Biomedical subjects

J L Ho

Publications and source records attributed to J L Ho.

At least 19 recordsLinked to original sources

PubMed2006

Levels of serum chemokines discriminate clinical myelopathy associated with human T lymphotropic virus type 1 (HTLV-1)/tropical spastic paraparesis (HAM/TSP) disease from HTLV-1 carrier state.

Approximately 5% of people infected with human T lymphotropic virus type 1 (HTLV-1) develop clinical myelopathy or tropical spastic paraparesis (HAM/TSP) that is associated with high-levels of Th1 cytokines, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. Chemokines are known to induce cytokine secretion and direct the trafficking of immune cells to sites of disease. The present study measured serum chemokines correlated with autonomously released IFN-gamma in cell cultures. HTLV-1 infection was defined by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot. Subjects included HTLV-1 carriers (n = 56), patients with HAM/TSP (n = 31) and healthy HTLV-1 seronegative volunteer controls (n = 20). Serum chemokines and IFN-gamma autonomously released by mononuclear cells in culture were quantified by ELISA. Compared to HTLV-1 carriers, serum chemokines in HAM/TSP patients showed significantly increased levels of CXCL9 and CXCL10, significantly diminished levels of CCL2 and similar amounts of CCL11 and CCL24. In contrast, CCL11 and CCL24 were significantly lower in serum of HAM/TSP patients than either control. IFN-gamma was positively correlated with CXCL9 and CXCL10 when HAM/TSP and HTLV-1 carriers were used as a combined group. However, despite a large proportion of HTLV-1 carriers having high IFN-gamma levels, these chemokines were not increased in carriers. This study showed that high levels of CXCL9 and CXCL10 in the systemic circulation and low serum CCL2 levels are features of HAM/TSP. HTLV-1 infection and Tax and/or additional viral encoded factor-mediated pathological processes triggering T cell activation with autogenous IFN-gamma release are probably involved in regulating chemokine release.

Biomarkers↗

PubMed2004

HTLV-1 modifies the clinical and immunological response to schistosomiasis.

The immunological response in HTLV-1 infected individuals is characterized by a prominent Type-1 cytokine response with high production of IFN-gamma and TNF-alpha. In contrast, helminthic infections and in particular chronic schistosomiasis are associated with a predominant production of IL-4, IL-5, IL-10 and IL-13. Liver fibrosis is the main pathological finding in schistosomiasis that occurs after many years of infection. This pathology is T cell dependent but the immune response mechanisms are not completely understood. The North-east region of Brazil is endemic for both HTLV-1 and schistosomiasis. In the present study the immune response, clinical severity, and therapeutic response to praziquantel of patients with schistosomiasis coinfected with HTLV-1 were compared with patients infected only with S. mansoni. Patients with HTLV-1 and S. mansoni had lower levels of IL-5 (P < 0.05) and higher levels of IFN-gamma (P < 0.05) in cultures stimulated with S. mansoni antigen and decreased S. mansoni antigen specific IgE levels when compared with patients with schistosomiasis without HTLV-1 coinfection. Liver fibrosis was mild in all HTLV-1 coinfected patients and efficacy of praziquantel was lower in patients dually infected than in patients infected only with S. mansoni.

Adult↗

PubMed2002

Colonoscopy in asymptomatic individuals with a family history of colorectal cancer.

BACKGROUND: This study was performed to evaluate the use of total colonoscopy as the optimal screening test in asymptomatic individuals with a family history of colorectal cancer (CRC). METHODS: Colonoscopy was performed in 249 asymptomatic individuals who had one or two first-degree relatives (FDRs) with CRC; individuals with three or more FDRs with CRC were excluded. RESULTS: Eighty-six colonic lesions were found in 51 individuals (51 of 249; 20.5%). Among these 51 subjects, 27 had neoplastic polyps (n = 38) and 29 had metaplastic polyps (n = 44). Although no invasive cancer was detected, in 14 individuals the lesions had a high malignancy potential because of their size and histopathology. We did not confirm a statistically significant difference in the incidence of neoplastic polyps according to the number of affected FDRs. Finally, the presence of metaplastic polyps was a very strong indication for the concomitant presence of metaplastic polyps (P <.0001). CONCLUSIONS: Total colonoscopy is the optimal screening procedure for the examination of asymptomatic individuals with a family history of CRC.

