Recombinant Beta Research Articles

Artificial Cell Membrane Polymersome-Based Intranasal Beta Spike Formulation as a Second Generation Covid-19 Vaccine.

Current parenteral coronavirus disease 2019 (Covid-19) vaccines inadequately protect against infection of the upper respiratory tract. Additionally, antibodies generated by wild type (WT) spike-based vaccines poorly neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. To address the need for a second-generation vaccine, we have initiated a preclinical program to produce and evaluate a potential candidate. Our vaccine consists of recombinant Beta spike protein coadministered with synthetic CpG adjuvant. Both components are encapsulated within artificial cell membrane (ACM) polymersomes, synthetic nanovesicles efficiently internalized by antigen presenting cells, including dendritic cells, enabling targeted delivery of cargo for enhanced immune responses. ACM vaccine is immunogenic in C57BL/6 mice and Golden Syrian hamsters, evoking high serum IgG and neutralizing responses. Compared to an ACM-WT spike vaccine that generates predominantly WT-neutralizing antibodies, the ACM-Beta spike vaccine induces antibodies that neutralize WT and Beta viruses equally. Intramuscular (IM)-immunized hamsters are strongly protected from weight loss and other clinical symptoms after the Beta challenge but show delayed viral clearance in the upper airway. With intranasal (IN) immunization, however, neutralizing antibodies are generated in the upper airway concomitant with rapid and potent reduction of viral load. Moreover, antibodies are cross-neutralizing and show good activity against Omicron. Safety is evaluated in New Zealand white rabbits in a repeated dose toxicological study under Good Laboratory Practice (GLP) conditions. Three doses, IM or IN, at two-week intervals do not induce an adverse effect or systemic toxicity. Cumulatively, these results support the application for a Phase 1 clinical trial of ACM-polymersome-based Covid-19 vaccine (ClinicalTrials.gov identifier: NCT05385991).

Interferon Beta-1a versus Combined Interferon Beta-1a and Oligodendrocyte-Specific FGFR1 Deletion in Experimental Autoimmune Encephalomyelitis.

Recombinant beta interferons-1 (IFNβ-1) are used as first line therapies in patients with relapsing multiple sclerosis (MS), a chronic inflammatory and neurodegenerative disease of the CNS. IFNβ-1a/b has moderate effects on the prevention of relapses and slowing of disease progression. Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) are known to play a key role in the pathology of MS and its model EAE. To investigate the effects of short-term treatment with s.c. IFNβ-1a versus the combined application of s.c. IFNβ-1a and oligodendrocyte-specific deletion of FGFR1 (Fgfr1ind−/− mice) in MOG35-55-induced EAE. IFNβ-1a (30 mg/kg) was applied s.c. from days 0–7 p.i. of EAE in controls and Fgfr1ind−/− mice. FGFR signaling proteins associated with inflammation/degeneration in MS/EAE were analyzed by western blot in the spinal cord. Further, FGFR1 in Oli-neu oligodendrocytes were inhibited by PD166866 and treated with IFNβ-1a (400 ng/mL). Application of IFNβ-1a over 8 days resulted in less symptoms only at the peak of disease (days 9–11) compared to controls. Application of IFNβ-1a in Fgfr1ind−/− mice resulted in less symptoms primarily in the chronic phase of EAE. Fgfr1ind−/− mice treated with IFNβ-1a showed increased expression of pERK and BDNF. In Oli-neu oligodendrocytes, treatment with PD166866 and IFNβ-1a also showed an increased expression of pERK and BDNF/TrkB. These data suggest that the beneficial effects in the chronic phase of EAE and on signaling molecules associated with ERK and BDNF expression are caused by the modulation of FGFR1 and not by interferon beta-1a. FGFR may be a potential target for therapy in MS.

