bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of DNA of Lymphotropic Herpesviruses in Plasma of Human Immunodeficiency Virus-Infected Patients: Frequency and Clinical Significance
Authors: Broccolo Francesco , Bossolasco Simona , Careddu Anna M. , Tambussi Giuseppe , Lazzarin Adriano , Cinque Paola
Published: November 2002

The frequency and clinical significance of detection of DNA of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, and HHV-8 in plasma were investigated by PCR. The plasma was obtained from 120 selected human immunodeficiency virus (HIV)-infected patients, of whom 75 had AIDS-related manifestations, 32 had primary HIV infection (PHI), and 13 had asymptomatic infections. Nested PCR analysis revealed that none of the lymphotropic herpesviruses tested were found in patients with PHI, in asymptomatic HIV-positive individuals, or in HIV-negative controls. By contrast, DNA of one or more of the viruses was found in 42 (56%) of 75 patients with AIDS-related manifestations, including CMV disease (CMV-D) or AIDS-related tumors. The presence of CMV DNA in plasma was significantly associated with CMV-D (
P
< 0.001). By contrast, EBV detection was not significantly associated with AIDS-related lymphomas (
P
= 0.31). Interestingly, the presence of HHV-8 DNA in plasma was significantly associated with Kaposi's sarcoma (KS) disease (
P
< 0.001) and with the clinical status of KS patients (
P
< 0.001). CMV (primarily), EBV, and HHV-8 were the viruses most commonly reactivated in the context of severe immunosuppression (
P
< 0.05). In contrast, HHV-6 and HHV-7 infections were infrequent at any stage of disease. In conclusion, plasma PCR was confirmed to be useful in the diagnosis of CMV-D but not in that of tumors or other conditions possibly associated with EBV, HHV-6, and HHV-7. Our findings support the hypothesis of a direct involvement of HHV-8 replication in KS pathogenesis, thus emphasizing the usefulness of sensitive and specific diagnostic tests to monitor HHV-8 infection.
Cytokine Profiles in Peripheral Blood Mononuclear Cells and Lymph Node Cells from Piglets Infected In Utero with Porcine Reproductive and Respiratory Syndrome Virus
Authors: Aasted B. , Bach P. , Nielsen J. , Lind P.
Published: November 2002

The aim of the present study was to investigate at 2, 4, and 6 weeks after birth cytokine expression by peripheral blood mononuclear cells and bronchial lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). Technically, by flow cytometry we were able to measure gamma interferon (γ-IFN), tumor necrosis factor alpha (TNF-α), interleukin-4 (IL-4), and IL-8 levels. In general, we found increases in the percentages of IL-4-, γ-IFN-, and TNF-α-producing lymphocytes in the infected piglets compared to the percentages in the uninfected control animals, while there was a decrease in the percentage of IL-8-producing monocytes. We believe that these findings reflect a general lymphocyte activation stage that is created due to the infection and that occurs in combination with impairment of the monocyte function, possibly due to the ongoing viral replication in these cells. Single-cell bronchial lymph node preparations exhibited very much the same cytokine profiles as peripheral blood mononuclear cells except for a lack of IL-8 production. When the levels of the individual cytokines in the three groups of PRRSV-infected piglets were compared, the levels of cytokine expression at 4 weeks diverged from those at 2 and 6 weeks, in that there was a significant decrease in the numbers of lymphocytes producing γ-IFN and TNF-α. This tendency was also observed among blood monocytes and lymph node macrophages. Possible reasons for this temporary immunosuppression in the piglets at 4 weeks are discussed.
Use of Coefficient of Variation in Assessing Variability of Quantitative Assays
Authors: Reed George F. , Lynn Freyja , Meade Bruce D.
Published: November 2002

We have derived the mathematical relationship between the coefficient of variation associated with repeated measurements from quantitative assays and the expected fraction of pairs of those measurements that differ by at least some given factor, i.e., the expected frequency of disparate results that are due to assay variability rather than true differences. Knowledge of this frequency helps determine what magnitudes of differences can be expected by chance alone when the particular coefficient of variation is in effect. This frequency is an operational index of variability in the sense that it indicates the probability of observing a particular disparity between two measurements under the assumption that they measure the same quantity. Thus the frequency or probability becomes the basis for assessing if an assay is sufficiently precise. This assessment also provides a standard for determining if two assay results for the same subject, separated by an intervention such as vaccination or infection, differ by more than expected from the variation of the assay, thus indicating an intervention effect. Data from an international collaborative study are used to illustrate the application of this proposed interpretation of the coefficient of variation, and they also provide support for the assumptions used in the mathematical derivation.
Adjuvant Activity of Emulsan, a Secreted Lipopolysaccharide from
Acinetobacter calcoaceticus
Authors: Panilaitis Bruce , Johri Atul , Blank Walter , Kaplan David , Fuhrman Juliet
Published: November 2002

