Serum IL-17 and TNFα as prognostic biomarkers in systemic sclerosis patients: a prospective study.

Recent reports have demonstrated that endothelial injury is critical in the pathogenesis of systemic sclerosis (SSc) and is associated with increased levels of circulating inflammatory biomarkers. This study aims to analyze the serum concentrations of selected cytokines and evaluate their relationship with SSc clinics and the long-term course of the disease. This study included 43 SSc patients and 24 matched healthy controls. In both groups, we measured serum levels of inflammatory cytokines related to the inflammatory response, such as tumor necrosis factor (TNF)α, interferon (IFN)γ, interleukin (IL)-4, IL-6, IL-10, and IL-17, and fibroblast activation protein (FAP). Additionally, in SSc patients, we evaluated the presence of four single nucleotide polymorphisms (SNPs) located in the promotor region of the TNFA gene, namely rs361525, rs1800629, rs1799964, and rs1799724, which might be related to increased TNFα concentrations. The main aim consisted of associating inflammatory cytokines with (1) clinical disease characteristics and (2) longitudinal observation of survival and cancer prevalence. SSc patients were characterized by a 17% increase in serum TNFα. There was no other difference in serum cytokines between the studied groups and diffuse vs. limited SSc patients. As expected, evaluated serum cytokines correlated with inflammatory biomarkers (e.g., IL-6 and C-reactive protein). Interestingly, patients with higher IL-17 had decreased left ventricle ejection fraction. During the median 5-year follow-up, we recorded four cases of neoplastic diseases (lung cancer in two cases, squamous cell carcinoma of unknown origin, and breast cancer with concomitant multiple myeloma) and nine deaths. The causes of death included lung cancer (n = 2), renal crisis (n = 1), multiple-organ failure (n = 1), and unknown reasons in five cases. Surprisingly, higher TNFα was associated with an increased cancer prevalence, while elevated IL-17 with death risk in the follow-up. Furthermore, the AG rs361525 genotype referred to higher TNFα levels than GG carriers. Both AG rs361525 and CT rs1799964 genotypes were associated with increased cancer risk. Higher serum concentrations of TNFα characterize the SSc patients, with the highest values associated with cancer. On the other hand, increased IL-17 in peripheral blood might predict poor SSc prognosis. Further research is needed to validate these findings.

Parallel online determination of ethylene release rate by Shaken Parsley cell cultures using a modified RAMOS device.

BACKGROUND: Ethylene is an important plant hormone that controls many physiological processes in plants. Conventional methods for detecting ethylene include gas chromatographs or optical mid-infrared sensors, which are expensive and, in the case of gas chromatographs, are hardly suitable for automated parallelized online measurement. Electrochemical ethylene sensors are cheap but often suffer from poor resolution, baseline drifting, and target gas oxidation. Thus, measuring ethylene at extremely low levels is challenging. RESULTS: This report demonstrates the integration of electrochemical ethylene sensors into a respiration activity monitoring system (RAMOS) that measures, in addition to the oxygen transfer rate, the ethylene transfer rate in eight parallel shake flasks. A calibration method is presented that is not prone to baseline drifting and considers target gas oxidation at the sensor. In this way, changes in ethylene transfer rate as low as 4 nmol/L/h can be resolved. In confirmatory experiments, the overall accuracy of the method was similar to that of gas chromatography-mass spectrometry (GC/MS) measurements. The RAMOS-based ethylene determination method was exemplified with parsley suspension-cultured cells that were primed for enhanced defense by pretreatment with salicylic acid, methyl jasmonate or 4-chlorosalicylic acid and challenged with the microbial pattern Pep13. Ethylene release into the headspace of the shake flask was observed upon treatment with salicylic acid and methyl jasmonate was further enhanced, in case of salicylic acid and 4-chlorosalicylic acid, upon Pep13 challenge. CONCLUSION: A conventional RAMOS device was modified for simultaneous measurement of the ethylene transfer rate in eight parallel shake flasks at nmol/L/h resolution. For the first time electrochemical sensors are used to provide a medium-throughput method for monitoring ethylene release by plants. Currently, this can only be achieved by costly laser-based detection systems and automated gas chromatographs. The new method is particularly suitable for plant cell suspension cultures. However, the method may also be applicable to intact plants, detached leaves or other plant tissues. In addition, the general principle of the technology is likely extendable to other volatiles or gases as well, such as nitric oxide or hydrogen peroxide.

Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments.

