Recombinant Interferon-γ Research Articles

Control of influenza infection is impaired by diminished interferon-γ secretion by CD4 T cells in the lungs of toddler mice.

Respiratory viral infections, such as influenza, can lead to delayed viral clearance in toddlers, possibly exacerbating disease morbidity. We hypothesized that defective CD4 T cells in toddlers may contribute to a failure to clear virus at a similar rate to adults. Thus, we developed a young mouse model to examine potential divergent responses between toddlers and adults. We determined that young mice (toddler mice, 21 d old) were actively generating and recruiting effector/memory T cells, whereas memory populations were firmly established in older, adult mice (8-10 wk old). We infected toddler and adult mice with influenza A/PR8/34 (H1N1) and found young mice had elevated morbidity, as measured by enhanced weight loss and lower partial pressure of oxygen levels, throughout the infection, thus, modeling the higher morbidity observed in children (<2 y old) during infection. Early viral loads were comparable to adult mice, but toddler mice failed to clear virus by 10 d postinfection. This delayed clearance corresponded to poor lung recruitment of CD4 T cells, lower antiviral T cell responses, and lower B cell/antibodies in the lungs. Mechanistically, diminished interferon-γ was detected in the lungs of toddler mice throughout the infection and corresponded to intrinsic, rather than extrinsic, CD4 T cell limitations in interferon-γ transcription. Moreover, defects in interferon-γ production appeared downstream from signal transducer and activator of transcription 4 in the interleukin-12 signaling pathway, suggesting maturational delays different from neonates. Importantly, recombinant interferon-γ supplementation rescued CD4 T cell numbers in the lungs and influenza-specific antibody formation. This study highlights the intrinsic limitations in CD4 T cell effector functions that may arise in toddlers and contribute to disease pathology.

Effect of monoamine oxidase- A on the anti- tumor immunity of tumor- associated macrophages in biliary tract cancers

Objective To investigate the effect of monoamine oxidase- A(MAO- A)in biliary tract cancers(BTCs) on the immunity of tumor- associated macrophages. Methods MAO- A expression plasmid and control plasmid were constructed and transiently transfected into BTC cell lines respectively. Macrophages were derived from peripheral blood mononuclear cells donated by healthy volunteers. Macrophages were then cocultured with aforementioned transfected cancer cell lines for 48 h respectively, Western blotting and enzyme linked immunosorbent assay(ELISA) were applied to assess the expression of immune effector proteins and the excretion of cytokines from cocultured macrophages.24 h priming of macrophages with human recombinant interferon-γ(IFN -γ) were performed after their first stage of coculture; then the second stage of coculture with corresponding transfected cancer cell lines for 48 h were followed.Flow cytometry was employed to detect necrosis and apotosis of cancer cells induced by cocultured macrophages. Results The expression of MAO- A was down- regulated in BTC cell lines, which polarized the cocultured macrophages into M2 type tumor- associated macrophages, and promoted their excretion of interleukin(IL)- 10 and expression of PD- L1, while suppressed their excretion of tumor necrosis factor-α(TNF-α), IL- 1βand IL-12p70(18.8±2.3 vs.31.7±1.9,75.0±0.4 vs.150.2±17.0 and 49.1±15.2 vs.135.2±1.0, respectively, P<0.05),as well as the expression of Human leukocyte antigen- DR(HLA- DR).On the contrary, however, macrophages cocultured with MAO- A overexpressing BTC cells exhibited pro- inflammatory phenotype(M1 type) which opposite to above suppressive immunity phenotype (M2 type).After priming of IFN-γ, macrophages cocultured with MAO- A overexpressing BTC cells induced tumor necrosis and apoptosis more effectively than those cocultured with control BTC cells[(84.85±5.66)% vs.(1.56±0.46)% and(76.73±6.31)% vs.(1.28±0.57)%, respectively, P<0.05]. Conclusion Down- regulation of MAO- A in BTC can promote the development of M2 type tumor- associated macrophages, while restoration of MAO- A in BTC can recover antitumor immunity of macrophages.Present study supports MAO- A as a promising target in the immunotherapy of BTC. Key words: Monoamine oxidase-A; Biliary tract cancer; Immune microenvironment; Tumor-associated macrophages; Tumor immune escape

