Quality Attributes and Sensory Acceptance of Different Botanical Coffee Co-Products.

Coffee processing is a major contributor to the creation of food and product waste. Using coffee co-products can play an essential role in addressing environmental problems and issues with nutritionally unbalanced foods, population growth, and food-related diseases. This research aimed to determine the quality and sensory parameters (aw, pH, dry matter, TAC, TPC, fat, fatty acids profile, fiber, caffeine, chlorogenic acids, color, and sensory analysis) of different botanical origins of cascara (coffee husks) and silverskin (thin layer). The results of this study show that silverskin and cascara are a good source of TAC (1S 58.17 ± 1.28%, 2S 46.65 ± 1.20%, 1C 36.54 ± 1.84%, 2C 41.12 ± 2.11%). Cascara showed the presence of polyphenols (2C 49.135 g GAE·kg-1). Coffee co-products are good sources of fiber. Silverskin had higher values of caffeine than cascara. Palmitic, stearic, oleic, linoleic, and arachidic acids were the most represented acids in the samples. Given the obtained results, cascara can be considered "low-fat" (1C 4.240 g·kg-1 and 2C 5.4 g·kg-1). Based on the sensory evaluation, no sample reached the acceptable index value of 70%. Understanding the link between the character, identification properties, and composition of coffee co-products of different botanical origins can enable their application in the food industry.

Variability of caffeine content in green and roasted Coffea arabica regarding the origin, post-harvest processing, and altitude, and overview of recommended daily allowance.

Caffeine content is a crucial attribute of coffee. Its concentration and thus maximum cups of Coffea arabica from Africa, Asia, Central America, and South America from different altitudes of growing areas, altitude, and process using different post-harvest processing (dry, wet, and pulped natural). Our results suggest that geographical origin might affect the alkaloid concentration in C. arabica. The caffeine concentration pattern in green samples was as follows: Central America > South America > Asia > Africa. Altitude affected the concentrations, lowlands > midlands > highlands, however, not significantly. Given caffeine is thermostable, the medium roasting process did not affect the concentration of caffeine directly, but a small increase was observed. Scientific opinion on the safety of habitual caffeine consumption of up to 400 mg per day does not raise safety concerns for non-pregnant adults. A cup (7 g coffee in 120 mL of water) was used for recalculation. Results suggest that mostly highlands and midlands coffee from Africa reached levels of caffeine that might be consumed in more than 5.5 cups a day.

Comparative Analysis of Selected Chemical Parameters of Coffea arabica, from Cascara to Silverskin.

Nowadays, there is an increased interest in coffee derivatives (green beans, roasted beans, and coffee by-products (Cascara and Silverskin)) due to their particular chemical composition. This study aimed to compare the content of dry matter, total fat, fatty acids, and fiber (ADF, NDF) of coffee by-products (Cascara and Silverskin) and coffee beans (green and roasted under different conditions). Coffee beans and their by-products were obtained from 100% C. arabica coffee cherries from Panama by dry process. The lowest concentrations of fat corresponded to Cascara 4.24 g·kg-1 and Silverskin 23.70 g·kg-1, respectively. The major fatty acids detected in all samples were palmitic, stearic, oleic, and linoleic acids, the latter two being essential fatty acids. LDA showed that 89.01% of the variability between beans and by-products was explained by lignoceric, myristic, behenic, tricosanoic, arachidic, and heneicosanoic acids. Silverskin appeared to be a good source of lignoceric, myristic, and behenic acids and had a higher concentration of dietary fiber (314.95 g·kg-1) than Cascara (160.03 g·kg-1). Coffee by-products (Silverskin and Cascara) are low-fat products enriched in dietary fiber. Their incorporation, after adjustment, into the global diet may contribute to nutrition security, the sustainability of the coffee sector, and human health.

Detection of Changes in Total Antioxidant Capacity, the Content of Polyphenols, Caffeine, and Heavy Metals of Teas in Relation to Their Origin and Fermentation.

