bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrile Hydratase-Catalyzed Production of Nicotinamide from 3-Cyanopyridine in
Rhodococcus rhodochrous
J1
Authors: Nagasawa Toru , Mathew Caluwadewa Deepal , Mauger Jacques , Yamada Hideaki
Published: December 2019

Nitrile hydratase, which occurs abundantly in cells of
Rhodococcus rhodochrous
J1 isolated from soil samples, catalyzes the hydration of 3-cyanopyridine to nicotinamide. By using resting cells, the reaction conditions for nicotinamide production were optimized. Under the optimum conditions, 100% of the added 12 M 3-cyanopyridine was converted to nicotinamide without the formation of nicotinic acid, and the highest yield achieved was 1,465 g of nicotinamide per liter of reaction mixture containing resting cells (1.48 g as dry cell weight) in 9 h. The nicotinamide produced was crystallized and then identified physicochemically. The further conversion of the nicotinamide to nicotinic acid was due to the low activity of nicotinamide as a substrate for the amidase(s) present in this organism.
Peptide Utilization Encoded by
Lactococcus lactis
subsp.
lactis
SSL135 Chromosomal DNA
Authors: Tynkkynen Soile , von Wright Atte , Syväoja Eeva-Liisa
Published: December 2019

A cloned chromosomal fragment of
Lactococcus lactis
subsp.
lactis
SSL135 on plasmid p VS8 in an
L. lactis
subsp.
lactis
MG1614 background enabled proteinase-negative strain MG1614 to grow in autoclaved milk. The strain (VS230) did not, however, degrade milk proteins and did not grow in pasteurized milk. In contrast, a strain (VS150) carrying p VS9, the proteinase plasmid of SSL135, in an MG1614 background degraded β-casein but did not grow in milk. VS230 was shown to utilize peptides produced by VS150 in growth experiments in pasteurized milk preincubated with the latter strain. The peptide utilization phenotype linked with p VS8 was further confirmed by growth of VS230 on tryptic peptide fractions, on which the plasmid-free but otherwise isogenic strain MG1614 failed to grow. Plasmid p VS8 produced 69-, 42-, 38-, and 36-kilodalton proteins, as determined by in vitro transcription-translation. At least three of these proteins affected the peptide utilization phenotype. We suggest that there could be a coupled peptidase-peptide transport system encoded by the chromosomal fragment.
Influence of body weight on response of Fischer 344 rats to anthrax lethal toxin
Authors: Ivins B E , Ristroph J D , Nelson G O
Published: December 2019

Groups of Fischer 344 rats were injected intravenously with Bacillus anthracis culture supernatant containing crude anthrax toxin. Times to death of rats given identical toxin preparations varied directly with the weights of the rats (P = 0.0001). In contrast to previous reports, the data indicate that rat weight must be taken into account during in vivo assays of anthrax lethal toxin activity.
Carbon Concentration and Carbon-to-Nitrogen Ratio Influence Submerged-Culture Conidiation by the Potential Bioherbicide
Colletotrichum truncatum
NRRL 13737
Authors: Jackson Mark A. , Bothast Rodney J.
Published: December 2019

We assessed the influence of various carbon concentrations and carbon-to-nitrogen (C:N) ratios on
Colletotrichum truncatum
NRRL 13737 conidium formation in submerged cultures grown in a basal salts medium containing various amounts of glucose and Casamino Acids. Under the nutritional conditions tested, the highest conidium concentrations were produced in media with carbon concentrations of 4.0 to 15.3 g/liter. High carbon concentrations (20.4 to 40.8 g/liter) inhibited sporulation and enhanced the formation of microsclerotiumlike hyphal masses. At all the carbon concentrations tested, a culture grown in a medium with a C:N ratio of 15:1 produced more conidia than cultures grown in media with C:N ratios of 40:1 or 5:1. While glucose exhaustion was often coincident with conidium formation, cultures containing residual glucose sporulated and those with high carbon concentrations (>25 g/liter) exhausted glucose without sporulation. Nitrogen source studies showed that the levels of
C. truncatum
NRRL 13737 conidiation were similar for all protein hydrolysates tested. Reduced conidiation occurred when amino acid and inorganic nitrogen sources were used. Of the nine carbon sources evaluated, acetate as the sole carbon source resulted in the lowest level of sporulation.
Limited Multiplication of Symbiotic Cyanobacteria of
Azolla
spp. on Artificial Media
Authors: Tang L. F. , Watanabe I. , Liu C. C.
Published: December 2019

We examined various media and conditions to isolate symbiotic cyanobacteria from the leaf cavities of
Azolla
spp. Cyanobacteria survived and multiplied to a limited extent on a medium with fructose, Casamino Acids, yeast extract, and NaNO
3
under 1% O
2
. These cyanobacteria were antigenically identical to the endosymbionts.
Selection of colony, plasmid, and virulence variants of Staphylococcus epidermidis NRC853 during growth in continuous cultures exposed to erythromycin
Authors: DeGuglielmo M A , George C G , Kloos W E
Published: December 2019

