ß-Catenin and NF-κB co-activation triggered by TLR3 stimulation facilitates stem cell-like phenotypes in breast cancer.

Cancer stem cells (CSCs) are responsible for tumor initiation and progression. Toll-like receptors (TLRs) are highly expressed in cancer cells and associated with poor prognosis. However, a linkage between CSCs and TLRs is unclear, and potential intervention strategies to prevent TLR stimulation-induced CSC formation and underlying mechanisms are lacking. Here, we demonstrate that stimulation of toll-like receptor 3 (TLR3) promotes breast cancer cells toward a CSC phenotype in vitro and in vivo. Importantly, conventional NF-κB signaling pathway is not exclusively responsible for TLR3 activation-enriched CSCs. Intriguingly, simultaneous activation of both ß-catenin and NF-κB signaling pathways, but neither alone, is required for the enhanced CSC phenotypes. We have further identified a small molecule cardamonin that can concurrently inhibit ß-catenin and NF-κB signals. Cardamonin is capable of effectively abolishing TLR3 activation-enhanced CSC phenotypes in vitro and successfully controlling TLR3 stimulation-induced tumor growth in human breast cancer xenografts. These findings may provide a foundation for developing new strategies to prevent the induction of CSCs during cancer therapies.

Comparison of interferon-γ-, interleukin (IL)-17- and IL-22-expressing CD4 T cells, IL-22-expressing granulocytes and proinflammatory cytokines during latent and active tuberculosis infection.

In this study, we investigated the role and expression of T helper type 17 (Th17) cells and Th17 cytokines in human tuberculosis. We show that the basal proportion of interferon (IFN)-γ-, interleukin (IL)-17- and IL-22-expressing CD4(+) T cells and IL-22-expressing granulocytes in peripheral blood were significantly lower in latently infected healthy individuals and active tuberculosis patients compared to healthy controls. In contrast, CD4(+) T cells expressing IL-17, IL-22 and IFN-γ were increased significantly following mycobacterial antigens stimulation in both latent and actively infected patients. Interestingly, proinflammatory IFN-γ and tumour necrosis factor (TNF)-α were increased following antigen stimulation in latent infection. Similarly, IL-1ß, IL-4, IL-8, IL-22 and TNF-α were increased in the serum of latently infected individuals, whereas IL-6 and TNF-α were increased significantly in actively infected patients. Overall, we observed differential induction of IL-17-, IL-22- and IFN-γ-expressing CD4(+) T cells, IL-22-expressing granulocytes and proinflammatory cytokines in circulation and following antigenic stimulation in latent and active tuberculosis.

Filter selection for five color flow cytometric analysis with a single laser.

Flow cytometry has evolved from single- and two-color analysis to the current use of 11-16 colors. The relatively bright excitation spectra of most fluorochromes have made color compensation a challenge especially when performed manually. We describe how by choosing filters with narrower bandwidths results in the color compensation values between FITC, PE, PE-TxR (ECD), PE-Cy5, and PE-Cy7 that range from 0 % to 50% depending on the combination of fluorochromes. Peripheral blood mononuclear cells were stained with alpha-CD4-FITC, alpha-CD27-PE, alpha-CD62L-ECD, alpha-CD45RA-PE-Cy5 and alpha-CD3-PE-Cy7. The samples were acquired on a MO Flo. The initial (first) and second filter sets for our experiments consisted of 530/30 or 519/20 for FITC, 580/30 or 575/20for PE, 630/30 or 630/22 for PE-TxR (ECD), 670/30 or 675/20 for PE-Cy5 and 740LP or 780/40 for PE-Cy7. Nonstained cells were used to adjust the threshold values of detection for each photo multiplier tube (PMT) for each filter set. The mean fluorescent intensity (MFI) of each fluorochrome was not reduced to any great extent by either filter set. However, the compensation value between PE and PE-TxR (ECD) with the first filter selection ranged from 84% to 89% and with the second set of filters it was 25-36%. In addition, the compensation between PE-TxR (ECD) and PE-Cy5 were reduced to 30.2% from 44.2% with the second filter set. The reduction of filter bandwidths that results in minimizing spectral overlaps without lost of signal provides a method by which discrimination of signals between PE containing fluorochromes can be achieved.