BACKGROUND: Media containing yeast extracts and other complex raw materials are widely used for the cultivation of microorganisms. However, variations in the specific nutrient composition can occur, due to differences in the complex raw material ingredients and in the production of these components. These lot-to-lot variations can affect growth rate, product yield and product quality in laboratory investigations and biopharmaceutical production processes. In the FDA's Process Analytical Technology (PAT) initiative, the control and assessment of the quality of critical raw materials is one key aspect to maintain product quality and consistency. In this study, the Respiration Activity Monitoring System (RAMOS) was used to evaluate the impact of different yeast extracts and commercial complex auto-induction medium lots on metabolic activity and product yield of four recombinant Escherichia coli variants encoding different enzymes. RESULTS: Under non-induced conditions, the oxygen transfer rate (OTR) of E. coli was not affected by a variation of the supplemented yeast extract lot. The comparison of E. coli cultivations under induced conditions exhibited tremendous differences in OTR profiles and volumetric activity for all investigated yeast extract lots of different suppliers as well as lots of the same supplier independent of the E. coli variant. Cultivation in the commercial auto-induction medium lots revealed the same reproducible variations. In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times. Only by online monitoring of the cultures, a distinct cultivation phase (e.g. glycerol depletion) could be detected and chosen for comparable and reproducible offline analysis of the yield of functional product. CONCLUSIONS: This work proves that cultivations conducted in complex media may be prone to significant variation in final product quality and quantity if the quality of the raw material for medium preparation is not thoroughly checked. In this study, the RAMOS technique enabled a reliable and reproducible screening and phenotyping of complex raw material lots by online measurement of the respiration activity. Consequently, complex raw material lots can efficiently be assessed if the distinct effects on culture behavior and final product quality and quantity are visualized.

Increased oxidative stress response in circulating blood of systemic sclerosis patients - relation to disease characteristics and inflammatory blood biomarkers.

BACKGROUND: Systemic sclerosis (SSc) is a rare connective tissue disorder of unknown etiology characterized by organ fibrosis and microcirculation dysfunction. Emerging evidence suggests that SSc is related to increased oxidative stress, which contributes to further tissue and vascular damage. METHODS: Oxidative stress response in the peripheral blood was assessed in patients with SSc (n = 55) and well-matched controls (n = 44) using real-time monitoring of protein hydroperoxide (HP) formation by the coumarin boronic acid (CBA) assay. We also analyzed the relationship between HP generation and SSc clinics, systemic inflammation, and cellular fibronectin, an emerging biomarker of endothelial damage. RESULTS: SSc was characterized by a significantly faster (2-fold) fluorescent product generation in the CBA assay and higher cumulative HP formation (3-fold) compared to controls (p<0.001, both). The dynamics of HP generation were not associated with the form of the disease (diffuse vs. limited SSc), current immunosuppressive therapy use, presence of abnormal nailfold capillaries, and autoantibody profile. Still, it was enhanced in patients with more severe illness and certain clinical manifestations (i.e., pulmonary hypertension, digital ulcers, and cyclophosphamide treatment) and in smokers (current or past). Higher serum CRP, blood eosinophil count, and cellular fibronectin with lower hemoglobin levels were independent determinants of increased HP formation. CONCLUSIONS: Our data indicate a pro-oxidant imbalance in SSc, likely related to systemic inflammation and endothelial injury. However, extensive prospective studies are needed to verify whether it is also associated with clinical disease progression.

Association between clinical features and course of systemic sclerosis and serum interleukin-8, vascular endothelial growth factor, basic fibroblast growth factor, and interferon alpha.

BACKGROUND: Certain mediators, such as soluble growth factors and cytokines, among others, are implicated in the immunopathogenesis of systemic sclerosis (SSc). OBJECTIVES: This study aimed to examine the association between serum levels of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), interferon alpha (IFN-α), and basic fibroblast growth factor (bFGF) and the clinical presentation and course of SSc. MATERIAL AND METHODS: This longitudinal, observational study included 43 patients with SSc and 24 healthy subjects. Serum concentrations of VEGF, IL-8, IFN-α, and bFGF were measured at baseline in patients previously treated for SSc. Medical history of patients was analyzed retrospectively at the time of cytokine measurement to infer clinical correlations, and during follow-up for a median of 5 years, assessing the incidence of death or cancer. RESULTS: The bFGF and IFN-α concentrations differed between SSc patients and controls (p < 0.01). In turn, organ involvement and SSc phenotypes did not impact studied cytokine concentrations, similar to systemic steroid and/or immunosuppressant use at enrollment. However, we have documented a positive correlation between the current oral steroid dose and serum levels of IL-8 and bFGF. Furthermore, patients with a VEGF level ≥95.7 pg/mL and IFN-α level ≥3.6 pg/mL required cyclophosphamide therapy more often, currently or in the past (approx. 3-fold and 4-fold, respectively). Substantially elevated VEGF and IFN-α concentrations at baseline were associated with higher cancer occurrence (n = 4) during follow-up, while elevated circulating IL-8 level was associated with an increased risk of death (n = 9). CONCLUSIONS: The SSc group was characterized by higher serum concentrations of bFGF and IFN-α compared to healthy controls. Patients treated with cyclophosphamide or receiving higher systemic steroid doses, thus suffering from a more severe disease type, had increased cytokine levels. Elevated circulating IFN-α and VEGF levels might be correlated with cancer, whereas raised IL-8 levels may be associated with an increased risk of death. However, further research is needed to verify our findings.

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains.

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315-327, 2018.