Removal Of Adherent Cells Research Articles

Dendritic cells loaded with HeLa-derived exosomes simulate an antitumor immune response.

The aim of the present study was to investigate the effect of loading dendritic cells (DCs) with HeLa-derived exosomes on cytotoxic T-lymphocyte (CTL) responses, and the cytotoxic effects of CTL responses on the HeLa cell line. Ultrafiltration centrifugation combined with sucrose density gradient ultracentrifugation was applied to isolate exosomes (HeLa-exo) from the supernatant of HeLa cells. Morphological features of HeLa-exo were identified by transmission electron microscopy (TEM), and the expression of cluster of differentiation (CD)63 was detected by western blotting. Next, monocytes were isolated from peripheral blood and cultured with the removal of adherent cells to induce DC proliferation. DCs were then phenotypically characterized by flow cytometry. Finally, MTT assays were performed to analyze the effects of DCs loaded with HeLa-exo on T cell proliferation and cytotoxicity assays to evaluate the effect of CTL responses on HeLa cells. TEM revealed that HeLa-exo exhibit typical cup-shaped morphology with a diameter range of 30-100 nm. It was also identified that the CD63 surface antigen is expressed on HeLa-exo. Furthermore, monocyte-derived DCs were able to express CD1a, suggesting that DC induction was a success. DCs exhibited hair-like protrusions and other typical dendritic cell morphology. Furthermore, DCs loaded with HeLa-exo could enhance CTL proliferation and the cytotoxic activity of CTLs compared with DCs without HeLa-exo (P<0.05). In conclusion, DCs loaded with HeLa-exo may promote T cell proliferation and induce CTL responses to inhibit the growth of cervical cancer cells in vitro.

Effect of peracetic acid on biofilms formed by Staphylococcus aureus and Listeria monocytogenes isolated from dairy plants

This research investigated the removal of adherent cells of 4 strains of Staphylococcus aureus and 1 Listeria monocytogenes strain (previously isolated from dairy plants) from polystyrene microtiter plates using peracetic acid (PAA, 0.5%) for 15, 30, 60, and 120 s, and the inactivation of biofilms formed by those strains on stainless steel coupons using the same treatment times. In the microtiter plates, PAA removed all S. aureus at 15 s compared with control (no PAA treatment). However, L. monocytogenes biofilm was not affected by any PAA treatment. On the stainless steel surface, epifluorescence microscopy using LIVE/DEAD staining (BacLight, Molecular Probes/Thermo Fisher Scientific, Eugene, OR) showed that all strains were damaged within 15 s, with almost 100% of cells inactivated after 30 s. Results of this trial indicate that, although PAA was able to inactivate both S. aureus and L. monocytogenes monospecies biofilms on stainless steel, it was only able to remove adherent cells of S. aureus from polystyrene microplates. The correct use of PAA is critical for eliminating biofilms formed by S. aureus strains found in dairy plants, although further studies are necessary to determine the optimal PAA treatment for removing biofilms of L. monocytogenes.

Growth of human mast cells from bone marrow and peripheral blood-derived CD34(+) pluripotent hematopoietic cells.

Human mast cells (HuMCs) are derived from CD34(+) pluripotent hematopoietic cells which are KIT (CD117)(+) and FcεRI(-), and lack lineage-specific surface markers. Bone marrow and peripheral blood are the two readily available sources for obtaining CD34(+) cells from which HuMCs can be cultured. CD34(+) cells are isolated and enriched by magnetic separation columns and stored under specific conditions until ready for use. Alternatively, enriched CD34(+) cells may be immediately cultured in serum-free culture media containing recombinant human (rh) stem cell factor (SCF), rhIL-6, and rhIL-3 (added only during the first week). Weekly hemidepletions and removal of adherent cells and/or debris enables the investigator to obtain HuMC cultures, identified by Wright-Giemsa and acidic toluidine blue stains, by 8-10 weeks.

A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.

