Biomedical subjects

A Facchini

Publications and source records attributed to A Facchini.

At least 109 records · Page 6Linked to original sources

PubMed1992

IgG subclass distribution of anti-HBs antibodies following vaccination with cDNA HBsAg.

Serum samples were obtained during follow-up of nine young adults vaccinated over 1 year with cDNA hepatitis B antigen. The absolute amounts of anti-HBs IgG subclass antibodies present in the sera were determined by comparing the optical densities (OD) obtained using an antigen-specific ELISA with those obtained by serial dilutions of known amounts of human IgG1-4. The calibration curves for each IgG subclass were corrected for the corresponding coating efficiency. Our data suggest that HBs antibody responses of vaccinated subjects occur in all IgG subclasses but IgG1 and IgG2 are the major subclasses involved.

Adult↗

PubMed1992

In situ hybridization in suspension and flow cytometry as a tool for the study of gene expression.

We describe a method to detect mRNA expression using in situ hybridization in suspension and flow cytometry. Our model was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in the leukemic cell line K562. A GAPDH cDNA probe was labeled with digoxigenin-11-dUTP and detected using an FITC-labeled anti-digoxigenin antiserum. We obtained good resolution in specific signals against background (GAPDH signal/control plasmid signal ratio +/- SE 3.5 +/- 0.9). The technique was optimized taking into account several hybridization variables, like fixation, hybridization time, effect of blocking agents, and stringency wash variations. This method also allowed us to quantitate the GAPDH RNA copy number/cell using a fluorescence standard; we obtained a figure of about 1200 copies/cell, which is in good agreement with the dot blot hybridization assay result. Flow cytometric analysis of in situ hybridization represents an original method to study gene expression. This technique has the potential to develop into a multiparametric tool for cell biology studies, examining specific mRNA production together with DNA content or membrane molecules expression, and offering the possibility to purify by sorting a cell population expressing a specific mRNA.

Base Sequence↗

PubMed1992

The impairment of natural killer function in the healthy aged is due to a postbinding deficient mechanism.

In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolytic activity which occurs during human aging, we examined by multiparametric flow cytometry the binding and lytic activities of human natural killer cells. The flow analysis revealed a striking increase of the CD16+8- subset, together with a significant decrease of CD8bright cells and total T cells (CD3+). Aging had no influence on the CD8dim subset. The total lytic activity expressed by PBL as well as their binding efficiency to K562 targets were moderately but not significantly increased in the elderly. In contrast, the cytotoxicity of the single target-bound natural killer cell (i.e., lytic efficiency) was deeply impaired in aged subjects, suggesting that the NK functional impairment observed in aging is located at postbinding level.

Adult↗

PubMed1992

Intracellular nucleotides of lymphocytes and granulocytes from normal ageing subjects.

Impaired lymphocyte and granulocyte function in the aged may, in part, reflect intrinsic aged-related biochemical alterations. In this study we compared the ribonucleotide contents of lymphocytes and granulocytes from young and old subjects evaluated by means of an HPLC-anion exchange method. We found that in general both populations from old subjects present higher levels of the various nucleotides, in particular: ATP, UDP, CTP, UDP-glucose in granulocytes, AMP, CTP, UDP-N-acetylglucosamine, UDP-glucose in lymphocytes. These data suggest that these molecules accumulate in aged subjects because of altered biochemical pathways. The increased pool of UDP-sugars, in particular, could be due to a depressed activity of some glycosyltransferases which therefore fail to glycosylate some plasma membrane cell proteins, thus accounting for their functional impairment.

Adult↗

PubMed1992

Cellular response and anti-HBs synthesis in vitro after vaccination with yeast-derived recombinant hepatitis vaccine.

Two groups of subjects receiving two different doses of yeast-derived recombinant hepatitis B surface antigen (rHBsAg) (10 micrograms Gen-HB-Vax, Merck Sharp and Dohme and 20 micrograms Engerix-B, Smith Kline and French) were investigated for in vitro specific humoral and cellular response to the native protein. In vitro proliferative response was dependent on the following critical variables: (1) antigen-specific precursor lymphocytes were present in the peripheral blood for a very short time; (2) the number of circulating specific precursors was dependent on the dose of HBsAg used for vaccination; (3) the presence of antigen-presenting cells was necessary to obtain a blastogenic response in vitro. In vitro proliferation was enhanced by the addition of recombinant interleukin 2 (rIL-2). Spontaneous and stimulated (anti-CD3, pokeweed mitogen) anti-HBs antibody production in vitro was obtained in only eight out of 20 subjects after the fourth boost. Although a different immunogenicity of the two vaccines cannot be excluded, these data strongly suggest that T and B cells responsive to HBsAg present different kinetics of recirculation in the peripheral blood, depending on the antigen dose used for immunization.