Tea (Camellia sinensis) is widely sought for beverages worldwide. Heavy metals are often the main aims of the survey of teas, given that the use of agricultural fertilization is very frequent. Some of these may affect the content of bioactive compounds. Therefore, in this study, we analyzed fermented and non-fermented teas of a single plant origin from Japan, Nepal, Korea, and China, and described mutual correlations and changes in the total antioxidant capacity (TAC), and the content of polyphenols (TPC), caffeine, and heavy metals in tea leaves, in relation to the origin and fermentation process. Using UV-VIS spectrophotometry and HPLC-DAD, we determined variations in bioactive compounds' content in relation to the fermentation process and origin and observed negative correlations between TAC and TPC. Heavy metal content followed this order: Mn > Fe > Cu > Zn > Ni > Cr > Pb > Co > Cd > Hg. Given the homogenous content of these elements in relation to fermentation, this paper also describes the possibility of using heavy metals as determinants of geographical origin. Linear Discriminant Analysis showed an accuracy of 75% for Ni, Co, Cd, Hg, and Pb, explaining 95.19% of the variability between geographical regions.

Association of HLA-G Polymorphisms in the 3'UTR Region and Soluble HLA-G with Kidney Graft Outcome.

Background: Human leukocyte antigen G (HLA-G) belongs to nonclassical HLA I molecule involving in the suppression of immune response. Besides its profound effect to induce fetal tolerance, HLA-G expression has been associated with allograft acceptance. For the regulation of HLA-G levels, polymorphic sites within the 3' untranslated region (3'UTR) are of crucial importance. The aim of the study was to analyze the association between several HLA-G 3'UTR variants (+3003T/C, +3010C/G, +3027C/A, +3035C/T, +3142G/C, +3187A/G, and +3196C/G), soluble HLA-G (sHLA-G) level, and kidney graft outcome in the Slovak Caucasian population. Methods: We investigated 69 kidney transplant recipients (45 males, 24 females) of age 27-65 years. Out of this group, 37 recipients developed acute rejection that was biopsy proven. Recipient's plasma was obtained at 1 day before transplantation and analyzed by ELISA. The HLA-G 3'UTR polymorphisms were typed by direct sequencing. Results: In the recipients with stable allograft function, significantly higher values of sHLA-G were found in the homozygous +3010GG, +3142CC, +3187GG, and +3196CC carriers in comparison to the acute rejection recipients (P = 0.01-0.05). Conclusion: The study demonstrated genetic association between HLA-G 3'UTR variants and sHLA-G level in kidney recipients leading to graft acceptance. We suggest to monitor the pretransplantation sHLA-G level as additional marker to predict kidney graft outcome. Abbreviations: AMR: Antibody-mediated rejection; APC: antigen-presenting cell; CD: cluster of designation; del: deletion; HLA: human leukocyte antigen; ILT: immunoglobulin-like transcript; ins: insertion; KIR: killer-cell immunoglobulin-like receptor; NK: natural killer; sHLA-G: soluble HLA-G; SNP: single nucleotide polymorphism; TCMR: T cell-mediated rejection; URR: upstream regulatory region; UTR: untranslated region.

Expression of HLA-G transcripts in graft biopsy samples of renal transplant recipients.

BACKGROUND: The HLA-G molecule has a high potential to modulate immune response towards the improvement of graft survival after transplantation. In this work, we have analyzed the total HLA-G mRNA expression in graft tissues of dysfunctional transplanted kidneys. MATERIAL AND METHODS: We examined 84 kidney biopsy samples obtained from 65 renal transplant recipients with dysfunctional graft (50 males, 15 females; average age 46.8 ± 11.9 years). 52 specimens were with signs of acute rejection and 32 without any rejection characteristics (diagnosed as glomerulonephritis, ATN and IFTA). Patients with acute rejection were divided into three groups: antibody-mediated rejection (AMR; n = 23), T cell mediated rejection (TCMR; n = 16) and combined antibody and T cell-mediated rejection (AMR + TCMR; n=13). The biopsy samples were taken from a dysfunctional graft at different time periods after kidney transplantation. The relative expression of total HLA-G mRNA in biopsy specimens was determined by real time RT-PCR. The correlation between HLA-G mRNA expression and dysfunctional graft state was investigated. The impact of different factors (post-transplantation interval, gender,mismatch, induction therapy and cold ischemia time) on relative expression of total HLA-G mRNA was also studied. RESULTS: We have found that the levels of HLA-G transcripts in kidneys with rejection were higher than those in non-rejected but dysfunctional grafts (P = 0.0003). The highest levels of HLA-G mRNA were detected at combined AMR + TCMR rejection (P= 0.005). The time-course analysis of total HLA-G mRNA expression was also studied. In both dysfunctional graft groups (rejected and non-rejected) the lower levels of HLA-G transcripts were detected during early post-transplant period (1­3 months), however a substantial increase of HLA-G mRNA expression was observed after an extended period of time(N3 months). It was also revealed that antibody induction therapy may reduce HLA-G expression (P=0.0004) and in female samples were higher levels of HLAG transcripts than those in male recipients (P=0.003). It was found no significant impact of age, cold ischemic time, PRA (Panel Reactive Antibody) score, and a number of HLA-mismatches on HLA-G mRNA expression. CONCLUSIONS: We have demonstrated that the expression of total HLA-GmRNA in renal grafts can be influenced by different factors such as clinical state of transplanted kidney, elapsed time after transplantation, gender and antibody induction therapy. We have proved that HLA-G mRNA expression was significantly higher in recipients with acute rejection in comparison to patients with dysfunctional but non-rejected grafts.