Adult↗

PubMed2002

The usefulness of serum adenosine deaminase 2 (ADA2) activity in adults for the diagnosis of pulmonary tuberculosis.

Rapid diagnosis of Mycobacterium tuberculosis remains an obstacle for therapy of tuberculosis (TB). Adenosine deaminase isoform 2 (ADA2) is produced by activated macrophages and has been used for diagnosis of TB from extra-pulmonary sites. However, few studies adequately address whether serum ADA2 activity is useful for diagnosis of active pulmonary tuberculosis (PTB). We prospectively measured serum ADA2 activity in 110 patients with pulmonary disease (65 cases with active PTB and 45 cases with other respiratory diseases) and 78 healthy volunteers (eight with tuberculin skin test positive). The serum ADA2 for the diagnosis of PTB had the sensitivity of 36.9%, the specificity of 84.5%, the positive predictive value of 10.9% and the negative predictive value of 96.2%. We concluded that serum ADA2 activity is neither useful to diagnosis of active PTB nor to differentiate from other respiratory diseases.

Adenosine Deaminase↗

PubMed2001

Characterization of the replication region of the Lactobacillus reuteri plasmid pTC82 potentially used in the construction of cloning vector.

A 3.2 kb DNA fragment containing the replication region (RR) from pTC82 was cloned, sequenced, and found to contain elements typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR), including double-strand origin (DSO), replication protein gene (rep), and single-strand origin (SSO). The DSO of pTC82 contains two domains showing 55.5% and 84.6% similarities in nucleotide (nt) sequence to the conserved functional elements bind and nic, respectively, which are required for the initiation of the leading strand typical of the pC194-RCR family. Although the predicted rep gene product of pTC82 (Rep82) shares little identity (less than 24%) with other known Reps, a region containing three motifs, characteristic of the pC194-family Reps, was identified, indicating the Rep82 as a novel Rep protein of this family. Downstream of the rep82 gene, strong similarity to the typical palT type-SSO could be detected. This is the first palT type-SSO to be identified from Lactobacillus. Through a series of deletion studies, the minimal replicon of the cloned RR was found to be 2.66 kb in size including the DSO region and rep gene. This RR was further identified as being highly stable in L. reuteri and also bearing a very narrow host-range property, suggesting it to be a good replicon potentially useful in vector construction for developing L. reuteri as a vaccine carrier.

Amino Acid Sequence↗

PubMed2000

Preferential recruitment of phagocytes into the lung of patients with advanced acquired immunodeficiency syndrome and tuberculosis.

Limited data are available on the cellular and immunocytological characteristics of bronchoalveolar lavage (BAL) fluid in individuals infected with the human immunodeficiency virus (HIV) and pulmonary tuberculosis (TB). The immune host response against tuberculosis in early HIV-infection may differ from that in later stages of HIV disease, as is strongly suggested by different clinical and radiographic patterns. We studied the cellular elements in the lungs of 15 HIV-infected patients with advanced immunosuppression and pulmonary tuberculosis (TB/AIDS). The findings were compared with data from four other groups: 1) 15 HIV-seronegative patients with pulmonary TB; 2) 12 HIV-seropositive TB patients without previous AIDS-defining illnesses and with CD4+ >200 cells mm(-3); 3) five AIDS patients without pulmonary lesions; and 4) five healthy controls. BAL fluid and differential cell counts, as well as lymphocyte subsets, were determined. Despite a low CD4/CD8 ratio, the TB/AIDS group had a higher absolute number of CD8+ lymphocytes in the BAL fluid than the other groups. Alveolar macrophages and neutrophils were significantly increased in TB/AIDS patients compared to control groups. The number of eosinophils was increased in TB/HIV--patients but not in TB/AIDS patients. We conclude that tuberculosis in late stage HIV-infected patients has a distinct inflammatory cell profile, suggesting an enhanced compensatory mechanism that amplifies the unspecific inflammatory reaction.

