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In addition to humans, only certain nonhuman primates are naturally susceptible to measles virus (MeV) infection. Disease severity is species dependent, ranging from mild to moderate for macaques to severe and even lethal for certain New World monkey species. To investigate if squirrel monkeys ( Saimiri sciureus ), which are reported to develop a course of disease similar to humans, may be better suited than macaques for the identification of virulence determinants or the evaluation of therapeutics, we infected them with a green fluorescent protein-expressing MeV. Compared to cynomolgus macaques ( Macaca fascicularis ) infected with the same virus, the squirrel monkeys developed more-severe immunosuppression, higher viral load, and a broader range of clinical signs typical for measles. In contrast, infection with an MeV unable to interact with the epithelial receptor nectin-4, while causing immunosuppression, resulted in only a mild and transient rash and a short-lived elevation of the body temperature. Similar titers of the wild-type and nectin-4-blind MeV were detected in peripheral blood mononuclear cells and lymph node homogenates, but only the wild-type virus was found in tracheal lavage fluids and urine. Thus, our study demonstrates the importance of MeV interactions with nectin-4 for clinical disease in the new and better-performing S. sciureus model of measles pathogenesis. IMPORTANCE The characterization of mechanisms underlying measles virus clinical disease has been hampered by the lack of an animal model that reproduces the course of disease seen in human patients. Here, we report that infection of squirrel monkeys ( Saimiri sciureus ) fulfills these requirements. Comparative infection with wild-type and epithelial cell receptor-blind viruses demonstrated the importance of epithelial cell infection for clinical disease, highlighting the spread to epithelia as an attractive target for therapeutic strategies., (Copyright © 2017 American Society for Microbiology.)

An open-label pharmacokinetics (PK) clinical trial was conducted to comparatively assess the PK and explore the pharmacodynamics (PD) of miltefosine in children and adults with cutaneous leishmaniasis (CL) in Colombia. Sixty patients, 30 children aged 2 to 12 years and 30 adults aged 18 to 60 years, were enrolled. Participants received miltefosine (Impavido) at a nominal dose of 2.5 mg/kg/day for 28 days. Miltefosine concentrations were measured in plasma and peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry of samples obtained during treatment and up to 6 months following completion of treatment, when therapeutic outcome was determined. Fifty-two patients were cured, 5 pediatric patients failed treatment, and 3 participants were lost to follow-up. Leishmania ( Viannia ) panamensis predominated among the strains isolated (42/46; 91%). Noncompartmental analysis demonstrated that plasma and intracellular miltefosine concentrations were, overall, lower in children than in adults. Exposure to miltefosine, estimated by area under the concentration-time curve and maximum concentration, was significantly lower in children in both the central and intracellular compartments ( P < 0.01). Leishmania persistence was detected in 43% of study participants at the end of treatment and in 27% at 90 days after initiation of treatment. Clinical response was not dependent on parasite elimination. In vitro miltefosine susceptibility was similar for Leishmania strains from adults and children. Our results document PK differences for miltefosine in children and adults with cutaneous leishmaniasis that affect drug exposure and could influence the outcome of treatment, and they provide bases for optimizing therapeutic regimens for CL in pediatric populations. (This study has been registered at ClinicalTrials.gov under identifier NCT01462500.)., (Copyright © 2017 Castro et al.)

