Biomedical subjects

J J Gaforio

Publications and source records attributed to J J Gaforio.

At least 19 recordsLinked to original sources

PubMed2005

External quality control program for semen analysis: Spanish experience.

PURPOSE: Results from an external quality control programme for semen analysis carried out in Spain are analysed. METHODS: Quality control materials were distributed and the following seminal parameters were determined: concentration, total motility, progressive motility, rapid progressive motility, morphology and sperm vitality. The between-laboratories coefficients of variation were assessed on different types of quality control material. RESULTS: The majority of participating laboratories utilised manual versus computer-assisted semen analysis methods. Some between-laboratories coefficients of variation ranges were: 20.8-33.8% for concentration (semen pool suspension); 13.9-19.2% for total motility (videotapes); 54.2-70.2% for sperm morphology (strict criteria using stained smears); and 9.8-41.1% for sperm vitality (stained smears). There was an inverse relation between mean percentage of sperm and coefficients of variation between laboratories for sperm motility, morphology and vitality. CONCLUSIONS: These data highlight the urgent need for improvement in the overall quality of andrology testing.

Clinical Laboratory Techniques↗

PubMed2005

International conference on the healthy effect of virgin olive oil.

1. Ageing represents a great concern in developed countries because the number of people involved and the pathologies related with it, like atherosclerosis, morbus Parkinson, Alzheimer's disease, vascular dementia, cognitive decline, diabetes and cancer. 2. Epidemiological studies suggest that a Mediterranean diet (which is rich in virgin olive oil) decreases the risk of cardiovascular disease. 3. The Mediterranean diet, rich in virgin olive oil, improves the major risk factors for cardiovascular disease, such as the lipoprotein profile, blood pressure, glucose metabolism and antithrombotic profile. Endothelial function, inflammation and oxidative stress are also positively modulated. Some of these effects are attributed to minor components of virgin olive oil. Therefore, the definition of the Mediterranean diet should include virgin olive oil. 4. Different observational studies conducted in humans have shown that the intake of monounsaturated fat may be protective against age-related cognitive decline and Alzheimer's disease. 5. Microconstituents from virgin olive oil are bioavailable in humans and have shown antioxidant properties and capacity to improve endothelial function. Furthermore they are also able to modify the haemostasis, showing antithrombotic properties. 6. In countries where the populations fulfilled a typical Mediterranean diet, such as Spain, Greece and Italy, where virgin olive oil is the principal source of fat, cancer incidence rates are lower than in northern European countries. 7. The protective effect of virgin olive oil can be most important in the first decades of life, which suggests that the dietetic benefit of virgin olive oil intake should be initiated before puberty, and maintained through life. 8. The more recent studies consistently support that the Mediterranean diet, based in virgin olive oil, is compatible with a healthier ageing and increased longevity. However, despite the significant advances of the recent years, the final proof about the specific mechanisms and contributing role of the different components of virgin olive oil to its beneficial effects requires further investigations.

Aging↗

PubMed2003

Biological variation of seminal parameters in healthy subjects.

BACKGROUND: A study was undertaken to assess the components of biological variation of seminal parameters in healthy subjects. METHODS: Twenty donor candidates were included in a 10-week follow-up study. Within- and between-subject biological variation, indices of individuality and heterogeneity, coefficient of reliability, critical differences, analytical goals and the lowest value observed with a <5% probability of having a true value less than the World Health Organization (1999) reference value were estimated for the following seminal parameters: concentration, total motility (WHO grades a + b + c), progressive motility (grades a + b), rapid progressive motility (grade a), sperm morphology and vitality. All analysis was performed by a single technician according to WHO 1999 guidelines for routine semen analysis. Analytical variation was assessed on different types of quality control material (frozen straws, sperm suspension, videotape, and slides) and at different (low, medium, high) quality levels. RESULTS: The analytical variation observed depended on the quality control material used and the level of semen quality. Concentration was the semen parameter with highest within- and between-subject variation, and vitality the lowest. Indices of individuality were all <0.7, and coefficients of reliability were high (0.68-0.84). The critical difference for sequential values significant at P < 0.05 for vitality, progressive motility and morphology (34.4, 49.2 and 58.0% respectively) were lower than for concentration (77.8%). CONCLUSIONS: The study results showed that conventional reference values for seminal parameters have little diagnostic value because of their marked individuality, though seminal parameters can be useful for assessing differences in an individual's serial results, in particular of progressive motility, morphology and vitality.