Monocyte-derived cytokines in multiple sclerosis.

MS is an inflammatory, presumably autoimmune, disease mediated by the activation of T cells, B cells and monocytes (MO). Inflammation is thought to occur early during the relapsing-remitting phase of MS (RRMS), whereas in the later phases of MS such as secondary progressive MS (SPMS), inflammation tends to diminish. Our objective was to compare the types and amounts of proinflammatory and regulatory cytokines produced by MO from relapsing-remitting patients with or without treatment with IFN-beta (RRMS+ therapy, RRMS- therapy), respectively, from secondary progressive patients (SPMS) and from healthy controls (HC). MO were isolated by a density-gradient technique and three different techniques (RNase protection assay, ELISA and intracellular cytokine staining) were used to assess cytokine levels. An increase in IL6, IL12 and TNF-alpha was observed by all three methods for RRMS- therapy and for SPMS patients compared to HC and RRMS+ therapy patients. We conclude that proinflammatory and regulatory monokines can be derived from MO of MS patients and that these levels are modulated by IFN-beta therapy. Although it is believed that inflammation tends to diminish in SPMS patients, our data show that inflammatory cytokines continue to be released at high levels, suggesting that IFN-beta or IL10 treatment may be beneficial for this group.

Occurrence of Legionella in groundwater: an ecological study.

The natural habitat of Legionella is the water environment. Little is known about their presence in groundwater in spite of the fact that many millions around the globe regularly rely on groundwaters. This pilot study was aimed at evaluating the occurrence of Legionella in groundwater samples (water and biofilms) collected from various sites. Water and biofilm samples from selected groundwater sources were examined for Legionella using culture media (selective and non-selective) and a semi-nested PCR assay. Innovative approaches such as immunomagnetic separation (IMS) in combination with cultivation and flow cytometry were also evaluated. The findings available thus far show that (a) Legionella could be readily recovered from groundwater samples by cultivation even though their numbers showed considerable variations, (b) surprisingly, the PCR methodology was not yet as sensitive as cultivation and (c) flow cytometry was not directly applicable on natural samples because of debris and the high number of heterotrophic associated microflora from which some members were likely to cross-react with the monoclonal antibody used for separation procedures (IMS).

Safe use of the CXCR4 inhibitor ALX40-4C in humans.

ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.

Differentiation of murine NK cells into distinct subsets based on variable expression of the IL-12R beta 2 subunit.

The cytokine IL-12 manifests its biological activity via interaction with a heterodimeric receptor (IL-12R) present on activated T and NK cells. The cDNAs for two IL-12R subunits have been cloned from human and mouse and designated IL-12Rbeta1 and IL-12Rbeta2. The expression of IL-12Rbeta2 on T cells is influenced by cytokines, particularly IL-4, IL-12, and IFN-gamma; however, little is known regarding regulation of IL-12R expression on NK cells. In this study we show that murine NK cells differentiate into IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets after in vitro stimulation with IL-2 in the absence of exogenous polarizing cytokines. Subset development occurs gradually as NK cells expand in vitro and is generally complete by 8-12 days of culture. Once established, IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets are highly stable in vitro and can be maintained for at least 20 days after FACS sorting. Formation of these NK subsets appears to be strain independent. Flow cytometric analyses demonstrate that both subsets express a number of NK-associated markers, including NK1.1, DX-5, Ly-49A, and Ly-49C, but that the Ly-49G2 class I inhibitory receptor is expressed predominantly on the IL-12Rbeta2(high) population. Both IL-12Rbeta2(low) and IL-12Rbeta2(high) NK cells respond to exogenous IL-12 by rapid production of high levels of IFN-gamma and increased lytic activity against NK-sensitive YAC-1 target cells. Analyses of cytokine gene expression by RNase protection assay indicated that similar to the recently described human NK1 subset, both IL-12Rbeta2(high) and IL-12Rbeta2(low) murine NK subsets expressed high levels of IFN-gamma, whereas neither subset expressed mRNA for the NK2-associated cytokines IL-5 and IL-13.