Fibroblast adhesion on unidirectional polymeric nanofilms

Nanotextured polymeric surfaces with inclined rods reveal highly anisotropic properties concerning wetting and adhesion. In this work, we report on the interaction of fibroblast cells with these highly anisotropic materials. The authors quantified removal of adherent cells from such surfaces by a laminar flow. The critical shear force needed for cell removal from the surface depends on the inclination direction. Based on electron microscopy cross sections we deduce that interactions of cellular filopodia extending into the nanotextured surface are causing the direction depending removal.

Development of microfabricated magnetic actuators for removing cellular occlusion

Here we report on the development of torsional magnetic microactuators for displacing biological materials in implantable catheters. Static and dynamic behaviors of the devices were characterized in air and in fluid using optical experimental methods. The devices were capable of achieving large deflections (>60°) and had resonant frequencies that ranged from 70 Hz to 1.5 kHz in fluid. The effect of long-term actuation (>2.5 × 108 cycles) was quantified using resonant shift as the metric (▵f < 2%). The cell-clearing capabilities of the devices were evaluated by examining the effect of actuation on a layer of aggressively growing adherent cells. On average, actuated microdevices removed 37.4% of the adherent cell layer grown over the actuator surface. The effects of actuation time, deflection angle and beam geometry were evaluated. The experimental results indicate that physical removal of adherent cells at the microscale is feasible using magnetic microactuation.

Separating the One From the Many-Microfabricated Arrays for Cell Separations

The separation of single or small groups of cells from within a heterogeneous population is a fundamental need in almost all areas of biomedical research. This effort is required in order to obtain unique cells possessing a desired characteristic for genetic studies, cloning, or other applications. Despite recent technological advances, selection and isolation of individual or small groups of live cells from a population remains a signicant challenge. Most live-cell separation methods require that cells be dispersed into a single-cell suspension, but removal of adherent cells from their growth surface may at times be undesirable. A microfabricated cell array platform composed of releasable elements in combination with either a pulsed laser or needle-based punch system has been developed for analyzing, sorting and collecting viable cells from a mixed population while the cells remain adherent to their growth surface. Both noadherent and adherent cells cultured on the array can be analyzed and selected using standard imaging methods. Target cells can then be collected with high viability and efficiently cloned by releasing the polymeric base or cup in which the cell resides. Benefits of this new approach include high cell viability, small sample size requirements, and broad cell selection criteria. Separation properties include fluorescence signals, morphology, and uniquely, time-dependent variables such as the transmission of properties to daughter cells, growth rate or signaling behavior. Mating the microarrays with image cytometry provides a high-throughput tool for selection and isolation of cells for biomedical and pharmaceutical applications.

Evidence that defective interferon-gamma production in dermatitis patients is due to intrinsic abnormalities

The in vitro production of interferon-gamma (IFN-gamma) in 19 atopic dermatitis (AD) patients was compared with that of 12 controls. IFN-gamma production by phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) was profoundly diminished in AD patients, whereas the proliferative response was similar to that of control PBMC. The addition of 40 U/ml of interleukin-2 (IL-2) to the cultures failed to restore IFN-gamma production. Similarly, removal of adherent cells also had no effect. Reduced IFN-gamma secretion was observed after stimulation with the CD3 monoclonal antibody OKT3, ionomycin + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with high levels of IL-2 (200 U/ml). There were increased proportions of CD4+ T helper/inducer cells and decreased proportions of CD8+ T cytotoxic-/suppressor cells and CD16+ natural killer (NK) cells in AD patients. This resulted in an increased CD4/CD8 ratio as compared with controls, but no correlation was observed between numbers of T cell subpopulations and IFN-gamma generation. However, a significant correlation was found between IFN-gamma generation in vitro and IgE serum concentration in AD patients. The data suggest that the decreased production of IFN-gamma by AD patients is due to intrinsic differences in capacity to produce this cytokine and is not the result of differences in regulatory cell interactions. Moreover, the findings indicate that decreased production of IFN-gamma may be an important factor in the pathogenesis of this disease.

