Interferon-y Research Articles

Production of CD87 Antigen-Specific CAR-T Lymphocytes and Assessment of Their In Vitro Functional Activity

Aim. To generate anti-CD87 CAR-T lymphocytes and to assess their in vitro functional activity. Materials & Methods. Т-lymphocytes isolated from healthy donor peripheral blood were transduced with the anti-CD87-CAR, T2A, and FusionRed gene coding lentiviral vector. Transduction efficacy assessed by reporter protein FusionRed signal, subpopulation structure, and functional status of CAR-T lymphocytes were determined by flow cytometry. Interferon-y (IFN-y) expression by CAR-T lymphocytes was analyzed using immunoassay. Cytotoxic activity of CAR-T lymphocytes was evaluated during their co-cultivation with HeLa target cells by means of xCELLigence real-time assay. Results. The efficacy of T-lymphocyte transduction was 8.4 %. The obtained CAR-T cells contained the markers of both CD27 and/or CD28 activation (92.91 % cases) and PD1 exhaustion (20.66 % cases). The population of CAR-T lymphocytes showed 98.51 % central memory T-cell phenotype and CD4/CD8 ratio of 1:7. IFN-y concentration in the medium after co-cultivation of CAR-T lymphocytes with target cells appeared to be significantly higher than in control samples. The study demonstrates that generated CAR-T lymphocytes manifest specific cytotoxicity towards target cells with both unmodified expression and overexpression of CD87 antigen in HeLa cell lines. Cytotoxicity proved to be more pronounced with respect to the cell line with CD87 antigen overexpression. Conclusion. Despite overexpression of PD1 exhaustion marker, CAR-T lymphocytes showed specific IFN-y secretion and pronounced cytotoxic activity in interaction with CD87 antigen on target cell membranes. Therefore, anti-CD87 CAR-T lymphocytes can be applied in the treatment of hematologic as well as solid tumors. Since the observed difference in cytotoxicity does not linearly correlate with CD87 antigen density on the surface of attacked cells, the in vivo administration of a CAR-T cell drug should be designed to prevent cytotoxic risk for CD87-expressing healthy cells.

Experimental Study of the In Vitro and In Vivo Functional Activity of NKG2D Chimeric Antigen Receptor

Aim. To study antitumor cytotoxic effect of CAR-T NKG2D and CAR-T anti-CD19 in vitro and in vivo in order to compare antitumor activity of chimeric antigen receptors (CAR) with different structural and functional properties. Materials & Methods. CAR constructions were produced by molecular cloning. CAR-T cell populations were obtained by transduction of healthy donor T-lymphocytes with recombinant lentiviral particles coding CAR NKG2D or CD19 target antigen CAR sequences. CAR-T cell proportion was assessed by FusionRed fluorescence and EGFR membrane receptor imaging. Specific in vitro cytotoxic activity of CAR-T effector cells was analyzed by Real-Time Cytotoxicity Assay (RTCA) during co-cultivation with HeLa_CD19 target cell line using xCELLigence. Interferon-Y (IFN-y) synthesis in vitro and in vivo along with the degree of cytotoxic effect were analyzed by immunoassay of culture medium of co-cultivated effector cells and target cells as well as isolated auto-plasma from the peripheral blood of mice. To assess the in vivo functional activity, CAR-T cell populations were infused into immunodeficient NSG-SGM3 mice (10 000 000 cells/mouse) 12 days after HeLa_CD19 cell injection and confirmation of engraftment and tumor growth. Upon euthanasia, tumors were removed and fixed in paraffin to prepare histological sections. CAR-T cell tumor infiltration was assessed by CD3 antigen immunohistochemical staining. Results. The highest ligand (molecules MICA, ULBP1/2/3/4/5/6) expression levels were detected in HeLa cell line. The obtained NKG2D CAR-T cells showed a considerable cytotoxic activity against HeLa_CD19 target line (cell index [CI] = 1.27), which was, however, twice as low as that of CAR-T anti-CD19 (CI = 0.60) (p = 0.0038). IFN-y level during co-cultivation of CAR-T anti-CD19 with HeLa_CD19 at the ratio of Е/Т = 1:1 was 64,852 pcg/mL, which was 3.5 times higher than IFN-y level during co-cultivation of CAR-T NKG2D with HeLa_CD19 (18,635 pcg/mL) (p = 0.0360). The degree of tumor infiltration by CAR-T anti-CD19 cells was higher than that by CAR-T NKG2D. The absence of NKG2D proliferating CAR-T cells in mice peripheral blood confirms their low persistence. IFN-y concentration in mice auto-plasma was 11.89 pcg/mL after CAR-T anti-CD19 infusion and 0.57 pcg/mL after CAR-T NKG2D infusion (p = 0.0079). The mean weight of tumor xenografts in experimental groups 10 days after CAR-T anti-CD19 injection was 0.72 g (p = 0.0142), after Т-lymphocyte and NKG2D CAR-T cell infusions it was 2.12 g and 1.2 g, respectively. Conclusion. CAR-T anti-CD19 cells are characterized by more pronounced cytotoxic effect under both in vitro and in vivo experimental conditions compared with CAR-T NKG2D cells. The degree of CAR-T anti-CD19 proliferation and their infiltration in mice xenograft models is considerably higher than the levels reached with NKG2D CAR-T cell injections. A single CAR-T NKG2D injection results only in short-term tumor reduction.