A novel cyclic dipeptide from deep marine-derived fungus Aspergillus sp. SCSIOW2

A novel cyclic dipeptide, 14-hydroxy-cyclopeptine (1), was purified from a deep sea derived fungal isolate identified as an Aspergillus sp. The structure was elucidated by detailed spectroscopic analyses using 1D and 2D NMR experiments and high resolution mass spectrometry. The absolute configuration of the amino acid was determined by Marfey's method. Two conformational isomers of 1 were established by ROE analyses. 1 inhibited nitric oxide production with IC50 values at 40.3 μg/mL in a lipopolysaccharide and recombinant mouse interferon-γ -activated macrophage-like cell line, RAW 264.7 and showed no cytotoxic effect in the tested dose range up to 100 μg/mL.

Coumarins from the Twigs of Juglans regia and Their Nitric Oxide Inhibitory Activities

In our investigation of in vitro anti-allergic screening of medicinal herbal extracts, the chloroform extract of the twigs of Juglans regia L. was observed to show inhibitory activity on nitric oxide (NO) production in a lipopolysaccharide (LPS) and recombinant mouse interferon-γ activated murine macrophage-like cell line RAW 264.7. Fractionation of the active extract led to the isolation of one new coumarin, juglanslactone (1), with four known compounds, isoscopoletin (2), osthol (3), psoralene (4), and ayapanin (5). The structures and configurations were elucidated by spectroscopic methods.

Isolation and functional characterization of buffalo (Bubalus bubalis) β-casein promoter for driving mammary epithelial cell-specific gene expression

Therapeutic proteins are produced in microbes, mammalian cell lines, and body fluids by applying recombinant DNA technology. They are required for compensating the deficiency of essential proteins in patients. Animal bioreactors producing such valuable bio-pharmaceuticals in body fluids have lately emerged as efficient and cost-effective expression systems. Promoters, along with other regulatory elements of genes coding for milk proteins, have been cloned from few species for directing the expression of desired proteins in the milk of farm animals. However, buffaloes, which are the second largest source of milk production in the world, have remained unexplored for such use. Since mammary epithelial cell-specific β-casein is the most abundantly expressed protein found in buffalo milk, we have isolated the promoter region and the transcriptional regulatory element along with exon 1, Intron 1 and partial exon 2 of the β-casein gene from the genome of the Indian river buffalo (Bubalus bubalis) and have characterized the same (GenBank accession no. KF612339). Mammary epithelial cells of buffalo and human (MCF7) expressed Enhanced green fluorescent protein (EGFP) upon transfection with the construct where egfp was cloned under the β-casein promoter. Transfected HEK-293 cells failed to express EGFP. Transgenic female mice generated using this construct expressed EGFP in the milk gland during lactation, without leaky expression in any other organs. This promoter also drove expression of recombinant human Interferonγ suggesting its use for expressing recombinant bio-pharmaceuticals in the milk of buffalo or other farm animals. Additionally, this may also allow breast gland-specific gene expression for remediation of breast gland-associated diseases.

Preparation of a novel weak cation exchange/hydrophobic interaction chromatography dual-function polymer-based stationary phase for protein separation using "thiol-ene click chemistry".

A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) microspheres was synthesized by thiol-ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96% can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2%, 92.4%, and 2.8 × 10(7) IU/mg, respectively, in IEC mode, and 83.4%, 95.2%, and 4.3 × 10(7) IU/mg, respectively, in HIC mode. The results indicate that the dual-function mixed-mode stationary phase prepared in this study may aid in the development of new two-dimensional liquid chromatography technology with a single column for intensive proteomic studies, and it may also find applications in recombinant protein drug production since it can save column costs and simplify the biotechnological processes.