Several promising adjuvant candidates have been studied over the past 75 years; however, only alum is currently approved for human use. The complex acylated polysaccharide emulsan, secreted from
Acinetobacter calcoaceticus
, represents a new candidate. Unique features of this family of polymers are their amenability to structural tailoring and their emulsification behavior. We demonstrate that emulsan activates macrophages in a dose-dependent manner. This activation is dependent on the presence of the fatty acid side chains that decorate the polysaccharide backbone, and, furthermore, the level of activation can be affected by changes in the chemical characteristics of emulsan structural variants. One emulsan variant was examined in a classical hapten carrier immunization protocol and demonstrated significant adjuvant activity as determined by hapten-specific antibody titers. This immune response was characterized by a high immunoglobulin G2a titer, consistent with a Th1 response. The significant immunopotentiation demonstrated by this complex polymer establishes emulsan as an exciting new candidate adjuvant. Furthermore, by manipulating the chemical structure of this compound, we can explore the physical basis of pattern recognition receptors and macrophage activation.
Quantitative Fluorescence Measures for Determination of Intracellular Perforin Content
Authors: Maher Kevin J. , Klimas Nancy G. , Hurwitz Barry , Schiff Richard , Fletcher Mary Ann
Published: November 2002

We present methodologic details and operating characteristics of a procedure with whole blood for the quantitative assessment of intracellular perforin within distinct lymphocyte subsets. Using this method, we analyzed 20 healthy controls and 2 individuals with an inherited deficiency of perforin. The mean ± standard deviation perforin contents of natural killer (NK) cells and cytotoxic T cells of healthy controls were 3,561 ± 1,157 and 500 ± 779 relative number of molecules (rMol) of antiperforin antibody bound per cell, respectively. The NK cell perforin contents of individuals with heterozygous and homozygous perforin deficiency (familial hemophagocytic lymphohistiocytosis) were 2,260 and 212 rMol of antiperforin antibodies per NK cell. While the homozygous deficiency was found to be associated with negligible antiperforin binding, the heterozygous condition was associated with a level of perforin binding that was below the 15th percentile for healthy individuals. Because 83% of this subject's NK cells were shown to bind to antiperforin antibodies by conventional flow cytometry (relative to the normal range of 81% ± 25%), quantitative cytometry may be more sensitive than conventional cytometric methods in identifying cytolytic defects.
Immunomodulatory Effects of CpG Oligodeoxynucleotides on Established Th2 Responses
Authors: Kitagaki Kunihiko , Jain Vipul V. , Businga Thomas R. , Hussain Iftikhar , Kline Joel N.
Published: November 2002

CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-γ) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-γ, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8+T lymphocytes. Although IFN-γ plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-γ was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-γ, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-γ was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-γ and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.
Identification of Pulmonary T-Lymphocyte and Serum Antibody Isotype Responses Associated with Protection againstRhodococcus equi
Authors: Lopez A. Marianela , Hines Melissa T. , Palmer Guy H. , Alperin Debra C. , Hines Stephen A.
Published: November 2002

Rhodococcus equiinfects and causes pneumonia in foals between 2 and 4 months of age but does not induce disease in immunocompetent adults, which are immune and remain clinically normal upon challenge. Understanding the protective response againstR. equiin adult horses is important in the development of vaccine strategies, since those mechanisms likely reflect the protective phenotype that an effective vaccine would generate in the foal. Twelve adult horses were challenged with virulentR. equiand shown to be protected against clinical disease. Stimulation of cells obtained from bronchoalveolar lavage fluid with eitherR. equior the vaccine candidate protein VapA resulted in significant proliferation and a significant increase in the level of gamma interferon (IFN-γ) expression by day 7 postchallenge. The levels of interleukin-4 expression were also increased at day 7 postchallenge; however, this increase was not antigen specific. Anamnestic increases in the levels of binding toR. equiand VapA of all immunoglobulin G (IgG) antibody isotypes [IgGa, IgGb, IgG(T)] examined were detected postchallenge. The levels ofR. equi- and VapA-specific IgGa and IgGb antibodies, the IgG isotypes that preferentially opsonize and fix complement in horses, were dramatically enhanced postchallenge. The antigen-specific proliferation of bronchoalveolar lavage fluid cells, the levels of IFN-γ expression by these cells, and the anamnestic increases in the levels of opsonizing IgG isotypes are consistent with stimulation of a memory response in immune adult horses and represent correlates for vaccine development in foals.
Unique Epitope of Bovine Immunodeficiency Virus Gag Protein Spans the Cleavage Site between p16
MA
and p2L
Authors: Lu Ming , Zheng Ling , Mitchell Kathy , Kapil Sanjay , Wood Charles , Minocha Harish
Published: November 2002

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are closely related bovine lentiviruses that are difficult to distinguish by presently available diagnostic methods. Recently, in our laboratory, a monoclonal antibody (MAb; MAb 10H1) against the BIV Gag protein identified a differential epitope, located at the 6.4-kDa N terminus of a 29-kDa Gag capsid protein, which was absent in JDV. To define the essential amino acids of the epitope, a series of primers within the 163 bp of DNA corresponding to the 6.4-kDa protein were designed. The full-length 163-bp DNA fragment and the smaller DNA fragments with deletions were amplified by PCR and then cloned into pQE32 vectors for protein expression studies. The expressed proteins were analyzed with MAb 10H1 by Western blotting. The differential epitope has been narrowed to a 26-amino-acid region (R121 to R146), which includes 6 residues of p16
MA
(where MA represents the matrix protein) and 20 residues of p2L. A synthetic peptide corresponding to the putative 26-amino-acid epitope blocked MAb 10H1 binding to the expressed peptide. These experiments revealed that the epitope spans the cleavage site between p16
MA
and p2L and presumably will be valuable in distinguishing the two viruses.
NK Cells Mediate Increase of Phagocytic Activity but Not of Proinflammatory Cytokine (Interleukin-6 [IL-6], Tumor Necrosis Factor Alpha, and IL-12) Production Elicited in Splenic Macrophages by Tilorone Treatment of Mice during Acute Systemic Candidiasis
Authors: Gaforio José Juan , Ortega Elena , Algarra Ignacio , Serrano María José , Alvarez de Cienfuegos Gerardo
Published: November 2002