Down regulation of CD4 expression following isolation and culture of human monocytes.

The down regulation of CD4 by cultured monocytes has been observed by our group and by other investigators. Flow cytometric experiments were done to examine which factors might influence this phenomenon. The addition of lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin-10 to monocyte cultures failed to inhibit the decrease in monocyte CD4 expression routinely observed following overnight culture. The down regulation was an adherence-independent phenomenon and was not influenced by the type of anticoagulant into which the peripheral blood was collected or by the presence or absence of lymphocytes within the cultures. The avoidance of the use of Ficoll-Paque to isolate peripheral blood mononuclear cells did not prevent monocyte CD4 down regulation. Finally, by tagging monocyte CD4 with an anti-CD4 phycoerythrin-conjugated monoclonal antibody prior to culture, we were able to determine that the down regulation observed was the result of the internalization of the molecule. At this time, we conclude that the observed down regulation of monocyte CD4 is probably due to the differentiation of blood monocytes into tissue culture-derived macrophages rather than to some artifact of the isolation procedure.

Mechanism of gammadelta T cell-induced human oligodendrocyte cytotoxicity: relevance to multiple sclerosis.

Gammadelta T cells may contribute to the pathogenesis of Multiple Sclerosis (MS) via cytotoxicity directed at the myelin-oligodendrocyte unit. We have previously demonstrated that peripheral blood-derived gammadelta T cells lyse fresh human oligodendrocytes in vitro. The present work extends these observations to gammadelta T cells derived from both peripheral blood (PBL) and cerebrospinal fluid (CSF) of MS and non-MS neurological disease controls and addresses the mechanism of cellular cytotoxicity. We found that MS patients contained increased proportions of Vdelta1+ gammadelta T cells in both CSF and PBL samples compared to other neurological disease (OND) controls. Although gammadelta T cells from all patients were cytotoxic towards Daudi, RPMI 8226, U937, Jurkat, oligodendroglioma and fresh human oligodendrocyte targets, OND-derived, Vdelta2+ rich, populations derived from the CSF exhibited greater cytotoxicity towards cell lines (Daudi, RPMI 8226) known to express high levels of heat shock proteins (hsp). To clarify the mechanism(s) of cytotoxicity used by gammadelta T cells, we first showed that cell-target contact was necessary by the use of physical barriers (transwells), which reduced target cell lysis by at least 75%. The use of Ca2+-free media reduced lysis by up to 50%, but fully blocking gammadelta T cell Perforin release and function by either Ca2+ chelation (Mg2EGTA) or the H+-ATPase inhibitor Concanamycin-A (CMA), completely abrogated the lysis of Fas-/hsp60high expressing targets (Daudi, U937). However, additional treatment with Brefeldin A was required for the complete inhibition of gammadelta T cell mediated killing of Fas+ expressing Jurkat targets and fresh human brain-derived oligodendrocytes. Inhibition of granzyme activity by an isocoumarin compound reduced cytolysis only slightly. The use of either Brefeldin A or an anti-Fas antibody alone did not significantly affect lysis. These findings suggest that in MS, gammadelta T cells may utilize either the Fas-mediated or Perforin-based cell cytotoxicity pathways in exerting oligodendrocyte damage, though the Perforin pathway is predominant.

Improvement in cell-mediated immune function during potent anti-human immunodeficiency virus therapy with ritonavir plus saquinavir.