Acquired Immunodeficiency Syndrome↗

PubMed2000

Mycobacterium tuberculosis lipid antigens: use of multi-antigen based enzyme immunoassay for free and complex dissociated antibodies.

OBJECTIVE: To test the sensitivity and specificity of four lipid antigens of Mycobacterium tuberculosis: BDA-TDA, DAT, SL-I, and PIMs, adsorbed in the same microplate well, to detect reactive IgG by enzyme-immunoassay (EIA) from plain serum (MA-EIA) and dissociated immune complexes (ICMA-EIA). DESIGN: IgG antibodies against four antigens, placed in the same microplate well, were evaluated in serum from 155 tuberculous (TB) cases non-infected with the human immunodeficiency virus (HIV): 78 patients with positive bacilloscopy and culture, 33 patients with positive culture and 44 patients diagnosed by clinical and radiological criteria; and from 211 HIV negative control subjects: 32 patients with other pulmonary diseases, 100 healthy people and 79 close contacts. RESULTS: MA-EIA had an overall sensitivity and specificity of 61% (94/155) and 95% (200/211), respectively. We further examined whether the dissociation of immune complexes increases the number of positive reactions in those initially found to be seronegative (SN). The subset of 112 (76 controls and 36 TB) MA-EIA SN samples tested using ICMA-EIA yielded an overall sensitivity and specificity of 83% and 100%. The ICMA-EIA results improved the overall sensitivity from 61 to 80% without changing specificity. CONCLUSION: These preliminary results suggest that MA-EIA followed by ICMA-EIA, for SN samples, might serve as a fast, cheap, and easy method for the diagnosis of TB in less than 48 hours.

Antibodies, Bacterial↗

PubMed1999

UV inducibility of rat proliferating cell nuclear antigen gene promoter.

Proliferating cell nuclear antigen (PCNA), also known as a cofactor of DNA polymerase delta, is required for eukaryotic cell DNA synthesis and nucleotide excision repair. Expression of PCNA gene is growth-regulated and UV inducible. In our previous study, we have observed that the rat PCNA promoter has the serum responsiveness. In this study, we demonstrate its UV inducibility in CHO.K1 cells. The UV induction of the rat PCNA promoter activity was dose-dependent in the cells synchronized at different phases. In addition, the sequences of the promoter responsible for the UV inducibility were delimited to the region between nucleotides -70 and +125, which contains an AP-1 site and a downstream proximal ATF/CRE site. While mutation of the AP-1 site abrogated the UV inducibility, mutation of the ATF/CRE site enhanced the UV inducibility, suggesting that the two sites play different roles in the UV induction of the promoter. In addition, the role of p53 in the UV induction of rat PCNA promoter was investigated. We found that exogenous p53 was unable to mimic the UV irradiation to induce rat PCNA promoter and that the UV induction of the rat PCNA promoter was seen in p53 deficient cells. Therefore, it is unlikely that the UV induction of the rat PCNA promoter is p53 dependent.

Animals↗

PubMed1998

Serum responsiveness of the rat PCNA promoter involves the proximal ATF and AP-1 sites.

We have previously shown that the rat PCNA (proliferating cell nuclear antigen) gene promoter is responsive to serum stimulation. In this study, the sequence of the promoter responsive to serum stimulation has been localized in the region between nucleotides -70 and +125 relative to the transcription initiation site. This region contains an ATF site (nucleotides -51 to -44) and an AP-1 site (nucleotides -64 to -58). Mutation at either the ATF or the AP-1 site reduced the serum responsiveness of the promoter. In gel mobility shift assays, nuclear extracts from serum stimulated cells, compared to those from quiescent cells, exhibit an increasing binding activity toward a promoter related oligonucleotide (-70 to -42) which includes the ATF site and the AP-1 site. Formation of the DNA:protein complexes requires the simultaneous involvement of ATF and AP-1 sites as either element can abrogate the complexes in the competition experiment. Both the distance and sequence are essential to complex formation. Moreover, ATF-1 but not ATF-2 (or CREB) has been identified as a major component of the complexes in the antibody supershift or interference experiment. The results of this study suggest that ATF-1 in association with other factors is involved in regulating the serum stimulation of the rat PCNA promoter activity via the proximal ATF and AP-1 sites.