Major histocompatibility complex class II-deficient (MHC-II KO; Aβ(-/-)) mice were used to assess the roles of MHC-II molecules in inducing protective immune responses to vaccination. After vaccination with influenza A/PR8 virus-like particle (VLP) vaccine, in vivo and in vitro vaccine antigen-specific IgG isotype antibodies were not detected in MHC-II KO mice, which is quite different from CD4 T cell-deficient mice that induced vaccine-specific IgG antibodies. The deficiency in MHC-II did not significantly affect the induction of antigen-specific IgM antibody in sera. MHC-II KO mice that were vaccinated with influenza VLP, whole inactivated influenza virus, or live attenuated influenza virus vaccines were not protected against lethal infection with influenza A/PR8 virus. Adoptive transfer of fractionated spleen cells from wild-type mice to MHC-II KO mice indicated that CD43(+) cell populations with MHC-II contributed more significantly to producing vaccine-specific IgG antibodies than CD43(-) B220(+) conventional B cell or CD4 T cell populations, as well as conferring protection against lethal infection. Bone marrow-derived dendritic cells from MHC-II KO mice showed a significant defect in producing interleukin-6 and tumor necrosis factor alpha cytokines. Thus, results indicate that MHC-II molecules play multiple roles in inducing protective immunity to influenza vaccination. Importance: Major histocompatibility complex class II (MHC-II) has been known to activate CD4 T helper immune cells. A deficiency in MHC-II was considered to be equivalent to the lack of CD4 T cells in developing host immune responses to pathogens. However, the roles of MHC-II in inducing protective immune responses to vaccination have not been well understood. In the present study, we demonstrate that MHC-II-deficient mice showed much more significant defects in inducing protective antibody responses to influenza vaccination than CD4 T cell-deficient mice. Further analysis showed that CD43 marker-positive immune cells with MHC-II, as well as an innate immunity-simulating adjuvant, could rescue some defects in inducing protective immune responses in MHC-II-deficient mice. These results have important implications for our understanding of host immunity-inducing mechanisms to vaccination, as well as in developing effective vaccines and adjuvants., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

CD40 and CD40 ligand (CD40L) have costimulatory effects as part of a complex series of events in host immunity. In this study, the expression of CD40 and CD40L on peripheral blood mononuclear cells (PBMCs) isolated from cattle with Johne's disease were measured on freshly isolated PBMCs and on cells cultured for 8, 24, and 72 h in the presence or absence of live Mycobacterium avium subsp. paratuberculosis and exogenous gamma interferon, interleukin 10, and transforming growth factor β. Results demonstrated greater CD40 and CD40L expression on fresh PBMCs obtained from animals in the clinical stage of disease (symptomatic) than those from healthy control animals or cows in the subclinical stage of disease (asymptomatic). A similar expression profile with greater magnitude was noted for cultured PBMCs, with increased CD40 expression after 8 and 24 h of culture and increased CD40L expression between 24 and 72 h on PBMCs obtained from clinically infected animals. The addition of live M. avium subsp. paratuberculosis to cell cultures resulted in downregulation of CD40L expression in naturally infected cows, regardless of the disease stage. In contrast, the addition of live M. avium subsp. paratuberculosis to cultures resulted in upregulation of CD40 expression on cells obtained from clinically infected animals, while a decrease in expression was noted for healthy and subclinically infected cows. No effects of exogenous cytokines on CD40 or CD40L expression were observed. These results clearly point for the first time to a disparity in the expression of these costimulatory molecules on immune cells from cattle in different stages of Johne's disease and suggest further investigation into their roles in paratuberculosis pathogenesis.

The current treatment of chronic hepatitis C is based on pegylated alpha interferon (PEG-IFN-α) and ribavirin. The aim of this study was to identify biological and clinical variables related to IFN therapy that could predict patient outcome. The study enrolled 47 patients treated with PEG-IFN and ribavirin combined therapy. The interferon concentration was measured in serum by a bioassay. The expression of 93 interferon-regulated genes in peripheral blood mononuclear cells was quantified by real-time quantitative reverse transcription-PCR (RT-PCR) before and after 1 month of treatment. The interferon concentration in the serum was significantly lower in nonresponders than in sustained virological responders. Moreover, a significant correlation was identified between interferon concentration and interferon exposition as well as body weight. The analysis of interferon-inducible genes in peripheral blood mononuclear cells among the genes tested did not permit the prediction of treatment outcome. In conclusion, the better option seems to be to treat patients with weight-adjusted PEG-IFN doses, particularly for patients with high weight who are treated with PEG-IFN-α2a. Although the peripheral blood mononuclear cell samples are the easiest to obtain, the measurement of interferon-inducible genes seems not be the best strategy to predict treatment outcome.

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β(2)-microglobulin (β(2)m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.

Staphylococcal enterotoxins are potent activators for human T cells and cause lethal toxic shock. Rapamycin, an immunosuppressant, was tested for its ability to inhibit staphylococcal enterotoxin B (SEB)-induced activation of human peripheral blood mononuclear cells (PBMC) in vitro and toxin-mediated shock in mice. Stimulation of PMBC by SEB was effectively blocked by rapamycin as evidenced by the inhibition of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-2, gamma interferon (IFN-gamma), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and T-cell proliferation. In vivo, rapamycin protected 100% of mice from lethal shock, even when administered 24 h after intranasal SEB challenge. The serum levels of MCP-1 and IL-6, after intranasal exposure to SEB, were significantly reduced in mice given rapamycin versus controls. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB.