LBA75 - 18-months relapse-free survival (RFS) and biomarker analyses of OpACIN-neo: A study to identify the optimal dosing schedule of neoadjuvant (neoadj) ipilimumab (IPI) + nivolumab (NIVO) in stage III melanoma

BackgroundPrimary analysis of the OpACIN-neo study testing 3 dosing schedules of neoadj IPI+NIVO identified 2 cycles IPI 1mg/kg + NIVO 3mg/kg (IPI1+NIVO3; arm B) as most favourable, with 20% grade 3-4 irAEs and a pathologic response rate (pRR) of 77%. After a median FU of 8.3mo none of the pts with a pathologic response versus 9/21 (43%) of the non-responders had relapsed. Here we present updated RFS and biomarker analyses. MethodsOpACIN-neo is a multicentre, randomized phase II trial in resectable stage III melanoma pts with ≥1 measurable lymph node metastasis (RECIST 1.1). 86 pts were randomized to arm A: 2x IPI3+NIVO1 Q3W (n=30); B: 2x IPI1+NIVO3 Q3W (n=30); or C: 2x IPI3 Q3W followed by 2x NIVO3 Q2W (n=26). Lymph node dissection was planned at wk 6. Primary endpoints were toxicity, radiologic RR and pRR; RFS and biomarker analyses were secondary endpoints. Mutational profiles, gene expression signatures (GES) and immune protein expression were examined in baseline biopsies by whole exome seq, RNA seq and digital spatial profiling (DSP) analysis. Pre- and post-treatment plasma samples were profiled for 92 proteins. ResultsAfter a median FU of 17.7mo, the median RFS was not reached in any of the arms. Estimated 18-mo RFS was 85% for all pts (95% CI 78%-93%), 90% for arm A (95% CI, 80%-100%), 82% for arm B (95% CI, 70%-98%) and 83% for arm C (95% CI, 70%-100%). Relapses were observed in 1/64 (2%) pathological responders versus 13/21 (62%) of the non-responders. High tumour mutational burden (TMB) and high interferon-y (IFN-y) signature were associated with pathologic response and favourable RFS. Cytokine and PD-1 levels in plasma increased post-treatment irrespective of response. Additional GES and DSP analysis will be presented. ConclusionsThe 18-mo FU confirms that durable RFS can be achieved with 2 cycles of neoadj IPI+NIVO without any additional adjuvant therapy. Pathologic response remains the strongest marker for RFS. TMB and IFN-y signature might serve as baseline markers identifying pts benefiting from neoadj IPI+NIVO. Neoadj 2 cycles IPI1+NIVO3 should be tested in a randomized phase III study versus adjuvant therapy. Clinical trial identificationNCT02977052. Legal entity responsible for the studyNetherlands Cancer Institute. FundingBMS. DisclosureE.A. Rozeman: Travel / Accommodation / Expenses: NanoString; Travel / Accommodation / Expenses: MSD. A.M. Menzies: Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Roche; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pierre Fabre. O. Krijgsman: Research grant / Funding (institution): BMS. T.M. Van: Travel / Accommodation / Expenses: NanoString. A.C.J. van Akkooi: Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: MSD-Merck; Advisory / Consultancy: Merck-Pfizer; Advisory / Consultancy: 4SC. R.A. Scolyer: Advisory / Consultancy: MSD; Advisory / Consultancy: Neracare; Advisory / Consultancy: Novartis. J. Hansson: Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Novartis. G.V. Long: Advisory / Consultancy: Aduro; Advisory / Consultancy: Amgen; Advisory / Consultancy: BMS; Advisory / Consultancy: Mass Array; Advisory / Consultancy: MSD; Advisory / Consultancy: Novartis; Advisory / Consultancy: Oncosec; Advisory / Consultancy: Pierre-Fabre; Advisory / Consultancy: Roche. C.U. Blank: Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Roche; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Lilly; Advisory / Consultancy: Pfizer; Advisory / Consultancy: GSK; Advisory / Consultancy: GenMap; Advisory / Consultancy: Pierre Fabre; Research grant / Funding (institution): NanoString. All other authors have declared no conflicts of interest.

