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PubMed2004

Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas.

BACKGROUND: Ocular adnexal lymphomas may be antigen-driven disorders; however, the source of the putative antigen or antigens is still unknown. Hence, we assessed whether Chlamydiae infection is associated with the development of ocular adnexal lymphomas. METHODS: The presence of Chlamydia psittaci, trachomatis, and pneumoniae DNA was investigated by polymerase chain reaction in 40 ocular adnexal lymphoma samples, 20 nonneoplastic orbital biopsies, 26 reactive lymphadenopathy samples, and peripheral blood mononuclear cells (PBMCs) from 21 lymphoma patients and 38 healthy individuals. Seven patients with chlamydia-positive PBMCs were treated with the antibiotic doxycycline, and objective response was assessed in four patients with measurable lymphoma lesions. Differences in Chlamydiae DNA detection between the case patients and the control subjects were analyzed using the Fisher exact test. All statistical tests were two-sided. RESULTS: Thirty-two of the 40 (80%) ocular adnexal lymphoma samples carried C. psittaci DNA, whereas all lymphoma samples were negative for C. trachomatis and C. pneumoniae. In contrast, none of the 20 nonneoplastic orbital biopsies (0% versus 80%; P<.001) and only three of 26 (12%) reactive lymphadenopathy samples (12% versus 80%; P<.001) carried the C. psittaci DNA. Nine of 21 (43%) patients with chlamydia-positive lymphomas carried C. psittaci DNA in their PBMCs, whereas none (0%) of the healthy PBMC donors carried C. psittaci DNA in their PBMCs (43% versus 0%; P<.001). One month after doxycycline treatment, chlamydial DNA was no longer detectable in the PBMCs of all seven treated patients, and objective response was observed in two of the four evaluable patients. CONCLUSION: Patients with ocular adnexal lymphoma had a high prevalence of C. psittaci infection in both tumor tissue and PBMCs. Persistent C. psittaci infection may contribute to the development of these lymphomas, as was also supported by the clinical responses observed in this study with C. psittaci-eradicating antibiotic therapy.

Adult↗
PubMed2004

Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon Alfa immunotherapy.

BACKGROUND: Regulation of gene expression by signal transducer and activator of transcription 1 (STAT1) within host tissues mediates the antitumor effects of interferon alfa (IFN alpha). We used a novel flow cytometric assay to examine phosphorylation-mediated activation of STAT1 within immune effector cell subsets following in vitro or in vivo IFN alpha treatments. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 17) or melanoma patients (n = 19) were treated in vitro with interferon alfa-2b (IFN alpha-2b) or phosphate-buffered saline (PBS) and subjected to multiparametric flow cytometry to measure the levels of phosphorylated STAT1 (P-STAT1) within immune cell subsets. We similarly analyzed PBMCs isolated from melanoma patients before and 1 hour after immunotherapy with IFN alpha-2b. All statistical tests were two-sided. RESULTS: P-STAT1 levels in all major immune cell subsets increased within 15 minutes of in vitro IFN alpha-2b treatment of PBMCs; the increase was most pronounced in T lymphocytes and monocytes. Relatively low doses of IFN alpha-2b (i.e., 10(2)-10(3) IU/mL) induced maximal STAT1 activation in vitro. Compared with melanoma patients, healthy donors had higher basal levels of P-STAT1 (specific fluorescence [Fsp]; i.e., Fsp(PBS), the level of P-STAT1 in PBS-treated cells) in total PBMCs, natural killer (NK) cells, and T cells (mean Fsp(PBS) in total PBMCs: 5.5 in healthy donors versus 1.6 in patients, difference = 3.9, 95% confidence interval [CI] = 1.4 to 6.5, P =.004; mean Fsp(PBS) in NK cells: 4.6 in healthy donors versus 0.9 in patients, difference = 3.7, 95% CI = 1.7 to 5.7, P =.001; mean Fsp(PBS) in T cells: 6.8 in healthy donors versus 0.9 in patients, difference = 5.9, 95% CI = 2.5 to 9.3, P =.002). P-STAT1 was detected in the NK and T cells of two patients who received IFN alpha-2b immunotherapy (20 MU/m2 [MU = million units], administered by intravenous injection). P-STAT1 levels in the PBMCs of a patient treated sequentially with 5 MU/m2 and 10 MU/m2 IFN alpha-2b (administered by subcutaneous injection) also increased in response to treatments with IFN alpha-2b but did not increase further with the increased dosage of IFN alpha-2b. CONCLUSION: This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN alpha immunotherapy.

