Quantification of Coffea arabica and Coffea canephora var. robusta concentration in blends by means of synchronous fluorescence and UV-Vis spectroscopies.

The potential of fluorescence, UV-Vis spectroscopies as well as the low- and mid-level data fusion of both spectroscopies for the quantification of concentrations of roasted Coffea arabica and Coffea canephora var. robusta in coffee blends was investigated. Principal component analysis was used to reduce data multidimensionality. To calculate the level of undeclared addition, multiple linear regression (PCA-MLR) models were used with lowest root mean square error of calibration (RMSEC) of 3.6% and root mean square error of cross-validation (RMSECV) of 7.9%. LDA analysis was applied to fluorescence intensities and UV spectra of Coffea arabica, canephora samples, and their mixtures in order to examine classification ability. The best performance of PCA-LDA analysis was observed for data fusion of UV and fluorescence intensity measurements at wavelength interval of 60nm. LDA showed that data fusion can achieve over 96% of correct classifications (sensitivity) in the test set and 100% of correct classifications in the training set, with low-level data fusion. The corresponding results for individual spectroscopies ranged from 90% (UV-Vis spectroscopy) to 77% (synchronous fluorescence) in the test set, and from 93% to 97% in the training set. The results demonstrate that fluorescence, UV, and visible spectroscopies complement each other, giving a complementary effect for the quantification of roasted Coffea arabica and Coffea canephora var. robusta concentration in blends.

GRP78-targeting subtilase cytotoxin sensitizes cancer cells to photodynamic therapy.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER)-resident chaperone and a major regulator of the unfolded protein response (UPR). Accumulating evidence indicate that GRP78 is overexpressed in many cancer cell lines, and contributes to the invasion and metastasis in many human tumors. Besides, GRP78 upregulation is detected in response to different ER stress-inducing anticancer therapies, including photodynamic therapy (PDT). This study demonstrates that GRP78 mRNA and protein levels are elevated in response to PDT in various cancer cell lines. Stable overexpression of GRP78 confers resistance to PDT substantiating its cytoprotective role. Moreover, GRP78-targeting subtilase cytotoxin catalytic subunit fused with epidermal growth factor (EGF-SubA) sensitizes various cancer cells to Photofrin-mediated PDT. The combination treatment is cytotoxic to apoptosis-competent SW-900 lung cancer cells, as well as to Bax-deficient and apoptosis-resistant DU-145 prostate cancer cells. In these cells, PDT and EGF-SubA cytotoxin induce protein kinase R-like ER kinase and inositol-requiring enzyme 1 branches of UPR and also increase the level of C/EBP (CCAAT/enhancer-binding protein) homologous protein, an ER stress-associated apoptosis-promoting transcription factor. Although some apoptotic events such as disruption of mitochondrial membrane and caspase activation are detected after PDT, there is no phosphatidylserine plasma membrane externalization or DNA fragmentation, suggesting that in DU-145 cells the late apoptotic events are missing. Moreover, in SW-900 cells, EGF-SubA cytotoxin potentiates PDT-mediated cell death but attenuates PDT-induced apoptosis. In addition, the cell death cannot be reversed by caspase inhibitor z-VAD, confirming that apoptosis is not a major cell death mode triggered by the combination therapy. Moreover, no typical features of necrotic or autophagic cell death are recognized. Instead, an extensive cellular vacuolation of ER origin is observed. Altogether, these findings indicate that PDT and GRP78-targeting cytotoxin treatment can efficiently kill cancer cells independent on their apoptotic competence and triggers an atypical, non-apoptotic cell death.

Application of proteomic methods for identification of sperm immunogenic antigens.