Antioxidant effects of immunomodulators in patients with recurrent pyelonephritis

The purpose of our work was to investigate the effect of immunomodulatory medicines on the intensity of oxidative stress (OS), the cytokines level and the activity of renospecific enzymes in patients with recurrent pyelonephritis (rPN).
 Methods. A prospective, randomized, open-label study involved of 100 women aged 33.4 ± 8.8 year old. According to the sensitivity of the detected pathogens all patients received antibacterial therapy for two weeks. Along with the main course of antibiotic therapy, 25 patients were assigned Sodium nucleinate at a dose of 0.25 g 4 times per day during 14 days, 18 patients were prescribed Galavit intramuscularly 2 ml per dayfor 10 days, and 27patients were prescribed Proteflazid according to the manufacturer’s instructions. The comparison group consisted of 30 women with rPN who received antibiotic therapy exclusively.Women were screened before and after the treatment. The content of malondialdehyde (MDA), ceruloplasmin (CP), transferrin (TF) and sulfhydryl groups (SH-groups) were determined in the blood by colorimetric method. Oxidative stress index (OSI) was calculated. The concentration of interleukins (IL) -1f, -4, -8, -10, -17, tumor necrosis factor a (TNF-a), transforming growth factor f (TGF-f), monocytic chemoactive protein-1 (MCP-1) and interferon y (IFN-y) were analyzed in the blood of the women using an ELISA. To evaluate the functional state of the renal parenchyma the activity of tubular lysosome enzymes a total f-N-acetylhexosaminidase and f-galactosidase were determined in urine.
 Results. The use of Sodium nucleinate decreased of the OS activity by reducing MDA level (p <0.001) and increasing the concentration of CP (p < 0.03). The serum levels ofIL-4 (p = 0.007), lL-17 (p = 0.04), TGF-f (p = 0.02) and MCP-1 (p = 0.03) were decrease. The use of Galavit contributed to a statistically significant decrease in the concentration of TNF-a (p <0.001), IL-8(p <0.001), IFN-y (p = 0.001) and TGF-f (p <0.001). The administration of Proteflazid resulted in a decrease in the concentrations of IL-8 and IFN-y, with a decrease in OSI (p =0.04) compared to pre-treatment. All applied immunomodulators partially reduced the activity of renospecific enzymes markers of kidney damage.
 Conclusions. The use of immunomodulators in the complex therapy of patients with rPN contributes to the partial normalization of functional activity of immune system by the decreasing ofthe production ofcytokines as its mediators and the reducing ofthe OS intensity.

Nanovaccine for immunotherapy and reduced hepatitis-B virus in humanized model

Chronic Hepatitis B Virus (HBV) infections are severe with weak antiviral immune responses. The lack of an appropriate small animal model for chronic hepatitis, a major hurdle for studying the immunotolerance and immunopathogenesis induced by hepatitis B viral (HBV) infection. In this study, for enhancing the antibody production efficiency the prepared polymeric HBsAg-loaded nanoparticles (nanovaccine) will be tested in immune-deficit mice, which suffer from chronic Hepatitis B virus. Vaccination of Balb/c mice by this prepared nanoparticles that were engrafted with peripheral blood mononuclear cells (PBMCs), which was already lethally irradiated and transplanted by the bone marrow of NOD (knockout mice) mice. In the present study, after the vaccination detected the high frequencies of immunoglobulin G (IgG)-secreting B cells and mitogen-responsive interferon-Y (IFN-Y) secreting T cells in serum, determined by specific ELISA technique. During the entire observation period, unvaccinated animals showed lower concentration of specific IgG secreting B cells and IFN-Y secreting T cells found in comparison to vaccinated mice group. Chronic HBV carrier PBMCs transplanted into the chimera failed to produce antigen and increased the antibodies production due to vaccination. Furthermore, another advantage was that the viral gene expression and viral DNA replication was no longer observed in vaccinated group. This prepared nanovaccine formulations is better for the cure of Hepatitis B viral infection carrier. Therefore, specific memory responses were elicited by vaccination with Hepatitis B virus surface (HBsAg) antigen of chimeric mice transplanted with PBMCs derived from HBV donors.