A continuous-culture system was developed to study changes in the structure of Staphylococcus epidermidis populations exposed to subminimum inhibitory concentrations of erythromycin. Continuous-culture experiments were carried out in a dextrose-free, tryptic soy broth medium supplemented with lactic acid and sodium lactate (MTSB-D). The multiresistant (penicillin-, tetracycline-, and erythromycin-resistant) S. epidermidis strain NRC853 was subjected to a series of experiments: (i) growth individually in continuous culture in the absence and presence of erythromycin and (ii) growth in mixed culture with the erythromycin-susceptible S. epidermidis strain NRC852 in the absence and presence of erythromycin. Strain NRC853 produced colony morphology variants during continuous culture in the presence of 0.05 and 0.1 microgram of erythromycin per ml. Variants (A, B, and C) were different from their wild-type parent on the basis of colony size, sector pattern, and/or the ability to transmit light. A variants rapidly lost a 2.7-MDa tetracycline resistance plasmid. B and C variants formed an ermC plasmid multimer series from unit size to a 16-mer and exhibited an approximately twofold increase in erythromycin MIC over that of the wild-type parent. They slowly lost the tetracycline resistance plasmid. The small-colony B variant demonstrated an increased virulence in the neonatal mouse weight gain test and an increase in fibronectin binding compared with the wild-type parent. The presence of a competing strain drastically increased the frequency of all variants.
Regulation of Proteolytic Activity in the Hyperthermophile
Pyrococcus furiosus
Authors: Snowden Lesley J. , Blumentals Ilse I. , Kelly Robert M.
Published: December 2019

Pyrococcus furiosus
was shown to grow on casein or peptides as the sole carbon, energy, and nitrogen sources, while maltose could be used as a carbon and energy source only if peptides were present in the medium. A mixture of all 20 single amino acids could not replace the peptide requirement. Specific intracellular proteolytic activity was induced under low casein or tryptone levels and was decreased by the addition of maltose to both peptide-limiting and peptide-rich media in batch and continuous cultures. In a peptide-limited chemostat, activity towards azocasein and MeO-Suc-Arg-Pro-Tyr-
p
-nitroanilide reached a maximum at a dilution rate of 0.28 h
-1
, while activity toward
l
-lysine-
p
-nitroanilide reached a maximum at 0.50 h
-1
. Under peptide-limiting conditions, levels of the 66-kDa protease (S66) were enhanced relative to those of other cell proteins. Preliminary evidence suggests that this protease is immunologically related to the eukaryotic multicatalytic proteinase complex (proteosome).
Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression
Authors: Bosworth A H , Williams M K , Albrecht K A , Kwiatkowski R , Beynon J , Hankinson T R , Ronson C W , Cannon F , Wacek T J , Triplett E W
Published: December 2019

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC -2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.
Comparison of Neutralizing and Hemagglutination-Inhibiting Antibody Responses to Influenza A Virus Vaccination of Human Immunodeficiency Virus-Infected Individuals
Authors: Benne C. A. , Kroon F. P. , Harmsen M. , Tavares L. , Kraaijeveld C. A. , De Jong J. C.
Published: December 2019

A neutralization enzyme immunoassay (N-EIA) was used to determine the neutralizing serum antibody titers to influenza A/Taiwan/1/86 (H1N1) and Beijing/353/89 (H3N2) viruses after vaccination of 51 human immunodeficiency virus (HIV) type 1-infected individuals and 10 healthy noninfected controls against influenza virus infection. Overall, the N-EIA titers correlated well with the hemagglutination-inhibition (HAI) titers that were observed in the same samples in a previous study (F. P. Kroon, J. T. van Dissel, J. C. de Jong, and R. van Furth, AIDS 8:469–476,1994). The N-EIA appeared to be more sensitive than the HAI test. Significantly more fourfold or higher rises in N-EIA titer and higher mean N-EIA titers occurred in HIV-infected individuals with ≥200 CD4
+
cells per μl than in those with <200 CD4
+
cells per μl.
Distribution of CCR5Δ32 in Human Immunodeficiency Virus-Infected Children and Its Relationship to Disease Course
Authors: Bakshi Saroj S. , Zhang Linqi , Ho David , Than Soe , Pahwa Savita G.
Published: December 2019

