared to be macrophages. Plaques were not detected with R2 or R3. No plaques were produced by spleen cells under similar experimental conditions. Bone marrow cells produced plaques with both sensitized and non-sensitized erythrocytes after prolonged incubation. Optimal plaque formation occurred when R1 or R4 was added before incubation. Plaques were not produced if the tissue cells were pre-incubated in suspension cultures for 2 hours or longer before plating. Treatment of the peritoneal exudate cells with KCN or with dinitrophenol prevented plaque formation. [ABSTRACT FROM AUTHOR]

By means of a modified plaque assay the numbers of cells producing 19S antibody (direct PFC) and 7S antibody (developed PFC) have been studied separately. Significant and somewhat surprising differences in the responses of the two kinds of antibody-producing cell have been noted. It is clear that the route of injection of the antigen is of importance in determining the response in the spleen. The response of both types of PFC was found to be biphasic. The total increase in spleen cell number after immunization was not readily accounted for by the increase in specific PFC. [ABSTRACT FROM AUTHOR]

The article presents a study where immune haemolysis by cold agglutinins allows the reactions of the components of complement after elution of antibody from the haemolytic complex. In the experiments to be described, the human cold agglutinin has been utilized to study the haemolytic complex of trypsinized human erythrocytes and human complement, taking advantage of the ease in eluting cold agglutinins from erythrocyte-antibody-complement complexes.

The article presents a discussion related to the administration of azathioprine and the development of malignant lymphomas in NZB mice. Azathioprine in doses that made NZB mice more anaemic did not cause reversal of their positive Coombs tests. When given to young NZB mice it did not delay development of the positive state of the test. It is the purpose of this paper to report that in a controlled therapeutic trial administration of azathioprine to NZB mice did not benefit their autoimmune haemolytic anaemia.