Antineoplastic Agents↗
PubMed1999

Effect of omeprazole, lansoprazole, and ranitidine on the DNA synthesis of mononuclear cells.

OBJECTIVE: To examine and compare the effects of omeprazole, lansoprazole, and ranitidine on the DNA synthesis of peripheral blood mononuclear cells. DESIGN: Ex vivo laboratory study. SETTING: Clinical research laboratory of an academic medical center. SUBJECTS: Healthy volunteers. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Venous blood was collected from normal subjects and peripheral blood mononuclear cells (PBMCs) were isolated using centrifugation techniques over a Ficoll-Hypaque density gradient. PBMCs were added to 12-well culture plates in four groups of media: a) control; b) control plus lansoprazole (25 microg/mL); c) control plus omeprazole (0.35 microg/mL); and d) control plus ranitidine (50 microg/mL). PBMCs were exposed to the drug for 96 hrs, with addition of phytohemagglutinin (2.5 microg/ mL) for the last 48 hrs, and 3H-thymidine (1 microCi) during the final 6 hrs. PBMCs were filtered onto glass-fiber filter paper and the radioactivity was determined by scintillation counting. Since radioactivity is measured only in those cells undergoing DNA synthesis or cell division, results are expressed as quantification of 3H-thymidine uptake. Median disintegrations per min (DPM)/number of PBMCs per well+/-SEM are reported: control 68.3+/-37.8; ranitidine 38.4 +/-94.2; lansoprazole 14.6+/-84.4; and omeprazole 15.1+/-48.9. There was a significant difference between lansoprazole vs. ranitidine (p< .01), and omeprazole vs. ranitidine (p< .05), and no significant difference between lansoprazole and omeprazole. CONCLUSIONS: This is the first study to compare the potential immunomodulating effects of these commonly used agents. Ranitidine caused increased DNA synthesis in PBMCs when compared with lansoprazole and omeprazole. This phenomenon may be an important, often disregarded, effect of histamine-2-receptor antagonists when used in postsurgical or trauma patients who have T-lymphocyte-mediated immune suppression.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1999

Hepatitis C virus in peripheral blood mononuclear cells from a chronically infected patient receiving liver graft from infected donor.

BACKGROUND: In hepatitis C virus (HCV)-positive patients receiving HCV-positive liver allografts either the donor or recipient strain overtakes the other strain. Whether these changes are reflected in peripheral blood mononuclear cell (PBMC)-associated virus is unknown. METHODS: We analyzed by single-strand conformation polymorphism and sequencing HCV RNA from serum and PBMCs from a liver transplant recipient whose indigenous strain was replaced by the donor strain. RESULTS: Only the recipient strain was detectable in serum and PBMCs 3 and 5 days after transplantation; at day 7 and 8, a mixture of both was present in the PBMCs, but only recipient strain was detectable in serum. This coincided with the peak presence of donor DNA in recipient PBMCs. From day 14 on, HCV sequences in serum and PBMCs were indistinguishable. CONCLUSIONS: Overtake phenomenon in the setting of liver transplantation from infected donors to infected recipients is manifested in PBMCs. Cells released from infected graft carry donor HCV strain.

Aged↗
PubMed2002

Reduction of human-to-pig cellular response by alteration of porcine MHC with human HLA DPW0401 exogenes.