IL-2 regulation of soluble IL-2 receptor levels following thermal injury.

In the immunosuppressed burn patient serum levels of both IL-2 and a soluble form of IL-2 receptor alpha (sIL-2R alpha) are significantly elevated. Strikingly, the production of these markers by the in vitro activated patients' cells is decreased. This study examines the role of IL-2 in the decreased production of the sIL-2R alpha in vitro in patients with major burns (n = 18, 30 to greater than 70% total body surface area). Peripheral blood mononuclear cell (PBMC) cultures from patients with highly elevated serum sIL-2R alpha, and from healthy controls (n = 12) were activated with concanavalin A (Con A) at initiation. In patients' cultures mitogen-induced increments of sIL-2R alpha levels were significantly lower. There was a significant negative correlation (r = 0.64, P less than 0.001) between a high serum sIL-2R alpha level and a decreased lectin-induced sIL-2R alpha release in vitro. Low levels of sIL-2R alpha in patients' samples were not normalized by increasing the number of T lymphocytes. Also exogenous rIL-1 was without effect, whereas rIL-3 increased sIL-2R alpha release in some cultures. However, sIL-2R alpha levels were significantly increased in patients' cultures by (i) addition of exogenous IL-2; (ii) removal of adherent cells; (iii) addition of cyclooxygenase inhibitor, indomethacin; (iv) bypassing cell surface activation by the combination of the calcium ionophore A23187 and the phorbol ester 12-o-tetradecanoyl acetate. The cyclic AMP-elevating drug, forskolin, abrogated the ability of exogenous IL-2 to increase sIL-2R alpha production. Thus, in the burn patient, the reduced in vitro sIL-2R alpha release appears to relate to abnormalities in IL-2 production and action mediated through its functional surface receptor. Elevated levels of sIL-2R alpha in vivo may, therefore, reflect systemic activation of T lymphocytes in response to biologically active IL-2.

Mononuclear cells from HIV-infected patients produce factors which enhance functional activity of polymorphonuclear neutrophils from healthy subjects.

The influence of mononuclear cell supernatants (MNCS) from nine healthy donors and 35 HIV-infected patients (17 with lymphoadenopathy syndrome (LAS), 15 with ARC and three with AIDS) on functional activity of polymorphonuclear neutrophils (PMN) from healthy donors was investigated. MNC after short-term cultivation (24 h) produced factors which enhanced chemiluminescence (CL) and chemotaxis of PMN. This augmentation did not depend on stimulation of MNC by mitogens (lipopolysaccharide Escherichia coli (LPS) and concanavalin A (Con A)) or on activation of PMN by FMLP. After 48 h of cultivation only MNC stimulated by LPS produced these factors. MNCS from HIV-infected patients provoked a more pronounced augmentation of PMN CL compared with MNCS from healthy subjects. This enhancement was observed in patients at all stages of infection, but was more pronounced in patients with LAS. MNCS impact on PMN CL was not connected with proliferative activity of MNC but was correlated with the level of CD4 cells. It was shown that removal of adherent cells from MNC fraction resulted in decreased MNCS impact. Treatment of MNCS by antibody to IL-1 beta, IL-8, interferon-alpha (IFN-alpha) and tumour necrosis factor-alpha (TNF-alpha) did not decrease MNCS impact on PMN CL.

In human immature BFU-E tumor necrosis factor-α not only downregulates CDK6 but also directly produces apoptosis which is prevented by stem cell factor