Evolution of In-Vitro Sensitisation to Cow's Milk Proteins Among Preterm Infants with Necrotising Enterocolitis

Background: Cow's milk protein (CMP) sensitisation in preterm (PT) infants with necrotising enterocolitis (NEC), and associated inflammatory and the regulatory components were described (1, 2). This study aimed to follow the evolution of this phenomenon in relation to the natural history of NEC. Methods: ELISPOTs was used to detect numbers of peripheral blood mononuclear cells (PBMCs) secreting interferon-y (IFN-y), interleukin (IL)-4, IL-10, and transforming growth factor-β1 (TGF-β1), in response to stimulation with CMPs (casein and β-lactoglobulin [β-lg]) in the acute-stage NEC, compared to septic and healthy controls, then at recovery, and at full feeding in PT infants with NEC stage II and III on the modified Bell's score Results: During acute NEC, a pattern of higher levels of cytokine secretion to CMPs (for IFN-γ, IL-4 and IL-10) than in septic and healthy controls was noted, with a minimal TGF-β1 response. Responses to β-lg and casein were significantly enhanced at recovery for IFN-γ (p=0.01, 0.06), IL-4 (0.02, 0.009) and IL-10 (p=0.005, 0.002) but less marked at full feeding to β-lg than to casein: IFN-γ (p=0.03, 0.002), IL-4 (0.03, 0.002) and IL-10 (p=0.11, 0.007). TGF-β1 secretion was only slightly enhanced at presentation but progressively enhanced at recovery (p=0.002, 0.01) and at full feeding (p=0.002, 0.002). Figure 1 Figure 2

Cross-talk of the related bioactivity mediators in serum after injection of soluble TNF-α receptor on silicosis model of rats

This study investigates cross-talk of the related bioactivity mediators in silica-induced pulmonary inflammatory and fibrosis on rats, which contributes to the preventive and therapeutic effect of soluble TNF-α receptor. Wistar rats received saline or 50 mg of quartz by intratracheal instillation. Rats in drug-treated groups were given soluble tumor necrosis factor-α (TNF-α) receptor (500 μg) by hypodermic injection on days 1, 5 and 8 after operation. At 7 days or 14 days after instillation, rats were killed to observe the degree of injury and expression of the related bioactivity mediators including nuclear factor KB (NF-KB), nitric oxide, interleukin-1β, interleukin-10, transforming growth factor beta 1 (TGF-β1), TNF-α, interferon-Y (IFN-Y) and granulocyte macrophage colony-stimulating factor (GM-CSF). The area percentages of type I and III collagens in intervention group were lower than those in silica group. The expression of NF-κB, TGF-β1, and COL I were lower in intervention group than in silica group(p < 0.05) and GM-CSF was significantly higher (p < 0.05) at 7 days after instillation, however, NF-κB, TGF-β1, and COL I were identically lower in intervention group than in silica group, and TNF-α, IFN-γ, and GM-CSF were higher at 14 days after instillation. It may be concluded that soluble TNF-α receptor upregulating or downregulating the expression of the related bioactivity mediators results in decreasing lung injury induced by silica.

Maternal cytotoxic T cell responses to fetal antigens during and after pregnancy

During human pregnancy there is the ‘transplacental traffic’of fetal cells into the maternal circulation. These cells can persist for decades, a phenomenon termed ‘fetal microchimerism’. Exposure to fetal cells may...

The Alternatively Activated Human—Redux

It has long been appreciated that, even in their nonactivated state, tissue-associated macrophages represent a diverse population of cells whose varied functional phenotypes are a product of their local environment. Although derived from the same bone marrow precursors, macrophages that take up residence in the liver, central nervous system, lung, spleen, and adipose tissues adopt distinct functions that are important in maintaining tissue homeostasis and defense against pathogens. Another layer of complexity is revealed when macrophages are activated during innate and adaptive immune responses. It has become clear that activated macrophages, similar to T cells, take on varied activation states that depend on the specific nature of the molecular signals they receive. Building on the initial observations that activated phagocytic cells are a critical component of effective antibacterial immunity [1-3], Nathan et al. [4] demonstrated that lymphocyte-derived interferon-y (IFN-y) is the factor that converts resting macrophages into cells with a markedly enhanced capacity to