Homozygosity for a 32-bp deletion in the CCR5 gene (CCR5Δ32) has been shown to confer resistance to infection with the macrophage-tropic strain of human immunodeficiency virus (HIV) type 1. We examined the distribution of CCR5Δ32 in 47 children (age range, 1.5 to 19 years), of whom 43 were infected with HIV, by the perinatal route (
n
= 41) or by the intravenous route (
n
= 2). The infected patients were classified as rapid progressors (RP) (
n
= 7) (CDC category C3 or death by 2 years of age), non-rapid progressors (NRP) (
n
= 17) (survival for ≥8 years after infection), or intermediate (
n
= 19). CCR5Δ32 heterozygosity was found in two HIV-infected children, both NRP. None of the subjects were homozygous for CCR5Δ32, and the remaining children had no evidence of CCR5Δ32. The presence of CCR5Δ32 heterozygosity in 4.8% of this, predominantly non-Caucasian population is consistent with the published distribution of the mutation. The finding that CCR5Δ32 was present only in NRP and not in any RP is in agreement with previous reports suggesting that heterozygosity for CCR5Δ32 may confer limited protection from disease progression.
Cytokine Production in Cell Culture by Peripheral Blood Mononuclear Cells from Immunocompetent Hosts
Authors: Katial Rohit K. , Sachanandani Doris , Pinney Carolyn , Lieberman Michael M.
Published: December 2019

The production of interleukin 2 (IL-2) gamma interferon, IL-4, tumor necrosis factor alpha (TNF-α), TNF-β, IL-5, and IL-10 in vitro by peripheral blood mononuclear cells cultured from healthy immunocompetent subjects after mitogen stimulation was determined. The mitogens used were concanavalin A, phytohemagglutinin, pokeweed mitogen, andStaphylococcus aureusCowen. The results obtained provide a normal range for the production of these cytokines under specified conditions in vitro.
Enhancement of Antibody-Dependent Cellular Cytotoxicity of Neonatal Cells by Interleukin-2 (IL-2) and IL-12
Authors: Nguyen Quoc H. , Roberts Robert L. , Ank Bonnie J. , Lin Syh-Jae , Lau Casey K. , Stiehm E. Richard
Published: December 2019

Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood MNCs was less than 50% that of adult MNCs in most assays prior to exposure to cytokines. Incubation with IL-2 (100 U/ml) or IL-12 (1 ng/ml) for 18 h increased the NK cell activity (using K562 target cells) of both cord blood and adult MNCs, and the combination of IL-2 and IL-12 increased cord blood cytotoxicity threefold, making the cytotoxicity of cord blood cells equivalent to that of adult cells treated with the same cytokines. In ADCC assays with chicken erythrocyte targets, the combination of IL-2 and IL-12 increased the cytotoxicities of both cord blood and adult MNCs, with greater enhancement again seen with cord blood cells. In assays with NK cell-resistant CEM cells coated with human immunodeficiency virus (HIV) gp120 antigen in the presence of hyperimmune anti-HIV immunoglobulin, ADCC of cord blood MNCs was about 50% that of adult MNCs; ADCC of cord blood MNCs increased two- to threefold with the addition of IL-2 and IL-12, whereas ADCC of adult MNCs did not increase. Incubation of cord blood cells, but not adult cells, with IL-2 or IL-12 for 1 week increased the percentage of CD16+/CD56+cells two- to fivefold and enhanced ADCC activity. Thus, IL-2 and IL-12 greatly enhance both the NK cell and ADCC activities of neonatal MNCs and increase the number of NK cells in longer-term culture.
Blinded Multiplex PCR Analyses of Middle Ear and Nasopharyngeal Fluids from Chinchilla Models of Single- and Mixed-Pathogen-Induced Otitis Media
Authors: Bakaletz Lauren O. , White Gregory J. , Post J. Christopher , Ehrlich Garth D.
Published: December 2019

Multiplex PCR analyses for both bacterial and viral pathogens were conducted in a blinded manner on 33 archival specimens, of known culture status, procured from chinchilla models of both single- and mixed-pathogen-induced otitis media and from a pediatric patient. These specimens had been maintained at −70°C for up to 6 years. Experimental specimens evaluated included middle-ear effusions, nasopharyngeal lavage fluids and middle-ear lavage fluids from animals which were immunologically naive, sham-immunized or actively immunized with nontypeableHaemophilus influenzaeantigens. Sampling times used ranged from the day of bacterial or viral challenge to 42 days after challenge. Initial PCR analyses of the 33 specimens matched the traditional culture data in 24 instances (73%), correctly identifying nontypeableH. influenzae,Moraxella catarrhalis,Streptococcus pneumoniae, or adenovirus as the causative agent. A PCR-positive signal for the microbe(s) inoculated was also obtained in four animal model specimens (12%) which were culture negative. One of two culture-negative human effusions was also PCR positive. Thus, overall, results obtained by blinded PCR were 85% concordant with traditional culture methods or correctly indicated the specific pathogen introduced in four specimens that were sterile. In no instance was a false-positive signal obtained for any of the five etiologic agents being evaluated. We conclude that the multiplex PCR analyses are rapid and accurate methodologies when they are used to retrospectively evaluate diverse archival specimens of limited volume from experimental models of otitis media.
Evaluation of an Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5 for Serological Diagnosis of Bovine Anaplasmosis in Venezuela
Authors: Reyna-Bello Armando , Cloeckaert Axel , Vizcaíno Nieves , Gonzatti Mary I. , Aso Pedro M. , Dubray Gérard , Zygmunt Michel S.
Published: December 2019