The APOBEC3H gene is polymorphic in humans, with four major population-dependent haplotypes that encode proteins with different levels of antiviral activity. Haplotype II, present most frequently in African populations, encodes the most stable protein and is most active against human immunodeficiency virus type 1 (HIV-1). In contrast to human APOBEC3G, which can be completely counteracted by HIV-1 Vif, the protein encoded by APOBEC3H haplotype II is only partially sensitive to Vif, while the protein encoded by APOBEC3H haplotype I is completely resistant to HIV-1 Vif. We mapped a residue on APOBEC3H that determines this partial Vif sensitivity. However, it is unclear how HIV-1 can replicate in vivo without the ability to neutralize APOBEC3H antiviral activity. In order to directly address this question, we cloned vif genes from HIV-1-infected individuals with different APOBEC3H genotypes and tested them for their ability to inhibit human APOBEC3H. We found that while the APOBEC3H genotype of infected individuals significantly influences the activity of Vif encoded by their virus, none of the Vif variants tested can completely neutralize APOBEC3H as well as they neutralize APOBEC3G. Consistent with this genetic result, APOBEC3H protein expression in human peripheral blood mononuclear cells was below our limit of detection using newly developed antibodies against the endogenous protein. These results demonstrate that human APOBEC3H is not as strong of a selective force for current HIV-1 infections as human APOBEC3G.

We have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55(gag) virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4(+) T cells in an ex vivo immunization assay. In the present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells (PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14(+) cell populations to an immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation induced by HIV-VLPs in CD14(+) circulating cells, which does not consistently correlate with an advanced status of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of "responsive" vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.

The extracellular adhesion protein (Eap) secreted by the major human pathogen Staphylococcus aureus is known to have several effects on human immunity. We have recently added to knowledge of these roles by demonstrating that Eap enhances interactions between major histocompatibility complex molecules and human leukocytes. Several studies have indicated that Eap can induce cytokine production by human peripheral blood mononuclear cells (PBMCs). To date, there has been no rigorous attempt to identify the breadth of cytokines produced by Eap stimulation or to identify the cell subsets that respond. Here, we demonstrate that Eap induces the secretion of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) by CD14(+) leukocytes (monocytes and macrophages) within direct ex vivo PBMC populations (note that granulocytes are also CD14(+) but are largely depleted from PBMC preparations). Anti-intercellular adhesion molecule 1 (CD54) antibodies inhibited this induction and implicated a role for this known Eap binding protein in cellular activation. IL-6 and TNF-alpha secretion by murine cells exposed to Eap was also observed. The activation of CD14(+) cells by Eap suggests that it could play a significant role in both septic shock and fever, two of the major pathological features of S. aureus infections.

