Density Of HLA-DR Research Articles

A reactive monocyte subset characterized by low expression of CD91 is expanded during sterile and septic inflammation.

This study was undertaken to assess CD91 expression on monocytes and changes in monocyte subset distribution during acute tissue damage and bloodstream infection (BSI). We investigated blood specimens from healthy individuals, trauma and cardiac surgery patients as a model of tissue damage, and patients with BSI, by flow cytometry using a panel of antibodies comprising CD45, HLA-DR, CD14, CD16 and CD91 for the identification of monocyte subsets. While infrequent in healthy subjects, CD91low/neg monocyte levels were markedly high in BSI, trauma and after cardiac surgery. This monocyte subset expanded up to15-fold in both patient cohorts, whereas CD14+CD16+ inflammatory monocytes were multiplied by a factor of 5 only. CD14+CD91low monocytes displayed a significantly lower density of HLA-DR and markedly reduced expression of CD300e, compared to the other subsets. They also expressed high levels of myeloperoxidase and showed robust phagocytic and oxidative burst activity. Expansion of CD91low monocytes is a sensitive marker of acute inflammatory states of infectious and non-infectious etiology.

Dampened Inflammatory Signalling and Myeloid-Derived Suppressor-Like Cell Accumulation Reduces Circulating Monocytic HLA-DR Density and May Associate With Malignancy Risk in Long-Term Renal Transplant Recipients

BackgroundMalignancy is a major cause of morbidity and mortality in transplant recipients. Identification of those at highest risk could facilitate pre-emptive intervention such as reduction of immunosuppression. Reduced circulating monocytic HLA-DR density is a marker of immune depression in the general population and associates with poorer outcome in critical illness. It has recently been used as a safety marker in adoptive cell therapy trials in renal transplantation. Despite its potential as a marker of dampened immune responses, factors that impact upon monocytic HLA-DR density and the long-term clinical sequelae of this have not been assessed in transplant recipients.MethodsA cohort study of stable long-term renal transplant recipients was undertaken. Serial circulating monocytic HLA-DR density and other leucocyte populations were quantified by flow cytometry. Gene expression of monocytes was performed using the Nanostring nCounter platform, and 13-plex cytokine bead array used to quantify serum concentrations. The primary outcome was malignancy development during one-year follow-up. Risk of malignancy was calculated by univariate and multivariate proportionate hazards modelling with and without adjustment for competing risks.ResultsMonocytic HLA-DR density was stable in long-term renal transplant recipients (n=135) and similar to non-immunosuppressed controls (n=29), though was suppressed in recipients receiving prednisolone. Decreased mHLA-DRd was associated with accumulation of CD14+CD11b+CD33+HLA-DRlo monocytic myeloid-derived suppressor-like cells. Pathway analysis revealed downregulation of pathways relating to cytokine and chemokine signalling in monocytes with low HLA-DR density; however serum concentrations of major cytokines did not differ between these groups. There was an independent increase in malignancy risk during follow-up with decreased HLA-DR density.ConclusionsDampened chemokine and cytokine signalling drives a stable reduction in monocytic HLA-DR density in long-term transplant recipients and associates with subsequent malignancy risk. This may function as a novel marker of excess immunosuppression. Further study is needed to understand the mechanism behind this association.

DenVar: density-based variation analysis of multiplex imaging data.

Multiplex imaging platforms have become popular for studying complex single-cell biology in the tumor microenvironment (TME) of cancer subjects. Studying the intensity of the proteins that regulate important cell-functions becomes extremely crucial for subject-specific assessment of risks. The conventional approach requires selection of two thresholds, one to define the cells of the TME as positive or negative for a particular protein, and the other to classify the subjects based on the proportion of the positive cells. We present a threshold-free approach in which distance between a pair of subjects is computed based on the probability density of the protein in their TMEs. The distance matrix can either be used to classify the subjects into meaningful groups or can directly be used in a kernel machine regression framework for testing association with clinical outcomes. The method gets rid of the subjectivity bias of the thresholding-based approach, enabling easier but interpretable analysis. We analyze a lung cancer dataset, finding the difference in the density of protein HLA-DR to be significantly associated with the overall survival and a triple-negative breast cancer dataset, analyzing the effects of multiple proteins on survival and recurrence. The reliability of our method is demonstrated through extensive simulation studies. The associated R package can be found here, https://github.com/sealx017/DenVar. Supplementary data are available at Bioinformatics Advances online.