BACKGROUND: In pig-to-human discordant xenotransplantation, the xenograft can be rejected by a formidable human xenogenic T-cell response, even if the graft has gone through hyperacute rejection or delayed xenograft rejection (acute vascular rejection). We therefore examined, in this study, whether the human-to-pig cellular response could be attenuated through the generation of a transgenic pig for human HLA II. METHODS: With the technique of microinjection, we produced the HLA DPw0401 transgenic pig. The expression of the HLA DPw0401 gene on peripheral blood mononuclear cells (PBMCs) of the transgenic pig was examined by reverse transcriptase-polymerase chain reaction and flow cytometry. The antigenicity of the transgenic HLA DPw0401 molecule was tested by the HLA DPw0401-primed lymphocyte test reagent. The cellular response was analyzed by xenogenic mixed lymphocyte culture. RESULTS: The mRNA and protein of HLA DPw0401 were expressed in the PBMCs of the transgenic pig. The PBMCs of the HLA transgenic pig induced a stronger cellular reaction to HLA DPw0401-primed lymphocyte test reagents than the nontransgenic littermate pig (n=7, P<0.01). In direct xenogenic mixed lymphocyte culture with responders from HLA DPw0401(+) humans, the PBMCs from the HLA DPw0401 transgenic pig, as compared with those from the normal pig, induced a lower degree of xenogenic cellular response to human PBMCs (n=4, P=0.08). CONCLUSIONS: Our preliminary data demonstrated the possibility that the human HLA DPw0401 phenotype can be transferred onto porcine cells through the generation of HLA transgenic pigs and make the PBMCs of humans more tolerant to porcine cells.

Animals↗
PubMed2006

The prognostic value of telomerase expression in peripheral blood mononuclear cells of head and neck cancer patients.

OBJECTIVE: This study investigates the expression of telomerase in the peripheral blood mononuclear cells (PBMCs) of head and neck cancer patients and the relationship between the telomerase expression in PBMCs and clinicopathological features with prognosis. METHODS: Peripheral blood samples were collected from 100 head and neck squamous cell cancer patients and 20 normal control group. The telomerase activity in peripheral blood mononuclear cells was measured by telomerase rapid amplification protocol (TRAP) assay using TRAPeze telomerase detection kit. Pearson chi test was used to analyze the correlation of telomerase expression with clinicopathologic parameters. Kaplan-Meier method and Cox proportional hazards model were used for prognostic analysis. RESULTS: Regarding the expression of telomerase in PBMCs of patients, 73 out of 100 (73.0%) were found to be positive, and the remaining 27 cases (27.0%) were negative. The difference to the telomerase expression of PBMCs in the normal and cancer patients was significant (P < 0.001). The expression of telomerase in PBMCs of patients was significant correlated with T classification (P = 0.005), N classification (P = 0.002), and AJCC stages (P < 0.001). On multivariate analysis, N classification (P = 0.007), AJCC stages (P = 0.02), and telomerase expression (P = 0.017) showed independent factors associated with poor survival. CONCLUSION: The detection of telomerase expression in PBMCs of head and neck cancer patients is a simple and very useful molecular marker for the progression and prognosis of head and neck cancer.

Adult↗
PubMed2006

Antihypertensive drugs clonidine, diazoxide, hydralazine and furosemide regulate the production of cytokines by placentas and peripheral blood mononuclear cells in normal pregnancy.

BACKGROUND: Antihypertensive drugs such as clonidine, diazoxide, hydralazine and furosemide are used in the hypertensive disorders of pregnancy to control blood pressure, but it is not clear if they modulate the production of placental or circulating cytokines. OBJECTIVE: To examine the effect of pharmaceutical doses of well known antihypertensive drugs used for blood pressure control on the production of the cytokines interleukin (IL)-6, IL-10 and tumour necrosis factor (TNF)-alpha in placental tissue and peripheral blood mononuclear cells (PBMCs) in normal pregnancy. DESIGN: Placental biopsies were taken from the decidual surface of placentas after delivery of normal pregnancies (n = 6) and PBMCs were separated from the whole blood of normal term pregnant women (n = 7). Both villous explants and PBMCs were cultured with increasing concentrations of antihypertensive drugs. The dose effect of drugs on the production of placental and circulating cytokines (IL-6, IL-10 and TNF-alpha) were examined by enzyme-linked immunosorbent assay. RESULTS: Placental production of IL-10 was not affected by clonidine, but decreased significantly after incubation of the tissue with diazoxide, hydralazine or furosemide. Production of IL-10 by PBMCs increased significantly: by from 3.4 +/- 2.7% [16.3 pg/ml (range 6.1-21.5 pg/ml)] to 24.5 +/- 3.3% [30.4 pg/ml (range 16.9-34.8 pg/ml)] with increasing concentrations of clonidine (0.08-1.3 microg/ml), and by 8.8 +/- 3.5% [4.1 pg/ml (range 3.0-17.8 pg/ml)] and 17.2 +/- 1.9% [22.6 pg/ml (range 13.2-23.2 pg/ml)] with lower doses of hydralazine (6.3 and 12.5 microg/ml) (all P values < 0.05). There was a stepwise reduction in production of TNF-alpha and IL-6 with increasing doses of diazoxide, hydralazine and furosemide by placentas and PBMCs from these women with normal pregnancies. CONCLUSION: Our data suggest that the antihypertensive drugs clonidine and hydralazine can stimulate production of the circulating anti-inflammatory cytokine IL-10, whereas furosemide and diazoxide inhibit the production of this cytokine and the proinflammatory cytokines TNF-alpha and IL-6 by placentas and PBMCs.