Inhibiting human immunodeficiency virus (HIV) replication with potent antiretroviral therapy may result in improved immune function, and this may lead to favorable outcomes, independent of changes in CD4+ lymphocyte count. The effect of combination protease inhibitor therapy (ritonavir plus saquinavir) on functional measures of cell-mediated immunity in 41 HIV-infected patients from one center of a multicenter trial was investigated. After 24 weeks, median plasma virus load decreased from 4.74 log10 copies/mL to below the detection limit of the assay (2.30 log10), and mean CD4+ lymphocyte count increased from 284 cells/microL to 413 cells/microL. Proliferative responses to phytohemagglutinin developed in 21 of 34 patients in whom responses were absent at baseline. Increases were observed in interleukin-2, -12, and -10 production and in the expression of CD28 on CD8+ lymphocytes. Initiation of potent anti-HIV therapy results in a degree of immune restoration, suggesting that HIV-induced immune suppression is a dynamic and potentially reversible process.

Relative alterations in blood mononuclear cell populations reflect radiation injury in mice.

Damage to the immune and hematopoietic systems following exposure to ionizing radiation, whether accidental or for therapeutic purposes, renders victims susceptible to opportunistic infections and diseases. Elucidating a reliable biological indicator or "biological dosimeter" to indicate rapidly the extent of injury sustained by an individual would be desirable. Total leukocyte count has been used historically as an indicator of immune damage, but it is not truly indicative of functional immunity post-irradiation. A flow cytometric (FCM) technique was developed to determine whether a rapid reproducible assay could be developed to assess the extent of radiation damage. To this end, peripheral blood leukocyte populations and subpopulations were monitored. C57BL/6 mice were exposed to 100-, 400-, or 700-cGy whole-body gamma-irradiation (WBI) and blood samples were collected from the retro-orbital sinus 1, 4, and 7 days post-irradiation. The blood samples were prepared for FCM by incubation with monoclonal antibodies (mAb) to various murine leukocyte CD surface markers. The results show that the proportion of CD4+ T lymphocytes increased approximately 2-fold on day 4 after 700 cGy, the proportion of B lymphocytes declined markedly at all doses relative to unirradiated controls, and natural killer (NK) cells rose dramatically (9-fold) by day 4 after 700 cGy. The patterns of alteration in the relative proportions of peripheral blood mononuclear cells (PBMC) populations observed post-irradiation, particularly in B lymphocytes and natural killer (NK) cells, seem to represent potent and consistent indicators of immune damage, allowing some inference as to the immune competence of the individual.

A quick, easy and inexpensive method for the isolation of human peripheral blood monocytes.

A commercial monocyte isolation technique based on the OptiPrep density-gradient medium was up-scaled with respect to sample and reagent volumes. The results of 7 isolations are reported in which the average purity ranged from 87.9 to 96.4%. In all but the initial isolation, monocytes were defined as CD15+ dim CD4+ dim as assessed by flow cytometric analysis; in the first isolation, monocytes were defined by the traditional CD14+ CD4+ dim combination. The mean yield (the number of isolated monocytes relative to the number present in the buffy coat) of all isolations was 26.1%, with the individual yields ranging from 10.8 to 41.4%. The mean number of isolated monocytes per experiment was 3.6 x 10(6) monocytes for those isolations performed using 14 ml of buffy coat/OptiPrep mixture (n = 4). The isolated cells were viable (> 95%) and were not activated, according to HLA-DR expression. This technique is a convenient, tast (less than 2 h), relatively simple, and inexpensive alternative to traditional monocyte isolation techniques. The up-scaled version of this method also results in significantly higher numbers of monocytes per isolation than some traditional techniques. Furthermore, this is the first literature report of the use of the OptiPrep density-gradient medium for the isolation of monocytes.

Radiation damage and immune suppression in splenic mononuclear cell populations.