T Cells and Tuberculosis: Beyond Interferon‐γ

The cytokine interferon-y (IFN-y) plays a pivotal role in protective immunity against intracellular pathogens. Specifically, in Mycobacterium tuberculosis (MTB) infection, IFN-y is an important mediator of macrophage activation [ 1 ] . Mice deficient in the gene for IFN-y are susceptible to fatal tuberculosis (TB) [1, 2], and humans deficient in either the gene for IFN-y or the IFN-y receptor show enhanced susceptibility to mycobacterial infections [3]. Measurement of the IFN-y response to MTB infection has been exploited in research and in clinics, to evaluate and develop new tools for the prevention, diagnosis, and treatment of infection. For example, the development of standardized quantitative ex vivo assays of T cell function, along with the discovery of antigens that are highly specific for MTB, led us [4, 5] and other investigators [6] to develop new diagnostic tools for the detection of MTB infection. These T cell IFN-y release

The Modulating Effect of 棺-1, 3/1, 6-glucan Supplementation in the Diet on Performance and Immunological Responses of Broiler Chickens

The object of this trial was to investigate the effect of dietary β-1,3/1,6-glucan supplementation on the performance and immunological response of broiler chickens. Two hundred and forty 1-day old male broilers (39±1 g) were separated into six treatments which were given six different feeds containing 0 (control), 25, 50, 75, 100 and 125 mg/kg dietary β-1,3/1,6-glucan supplementation. On days 21 and 42, body weight gain, feed consumption and feed conversation rate were recorded as measures of growth performance. The levels of key cytokines in the immuno-regulating pathway: interleukin-1 (IL-1), interleukin-2 (IL-2), interferon y (IFN-y), tumor necrosis factor a (TNF-a), and the concentrations of signal molecules: peripheral blood plasma globulin, serum Immunoglobulin G (IgG) and intestinal secretary Immunoglobulin A (sIgA), were measured as indices of the immune response to determine suitable levels of dietary β-1,3/1,6-glucan supplementation. The results indicated that performance was elevated quadratically with dietary β-1,3/1,6-glucan supplementation. Maximal growth performance and an enhanced immunological response were obtained at a supplemented level of 50 mg/kg.

New Lignans from Kadsura coccinea and Their Nitric Oxide Inhibitory Activities

In vitro anti-allergic screening of medicinal herbal extracts revealed that the chloroform extract of the rhizoma of Kadsura coccinea inhibited nitric oxide (NO) production in a lipopolysaccharide (LPS) and recombinant mouse interferon-y (IFN-y) activated murine macrophage like cell line, RAW264.7. Further fractionation of the chloroform extract led to the isolation of two new lignans kadsuralignans D (1) and E (2) with other two known dibenzocyclooctadiene lignans kadsulignan N (3) and neokadsuranin (4).