The aim of this study was to reveal the mechanisms by which tumor necrosis factor-alpha (TNF-alpha) inhibits immature human day-4 burst-forming units-erythroid (BFU-E) and the effect of stem cell factor (SCF) on this process. Sequential density-gradient centrifugation, depletion of lymphocytes, removal of adherent cells, and negative selection with CD2, CD11b, CD16, and CD45 monoclonal antibodies were used to purify day-1 BFU-E, which were then incubated for 3 days to generate day-4 cells. The day-4 cells were incubated with TNF-alpha, and/or SCF, and the extent of apoptosis was gauged by morphologic observations, TUNEL assays, and Western blots. The cell number and the number and size of erythroid colonies were significantly reduced when day-4 cells were incubated with TNF-alpha. Apoptosis was observed in single-cell plasma clot assays. TUNEL assays showed 20% +/- 6% apoptotic cells with TNF-alpha while controls had 2.8% +/- 2.2%. Caspases 3 and 8 were strongly activated while the amount of CDK6 was reduced by TNF-alpha. When SCF, a potent stimulator of cell growth, was added with TNF-alpha, cell growth inhibition was reduced and the apoptotic cells decreased to 0.9% +/- 1.2%. The activations of caspase 3 and caspase 8 were almost completely blocked by SCF while CDK6 and the FLICE-inhibitory protein (FLIP) were increased. Our results indicate that in immature human BFU-E, TNF-alpha downregulates CDK6 but also directly produces apoptosis which is prevented by SCF.

Immunosuppression in hamsters with progressive visceral leishmaniasis is associated with an impairment of protein kinase C activity in their lymphocytes that can be partially reversed by okadaic acid or anti-transforming growth factor beta antibody.

Progressive visceral infection of golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative responses of their splenic or peripheral blood mononuclear cells (SPMC or PBMC, respectively) to in vitro stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals (I-SPMC or I-PBMC) was earlier shown to restore almost completely their lymphoproliferative responses to PMA plus Io. The present study was directed to evaluate the status of protein kinase C (PKC), a molecule(s) known to play a key role in the lymphoproliferative process. Our results demonstrate that PKC activities (Ca(2+), phosphatidyl serine, and diacyl glycerol dependent) in the cytosolic fraction of untreated nonadherent I-SPMC or I-PBMC as well as in the membrane fraction of PMA-treated cells were decreased significantly relative to those for normal controls. However, removal of adherent cells from I-SPMC or I-PBMC and subsequent overnight in vitro cultivation of nonadherent cells (lymphocytes) resulted in significant restoration of PKC activity in the cytosolic or membrane fraction of untreated or PMA-treated cells, respectively. Partial, though significant, restoration of PKC activity could also be achieved in the membrane fraction of PMA-treated cells following overnight in vitro treatment of I-SPMC or I-PBMC with the Ser/Thr phosphatase inhibitor okadaic acid (OA) or an anti-transforming growth factor beta (anti-TGF-beta) neutralizing antibody. These results correlated well with the ability of OA or the anti-TGF-beta antibody to restore the lymphoproliferative response of I-SPMC or I-PBMC following stimulation with PMA plus Io. Interestingly enough, immunoblotting experiments failed to show any reduction in the level or translocation (following PMA treatment) of conventional PKC isoforms in the SPMC or PBMC of infected animals compared to those of normal controls. The results presented in this study suggest that the adherent cells generated in the SPMC or PBMC of infected animals exert a suppressive effect on the proliferative response of nonadherent cells (lymphocytes) which is likely to be mediated through the downregulation of the activation pathway involving PKC and its downstream molecules such as mitogen-activated protein kinases. Further, the observed suppression of PKC activity and subsequent lymphoproliferative responses can be attributed to alternations in the intracellular phosphorylation-dephosphorylation events. The relevance of these results is discussed in relation to the role of TGF-beta, levels of which are known to be elevated in visceral leishmaniasis.

Oxidative stress response induced in rat primary hepatocyte monolayers by mechanical removal of adherent cells.

We show that mechanical separation of adherent rat primary hepatocytes after the monolayer-forming stage causes the induction of the oxidative stress genes HO-1 (haem oxygenase) and MnSOD (manganese superoxide dismutase). The procedures for enzymatically breaking up liver tissue structure and isolating hepatocytes do not cause HO-1 and MnSOD activation. Only after a 3-h incubation, during which hepatocytes form a monolayer on culture dishes, does the hydrodynamic shearing away of necrotic cells sticking to the monolayer surface activate these two genes. Analysis of this injury-response pathway shows that oxidative stress and mitochondrial dysfunction play a role, as activation can be repressed by antioxidants and by respiratory inhibitors. Recovery of the cells takes a further 24-h incubation during which HO-1 and MnSOD expression returns to basal levels.