Activating Transcription Factors↗

PubMed1998

Functional activity of alveolar and peripheral cells in patients with human acquired immunodeficiency syndrome and pulmonary tuberculosis.

We compared the peripheral and pulmonary response to assess the phagocytic activity of monocytes/macrophages and neutrophils and the lymphoproliferative response (LPR) against Mycobacterium tuberculosis antigens from 21 AIDS patients, presenting at diagnosis with active pulmonary tuberculosis (TB), other non-TB pulmonary infection, or no pulmonary infection, as well as patients with active pulmonary TB and healthy control subjects. Alveolar lymphocyte analysis demonstrated that AIDS/TB patients had more markedly reduced percentages of CD4(+) lymphocytes than AIDS/TB patients and an increase in the percentage of CD8(+) lymphocytes, probably reflecting the impairment of CD4(+) T lymphocytes in peripheral blood at the lungs. Moreover, alveolar lymphocytes from AIDS/TB patients demonstrated a two- to fourfold decrease in LPR against M. tuberculosis antigens. Interestingly, it was observed an enhanced migration of natural killer cells to the lungs in all patients group. The phagocytic activity in alveolar macrophages and neutrophils showed that AIDS/TB patients had a twofold decreased capacity to ingest inert particles compared with AIDS patients. Comparing the alveolar and peripheral lymphocyte number and functional activity to M. tuberculosis-antigens it was possible to demonstrate that in both sites these cells had similar profile. However, the innate immune response in lungs showed a reduced activation in the presence of HIV infection, regarding the M. tuberculosis coinfection. These findings suggest that the advanced impairment of CD4(+) T lymphocyte in HIV-1 infection may lead to a deactivation of alveolar macrophages, enhancing bacilli burden and HIV replication in the lungs and furthering dissemination.

AIDS-Related Opportunistic Infections↗

PubMed1998

Induction of in vitro human macrophage anti-Mycobacterium tuberculosis activity: requirement for IFN-gamma and primed lymphocytes.

Mycobacterium tuberculosis (Mtb) is the world's leading infectious cause of mortality. Despite the overwhelming data supporting the critical role of cellular immunity, little is known of the early microbial and immune cell interactions and whether human macrophages can be activated to express anti-Mtb activity. We report the reconstitution of an in vitro system whereby human macrophages express anti-Mtb activity only in coculture with PBL and with IFN-gamma. Omission of IFN-gamma in the cocultures or Mtb lysate/IFN-gamma-primed lymphocytes was associated with high growth of Mtb, high IL-10 and IL-12 p40, nearly undetectable IL-12 p70 levels, and the highest percentages of CD4 and CD8 T cells. In contrast, IFN-gamma treatment of cocultures containing Mtb lysate/IFN-gamma-primed PBL reduced bacilli count by approximately 2.5 log, decreased the production of IL-10 by 5.7-fold, increased IL-12 p70 by approximately 50-fold, and reduced the percentages of CD4 and CD8 T cells. Activation of anti-Mtb activity was time and dose dependent. At 2000 U/ml of IFN-gamma, bactericidal activity was achieved (10-fold reduction from initial inoculum). Anti-Mtb activity against several strains of M. tuberculosis (H37Ra and H37Rv, and C, a clinical isolate) was observed and was associated with expression of inducible nitric oxide synthase. These data suggest that induction of human macrophage anti-Mtb activity required dual signaling from PBL and IFN-gamma. Thus, the development of an in vitro human system may greatly facilitate studies to delineate immune cells, cytokines, and effector functions/genes critical in controlling Mtb. Defining the mechanisms may also provide novel treatment strategies for tuberculosis.