The participation of NK cells in the activation of splenic macrophages or in resistance to systemic candidiasis is still a matter of debate. We had previously reported that there is a correlation between natural killer cell activation and resistance to systemic candidiasis. In those experiments we had used tilorone to boost NK cell activity in mice. Here we show a mechanism elicited by tilorone in splenic macrophages which could explain their effect on mouse survival during acute disseminatedCandida albicansinfection. The results demonstrate that tilorone treatment elicits, by a direct effect, the production of proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α], and IL-12) by splenic macrophages. In addition, it increases the capacity of splenic macrophages to phagocytizeC. albicansthrough activation of NK cells. We also demonstrate that the presence of NK cells is essential for maintaining a basal level of phagocytic activity, which characterizes splenic macrophages of naïve control mice. The results demonstrate that it is possible to identify two phenotypically and functionally peculiar cell populations among splenic macrophages: (i) cells of the “stimulator/secretor phenotype,” which show high levels of major histocompatibility complex (MHC) class II surface expression, are poorly phagocytic, and synthesize the proinflammatory cytokines IL-6, TNF-α, and IL-12, and (ii) cells of the “phagocytic phenotype,” which express low levels of MHC class II molecules, are highly phagocytic, and do not secrete proinflammatory cytokines.
Celiac Disease and Immunoglobulin A Deficiency: How Effective Are the Serological Methods of Diagnosis?
Authors: Kumar V. , Jarzabek-Chorzelska M. , Sulej J. , Karnewska Krystyna , Farrell T. , Jablonska S.
Published: November 2002

Immunoglobulin A (IgA) deficiency is 10 to 15 times more common in patients with celiac disease (CD) than in healthy subjects. Serological tests have become the preferred methods of diagnosing CD in both symptomatic and asymptomatic patients. However, commercially available serological methods are limited in that they detect only the IgA isotype of antibodies (with the exception of IgG gliadin assays); hence, IgA-deficient patients with CD may yield false-negative serology. Fifteen pediatric patients with CD and 10 IgA-deficient pediatric patients without CD were examined for IgA and IgG antibodies to endomysium, gliadin, and tissue transglutaminase. Twenty-five specimens from patients with IgA deficiency were examined. Fifteen were from patients with CD, and 10 were patients without CD. All 15 IgA-deficient patients with CD were positive for endomysium antibodies of the IgG isotype and for IgG gliadin antibodies. All but one of the IgA-deficient patients with CD were also positive for IgG tissue transglutaminase antibodies. None of the IgA-deficient patients without CD were positive for any of the antibody markers. All the specimens examined were also negative for IgA-specific antibodies to endomysium, gliadin, and tissue transglutaminase. IgG-specific antibody tests for endomysium, gliadin, and tissue transglutaminase are useful for the identification of IgA-deficient patients with CD. IgG antibody tests along with tests routinely being used in clinical laboratories can reliably detect all active patients with CD. In addition, the levels of these CD-specific IgG antibodies could be used to monitor patient dietary compliance.
Conformational Dependence and Conservation of an Immunodominant Epitope within the
Babesia equi
Erythrocyte-Stage Surface Protein Equi Merozoite Antigen 1
Authors: Cunha Cristina W. , Kappmeyer Lowell S. , McGuire Travis C. , Dellagostin Odir A. , Knowles Donald P.
Published: November 2002

Equi merozoite antigen 1 (EMA-1) is an immunodominant
Babesia equi
erythrocyte-stage surface protein. A competitive enzyme-linked immunosorbent assay (ELISA), based on inhibition of monoclonal antibody (MAb) 36/133.97 binding to recombinant EMA-1 by equine anti-
B. equi
antibodies, detects horses infected with strains present throughout the world. The objectives of this study were to define the epitope bound by MAb 36/133.97 and quantify the amino acid conservation of EMA-1, including the region containing the epitope bound by MAb 36/133.97. The alignment of the deduced amino acid sequence of full-length EMA-1 (Florida isolate) with 15 EMA-1 sequences from geographically distinct isolates showed 82.8 to 99.6% identities (median, 98.5%) and 90.5 to 99.6% similarities (median, 98.9%) between sequences. Full-length and truncated recombinant EMA-1 proteins were expressed and tested for their reactivities with MAb 36/133.97. Binding required the presence of amino acids on both N- and C-terminal regions of a truncated peptide (EMA-1.2) containing amino acids 1 to 98 of EMA-1. This result indicated that the epitope defined by MAb 36/133.97 is dependent on conformation. Sera from persistently infected horses inhibited the binding of MAb 36/133.97 to EMA-1.2 in a competitive ELISA, indicating that equine antibodies which inhibit binding of MAb 36/133.97 also recognize epitopes in the same region (the first 98 residues). Within this region, the deduced amino acid sequences had 85.7 to 100% identities (median, 99.0%), with similarities of 94.9 to 100% (median, 100%). Therefore, the region which binds to both MAb 36/133.97 and inhibiting equine antibodies has a median amino acid identity of 99.0% and a similarity of 100%. These data provide a molecular basis for the use of both EMA-1 and MAb 36/133.97 for the detection of antibodies against
B. equi
.
Primary Role of Interleukin-1α and Interleukin-1β in Lipopolysaccharide-Induced Hypoglycemia in Mice
Authors: Oguri Senri , Motegi Katsutoshi , Iwakura Yoichiro , Endo Yasuo
Published: November 2002