High sensitivity acidic N-glycan profiling with MS-enhancing derivatization and mixed mode chromatography

Acidic N-linked glycan content is often associated with a protein drug’s stability, efficacy and immune response. It has often been a challenge to analyze these types of glycans, including those that are differentiated by the incorporation of N-acetyl (NANA) and N-glycolyl neuraminic acid (NGNA) residues. In this study, a strategy for rapid N-glycan profiling by mixed mode chromatography is proposed as a complement to established HILIC methodologies. Hybrid silica chromatographic surfaces are used to improve recoveries during a column’s initial use and to eliminate the need for column conditioning. In addition, the loss of labeled acidic glycans, especially phosphorylated glycan species, during SPE purification is addressed through the use of a citrate containing eluent. Yields for both singly and doubly phosphorylated glycan species are markedly improved. Combined with a mixed mode anion exchange reversed phase separation, these advances afford a class separation of glycans derivatized with labels designed to enhance positive ion mode MS detection. These labeled glycan species are separated according to their charge and with an added level of resolution imparted by the reversed phase retention mechanism. The separation technique itself can be accomplished with a low ionic strength gradient running from 0 to 22 mM ammonium formate such that high sensitivity detection can be achieved by both fluorescence and mass spectrometry. Using analytical scale chromatography, features in an N-glycan profile were easily interrogated to well below a 0.1% relative abundance. As such, it became possible to characterize N-glycans from recombinant beta glucuronidase and to quickly identify a number of unique phosphorylated glycan species.

Validation of a test procedure for determination of specific activity of human recombinant interferon beta (Infibeta®) using various cell/virus combinations

Interferon beta preparations have demonstrated efficacy in the treatment of multiple sclerosis. One of the most important quality attributes that support efficacy and safety of interferon beta preparations is specific antiviral activity. Interferon beta activity is determined by the biological test method which can lead to an erroneous final result due to the system uncertainties that contribute to the overall uncertainty. Accurate assessment of specific activity plays an important role in adequate determination of the product’s therapeutic dose, therefore the test method standardisation and validation are of particular relevance. The aim of the study was to validate a test procedure for assessing specific activity of human recombinant interferon beta preparations, using various cell/virus combinations, as illustrated by the example of Infibeta®. Materials and methods: WISH, Vero, A-549, and MDBK cells in combination with mouse encephalomyocarditis virus were used in the study. The testing was performed using the biological test method based on the interferon ability to suppress virus-induced cytopathic effects in cell cultures. The results were processed using methods of mathematical statistics and the GraphPadPrism, Statistical 2/0 software package. Results: the paper compares the results of specific activity determination of Infibeta® (interferon beta-1b) using various cell/virus combinations. The dose-response curves were used to compare the test results. It was demonstrated that all the tested cell lines could be used in the biological test procedure for determination of interferon beta specific activity. However, the best results were obtained with A-549, WISH cells in combination with mouse encephalomyocarditis virus. The following validation characteristics were determined in the cell/virus systems: specificity, linearity, precision, and robustness. Conclusions: the study validated the test procedure that enables measurement of interferon beta specific activity in the range of 4.8–11.2 IU/mL at a satisfactory accuracy level, which guarantees reliable test results. The study demonstrated robustness, intermediate precision, and reproducibility of the test procedure.

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens.

The alpha (CPA), beta (CPB) and epsilon (ETX) toxins of Clostridium perfringens are responsible for causing diseases that are difficult to eradicate and have lethal potential in production animals. Vaccination of herds is still the best control strategy. Recombinant clostridial vaccines have shown good success at inducing neutralizing antibody titers and appear to be a viable alternative to the conventional production of commercial clostridial toxoids. Research is still needed on the longevity of the humoral immune response induced by recombinant proteins in immunized animals, preferably in target species. The objective of this study was to measure the humoral immune response of cattle immunized with trivalent vaccines containing the recombinant proteins alpha (rCPA), beta (rCPB) and epsilon (rETX) of C. perfringens produced in Escherichia coli at three different concentrations (100, 200, and 400 µg) of each protein for 12 months. The recombinant vaccines containing 200 (RV2) and 400 µg (RV3) yielded statistically similar results at 56 days. They performed better throughout the study period because they induced higher neutralizing antibody titers and were detectable for up to 150 and 180 days, respectively. Regarding industrial-scale production, RV2 would be the most economical and viable formulation as it achieved results similar to RV3 at half the concentration of recombinant proteins in its formulation. However, none of the vaccines tested induced the production of detectable antibody titers on day 365 of the experiment, the time of revaccination typically recommended in vaccination protocols. Thus, reiterating the need for research in the field of vaccinology to achieve greater longevity of the humoral immune response against these clostridial toxins in animals, in addition to the need to discuss the vaccine schedules and protocols adopted in cattle production.

Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months.

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry’s production of vaccines against the action of C. perfringens in equine species.

What are the effects of recombinant interferon beta for delaying conversion of the first demyelinating event to multiple sclerosis (MS)?