1648. CD4 T Lymphocyte and Monocyte Activation within One Year After Treatment Completion for Pulmonary Tuberculosis: A Pilot Study

BackgroundPersons with active tuberculosis (TB) have increased immune activation, which declines with TB treatment. Whether residual immune activation is present after completion of TB treatment is unknown. We conducted immunophenotyping of T cells and monocytes from individuals who had completed TB treatment, and compared them to controls without history of active TB.MethodsCross-sectional study of HIV-uninfected individuals 40 to 70 years old recruited in Lima, Peru. For this analysis, we included 6 individuals who had completed treatment for pulmonary TB within the past year, and 10 healthy controls without history of active TB (6 QuantiFERON®-TB positive; 4 QuantiFERON®-TB negative). Participants provided blood for T cell and monocyte immunophenotyping using multi-parameter flow cytometry, including expression of cell surface activation markers.ResultsThere were no significant differences in age, sex, or comorbidities between patients previously treated for TB and controls without history of active TB. Median time since TB treatment completion was 4 months (range, 2 – 9) for the previously treated group. Compared to controls, persons previously treated for TB had increased CD4+ to CD8+ ratio (2.4 vs. 1.3; p=0.03), increased proportion of CD4+ T cells co-expressing HLA-DR and CD38 activation markers (5.8% vs. 3.1%; p=0.02), increased CD4+ T cell ICAM-1 expression (56.3% vs. 33.2%; p=0.03), as well as increased density of HLA-DR in monocytes (HLA-DR MFI; 7572 vs. 3917; p=0.03).ConclusionIn this pilot study, individuals who recovered from pulmonary TB exhibited increased CD4+ T cell and monocyte activation phenotypes within one year of TB treatment completion. These results suggest residual immune activation after clinical TB cure.Disclosures Carl Fichtenbaum, MD, Amgen (Grant/Research Support, outside the submitted work)Clinical Care Options (Other Financial or Material Support, outside the submitted work)Cytodyn (Grant/Research Support, outside the submitted work)Gilead Sciences (Grant/Research Support, outside the submitted work)Janssen (Grant/Research Support, outside the submitted work)Merck (Grant/Research Support, outside the submitted work)ViiV Healthcare (Research Grant or Support, outside the submitted work)

Monocyte activation in persons living with HIV and tuberculosis coinfection.

To characterize monocyte subsets and activation in persons living with HIV (PLWH) with tuberculosis coinfection. Cross-sectional study within a cohort of PLWH and HIV-uninfected participants at the Joint Clinical Research Centre in Kampala, Uganda. Participants were at least 45 years old with at least one cardiovascular risk factor. PLWH had an HIV viral load 1000 copies/ml or less on stable antiretroviral therapy prior to cohort entry. QuantiFERON-TB testing was performed to define latent tuberculosis infection (LTBI). Prior active TB was defined by self-report and verified by medical records. Blood was stained with monocyte subset markers (CD14+, CD16), CD62p, CD69, CX3CR1, HLA-DR, and tissue factor, and examined with flow cytometry. One hundred and twenty-five participants (83 PLWH and 42 without HIV) were included. Median CD4+ count was 582 cells/μl in PLWH. PLWH had a higher frequency of total monocytes (4.3% vs. 3.2%; P < 0.001) and inflammatory monocyte subset (15.5% vs. 11.7%; P = 0.016) compared with HIV-uninfected individuals. No differences in the frequency of monocyte subsets were observed by TB status. Among PLWH, prior active TB was associated with increased frequency of total monocytes compared with LTBI (5.1% vs. 3.7%; P = 0.013). HLA-DR density on monocytes was three-fold higher in PLWH with LTBI or prior TB compared with PLWH without LTBI (P = 0.002). In multivariate analysis, a higher monocyte HLA-DR density remained associated with LTBI or prior TB in PLWH (log-MFI; b = 1.17; P < 0.001). Our findings indicate enhanced monocyte activation in PLWH with LTBI or prior active TB, which may contribute to the pathogenesis of noncommunicable diseases in HIV.