Antigen Presentation↗

PubMed1998

Leishmania lipophosphoglycan reduces monocyte transendothelial migration: modulation of cell adhesion molecules, intercellular junctional proteins, and chemoattractants.

We previously identified the structural requirement for the inhibitory activity of Leishmania lipophosphoglycan (LPG) to block endothelial adhesion to monocytes. Here we showed that LPG reduces transendothelial migration of monocytes. LPG pretreatment of endothelial cells (2 microM, 1 h) reduced monocyte migration across endothelial cells activated by bacterial endotoxin (LPS) or IL-1beta (60 and 46%, respectively). A fragment of LPG (i.e., repeating phosphodisaccharide (consisting of galactosyl-mannose)) and LPG coincubated with LPG-neutralizing mAb lacks inhibitory activity on monocyte migration. Pretreatment of monocytes with LPG (2 microM, 1 h) also did not affect monocyte migration through control or LPS-activated endothelial cells. FACS analysis reveals that LPG treatment blocked the LPS-mediated expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells and monocyte adhesion without altering the integrity of the endothelial monolayer. LPG (2 microM, 1 h) alone was capable of altering the expression and distribution of two junctional adhesion molecules, CD31 and vascular endothelium cadherin, as well as reversing the effects of LPS on these proteins. The induction of endothelial cells by LPS to transcribe and release monocyte chemoattractant protein-1 (MCP-1) was significantly reduced by LPG (40-65%). LPG treatment of nonactivated endothelial cells also suppressed by 55 to 75% the monocyte migration triggered by a MCP-1 chemoattractant gradient, and coincubation of LPG with neutralizing mAb abrogated the inhibitory activity. Together, these data point to a novel anti-inflammatory function of LPG in reducing monocyte migration across endothelial cells via a mechanism of inhibition of endothelial expression of cell adhesion molecules, modulation of intercellular junctional proteins, and synthesis of MCP-1.

Animals↗

PubMed1996

Lipophosphoglycan from Leishmania suppresses agonist-induced interleukin 1 beta gene expression in human monocytes via a unique promoter sequence.

Leishmania are parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid of Leishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1 beta steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1 beta mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor alpha and Staphylococcus induction of IL-1 beta gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1 beta mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the -310 to -57 nucleotide region of the IL-1 beta promoter was found to mediate LPG's inhibitory activity. The requirement for the -310 to -57 promoter gene sequence for LPG's effect is demonstrated by the abrogation of LPG's inhibitory activity by truncation or deletion of the -310 to -57 promoter gene sequence. Furthermore, the minimal IL-1 beta promoter (positions -310 to +15) mediated LPG's inhibitory activity with dose and kinetic profiles that were similar to LPG's suppression of steady-state IL-1 beta mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a "gene silencer", a function, to our knowledge, not previously described. LPG's inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.

Animals↗

PubMed1996

Structure-function analysis of Leishmania lipophosphoglycan. Distinct domains that mediate binding and inhibition of endothelial cell function.