What are the effects of recombinant interferon beta for delaying conversion of the first demyelinating event to multiple sclerosis (MS)?

TRAILR1 (rs20576) and GRIA3 (rs12557782) are not associated with interferon-β response in multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune-type inflammatory disorder in human central nervous system. Recombinant interferon beta (IFN-β) decreases the number of relapses and postpones disability progression in MS. However, up to 50% of patients treated with interferon beta continue experiencing relapses and/or worsening disability. Single nucleotide polymorphisms in different genes have been known to show significant associations with response to IFN-β in MS patients. In the present work, we examined the potential role of TRAILR1 and GRIA3 genes polymorphisms on response to IFN-β therapy in Iranian MS patients. The DNA was extracted from blood samples by standard procedures from 73 patients diagnosed with Multiple Sclerosis that were either responded to IFN-β or did not. We carried out RFLP -PCR and tetra-primer ARMS-PCR methods to study of rs20576 and rs12557782, respectively. All results were analyzed using the SPSS software. TRAILR1 rs20576 genotype frequencies in responders and non-responders were similar (χ2 = 0.26, P = 0.87, Fisher, s Exact test). Our results showed that response to IFN-β has not association with sex (p = 0.73). Also, genotypic frequencies of GRIA3 rs12557782 had no significant differences between two groups of female population (χ2 = 3.75, p = 0.15). Furthermore, it had not been any statistical differences between responder and non-responder males (χ2 = 0.7, p = 0.4) related to the SNP. Our results analysis revealed no significant association between the studied SNPs (TRAILR1 rs20576 and GRIA3rs 12,557,782) and response to IFN-β in Iranian MS patients.

Effects of ackA, pta and poxB inhibition by antisense RNA on acetate excretion and recombinant beta interferon expression in Escherichia coli

Effects of ackA, pta and poxB inhibition by antisense RNA on acetate excretion and recombinant beta interferon expression in Escherichia coli

SIGNIFICANCE OF GENOTYPIC ALPHA GALACTOSIDASE A MUTATIONS IN FABRY DISEASE TREATMENT

Fabry disease (FD) is a rare inherited X-linked lysosomal storage disorder caused by a deficiency in alfa-galactosidase A (a-GAL). It is resulting in the accumulation of glycosphingolipids that leads to multiple organ dysfunction and ultimately signs and symptoms of the disease. The aim of this study was to examine the significance of genotypic a-GAL mutations in the treatment of FD. The disease can be divided into a severe, classical phenotype, and a milder nonclassical phenotype. Numerous a-GAL mutations are described in gene mutation databases. Missense, nonsense, consensus splice site, cryptic splicing, and frameshift mutations are reported. Enzyme replacement therapy (ERT) can lead to a significant clinical improvement. Depending on the a-GAL mutation, there are various recommendations for initiation of ERT in adult male and female patients with classic Fabry mutations, later-onset Fabry mutations or aGAL variants of unknown significance. ERT with recombinant human agalsidase alfa and agalsidase beta is currently available therapy. Although there are no uniform guidelines, development of signs or symptoms related to FD should be an indication to start ERT. Treatment with ERT should be combined with adjuvant treatments for specific disease manifestations. Migalastat is a new oral pharmacological molecule developed as an alternative treatment to intravenous ERT for patients with FD and amenable mutations. Migalastat and ERT have similar effects on renal function in patients with FD. Long-term treatment of adult Fabry patients should involve timely ERT, regular assessment of disease progression in all patients, use of appropriate adjunctive therapies and multidisciplinary team approach.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid beta 1-42.

Amyloid plaques found in the brains of Alzheimer’s disease patients primarily consists of amyloid beta 1-42 (Aβ42). Commercially, Aβ42 is synthesized using high-throughput peptide synthesizers resulting in the presence of impurities and the racemization of amino acids that affects its aggregation properties. Furthermore, the repeated purchase of even a small quantity (~1 mg) of commercial Aβ42 can be expensive for academic researchers. Here, we describe a detailed methodology for robust expression of recombinant human Aβ(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli using standard molecular biology techniques with refined and rapid one-step analytical purification techniques. The peptide is isolated and purified from transformed cells using an optimized reverse-phase high-performance liquid chromatography (HPLC) protocol with commonly available C18 columns, yielding high amounts of peptide (~15–20 mg per 1 L culture) within a short period of time. The recombinant human Aβ(M1-42) forms characteristic aggregates similar to synthetic Aβ42 aggregates as verified by western blotting and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique produces pure recombinant human Aβ(M1-42) that may be used to synthesize chemical probes and in several downstream in vitro and in vivo assays to facilitate Alzheimer’s disease research.

Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain.

Clostridium perfringens beta toxin is only produced by types B and C and plays an important role in many human and animal diseases, causing fatal conditions that originate in the intestines. We compared the expression of C. perfringens type B vaccine strain recombinant beta toxin gene in the Escherichia coli strains RosettaTM(DE3) and BL21(DE3). The beta toxin gene was extracted from pJETβ and ligated with pET22b(+). pET22β was transformed into E. coli strains BL21(DE3) and RosettaTM(DE3). Recombinant protein was expressed as a soluble protein after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction in strain RosettaTM(DE3) but not in BL21(DE3). Expression was optimised by growing recombinant cells at 37 °C and at an induction of 0.5 mM, 1 mM, 1.5 mM IPTG. Expression was evaluated using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified via Ni-NTA and was analysed using western blot. We concluded that E. coli strain RosettaTM(DE3) can enhance the expression of C. perfringens recombinant beta toxin.

SELECTION OF THERAPEUTIC TACTICS FOR EARLY ANEMIA OF PREMATURE INFANTS WITH VERY LOW OR EXTREMELY LOW BIRTH WEIGHT: RETROSPECTIVE STUDY RESULTS

Background: Early anemia of prematurity (EAP) is a serious issue of contemporary neonatology due to increasing survivability of premature infants. One of the most efficient preventive methods is extreme prematurity prevention. At the same time, efficacy of different methods of EAP therapy remains undetermined. Objective: Our aim was to compare efficacy of erythrocyte-containing donor blood components (packed red cells, erythrocytic suspension) and recombinant epoetin beta for treating premature infants with EAP. Methods: The retrospective cohort study provided analysis of data of premature infants (gestation age — 27–32 weeks) born with very low (VLBW) and extremely low birth weight (ELBW) and EAP (hemoglobin < 110 g/l, hematocrit < 27%, reticulocyte share < 20‰, normochromia, normocytosis; all criteria must have been met). We determined primary hematological morphological parameters and population structure of umbilical (at birth) and peripheral blood (after 30 days of treatment) leukocytes. Results: 32 premature infants with EAP underwent donor blood component transfusions, 26 — recombinant epoetin beta injections. The groups were comparable in terms of the primary clinical laboratory parameters. In the donor blood component group, we observed lower hemoglobin concentration (108 ± 17 vs. 119 ± 16 g/l in the epoetin beta group; p = 0.07) and reticulocyte share (3.7 ± 1.5 vs. 8.7 ± 1.7‰, respectively; p = 0,053) at the age of 1 month. We revealed a drop in cell immunity parameters in the setting of donor blood component transfusions: CD4+ lymphocyte share — down to 32,2 ± 14,0% (in the epoetin beta group; p = 0.055); CD8+ lymphocyte share — down to 21,2 ± 7,1% (in the epoetin beta group; p = 0.050). Conclusion: Epoetin beta is more efficient in terms of EAP correction in premature infants with VLBW and ELBW that erythrocyte-containing donor blood component transfusions. The latter are characterized by adverse effect on cell immunity parameters.

Preparation of Immobilized Recombinant Tubulin Beta(TuBb) on Chitosan Nanoparticles by Covalent Binding Method

Objective In order to screen TuBb inhibitors, this paper describes the preparation of immobilized TuBb on chitosan nanoparticles. Methods TuBb was immobilized onto chitosan nanoparticles by covalent binding method. Results The results of the univariate test indicated that the highest immobilized yield can be obtained when the optimal immobilization condition was 1 mg of TuBb, 0.5 mol/L of buffer solution with pH 6.5, immobilization 30 min and immobilization at 0-4 °C. Conclusions The authors conclude that the immobilized TuBb maintain the catalysis properties and can be used as the screening of TuBb inhibitors.