Abstract 2798: High density of CD68+HLA-DR- macrophages in the stroma of primary melanoma correlates with an unfavorable immune microenvironment as assessed by Digital Spatial Profiling

Abstract Introduction: The role of macrophages (Mϕ) in melanoma progression is controversial, as they have been shown to both favor and inhibit anti-tumor immunity. Density of Mϕ at the leading tumor edge has been shown to be a marker of poor prognosis. In previous work, we used quantitative multiplexed immunofluorescence (qmIF) to discover the ratio of CD8+ T lymphocytes (CTLs) to CD68+ Mϕ in the peritumoral stroma predicts a favorable prognosis. Further, we found that increased proximity of HLA-DR- Mϕ to CTLs in the stroma predicts poor prognosis. These findings highlight the importance of the location of Mϕ within the tumor microenvironment (TME). Here, we further analyze and compare the TME of patients with high and low stromal densities of HLA-DR- Mϕ. Methods: From a cohort of 104 patients with primary stage II-III melanoma and known survival information, we selected 8 patients for Digital Spatial Profiling (DSP) analyses. Of this subcohort, 4 patients had a high density of HLA-DR- Mϕ and close proximity of HLA-DR- Mϕ to CTLs in the stroma while the other 4 had a high CTL to Mϕ ratio with low HLA-DR- Mϕ density, as determined by qmIF (methods published). FFPE slides were stained with antibodies conjugated to UV-photocleavable DNA barcodes and specific to 34 proteins, including CD45, CD4, CD8, CD68, PD-1, and PD-L1. Twelve regions of interest (ROIs) per patient were selected based on high Mϕ density as determined by qmIF. ROIs were then analyzed using UV excitation, which releases DNA barcodes for downstream quantitation on the nanoString nCounter® platform. Protein expression was compared between patients and statistical analysis performed using Mann-Whitney test. Results: We found that ROIs from patients with higher density of HLA-DR- Mϕ had a lower immune infiltration overall as assessed by quantitation of CD45 per ROI (p&amp;lt;0.0001). As expected, the ratio of CD68 to CD45 was higher in these patients than in patients with lower density of HLA-DR- Mϕ (p&amp;lt;0.0001). Interestingly, patients with higher density of HLA-DR- Mϕ also had a significantly higher ratio of CD4 to CD45 (p&amp;lt;0.0001), but a similar CD8A to CD45 ratio. Patients with a higher HLA-DR- Mϕ density had a higher CD4 to CD8A ratio (p&amp;lt;0.0001). Further, PD-L1 and PD1 levels per CD45 were significantly higher in patients with higher HLA-DR- Mϕ density (p&amp;lt;0.0001 and p=0.0002, respectively). Conclusion: Patients with higher densities of HLA-DR- Mϕ in the stroma and increased proximity of HLA-DR- Mϕ to CTLs in the tumor stroma have lower levels of CD45, a higher ratio of CD4 to CD8, and a higher ratio of PDL1 and PD1 to CD45 by assessment of Mϕ-rich areas using DSP. These findings highlight the close relation between Mϕ and the local immune microenvironment in primary stage II-III melanoma. Citation Format: Emanuelle M. Rizk, Andrew Chen, Andrew M. Silverman, Douglas K. Marks, Raul Rabadan, Kit Fuhrman, Alison VanSchoiack, Yan Liang, Joseph Beechem, Yvonne M. Saenger, Robyn D. Gartrell. High density of CD68+HLA-DR- macrophages in the stroma of primary melanoma correlates with an unfavorable immune microenvironment as assessed by Digital Spatial Profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2798.