A study on cloning, expressing and investigating biological activity of recombinant chicken interferon produced from yeast Pichia pastoris

Virus-infectious diseases are permanent threats and cause serious lost for chicken industry of our country. Thus, the development of a bio-product enhancing the immune system against viral-caused diseases on chicken is highly necessary. Aims of this study were to clone, to express and to investigate anti-viral activity of recombinant chicken interferon-α and interferon-γ (ChIFN) produced from yeast Pichia pastoris. Gene fragments encoding ChIFN-α and ChIFN-γ were obtained by PCR and PCR/SOE (Splicing Overlapping Extension) based on the chicken genome DNA. Expressing vectors pPIC9K bearing gene-encoding ChINF-α or ChIFN-γ were cloned and two clones of P. pastoris stably expressing ChIFN-α and ChIFN-γ were generated. The expression of ChIFN-α and ChIFN-γ in P. pastoris culture supernatant was identified by Tricine SDS-PAGE. The biological activity of recombinant ChIFN-α was investigated on the model of chicken embryo fibroblast infected with IBDV and NDV. In case of IBDV-infected cells, the survival rate of cells treated by 1.6 µg/ml ChIFN-α was 79-88%, significantly higher than that of cells treated by PBS which was 33-34%. With NDV-infected cells, the survival rate of cells treated by 1.6 µg/ml ChIFN-α was 81-90%, also obviously higher than that of cells treated by PBS which was 27-32%. In conclusion, recombinant ChIFN-α and ChIFN-γ were expressed successfully on yeast P. pastoris and ChIFN-α was functional to stimulate anti- IBDV and NDV activities of cells.

Optimization of conditions for expression of recombinant interferon-γ in E.coli.

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.

Sera from early-onset, severely preeclamptic women directly modulate HLA-E expression in the EA.hy296 endothelial cell line

The expression of endothelial HLA-E in the context of the systemic inflammatory response observed in preeclampsia has not been established. An experimental study was designed to determine the effect of the sera of pregnant women on the expression of HLA-E in EA.hy296 endothelial cells. First, measurements of protein fractions were performed in sera from early-onset, severely preeclamptic women without HELLP syndrome, in which there was no significant difference in total proteins between the groups, but a reduced level of plasma albumin and an increase in α1-globulin were observed in both groups of pregnant women compared with non-pregnant women. Measurements of colloid osmotic pressure (COP) using a recalculated albumin/globulin ratio formula determined only a significant decrease in COP in all pregnant groups compared with non-pregnant women. The expression of membrane HLA-E was increased in EA.hy296 endothelial cells stimulated with sera of early-onset, severely preeclamptic women, while recombinant interferon-γ (IFN-γ) significantly reduced the expression of membrane HLA-E. Pro-inflammatory cytokines were measured by Luminex in the serum samples, and increased levels of tumor necrosis factor (TNF) and decreased levels of IFN-γ were observed in early-onset, severe preeclampsia compared with normal pregnancy. Moreover, soluble HLA-E was detected in these serum samples by Western blot and ELISA, but no significant difference was found. This raises the possibility that a systemic inflammatory response promotes a compensatory mechanism of COP balance in severe preeclampsia by release of inflammation-induced factors, including endothelial HLA-E. Evidence is now provided regarding HLA-E expression by EA.hy296 cells.

Immunomodulatory Intervention with Interferon-γ in Escherichia coli Pyelonephritis

We investigated the efficacy of recombinant human interferon-γ in experimental pyelonephritis due to Escherichia coli. Pyelonephritis was induced by intrapelvic inoculation ofbacteria after ureteral ligation in 38 rabbits assigned to 1 of 3 groups, including group 1-16 controls, group 2-14 rabbits treated with intravenous recombinant human interferon-γ and group 3-8 rabbits treated with intravenous recombinant human interferon-γ plus amikacin. Bacterial counts, cytokines and malondialdehyde were measured in blood. Peripheral blood mononuclear cells were isolated to measure TNFα transcripts, cytokine stimulation and apoptosis. Survival was recorded, and the tissue bacterial load and myeloperoxidase activity were measured after sacrifice. The mortality rate in groups 1, 2 and 3 was 66.7%, 25% and 12.5%, respectively. The circulating bacterial count and tissue bacterial load were less in group 2 than in group 1. Circulating malondialdehyde negatively correlated with the bacterial load of the spleen. Although the number of TNFα transcripts in circulating peripheral blood mononuclear cells did not differ, peripheral blood mononuclear cells isolated from group 2 at 48 hours produced much greater concentrations of tumor necrosis factor-α after stimulation with Pam3Cys. In parallel, the apoptosis rate of circulating monocytes was increased in group 2 at48 hours. Lung myeloperoxidase activity at 24 hours, serving as indirect evidence of neutrophil infiltration, was decreased in group 2. Recombinant human interferon-γ administration prolonged survival in rabbits with experimental E. coli urosepsis. Its action was probably related to increased bacterial phagocytosis after modulation of oxidant status and reversal of monocyte immunoparalysis.