Adult↗

PubMed2002

Phagocytosis of apoptotic cells assessed by flow cytometry using 7-Aminoactinomycin D.

BACKGROUND: Apoptotic cells are recognized specifically by macrophages and are cleared rapidly by phagocytosis. However, the recognition mechanisms involved in the clearance of apoptotic cells by macrophages are still not fully understood. Therefore, new methods must be designed to better our understanding of the mechanisms of interaction between macrophages and apoptotic cells. 7-Aminoactinomycin D (7-AAD) is a fluorescent DNA-binding stain usually used as a single agent to detect apoptotic cells by flow cytometry. We propose the use of 7-AAD-stained apoptotic cells as targets for a new flow cytometry phagocytosis assay. METHODS: Murine T-cell lymphoma YAC-1 cells were treated with etoposide to induce apoptosis. Etoposide-treated YAC-1 target cells were stained subsequently with 7-AAD and then coincubated with resident peritoneal macrophages to allow phagocytosis. The samples were analyzed by flow cytometry. Macrophages that had phagocytosed 7-AAD-stained apoptotic cells were identified by their bright red fluorescence and the resulting values were expressed as the percentage of cells. RESULTS: The phagocytic cells appeared as a distinct population characterized by bright fluorescence, which could not be detected in the negative controls. The effects of a phagocytic enhancer (interferon-gamma [IFN-gamma]) or inhibitor (incubation at 4 degrees C) were assessed accurately with this flow cytometric method. CONCLUSIONS: We describe the use of 7-AAD in an assay that is easy and quick to perform. This flow cytometric-based assay allows the quantification of phagocytosis of apoptotic cells by macrophages.

Animals↗

PubMed2002

Suppression of splenic macrophage Candida albicans phagocytosis following in vivo depletion of natural killer cells in immunocompetent BALB/c mice and T-cell-deficient nude mice.

The resistance of mice to systemic infections caused by Candida albicans is associated with activated splenic macrophages. In addition, there is a correlation between natural killer (NK) cell activation and the resistance to systemic candidiasis. The present study was designed to clarify the role of NK cells in the control of splenic macrophage C. albicans phagocytosis by either depleting NK cells (anti-asialo GM(1) treatment) or maintaining them in an activated state (tilorone treatment) in both immunocompetent BALB/c mice and T-cell-deficient nude mice. The results of the in vitro phagocytosis assays were analyzed by flow cytometry and demonstrate the pivotal role of NK cells in controlling the capacity of splenic macrophages to phagocytose C. albicans. In summary, these data provide evidence that the NK cells are the main inducers of phagocytic activity of splenic macrophages and that they mediate the protection against C. albicans systemic infection.

Animals↗

PubMed2002

Use of SYTOX green dye in the flow cytometric analysis of bacterial phagocytosis.

BACKGROUND: Fluorescein isothiocyanate (FITC) is used widely to label the targets used in flow cytometric phagocytosis assays. Unfortunately, the fluorescence intensity of phagocytosed FITC-labeled targets is influenced by changes in intracellular pH level, making quantitative measurements with this fluorophore problematic. We describe the use of SYTOX green nucleic acid stain to measure phagocytosis by flow cytometry. METHODS: Suspensions of isopropyl alcohol-permeabilized Escherichia coli DH5alpha were stained with the SYTOX green dye and then incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytosis was determined by gating the cells. RESULTS: Results are expressed as percentage of phagocyte-associated green fluorescent cells. The validity of the method was shown by the effects of a phagocytosis inhibitor (incubation at 4 degrees C) or enhancer (gamma interferon [IFN- gamma] treatment) being accurately assessed with this assay. CONCLUSIONS: The method described was reproducible and provides an advantageous alternative to the use of FITC to label bacteria for the flow cytometric measurement of target uptake by phagocytic cells.

