Human lymphoid cell lines as targets for DRw.

Cultured human lymphoid cell lines (LCL) are useful as a source of target cells in several immunologic assays. More recently such cells have been used for the serological characterizations of the HLA-DR antigens. Typing of the same LCL in various laboratories during the VII Histocompatibility Workshop has given comparable results with a discordancy rate of less than 10%. This discordancy is likely to reflect the different sources of complement that can greatly alter the results of cytotoxic assays. The presence of naturally occurring antibody in rabbit complement to human cells can be avoided by: (a) absorbing with human cells at 0 degrees C; (b) dilution with human serum; (c) dilution with heat-inactivated rabbit serum; (d) repeated freeze-thawing of the complement; or (e) careful selection of complement by screening procedures. Comparison of the results of HLA-DR typing of LCL with peripheral B-cells of the same donor show good correlations. However, LCL will occasionally give extra reactions perhaps due to the expression of new antigens. LCL can be coated with F(ab')2 fragments from antihuman beta2-microglobulin antibodies that block reactions of HLA-A, -B and -C antibodies allowing for discrimination of anti-DRw activity.     

Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8

To study the fine specificity of the HLA-D region, a panel of human T-lymphocyte clones (TLCs) was generated against alloantigens associated with HLA-DR1 through DRw8. HLA-DR-homozygous peripheral blood lymphocytes (PBLs) were stimulated with DR-heterozygous PBLs in primary mixed lymphocyte cultures for 4 days. Blasts were cloned by limiting dilution at 0.3 cells/well in the presence of 20% T-cell growth factor and irradiated stimulator cells. Viable clones were subsequently tested in proliferation assays against the original stimulator and a limited panel of stimulators bearing relevant DR specificities. Initial primings produced approximately 800 clones; some recognized DR-associated antigens, 70 recognized only their original stimulator, and approximately 50% were nonresponsive. Analysis on extended stimulator panels revealed alloantigenic complexity within similar DR-associated antigens as recognized by TLCs. The data are consistent with evidence that extreme heterogeneity exists within the HLA-D region.Abbreviations used in this paper cpm counts per minute - DNV double normalized value - EBV Epstein-Barr virus - FCS fetal calf serum - HLA human major histocompatibility complex - HTC homozygous typing cell - LCL lymphoblastoid cell line - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PLT primed lymphocyte typing - TCGF T-cell growth factor - TLC T-lymphocyte clone - T-max maximized T-test analysis     

Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates

The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)—based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galβ1-3GalNAcα- and Galβ1-3GalNAcβ-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcα-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC₅₀ values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 × 10⁻⁸ M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcβ and GalNAcβ1-3GalNAcβ ligands was found in human serum. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.     

Bacillus thuringiensisδ-Endotoxin Proteins Show a Correlation in Toxicity and Short Circuit Current Inhibition Against Helicoverpa zea

Pesticidal activity of Bacillus thuringiensisδ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A, was determined by using the force-feeding bioassay method to 4^th instar larvae of Helicoverpa zea. H. zea was susceptible to Bt toxins in the order Cry1Ac > Cry1Ab > Cry1Aa > Cry2A with 63.60, 89.04, 159.65, and 375.78 ng/larvae respectively. The abilities of selected Bacillus thuringiensis toxins to inhibit short circuit current (I_SC) in midgut epithelia of H. zea were also investigated by voltage clamp assay. The voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short circuit current (I_SC) by activated toxin. A Cry1Aa toxin dilution of 33.3 and 500 ng/ml resulted in inhibition of I_SC of −2.29 μA/min (lag time 15 min) and −4.48 μA/min (lag time, 2 min) respectively. The Cry1Ab dilution of 25 ng/ml inhibited I_SC to −1.39 μA/min, a lag time of 14 min, and 333.3 ng/ml dilution resulted in decay of I_SC−2.49 μA/min, lag time 1 min respectively. The Cry1Ac lower dilution 16.7 ng/ml inhibited I_SC to −1.39 μA/min, lag time 4 min, and a high dilution 333.3 ng/ml decay I_SC to −2.44 μA/min, lag time 1 min. The inhibition of I_SC (−1.10 μA/min, lag time 25) at lower dilution (33.3 ng/ml) and high dilution (500 ng/ml), decay (−2.38 μA/min, lag time 5 min), showed a correlation between toxin concentration and inhibitory response with Cry2A toxin. The lag time decreased with increasing concentration of toxin applied, which is additional evidence of dose response besides direct correlation of toxicity assays and I_SC. Received: 7 April 2000 / Accepted: 2 May 2000     

Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans

Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses.     

Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor

Continuous hydrogen gas evolution by self-flocculated cells of Enterobacter aerogenes, a natural isolate HU-101 and its mutant AY-2, was performed in a packed-bed reactor under glucose-limiting conditions in a minimal medium. The flocs that formed during the continuous culture were retained even when the dilution rate was increased to 0.9 h⁻¹. The H₂ production rate increased linearly with increases in the dilution rate up to 0.67 h⁻¹, giving maximum H₂ production rates of 31 and 58 mmol l⁻¹ h⁻¹ in HU-101 and AY-2 respectively, at a dilution rate of more than 0.67 h⁻¹. The molar H₂ yield from glucose in AY-2 was maintained at about 1.1 at dilution rates between 0.08 h⁻¹ and 0.67 h⁻¹, but it decreased rapidly at dilution rates more than 0.8 h⁻¹. Received: 27 August 1997 / Received revision: 11 November 1997 / Accepted: 14 December 1997     

Alloantigens expressed on a subset of unstimulated human T lymphocytes that generate suppressor function

Human alloantisera were tested for antibodies reacting with T-cell subpopulations. T-cell subsets were separated using the monoclonal antibodies OKT4 and OKT8. Five sera reacting with the T4-T8+ subset and two sera reacting with T4+T8- lymphocytes were identified. Serum Z. G. reacted with T4-T8+ cells from 8 of a panel of 19 donors. T cells treated with Z. G. serum and rabbit complement lost the capacity to generate suppressor cells but showed no decrease in the development of cytotoxic effector cells. ZG antigens were demonstrated by absorption also on monocytes but not on B cells. Their reactions on T cells were blocked by chicken anti-human la serum, but not by turkey anti-₂-microglobulin or by a monoclonal anti-human DR (L227). Studies in four informative families suggested that the ZG determinants are inherited in linkage with HLA. Although the similarities between ZG antigens and mouse I-J products are striking, structural studies are needed to establish their homology.     

A human autoreactive T cell line specific for minor histocompatibility antigen(s) isolated from a bone marrow-grafted patient

A human autoreactive T cell line named Bur-1 has been obtained from a woman 4 mo after an allogeneic bone marrow transplantation (BMT) from one of her HLA-identical brothers. The phenotype of the cell line is 100% T11+ and over 90% T4+, and the karyotype confirms its donor (male) origin. These donor T cells proliferate specifically in the presence of donor's peripheral blood monocytes (PBM) but not recipient's cells, and they kill specifically donor's but not recipient's Epstein-Barr virus (EBV)-induced lymphoblastoid cell lines (LCL). PBM from another HLA-identical brother and from several unrelated donors also stimulate Bur-1 cells, and EBV-induced LCL from the same donors are killed in cytotoxicity assays. All of these donors share HLA-DR5 or HLA-DRw11 (the major split of HLA-DR5) with Bur-1 cells. However, some but not all of the PBM sharing HLA-DR5 with Bur-1 cells are recognized. Therefore, in contrast with the previously described autoreactive T cells, Bur-1 cells are not directed against self-MHC antigens but rather recognize autologous minor histocompatibility (mH) antigens in the context of autologous HLA class II molecules. Because both male and female cells can be recognized, the reacting minor antigen could not be the male-specific HY antigen. It is suggested that autoreactivity against mH antigens can be observed in bone marrow-grafted patients due to the education of bone marrow donor precursors in the recipient thymus not allowing tolerance to autologous (donor) mH antigens not shared by the recipient.     

Some serological properties ofActinobacillus pleuropneumoniae strains of serotypes 1 through 5

Rabbits were hyperimmunized with live, formalin-killed, and heat-treated antigen preparations of the reference strains of serotypes 1 through 5 ofActinobacillus pleuropneumoniae in order to study the antibody response to both soluble and particulate antigens. The antibody response was studied by means of precipitation, agglutination, coagglutination, indirect hemagglutination, and complement fixation tests.Serotyping ofA. pleuropneumoniae strains was done by ring precipitation (RP) and coagglutination (CoA) tests with unheated and heated cell-saline extract as antigens and rabbit hyperimmune sera produced against either live cultures or formalin-killed whole-cell suspensions. The results showed that live cultures provoked more cross-reactive antibodies in rabbits, thus making the antisera unsuitable for use in serotyping by the RP test when unheated wholecell saline extract was used as antigen. Rabbit hyperimmune serum produced against formalinkilled bacterial suspension gave serotype-specific reactions in the RP test. Boiled or autoclaved cell-saline extracts gave serotype-specific reactions in the RP test even when rabbit anti-livecell sera were used. Serotype-specific reactions were obtained in the CoA test in both rabbit anti-live or anti-formalin-killed cell sera with either unheated or heated bacterial cell suspensions as antigens.Live and formalin-killed whole-cell suspensions as well as their saline extracts provoked a high antibody response in rabbits. Heating the cell suspension at 100°C for 1 h caused a significant reduction in their immunogenic potency, whereas autoclaving (121°C) of the cell suspension for 1 h almost completely destroyed their serotype-specific immunogenic properties, since the antibody response was either absent or very poor and not type-specific. However, neither boiling nor autoclaving of the cell suspensions caused significant reduction in their ability to react with preformed antibodies. Phenol-water-extracted antigens gave the highest degree of serotype specificity in the complement fixation test.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Tumour-specific MHC-class-II-restricted responses after in vitro sensitization to synthetic peptides corresponding to gp100 and Annexin II eluted from melanoma cells