A dermocosmetic containing bakuchiol, Ginkgo biloba extract and mannitol improves the efficacy of adapalene in patients with acne vulgaris: result from a controlled randomized trial.

BACKGROUND: Acne vulgaris is an inflammatory disorder of the pilosebaceous unit. AIM: To confirm that BGM (bakuchiol, Ginkgo biloba extract, and mannitol) complex increases the established clinical efficacy of adapalene 0.1% gel in patients with acne. METHODS: A clinical trial was conducted in acne patients. A total of 111 subjects received adapalene 0.1% gel and BGM complex or vehicle cream for 2 months. Assessments comprised Investigator Global Assessment (IGA), global efficacy, seborrhea intensity, inflammatory and non-inflammatory lesions, and subject perception, as well as overall safety and local tolerance and quality of life. RESULTS: At the end of the trial, inflammatory and non-inflammatory lesions, IGA, global efficacy, and seborrhea intensity had significantly improved in both treatment groups. Differences were statistically significant (P<0.05) in favor of BGM complex for inflammatory lesions as well as IGA and seborrhea intensity. Global efficacy assessments and subject perception confirmed the superiority of BGM complex-including treatment over the comparative combination. Quality of life had improved more with the active combination than with the vehicle combination. In the active group, four subjects had to interrupt temporarily BGM complex and 12 adapalene compared to seven subjects interrupting the vehicle and eleven adapalene in the vehicle group. One subject withdrew from the trial due to an allergy to adapalene. The majority of all events were mild. CONCLUSION: BGM complex improves the treatment outcome of adapalene 0.1% gel in patients with acne vulgaris. Overall, safety and local tolerance of BGM complex were good.

Genetically engineered mesenchymal stromal cells producing TNFα have tumour suppressing effect on human melanoma xenograft.

BACKGROUND: Mesenchymal stromal cells (MSC) are a promising tool for targeted cancer therapy due to their tumour-homing ability. Intrinsic resistance enables the MSC to longer tolerate therapeutic factors, such as prodrug converting enzymes, cytokines and pro-apoptotic proteins. Tumour necrosis factor alpha (TNFα) is known to be cytotoxic to a variety of cancer cells and exert a tumour-destructive capacity. METHODS: MSC were retrovirally transduced to stable express an exogenous gene encoding the desired therapeutic agent hTNFα. The effect of a TNFα-producing adipose tissue-derived MSC (AT-MSC/hTNFα) was tested on the tumour cell lines of different origins: melanoma (A375), breast carcinoma (SKBR3, MDA-MB-231), colon carcinoma (HT29), ovarian carcinoma (SKOV3) and glioblastoma (U87-MG) cells. The tumour suppressing effect of AT-MSC/hTNFα on A375 melanoma xenografts was monitored in an immunodeficient mouse model in vivo. RESULTS: Engineered AT-MSC are able to constitutively secrete human TNFα protein, induce apoptosis of tumour cell lines via caspase 3/7 activation and inhibit the tumour cell proliferation in vitro. Melanoma A375 and breast carcinoma SKBR3 cells were the most sensitive, and their proliferation in vitro was reduced by conditioned media produced by AT-MSC/hTNFα to 60% and 40%, respectively. The previously reported tumour supportive effect of AT-MSC on subcutaneous A375 melanoma xenograft growth was neutralised and suppressed by engineered AT-MSC stably producing hTNFα. When AT-MSC/hTNFα were coinjected with A375 melanoma cells, the tumour mass inhibition was up to 97.5%. CONCLUSIONS: The results of the present study demonstrate that tumour cells respond to hTNFα-based treatment mediated by genetically engineered AT-MSC/hTNFα both in vitro and in vivo.