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of bovine anaplasmosis with purified recombinant major surface protein 5 (MSP5) ofAnaplasma marginaleproduced inEscherichia coli. Serum antibody responses against MSP5 were detected in calves experimentally infected withA. marginaleas early as 21 days postinfection and reached maximum titers at 28 days postinfection. The MSP5 ELISA performed with serum samples taken from field cattle from different regions of Venezuela showed a seroprevalence of 47%, which seems to be in accordance with the reported epidemiological status of bovine anaplasmosis in Venezuela. Positive results obtained in the MSP5 ELISA were further confirmed by immunoblotting, with the recombinant MSP5 as the antigen. Thus, these results confirmed the importance of MSP5 as a suitable antigen for the serological diagnosis of bovine anaplasmosis.
Analysis of the Humoral Immune Response toChlamydiaOuter Membrane Protein 2
Authors: Mygind Per , Christiansen Gunna , Persson Kenneth , Birkelund Svend
Published: December 2019

The humoral immune response toChlamydiaouter membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of allChlamydiaspecies, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2aa23-aa93,Chlamydia psittaci-Omp2aa23-aa94, andChlamydia trachomatis-Omp2aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547). By an enzyme-linked immunosorbent assay with serologically defined patient sera, Omp2 was found to be a major immunogen of bothC. pneumoniaeandC. trachomatisinfections (P≪ 0.0001). The humoral immune responses were not confined to any particular region of the Omp2 protein, and no species-specific anti-Omp2 immunoglobulins were detected.
Clinical Evaluation of a Rapid Immunochromatographic Test for the Diagnosis of Dengue Virus Infection
Authors: Sang Chew Theng , Hoon Lim Siew , Cuzzubbo Andrea , Devine Peter
Published: December 2019

A rapid immunochromatographic test was compared to the hemagglutination inhibition assay for separate determinations of dengue virus-specific immunoglobulin M (IgM) and IgG levels in paired serum specimens from 92 patients (34 with primary dengue virus infection, 35 with secondary dengue virus infection, and 23 without dengue virus infection). The rapid test showed 99% sensitivity in the diagnosis of dengue virus infection. The majority (30 of 34 [88%]) of patients with primary infection showed positive IgM but negative IgG, while 34 of 35 (97%) patients with secondary infection showed positive IgG with or without IgM. Specificity in nonflavivirus infections was 96% (1 of 23 positive). The rapid test should be a useful aid in rapid diagnosis of dengue virus infection.
Effect of Cytokines on Anticryptococcal Activity of Human Microglial Cells
Authors: Lipovsky Myriam M. , Juliana Amadu E. , Gekker Genya , Hu Shuxian , Hoepelman Andy I. M. , Peterson Phillip K.
Published: December 2019

The effect of selected cytokines on the antifungal activity of human microglia was studied with encapsulated and acapsular strains ofCryptococcus neoformans. None of the cytokines tested increased the fungistatic activity of microglia, suggesting that killing of cryptococci within the central nervous system is dependent on other host defense mechanisms.
A Modified Agglutination Test forNeospora caninum: Development, Optimization, and Comparison to the Indirect Fluorescent-Antibody Test and Enzyme-Linked Immunosorbent Assay
Authors: Packham Andrea E. , Sverlow Karen W. , Conrad Patricia A. , Loomis Emily F. , Rowe Joan D. , Anderson Mark L. , Marsh Antoinette E. , Cray Carolyn , Barr Bradd C.
Published: December 2019

Current serologic tests used to detect antibodies toNeospora caninumrequire species-specific secondary antibodies, limiting the number of species that can be tested. In order to examine a wide variety of animal species that may be infected withN. caninum, a modified direct agglutination test (N-MAT) similar to theToxoplasma gondiimodified direct agglutination test (T-MAT) was developed. This test measures the direct agglutination of parasites byN. caninum-specific antibodies in serum, thus eliminating the need for secondary host-specific anti-isotype sera. The N-MAT was compared to the indirect fluorescent-antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA) with a “gold standard” serum panel from species for which secondary antibodies were available (n= 547). All positive samples tested were from animals with histologically confirmed infections. Up to 16 different species were tested. The N-MAT gave a higher sensitivity (100%) and specificity (97%) than the ELISA (74 and 94%, respectively) and had a higher sensitivity but a lower specificity than the IFAT (98 and 99%, respectively). The reduced specificity of the N-MAT was due to false-positive reactions in testing fetal fluids with particulate matter or severely hemolyzed serum. Overall, the N-MAT proved to be highly sensitive and specific for both naturally and experimentally infected animals, highly reproducible between and within readers, easy to use on large sample sizes without requiring special equipment, and useful in testing serum from any species without modification.
Effect of Ovalbumin Aerosol Exposure on Colonization of the Porcine Upper Airway by
Pasteurella multocida
and Effect of Colonization on Subsequent Immune Function
Authors: Hamilton T. D. C. , Roe J. M. , Hayes C. M. , Webster A. J. F.
Published: December 2019