The primary objective of this study was to measure atazanavir-ritonavir and tenofovir pharmacokinetics when the drugs were used in combination in young adults with human immunodeficiency virus (HIV). HIV-infected subjects > or =18 to <25 years old receiving (> or =28 days) 300/100 mg atazanavir-ritonavir plus 300 mg tenofovir disoproxil fumarate (TDF) plus one or more other nucleoside analogs underwent intensive 24-h pharmacokinetic studies following a light meal. Peripheral blood mononuclear cells were obtained at 1, 4, and 24 h postdose for quantification of intracellular tenofovir diphosphate (TFV-DP) concentrations. Twenty-two subjects were eligible for analyses. The geometric mean (95% confidence interval [CI]) atazanavir area under the concentration-time curve from 0 to 24 h (AUC(0-24)), maximum concentration of drug in serum (C(max)), concentration at 24 h postdose (C(24)), and total apparent oral clearance (CL/F) values were 35,971 ng x hr/ml (30,853 to 41,898), 3,504 ng/ml (2,978 to 4,105), 578 ng/ml (474 to 704), and 8.3 liter/hr (7.2 to 9.7), respectively. The geometric mean (95% CI) tenofovir AUC(0-24), C(max), C(24), and CL/F values were 2,762 ng.hr/ml (2,392 to 3,041), 254 ng/ml (221 to 292), 60 ng/ml (52 to 68), and 49.2 liter/hr (43.8 to 55.3), respectively. Body weight was significantly predictive of CL/F for all three drugs. For every 10-kg increase in weight, there was a 10%, 14.8%, and 6.8% increase in the atazanavir, ritonavir, and tenofovir CL/F, respectively (P < or = 0.01). Renal function was predictive of tenofovir CL/F. For every 10 ml/min increase in creatinine clearance, there was a 4.6% increase in tenofovir CL/F (P < 0.0001). The geometric mean (95% CI) TFV-DP concentrations at 1, 4, and 24 h postdose were 96.4 (71.5 to 130), 93.3 (68 to 130), and 92.7 (70 to 123) fmol/million cells. There was an association between renal function, tenofovir AUC, and tenofovir C(max) and intracellular TFV-DP concentrations, although none of these associations reached statistical significance. In these HIV-infected young adults treated with atazanavir-ritonavir plus TDF, the atazanavir AUC was similar to those of older adults treated with the combination. Based on data for healthy volunteers, a higher tenofovir AUC may have been expected, but was not seen in these subjects. This might be due to faster tenofovir CL/F because of higher creatinine clearance in this age group. Additional studies of the exposure-response relationships of this regimen in children, adolescents, and adults would advance our knowledge of its pharmacodynamic properties.

APOBEC3F and APOBEC3G (hA3F and hA3G) are part of an innate mechanism of antiretroviral defense. The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif targets both proteins for proteasomal degradation. Using mRNA from peripheral blood mononuclear cells of 92 HIV-infected subjects not taking antiretroviral therapy and 19 HIV-uninfected controls, we found that hA3F (P < 0.001) and hA3G (P = 0.016) mRNA levels were lower in HIV-infected subjects and were positively correlated with one another (P = 0.003). However, we found no correlation in the abundance of either hA3F or hA3G mRNA with either viral load or CD4 counts in HIV-infected subjects.

Previously, we have shown that macrophage migration inhibitory factor (MIF) was highly elevated in the placental intervillous blood (IVB) of Plasmodium falciparum-infected women. Here, we compared the expression of MIF in placental tissues obtained from P. falciparum-infected and -uninfected women. Immunoperoxidase staining showed a consistent pattern of MIF expression in syncytiotrophoblasts, extravillous trophoblasts, IVB mononuclear cells, and amniotic epithelial cells, irrespective of their malaria infection status. Cytotrophoblast, villous stroma, and Hofbauer cells showed focal staining. Only amniotic epithelial and IVB mononuclear cells from P. falciparum-infected placentas exhibited significantly higher level of MIF expression than uninfected placentas. Stimulation of syncytilized human trophoblast BeWo cells with P. falciparum-infected erythrocytes that were selected to bind these cells resulted in significant increases in MIF secretion, whereas control erythrocytes, lipopolysaccharides, and synthetic beta-hematin had minimal effect. These findings suggest that placental malaria modulates MIF expression in different placental compartments.

Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9). However, the nature and cellular localization of TLR9 in primary human cells remain controversial. Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface. The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed. Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9(+) cells were confined to the major histocompatibility complex (MHC) class II(+) CD19(-) populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II(+) CD19(+) cells. Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10%. In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression. Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization. Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.