Within a few hours of its injection into mice, lipopolysaccharide (LPS) induces hypoglycemia and the production of various cytokines. We previously found that interleukin-1α (IL-1α), IL-1β, and tumor necrosis factor alpha (TNF-α) induce hypoglycemia and that the minimum effective dose of IL-1α or IL-1β is about 1/1,000 that of TNF-α. In the present study, we examined the contribution made by IL-1 to the hypoglycemic action of LPS. Nine other cytokines tested were all inactive at inducing hypoglycemia. LPS produced hypoglycemia in mice deficient in either IL-1α or IL-1β but not in mice deficient in both cytokines (IL-1α and -1β knockout [IL-1α/β KO] mice). IL-1α, IL-1β, and TNF-α induced hypoglycemia in IL-1α/β KO mice, as they did in normal control mice. The LPS-induced elevation of serum cortisol was weaker in IL-1α/β KO mice than in control mice, and, in the latter, serum cortisol was markedly raised while blood glucose was declining. IL-1α decreased blood glucose both in NOD mice (which have impaired insulin production) and in KK-Ay mice (insulin resistant). These results suggest that (i) cortisol may not be involved in mediating the resistance of IL-1α/β KO mice to the hypoglycemic action of LPS, (ii) as a mediator, IL-1 is a prerequisite for the hypoglycemic action of LPS, (iii) IL-1α and IL-1β perform mutual compensation, and (iv) IL-1 plays a role as the primary stimulator of the many anabolic reactions required for the elaboration of immune responses against infection.
Responses of Endoscopy Patients in Ladakh, India, to
Helicobacter pylori
Whole-Cell and CagA Antigens
Authors: Romero-Gallo Judith , Pérez-Pérez Guillermo I. , Novick Richard P. , Kamath Patrick , Norbu Tsering , Blaser Martin J.
Published: November 2002

Although
Helicobacter pylori
is a cosmopolitan colonizer of the human stomach, the responses among persons in remote populations from whom
H. pylori
was cultured have not been studied. We report on studies of 189 persons in the Ladakh region of India in whom serum immunoglobulin G responses to
H. pylori
whole-cell and CagA antigens were measured.
H. pylori
was isolated from 68 of these patients. An
H. pylori
whole-cell antigen derived from Ladakhi strains outperformed a similar antigen from U.S. strains, as determined by antigen-specific enzyme-linked immunosorbent assays. In total, 95% of the population was seropositive, including individuals responding only to the CagA antigen. Correlation with culture results showed that these were true positives and, therefore, that the
H. pylori
whole-cell serology was falsely negative in some cases. In addition to establishing a collection of
H. pylori
isolates from a remote area in the world, we show that use of
H. pylori
whole-cell and CagA serology together increases the sensitivity for the detection of colonization.
Evaluation of Immunomodulatory Effects of Lactic Acid Bacteria in Turbot (Scophthalmus maximus)
Authors: Villamil L. , Tafalla C. , Figueras A. , Novoa B.
Published: November 2002

In the present work, the effects of several lactic acid bacteria on the immune response of turbot (Scophthalmus maximus) macrophages have been studied both in vitro and in vivo. Out of six lactic acid bacterial strains tested, only heat-killedLactococcus lactissignificantly increased the turbot head kidney macrophage chemiluminescent (CL) response after 24 h of incubation. Nitric oxide (NO) was also significantly enhanced by this bacterium after 72 h of incubation with either viable (103and 106cells/ml) or heat-killed (106cells/ml) bacteria. ViableLeuconostoc mesenteroides(106cells/ml) was also capable of significantly increasing NO production. SinceL. lactisproved to be the strain with more effects on the host immune function, further in vivo and in vitro experiments were conducted with this bacterium. The in vitro capacity ofL. lactisto adhere to turbot intestinal mucus was positively confirmed. When orally administered,L. lactissignificantly increased the macrophage CL response and the serum NO concentration after 7 days of daily administration. The antibacterial effect of the extracellular products from the six LAB strains against the fish-pathogenic bacteriumVibrio anguillarumwas also demonstrated in vitro.
Patterns of Cytokines and Soluble Cellular Receptors in the Sera of Children with Acute Chagas' Disease
Authors: Moretti Edgardo , Basso Beatriz , Cervetta Liliana , Brigada Ana , Barbieri Gustavo
Published: November 2002