HLA-G5 in the blood of leukemia patients and healthy individuals.

In this work we focused on analysis of HLA-G5 molecules in the blood of patients with B-CLL leukemia and healthy individuals. Using sandwich ELISA, we found total soluble HLA-G, represented by sHLA-G1 and HLA-G5 in most of B-CLL patients while HLA-G5 alone was present only in few cases in both groups. These results lead us to assume that the majority of soluble HLA-G in blood is generated by proteolytic cleavage and shedding of membrane-bound HLA-G1. There is no correlation between the presence of HLA-G5 in blood of B-CLL patients and the stage of disease, age, and gender.

MR enterography with a new negative oral contrast solution containing maghemite nanoparticles.

AIM: The aim of this study was to test an oral contrast solution with maghemite for the magnetic resonance imaging of small bowel diseases. PATIENTS AND METHODS: The study sample included 3 cohorts: 17 healthy volunteers (group A), 22 patients with small bowel disease (group C). Both groups underwent MR enterography and 24 patients with small bowel disease (group B) underwent magnetic resonance cholecystopancreaticography. Various concentrations in 1000 ml vs 500 ml of experimental solution were tested. All cohorts completed questionnaires evaluating the solution characteristics and side-efects during and after drinking. RESULTS: A maghemite concentration of 800 mg /4 g bentonite in 1000 ml solution was sufficient for proper intraluminal lay-out. An experimental solution of 500 ml was sufficient for magnetic resonance cholecystopancreaticography and 1000 ml for MR enterography. There were no statistically significant differences between groups for taste, taste characteristic or appearance of the experimental solution. Side-effects experienced during drinking were: nausea (29.4%) and eructation (29.4%) in group A, in group B (42%) and diarrhoea (27.3%) in group C. Side-effects 2 h after drinking occured in group A (nausea 17.6%) and in group C (diarrhoea 47%). The best tolerance of experimental solution was found in group B with a higher median patient age than groups A and C. The experimental solution was evaluated more favorably in the older subjects (age over 50 years). CONCLUSION: The experimental oral solution with maghemite was well tolerated in all 3 groups. Our study supports its use in magnetic resonance practice.

A DNA vaccine expressing PB1 protein of influenza A virus protects mice against virus infection.

Although influenza DNA vaccine research has focused mainly on viral hemagglutinin and has led to promising results, other virion proteins have also shown some protective potential. In this work, we explored the potential of a DNA vaccine based on the PB1 protein to protect BALB/c mice against lethal influenza A virus infection. The DNA vaccine consisted of pTriEx4 plasmid expressing PB1. As a positive control, a pTriEx4 plasmid expressing influenza A virus HA was used. Two weeks after three subcutaneous doses of DNA vaccine, the mice were challenged intranasally with 1 LD50 of A/Puerto Rico/8/34 (H1N1) virus, and PB1- and HA-specific antibodies, survival rate, body weight change, viral mRNA load, infectious virus titer in the lungs, cytokines IL-2, IL-4 and IL-10, and granzyme-B were measured. The results showed that (i) the PB1-expressing DNA vaccine provided a fair protective immunity in the mouse model and (ii) viral structural proteins such as PB1 represent promising antigens for DNA vaccination against influenza A.

Antibodies to PB1-F2 protein are induced in response to influenza A virus infection.

PB1-F2 is a small influenza A virus (IAV) protein encoded by an alternative (+1) reading frame of the PB1 gene. While dispensable for IAV replication in cultured cells, PB1-F2 has been implicated in IAV pathogenicity. To better understand PB1-F2 expression in vivo and its immunogenicity, we analyzed anti-PB1-F2 antibodies (Abs) in sera of mice infected intranasally (i.n.) with A/PR/8/34 (H1N1) virus and human acute and convalescent sera collected from the influenza H3N2 winter 2003-2004 epidemic. We explored a number of methods for detecting anti-PB1-F2 Abs, finding that PB1-F2-specific Abs could clearly be detected via immunoprecipitation or immunofluorescence assays using both immune mouse and human convalescent sera. Importantly, paired human sera exhibited similar increases in HI titers and PB1-F2-specific Abs. This study indicates that PB1-F2 is expressed in sufficient quantities in mice and humans infected with IAV to elicit an Ab response, supporting the biological relevance of this intriguing accessory protein.