The aim of this study was to investigate the relationship between lymphocyte P-glycoprotein (P-gp) expression and genotype in vivo and the expression of lymphocyte receptors critical in the life cycle of human immunodeficiency virus type 1 (HIV-1), i.e., CD4, CCR5, and CXCR4. Using flow cytometry to quantify each membrane receptor/transporter, we demonstrate a highly significant correlation between P-gp protein expression and the expression of CXCR4 (rho = 0.874; P < 0.0001). Furthermore, confocal microscopy showed colocalized expression of CXCR4 and P-gp in the lymphocyte membrane. This significant relationship was also apparent at the mRNA level by use of reverse transcription-PCR (rho = 0.61; P < 0.005) and was present in both phytohemagglutinin-stimulated and unstimulated peripheral blood mononuclear cells. Genotypic analysis of the C3435T single-nucleotide polymorphism of P-gp confirmed significantly higher levels of P-gp in C (range, 2.45 to 11.00 relative fluorescence units [RFU])- than in T (range, 0.25 to 5.00 RFU)-homozygous individuals (P = 0.0088; 95% confidence interval [95% CI], 0.7 to 6.3 RFU). An equivalent association between CXCR4 levels and C (range, 12.7 to 44.1 RFU) versus T (range, 3 to 18.9 RFU) genotype was also demonstrated (P = 0.0019; 95% CI, 5.4 to 23.7). Functionally, although these correlates had no impact on HIV-1 production from either X4- or R5-tropic virus, expression correlated significantly with the activity of the HIV-1 protease inhibitor (PI) saquinavir for both P-gp (rho = 0.75; P = 0.0019) and CXCR4 (rho = 0.71; P = 0.0041). This study defines an association between P-gp (expression and genotype) and CXCR4 that may have implications for the selection of viral tropism and the access of drugs to protease for specific tropic types. The interplay between these two proteins may also influence the viral genotypes which escape effective chemotherapy and which therefore have the opportunity to evolve resistance to PIs.

This report describes the presence and activity of 1,25-dihydroxyvitamin D3 (1,25-D3) in experimental bovine tuberculosis. Animals that went on to develop tuberculous lesions exhibited a rapid transient increase in serum 1,25-D3 within the first 2 weeks following infection with Mycobacterium bovis. 1,25-D3-positive mononuclear cells were later identified in all tuberculous granulomas by immunohistochemical staining of postmortem lymph node tissue. These results suggest a role for 1,25-D3 both at the onset of infection and in the development of the granuloma in these infected animals. Using a monoclonal antibody to the vitamin D receptor (VDR) as a VDR agonist, we confirmed that activation of the vitamin D pathway profoundly depresses antigen-specific, but not mitogenic, bovine peripheral blood T-cell responses (proliferation and gamma interferon production). Investigation of the mechanism of this suppression showed that the VDR antibody modified the expression of CD80 by accessory cells, such that a significant positive correlation between T-cell proliferation and accessory cell CD80 emerged.

CXCR4 expression on feline peripheral blood mononuclear cells (PBMC) was analyzed. While monocytes and B lymphocytes expressed CXCR4, no CXCR4 was detected on T lymphocytes, in stark contrast to the expression pattern on T lymphocytes from humans. In spite of the important role that CXCR4 plays in infection with feline immunodeficiency virus, expression on PBMC in vivo was unaffected by infection with either a primary or a cell culture-adapted virus strain.

Resistance to HIV-1 infection and delayed disease progression have been associated with a 32-bp deletion (Delta32) in the gene encoding the CCR5 chemokine receptor. In the present study we describe the modification of a nucleic acid sequence-based amplification (NASBA)-based CCR5 genotyping assay for a NucliSens Basic Kit (Organon Teknika, Durham, N.C.) format using a new target-specific sandwich oligonucleotide detection methodology. The new method permitted the use of generic electrochemiluminescent probes supplied in the NucliSens Basic Kit, whereas the original NASBA method required expensive target-specific ruthenium detection probes. The Basic Kit CCR5 Delta32 genotypic analysis was in 100% concordance with both the original NASBA assay and DNA PCR results. This study also evaluated the use of multiple specimen types, including peripheral blood mononuclear cells (PBMC), whole blood, dried blood spots, buccal scrapings, and plasma, for CCR5 genotype analysis. The sensitivities of the three assays were comparable when PBMC or whole blood was the specimen source. In contrast, when dried blood spots, buccal scrapings, or plasma was used as the sample source, the sensitivity of DNA PCR was 80.95, 42.8, or 0%, respectively, compared to 100% sensitivity obtained with the original NASBA and Basic Kit NASBA assays. Our study indicates that the NucliSens Basic Kit NASBA assay is very sensitive and specific for CCR5 Delta32 genotyping using multiple sample types.