We have examined alterations in all of the major splenic mononuclear cell (SMNC) populations in C57B1/6 mice following whole-body irradiation (0-700 cGy) in order to determine which populations may play a role in active immune suppression and/or haematopoietic recovery. A protocol has been established for characterization and differentiation by flow cytometric analysis (FCA) of the major MNC populations in the mouse spleen: T lymphocytes (CD4+ and CD8+ cells), B lymphocytes, natural killer (NK) cells, and monocytes/macrophages. Ionizing radiation caused decreased spleen cellularity and decreased ability of surviving SMNC to respond to mitogen. FCA revealed alterations in the relative composition of the constituent splenic cell populations following irradiation, reflecting differential radiosensitivity, with selective enrichment of NK cells (seven-fold) and CD4+ T lymphocytes (three-fold). Enrichment developed during the 7-day post-irradiation period. In addition, some MNC became activated in a dose- and time-dependent fashion following whole-body irradiation, as indicated by expression of CD71, the transferrin receptor. These cells were CD34+ and Thy 1.2+, but were CD4- or CD8- as well as CD45- (B cell). The observed increase in NK cells corresponds with a previously reported increase in natural suppressor (NS) cells following total-lymphoid irradiation (TLI). The balance of recovery-inhibiting NK cells and recovery-enhancing CD4+ T lymphocytes following irradiation may reflect or influence the degree of haematopoietic recovery, and may provide an indication of the extent of damage (biological dosimetry).

Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C.

ALX40-4C is an antiretrovirus agent that has been found to have some inhibitory properties against human immunodeficiency virus (HIV) replication in vitro. The compound was designed as a competitor of the HIV Tat protein for TAR binding. In addition to its anti-HIV properties, it has demonstrated the ability to inhibit in vitro replication of herpes simplex virus types 1 and 2 as well as human cytomegalovirus. Subsequently, in vivo pharmacokinetic evaluation of ALX40-4C necessitated the establishment of a detection system for the measurement of ALX40-4C in subject serum. For this purpose, an indirect-competition enzyme-linked immunosorbent assay with generated rabbit anti-ALX40-4C antiserum was developed. The original assay took 12 h to complete and required many manipulations. Herein, we describe alterations to the system that resulted in the overall reduction in assay time and manipulation. We demonstrate that our alterations do not affect the specificity or sensitivity of the assay compared to that of the original system. ALX40-4C levels in spiked serum samples as well as drug levels from patient samples were used to validate the assay.

Inducible immunity to Trichomonas vaginalis in a mouse model of vaginal infection.

We studied the protective effect of subcutaneous immunization with Trichomonas vaginalis in a mouse model of vaginal infection. BALB/c mice were immunized with various doses of T. vaginalis (4.5 x 10(5), 9 x 10(6), and 1 x 10(8) organisms per ml) suspended in Freund's complete adjuvant 56 days prior to vaginal infection and were given booster injections of the same doses of T. vaginalis in Freund's incomplete adjuvant 4 weeks later. Control mice were immunized and given booster injections of phosphate-buffered saline suspended in Freund's complete and incomplete adjuvants. The mice were tail bled and vaginal washes were performed at weekly intervals for 4 weeks to determine the isolation of T. vaginalis and the serum and vaginal antibody reactivity. Mice which had been immunized and given booster immunizations had significantly fewer intravaginal infections and had increased serum and vaginal antibody responses compared with those of control mice (P < 0.01). Mice that were vaginally infected, treated with metronidazole, and then reinfected vaginally did not develop protective immunity. Subcutaneous immunization with whole T. vaginalis organisms appears to confer protection against intravaginal challenge with T. vaginalis, protection which is not achieved as a result of prior vaginal infection.

Evaluation of humoral and cell-mediated inducible immunity to Haemophilus ducreyi in an animal model of chancroid.