Cytokines and soluble cellular receptors are involved in inflammatory processes and probably in the pathogenesis of parasite and bacterial diseases. In a previous study, we reported increased levels of soluble receptors of interleukin-2 (sIL2-R) in children with acute Chagas' disease, one of the main parasitic infections that is endemic in Latin America. We sought to analyze the pattern of different cytokines and soluble receptors in the sera of children with chagasic infection. Children with acute and indeterminate stages of Chagas' disease, as well as nonchagasic children, were studied. Sera were assayed by enzyme-linked immunosorbent assay to measure the levels of tumor necrosis factor alpha (TNF-α), IL-6, IL-2, IL-8, IL-12, sIL-2R, and the soluble receptors of CD8 and CD4 (sCD8 and sCD4). sIL-2R and sCD8 showed the highest levels in serum in acutely infected children, decreasing after specific antiparasite therapy. Chronic children showed a pattern similar to the one of nonchagasic children. Although they were not statistically significant, TNF-α, IL-6, and sCD4 showed a tendency to reach high levels in the acutely infected group, whereas IL-2, IL-8, and IL-12 did not reveal changes with respect to the noninfected children. In summary, we report here the patterns of cytokines and soluble receptors in in the sera of children infected with
Trypanosoma cruzi
; we found significantly increased levels of sIL-2R and sCD8 in acute infection that decreased after therapy, and high levels of TNF-α, IL-6, and sCD4 in some of the acute patients. The measurement of sIL-2R and sCD8 may provide a useful tool in the follow-up of children with Chagas' disease.
Antimicrobial Susceptibilities and Serotype Distribution of
Streptococcus pneumoniae
Isolates from a Low Socioeconomic Area in Lima, Peru
Authors: Cullotta Anna R. , Kalter H. D. , Delgado Jose , Gilman Robert H. , Facklam Richard R. , Velapatino Billie , Coronel Jorge , Cabrera Lilia , Urbina M.
Published: November 2002

Streptococcus pneumoniae
isolates were obtained from nasopharyngeal swabs taken from children living in a low socioeconomic area of Lima, Peru, to determine the rates of antimicrobial resistance and serotype distribution. A total of 146 nasopharyngeal isolates were collected from children from 3 to 38 months of age. Twenty-one clinical laboratory isolates from both sterile and nonsterile sites were obtained from a local hospital. Isolates with reduced susceptibilities to penicillin represented 15.1 and 42.9% of the nasopharyngeal and clinical isolates, respectively. For neither group of isolates did penicillin MICs exceed 1.5 μg/ml, indicating only intermediate resistance. Thirty-two different serotypes were identified from the 146 nasopharyngeal isolates. The serotypes of the clinical isolates were represented among those 32 types. Isolates with reduced susceptibility to multiple antimicrobial agents were present in both settings. These findings indicate some of the highest rates of antimicrobial resistance in the region as well as a slightly different serotype distribution pattern from those of other South American countries. The 7-valent conjugate pneumococcal vaccines would only have a limited effect, providing coverage for about half of all isolates. Increasing rates of resistance in Peru necessitate an awareness of antimicrobial treatment practices and vaccination strategies.
Comparative Study of the Presence of
Chlamydia pneumoniae
in Cerebrospinal Fluid of Patients with Clinically Definite and Monosymptomatic Multiple Sclerosis
Authors: Sriram Subramaniam , Yao Song-yi , Stratton Charles , Calabresi Peter , Mitchell William , Ikejima Hideaki , Yamamoto Yoshimasa
Published: November 2002

There is considerable controversy concerning the evidence for the presence of
Chlamydia pneumoniae
in the cerebrospinal fluid (CSF) of both multiple sclerosis (MS) patients and patients with other neurological diseases (OND). In order to clarify this issue, the laboratories at Vanderbilt University Medical Center (VUMC) and the University of South Florida (USF) examined the reproducibility of their respective PCR assays for the detection of
C. pneumoniae
DNA in the CSF of a common group of MS patients and OND controls. The two laboratories used different DNA extraction and PCR techniques in order to determine the prevalence of the
C. pneumoniae
genome in both monosymptomatic and clinically definite MS patients as well as in OND controls. In clinically definite MS patients, the VUMC and USF detection rates were 72 and 61%, respectively, and in patients with monosymptomatic MS, the VUMC and USF detection rates were 41 and 54%, respectively. The PCR signal was positive for 7% of the OND controls at VUMC and for 16% at USF. These studies confirm our previous reports concerning the high prevalence of
C. pneumoniae
in the CSF of MS patients. The presence of
C. pneumoniae
in patients with monosymptomatic MS would also suggest that infection with the organism occurs early in the course of the disease.
Efficiency of Reconstitution of Immunoglobulin G from Blood Specimens Dried on Filter Paper and Utility in Herpes Simplex Virus Type-Specific Serology Screening
Authors: Hogrefe Wayne R. , Ernst Carolyn , Su Xin
Published: November 2002

The performance of studies using sera from remote locations is greatly facilitated if whole-blood samples dried on filter paper are shown to be compatible with the serologic assay being employed. Since dried blood samples do not require immediate refrigeration, occupy little space, and are easily transported, they may be used for evaluating the seroprevalence of herpes simplex virus type 1 (HSV-1) and HSV-2 in geographic locations where laboratory resources are limited. We evaluated the utility of dried blood samples for the detection of type-specific HSV antibodies. The efficiency of using immunoglobulin G (IgG) eluted from dried blood samples was found to be consistent with measurement of IgG concentrations in most corresponding serum samples. The ratio of the mean IgG concentration for all dried blood samples to the mean IgG concentration for the corresponding sera was 1:29. When the 1:29 ratio was applied to each of the 22 pairs of samples, there was a deviation of less than 15% between concentrations in the dried blood sample and in the corresponding serum sample in 19 of the pairs. No positive or negative bias was detected for the IgG eluted from dried blood. The presence of HSV-1 and HSV-2 antibodies was determined in the paired dried blood and serum samples, and no differences in the HSV serostatuses were detected for 43 of the 44 pairs. One pair's serostatus varied, with the serum sample being weakly positive for HSV-1 and the dried blood sample results being equivocal. The detection of HSV antibodies was generally consistent for dried blood samples stored frozen for over 1 year or at room temperature for 30 days, although decreased reactivities were found in a few samples.
Characterization ofToxoplasma gondiiSAG2 Expressed in Insect Cells by Recombinant Baculovirus and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay
Authors: Huang Xiaohong , Xuan Xuenan , Suzuki Hiroshi , Sugimoto Chihiro , Nagasawa Hideyuki , Fujisaki Kozo , Mikami Takeshi , Igarashi Ikuo
Published: November 2002