Treatment of eosinophilic gastroenteritis with interferon alpha-2b

Treatment of Eosinophilic Gastroenteritis With Interferon li,JI Alpha-2b Richard E Crockett, Dennis K Ledford University of South Florida, Tampa, FL Interferon alpha (IFN~) has been used to successfully treat a variety of eosinophilic diseases including hypereosinophilic syndrome, eosinophilic leukemia and eosinophilic pustular folliculitis. This is the first published case report of successful treatment of eosinophilic gastroenteritis with IFNt~. The patient developed severe, sub-acute epigastric pain and persistent vomiting at age 15. The pain was worsened by eating any foods and was not relieved by ranitidine 300 mg twice daily or multiple over-the-counter antacid medications. Endoscopy revealed esophagitis, gastritis and ulceration of the pyloric channel and duodenum. A biopsy of the ulcer showed nonspecific inflammation, but subsequent biopsies one month later showed dense eosinophilic mucosal and submucosal infiltration of the esophagus, stomach and duodenum. Colonoscopy and terminal ileoscopy were normal. Ulcer biopsies and serum antibodies were negative for H. pylori. The patient was not taking NSAIDS. Gastrin levels were normal. Three stool samples showed no ova or parasites. The absolute eosinophil count ranged from 600-1200 cells/ktL and total IgE was 1012 IU/mL. In vitro specific IgE was positive to wheat, egg, milk and fish. Echocardiogram and bone marrow biopsy were normal. Intermittent abdominal pain persisted despite medical management with omeprazole, prednisone and elimination diet, and surgical treatment with partial gastrectomy and selective vagotomy. One year later, exclusive enteral feeding with an elemental formula (Neocate One Plus ®) was initiated through a gastrostomy tube. Abdominal pain and vomiting recurred with any oral ingestion of food or when prednisone was tapered below 20 mg daily. Eosinophil counts remained between 330670 cells/~L on prednisone therapy. Two years after onset of symptoms, an immunology consult was requested to suggest alternative therapies to minimize corticosteroid side effects. Subcutaneous IFNot-2b 500,000 units daily was initiated. Within 2 weeks, the eosinophil count decreased to 146 cells/gtL and symptoms improved. Prednisone dosage was tapered over 21⁄2 years, and a normal diet was gradually reintroduced, except for dairy and eggs. IFNt~ was tapered over 4 months after prednisone was discontinued. The patient has not taken prednisone for 9 months or IFNct for 5 months. He is tolerating a regular diet and has gained 25 pounds, without abdominal discomfort or diarrhea. Eosinophil counts without therapy are remaining normal. 1 ? Use of Interferon7 in a Patient With Severe Molluscum Conta/ giosum, Severe Atopic Dermatitis and Undefined ToCell Deftciency Fabienne Dayer Pastore*, Javier Chinen*, WT Shearer§, SE Pacheco§ *Baylor College of Medicine, Houston, TX §Baylor College of MedicineTexas Children's Hospital, Houston, TX INTRODUCTION: Immunodeficiencies like Omenn syndrome, hyper-IgE syndrome or HIV infection may manifest an imbalance of Thl/Th2 type immune phenotype. Skin conditions such as molluscum contagiosum (MC) or atopic dermatitis tAD) are manifestations of a Th2 imbalance. Among our population of immunodeficient patients in the Allergy/Immunology Service at Texas Children's Hospital, we have seen severe MC in patients with Leukocyte Adhesion Deficiency, hyper-IgE syndrome and twins with an ill-defined T-cell deficiency. MC infection is caused by a defective pox virus that produces IL18 binding proteins. IL18 is an important cytokine regulating interferon-y (IFN-y) production and therefore its binding leads to decreased levels of IFN-'/. RATIONALE: IFN-ytherapy has been described to be effective in AD through immunomodulation and counteraction of Th2 immune response. In the case of MC, decreased IFN-yproduction leads to a shift towards Th2 immunity. Therefore, we hypothesized that treatment with IFN7 would be beneficial in patients with AD and MC, through immunomodulation and counteraction of Th2 related cytokines. STUDY: A 9-year-old Latin-American female with severe MC, severe AD, undefined T-cell deficiency and asthma had been hospitalized several times per year, mainly for bacterial skin superinfections of AD and MC. Baseline laboratory studies demonstrated high eosinophil count and very high IgE level (see table). In November 2000, she was admitted for severe bacterial superinfection of MC lesions. Subcutaneous injection of IFN-y at 50 lag/m2/dose three times/week was initiated in addition to conventional antibiotic therapy and atopic dermatitis skin care. Ten months after initialization of IFN7 treatment the patient has had great improvement in AD symptoms and minor decrease in MC lesions. She has not required hospitalizations related to AD or MC. A decrease in eosinophil number and IgE level has been observed (see table). CONCLUSION: IFN-Ttherapy may have been beneficial in controlling AD and MC in this immunodeficient patient because of its immunomodulatory effect and counteraction of the Th2 immune response. (Supported by the David Fund of Texas Children's Hospital) Test IgE (0-90 IU/ml) Eosinophil count (cells/ram3) 11106100 41,66

Cytokine Message and Protein Expression During Lung Granuloma Formation and Resolution Induced by the Mycobacterial Cord Factor Trehalose-6,6′-Dimycolate

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.

Defective Th1 responses and augmented IL-4 production in patients with Whipple's disease