Although the immunogenic properties of sperm have been explored for a few decades, none of the antigens studied so far appears to be an effective target, to inhibit the fertilization process or shown the full spectrum of sperm antigenic potential. Antisperm antibodies (ASA) collected from infertile individuals and prepubertal boys with cryptorchidism together with two-dimensional (2D) electrophoresis have been employed. Immunoreactive antigens were cored from silver stained 2D gels and analyzed by mass spectrometry (MS). The obtained sequences were searched in the published protein databases. Altogether, 35 different sperm entities were identified in accessible protein databases, out of which 10 appeared to be sperm-specific. Additionally, 6 amino acid sequences indicated novel (hypothetical) proteins. Seventeen sperm entities were detected in sera samples from immune infertile males and 18 entities in ASA-positive seminal plasma (SP). Interestingly, we identified a few sperm structures, none of them sperm specific in sera samples from infertile females. Although, infertile males from whom the ASA-positive SP samples were obtained, did not have ASA in their circulation, the range of sperm antigens detected by systematic and local antibodies overlapped to a great extent (six identical entities). Sera samples from prepubertal boys allowed to show antigens, previously thought to be only present on mature sperm. Three out of four detected were sperm-specific. Using serum and SP of ASA-positive infertile adults and sera samples of prepubertal boys with testicular failure, we have extended the range of known, immunogenic sperm proteins as well as identified some novel antigens (n=6) of human sperm for further characterization.

Complex nature of the human antisperm antibody response in SCID mice.

Human peripheral blood mononuclear (PBMs) cells were introduced into the peritoneal cavity of severely-combined immunodeficient (SCID) mice in concentrations of 2.5-4.0 x 10(7) cells per mouse. Whole mononuclear cell suspensions were used either unstimulated or following primary in vitro culture with human spermatozoa. In some experiments, immunodepletion of CD8(+) cells was carried out prior to grafting. Lymphocytes were obtained from nonsensitized (to antigen) human subjects or from individuals who were primed in vivo (vasectomized individuals in case of sperm antigens). An enzyme-linked immunosorbent assay was employed to assess total human immunoglobulin (G or M) levels as well as the specificity of the antibodies generated. We have been successful by generating primary and secondary immune responses with 'naïve' human lymphocytes, challenged with chlamydia or ovalbumin but without adjuvant or CD8(+) immunodepletion; however, we were unable to induce specific antibodies to spermatozoa under this regime in SCID male mice. We then employed female SCID mice, treated with sperm antigen extracts (glycosylated or deglycosylated) encapsulated in liposomes and human lymphocytes obtained from 'naïve' or pre-sensitized in vivo subjects. It was found that the most pronounced humoral response to sperm antigens was obtained with deglycosylated antigens and PBMs from vasectomized (in vivo pre-primed to spermatozoa) individuals. A presented SCID mice model can be helpful at understanding of antisperm antibody development and the molecular nature of generated antibodies to modified sperm antigenic entities.

CD52 antigen--a review.

Human CD52 is a glycosylphosphatidylinositol (GPI)--anchored antigen expressed on the all lymphocytes as well as within the male genital tract, where it can be produced by epithelial cells of the distal epididymis and duct deferens. CD52 can be shed into seminal plasma and then acquired by sperm cells during their passage through the genital tract, thus it is detectable on the surface of epididymal sperm and in the ejaculate but not on either spermatogenetic cells or testicular spermatozoa. Protein core of the sperm and lymphocyte CD52 is identical and it is a product of a single copy gene located on chromosome 1. However, N-linked carbohydrate side chains (attached to Asn-3) and GPI-anchor structure are different. Physiological role of CD52 on lymphocytes is unclear, although antibody directed to CD52, CAMPATH-1, is capable of complement activation and it can be clinically useful to treat lympho-proliferative disorders and to deplete lymphocytes in bone marrow transplants. Monoclonal antibodies directed against sperm CD52 may cause sperm immobilisation and agglutination and affect hamster oocyte penetration suggesting an association of this molecule with fertilisation.

[Reconstruction of humoral response to sperm antigens in scid mice]. / Rekonstrukcja in situ ludzkiej odpowiedzi humoralnej w myszach z uposledzona odpornoscia wobec mieszaniny antygenów plemnikowych.