In a search for potentially tumour-specific MHC-class-II-restricted antigens, the immunogenicity of endogenous peptides that had been eluted from HLA-DR molecules of the human melanoma cell line FM3 (HLA-DRB1*02x, DRB1*0401) was tested in vitro. Two 16-mers representing gp100 positions 44–59, and annexin II positions 208–223 bound well to isolated DRB1*0401 molecules and are discussed here. HLA-DR-matched normal donors' T cells were cultured with peptide-pulsed artificial antigen-presenting cells (CHO cells cotransfected with genes for HLA-DRB1*0401 and CD80 and coexpressing high levels of both human molecules). Specific sensitization was achieved against both peptides, as measured in assays of autocrine proliferation and interleukin-2 secretion. Moreover, responses to native autologous melanoma cells but not to autologous B cells were also observed. In view of the expression of fas by the activated T cells and of fas ligand by the melanoma cells, blockade of potential fas/fas-ligand interactions was undertaken using monoclonal antibodies (mAb). The antagonistic fas-specific mAb M3, but not the fas agonist M33, caused a markedly enhanced T cell response to FM3 cells. These results demonstrate that synthetic peptide antigens are able to sensitize T cells in vitro for effective MHC-class-II-restricted recognition of melanoma cells. Received: 12 April 1998 / Accepted: 23 April 1998     

HLA-DRB1 amino acid disparity is the major stimulus of interleukin-2 production by alloreactive helper T-lymphocytes

 The generation of interleukin-2 (IL-2)-mediated helper activity is a central step in the immune response induced by allogeneic histocompatibility antigens, and IL-2-producing helper T-lymphocyte precursor (HTLp) frequencies have been proposed as a measure of alloreactivity in transplant recipients. We analyzed the influence of HLA-matching on the alloresponse of HTLp in limiting dilution assays derived from healthy individuals. Mean HTLp frequencies were significantly higher in HLA-DR antigen-mismatched than HLA-DR-matched combinations. Significant differences in the effect of one or two mismatched HLA-DR antigens on mean HTLp frequencies were also detected. Mean HLA class I (HLA-A, -B, -Cw) mismatches were not significantly different in each group and had no apparent influence on HTLp frequencies. Analysis of HLA protein sequence disparities revealed no significant difference in the number of mismatched amino acid residues at the HLA-DRB1 locus between one and two HLA-DR antigen-mismatched combinations but correlated strongly with HTLp frequency. The positive correlation was evident with mismatched residues in the beta sheet and alpha helical regions of the HLA-DRB1 molecule, suggesting a predominant influence of bound peptides in the stimulation of alloreactive helper cells. This finding was supported by analysis of the location of individual residue mismatches. Evidence of an effect of polymorphism in the CD4-binding region in the β-2 domain of HLA-DRB1 molecules was also found. Our results demonstrate the major influence of HLA-DR amino acid sequence mismatching on alloreactive HTLp frequencies but also suggest that additional genetic or environmental influences affect the alloreactive helper T-cell repertoire. Received: 2 September 1997 / Revised: 29 September 1997     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.     

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

The cystine-glutamate exchanger, system x_c ⁻, mediates the Na⁺-independent exchange of cystine into cells, coupled to the efflux of intracellular glutamate. System x_c ⁻ plays a critical role in glutathione homeostasis. Early studies of brain suggested that system x_c ⁻ was present primarily in astrocytes but not neurons. More recent work indicates that certain brain neurons have an active system x_c ⁻. In the retina, system x_c ⁻ has been demonstrated in Müller and retinal pigment epithelial cells. We have recently suggested that two protein components of system x_c ⁻, xCT and 4F2hc, are present in ganglion cells of the intact retina. Here, we have used (1) molecular and immunohistochemical assays to determine whether system x_c ⁻ is present in primary ganglion cells isolated from neonatal mouse retinas and (2) functional assays to determine whether its activity is regulated by oxidative stress in a retinal ganglion cell line (RGC–5). Primary mouse ganglion cells and RGC–5 cells express xCT and 4F2hc. RGC–5 cells take up [³H]glutamate in the absence of Na⁺, and this uptake is blocked by known substrates of system x_c ⁻ (glutamate, cysteine, cystine, quisqualic acid). Treatment of RGC–5 cells with NO and reactive oxygen species donors leads to increased activity of system x_c ⁻ associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. This is the first report of system x_c ⁻ in primary retinal ganglion cells and RGC–5 cells. Oxidative stress upregulates this transport system in RGC–5 cells, and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or protein. This work was supported by National Institutes of Health grants EY014560 and EY012830.     