Successful management of acute myeloid leukemia transformed from myelodysplastic syndromes in an elderly patient aged over 80 years old by ultralow dose decitabine combined with amifostine and autologous CIK cells

Dear Editor, The myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell diseases that are characterized by cytopenia, dysplasia in one or more of the major myeloid cell lines, and a tendency to transform into acute myeloid leukemia (AML). Approximately, one third of MDS patients will undergo transformation into AML, which is resistant to treatment and ultimately fatal. We describe here a case of MDS in an 85-year-old male patient with transformation AML from MDS who was successfully treated with ultralow dose decitabine, amifostine, and infusion of autologous cytokineinduced killer (CIK) cells (dubbed the DAC regimen). The patient was admitted on March 11, 2010 due to pancytopenia. Past history was unremarkable except hypertension and COPD. His routine blood chemistries were as follows: white blood cell count, 2.9×10/L (neutrophils, 40 %; lymphocytes, 50 %; monocytes, 4.5 %); platelets, 67×10/L; and hemoglobin, 98 g/L. Bone marrow (BM) aspirate revealed 5.2 % blasts and trilineage dysplasia (Fig. 1a). Cytogenetic analysis demonstrated 46, XY[13]/t(7;11)(q35;q14)[10]/ del(4)(p10),t(7;11)(q35;q14)[2]/subdiploid(44)[2]. He was diagnosed with MDS-RAEB-I, IPSS intermediate-2 risk. The patient was given amifostine and erythropoietin, but hemoglobin progressively declined and he became transfusion dependent. BM aspirate on December 17, 2011 revealed 20.2 % blasts and flow cytometry demonstrated transformation AML-M4 (FAB classification) (Fig. 1b). One week later, the patient was started on the DAC regimen. Decitabine (10 mg; Chia-Tai Tianqing Pharmacy Co., Jiangsu, China) was given on d1, 3, 5, 7, and 9. Autologous CIK cells were prepared as previously described [1]. Briefly, on d0, 54 mL venous blood was collected from the median cubital vein of the patient, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density–gradient centrifugation. PBMCs were then washed and cultured in the presence of anti-human CD3 monoclonal antibody, recombinant human interferon-γ, and recombinant human interleukin-2 for 14 days. On d14, 2–6×10 cells were infused. Amifostine (0.4 g) was given on d14–18 and d21–25. The patient received a total of three courses of the regimen. The main side effects included Bo Yang, Hai-tao Wang, Li-li Cai, Yu Zhao, Xiao-hua Chi, Hong-li Zhu, Hai-hong Ran, Yang Yang, Rui-li Yu, Song-wei Li and Xue-chun Lu contributed equally to this work.

PRODUCTION, PURIFICATION, AND CHEMICAL STABILITY OF RECOMBINANT HUMAN INTERFERON-γ IN LOW OXYGEN TENSION CONDITION: A FORMULATION APPROACH

The low stability of recombinant human interferon-γ (rhIFN-γ) therapeutic protein imposes some restrictions in its medical applications. In the current study, the effect of oxygen tension on the stability of purified rhIFN-γ was investigated. The rhIFN-γ was purified (>99%) by a two-step chromatographic process. Storage vials were filled by purified formulated product under normal atmospheric oxygen and low oxygen tension conditions. At different time intervals, the amounts of rhIFN-γ covalent dimers and deamidated forms were analyzed using analytical high-performance liquid chromatography (HPLC; size exclusion and cation exchange) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods. To determine the biological activity of purified rhIFN-γ, an antiviral activity assay against vesicular stomatitis virus (VSV) was performed. Upon rhIFN-γ long-term storage in a low oxygen tension condition, the amounts of rhIFN-γ covalent dimers and deamidated forms and also the biological activity of rhIFN-γ changed a little. In contrast, by 9 months of storage of rhIFN-γ preparations under normal atmospheric condition, the amount of covalent dimers and deamidated forms increased with time and reached to approximately 3.5% and 11.5% of the initial amount, respectively. The antiviral specific activity of 9-month-old rhIFN-γ preparations decreased to 41% of the initial amount at normal storage condition, while no significant reduction was seen at the low oxygen tension condition. In conclusion, oxygen tension during storage could have a significant impact on rhIFN-γ stability and finally on the quality of pharmaceutical rhIFN-γ product.