Seventy-three piglets were weaned at 1 week of age, randomly assigned to 10 groups (A to J), accommodated in stainless steel exposure chambers, and exposed continuously to a controlled environment containing aerosolized ovalbumin. The concentrations of ovalbumin dust were as follows (milligrams per cubic meter): A and F, 16.6; B and G, 8.4; C and H, 4.2; D and I, 2.1; E and J, 0. At weekly intervals, the pigs were bled via venipuncture and anesthetized for nasal lavage and tonsilar biopsies performed for subsequent bacteriologic analysis. At 2 weeks of age, the pigs in groups A to E were challenged with toxigenic
Pasteurella multocida
(10
8
CFU pig
−1
), and at 6 weeks of age, the pigs were euthanatized. At postmortem, the extent of turbinate atrophy was assessed on the snout sections by using a morphometric index. Exposure to aerial ovalbumin resulted in a dose-dependent increase in serum antiovalbumin immunoglobulin G (IgG;
P
< 0.001) and serum antiovalbumin IgA (
P
< 0.001). Exposure also caused a significant increase in the numbers of
P. multocida
organisms isolated from the upper respiratory tract (
P
< 0.001) and a corresponding increase in turbinate atrophy, as judged by the morphometric index (
P
< 0.001). Concurrent challenge with
P. multocida
and ovalbumin resulted in a significant decrease in both the IgG and IgA responses to ovalbumin (
P
< 0.001). These results show that ovalbumin exposure increases pig susceptibility to
P. multocida
colonization and that toxigenic
P. multocida
modifies the serum IgG and IgA responses to ovalbumin in the pig. Both of these effects may enhance the virulence of this respiratory pathogen and so influence the pathogenesis of atrophic rhinitis in pigs.
Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection ofMycobacterium tuberculosisInfection
Authors: Desem Nuket , Jones Stephen L.
Published: December 2019

A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.
Effects of Colloidal Resuscitation Fluids on Reticuloendothelial Function and Resistance to Infection after Hemorrhage
Authors: van Rijen Ed A. M. , Ward Jason J. , Little Roderick A.
Published: December 2019

The effects of three resuscitation fluids, hydroxyethyl starch (HES), Haemaccel, and fresh autologous blood, on reticuloendothelial system phagocytic and catabolic functions and resistance to infection after 40% hemorrhages in BALB/c mice were studied. The mice, anesthetized with isoflurane, were bled over a 10-min period, left hypovolemic for 30 min, and then resuscitated with their shed blood or the same volume of asanguineous fluid. Normothermia was maintained throughout the experiments. The uptake and catabolism of intravenously injected double-labelled sheep erythrocytes (
51
Cr-
125
I-SRBC) in liver and spleen were determined at 1 and 48 h after hemorrhage. No significant changes in the uptake or catabolism of SRBC in liver or spleen were found at 1 h after hemorrhage and resuscitation with any of the fluids. However, at 48 h a significant increase in liver uptake of SRBC was seen in animals resuscitated with either Haemaccel or HES compared to that in animals resuscitated with shed blood or in animals subjected to a sham operation. The increase in liver uptake was accompanied by a small decrease in spleen uptake in animals resuscitated with Haemaccel but not with HES. No great changes in catabolic activity were seen at 48 h, although activity levels tended to be higher in animals resuscitated with Haemaccel. Separate groups of animals were challenged by an intraperitoneal injection with live
Escherichia coli
at 1 or 48 h after hemorrhage and resuscitation. Sixty-four percent of the animals resuscitated with shed blood survived the challenge with
E. coli
at 1 h after hemorrhage, whereas only 10 and 0% survival was seen for animals resuscitated with Haemaccel and HES, respectively. At 48 h survival was 80% for shed-blood-resuscitated animals and 60 and 70% for Haemaccel- and HES-resuscitated animals, respectively.
Measurement of Induced Cytokines in AIDS Clinical Trials Using Whole Blood: A Preliminary Report
Authors: Wallis Robert S. , Lederman Howard M. , Spritzler John , Devers Jennifer L. , Georges Daniel , Weinberg Adriana , Stehn Susan , Lederman Michael M. , Group the Actg Inducible Cytokines Focus
Published: December 2019