Characterization of a t cell lineage marker in carp ( Cyprinus carpio L.)

A monoclonal antibody against thymocytes (WCL9; of IgG1 class) was produced by immunization of mice with isolated membrane molecules of carp thymocytes. Flow cytometric and fluorescence microscopic analysis showed that WCL9 was reactive with 30–50% of thymocytes and not with lymphoid cells from blood, pronephros, spleen and intestine. Cryo-sections of thymus showed a WCL9 + and WCL9 − region in 3-month-old fish and only WCL9 + cells in 1-week-old fish. Because the WCL9 − region is more medulla-like, the WCL9 + cells can be considered as cortical thymocytes. The majority of WCL9 + thymocytes appeared to have a higher density (1.06–1.07 g/mL) than the WCL9 − cells (1.02–1.06 g/mL). Immunogold labelling or comparison of both density fractions did not show clear ultrastructural differences between WCL9 + and WCL9 − thymocytes. The WCL9 − fraction could be stimulated much better with PHA than the WCL9 + fraction. Removal of adherent cells or adding adherent accessory cells did not influence this result. Immunochemical analysis showed that WCL9 reacted with a protein determinant present on two molecules ( M r: 200 and 155 kDa) under reduced and non-reduced conditions. These results, together with the absence of WCL9 + cells in other lymphoid organs, strongly suggest that WCL9 is a specific marker for early thymocytes.

The tetrapeptide AcSDKP specifically blocks the cycling of primitive normal but not leukemic progenitors in long-term culture: evidence for an indirect mechanism

In the present study, we investigated the ability of the tetrapeptide NAc-Ser-Asp-Lys-Pro-OH (AcSDKP), a reported inhibitor of primitive hematopoietic cells, to influence the proliferative behavior of primitive normal and chronic myeloid leukemia (CML) progenitor cells in the adherent layer of long-term cultures (LTCs). Addition of &gt; or = 50 ng/mL of AcSDKP to LTCs of normal cells at the time of the regular weekly half-medium change selectively and reversibly decreased the proportion of high proliferative potential erythroid and granulopoietic progenitors in the adherent layer that were in S-phase without changing their numbers, but had no effect on either the cycling activity or number of analogous (neoplastic) cells in the adherent layer of CML LTCs. Specificity of the effect of AcSDKP on primitive normal progenitors was demonstrated by the finding that a similar addition of either the control peptide, AcSDKE, or 100 ng/mL of tumor necrosis factor-alpha (TNF-alpha, which contains the SDKP sequence), or SDKP itself (at 300 ng/mL) did not inhibit the proliferation of primitive normal progenitors in LTC adherent layers. Incorporation of &gt; or = 30 ng/mL of AcSDKP (but not the related control peptide, AcSDKE) directly into methylcellulose cultures of normal marrow cells resulted in a dose- dependent suppression of colony formation, which was not seen in similar studies with CML marrow or after removal of adherent cells from normal marrow. Additional experiments showed that the inhibitory effect of AcSDKP on primitive normal progenitor cycling in the LTC system could be overcome by the simultaneous addition of macrophage inflammatory protein-1 beta (MIP-1 beta); an antagonist of MIP-1 alpha. The apparent differential effect of AcSDKP on primitive normal and CML progenitors may thus be a secondary consequence of the differential responsiveness of these cells to MIP-1 alpha for another molecule antagonized by MIP-1 beta), whose production or release by adherent marrow cells is inducible by AcSDKP. Such a mechanism may offer a method for obtaining localized increases in vivo of cytokines like MIP- 1 alpha, suggesting novel and perhaps less toxic strategies for protecting primitive normal progenitors during repeated treatments with cycle-active chemotherapeutic agents where escalating the dose of drug given would be desirable.