A baculovirus carrying the SAG2 gene ofToxoplasma gondiiwas constructed, and recombinant SAG2 protein (S-rSAG2) was expressed in insect cells. S-rSAG2 was recognized by sera from cats and pigs infected withT. gondii. Mice immunized with S-rSAG2 produced high titers of specific immunoglobulin G2a (IgG2a) and IgG1 antibodies. In an indirect fluorescent antibody test, all mouse antisera against S-rSAG2 reacted strongly to the natural parasites, but those against rSAG2 expressed inEscherichia coli(E-rSAG2) only showed very weak reaction, although no markedly difference was found in the reaction to denatured antigen,T. gondiilysate, in Western blot analysis. The results suggest that S-rSAG2 is better than E-rSAG2 in both antigenicity and immunogenicity. Enzyme-linked immunosorbent assay (ELISA) with S-rSAG2 could differentiate clearly between sera from 30 specific-pathogen-free cats and 4 experimentally infected cats. Serum samples from domestic cats in Japan were tested by the ELISA and compared with a latex agglutination test (LAT) and ELISA with E-rSAG2. Of 187 samples, all 35 LAT-positive sera had strong reactions to S-rSAG2 and E-rSAG2. Of the 152 LAT-negative sera, 18 were positive in the ELISA with S-rSAG2, whereas only 2 were positive in the ELISA with E-rSAG2. Although there were significant correlations among the three methods, the ELISA with S-rSAG2 was more sensitive than the others, which could be attributed to the fact that S-rSAG2 shares some common conformational structure with the native antigen. The results suggest that S-rSAG2 would be a useful reagent for the detection ofT. gondiiinfection in cats.
Humoral Immune Response Associated with Lyme Borreliosis in Nonhuman Primates: Analysis by Immunoblotting and Enzyme-Linked Immunosorbent Assay with Sonicates or Recombinant Proteins
Authors: Pachner A. R. , Dail D. , Li L. , Gurey L. , Feng S. , Hodzic E. , Barthold S.
Published: November 2002

The immune response to
Borrelia burgdorferi
, the causative agent of Lyme disease, is complex. We studied the immunoglobulin M (IgM) and IgG antibody response to N40Br, a sensu stricto strain, in the rhesus macaque(nonhuman primate [NHP]) model of infection to identify the spirochetal protein targets of specific antibody. Antigens used in enzyme-linked immunosorbent assays were whole-cell sonicates of the spirochete and recombinant proteins of
B. burgdorferi
. Immunoblotting with a commercially available strip and subsequent quantitative densitometry of the bands were also used. Sera from four different groups of NHPs were used: immunocompetent, transiently immunosuppressed, extended immunosuppressed, and uninfected. In immunocompetent and transiently immunosuppressed NHPs, there was a strong IgM and IgG response. Major proteins for the early IgM response were P39 and P41 and recombinant BmpA and OspC. Major proteins for the later IgG response were P39, P41, P18, P60, P66, and recombinant BmpA and DbpA. There was no significant response in the NHPs to recombinant OspA or to Arp, a 37-kDa protein that elicits an antibody response during infection in mice. Most antibody responses, except for that to DbpA, were markedly diminished by prolonged dexamethasone treatment. This study supports the hypothesis that recombinant proteins may provide a useful adjunct to current diagnostic testing for Lyme borreliosis.
Fluorescence Polarization Assay for Detection of
Brucella abortus
Antibodies in Bulk Tank Bovine Milk Samples
Authors: Gall D. , Nielsen K. , Bermudez M. R. , Moreno F. , Smith P.
Published: November 2002

A simple, rapid, inexpensive fluorescence polarization assay for the detection of antibodies to
Brucella abortus
in bulk tank milk samples at the farm level or at dairies with a sensitivity and specificity of 100 and 95.9%, respectively, is described. The assay detects antibodies to
B. abortus
in 15 min by testing undiluted whey produced by chemical and physical manipulation of milk from bulk tanks. This sampling is noninvasive and therefore costs less and is less stressful than blood-based tests. The assay is specific and can detect antibodies at levels below that of the indirect enzyme immunoassay for milk and the fluorescence polarization assay for individual milk samples. Use of this test would make programs for surveillance of dairy animals and eradication of
B. abortus
more cost-effective.
Interpretation of a Commercial Bovine Paratuberculosis Enzyme-Linked Immunosorbent Assay by Using Likelihood Ratios
Authors: Collins M. T.
Published: November 2002