Antihypertensive Agents↗
PubMed2003

Relative cytokine and cytokine inhibitor production by mononuclear cells and neutrophils.

Mononuclear cells represent the major source of cytokines in blood; however, it has been postulated that neutrophils may produce and/or release modest amounts of cytokines. In this study, we compared the production of cytokines and cytokine inhibitors in lipopolysaccharide (LPS)-stimulated whole blood, peripheral blood mononuclear cells (PBMCs), and neutrophils (PMNs) separated by density gradient centrifugation. Contamination of PBMCs in the isolated PMNs was less than 0.1% as determined by morphological analysis and flow cytometry. LPS (50 ng/mL) induced a strong increase of the proinflammatory cytokines tumor necrosis factor, interleukin (IL)-1beta, IL-8, and IL-6 in whole blood. In the isolated cell preparations, proinflammatory cytokine production was significantly greater in the PBMCs compared with the PMNs. On a per cell basis, PMNs produced less than 1.5% of these cytokines compared with PBMCs. For cytokine inhibitors, TNF-soluble receptor type II and IL-1 receptor antagonist were increased by LPS stimulation in whole blood, PBMCs, and PMNs. On a per cell basis, LPS-induced TNF-soluble receptor type II and IL-1 receptor antagonist production by PMNs was 9.8% and 15.4% of those of PBMCs, respectively. These data show that a highly purified population of PMNs makes a relatively minor to nonexistent contribution to the production of proinflammatory cytokines.

Antigens, CD↗
PubMed2001

Human antibodies isolated from plasma by affinity chromatography increase the coxsackievirus B4-induced synthesis of interferon-alpha by human peripheral blood mononuclear cells in vitro.

Coxsackievirus B4 (CVB4) can be found in circulating blood of patients; however, the interaction of CVB4 with peripheral blood mononuclear cells (PBMCs) is poorly understood. CVB4 induced low levels of IFN-alpha synthesis in PBMCs from healthy donors. In contrast, preincubation of infectious CVB4 with plasma from these donors containing anti-CVB4 antibodies strongly enhanced the synthesis of IFN-alpha. IgG obtained from plasma by chromatography formed immune complexes with CVB4 and increased significantly the CVB4-induced production of IFN-alpha by PBMCs. These antibodies did not have a neutralizing effect on CVB4 infection of Hep-2 cells. The role of CVB and adenovirus receptor (CAR), FcgammaRII and FcgammaRIII in the increased synthesis of IFN-alpha induced by CVB4 preincubated with IgG was shown by inhibition with specific antibodies. The major interferon-alpha-producing cells in response to CVB4-IgG complexes were CD14(+) cells and monocyte-enriched PBMCs. With the latter, detection of IFN-alpha by immunostaining was positive whereas in monocyte-depleted PBMCs it was not. This study shows that CVB4-induced synthesis of IFN-alpha by PBMCs can be enhanced by an antibody-dependent mechanism through interactions between the virus, non-neutralizing antivirus antibodies, FcgammaRII and III and CAR.

Adolescent↗
PubMed1998

Genomic analysis of the viral population in genital secretions early after infection of simian immunodeficiency viruses in macaque monkeys.