Measures of immune function have become increasingly important as endpoints in AIDS clinical trials, with respect to both modulation and reconstitution of immunity by experimental therapies. Measurement of immune function in this setting requires the development of robust analytic approaches suitable for the clinical laboratory. Experiments were performed to evaluate the suitability of using cultured heparinized (“whole”) blood for induction of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), two cytokines critical in AIDS pathogenesis. TNF-α expression ranged from 229 to 769 pg/ml in lipopolysaccharide (LPS)-stimulated cultures and was not detected in unstimulated cultures. IFN-γ expression ranged from 0 to 112,000 pg/ml in phytohemagglutinin A (PHA)-stimulated cultures and from 0 to 789 pg/ml in antigen-stimulated cultures. The mean coefficient of variation observed in three weekly determinations was 0.47 for TNF-α and ranged from 0.12 to 1.73 for IFN-γ. These values indicate that sample sizes of 8, 24, and 29 subjects would be sufficient to detect twofold changes in LPS-induced TNF-α and in PHA- and antigen-induced IFN-γ, respectively, if two baseline and two treatment determinations were obtained, and if the interpatient variability of changes in true levels from baseline to follow-up is negligible compared to the variability in the three weekly measurements. Measurement of LPS-induced TNF-α and mitogen- or antigen-induced IFN-γ can be performed simply and reproducibly in human immunodeficiency virus-infected persons by the whole-blood culture method. Further studies are warranted to determine the effect of overnight shipping on assay reproducibility and to determine the extent to which responses can be reliably detected in subjects with low CD4 cell numbers.
Increased Interleukin-6 Levels in Nasal Lavage Samples following Experimental Influenza A Virus Infection
Authors: Gentile Deborah , Doyle William , Whiteside Theresa , Fireman Philip , Hayden Frederick G. , Skoner David
Published: December 2019

Interleukin-6 (IL-6) is a pleotropic cytokine implicated in the pathogenesis of local inflammation during viral upper respiratory infections. This study determined if experimental influenza A virus infection causes local IL-6 production. Seventeen healthy, adult subjects were intranasally inoculated, by course drops, with a safety-tested strain of influenza A/Kawasaki/86 (H1N1) virus. Nasal lavage samples were collected, symptoms were recorded, and expelled nasal secretions were weighed once before and then daily for 8 days after the virus inoculation. Lavage samples were submitted for virus culture and were examined for IL-6 and IL-4 by enzyme-linked immunosorbent assay. The IL-6, but not IL-4, levels were significantly increased in the nasal lavage samples of the 12 subjects who shed virus but not in those of the 5 subjects who did not shed virus. Moreover, the elevations in IL-6 levels were related temporally to the development of nasal symptoms and secretions but not to systemic symptoms. These results suggest a role for locally produced IL-6 in the pathogenesis and expressed symptomatology of influenza A virus infection.
Selection of Recombinant, Library-Derived Antibody Fragments against p24 for Human Immunodeficiency Virus Type 1 Diagnostics
Authors: de Haard Hans J. W. , Kazemier Bert , Koolen Marck J. M. , Nijholt Liekle J. , Meloen Rob H. , van Gemen Bob , Hoogenboom Hennie R. , Arends Jan-Willem
Published: December 2019

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VHand Vκregions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.
A Competitive Enzyme-Linked Immunosorbent Assay for Measuring the Levels of Serum Antibody toHaemophilus influenzaeType b
Authors: Mariani Massimo , Luzzi Enrico , Proietti Daniela , Mancianti Silvia , Casini Daniele , Costantino Paolo , van Gageldonk Pieter , Berbers Guy
Published: December 2019

A competitive ELISA method is described for the measurement of total antibodies to the capsular polysaccharide ofHaemophilus influenzaetype b (HibCPS) in human sera. The competitive method showed an excellent correlation to the radioantigen binding assay (RABA, or Farr assay) and improved correlation of sera with low titers with respect to the more conventional noncompetitive method. Overestimation of samples in the low concentration range was no longer observed with the competitive ELISA method. The free HibCPS competition allowed us to eliminate the day-to-day background variation typical of some sera; thus, only values representing the true anti-HibCPS response were determined. The use of precoated microplates, which could be stored up to 8 months, greatly improved the speed of the procedure. An overall correlation coefficient of 0.9660 was found when 407 serum samples with a wide variety of anti-HibCPS antibody levels were tested with the competitive ELISA and RABA. The regression line was very close to the ideal line, with a slope of 1.0045 and an intercept of −0.1996. A subset of 96 serum samples representative of all pre- and postimmunization samples was used to compare the competitive ELISA with a previously described ELISA method. The competitive method performed in two laboratories in different countries showed a better correlation with the RABA. The correlation factors were 0.9770 and 0.9816, respectively, while a factor of 0.9547 was found with the previously described noncompetitive procedure, which was better for this method than previously reported (r= 0.917). Therefore, the competitive ELISA is proposed for the assay of anti-HibCPS titers in sera from vaccinated subjects.
A Rapid Flow Cytometric Method to Explore Cellular Immunity againstToxoplasma gondiiin Humans
Authors: Kahi Sandrine , Cozon Grégoire J. N. , Greenland Timothy , Wallon Martine , Gay-Andrieu Françoise , Peyron François
Published: December 2019