We have shown that Leishmania lipophosphoglycan (LPG) inhibits IL-1 beta gene expression in human monocytes. Here, we show that LPG can bind in a time-dependent manner and suppress endothelial cell activation, possibly via specific LPG domains. Endotoxin (10 ng/ml, 4 h) consistently caused endothelium to increase monocyte adhesion (approximately 20-fold). LPG pretreatment (2 microM, 2 h) completely blocked endotoxin-mediated monocyte adhesion. LPG did not grossly suppress endothelial functions because TNF-alpha- and IL-1 beta-mediated adhesion toward monocytes were not affected. Using four highly purified LPG fragments (namely, repeating phosphodisaccharide (PGM), phosphoglycan, phosphosaccharide core-lyso-alkyl-phosphatidylinositol (core-PI), and lyso-alkyl-phosphatidylinositol (lyso-PI)), we examined whether these fragments can independently inhibit endothelial adhesion. In contrast to that of intact LPG, neither the four LPG fragments (2 microM, 2 h) independently nor the co-addition of phosphoglycan and core-P1 fragments blocked the endotoxin-mediated adhesion to monocytes. To determine whether the fragments can reverse the effect of intact LPG, endothelial cells were first pretreated with the LPG fragments (10 microM, 15 min), followed by the addition of LPG (2 microM). All four LPG fragments fully reversed the effect of LPG. Simultaneous addition of LPG fragments and intact LPG caused only partial suppression (approximately 45%), while the addition of LPG fragments 14 min later had no reversal effect. Flow cytometry revealed that only core-P1 and lyso-P1 competitively inhibited (approximately 30%) LPG binding. Conversely, LPG competed with the binding of [3H]lyso-P1 (approximately 30%). Furthermore, mAb against the PGM reversed (approximately 70%) the effect of LPG. Thus, the lyso-P1 domain on LPG mediates binding to endothelial cells, whereas the PGM domain mediates the cell inhibitory effect.

Animals↗

PubMed1996

Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis.

The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.

Amino Acid Sequence↗

PubMed1996

Differential effects of interleukin-12, interleukin-15, and interleukin-2 on human immunodeficiency virus type 1 replication in vitro.

Cytokines may have clinical utility as therapeutic agents for human immunodeficiency virus type 1 (HIV-1) infection and as an adjuvant for vaccines. The effect of interleukin-12 (IL-12) and IL-15 on in vitro HIV-1 replication was investigated. IL-12 and IL-15 at doses up to 10 ng/ml had little effect on basal HIV-1 p24 antigen production by chronically HIV-infected T (ACH-2) and monocytic (U1) cell lines. For ACH-2 cells stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml), IL-12 and IL-15 significantly increased p24 antigen production by 20 and 30%, respectively (n = 6). In contrast, IL-12 and IL-15 (10 ng/ml) treatment of PMA-stimulated U1 cells decreased p24 antigen production by 16 and 15%, respectively (n = 6). We next studied the effect of IL-12 and IL-15 on HIV-infected peripheral blood mononuclear cells (PBMCs). In 10 HIV-seropositive patients' PBMCs cocultured with mitogen-activated HIV-seronegative donor cells, two patterns of p24 antigen production were observed in response to IL-2: low (p24 antigen production < 10(3) pg/ml; n = 8) and high (p24 antigen production > 10(3) pg/ml; n = 2) response. For the low-response pattern, IL-12 and IL-15 increased viral replication by 97-fold and 100-fold, respectively (P = 0.05 and 0.004, respectively). For the high-response pattern, both IL-12 and IL-15 suppressed HIV replication. The effect of IL-2, IL-12, and IL-15 on acute in vitro infection by HIV-1JRCSF was also examined. IL-12 did not increase p24 antigen production above basal levels while IL-2 and IL-15 significantly enhanced p24 antigen production (by approximately 2-fold). In conclusion, IL-12 and IL-15 may have differential effects on latent and acute HIV infection, and their ability to enhance HIV production may depend on cell activation. Thus, the use of these cytokines may be dictated by the clinical state of the patient.

Cells, Cultured↗

PubMed1995

Neutrophils from human immunodeficiency virus (HIV)-seronegative donors induce HIV replication from HIV-infected patients' mononuclear cells and cell lines: an in vitro model of HIV transmission facilitated by Chlamydia trachomatis.