Adult↗

PubMed1992

Control of H-2 expression in transformed nonhaemopoietic cells by autocrine interferon.

The relationship between autocrine interferon (IFN) production and the expression of class I Major Histocompatibility Complex (MHC) membrane glycoproteins in vitro was investigated in a panel of murine transformed cells of nonhaemopoietic origin. The panel included 11 cell lines of H-2Kb haplotype derived from fibrosarcomas, carcinomas and melanoma, and from transformed fibroblasts. IFN activity was detected in the conditioned medium of nine cell lines; fibrosarcomas were among the high IFN producers, while the non-producers were a melanoma clone and a lung carcinoma cell line. A significant correlation was found between IFN production and the expression of H-2K/D glycoproteins, thus suggesting that long-term maintainment of MHC glycoprotein expression in vitro could be mediated by self produced IFN. Two IFN producer cell lines, MN/MCA1 and R80/17, were cultured in the presence of a blocking antiserum against IFN-alpha/beta: a significant decrease in H-2b expression was observed, thus indicating the existence of an autocrine IFN circuit. Taken together these findings suggest that release of IFN is a frequent event among transformed nonhaemopoietic cells, and that self-produced IFN contributes to the regulation of MHC antigen levels in solid tumours.

Carcinoma↗

PubMed1992

T-cell receptor gamma-delta positive lymphocytes in synovial membrane.

The distribution of T-lymphocytes expressing CD3 and T-cell receptor gamma-delta (T gamma/delta) has been examined by immunocytochemistry in the synovial membrane of eight patients with inflammatory arthritis (six rheumatoid arthritis, two spondyloarthritis) and eight with non-inflammatory arthritis (four osteoarthritis, four post-traumatic arthritis). T gamma/delta cells were present in eight out of eight inflammatory arthritis synovial membranes, but in only one out of eight non-inflammatory membranes (P less than 0.005). The mean T gamma/delta percentage (of total T-cells) in inflammatory arthritis was 14% (range 7-25%). T gamma/delta cells were found mainly in the transitional area of the synovial membrane with a scattered distribution as single cells or couplets. No relation was found between the presence and percentage of T gamma/delta cells and disease duration or steroid treatment.

Adolescent↗

PubMed1992

Immune defects in families and patients with xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by photosensitivity, a high incidence of cancer in sun-exposed portions of the skin and a reduced capacity to repair the u.v.-induced DNA damage. One of the XP mutations (XP-D) has also been identified in patients affected by trichothiodystrophy (TTD), a rare autosomal recessive disease characterized by brittle hair, mental and physical retardation, peculiar face and ichthyosis. However, in these patients there is no evidence of increased skin tumour incidence. Since an impairment of cell-mediated immunity has been proposed as a co-factor in the cancer proneness of XP patients, we investigated the involvement of immune defect(s) in five XP patients, five TTD patients, their parents, and 24 TTD relatives. We evaluated the phenotype of circulating lymphocytes, natural killer (NK) cell lytic activity, target cell binding of NK cells at single cell level and the effect of interferons (IFN) alpha and beta on NK cell activity. The relative proportion of CD3+ and CD4+ circulating lymphocytes was reduced in XP but not in TTD patients. NK cell lytic activity was decreased in XP patients and their mothers, but their fathers showed normal lytic activity. NK activity varied among TTD families: four out of five patients and their relatives presented low NK cell activity, and one family was normal. In TTD family members, NK activity increased after incubation with IFN-alpha or IFN-beta, but never reached normal values. In contrast, in XP patients and their mothers, the defect was almost completely corrected after in vitro incubation with IFN-alpha or IFN-beta. Our study indicates impaired NK lytic activity in the majority of TTD and XP patients and that this defect is present also in members of their families. In addition, XP patients present a low number of circulating T cells. These multiple abnormalities, together with DNA repair defects, could be related to the increased cancer risk in XP patients.