Recombinant beta interferon could clear the low-dose infected porcine reproductive and respiratory syndrome virus (PRRSV) in MARC-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes one of the most economically devastating and pandemic porcine diseases. Previous study has shown that MARC-145 cells pretreated with recombinant IFN-β (rIFN-β) couldn't develop cytopathic effect (CPE) of PRRSV. However, up to date, it is not clear whether MARC-145 cells post-treated with rIFN-β could develop CPE of PRRSV. The present work showed that the MARC-145 cells didn't develop the CPE at 120 hr post-infection (p.i.) with low-dose of PRRSV when the cells were pre-treated with rIFN-β (Group 1), post-treated with rIFN-β at 4 hr p.i. (Group 2), or post-treated with rIFN-β at 8 hr p.i. (Group 3), while the MARC-145 cells could develop CPE when the cells were infected with high-dose PRRSV and then treated with rIFN-β at 24 hr p.i.. Furthermore, the indirect immunofluorescence assay confirmed that there were a few N protein-positive cells in the high-dose infected cells in Group 1, Group 2 and Group 3, while there were no N protein-positive cells in the low-dose infected cells in all rIFN-β treatment groups. In addition, the numbers of N protein-positive cells in high-dose infected cells (MOI = 10) in Group 1 were lower than that in Group 2 and Group 3. The results above demonstrated that both pre-treatment with rIFN-β and an earlier post-treatment with rIFN-β could inhibit the PRRSV replication and could clear the low-dose infected PRRSV, which indicated that the rIFN-β had efficient antiviral activities when the cells have been infected with PRRSV.

Protective antibody and cytokine responses in mice following immunization with recombinant beta-tubulin and subsequent Trypanosoma evansi challenge.

BackgroundTrypanosomosis or Surra, caused by the flagellated hemoprotozoan parasite Trypanosoma evansi, is a disease of economic importance through its wide prevalence in domestic livestock in tropical countries. In the absence of a protective vaccine, management of the disease relies on a few available chemotherapeutic agents. Although humoral immunity is the mainstay of resistance to T. evansi, the ability of the parasite to vary its immunodominant surface proteins to subvert the immune system has forced vaccine efforts to target a variety of invariant epitopes. Beta tubulin, an integral component of the trypanosome cytoskeleton, was therefore targeted using the recombinant form of the protein for immunization.MethodsThe 1329 bp coding sequence of beta tubulin gene was PCR amplified and cloned in pQE-TriSystem expression vector. Recombinant beta tubulin was heterologously expressed in Escherichia coli as a 46 KDa fusion protein and used for immunization of mice. The Ig response was studied by ELISA, whereas the cytokine response was measured using a cytometric bead-based assay quantified by flow cytometry.ResultImmunization with recombinant beta (β)-tubulin protein induced a beta-tubulin specific humoral immune response of predominantly IgG2a isotype. Lethal challenge with T. evansi blood-form trypomastigotes post-immunization elicited a robust anamnestic response. An abundance of IFN-γ further confirmed the Th-1 bias of the protective response. We also observed extended survival and better control of the challenge infection in the immunized mice.ConclusionsA robust anamnestic response following challenge including a Th-1 serum cytokine profile coupled with increased survival is indicative of protective immunity in the immunized mice. These observations suggest that β-tubulin of T. evansi is a viable antigenic target for development of a vaccine against this important livestock pathogen.

Abstract 148: Stat2 Deficiency Does Not Protect From Atherosclerosis in Ldlr Knockout Mice Fed a Western Diet

Signal Transducer and Activator of Transcription ( Stat2 ) is a transcription factor that plays a critical role in type I interferon (IFN) signaling. Recent evidence supports a role for the type I IFN pathway in atherosclerosis, since recombinant IFN alpha and IFN beta both promote atherosclerosis in mice. We tested the hypothesis that constitutive type I IFN signaling through STAT2 may promote atherosclerotic lesion formation. Male Ldlr KO and Stat2/Ldlr DKO mice on a C57BL6/J background were fed a western diet for 16 weeks. Total cholesterol levels were indistinguishable at baseline ( Ldlr KO: 232 ± 24 vs. Stat2/Ldlr DKO: 239 ± 36 mg/dl, p=0.39), but significantly higher in the Stat2/Ldlr DKO mice after 16 weeks of western diet ( Ldlr KO: 856 ± 203 Vs Stat2/Ldlr DKO: 1015 ± 176 mg/dl, p=0.004). Despite consuming less food than Ldlr KO controls, the Stat2/Ldlr DKO animals weighed significantly more than controls and had greater fat mass in the inguinal subcutaneous and perigonadal depots respectively ( Ldlr KO: 0.45±0.23 Vs Stat2/Ldlr DKO: 0.69 ± 0.27 g, p=0.003 and Ldlr KO: 0.89 ± 0.50 Vs Stat2/Ldlr DKO: 1.39 ± 0.56 g, p=0.003). Surprisingly, no significant differences were observed in percent lesional area of aortae ( Ldlr KO: 9.7% ± 5.6% Vs Stat2/Ldlr DKO: 12.5% ± 6.8%, p=0.156). In conclusion, STAT2 does not appear to have a major impact on atherosclerosis in the absence of exogenous type I IFNs or other agonism of the pathway.