Infection with a sexually transmitted disease (STD) increases the risk for human immunodeficiency virus (HIV) infection. Polymorphonuclear leukocytes (PMNs) are recruited into the genital tract by STD pathogens, such as Chlamydia trachomatis. Semen of HIV-infected men contains HIV associated with mononuclear cells. This study investigated the interaction among PMNs from HIV-uninfected persons, C. trachomatis, and HIV-infected cells and examined the mechanisms for enhanced HIV replication. We demonstrated that PMNs from HIV-seronegative donors induced HIV replication in mononuclear cells from 17 HIV-infected patients in medium without exogenous IL-2. HIV in the cell-free supernatants from cocultures of PMNs and patients' peripheral blood mononuclear cells (PBMCs) was replication competent, as indicated by their capacity to propagate HIV in a second round of culture using PBMCs from HIV-seronegative individuals and by the fact that proviral DNA was found in these cells. PMNs from HIV-seronegative donors increased HIV replication over 100-fold in chronically HIV-infected cell lines of the monocytic, T, and B cell lineages. Moreover, PMNs increased U1 cells' production of p24 antigen by as much as ninefold when compared with U1 cells cocultured with PBMCs. The addition of C. trachomatis to PMN and U1 coculture increased HIV replication by an additional ninefold at 24 h, whereas C. trachomatis alone had no effect on p24 antigen production by U1 cells. Thus, C. trachomatis serves not only to recruit PMNs, but also to interact with PMNs to increase HIV replication. HIV replication is triggered by contact of HIV-infected cells with PMNs, by the generation of reactive oxygen intermediates (ROIs), and by soluble factors such as TNF-alpha and IL-6. This is based on the findings that production of p24 antigen, IL-6, and TNF-alpha induced by PMNs is abrogated by disrupting or partitioning PMNs from HIV-infected cells; is inhibited by superoxide dismutase and catalase, enzymes that destroy ROIs; is enhanced by differentiated HL60 cells capable of producing ROIs; and is induced by PMNs tested negative for CMV. Furthermore, the production of ROIs is independent of HIV infection of mononuclear cells, since PMNs cocultured with HIV-uninfected parental monocytic and T cell lines generated ROIs. Therefore, the increased risk for acquiring HIV infection associated with chlamydia cervicitis may be related to the local recruitment of PMNs by C. trachomatis and the induction of infectious virus from mononuclear cells present in semen. These observations provide a rationale for strategies to reduce HIV transmission by control of STD.

B-Lymphocytes↗

PubMed1994

Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.

Interleukin 4 (IL-4) diminishes cytokine activation of human macrophage. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacyglycerol (DAG), an activator of PKC, from membrane phospholipids was investigated to define the proximal events of IL-4R signaling. IL-4 induced a statistically significant time-and dose-dependent generation of DAG. The IL-4-triggered production of DAG was not derived from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in response to IL-4. Experiments were performed using [14C-methyl]choline-labeled U937 cells and monocytes to determine whether IL-4R activated phospholipase C (PLC), PLD, or PLA2 to use membrane phosphatidylcholine (PC) to form DAG. IL-4 induced a time- and dose-dependent increase of phosphocholine (pchol) with concomitant degradation of membrane PC (p < 0.05 compared with control). The finding that the peak reduction of PC was equivalent to peak production of pchol suggested that IL-4R signaling involved the activation of a PC-specific PLC. Changes in choline (chol) or lyso-PC and glycerolphosphocholine, the respective products of PC cleavage by PLD or PLA2, were not detected in IL-4-treated cells. In contrast, exogenous PLD induced an increase in chol and concomitant loss of membrane PC. Additional investigation suggested that IL-4R signaling does not involve PLD. In cells labeled with L-lyso-3-PC 1-[1-14C]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with ethanol. Propranolol, an inhibitor of phosphatidate phosphohydrolase, and calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG production in response to FMLP but not to IL-4. In propranolol pretreated cells, PMA but not IL-4 triggered the production of PA and lowered the amount of DAG. Evidence that PLA2 is not coupled to IL-4R is the detection of arachidonate production in response to FMLP but not to IL-4. Furthermore, IL-4R is not coupled to sphingomyelinase (SMase) since IL-4, unlike exogenous SMase, did not generate ceramide but induced the hydrolysis of PC to pchol that was comparable to exogenous PLC. In summary, IL-4R signaling in monocytes and U937 cells involves PLC and not PLD, PLA2, or SMase, and it uses PC and not PIP2 to form DAG.

Arachidonic Acid↗