Perturbations of Monocyte Subsets and Their Association with T Helper Cell Differentiation in Acute and Chronic HIV-1-Infected Patients.

Monocytes have been recently subdivided into three subsets: classical (CD14++CD16−), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) subsets, but phenotypic and functional abnormalities of the three monocyte subsets in HIV-1 infection have not been fully characterized, especially in acute HIV-1 infection (AHI). In the study, we explored the dynamic changes of monocyte subsets and their surface markers, and the association between monocyte subsets and the IFN-γ, interleukin (IL)-4, IL-17, and TNF-α producing CD4+ T cells in acute and chronic HIV-1-infected patients. We found that, in the acute HIV-1-infected individuals, the frequency of the intermediate CD14++CD16+ monocyte subsets, the CD163 density and HLA-DR density on intermediate CD14++CD16+ monocytes, and plasma soluble form of CD163 (sCD163) were significantly higher than that in healthy controls. Intermediate CD14++CD16+ monocyte subsets and their HLA-DR expression levels were inversely correlated with the CD4+ T cell counts, and the intermediate CD14++CD16+ monocytes were positively correlated with plasma sCD163. In contrast to the non-classical CD14+CD16++ and classical CD14++CD16− monocyte subsets, the frequency of the intermediate CD14++CD16+ monocytes was positively associated with the frequency of IFN-γ and IL-4 producing CD4+ T cells in HIV-1-infected patients. Taken together, our observations provide new insight into the roles of the monocyte subsets in HIV pathogenesis, particularly during AHI, and our findings may be helpful for the treatment of HIV-related immune activation.

Sepsis-induced long-term immune paralysis--results of a descriptive, explorative study.

BackgroundLong-lasting impairment of the immune system is believed to be the underlying reason for delayed deaths after surviving sepsis. We tested the hypothesis of persisting changes to the immune system in survivors of sepsis for the first time.MethodsIn our prospective, cross-sectional pilot study, eight former patients who survived catecholamine-dependent sepsis and eight control individuals matched for age, sex, diabetes and renal insufficiency were enrolled. Each participant completed a questionnaire concerning morbidities, medications and infection history. Peripheral blood was collected for determination of i) immune cell subsets (CD4+, CD8+ T cells; CD25+ CD127- regulatory T cells; CD14+ monocytes), ii) cell surface receptor expression (PD-1, BTLA, TLR2, TLR4, TLR5, Dectin-1, PD-1 L), iii) HLA-DR expression, and iv) cytokine secretion (IL-6, IL10, TNF-α, IFN-γ) of whole blood stimulated with either α-CD3/28, LPS or zymosan.ResultsAfter surviving sepsis, former patients presented with increased numbers of clinical apparent infections, including those typically associated with an impaired immune system. Standard inflammatory markers indicated a low-level inflammatory situation in former sepsis patients. CD8+ cell surface receptor as well as monocytic HLA-DR density measurements showed no major differences between the groups, while CD4+ T cells tended towards two opposed mechanisms of negative immune cell regulation via PD-1 and BTLA. Moreover, the post-sepsis group showed alterations in monocyte surface expression of distinct pattern recognition receptors; most pronouncedly seen in a decrease of TLR5 expression. Cytokine secretion in response to important activators of both the innate (LPS, zymosan) and the adaptive immune system (α-CD3/28) seemed to be weakened in former septic patients.ConclusionsCytokine secretion as a reaction to different activators of the immune system seemed to be comprehensively impaired in survivors of sepsis. Among others, this could be based on trends in the downregulation of distinct cell surface receptors. Based on our results, the conduct of larger validation studies seems feasible, aiming to characterize alterations and to find potential therapeutic targets to engage.

Altered Monocyte Phenotype in HIV-1 Infection Tends to Normalize with Integrase-Inhibitor-Based Antiretroviral Therapy.