To study the mechanisms of inducible immunity to Haemophilus ducreyi infection in the temperature-dependent rabbit model of chancroid, we conducted passive immunization experiments and characterized the inflammatory infiltrate of chancroidal lesions. Polyclonal immunoglobulin G was purified from immune sera raised against H. ducreyi 35000 whole-cell lysate or a pilus preparation and from naive control rabbits. Rabbits were passively immunized with 24 or 48 mg of purified polyclonal immunoglobulin G intravenously, followed 24 h after infusion by homologous titered infectious challenge. Despite titratable antibody, no significant difference in infection or disease was observed. We then evaluated the immunohistology of lesions produced by homologous-strain challenge in sham-immunized rabbits and those protectively vaccinated by pilus preparation immunization. Immunohistochemical stains for CD5 and CD4 T-lymphocyte markers were performed on lesion sections 4, 10, 15, and 21 days from infection. Lesions of pilus preparation vaccinees compared with those of controls had earlier infiltration with significantly more T lymphocytes (CD5+) and with a greater proportion of CD4+ T lymphocytes at day 4 (33% +/- 55% versus 9.7% +/- 2%; P = 0.002), corroborating earlier sterilization (5.0 +/- 2 versus 13.7 +/- 0.71 days; P < 0.001) and lesion resolution. Intraepithelial challenge of pilus-vaccinated rabbits with 100 micrograms of the pilus preparation alone produced indurated lesions within 48 h with lymphoid and plasmacytoid infiltration, edema, and extravasation of erythrocytes. We conclude that passive immunization may not confer a vaccine effect in this model and that active vaccination with a pilus preparation induces a delayed-type hypersensitivity skin test response and confers protection through cell-mediated immunity seen as an amplified lymphocytic infiltrate and accelerated maturation of the T-lymphocyte response.

Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients.

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type 1 (Th1) (interferon-gamma (IFN-gamma)) and Th2 (IL-4, IL-10) type cytokines in peripheral blood lymphocytes (PBL) from HIV+ patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that IFN-gamma mRNA in unstimulated PBL was significantly decreased and IL-10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n = 30) as compared to patients with > 400 CD4+ T cells/mm3 (n = 6) and normal controls (n = 16). In addition, IL-10 mRNA levels were inversely associated with IFN-gamma expression. Similar results were obtained by measuring IL-10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL-4 and IFN-gamma produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV+ individuals based on IL-10 production. PBL from one set of individuals produced low levels of IL-10 (low IL-10 producers) whereas the other group produced IL-10 comparable to that of normal controls (IL-10 producers). Production of IL-4 was significantly reduced in HIV+ individuals with < 400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN-gamma by mitogen-stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulated and mitogen-stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.

Inducible immunity with a pilus preparation booster vaccination in an animal model of Haemophilus ducreyi infection and disease.

Using the temperature-dependent rabbit model of Haemophilus ducreyi infection as a quantitative virulence assay, we tested the abilities of two bacterial antigen preparations to induce protection against subsequent infection and disease. Lipooligosaccharide (LOS) and a pilus preparation were purified from H. ducreyi 35000 and were used in a booster immunization procedure. The serologic response to each immunogen was monitored by enzyme immunoassay. H. ducreyi virulence was assayed by intraepithelial inoculation and subsequent measurement of disease for homologous strain 35000 or clinical isolate RO-34. LOS and the pilus preparation induced humoral responses. The kinetics of the LOS antibody response suggest a type 1 T-independent response, whereas the pilus preparation induced an anamnestic response. An inoculum of 10(5) CFU of H. ducreyi 35000 or RO-34 consistently produced ulcerative chancroidal lesions in naive rabbit controls. Immunization with LOS did not modify the virulence of H. ducreyi 35000. Immunization with the strain 35000 pilus preparation significantly reduced the severity of disease and the duration of infection and disease compared with controls, with either homologous or heterologous strain infection. The histology of lesions from pilus preparation-vaccinated rabbits compared with that of lesions from controls revealed accelerated lymphoid cell recruitment, more prominent plasma cell infiltrate, and reduction in subsequent histiocytic infiltration. We conclude that both LOS and the pilus preparation are immunogenic and that the latter induces homologous and heterologous strain protection in this animal model of infection and disease.