To clarify the change in the viral population during passage from the vaginal cavity to blood circulation and vice versa, we examined the viral clones detected in cells in vaginal washes (VWCs) early after inoculation and after systemic infection with polyclonal SIV. In two intravaginally inoculated monkeys, the viral clones found in VWCs at 18 days p.i. were shown to be some of those contained in the inoculum, whereas the viral population in the peripheral blood mononuclear cells (PBMCs) was a monotype. This gradual decrease of viral clones suggested the possible existence of two barriers, one at the genital tract and the other between the genital tract and the blood. Later, at one month p.i., the viral clones in VWCs became rather restricted, whereas those in PBMCs diverged from a single clone to several clones. This suggested that different mechanisms affect the viral populations in PBMCs and VWCs. In order to examine how the viral population was affected by passage from the blood to the vaginal cavity, a monkey was intravenously inoculated and the viral clones in VWCs were analyzed at 14 days p.i., at a time of the heterogeneous population in PBMCs. The viral population in VWCs was found to be a single clone and this clone was a minor type in PBMCs, suggesting that the major clone in PBMCs was not always secreted to the vaginal cavity.

Animals↗
PubMed2002

Enhanced anti-tumor effect of trastuzumab in combination with cisplatin.

The oncogenenic transmembrane tyrosine kinase receptor HER-2/neu is a promising target for treatment of HER-2-overexpressing cancers. The humanized anti-HER-2/neu antibody Trastuzumab is under clinical evaluation in combination with chemotherapy against breast cancer. The combination of Trastuzumab and cisplatin is expected to be active against HER-2 / neu-expressing tumors. We examined the mechanisms of this combination effect against human solid tumor cells in the presence of human peripheral blood mononuclear cells (PBMCs) using an in vitro MTT assay. The growth-inhibitory effects of cisplatin (CDDP) on the tumor cells were not significantly affected by Trastuzumab in the absence of effector cells. CDDP alone at a dose of less than 12.5 mM did not affect the viability of PBMCs, as determined by MTT assay, suggesting that PBMCs could exert antibody-dependent cell-mediated cytotoxicity (ADCC) at this CDDP concentration. The combination of Trastuzumab and CDDP showed higher cytotoxic effects against the tumor cells in the presence of PBMCs. The CDDP concentration required to inhibit tumor cell growth by 50% was reduced to approximately 20% by Trastuzumab in the presence of PBMCs at an effector/target ratio of 10. It may be important to select combined chemotherapeutic agents which do not diminish the ADCC activity of Trastuzumab via PBMCs. Both the expression of HER-2 / neu and the ADCC activity may be important determinants of the therapeutic benefit of the Trastuzumab/CDDP combination.

Antibodies, Monoclonal↗
PubMed2006

Inhibitory effects of fluvastatin on cytokine and chemokine production by peripheral blood mononuclear cells in patients with allergic asthma.

BACKGROUND: Statins have anti-inflammatory effects on immune cells. OBJECTIVE: To investigate the immunomodulatory effects of fluvastatin on peripheral blood mononuclear cells (PBMCs) after allergen-specific and non-allergen-specific stimulation in patients with asthma and in healthy subjects. METHODS: PBMCs from seven patients with asthma who showed elevated immunoglobulin (Ig)E to house dust mite were isolated and stimulated with Dermatofagoides farinae, purified protein derivative, and phytohaemagglutinin (PHA) in the presence or absence of fluvastatin. PBMCs from seven healthy subjects were stimulated with PHA. The effects of fluvastatin on cell proliferation and production of cytokines (interferon [IFN]-gamma and interleukin [IL]-5) and chemokines (chemokine CXC motif, ligand [CXCL10], and CC chemokine ligand [CCL17]) were measured. Migration of T helper (Th)1 and Th2 cell lines was also investigated. The expression of CXCR3 and CCR4 was analysed with flow cytometry. Steroid-insensitive PBMCs induced by preculture with IL-2 and IL-4 were also evaluated. Some experiments were performed in the presence of mevalonic acid. RESULTS: Fluvastatin inhibited the proliferation of PBMCs and decreased the production of IL-5, IFN-gamma, CCL17, and CXCL10 after allergen-specific and non-allergen-specific stimulation; all these effects, except for decreased CXCL10 production, were partially reversed by mevalonic acid. Culture supernatants obtained in the presence of fluvastatin prevented the migration of Th1 and Th2 cell lines in a dose-dependent manner. In addition, CCR4 and CXCR3 expression on CD4(+) T cells was not affected by the presence of fluvastatin. Fluvastatin inhibited the proliferative response of steroid-insensitive PBMCs to phytohaemagglutinin. CONCLUSION: Fluvastatin has inhibitory effects on cytokine and chemokine production, and thus might be used as a potential therapeutic agent in severe asthma.