Escherichia coli↗

PubMed2001

The use of 7-amino-actinomycin D in the analysis of Candida albicans phagocytosis and opsonization.

We describe the use of 7-amino-actinomycin D (7AAD) to measure phagocytosis and the opsonizing capacity of serum. Heat-inactivated Candida albicans was previously stained with 7AAD and incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytic cells were identified by their bright red fluorescence. This is a rapid, reproducible and reliable one-step procedure and provides a means of evaluating low levels of phagocytosis.

Animals↗

PubMed2001

Immunoselection by T lymphocytes generates repeated MHC class I-deficient metastatic tumor variants.

Alteration of MHC class I molecule expression is a widespread mechanism used by tumor cells to evade T cell responses. It has long been proposed that the origin of these MHC class I-negative or -deficient tumor variants is T cell immune selection. However, there are no experimental or clinical data to substantiate this hypothesis, and this issue is currently the subject of debate. Here we report that an H-2 class I-negative fibrosarcoma tumor clone generated MHC class I-negative spontaneous lung metastases in immunocompetent syngeneic BALB/c mice. Interestingly, the same B9 clone generated MHC class I-positive metastatic nodes, under basal conditions, in athymic nu/nu BALB/c mice. This phenomenon was observed in the metastatic nodules generated after a period of in vivo growth but not in the primary tumors growing locally in the footpad. These findings support the hypothesis that the H-2 phenotype of metastatic nodes is influenced by the T cell repertoire of the host, since in the absence of this T cell pressure (i.e., in nude mice) the metastatic nodes 'recovered' H-2 class I expression. In addition, 2 different phenotypes were found when the metastatic nodules obtained from immunocompetent mice were treated with IFN-gamma. One phenotype, present in 83% of the colonies, was characterized by resistance of the Ld molecule to IFN-gamma induction, due to a deletion involving the Ld gene. The second phenotype (17% of the colonies) was similar to the original B9 clone and was characterized by the response of K, D and L class I genes to IFN-gamma. These data provide evidence that the changes in MHC class I expression during tumor development might not be random but could be predictable.

Animals↗

PubMed2000

Modification of acquired immunity in BALB/c mice by aztreonam.

Recent studies have suggested that antibiotics may act as biological response modifiers. In this study we investigated the effect of aztreonam, a monobactam antibiotic, on different parameters of acquired immunity in BALB/c mice. Different dosages of aztreonam injected into mice induced an increase in the lymphoproliferative response to specific mitogens and in the production of interleukin-2 by splenic cells, as well as a decreased response of this immune population to sheep erythrocytes lower total blood cell counts and a lower percentage of monocytes than in untreated mice. These results show a modulatory action of aztreonam on different immune parameters, which is independent of its antimicrobial activity and that could be of interest in human therapy.

Animals↗

PubMed2000

Determination of natural resistance of mice fed dietary lipids to experimental infection induced by Listeria monocytogenes.

Current understanding based on the effect of dietary lipid manipulation upon immune system function indicates that fatty acids are involved in the modulation of the immune response through different and complex pathways. Reduction of several immune parameters by fatty acid action may be applied in the treatment of diseases characterised by an overactivation of the immune system. As a consequence, a reduction of host resistance against infectious agents has been reported in animals fed dietary lipids. The present study confirms the action of dietary lipids on the survival of mice infected with the pathogenic bacterium Listeria monocytogenes. A significant increase in peritoneal cells from mice fed a hydrogenated coconut oil diet was found, while a significant reduction of bacterial recovery from spleens of these mice was observed in this group. In addition, both eicosanoid and phospholipase inhibitors did not promote any modification of lymphocyte proliferation from mice fed olive oil or fish oil.