1440720 Background: An impaired monocyte interleukin-12 (IL-12) and T cell interferon-y (IFN-y) production has been suggested in several patients as possible pathogenetic factor in Whipple s disease (WD) and cause for the delayed elimination of the causative bacteria, Tropheryma whippelii. Methods and Results: In the present study we found in a series of 15 WD patients with various disease activity an increased in vitro production of IL-4 (31.5 ::':: 6.2 pglml for WD patients versus 19.7 ::':: 3.8 pg/ml for controls; p<0.05) by peripheral mononuclear blood cells (PBMC) but reduced secretion of IFN-y (476 ::':: 135 vs. 1307 ::':: 395 pglml; p<O.01) and IL-2 (175 ::':: 40 vs. 253 ::':: 78; p<0.05) as compared to age and sex matched healthy controls. T cell proliferation in response to lectins was reduced in all patient groups versus the controls. For cytokines of monocyte origin, we observed a significantly reduced IL-12 production in response to various stimuli in WD patients whereas other cytokines such as secretion of tumor necrosis factor a (TNF-cx) were comparable to controls. Data of mucosal (lamina propria) cytokine secretion in four patients with WD showed similar reduction of IL-12 and IFN-y. These immunological alterations were even present in WD patients without clinical or histological signs of disease activity. Conclusions: Our data demonstrate a persistent defective cellular immune response and an enhancement of IL-4 secretion in a large series of WD patients. This polarized Th l/Th2 cytokine pattern is likely to facilitate engagement and survival of the causative bacteria and presents a pathogenetic basis for immunomodulatory therapy in patients with refractory WD.

16-Epiestriol, a novel anti-inflammatory nonglycogenic steroid, does not inhibit IFN-gamma production by murine splenocytes.

All the steroidal anti-inflammatory drugs currently available are glucocorticoids. The desired anti-inflammatory activities of glucocorticoids frequently are accompanied by adverse side effects, notably glycogenic activities and profound immunosuppression, that can limit clinical use. We recently identified 16-epiestriol, a naturally occurring steroid, as exhibiting significant anti-inflammatory activity without glycogenic activity. In the present study, we compared the effects of 16-epiestriol and hydrocortisone on the capacity of murine splenocytes to produce interferon-y (IFN-y). We injected young adult male BDF1 mice once with 20 mg/kg hydrocortisone or 20, 5, or 1 mg/kg 16-epiestriol and 4 h later harvested the splenocytes. Flow cytometric analysis confirmed that 16-epiestriol did not alter the number of CD3+ T cells in the spleen. In contrast to the suppressive effects of hydrocortisone, none of the 16-epiestriol concentrations inhibited concanavalin A-stimulated IFN-gamma production by spleen cells, as determined by ELISA. Incubating spleen cells from untreated mice in concentrations of 16-epiestriol ranging from 1 mg/ml to 100 pg/ml did not alter profiles of IFN-gamma production, in contrast to the suppressive dose-response effects of hydrocortisone. Collectively, these results support the contention that 16-epiestriol may be a clinically useful safe anti-inflammatory steroid without profound immunosuppressive activities.

Neopterin Induces Nitric Oxide-Dependent Apoptosis in Rat Vascular Smooth Muscle Cells

Numerous studies indicate that proinflammatory substances like tumor necrosis factor a (TNF(x) and interferon y (IFN-y) as well as macrophage-derived neopterin are increased in atherosclerotic tissue and thus are potentially involved in the process of atherogenesis.Since apoptotic death of vascular smooth muscle cells (VSMC) is reported to occur in atherosclerotic lesions, we investigated the effects of neopterin, TNF-a, and IFN-y on apoptosis in cultured VSMC.Morphological changes characteristic of apoptosis as well as DNA fragmentation were detected in cells treated with neopterin, TNF-a/IFN-y, and neopterin + TNF-oc/IFN-'y.Simultaneously, neopterin, TNF-oc/IFN-y, and neopterin + TNF-a/IFN-y led to inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) synthesis.NO generation was significantly reduced when cells were cotreated with the competitive iNOS inhibitor aminoguanidine.This was accompanied by decreased percentual apoptosis as detected by FACS analysis using all kinds of stimuli.We conclude that neopterin as well as TNF-a/IFN-y are potent mediators of apoptotic death in VSMC which is at least in part triggered by NO synthesis induced by these proinflammatory mediators.