Adult↗
PubMed2004

Expression of CTLA-4 in nonhuman primate lymphocytes and its use as a potential target for specific immunotoxin-mediated apoptosis: results of in vitro studies.

T-cell-mediated immunoregulation is one of the main mechanisms implicated in induction and maintenance of transplantation tolerance. In this regard, deletion or modulation of xeno/alloantigen-specific T cells, as well as blocking of their interactions with other cell populations, are currently being pursued for tolerance induction in humans as well as nonhuman primates. In order to investigate whether cytotoxic T-lymphocyte antigen-4 (CTLA-4) may represent a suitable target for a T cell depletion approach in nonhuman primate models, we analysed CTLA-4 expression in peripheral blood mononuclear cells (PBMCs) from nonhuman primates and the potential role of two anti-CTLA-4 saporin-conjugated immunotoxins. The analysis was performed in PBMCs from 8 cynomolgus monkeys from Philippines and from Mauritius both at protein level by flow cytometry and at transcriptional level by RT-PCR. In addition, the apoptotic role of the immunotoxins was investigated. The results showed that CTLA-4 was expressed at variable levels depending on the origin of the cynomolgus monkeys and the resting or activated cell condition. CTLA-4 was not expressed on resting Mauritius PBMCs and showed a lower up-regulation upon PMA/PHA activation compared to the Philippines PBMCs that expressed CTLA-4 also before activation. Two CTLA-4 RNA transcripts (672 and 550 bp) were detected with levels variations after cell stimulation. Two anti-CTLA-4 immunotoxins induced in vitro apoptosis of activated PBMCs from both sources of cynomolgus monkeys. This is the first report that documents CTLA-4 expression both at protein and transcriptional level by nonhuman primate PBMCs and provides novel perspectives of xeno/allograft rejection immunotherapy based on CTLA-4 targeting.

Animals↗
PubMed1995

IgG2 deficiency associated with defects in production of interferon-gamma; comparison with common variable immunodeficiency.

We report a novel mechanism of IgG2 deficiency. Several investigators have reported patients with IgG subclass deficiencies due to homozygous deletion of immunoglobulin heavy chain constant region genes. However, it is unclear what mechanism is responsible for IgG subclass deficiency in cases where no gene deletions have been detected and which are accompanied by recurrent infections due to aberrant immunoregulation. In the present study, we have focused our attention on production by peripheral blood mononuclear cells (PBMCs) of interferon-gamma (IFN-gamma), which is known to induce IgG2 expression. PBMCs from four patients with IgG2 deficiency and their families were studied. Mitogeninduced IFN-gamma production by PBMCs was decreased in all of the patients, although the proliferative responses of PBMCs and the percentages of CD3, CD4, and CD8 T cell subsets were not decreased. IgG2 production by PBMCs was restored upon addition of IFN-gamma and mitogen to the PBMCs of the patients with IgG2 deficiency though it was not restored in the patients with common variable immunodeficiency. We conclude that defects in production of IFN-gamma play an important role in IgG2 deficiency.

Adult↗
PubMed1999

Late disappearance of hepatitis C virus RNA from peripheral blood mononuclear cells in patients with chronic hepatitis C in sustained response after alpha-interferon therapy.