To study what determines the arthritogenicity of bacterial cell walls, cell wall-induced arthritis in the rat was applied, using four strains of Lactobacillus. Three of the strains used proved to induce chronic arthritis in the rat; all were Lactobacillus casei. The cell wall of Lactobacillus fermentum did not induce chronic arthritis. All arthritogenic bacterial cell walls had the same peptidoglycan structure, whereas that of L. fermentum was different. Likewise, all arthritogenic cell walls were resistant to lysozyme degradation, whereas the L. fermentum cell wall was lysozyme sensitive. Muramic acid was observed in the liver, spleen, and lymph nodes in considerably larger amounts after injection of an arthritogenic L. casei cell wall than following injection of a nonarthritogenic L. fermentum cell wall. The L. casei cell wall also persisted in the tissues longer than the L. fermentum cell wall. The present results, taken together with those published previously, underline the possibility that the chemical structure of peptidoglycan is important in determining the arthritogenicity of the bacterial cell wall.

An analytical methodology combining solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) was developed to quantitate the intracellular active 5'-triphosphate (TP) of beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (emtricitabine) (FTC) in human peripheral blood mononuclear cells (PBMCs). The FTC nucleotides, including 5'-mono-, di-, and triphosphates, were successively resolved on an anion-exchange SPE cartridge by applying a gradient of potassium chloride. The FTC-TP was subsequently digested to release the parent nucleoside that was finally analyzed by HPLC with UV detection (HPLC-UV). Validation of the methodology was performed by using PBMCs from healthy donors exposed to an isotopic solution of [(3)H]FTC with known specific activity, leading to the formation of intracellular FTC-TP that was quantitated by an anion-exchange HPLC method with radioactive detection. These levels of FTC-TP served as reference values and were used to validate the data obtained by HPLC-UV. The assay had a limit of quantitation of 4. 0 pmol of FTC-TP (amount on column from approximately 10(7) cells). Intra-assay precision (coefficient of variation percentage of repeated measurement) and accuracy (percentage deviation of the nominal reference value), estimated by using quality control samples at 16.2, 60.7, and 121.5 pmol, ranged from 1.3 to 3.3% and -1.0 to 4. 8%, respectively. Interassay precision and accuracy varied from 3.0 to 10.2% and from 2.5 to 6.7%, respectively. This methodology was successfully applied to the determination of FTC-TP in PBMCs of patients infected with human immunodeficiency virus after oral administration of various dosing regimens of FTC monotherapy.

ESAT-6 (for 6-kDa early secreted antigenic target) is a secreted antigen found almost exclusively in organisms of the Mycobacterium tuberculosis complex. We compared in vitro gamma interferon (IFN-gamma) responses by peripheral blood mononuclear cells to this antigen in patients with pulmonary disease due to either Mycobacterium avium complex (MAC) or Mycobacterium tuberculosis with those in healthy, skin test-negative, control subjects. Significant IFN-gamma responses to ESAT-6 were detected in 16 (59%) of 27 M. tuberculosis pulmonary disease patients, 0 (0%) of 8 MAC disease patients, and 0 (0%) of 8 controls. Significant IFN-gamma responses to M. tuberculosis purified protein derivative were detected in 23 (85%) of 27 M. tuberculosis disease patients, 2 (25%) of 8 MAC disease patients, and 5 (63%) of 8 healthy controls. M. avium sensitin was recognized in 24 (89%) of 27 M. tuberculosis disease patients, 4 (50%) of 8 MAC disease patients, and 1 (13%) of 8 controls. IFN-gamma responses to ESAT-6 are specific for disease due to M. tuberculosis and are not observed in patients with MAC disease or in healthy controls.

Tumor necrosis factor alpha (TNF-alpha) is a critical cytokine that mediates the toxic effects of bacterial superantigens like staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST-1). Pentoxifylline, an anti-inflammatory agent that inhibits endotoxemia and lipopolysaccharide (LPS)-induced release of TNF-alpha, was tested for its ability to inhibit SEB- and TSST-1-induced activation of human peripheral blood mononuclear cells (PBMCs) in vitro and toxin-mediated shock in mice. Stimulation of PBMCs by SEB or TSST-1 was effectively blocked by pentoxifylline (10 mM), as evidenced by the inhibition of TNF-alpha, interleukin 1beta (IL-1beta), gamma interferon (IFN-gamma), and T-cell proliferation. The levels of TNF-alpha, IL-1alpha, and IFN-gamma in serum after an SEB or TSST-1 injection were significantly lower in mice given pentoxifylline (5.5 mg/animal) versus control mice. Additionally, pentoxifylline diminished the lethal effects and temperature fluctuations elicited by SEB and TSST-1. Thus, in addition to treating endotoxemias, the cumulative in vitro and in vivo data suggest that pentoxifylline may also be useful in abrogating the ill effects of staphylococcal enterotoxins and TSST-1.