Immune response to sulfamethoxazole in patients with AIDS.

Antibody- and cell-mediated responses to sulfamethoxazole (SMX) were analyzed in AIDS patients with or without a history of hypersensitivity and in negative controls. In 20 of 20 (P < 0.01) human immunodeficiency virus (HIV)-seropositive patients with skin reactions to cotrimoxazole, we found SMX-specific antibodies, while only 9 of 20 and 17 of 20 HIV-seropositive patients without a history of hypersensitivity to cotrimoxazole had SMX-specific immunoglobulin M (IgM) and IgG, respectively. The levels of specific IgM and IgG were higher in patients with skin reactions than in patients without reactions (IgM, 1.0 +/- 0.19 versus 0.47 +/- 0.23 [P < 0.001]; IgG, 0.68 +/- 0.15 versus 0.47 +/- 0.14 [P < 0.001] [mean optical density values +/- standard deviations]). Seronegative controls with no history of exposure to sulfa compounds did not have SMX-specific IgG or IgM antibodies, and controls with a history of intake of SMX with or without reactions had low levels of IgG and IgM. The SMX-specific IgG subclasses were exclusively IgG1 and IgG3. None of the patients had detectable SMX-specific IgE or IgA antibodies nor did they exhibit a cell-mediated response as measured by a lymphocyte proliferation assay. Antibodies to SMX recognized N-acetyl-sulfonamide, N-(2-thiazolyl)-sulfanilamide, sulfadiazine, and sulfisoxazole but did not recognize sulfanilamide or 3-amino-5-methyl isoxazole in an inhibition assay. It is not known whether the SMX-specific antibodies associated with hypersensitivity reactions to SMX in HIV-seropositive patients have a pathogenic role in these reactions. Sulfanilamide or 3-amino-5-methyl isoxazole, on the other hand, could be potential alternative therapies in HIV-seropositive patients with a history of skin reactions to SMX.

Blood monocytes from most human immunodeficiency virus type 1-infected patients do not carry proviral DNA.

In blood, the CD4+ T cells of patients with human immunodeficiency virus type 1 (HIV-1) harbor HIV-1; however, whether the CD4+ blood monocytes carry the virus is controversial. Tissue macrophages are known to be infected. To determine in blood monocytes from HIV-1-seropositive patients contain HIV-1, we separated monocytes and T-cell subsets by using monoclonal antibodies bound to magnetic beads and by monocyte adherence to glass. Monocytes were cultured with macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3. After 14 days in culture, cells were analyzed for the presence of HIV-1 antigen and multinucleated giant cells (MGCs). Freshly isolated cell subsets were analyzed for HIV-1 proviral DNA by PCR with modified env (SK68i and SK69i2) and gag (SK145i and SK150) primers. We found that (i) monocytes cultured without depletion of CD4+ T cells (11 of 11 patients) were HIV-1 antigen positive and showed dramatically increased spontaneous formation of MGCs (ii) monocytes cultured after depletion of CD4+ T cells (three experiments) were HIV-1 antigen negative and showed markedly decreased MGC formation, and (iii) in specimens from 14 patients subsequently analyzed by PCR, purified CD4+ T cells were positive for HIV-1 proviral DNA in all patients. In 11 of 14 patients (79%), the monocyte fractions were HIV-1 proviral DNA negative, while in the remaining 3 patients, the monocytes were positive for HIV-1 proviral DNA. In conclusion, the major reservoir for HIV-1 infection in human peripheral blood is the CD4+ T cell (14 of 14 cases).(ABSTRACT TRUNCATED AT 250 WORDS)