OBJECTIVE: We aimed to investigate the modifications of HCV RNA (genomic and antigenomic strands) in peripheral blood mononuclear cells (PBMCs) of long-term responder patients to alpha-interferon therapy, and their usefulness as criteria of definitive HCV eradication. METHODS: We studied 10 patients with chronic hepatitis C with > 1 yr of sustained response after alpha-interferon therapy (normal alanine aminotransferase [ALT] and negative serum HCV RNA). Serum HCV RNA and genotyping were determined. Approximately 2 and 4 yr after completion of treatment we investigated the presence of HCV RNA (genomic and antigenomic strands) in PBMCs. Eight of 10 patients were rebiopsed 2 yr after discontinuation of treatment. RESULTS: The mean follow-up was 46.6 +/- 4.6 months (range, 39-51 months). In this period, all patients remained in sustained response. In the first determination, all patients had HCV RNA genomic strands and two patients had antigenomic strands detectable in PBMCs. Two years later only two patients had genomic and none had antigenomic strands detectable. After 4 yr of sustained response, eight of 10 patients lost HCV RNA from PBMCs. CONCLUSIONS: In the long-term follow-up, the majority of patients with chronic hepatitis C with sustained response after alpha-interferon therapy progressively lost HCV RNA from PBMCs. This determination in PBMCs is not a predictor of response.

Adolescent↗
PubMed1986

Enhancement of in vitro erythropoiesis by peripheral blood mononuclear cells from allogeneic marrow recipients in the early post-transplant period.

The effect of peripheral blood mononuclear cells (PBMCs) from bone marrow recipients on in vitro growth of erythroid burst-forming units (BFU-E) was studied. PBMCs were obtained from 5 allogeneic, 1 syngeneic and 1 autologous bone marrow recipient(s) at different intervals after transplantation. The number of BFU-E was significantly increased when donor bone marrow cells were co-cultured with PBMCs obtained from allogeneic marrow recipients in the early post-transplant period. No effect was observed using PBMCs obtained in the later post-transplant period, PBMCs from a syngeneic marrow recipient, or PBMCs from an autologous marrow recipient. The BFU-E enhancing activity was present among T cells and was abolished by treating them with OKT3 or OKT4 antibody and complement. These observations suggest that chimeric T lymphocytes, probably of the helper/inducer subset, from allogeneic marrow recipients in the early post-transplant period have a potent enhancing effect on in vitro erythropoiesis.

Adolescent↗
PubMed1992

Intracellular zidovudine (ZDV) and ZDV phosphates as measured by a validated combined high-pressure liquid chromatography-radioimmunoassay procedure.

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.

Cells, Cultured↗
PubMed2006

Homologous and cross-reactive immune responses to enterotoxigenic Escherichia coli colonization factors in Bangladeshi children.

We have studied homologous (HoM) and cross-reacting (CR) immunoglobulin A (IgA) antibody responses to colonization factors (CFs) in Bangladeshi children with diarrhea due to enterotoxigenic E. coli (ETEC) strains of the CF antigen I (CFA/I) group (CFA/I, n = 25; coli surface antigen 4 [CS4], n = 8; CS14, n = 11) and the CS5 group (CS5, n = 15; CS7, n = 8), respectively. The responses to the HoM, CR, and heterologous (HeT) CF antigens in each group of patient were studied and compared to that seen in healthy children (n = 20). In the CFA/I group (CFA/I and CS14), patients responded with antibody-secreting cell (ASC) responses to HoM CFs (geometric mean, 156 to 329 ASCs/10(6) peripheral blood mononuclear cells [PBMCs]) and to CR CFs ( approximately 15 to 38 ASCs/10(6) PBMCs) but least of all to the HeT CS5 antigen (2 to 4 ASCs/10(6) PBMCs). For the CS5 group of patients with ETEC (CS5 and CS7), likewise, responses to HoM CFs (230 to 372 ASCs/10(6) PBMCs) and CR CFs (27 to 676 ASCs/10(6) PBMCs) were seen, along with lower responses to the HeT CFA/I antigen (9 to 38 ASCs/10(6) PBMCs). Both groups of patients responded with CF-specific IgA antibodies to HoM and CR antigens in plasma but responded less to the HeT CFs. The responses in patients were seen very soon after the onset of diarrhea and peaked around 1 week after onset. Vaccinees who had received two doses of the oral, killed whole-cell ETEC vaccine (CF-BS-ETEC) responded with plasma IgA antibodies to CFA/I, a component of the vaccine, but also to the CR CS14 antigen, which was not included in the vaccine, showing that antibody responses can be stimulated by a CFA/I-containing ETEC vaccine to a CR-reacting antigen in individuals in countries where ETEC is endemic.

Antibodies, Bacterial↗