BackgroundMonocytes are increasingly implicated in the inflammatory consequences of HIV-1 disease, yet their phenotype following antiretroviral therapy (ART) initiation is incompletely defined. Here, we define more completely monocyte phenotype both prior to ART initiation and during 48 weeks of ART.MethodsCryopreserved peripheral blood mononuclear cells (PBMCs) were obtained at baseline (prior to ART initiation) and at weeks 12, 24, and 48 of treatment from 29 patients participating in ACTG clinical trial A5248, an open label study of raltegravir/emtricitibine/tenofovir administration. For comparison, cryopreserved PBMCs were obtained from 15 HIV-1 uninfected donors, each of whom had at least two cardiovascular risk factors. Thawed samples were stained for monocyte subset markers (CD14 and CD16), HLA-DR, CCR2, CX3CR1, CD86, CD83, CD40, CD38, CD36, CD13, and CD163 and examined using flow cytometry.ResultsIn untreated HIV-1 infection there were perturbations in monocyte subset phenotypes, chiefly a higher frequency and density (mean fluorescence intensity–MFI) of HLA-DR (%-p = 0.004, MFI-p = .0005) and CD86 (%-p = 0.012, MFI-p = 0.005) expression and lower frequency of CCR2 (p = 0.0002) expression on all monocytes, lower CCR2 density on inflammatory monocytes (p = 0.045) when compared to the expression and density of these markers in controls’ monocytes. We also report lower expression of CX3CR1 (p = 0.014) on patrolling monocytes at baseline, compared to levels seen in controls. After ART, these perturbations tended to improve, with decreasing expression and density of HLA-DR and CD86, increasing CCR2 density on inflammatory monocytes, and increasing expression and density of CX3CR1 on patrolling monocytes.ConclusionsIn HIV-1 infected patients, ART appears to attenuate the high levels of activation (HLA-DR, CD86) and to increase expression of the chemokine receptors CCR2 and CX3CR1 on monocyte populations. Circulating monocyte phenotypes are altered in untreated infection and tend to normalize with ART; the role of these cells in the inflammatory environment of HIV-1 infection warrants further study.

Abstract 370: Dendritic Cells Express Triggering Receptor Expressed on Myeloid Cells-1 and Correlate with Plaque Stability in Symptomatic and Asymptomatic Patients with Carotid Stenosis

Aim: Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory infiltrates. Immune cells, including T-lymphocytes, dendritic cells (DCs) and macrophages recruited from blood stream into atherosclerotic lesions, are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. Upon activation, DCs, macrophages, and neutrophils produce several inflammatory cytokines and growth factors. In atherosclerotic tissue, the number of DCs increases with the progression of the lesion. In this study, we examined the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in plaque DCs and compared between the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. Methods and Results: Surgical specimens of human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy. The expression patterns of TREM-1, HLA-DR, and fascin (DC) were determined by immunofluorescence with α-actin as specific marker to identify smooth muscle cells (VSMCs). There was a marked decrease in VSMCs and an increased density of HLA-DR+ cells in the plaques of S than AS patients. Fascin expression to show DCs was undetectable in normal carotid artery specimens but significantly increased in the carotid plaques of S compared to AS. TREM-1 expression was increased in S and localized with HLA-DR and fascin. However, TREM-1 was increased in S plaques containing less number of VSMCs. The mRNA expression of MMP-9, MMP-1 was increased while Col 1 (α1) and Col III (α1) mRNA transcripts were decreased in S plaques. Conclusions: Increased expression of TREM-1 in plaque DCs of S compared to AS patients suggests a potential role of TREM-1 in atherosclerotic plaque vulnerability. Collectively, these data demonstrate that selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing atherosclerotic plaques.

B lymphocytes can regulate the maturation and function of human dendritic cells but are partially inefficient in systemic lupus erythematosus

Backgroundand objectivesMature dendritic cells (DCs) are stimulators of T cell immune response, whereas immature DCs support T cell tolerance. Murine B cells can inhibit the production of interleukin 12 (IL-12)...