Evidence-based medicine encourages the use of quantitative diagnostic test results to estimate the probability of a particular diagnosis. Likelihood ratios (LRs) are among the best tools for maximizing the diagnostic information gained from diagnostic assays that provide results on a continuous scale. They provide the odds that an animal with a particular test result actually has the disease in question based on the magnitude of the test result. A commercial enzyme-linked immunosorbent assay (ELISA) was used to test sera from 143 dairy cattle infected with
Mycobacterium paratuberculosis
and 2,974 cattle free of this infection. This assay transforms ELISA reader optical density values into sample-to-positive (S/P) ratios. The LR was calculated for S/P results from 0.00 to 1.00 at 0.05-S/P unit intervals. LRs were directly but not linearly correlated with ELISA S/P ratios (
r
2
= 0.94). The mathematical function describing the relationship between the ELISA S/P ratio and the LR was LR = 265 × (S/P value)
2.03
. LRs were also directly related to the frequency of animals testing positive for paratuberculosis by fecal culture and other serologic tests. Based on these LRs, guidelines for interpretation and application of this ELISA for the diagnosis and control of paratuberculosis in dairy cattle herds are recommended.
Concentrations of Cytokines, Soluble Interleukin-2 Receptor, and Soluble CD30 in Sera of Patients with Hepatitis B Virus Infection during Acute and Convalescent Phases
Authors: Monsalve-de Castillo Francisca , Romero Tania A. , Estévez Jesús , Costa Luciana L. , Atencio Ricardo , Porto Leticia , Callejas Diana
Published: November 2002

The immunoregulatory roles of interleukin-2 (IL-2), IL-4, IL-10, gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), the soluble form of the IL-2 receptor (sIL-2R), and the soluble form of CD30 (sCD30) were evaluated in patients with hepatitis B virus (HBV) infection. Two groups of subjects were studied: 15 healthy individuals without hepatitis antecedents and 15 patients with HBV infection. Blood samples were taken during the acute and convalescent phases. The analysis of the samples was done by the enzyme-linked immunosorbent assay technique. IFN-γ and TNF-α levels decreased in the convalescent phase. IL-10, IL-2, and sIL-2R levels increased in the acute and convalescent phases, while sCD30 levels increased during the acute phase. The IL-4 concentrations decreased in both phases. During the acute phase, IFN-γ and TNF-α induced increases in IL-2, sIL-2R, IL-10, and sCD30 levels in serum, which allowed the development of immunity characterized by the nonreactivity of the HBV surface antigen, the onset of antibodies to the HBV surface antigen (anti-HBs), and normal alanine aminotransferase levels during the convalescent phase. Increased IL-2 levels during the acute phase would stimulate the activities of NK cells and CD8
+
lymphocytes, which are responsible for viral clearing. The raised sIL-2R levels reveal activation of T lymphocytes and control of the IL-2-dependent immune response. The sCD30 increment during the acute phase reflects the greater activation of the Th2 cellular phenotype. Its decrease in the convalescent phase points out the decrease in the level of HBV replication. The increase in IL-10 levels could result in a decrease in IL-4 levels and modulate IFN-γ and TNF-α levels during both phases of disease, allowing the maintenance of anti-HBs concentrations.
Defibrination of Blood Plasma for Use in Serological Tests for Syphilis
Authors: Castro Arnold R. , Kikkert Susan E. , Fears Martha B. , Pope Victoria
Published: November 2002

Syphilitic plasma can be salvaged from discarded blood donations and converted to serum by defibrination. Sixty-nine units of plasma were treated with a stock solution of 100 U of thrombin per ml in 1 M calcium chloride and then with a 10% (wt/vol) solution of kaolin. Fibrinogen concentrations detected in initial plasma samples ranged from 94 to 4,970 mg/liter (mean, 2,532 mg/liter) for samples that were reactive by the rapid plasma reagin circle card test (RPR) and from 314 to 2,742 mg/liter (mean 1,528 mg/liter) for samples that were not reactive by the RPR. The treated samples showed no measurable fibrinogen remaining after the defibrination process. In the nontreponemal RPR for syphilis, 86% of the treated plasma samples retained the same endpoint titer as that of the initial plasma sample. When the
Treponema pallidum
passive-particle-agglutination test was used, 98% retained the same reactivity. In the Captia Syphilis-G enzyme immunoassay, 89% of the treated samples demonstrated no change in reactivity index, and in the fluorescent treponemal antibody absorption test, 96% showed no reduction in fluorescence. Human sera containing antibodies to syphilis are used at the Centers for Disease Control and Prevention for the preparation of reference controls or as samples for proficiency testing. Finding reactive sera is becoming more difficult due to the general decline of syphilis cases in the United States. The decreasing availability of these sera can be alleviated by salvaging plasma and converting it to serum.
Use of a Single Monoclonal Antibody To Determine the Susceptibilities of Herpes Simplex Virus Type 1 and Type 2 Clinical Isolates to Acyclovir
Authors: Chutkowski Christine , Olson Betty , McDonough Ann , Mahoney James , McSharry James J.
Published: November 2002

This report describes a flow cytometry drug susceptibility assay that uses a single fluorochrome-labeled monoclonal antibody to determine the acyclovir susceptibilities of herpes simplex virus (HSV) type 1 or type 2 clinical isolates. This assay yields 50% effective doses (drug concentrations that reduce the number of antigen-positive cells by 50%) for HSV clinical isolates that are equivalent to those obtained with the plaque reduction assay.
Differential Immune Response to the Variable Surface Loop Antigen of P66 of
Borrelia burgdorferi
Sensu Lato Species in Geographically Diverse Populations of Lyme Borreliosis Patients
Authors: Ornstein Katharina , Östberg Yngve , Bunikis Jonas , Noppa Laila , Berglund Johan , Norrby Ragnar , Bergström Sven
Published: November 2002