In vitro and in vivo immunostimulatory effects of hot water extracts from the leaves of Artemisia princeps Pampanini cv. Sajabal

Ethnopharmacological relevanceArtemisia princeps Pampanini (Asteraceae) is used as a traditional medicine to immune function-related diseases, such as dysmenorrhea, inflammation, cancer, and ulcers. Aim of the studyThe purpose of this study is to evaluate the immunostimulatory effects of the hot water extract from the leaves of Artemisia princeps Pampanini (WAPP) in recombinant interferon-γ (rIFN-γ)-primed RAW 264.7 macrophages and in cyclophosphamide (20mg/kg, i.p.)-induced immunesuppressed Sprague-Dawley rats. Materials and methodsRAW 264.7 macrophages were treated with WAPP and production and expressions of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) via nuclear factor-κB (NF-κB) were detected by immunoassay, western blot, qRT-PCR and reporter gene assay. In addition, in vivo immunomodulatory activity was studied by cyclophosphamide-induced myelosuppression in rats. ResultsIn rIFN-γ-primed RAW 264.7 macrophages, pretreatment with WAPP increased the productions of nitric oxide (NO) and tumor necrosis factor-α (TNF-α),and increased the expressions of inducible nitric oxide synthase (iNOS) at the protein level and of iNOS and TNF-α at the mRNA level. Molecular data revealed that WAPP upregulated the transcriptional activity and translocation of nuclear factor-κB (NF-κB) by activating inhibitory kappa B-α (IκB-α) degradation and phosphorylation. Furthermore, WAPP upregulated the phosphorylations of p38 MAP kinase, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase1/2 (ERK1/2). In cycloheximide-induced immunosuppressed rats, pretreatment with WAPP (100, 200, or 400mg/kg, p.o.) increased the serum levels of albumin and globulin, and reduced immobility times. ConclusionOur results suggest that upregulations of the expressions of iNOS and TNF-α via the activations of NF-κB and MAPK are responsible for the immunostimulatory effects of WAPP.

Sesquiterpene Coumarins from &lt;i&gt;Ferula fukanensis&lt;/i&gt; and Their Pro-inflammatory Cytokine Gene Expression Inhibitory Effects

Six new sesquiterpene coumarin derivatives, fukanefuromarin H-M (1-6), were isolated from an 80% aqueous methanol extract of the roots of Ferula fukanensis. The structures were elucidated on the basis of spectroscopic evidence, particularly heteronuclear multiple-bond connectivity (HMBC) and high-resolution MS. The sesquiterpene coumarin derivatives inhibited nitric oxide (NO) and inducible NO synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) gene expression in a murine macrophage-like cell line (RAW264.7), which was activated by lipopolysaccharide (LPS) and recombinant mouse interferon-γ (IFN-γ).

Maximizing Production of Human Interferon-γ in HCDC of Recombinant E. coli.

Tuning recombinant protein expression is an approach which can be successfully employed for increasing the yield of recombinant protein production in high cell density cultures. On the other hand, most of the previous results reported the optimization induction conditions during batch and continuous culture of recombinant E. coli, and consequently fed-batch culture have received less attention. Hence, in this research induction conditions for the over-production of recombinant interferon-γ including the amount of inducer, induction time and post-induction duration during chemical induction were optimized. E. coli BL21 (DE3) (pET3a-hifnγ) was used to over-express human interferon-gamma (hIFN-γ) in an exponential fed-batch procedure with a maximum attainable specific growth rate of 0.55 h(-1) at the beginning of feeding and 0.4 h(-1) in induction time. The factors were considered as the amount of inducer (IPTG) in the range of 0.565-22 mg g(-1) L(-1) at seven levels, cell density at induction time as 53, 65 and 75 g (dry cell weight) L(-1), induction duration at different intervals of 3, 4, and 5 h after induction time. The final concentration of biomass and interferon gamma reached to 127 g L(-1) (DCW) and 51 g (hIFN-γ) L(-1) after 17 h, and also the final specific yield and overall productivity were obtained 0.4 g (hIFN-γ) g(-1) DCW and 3 g (hIFN-γ) L(-1) h(-1), respectively, which are the highest amounts of reported specific yield and productivity for recombinant proteins production.