Maturation and function of human dendritic cells are regulated by B lymphocytes

AbstractMature dendritic cells (DCs) are stimulators of T-cell immune response, whereas immature DCs support T-cell tolerance. Murine B cells can inhibit the production of IL-12 by DCs and thereby hinder the inflammatory response. Notwithstanding the importance of this modulation, only a few studies are available in humans. Here, we have developed an in vitro model of cocultures to assess its significance. We establish that human activated B cells restrained the development of monocytes into immature DCs and their differentiation into mature DCs. In addition, they decreased the density of HLA-DR from mature DCs, the expression of CD80 and CD86 coactivation molecules, the production of IL-12p70 required for antigen presentation and Th1 differentiation, and inhibited the DC-induced T-cell proliferation. These modulations were mediated by CD19+IgDlowCD38+CD24lowCD27− B cells and needed direct cell-to-cell contacts that involved CD62L for the control of CD80 and CD86 expression and a soluble factor for the control of IL-12 production. Moreover, mature DCs from patients with systemic lupus erythematosus displayed insensitivity to the regulation of IL-12. Overall, it appears that human B cells can regulate DC maturation and function and that inefficient B-cell regulation may influence an improper balance between an effector inflammatory response and tolerance induction.

Bronchial response pattern of antigen presenting cells and regulatory T cells in children less than 2 years of age

Background:In early childhood, the ability to mount protective immune responses in the airways is impaired, with increased risk of allergic sensitisation to inhaled allergens. Antigen presenting cells (APC) and regulatory...

Baculovirus-based Vaccination Vectors Allow for Efficient Induction of Immune Responses Against Plasmodium falciparum Circumsporozoite Protein

Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells. We found that baculovirus vectors can transduce professional antigen-presenting cells and trigger their maturation, which is a prerequisite for efficient antigen presentation. Upon intramuscular injection into mice, the vector that both displayed and expressed CS induced higher anti-CS antibody titers (of the immunoglobulin (IgG)1 and IgG2a type) and a higher frequency of interferon-gamma-producing T cells specific to CS, than the vectors which either only displayed or only expressed CS. The baculovirus CS display/expression vector was also superior in inducing CS-specific CD4(+) and CD8(+) T-cell responses in vitro using human peripheral blood mononuclear cells from naive donors. This, together with the absence of pre-existing immunity to baculoviruses in humans, the absence of viral gene expression in mammalian cells, and the relative low immunogenicity of baculovirus virions, makes these vectors promising tools for vaccination. Furthermore, the ability to produce large amounts in serum-free medium at a low cost adds a further advantage to this vector system.

Effects of simvastatin on human T cells in vivo

Objective The use of statins has shown several anti-inflammatory actions, including modulatory effects on T cells in vitro. Since the effects on human T cells in vivo are less clarified, our aim was to investigate the effects of simvastatin on human T cells in vivo and ex vivo. Methods and results A randomized, double-blind, placebo-controlled study design was applied. Eighty volunteers with mild to moderate hypercholesterolemia received either simvastatin 40 mg or placebo for 6 weeks. The serum levels of C-reactive protein (CRP) were significantly reduced by simvastatin. The proportions of CD4+ and CD8+ T cell subsets expressing early (CD25) or late (HLA-DR) activation markers, as assessed by flow cytometry, were not changed by simvastatin. However, simvastatin tended to increase the density of HLA-DR and L-selectin per CD8+ T cell. The T helper(h)1/Th2 response was evaluated by stimulatory assays followed by intra-cellular staining of interferon-γ and interleukin-4. Simvastatin treatment did not affect the Th1 response but the results indicated a potential to suppress Th2. Conclusion Simvastatin treatment resulted in a few discrete changes as regards peripheral T cells. However, the findings do not provide evidence that simvastatin-induced anti-inflammatory actions are related to any significant modulatory effects on human T cells in clinically healthy men with hypercholesterolemia.