The Tetrapeptide AcSDKP Specifically Blocks the Cycling of Primitive Normal But Not Leukemic Progenitors in Long-Term Culture: Evidence for an Indirect Mechanism

In the present study, we investigated the ability of the tetrapeptide NAc-Ser-Asp-Lys-Pro-OH (AcSDKP), a reported inhibitor of primitive hematopoietic cells, to influence the proliferative behavior of primitive normal and chronic myeloid leukemia (CML) progenitor cells in the adherent layer of long-term cultures (LTCs). Addition of > or = 50 ng/mL of AcSDKP to LTCs of normal cells at the time of the regular weekly half-medium change selectively and reversibly decreased the proportion of high proliferative potential erythroid and granulopoietic progenitors in the adherent layer that were in S-phase without changing their numbers, but had no effect on either the cycling activity or number of analogous (neoplastic) cells in the adherent layer of CML LTCs. Specificity of the effect of AcSDKP on primitive normal progenitors was demonstrated by the finding that a similar addition of either the control peptide, AcSDKE, or 100 ng/mL of tumor necrosis factor-alpha (TNF-alpha, which contains the SDKP sequence), or SDKP itself (at 300 ng/mL) did not inhibit the proliferation of primitive normal progenitors in LTC adherent layers. Incorporation of > or = 30 ng/mL of AcSDKP (but not the related control peptide, AcSDKE) directly into methylcellulose cultures of normal marrow cells resulted in a dose-dependent suppression of colony formation, which was not seen in similar studies with CML marrow or after removal of adherent cells from normal marrow. Additional experiments showed that the inhibitory effect of AcSDKP on primitive normal progenitor cycling in the LTC system could be overcome by the simultaneous addition of macrophage inflammatory protein-1 beta (MIP-1 beta); an antagonist of MIP-1 alpha. The apparent differential effect of AcSDKP on primitive normal and CML progenitors may thus be a secondary consequence of the differential responsiveness of these cells to MIP-1 alpha for another molecule antagonized by MIP-1 beta), whose production or release by adherent marrow cells is inducible by AcSDKP. Such a mechanism may offer a method for obtaining localized increases in vivo of cytokines like MIP-1 alpha, suggesting novel and perhaps less toxic strategies for protecting primitive normal progenitors during repeated treatments with cycle-active chemotherapeutic agents where escalating the dose of drug given would be desirable.

Suppression of erythroid progenitors in ponies infected with equine infectious anaemia virus

Six clinically normal ponies were infected intravenously with equine infectious anaemia virus (EIAV) to determine the effect of EIAV on numbers of erythroid, granulocyte/monocyte and fibroblastic progenitors in the bone marrow. Bone marrow progenitor assays were performed at weeks 1, 2, 3, 4 and 8 postinfection. The late erythroid progenitors, colony-forming units-erythroid (CFU-E), were suppressed at each time point postinfection with the maximal suppression occurring at week 2 postinfection. The maximal suppression corresponded to the peak of plasma virus concentration. The maximal suppression of the early erythroid progenitors, burst-forming units-erythroid (BFU-E), also occurred at week 2 postinfection. Removal of adherent cells from the bone marrow mononuclear cells (BMMC) abrogated the suppression. Neither granulocyte/monocyte progenitors (CFU-GM) nor fibroblastic progenitors (CFU-F) were affected by EIAV infection.