We have studied the immune response to a variable surface-exposed loop region of the P66 outer membrane protein from
Borrelia burgdorferi
sensu lato by using an enzyme immunoassay. Lyme borreliosis populations found in North America and Sweden were preferentially more seroreactive to P66 from their respective regional species, namely,
B. burgdorferi
sensu stricto and
B. garinii
and
B. afzelii
, respectively.
Fully Automated Detection of Hepatitis C Virus RNA in Serum and Whole-Blood Samples
Authors: Kessler Harald H. , Clarici Alexandra M. K. , Stelzl Evelyn , Mühlbauer Gerhard , Daghofer Elisabeth , Santner Brigitte I. , Marth Egon , Stauber Rudolf E.
Published: November 2002

In this study, we established a fully automated molecular assay for qualitative detection of hepatitis C virus (HCV) in serum and whole-blood samples and compared it with conventional molecular assays, including manual HCV RNA extraction protocols. Whole-blood samples were collected from patients with and without chronic HCV infection in EDTA tubes and nucleic acid stabilization tubes (NASTs). Prior to HCV RNA extraction, the HCV Internal Control (IC), derived from the COBAS AMPLICOR HCV test, version 2.0 (Roche Molecular Diagnostics), was added. The new assay was based on an automated extraction protocol on the MagNA Pure LC instrument (Roche Applied Science), followed by automated reverse transcription, amplification, hybridization, and detection on the Cobas Amplicor analyzer (Roche Molecular Diagnostics). The detection limit of the new assay was found to be similar to those of conventional molecular assays. In clinical samples, 100% agreement between the new assay and conventional methods was observed. The introduced amount of IC was detected in 45 of 45 serum samples, 41 of 45 EDTA tube whole-blood samples, and 43 of 45 NAST whole-blood samples. Retesting led to more frequent IC detection. The fully automated molecular assay was found to be suitable for detection of HCV RNA in different kinds of sample materials. It may be recommended for use in the high-throughput routine molecular diagnostic laboratory.
Prophylactic Effect ofMycobacterium bovisBCG Vaccination against Osteomyelitis in Children withMycobacterium ulceransDisease (Buruli Ulcer)
Authors: Portaels F. , Aguiar J. , Debacker M. , Steunou C. , Zinsou C. , Guédénon A. , Meyers W. M.
Published: November 2002

Mycobacterium ulceransdisease, or Buruli ulcer (BU), causes significant morbidity in West Africa. In 233 consecutive, laboratory-confirmed samples from BU patients in Benin whoseMycobacterium bovisBCG scar status was known, 130 children (<15 years old) and 75 adults had a neonatal BCG vaccination scar. Of 130 children with BCG scars, 10 (7.7%) had osteomyelitis, while 3 of 9 children without BCG scars (33.3%) had osteomyelitis. Our observations support the conclusion that having a BCG vaccination scar provides significant protection againstM. ulceransosteomyelitis in children with BU disease.
In Vitro Detection of Apoptosis in Monocytes/Macrophages Infected with Human Coronavirus
Authors: Collins Arlene R.
Published: November 2002

Human coronavirus (HCoV) strain 229E infection, but not HCoV strain OC43 infection, of monocytes/macrophages from healthy donors and patients with multiple sclerosis in remission resulted in increased apoptosis, as measured by DNA changes and annexin V staining. Apoptosis correlated with the differential release of infectious virus. HCoV strain 229E titers were 10
3.5
to 10
6
50% tissue culture-infective doses (TCID
50
)/ml, and HCoV strain OC43 titers were only 10
1.2
to 10
2.7
TCID
50
/ml.
Dichotomal Effect of the Coumadin Derivative Warfarin on Inflammatory Signal Transduction
Authors: Kater Arnon P. , Peppelenbosch Maikel P. , Brandjes Dees P. M. , Lumbantobing Mika
Published: November 2002

Warfarin, a widely prescribed drug for preventing thrombosis, is thought to act solely through inhibition of vitamin K-dependent coagulation factors. Low concentrations of warfarin inhibit interleukin-6 production and phosphorylation of I-κB but not activation of p38 mitogen-activated protein kinase. Thus, warfarin inhibits inflammatory signal transduction, and this may contribute to clinical effects of warfarin.
Coombs Antiglobulin Test Using
Brucella abortus
99 as Antigen To Detect Incomplete Antibodies Induced by
B. abortus
RB51 Vaccine in Cattle
Authors: Ciuchini Franco , Adone Rosanna , Pasquali Paolo
Published: November 2002

This study showed that vaccination of cattle with
Brucella abortus
rough strain RB51 induces incomplete antibodies that can be detectable by a Coombs antiglobulin test using the
B. abortus
99 smooth strain.
Peptides Containing Cyclin/Cdk-Nuclear Localization Signal Motifs Derived from Viral Initiator Proteins Bind to DNA When Unphosphorylated
Authors: Kim Ronald J. , Moine Stephanie , Reese Danielle K. , Bullock Peter A.
Published: November 2002

A single phosphorylation event at T-antigen residue Thr124 regulates initiation of simian virus 40 DNA replication. To explore this regulatory process, a series of peptides were synthesized, centered on Thr124. These peptides contain a nuclear localization signal (NLS) and a recognition site for cyclin/Cdk kinases. When unphosphorylated, the “CDK/NLS” peptides inhibit T-antigen assembly and bind non-sequence specifically to DNA. However, these activities are greatly reduced upon phosphorylation of Thr124. Similar results were obtained by using peptides derived from the CDK/NLS region of bovine papillomavirus E1. Related studies indicate that residues in the NLS bind to DNA, whereas those in the CDK motif regulate binding. These findings are discussed in terms of the control of T-antigen double hexamer assembly and initiation of viral replication.