Direct Antilymphoma Effects on Human Lymphoma Cells of Monotherapy and Combination Therapy with CD20 and HLA-DR Antibodies and 90Y-Labeled HLA-DR Antibodies

Monoclonal antibodies (mAb) in combination and mAbs combined with a radionuclide (radioimmunotherapy) have both been more effective in patients than mAb monotherapy. Using assays of cell growth and viability, the dose response and temporal characteristics of CD20 (rituximab) and HLA-DR (Lym-1) mAbs, singly and in combination, and of 90Y-conjugated Lym-1 mAb have been characterized in five human lymphoma cell lines (B35M, Raji, SU-DHL-4, SU-DHL-6, and Ramos) spanning Burkitt's to diffuse large cell lymphoma. Although Ramos had a lower HLA-DR density, these cell lines were otherwise selected because of high cell surface CD20 and HLA-DR abundance. Assays of cell growth and death were done using microscopy and trypan blue dye. Lym-1 and rituximab, used singly, showed direct antilymphoma effects; those of Lym-1 were often more potent than those of rituximab. Combinations of these mAbs were more effective, sometimes synergistic, than either mAb singly, even in more resistant SU-DHL-4 cells. Conjugation of 90Y to Lym-1 also augmented potency in all cell lines and overcame resistance to both Lym-1 and rituximab in Ramos cells. Lym-1 exhibited substantially greater direct antilymphoma effects than rituximab in lymphoma cells in culture. Combination of Lym-1 with rituximab or 90Y increased potency and overcame treatment resistance in lymphoma cells. Greater use of combination therapies of this type to increase potency and range of effectiveness seems likely to improve patient outcome.

Monocyte Anergy Is Present in Patients With Severe Acute Pancreatitis and Is Significantly Alleviated by Granulocyte-Macrophage Colony-stimulating Factor and Interferon-?? In Vitro

Severe acute pancreatitis (AP) is frequently associated with immune suppression, which increases the risk of infections, organ failure, and death. Our aims were to measure monocyte function (ie, HLA-DR expression and tumor necrosis factor-alpha [TNF-alpha] production as markers of immune suppression) in patients with severe AP and to determine whether treatment of blood samples with granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interferon-gamma (IFN-gamma) corrected the functional defects of monocytes in vitro. The study consisted of 28 patients with severe AP who were treated at intensive care unit and in whom the proportion of HLA-DR-positive monocytes in the circulation was less than 70%, and 28 matched control subjects who were selected from healthy laboratory personnel. HLA-DR density was determined by whole blood flow cytometry. Monocyte TNF-alpha production in response to bacterial lipopolysaccharides (LPSs) was studied in a whole blood assay. Aliquots of blood were supplemented with IFN-gamma (all 28 patients), GM-CSF (the last 24 patients), or both (the last 12 patients). The median proportion of HLA-DR-positive monocytes was 45% in patients (range, 18%-73%) and was 98% in controls (range, 86%-100%; P < 0.001). TNF-alpha levels in response to LPSs were lower in patients (545 pg/mL; range, 84-1990 pg/mL) than in controls (1415 pg/mL; range, 660-5490 pg/mL; P < 0.001). The proportion of HLA-DR-positive cells correlated positively with TNF-alpha levels (r = 0.56; P < 0.01). Both GM-CSF and IFN-gamma increased HLA-DR expression of monocytes in patients (98%; range, 74%-100% for GM-CSF; 99%; range, 86%-100% for IFN-gamma; both P < 0.001). The combination restored monocyte HLA-DR expression (99%; range, 96%-100%; P = 0.002). Compared with basal levels, GM-CSF increased TNF-alpha production of monocytes both in blood samples from patients (median, 1320 pg/mL; range, 35-8015 pg/mL) and controls (median, 3450 pg/mL; range, 1040-9835 pg/mL; both P < 0.001). IFN-gamma increased TNF-alpha production by monocytes in patients (683 pg/mL; range, 186-2705 pg/mL; P < 0.05) but not in controls (1658 pg/mL; range, 765-4755 pg/mL; P = 0.31). With the combination of GM-CSF and IFN-gamma, the TNF-alpha levels of monocytes in patients (3185 pg/mL; range, 545-8280 pg/mL) and in controls (2800 pg/mL; range, 1080-6860 pg/mL) were comparable. The proportion of HLA-DR-positive monocytes correlates with TNF-alpha production, and they both reflect the degree of immune suppression. The low proportion of HLA-DR-positive monocytes in AP can be reversed in vitro by GM-CSF and/or IFN-gamma. The GM-CSF and IFN-gamma treatments also increase LPS-induced TNF-alpha production. By the combination of GM-CSF and IFN-gamma, but not by either agent alone, LPS-induced TNF-alpha production of monocytes was equally high in patients and in controls.