OBJECTIVE AND DESIGN: Production of specific human antisperm antibodies by using human-SCID mice model with deposited peripheral blood lymphocytes. MATERIALS AND METHODS: Human peripheral blood lymphocytes (PBL's; CD8(+)-negative cell fraction) were grafted to the peritoneal cavity of severely-combined immunodeficient (SCID) mice at concentration of 20-35 x 10(6) cells per mouse. Lymphocytes were obtained from non-sensitized individual (to sperm antigen) and from in vivo primed males (vasectomized). Two sets of experiments were carried out, with 'native' (glycosylated) and enzymatically deglycosylated sperm antigenic extracts. In all applied variants, sperm antigens were administered with Complete and then with Incomplete Freund adjuvant to improve an immune response. RESULTS AND CONCLUSION: This approach allowed us to obtain better pronounced humoral antisperm response, specific to sperm deglycosylated antigens when PBL's were obtained from individuals in vivo sensitized to sperm (after vasectomy).

Sperm antigens recognized by antisperm antibodies present in sera of infertile adults and prepubertal boys with testicular failure.

Immunoblotting of a repertoire of sperm antigens reacting with antisperm antibodies present in sera of infertile adults and prepubertal boys with testicular failure was performed. In the subgroups selected for this study, 55% of examined infertile women, 65% of infertile men and 64% of prepubertal boys with gonadal failure gave positive results by Western blotting with extracted sperm antigens. Sperm antigens with molecular weights of 57, 58, 62, 63 and 66 kDa were the most immunodominant entities recognized by antisperm antibodies from prepubertal boys. No positive reactions were detected by Western blotting in a control population of fertile adults, whereas in a group of prepubertal healthy boys only one sample revealed reactivity against sperm antigens of 58 and 70 kDa.

Antisperm antibodies in prepubertal boys and their reactivity with antigenic determinants on differentiated spermatozoa.

PROBLEM: Antisperm antibodies induced in prepubertal boys with testicular failures were characterized by using four techniques of antibody detection. The reactivity of circulating antisperm antibodies in prepubertal boys and the reactivity of antibodies in sera samples of adult fertile and infertile males were compared against the same sperm antigenic pools (live or fixed spermatozoa, or sperm antigenic extracts). METHOD OF STUDY: The incidence of antisperm antibodies in sera samples of 69 prepubertal boys with testicular failures and 21 samples obtained from adult, male individuals was assessed by indirect immunobead binding test (IDIBT), flow cytometry measurement, enzyme-linked immunosorbent assay, and Western blotting. Immunoblot analysis was performed by using sperm extracts of glycosylated and deglycosylated solubilized membrane antigens. RESULTS: Sera samples were studied in a group composed of healthy prepubertal boys (n = 7) and prepubertal boys with testicular failures (n = 69). Applied tests of antibody detection revealed striking differences in a group of boys with testicular pathology. With IDIBT, 7% of the sera samples were found positive, whereas with flow cytometry measurement, 48% of the sera samples were positive. Immunosorbent assay (fixed sperm) indicated 32% positive cases in the same group. The sera samples were found to be positive in 65% of immunoblotting reactions with glycosylated antigens and in 70% of immunoblotting reactions with deglycosylated antigens. All applied detection assays were clearly negative on sera samples from fertile, adult males. Western immunoblotting indicated an immunodominant antigenic determinant of 58 kDa. CONCLUSIONS: Tests of antibody detection with the use of live sperm (IDIBT and flow cytometry measurements) presented low sensitivity (8% and 48%, respectively) in a group of prepubertal boys. This observation underlines the difficulties in assigning the prospective prognosis of future fertility status in prepubertal boys with antisperm antibodies.

Immunological characteristics of cervical mucus in infertile women.

The presence of local or circulating antisperm antibodies (ASA) in some women may cause infertility. We have examined 43 cervical mucus (CM) samples. In order to determine the total amount of immunoglobulins of individual subclasses we used the rocket immunoelectrophoresis of Laurell. To determine the specificity of the immunoglobulins to sperm surface antigens we employed the immunobead-binding test (IBT). Seventeen CM samples (39.5%) revealed high levels (> 0.22 microgram/microliter) of immunoglobulins. They were mainly of the IgA class, however IgG's were also present. We have found IgM in only two test samples (4.6%). Only two CM samples (4.6%) yielded positive results in indirect IBT. The obtained results show a complex immunological status of the human uterine cervix. Immunological response developed in this compartment may be directed against sperms as well as other antigens.