MONOPEGYLATED HUMAN INTERFERON BE- TA-1A PREPARATION: OPTIMIZATION OF CONDITIONS FOR N-TERMINAL PEGYLATION

A modification of recombinant human IFN beta- 1a by polyethylene glycol at a-amino group of N-terminal methionine has been performed. The PEGylation was conducted using activated butyraldehyde PEG derivative with molecular mass of 30000 Da. As a result of the multifactorial experiment, the dependence of monoPEGylated protein yield on the reaction conditions was demonstrated, and optimal PEGylation conditions were established. We developed a one-step chromatographic purification scheme allowing to obtain the monoPEG-IFN beta-1a conjugate with the above 98% purity . Mass-spectrometric studies showed that the molecular mass of the conjugate is 54130 Da, wherein the N-terminal methionine is associated with one PEG molecule. The obtained PEG-IFN beta-1a has specific antiviral activity, and it can be considered as a perspective candidate to the design of long-acting medicine for the prolonged treatment of multiple sclerosis.

TGF beta Exposure Promotes Acquisition of Epithelial-like Phenotype in Breast Cancer Spheres

The epithelial-to-mesenchymal transition (EMT) is considered as a critical developmental process in cancer biology. TGF beta has been shown to play an important role in the induction of EMT via down-regulating E-cadherin and up-regulating vimentin expression in cancer cells. However, whether TGF beta induces EMT in cancer spheres has not been determined yet. To address this question, we generated breast cancer spheres from MCF7 and MDAMB- 231 breast cancer cell lines. These cancer spheres then were treated with recombinant human TGF beta for 24 and 48 hours, respectively in order to examine the expression of epithelial marker E-cadherin and mesenchymal marker vimentin. We find that breast cancer spheres enriched with E-cadherin and exhibit characteristics of epithelial cells by TGF beta stimulation. In addition, we did not observe TGF beta induces EMT in breast cancer spheres such as lose E-cadherin expression and gain vimentin expression. Our data suggested that TGF beta plays a different role in breast cancer spheres and cancer cells during EMT induction.

Human intestinal beta defensins inhibit viral replication and are diminished in chronic untreated HIV infection

Background One of the hallmarks of HIV infection is an early and dramatic depletion of CD4 T cells in the intestinal lamina propria. This loss of T cells is accompanied by intestinal epithelial cell disruption resulting in the translocation of luminal bacterial products, persistent systemic immune activation and resultant disease progression. Beta defensins are cationic antimicrobial polypeptides produced by intestinal epithelial cells and phagocytes that are a part of the innate immune response. They have ap ivotal role in maintenance of immune homeostasis in the gut. Although beta defensins have been reported to inhibit bacterial and viral replication in vitro, their role in HIV infection has been incompletely characterized. Methods We investigated anti-viral activity and production of defensins in intestinal mucosal biopsies, stool and plasma from individuals with chronic untreated HIV (chronic progressors), immunologically controlled HIV (elite controllers) and HIV uninfected individuals using ELISA, immunohistochemistry, quantitative PCR and viral inhibition assays. Results Plasma levels of beta defensins were increased in the HIV controller group compared to HIV progressors, whereas levels of beta defensins were significantly decreased in biopsies from chronic progressors. Beta defensin release was induced by HIV in ex vivo cultured intestinal cells. Recombinant beta defensins inhibited HIV replication in vitro. Conclusion Beta defensins are upregulated by HIV and inhibit viral replication in vitro. Chronic progressors, however, had diminished levels of beta defensins in gut biopsies and in the plasma. We propose that beta defensins are induced in HIV infection and have a role in inhibiting viral replication and preventing intestinal epithelial cell disruption. In chronic infection, however, their release is diminished, likely due to epithelial damage in the setting of persistent viremia and mucosal inflammation.