To assess cell-mediated immunity toToxoplasma gondii, we evaluated the expression of the activation antigens CD69, CD71, and CD25 on T lymphocytes by flow cytometry after specific in vitro stimulation of whole blood from 127T. gondii-positive and 63T. gondii-negative patients. T lymphocytes from many seropositive individuals did not express CD69 at 24 h afterT. gondiiantigen stimulation, but CD71 and CD25 were easily detectable on T cells from seropositive individuals 7 days after specific activation. CD25 was mainly expressed by stimulated CD4+T cells, and its detection on total T cells was both a sensitive (98%) and a specific (97%) indicator of priorT. gondiiinfection. These results make flow cytometric detection of CD25 an excellent candidate for screening cell-mediated immunity toT. gondiiin vitro and an interesting tool for the diagnosis of congenital infection.
Evaluation of a Novel Mononuclear Cell Isolation Procedure for Serological HLA Typing
Authors: Schlenke Peter , Klüter Harald , Müller-Steinhardt Michael , Hammers Hans-Jörg , Borchert Kerstin , Bein Gregor
Published: December 2019

Despite recent advances in DNA-based genotyping, the microcytotoxicity test is still broadly used for the determination of human leukocyte class I antigens in patients as well as organ donors and also for the detection of HLA antibodies. Excellent purity and viability of peripheral blood mononuclear cells (PBMC) are essential for reliable HLA typing results. Background staining and cell loss can contribute to impaired typing results or even cause misinterpretations. A novel isolation procedure using cell preparation tubes (CPT) with prefilled Ficoll was compared with the standard Ficoll gradient. We determined the recovery, purity, and viability of the PBMC after several periods of storage. Finally, the isolated cells were used for HLA class I typing, and background reactivities were scored. By using the CPT method, the recovery of PBMC was significantly higher than recovery with the standard technique (
P
≤ 0.001). Contamination by granulocytes increased considerably during the storage time for the standard protocol, whereas purity remained stable when CPT were used (
P
≤ 0.001). With both methods, lymphocyte viability declined markedly over time. We found significantly more dead cells by using the CPT methods. Due to high background scores, HLA typing was impossible after 48 h. The isolation of PBMC by the CPT method resulted in a higher yield and improved purity compared to those obtained with the standard gradient technique. The decreasing viability after 48 h limits the use of both methods for HLA typing and HLA antibody screening.
Preparation of a Human Standard for Determination of the Levels of Antibodies to Oxidatively Modified Low-Density Lipoproteins
Authors: Koskinen Sinikka , Enockson Candace , Lopes-Virella Maria F. , Virella Gabriel
Published: December 2019

As part of our ongoing effort to develop a standardized competitive enzyme immunoassay for human autoantibodies to oxidized low-density lipoprotein (oxLDL), we have generated a reference human antibody standard and revised some of the conditions of the assay. The preparation of the standard involved purification of human anti-oxLDL antibodies by affinity chromatography using immobilized oxLDL. The total concentration of antibody in this purified human oxLDL antibody was established by adding the concentrations of immunoglobulin G (IgG), IgA, and IgM determined in the standard by radial immunodiffusion. The isolated antibody was used to calibrate a whole human serum standard, which was used to calibrate the assays to detect antibody in serum samples. We also revisited the general conditions for performance of our competitive assay. We determined that 1/20 was the ideal dilution for performing the absorption step, and that 1/20 and 1/40 were optimal dilutions to assay oxLDL antibody in unknown serum samples. We also established that the optimal concentration of oxLDL for absorption of free antibody in serum samples was 200 μg of oxLDL/ml; no significant decrease in the reactivity of samples with immobilized oxLDL was observed when higher concentrations of oxLDL were used for absorption. The minimum detection level of the assay is 0.65 mg/liter. Because serum samples are diluted 1/20 and 1/40 for the assay, the minimal concentration of antibody detectable in serum is 20-fold higher, i.e., 13 mg/liter. The intraassay coefficient of variation calculated from seven determinations of three samples containing antibody concentrations of 240, 340, and 920 mg/liter ranged from 8 to 6.1%. The interassay coefficients of variation for sera with antibody levels of 100 to 594 mg/liter varied from 9.2 to 7.0%, and for isolated antibodies with concentrations of 52 to 111 mg/liter, the coefficients varied from 5.8 to 3.9%.
Proposed Standardization of
Neisseria meningitidis
PorA Variable-Region Typing Nomenclature
Authors: Sacchi Claudio T. , Lemos Ana P. S. , Brandt Mary E. , Whitney Anne M. , Melles Carmo E. A. , Solari Claude A. , Frasch Carl E. , Mayer Leonard W.
Published: December 2019