[Immunologic response against spermatozoa in the human genital tract]. / Odpowiedz immunologiczna przeciw plemnikom w ukladzie rozrodczym czlowieka.

Spermatozoa are immunogenic and may induce an immune response in reproductive compartments. There have been controversies concerning the effect of naturally existing antisperm antibodies on fertility of males and females. It is known, that men or women suffering from unexplained infertility have often antisperm antibodies in their urogenital secretions and/or serum. Systemic and local immune responses to sperm antigens differ between each other and they may differently affect the fertilisation process. Physiologically, females are protected from an immune response against "foreign", spermatozoal antigens. Nevertheless under local pathological conditions antisperm antibodies can be induced and may interfere with fertilisation.

[Immunologic reactions in the male and female reproductive system]. / Reakcje immunologiczne zachodzace w zenskim i meskim ukladzie rozrodczym.

This review describes presence and significance of antisperm antibodies (ASA) in male and female reproductive compartments. We described their relevance to infertility in humans. A structure and function of cervical mucus as the environment of females defensive reactions against all the invasive factors is outlined. There are also briefly characterized techniques of ASA detection.

Failure to demonstrate transmission of enzootic bovine leukemia virus infection from cows to sheep by use of common injection needles.

Four bovine leukemia virus (BLV)-seropositive and 2 BLV-seronegative cows were used as donors in a study to provide evidence whether IM injection with common needles is a means of spreading bovine leukemia. Sheep were used as recipients. Of the 4 BLV-seropositive cows, 2 had high virus expression (VE; 43% and 28% of their lymphocyte thin sections had associated BLV-particles), whereas the other 2 cows did not have observed VE. After each of the 4 cows was given an injection of a 5-antigen Leptospira bacterin, a BLV-seronegative sheep was immediately given an injection of the same bacterin with the same needle. None of these sheep seroconverted, nor did either of 2 sheep given only the bacterin (with a previously unused needle). Sheep inoculated IM with 0.2 ml of whole blood from both of the cows with high VE and from 1 of the 2 BLV-seropositive cows that did not have observed VE did seroconvert. In contrast, the sheep inoculated with 0.2 ml of blood from the remaining BLV-seropositive (0% VE) cow and from the 2 BLV-seronegative cows remained seronegative. These results were interpreted to indicate that the quantity of infective lymphocytes passed during injection with common needles is too small to induce infection.

In vitro viral expression as a criterion for development of control procedures for enzootic bovine leukosis.

In a university beef herd of 304 cattle in which six died of lymphosarcoma between 1980 and 1984, 77% of the Angus and 26% of the Charolais cattle were determined to be infected with bovine leukemia virus (BLV). Changes in iatrogenic procedures were initiated as early control measures. In vitro viral expression (VE) was used as a criterion to identify cattle for subsequent segregation or culling. This involved determinations of percentages of BLV-associated lymphocyte profiles among thin-sectioned Ficoll-Paque-isolated blood lymphocytes that were processed into plastic after culture for 48 h. Cattle retained until completion of nutritional studies or as breeding stock were separated into two groups. The BLV-seronegative cattle, BLV-seropositive cattle with 0% VE, and BLV-seropositive cattle with 1% to 4% VE were placed in group 1. Seropositive cattle with greater than or equal to 5% VE were placed in group 2. In 1985, evaluation of in vitro VE in 108 mature BLV-seropositive cattle retained for breeding revealed 36 (33%) had no observable VE. In 1986, 58 of 108 cattle were available to be reexamined, and 21 (36%) had 0% VE in both years. The VE expression values for individual cattle were generally comparable over the 2-year period. Of 48 initial seronegative breeding stock housed in group 1 with BLV-seropositive cattle with low or no VE, 21 (44%) seroconverted during 1985 to 1986. A positive correlation of 0.585 was found between VE and age-related absolute lymphocyte number.