Preterm infants have an increased incidence of infection, which is principally due to deficiencies in neonatal host defense mechanisms. Monocyte adherence is important in localizing cells at sites of infection and is associated with enhanced antimicrobial functions. We isolated cord blood monocytes from preterm and full-term infants to study their adhesion and immune functions, including superoxide (O2-) generation, degranulation, and cytokine secretion and their adhesion receptors. O2- production and degranulation were significantly diminished, by 28 and 37%, respectively, in adherent monocytes from preterm infants compared to full-term infants (P < 0. 05); however, these differences were not seen in freshly isolated cells. We also observed a significant decrease of 35% in tumor necrosis factor alpha secretion by lipopolysaccharide-stimulated adherent monocytes from preterm infants compared to full-term infants (P < 0.05); however, this difference was not observed in interleukin-1beta or interleukin-6 production by the monocytes. The cell surface expression of the CD11b/CD18 adhesion receptor subunits was significantly decreased (by 60 and 52%, respectively) in monocytes from preterm infants compared to full-term infants (P < 0. 01). The cascade of the immune response to infection involves monocyte upregulation and adherence via CD11b/CD18 receptors followed by cell activation and the release of cytokines and bactericidal products. We speculate that monocyte adherence factors may be important in the modulation of immune responses in preterm infants.

A new sensitive method for the measurement of lamivudine triphosphate (3TC-TP), the active intracellular metabolite of lamivudine in human cells in vivo, has been established. The procedure involves rapid separation of 3TC-TP by using Sep-Pak cartridges, dephosphorylation to 3TC by using acid phosphatase, and measurement by radioimmunoassay using a newly developed anti-3TC serum. The radioimmunoassay had errors of less than 21% and a cross-reactivity of less than 0.016% with a wide variety of other nucleoside analogs. The limit of quantitation of the assay for intracellular 3TC-TP was 0.195 ng/ml (0.212 pmol/10(6) cells), and a cell sample of only 4 million cells was ample for the assay. This procedure, combined with our previously developed method for measuring zidovudine (ZDV) metabolite levels, proved capable of measuring 3TC-TP, ZDV monophosphate (ZDV-MP) and ZDV triphosphate (ZDV-TP) in human immunodeficiency virus (HIV)-infected subjects treated with combination 3TC and ZDV therapy. In seven subjects, intracellular 3TC-TP levels ranged from 2.21 to 7.29 pmol/10(6) cells, while intracellular ZDV-MP and ZDV-TP levels ranged from <0. 01 to 1.76 and 0.01 to 0.07 pmol/10(6) cells, respectively. Concentrations of 3TC in plasma determined in these subjects ranged from 0.34 to 9.40 microM, which was about fivefold higher than ZDV levels in plasma of 0.04 to 1.4 microM. This is the first study to determine the intracellular levels of the active metabolites in HIV-infected subjects treated with this combination. These methods should prove very useful for in vivo pharmacodynamic studies of combination therapy.

The production of interleukin 2 (IL-2) gamma interferon, IL-4, tumor necrosis factor alpha (TNF-alpha), TNF-beta, 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, and Staphylococcus aureus Cowen. The results obtained provide a normal range for the production of these cytokines under specified conditions in vitro.

We compared the immunological responses of leukocytes taken from healthy negative controls, laboratory workers immunized with the phenol-insoluble French vaccine against brucellosis, patients acutely ill with brucellosis, and patients chronically infected with Brucella melitensis. A salt-extractable antigen (protein-rich but with traces of lipopolysaccharide) and a sonicated suspension from B. melitensis 16M were used as antigens for in vitro lymphocyte proliferation test. Quantitation of T cells showed that the ratio of CD4+/CD8+ cells decreased as the condition of the patient deteriorated. An assay to quantitate the cell-mediated immunity showed that the activities of mononuclear cells stimulated with concanavalin A increased as the disease progressed as well.