Interleukin-12 Overcomes Paclitaxel-Mediated Suppression of T-Cell Proliferation

The antineoplastic agent paclitaxel (TAXOL®) is a potent inhibitor of tumor cell division and a useful chemotherapeutic for the treatment of refractory ovarian and breast carcinoma. Multiple immune system actions have been ascribed to paclitaxel, including the capacity to induce macrophage antitumor cytotoxic molecule production. However, T-cells are susceptible to paclitaxel's cytostatic functions, and no studies have investigated the effects of direct paclitaxel administration on lymphocyte function in the tumor-bearing host (TBH). Because paclitaxel is currently used as an antitumor chemotherapeutic agent and tumor growth alters leukocyte functions, we assessed T-cell function following chemotherapeutic-type paclitaxel treatment. Paclitaxel administration significantly compromised the proliferative capacity of both normal host and TBH lymphocytes in vitro. Although tumor growth impaired T-cell interferon-y (IFN-y) production, paclitaxel treatment did not alter IFN-γ. We speculate that the immunostimulatory cytokine interleukin-12 (IL-12), which promoted T-cell activation and proliferation, was capable of reversing paclitaxel-mediated immunosuppression. Exogenous IL-12 fully reconstituted proliferative reactivity and enhanced IFN-y production by both normal host and TBH lymphocytes in vitro. Collectively, these data suggest that chemotherapeutic paclitaxel regimens impart significant but reversible inhibition of lymphocyte populations, and IL-12 may be a useful ancillary immunotherapeutic to overcome paclitaxel-induced modulation of lymphocyte activities.

Effect of acute endotoxin tolerance on NO production by isolated hepatic parenchymal and nonparenchymal cells and alveolar macrophages in rats

The changes in NO production induced by endotoxin (LPS) tolerance are controversial. The aim of this study was to explore modulation of NO production by LPS tolerance in different liver cell types and alveolar macrophages. Such cells were studied in three groups of male Sprague Dawley rats: non-tolerant rats (sal-LPS) received saline or no treatment 48 h before a 3 mg/kg LPS injection, tolerant rats received low dose LPS (0.5 mg/kg) 48 h before a second injection of saline (LPS-sal) or LPS 3 mg/kg (LPS-LPS). All injections were delivered i.v. Animals were studied 1 and 6 h after the second injection. NO production (assessed by nitrite release) by hepatocytes, Kupffer cells, endothelial cells and alveolar macrophages was simultaneously determined after 20 h of culture in the presence or in the absence of LPS, interferon-y (IFN) or both. Basal NO production by hepatocytes of tolerant and nontolerant LPS injected rats was high 1 h after the second injection, and was dramatically reduced 6 h after the second injection. Hepatocytes of tolerized LPSinjected (LPS-LPS) rats were significantly less sensitive to in vitro stimulation by LPS and IFN at 1 h than hepatocytes of tolerized saline-injected rats and this difference disappeared by 6 h. In Kupffer cells of tolerant rats 6 h after the second LPS injection, basal NO generation was significantly less than in nontolerant rats. In both cell types of tolerant LPS-LPS rats, in vitro stimulated NO production was moderately upregulated at 1 h and then highly upregulated at 6 h, whereas in nontolerant (sal-LPS) animals, stimulated NO production was only slightly upregulated or not at all. Sensitivity to LPS and IFN stimulation of Kupper cells of LPS-LPS rats was not different from Kupffer cells of LPS-sal rats at 1 h, but became significantly higher at 6 h relative to both LPS-sal and sal-LPS animals. In endothelial cells of tolerant saline-injected (LPS-sal) rats, basal NO production was significantly less than in the sal-LPS group both at 1 and 6 h after the second injection. In endothelial cells of tolerant LPS-LPS animals, a significant upregulation of stimulated NO production higher than in the other groups was observed only at 1 h. No difference was evident in basal or stimulated NO production by alveolar macrophages of the different treatment groups, except for a significant increase in basal NO production in tolerant rats (LPS-LPS) at 6 h relative to 1 h after the second LPS injection.

Cross‐reactive cellular immune responses in chickens vaccinated with live infectious bronchitis virus vaccine

Summary Two‐week‐old chickens were vaccinated intra‐nasally with a live infectious bronchitis virus (IBV) vaccine (H120). On days 4, 7, 11 and 14 post‐vaccination (p.v.) spleen mononuclear cells (MNC) prepared from control and vaccinated chickens were stimulated in vitro with homologous (strain M41) and heterologous (strains 7 and 793/B) virus antigens. Antigen‐specific lymphoproliferation and interleukin‐2 (IL‐2) and interferon‐y (IFN) production were used to measure cross‐reactive cell mediated immune responses. In antigen‐specific lymphoproliferation assays, it was found that while 4/16 vaccinated birds responded to the homologous antigen, only one responded to an heterologous antigen (strain 7). However, IL‐2 production was seen in the supernatants of spleen MNC from vaccinated chickens stimulated with all three antigens. Production of IFN was also demonstrated in samples stimulated with the homologous and one heterologous (strain 7) antigen. Thus it appears that, following vaccination of chickens with live IBV vaccine, cross‐reactive cellular immune responses occur that vary in magnitude with the strain of IBV used for in vitro stimulation.