Animals↗

PubMed2000

Enhanced resistance to experimental systemic candidiasis in tilorone-treated mice.

Candida albicans is an increasingly important opportunistic fungal pathogen in immunocompromised patients. Natural killer (NK) cells constitute an important immune effector mechanism and are involved in the response to different pathological disorders. We wished to determine if this immune mechanism is involved in the specific response to C. albicans. Tilorone hydrochloride and related compounds have been described to display antiviral and antitumoral activity, as well as to enhance NK cell activity. In this study, we show the antimicrobial activity of different tilorone analogues and the enhanced resistance of tilorone-treated mice in experimental systemic candidiasis. We also present data suggesting that there is a correlation between NK cell activation and the resistance to experimental systemic candidiasis. Thus, it seems that the immunosurveillance of metastatic spread and the infection by C. albicans share some immune effector mechanisms, in particular activation of NK cells.

Animals↗

PubMed1999

Selective upregulation of MHC class I expression in metastatic colonies derived from tumor clones of a murine fibrosarcoma.

Eighteen metastatic nodes derived from the wild-type (GR9) and from 4 different clones (G2, D8, B10, and B9) obtained from a fibrosarcoma were analyzed for H-2 class I and II expression, as well as for adhesion molecules (CD44, CDIIb, CD18, CD49, and CD54). When metastatic nodes were cultured, typed for H-2 antigens, and compared with the H-2 expression of the inducing tumor cell, H-2 Kd and Dd class I expression was greater in most nodes analyzed. In contrast, the Ld molecule remained negative, or showed a minor increase. Class II expression was negative in the wild-type and the tumor clones, and remained so in the metastatic colonies. Analysis of the adhesion molecules revealed no differences between the inducing tumor cells and the metastatic nodes. The only molecule expressed was CD44, which was present in all cells studied and was also inducible by interferon-gamma. The increase in H-2K and H-2D expression was associated with resistance to natural killer cytotoxicity, as observed in the G2 tumor clone and some autologous metastases, such as B9MP2, G2MK2, and G2MLI. In three independent clones of this tumor system (D8, BIOMP6, and B9MP6) we found that tumor cells treated with interferon-gamma had the same altered phenotype, i.e., a selective lack of response of the Ld molecule to induction. These findings add a cautionary note to the well-established idea that tumor cells may lose all class I antigens during tumor progression, and suggest that sometimes this may not be the case. The selective downregulation of Ld and upregulation of Kd and Dd class I expression may give some tumor cells means of escaping both cytotoxic lymphocyte and natural killer immune surveillance.

Animals↗

PubMed1999

Modification of acquired immunity in mice by imipenem/cilastatin.

The immunomodulating properties of antimicrobial drugs may have important implications for clinical practice, particularly for those patients whose immune system has been compromised. In this study, we assessed the influence of different treatments with a beta-lactam antibiotic (imipenem/cilastatin) on several acquired immune responses of BALB/c mice; splenocyte responses to specific mitogens and to sheep red blood cells, IL-2 production and proportions of the different lympho-monocytic populations. Impenem/cilastatin was shown to modify some lymphocyte-associated immune functions and it would be useful to investigate whether immunomodulatory effects also occur in humans.

Animals↗

PubMed1999

Evaluation of immunomodulatory effects of nisin-containing diets on mice.

The effect of nisin on the immune response of mice was studied. Nisin (in the form of the commercial preparation Nisaplin) was incorporated in the diet of experimental mice which were fed for 30, 75 or 100 days. Short-term administration of diets containing Nisaplin induced an increase of both CD4 and CD8 T-lymphocyte cell counts and also a decrease of B-lymphocyte counts. After prolonged diet administration, T-cell counts returned to control levels. Normal levels of B-lymphocytes were also reached after prolonged administration of the lower (but not the higher) Nisaplin concentration. The macrophage/monocyte fraction isolated from peripheral blood became significantly increased after long-term administration (100 days) of Nisaplin-containing diets in a concentration-dependent way. Although the number of peritoneal cells was not affected by the diets, the phagocytic activity of peritoneal cells decreased after prolonged administration of low (but not high) Nisaplin doses.