The cellular tropism of hepatitis C virus (HCV) was studied in vivo in samples from patients with persistent HCV infection. Plasma, liver, peripheral blood mononuclear cell (PBMC), and bone marrow cell (BMC) samples from 15 subjects positive for anti-HCV antibodies were tested for the presence of HCV RNA sequences by reverse transcription PCR. Virus-specific RNA sequences were found to be present in liver samples from all subjects (100%), in plasma samples from 13 of 15 patients (86.7%), in PBMC samples from 3 patients (20%), and in BMC samples from 9 (60%) of the 15 anti-HCV-positive patients enrolled in this study. The presence of the molecular intermediate of HCV replication (the negative-stranded HCV RNA) was evident in the two of the three PBMC and in five of the nine BMC HCV RNA-positive samples. Finally, we studied the nucleotide sequence of a large portion (-270 to -59) of the 5'untranslated region of HCV amplified from plasma samples of 12 of the 15 patients with and without HCV in BMCs; the degree of heterogeneity compared with the prototype HCV sequence was similar in both groups. The data principally indicate that HCV infection of PBMCs and BMCs is frequent in persistently infected patients, as shown by the occurrence of positive- and negative-stranded HCV RNA, thus suggesting the possibility of extrahepatic replication of HCV.

In vitro studies of zidovudine (ZDV) phosphorylation may not accurately reflect the in vivo dose-response relationship, which is crucial to determining the relationship between ZDV exposure, efficacy, and toxicity. However, measurement of ZDV phosphorylated anabolites in peripheral blood mononuclear cells (PBMCs) from ZDV-treated human immunodeficiency virus (HIV)-infected patients would be extremely useful in the more appropriate utilization of ZDV in the treatment of HIV infection. We developed a specific and sensitive combined high-pressure liquid chromatography (HPLC)-radioimmunoassay (RIA) procedure for the determination of ZDV, ZDV-monophosphate, ZDV-diphosphate, and ZDV-triphosphate in PBMCs taken from ZDV-treated HIV-infected patients. ZDV and its anabolites were extracted from washed, Ficoll-Paque-isolated PBMCs and then separated by HPLC using a strong anion-exchange column. The anabolites were then hydrolyzed to ZDV with acid phosphatase. ZDV was then measured by using a modified commercially available RIA protocol. Our method was validated by measuring [3H]ZDV anabolites generated in Molt-4 cells radioisotopically and simultaneously by the combined HPLC-RIA procedure. The ZDV determinations correlated well (r2 = 0.97) over the range of 0.037 to 5.2 pmol (10 to 1,400 pg) per assay tube. Furthermore, we defined the stability of ZDV anabolites during ficoll isolation and the recovery after extraction and cleanup. We then measured intracellular parent ZDV and its phosphorylated anabolites in PBMCs from six ZDV-treated HIV-infected patients (PBMCs were taken 2 h after a 300-mg oral dose). The mean concentrations ( +/- standard deviations) of parent and of mono-, di-, and triphosphates were 0.15 +/- 0.08, 1.4 +/-, 0.082 +/- 0.02, and 0.081 +/- 0.03 pmol/10(6) PBMC, respectively (one pmol/10(6) PBMC represents a concentration of approximately 1 microm). Concurrent serum ZDV concentrations were between 1.3 and 7.1 microm. This method should provide a useful tool for evaluating in vivo pharmacokinetics of ZDV anabolites in PBMCs and possibly other cell types, even at the low doses of ZDV currently administered therapeutically.

Gene amplification of virus-specific sequences is widely used as a method to detect or confirm human immunodeficiency virus (HIV) infection. In this study we used an enzyme-linked affinity assay to quantify polymerase chain reaction products from whole blood, plasma, and separated mononuclear cells collected in the presence of four common anticoagulants: acid citrate dextrose, sodium EDTA, potassium oxalate, and sodium heparin. Attenuation of the product signal was observed after amplification of nucleic acid extraction from whole blood, washed mononuclear cells, and plasma from specimens collected in sodium heparin. These inhibitory effects on gene amplification could be reversed with heparinase. The addition of as little as 0.05 U of heparin completely inhibited amplification of an HLA-DQa sequence from placental DNA. We conclude that heparin can cause attenuation or inhibition of gene amplification. Acid citrate dextrose and EDTA, which lack inhibitory activity, are the most appropriate anticoagulants for clinical blood samples when polymerase chain reaction amplification is anticipated.