Glucocorticoid effects on natural and humoral immunity in mallards

Two studies were conducted to determine the effects of dexamethasone (DEX) on immune function in mallard ducks. Each day ducks were injected intramuscularly with DEX at doses ranging from 0.2–4.0 mg/kg for 28–30 days. Physiologic effects consistent with high dose glucocorticoid (GC) treatment were observed at the 4 mg/kg dose, and included significant body weight loss, lowered hematocrit, and elevated alanine aminotransferase (ALT) activity. At all doses, effects of DEX on the immune system were observed. When DEX was given at 0.2 mg/kg/day, significant suppression of primary IgG antibody titers to sheep erythrocytes (SRBC) was observed. At 1 mg/kg/day, primary IgM and secondary IgM and IgG titers were suppressed as well. These doses of DEX also produced significant elevation in natural killer cell (NKC) activity of peripheral blood mononuclear cells (PBMNC). Removal of adherent cells from the PBMNC prior to NKC assay eliminated the enhancement in NKC activity. Based on these results, it was postulated that the elevation in NKC activity may be due to suppression by DEX of monocyte production of prostaglandin-E2 (PGE-2) resulting in the release of NKC activity from the inhibitory effects of PGE-2. This hypothesis was supported by a measured decrease in PGE-2 production during the NKC assay by cells from DEX-treated birds. Furthermore, an enhanced NKC activity could be reproduced in vitro with the addition of indomethacin or DEX to NKC cultures containing adherent cells from PBMNC. Direct effects of DEX on nonadherent cell NKC activity and lymphocyte viability were only observed at high concentrations (10 −4 M) of DEX, while the phagocytic activity of adhered blood monocytes was inhibited at 10 −6 M DEX. The suppressed phagocytic activity may contribute to the suppressed antibody responses observed in DEX-treated birds. Together, these results support an indirect immunomodulatory effect of DEX on NKC activity and perhaps antibody responses in vivo via altered monocyte function in mallard ducks.

Impaired NK Response of Cancer Patients to IFN‐α but Not to IL‐2: Correlation with Serum Immunosuppressive Acidic Protein (IAP) and Role of Suppressor Macrophage

In vitro NK responses of cancer patients (N = 21) to rIFN-alpha A and rIL-2 were examined. The serum concentration of IAP (immunosuppressive acidic protein) was determined in parallel. Five out of seven patients whose serum IAP contents were within the normal range (270 micrograms/ml to 470 micrograms/ml), had their NK activities significantly augmented by rIFN-alpha A and rIL-2. On the other hand, NK cells from ten out of fifteen patients whose serum IAP concentrations were 650 micrograms/ml or more, were not activated by rIFN-alpha A. NK cells of these fifteen patients yet were capable of responding to rIL-2. NK cells from cancer patients, however, became responsive to rIFN-alpha A by either removal of adherent cells or treatment with indomethacin. Therefore, macrophages in PBMC of cancer patients with high serum IAP levels seem to selectively suppress NK response to rIFN-alpha A by an indomethacin-sensitive mechanisms. It was further shown that PGE2 was not the mediator of this suppression.

Increased ability of peripheral blood B cells from patients with rheumatoid arthritis to produce interleukin 1 in vitro.

Twenty-four patients with rheumatoid arthritis (RA) and 20 normal controls were examined for the ability of their peripheral blood B cells to produce interleukin 1 (IL-1) with or without lipopolysaccharide (LPS). B cells were purified from peripheral blood by negative selection methods (i.e., removal of adherent cells and sheep red blood cell rosette-forming cells, followed by treatment with monoclonal antibodies (OKT3 and OKM1) and complement). The amount of IL-1 in B cell culture supernatants (SN) was measured by thymocyte and fibroblast proliferation assays and an enzyme-linked immunosorbent assay for IL-1 alpha and beta. As a group, cultured B cells from patients with RA, both spontaneously and when stimulated with LPS, produced higher levels of IL-1 than those from normal controls. IL-1 production by RA B cells with LPS had a weak but positive correlation with disease activity. Moreover, RA B cell culture SN with elevated levels of IL-1 had a synergistic effect on the growth of anti-human IgM (anti-mu) stimulated B cells. In separate experiments, the growth of RA B cells was significantly promoted by IL-1 beta both with and without anti-mu stimulation. These results suggest that B cell-derived IL-1 may be involved in the B cell clonal expansion of RA through its own activity as a B cell stimulatory factor.