Decreased HLA (human leucocyte antigen)-DR expression on peripheral blood monocytes predicts the development of organ failure in patients with acute pancreatitis.

Immune suppression plays an important role in the pathogenesis of acute pancreatitis. Monocyte expression of HLA (human leucocyte antigen)-DR, a cellular marker of immune suppression, was determined in relation to the development of organ dysfunction in patients with acute pancreatitis. A total of 310 consecutive patients with acute pancreatitis, admitted to a university hospital within 72 h of pain onset, were studied; 194 (63%) had mild disease (group I), 87 (28%) had severe disease without organ dysfunction (group II), and 29 (9%) had severe disease with organ dysfunction (group III). HLA-DR expression, defined both as the proportion of monocytes that were HLA-DR-positive and as monocyte HLA-DR fluorescence intensity, was determined at admission, using whole-blood flow cytometry. Of the patients in group III, 13 (45%) developed organ dysfunction within 24 h of admission. The proportion of HLA-DR-positive monocytes and monocyte HLA-DR density were both related to the severity of pancreatitis (P<0.001 for linear trend). In predicting organ dysfunction, the sensitivity, specificity and positive-likelihood ratio for the proportion of HLA-DR-positive monocytes were 83% [95% CI (confidence interval) 64-94%], 72% (67-77%) and 3.0 respectively, and for monocyte HLA-DR density the respective values were 69% (49-85%), 84% (79-88%) and 4.3. In conclusion, monocyte HLA-DR expression predicts the development of organ dysfunction that occurs early in patients with acute pancreatitis.

Characteristics of human dendritic cells generated in a microgravity analog culture system.

Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR+ and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytose Aspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of DC expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.

The conjunctival epithelium in dry eye subtypes: Effect of preserved and non-preserved topical treatments

Purpose. To determine the effect of topical treatments on the conjunctiva in dry eye. Methods. N = 134 dry eye subjects were diagnosed using a protocol of McMonnies dry eye symptom survey score > 14, fluorescein break up time (FBUT) < 10s and presence of rose Bengal staining. Differential diagnosis of dry eye subtypes was based on biomicroscopic signs and ocular/medical history. Superficial perilimbal bulbar conjunctival epithelial samples were collected using impression cytology. The nucleo-cytoplasmic ratio (N/C), goblet cell density (GCD) and expression of monoclonal antibodies HLA DR and CD23 were determined. The ocular surface characteristics of untreated subjects, those receiving preserved dry eye treatments and those receiving non-preserved treatments were compared with each other and with controls. Ocular surface characteristics of dry eye subtypes were also examined. Results. An increase in N/C (p = 0.011), reduction in GCD (p = 0.0001) and increase in expression of HLA DR (p = 0.0001) and CD23 (p = 0.0001) were detected in the untreated group compared to controls. No significant differences were found between the group receiving non-preserved dry eye treatments and untreated dry eye group. The group receiving preserved treatments had a reduced GCD (p = 0.0003) and increased expression of HLA DR (p = 0.0003) and CD23 (p = 0.0001) compared to the group receiving non-preserved treatments. Dry eye subtype specific differences in HLA DR and CD23 expression were noted. Conclusions. The conjunctival inflammation and reduced goblet cell density of dry eye is exacerbated by use of preserved topical agents, and is not significantly improved by use of non-preserved artificial tear supplements alone. Therapeutic strategies for dry eye should aim to increase goblet cell density and control ocular surface inflammation.