The 4H84 monoclonal antibody detecting beta2m free nonclassical HLA-G molecules also binds to free heavy chains of classical HLA class I antigens present on activated lymphocytes.

We have found that 4H84 monoclonal antibody (mAb) used for detection of beta2m free HLA-G molecules also binds to free heavy chains of classical HLA class I antigens generated on the cell surface by mild acid treatment. Here we demonstrate that beta2m free classical HLA class I molecules induced on the surface of activated lymphocytes not expressing HLA-G also bind 4H84 mAb. These results demonstrate that 4H84 mAb should be used for detection of HLA-G in cells and tissues with backing by other HLA-G specific mAbs.

Development of a quantitative cell-based intracellular ELISA for the screening of B cell hybridoma supernatants: a novel rapid assay to detect positive clones.

The primary screening of hybridoma clones secreting monoclonal antibodies (MAbs) requires the testing of a large number of hybridoma culture supernatants within a short time and is very labor-intensive. In addition, the type of antigen and its location in the cell have to be considered when selecting the appropriate screening procedure, but relatively few reagents are available for analyzing these molecules. We have developed an intracellular and cell surface ELISA technique for screening hybridoma supernatants that hastens the screening procedure of a large number of clones in a short period of time, as the supernatants of fused cells grown in 96-well plates are used directly in the assay. This novel screening technique is rapid, sensitive, specific, and applicable to MAbs specific for a wide variety of intracellular and/or cell surface proteins.

Analysis of HLA-G expression in malignant hematopoetic cells from leukemia patients.

It has been suggested that HLA-G antigens may provide tumor cells with an effective immune escape mechanism. So far mostly solid tumors have been analyzed; HLA-G antigen was only exceptionally detected. To further examine HLA-G expression, patients were chosen with different forms of leukemia: AML (25), CML (4), ALL (9), CLL (8), HCL (2) and NHL (3). Using flow cytometry with three HLA-G specific mAbs (87G, 01G and MEM-G/9), western blotting with two specific mAbs (4H84 and MEM-G/1) and RT-PCR, neither HLA-G antigen nor mRNA for any HLA-G isoform was detected. These results strongly suggest that HLA-G antigen is not expressed in freshly isolated human leukemia cells and therefore is not involved in their escape from immune attack.

Mild acid treatment induces cross-reactivity of 4H84 monoclonal antibody specific to nonclassical HLA-G antigen with classical HLA class I molecules.

Mild acid treatment by releasing beta(2)m and antigenic peptides leaves human leukocyte antigen (HLA) class I free heavy chains attached to the cell surface. Acid treatment thus allows detection of the cell surface class I antigens by monoclonal antibodies (mAbs) specific to HLA-free heavy chains. We found that acid treatment also enables detection of the cell surface non-classical HLA-G class I antigen with mAbs specific for HLA-G free heavy chains, including 4H84 mAb recognizing all isoforms. Furthermore, we found that 4H84 mAb, but not other mAbs specific to HLA-G free heavy chains, binds to the surface of 8 out of 16 acid-treated leukemia cell lines. Nevertheless, HLA-G antigen is not present in any of these leukemia cells. This was demonstrated by failure to detect any antigen with 4H84 mAb in immunoblotting as well as by inability to detect HLA-G mRNA by RT-PCR. The antigen recognized by 4H84 mAb in some acid treated leukemia cells was identified by immunoprecipitation as a 45 kDa protein. A number of observations indicate that 45 kDa proteins are none other than classical class I heavy chains. Acid treatment thus induces the ability of the 4H84 mAb to recognize some classical HLA class I molecules. Remarkably, 4H84 determinant on HLA-G is linear but corresponding determinant present on some partially folded classical HLA class I free heavy chains is conformational. In view of the unexpected cross-reactivity, detection of HLA-G with this mAb must be carefully evaluated to avoid false detection.