Animals↗

PubMed1996

Effect of in vivo activation of natural killer (NK) cells by a tilorone analogue on the survival of mice injected intravenously with different experimental murine tumours.

We studied the effect of a tilorone analogue (RMI 10,874DA) and anti-asialo GM(1) serum on the survival of BALB/c and C57B1/6 mice after i.v. injections of different syngeneic murine tumour cells. Tumour lines used were different clones from chemically (GR9 wild type, GR9.B9, B7.1.B4, B7.1.B5, B7.2.38), and ultraviolet light (GRUV3)-induced sarcomas; B16 melanoma and LSTRA and YC8 lymphomas. Pretreatment of mice with tilorone inhibited metastatic colonization and increased survival significantly in all cases. In some tumour systems, the effect was attenuated when high numbers of cells were injected. Abrogation of NK cells with anti-asialo GM(1) serum significantly decreased (in all tumours and at different cell doses) survival in comparison with untreated mice injected with tumours, regardless of cell dose used. These results clearly suggest that NK cell activation in vivo by the tilorone analogue we tested prolongs survival and inhibits metastasis formation in mice, even when pretreatment consists of a single dose of the analogue.

Adjuvants, Immunologic↗

PubMed1994

Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, that codes for one of these antigens named MZ2-E. We show here that antigen MZ2-D, which is present on the same tumor, is encoded by another member of the MAGE gene family named MAGE-3. Like MAGE-1, MAGE-3 is composed of three exons and the large open reading frame is entirely located in the third exon. Its sequence shows 73% identity with MAGE-1. Like MZ2-E, antigen MZ2-D is presented by HLA-A1. The antigenic peptide of MZ2-D is a nonapeptide that is encoded by the sequence of MAGE-3 that is homologous to the MAGE-1 sequence coding for the MZ2-E peptide. Competition experiments using single Ala-substituted peptides indicated that amino acid residues Asp in position 3 and Tyr in position 9 were essential for binding of the MAGE-1 peptide to HLA-A1. Gene MAGE-3 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes. It is expressed in a larger proportion of melanoma samples than MAGE-1. MAGE-3 encoded antigens may therefore have a wide applicability for specific immunotherapy of melanoma patients.

Adult↗

PubMed1994

Structure, chromosomal localization, and expression of 12 genes of the MAGE family.

We reported previously that human gene MAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with a MAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12 MAGE genes are located in the q terminal region of chromosome X. Like MAGE-1, the 11 additional MAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that of MAGE-1. The coding sequences of the MAGE genes predict the same main structural features for all MAGE proteins. In contrast, the promoters and first exons of the 12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of each MAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of the MAGE family including MAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

Amino Acid Sequence↗

PubMed1994

In vivo activation of NK cells induces inhibition of lung colonization of H-2 positive and H-2 negative fibrosarcoma tumor clones.

The role of different tilorone analogs in the abrogation of the metastatic spread of H-2 positive and H-2 negative tumor clones was studied. Pre-treatment of BALB/c mice with RMI 10,874DA compound completely abolished lung colonization of an H-2 negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. This effect was also evident when clones were treated with other tilorone analogs (R11,567DA or R11,513DA). Other H-2 positive and H-2 negative chemically induced fibrosarcoma clones were also tested. The effect was not due to direct toxicity of the tilorone analog on tumor cells, but instead was dependent on NK cells; this was suggested by the finding that treatment of mice with anti-asialo GM1 abrogated the effect of the tilorone analog (RMI 10,874DA compound). Interestingly, the inhibition of lung colonization after intravenous injection was again observed regardless of the H-2 phenotype of the tumor clones, and H-2+ and H-2- clones were similarly inhibited. In vitro assays of NK sensitivity of tumor clones showed that lysis varied depending on the H-2 phenotype of tumor clones, indicating an absence of correlation between in vivo and in vitro results.

Animals↗