Alternative Ii-independent antigen-processing pathway in leukemic blasts involves TAP-dependent peptide loading of HLA class II complexes

During HLA class II synthesis in antigen-presenting cells, the invariant chain (Ii) not only stabilizes HLA class II complexes in the endoplasmic reticulum, but also mediates their transport to specialized lysosomal antigen-loading compartments termed MIICs. This study explores an alternative HLA class II presentation pathway in leukemic blasts that involves proteasome and transporter associated with antigen processing (TAP)-dependent peptide loading. Although HLA-DR did associate with Ii, Ii silencing in the human class II-associated invariant chain peptide (CLIP)-negative KG-1 myeloid leukemic cell line did not affect total and plasma membrane expression levels of HLA-DR, as determined by western blotting and flow cytometry. Since HLA-DR expression does require peptide binding, we examined the role of endogenous antigen-processing machinery in HLA-DR presentation by CLIP⁻ leukemic blasts. The suppression of proteasome and TAP function using various inhibitors resulted in decreased HLA-DR levels in both CLIP⁻ KG-1 and ME-1 blasts. Simultaneous inhibition of TAP and Ii completely down-modulated the expression of HLA-DR, demonstrating that together these molecules form the key mediators of HLA class II antigen presentation in leukemic blasts. By the use of a proteasome- and TAP-dependent pathway for HLA class II antigen presentation, CLIP⁻ leukemic blasts might be able to present a broad range of endogenous leukemia-associated peptides via HLA class II to activate leukemia-specific CD4⁺ T cells.     

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

Production of antibodies specific to human chorionic gonadotropin in mice immunized against its chemical analogs

Mice immunized against DS₅-hCG-Β and DS₆-hCG-Β, chemical analogs of Β-subunit of human choriogonadotropin (hCG-Β) in which 5 and 6 disulphide bonds respectively were reduced and alkylated, were found to produce antibodies specific to hCG without significant crossreactivity with human lutropin (hLH) as tested in a radioimmunoassay. In contrast, mice immunized against the native hCG-Β subunit produced hLH crossreacting antibodies. While the anti-DS5, DS6-hCG-Β serum was capable of selectively blocking the binding of [¹²⁵I]-hCG to rat testicular LH/hCG receptors, it failed to inhibit the binding of [₁₂₅I]-hLH to the same receptors. The radioimmunoassay for hCG using the mouse anti-DS₅, DS₆-hCG-Β serum was not as sensitive as that employing rabbit anti-DS₅, DS₆-hCG-Β serum. The minimal detection limit was 5 ng/ml for the mouse antibody as compared to 1 ng/ml for the rabbit antibody. Supported by U.S. Public Health Service Grant HD 08766 to OPB. An erratum to this article is available at .     

Microbial sensor system which usesMethylomonas sp. for the determination of methane

Summary Pseudomonas putida (ATCC 111 72) was studied in a continuous culture at various dilution rates with asparagine as the sole carbon source and limiting factor. Under the experimental conditions applied, a considerable number of the cells became attached to the fermentor walls and equipment. The viable count of the attached cells was of the same magnitude as those in suspension. The following steady-state characteristics were obtained: The cell-mass (OD₆₂₀ and dry weight) versus dilution rate (D) had maxima at 0.63 and 1.1 h⁻¹. The corresponding plot of viable count had a minimum at 0.94 h⁻¹ whereafter it reached a maximum at 1.3 h⁻¹. Largest yield coefficient obtained was 0.44 g dry weight/g asparagine (D=1.1 h⁻¹). The productivity of the culture increased with D up to 1.1 h⁻¹, which is far above the D corresponding to the maximum specific growth rate (^μmax) of a batch culture (0.59 h⁻¹). The cell mass was not completly washed-out of the fermentor even at a D of 2.2 h⁻¹. The influence of attached growth for the steady-state characteristics, and the significance of the results in relation to chemostate as an instrument for testing environmental factors, are discussed. It is suggested that the attached cells had a significantly higher (^μmax) value than the suspended ones.     

Inhibition of T-lymphocyte rosetting by HL-A alloantisera and complement.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.