Neisseria meningitidis
isolates are conventionally classified by serosubtyping, which characterizes the reactivities of the PorA outer membrane protein variable-region (VR) epitopes with monoclonal antibodies (MAbs). A newer method (PorA VR typing) uses predicted amino acid sequences derived from DNA sequence analysis. The resulting classification schemes are not standardized, offering conflicting and sometimes irreconcilable data from the two methods. In this paper, we propose a standardization of the PorA VR typing nomenclature that incorporates serologic information from traditional PorA serosubtyping with molecular data from predicted VR sequences. We performed a comprehensive literature and database search, generating a collection of strains and DNA sequences that reflects the diversity within PorA that exists to date. We have arranged this information in a comprehensive logical model that includes both serosubtype and PorA VR type assignments. Our data demonstrate that the current panel of serosubtype-defining MAbs underestimates PorA VR variability by at least 50%. Our proposal for VR typing is informative because amino acid sequence and serologic information, when serosubtype-defining MAbs are available, can be deduced simultaneously from the PorA VR designation. This scheme will be useful in future classification and applied epidemiologic studies of
N. meningitidis
, being a systematic way of selecting PorA vaccine candidates and analyzing vaccine coverage and failure.
Differentiation betweenMycobacterium bovisBCG-Vaccinated andM. bovis-Infected Cattle by Using Recombinant Mycobacterial Antigens
Authors: Buddle Bryce M. , Parlane Natalie A. , Keen Denise L. , Aldwell Frank E. , Pollock John M. , Lightbody Ken , Andersen Peter
Published: December 2019

Tuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection withMycobacterium tuberculosisorMycobacterium bovisand vaccination withM. bovisBCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins ofM. bovis—MPB59, MPB64, MPB70, and ESAT-6—were tested in a whole-blood gamma interferon (IFN-γ) assay for differentiation between cattle vaccinated with BCG and those experimentally infected withM. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected withM. bovisin BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-γ responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which wereM. bovisculture positive following challenge produced IFN-γ responses to PPDB and ESAT-6 which were significantly stronger than those observed in the correspondingM. bovisculture-negative animals. IFN-γ responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-γ assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.
Giant Syncytia and Virus-Like Particles in Ovarian Carcinoma Cells Isolated from Ascites Fluid
Authors: Rakowicz-Szulczynska Eva M. , McIntosh David G. , Smith McClure L.
Published: December 2019

Ovarian cancer cells were isolated from ascites fluid of 30 different patients diagnosed with cystadenocarcinoma of ovaries. Large colonies of malignant ASC cells were observed during the first week of cell growth in vitro. Colony formation was followed by fusion of cells and formation of large multinucleated and highly vacuolated syncytia. In contrast, cells isolated from the ascites fluid produced by patients with benign mucinous cystadenoma of ovaries did not form syncytia. Nonmalignant Brenner tumor cells, isolated from the ascites fluid, also did not form syncytia. Syncytia, but not the nonmalignant tumor cells, were immunofluorescence stained with an anti-human immunodeficiency virus type 1 (HIV-1) gp120 monoclonal antibody (MAb) and MAb RAK-BrI. Both MAbs recognized cancer-associated antigens RAK (for Rakowicz markers) p120, p42, and p25. Exposure of ASC cells to either the anti-HIV-1 gp120 MAb or MAb RAK-BrI inhibited syncytium formation. PCR with HIV-1 Env-derived primers revealed DNA sequences with over 90% homology to HIV-1 gp41 in syncytia and in ovarian cancer cells but not in normal ovary cells. Electron microscopic analysis revealed viral particles, hexagonal in shape (90 nm in diameter), with a dense central core surrounded by an inner translucent capsid and dense outer shell with projections. Negative staining detected membrane-covered particles (100 to 110 nm in diameter) in the cell culture medium. Incubation of normal breast cells with viral particles resulted in drastic morphological changes and syncytium formation by the transformed breast cells. The cytopathic effects of the identified virus resembled those of spumaviruses, which, in addition to their epitopic and genetic homology to HIV-1, might suggest a common phylogeny.
Viability and Recovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study
Authors: Kleeberger Cynthia A. , Lyles Robert H. , Margolick Joseph B. , Rinaldo Charles R. , Phair John P. , Giorgi Janis V.
Published: December 2019

The Multicenter AIDS Cohort Study (MACS), an ongoing prospective study of the natural history of human immunodeficiency virus (HIV), has stored biologic specimens, including peripheral blood mononuclear cells (PBMC), from 5,622 participants for up to 12 years. The purpose of the present analysis was to evaluate the quality of the PBMC in the MACS repository in order to test the validity and feasibility of nested retrospective studies and to guide the planning of future repositories. PBMC were collected from MACS participants at four centers at 6-month intervals from 1984 to 1995, cryopreserved, and transported to a central repository for storage. A total of 596 of these specimens were subsequently tested for viability and used to evaluate cell function, to conduct immunophenotype analysis, or to isolate HIV. Simple linear regression models were applied to evaluate trends in recovery and viability over time and by center. Results indicated that from a nominal 10
7
cells cryopreserved per vial at all four centers, the median number of viable cells recovered was at least 5 × 10
6
(50% of the number stored) and the median viability was at least 90%. Results suggested that cryopreserved cells can be stored for at least 12 years with no general tendency toward cell loss over time. Furthermore, there were no statistically significant changes in the percent cell viability according to the length of time frozen, regardless of HIV serostatus or the level of CD4
+
lymphocytes. Storing 10
7
PBMC per vial yields sufficient viable cells for phenotypic and/or functional analysis. Results from the MACS provide the basis for the planning of future repositories for use by investigators with similar research goals.