Renal Allograft Recipients Fail to Increase Interferon-γ During Invasive Fungal Diseases

Invasive fungal diseases are a major cause of death in renal allograft recipients. We previously reported that adjunctive recombinant human interferon-γ therapy has clinical utility for invasive fungal diseases after renal transplantation. We have now developed a rapid peripheral blood-based quantitative real-time PCR assay that enables accurate profiling of cytokine imbalances. Our preliminary studies in renal transplant patients with invasive fungal diseases suggest that they fail to mount an adequate interferon-γ response to the fungal infection. In addition, they have reduced IL-10 and increased TNF-α when compared to stable renal transplant patients. These preliminary cytokine profiling-based observations provide a possible explanation for the therapeutic benefit of adjunctive human interferon-γ therapy in renal allograft recipients with invasive fungal diseases.

Successful salvage therapy of refractory HIV-related cryptococcal meningitis with the combination of liposomal amphotericin B, voriconazole, and recombinant interferon-γ

We present 2 cases of HIV-related cryptococcal meningitis, persisting after 3 and 9 months, respectively, of standard treatment. Both patients were treated successfully with a salvage regimen consisting of the combination of liposomal amphotericin B (3 mg/kg), intravenous voriconazole, and subcutaneous recombinant interferon γ-1b (200 μg thrice weekly). Voriconazole was administered at an increased dose (5 mg/kg, twice daily) to overcome interactions with co-administered ritonavir. In both patients, resolution of clinical signs and symptoms, as well as sterilization of cerebrospinal fluid cultures occurred after 10 weeks of salvage therapy. No major side effects were encountered. At the end of treatment, both patients were placed on maintenance therapy with oral fluconazole; no recurrence has been observed after 4 years of follow-up.

Recombinant Interferon-γ Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo

Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P = 0.005–0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P = 0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

Effects of immunomodulators on liquefaction and ulceration in the rabbit skin model of tuberculosis

To better control tuberculosis (TB) epidemics in developing countries a real need exists to study the liquefaction and cavity formation that occur in pulmonary TB lesions. This report is the first to evaluate the effects of immunomodulators on these two processes in a rabbit skin model. The effects of recombinant human interferon-γ (rIFN-γ), recombinant human interleukin-2 (rIL-2), dexamethasone and cyclophosphamide (CTX) were evaluated in TB lesions produced by intradermal injection of 5 × 10(6) viable BCG bacilli. Recombinant IL-2 and rIFN-γ accelerated the liquefaction and healing of the lesions, and reduced the bacterial load. In contrast, dexamethasone inhibited the liquefaction of the lesions, and increased the bacterial load. The effect of CTX was similar to dexamethasone but not as pronounced. Serum levels of IL-2 were higher during the liquefaction and healing phases in the rIL-2 and rIFN-γ groups. Therefore, immunomodulators affect both the development of TB lesions and the survival of the mycobacteria within them. This study suggests that the rabbit skin model can be a valuable method to select therapeutic agents that could inhibit liquefaction and cavity formation in pulmonary tuberculosis.

Dibenzocyclooctadiene lignans from Kadsura coccinea

In vitro anti-allergic screening of medicinal herbal extracts revealed that the EtOAc extract of the rhizoma of Kadsura coccinea inhibited nitric oxide (NO) production in a lipopolysaccharide and recombinant mouse interferon-γ activated murine macrophage-like cell line, RAW264.7. Further fractionation of the EtOAc extract led to the isolation of two new dibenzocyclooctadiene lignans kadsuralignan G (1) and kadsuralignan L (2), and three known analog compounds. The absolute stereostructures were established by circular dichroism spectra. Both 1 and 2 showed moderate NO production inhibitory activities.