Abnormalities, Multiple↗

PubMed1992

Increased serum concentrations of tumour necrosis factor in beta thalassaemia: effect of bone marrow transplantation.

AIMS: Serum concentrations of tumour necrosis factor-alpha (TNF) were determined in beta thalassemic patients before and after bone marrow transplantation (BMT) to evaluate whether changes in TNF concentrations after BMT were related to immune mediated complications. METHODS: Serum TNF concentrations were determined by enzyme linked immunoassay (EIA) in paired samples from 71 patients with beta thalassemia before and after BMT. Serial samples from 13 patients were also studied for up to six months after BMT. Forty one normal healthy children matched for sex and age were studied as controls. RESULTS: beta thalassemic patients had high serum TNF concentrations before transplantation compared with controls. These were not related to sex, age, duration of disease, number of blood transfusions, transferrin concentrations or splenectomy. DQw1 positive patients showed significantly lower TNF concentrations than non-DQw1 cases. Patients with severe liver fibrosis had significantly higher TNF concentrations. No correlation was found between TNF values and BMT outcome before transplantation but TNF alpha values fell significantly after BMT. The decrease persisted only in patients with successful engraftment. In serial samples studied for up to six months after BMT, TNF values decreased but in four out of five patients with graft rejection and in all five with acute graft versus host disease (GVHD) sharp increases occurred at the time of clinical symptoms. No correlation was found between the degree of GVHD and serum TNF-alpha concentrations nor between TNF-alpha concentrations after BMT and the presence of bacterial, viral, and fungal infections. CONCLUSIONS: About 50% of beta thalassemic patients have increased serum TNF, and the changes after BMT are related to the occurrence of immune mediate complications. The persistence of low TNF concentrations after successful engraftment may be due to the preparative regimen and the lack of adverse immune reactions.

Bone Marrow Transplantation↗

PubMed1992

Serum amyloid A protein concentration in bone marrow transplantation for beta thalassaemia.

AIMS: To investigate whether serum amyloid A protein (SAA) and C-reactive protein (CRP) concentrations could be used in the management of beta thalassaemic patients undergoing bone marrow transplantation (BMT). METHODS: Serum SAA and CRP concentrations were determined in paired samples from 66 patients with beta thalassaemia before and after BMT. Serum SAA concentrations were determined by an enzyme linked immunoassay (EIA); serum CRP concentrations were determined by a nephelometric assay. RESULTS: Serum SAA concentrations before transplantation were significantly higher in the group that subsequently rejected the transplant than the group without complications. SAA concentrations increased after BMT in acute graft versus host disease (GvHD) and rejection. No significant increase in SAA or CRP was found in chronic GvHD. Increases in serum in SAA and CRP concentrations were not related to concomitant infection episodes. CONCLUSIONS: The different acute phase response in acute GvHD and rejection compared with chronic GvHD suggests that different immunopathogenic mechanisms are responsible.

Acute Disease↗

PubMed1992

The human anterior cruciate ligament: histological and ultrastructural observations.

In transverse and longitudinal paraffin-embedded sections, the human anterior cruciate ligament (ACL) is made up of wavy bundles of collagen fibres arrayed in various directions, the majority around the axis of the ligament with a few running parallel to it. The fascicles making up the larger bundles are also characterised by this undulating appearance. In thin sections 2 types of collagen fibrils are observed: small (with a single diameter peak at 45 nm) and large (3 peaks at 35, 50 and 75 nm respectively), organised into distinct areas made up of either large or small bundles of fibrils. The numerous fibroblasts that are present appear elongated in the direction of the bundles with branches and short cytoplasmic processes. The elastic system is made up of both elastic and oxytalan fibres. The varied orientation of the bundles in the ACL, the complex ultrastructural organisation and the abundant elastic system make it very different from other ligaments and tendons, providing a structure able to withstand the multiaxial stresses and varying tensile strains imposed upon it.

Anterior Cruciate Ligament↗

PubMed1992

In vitro methods to evaluate metal-cell interactions.

The aim of this study was to test different metals, widely employed in constructing prosthetic devices, by in vitro methods. Biological effects of such materials were analyzed through four different assays on human lymphocytes and granulocytes. The lymphocyte proliferation assay gave quantitative results, while the viability test showed the morphological appearance of the cells correlated well with previous results. NK cytotoxicity and granulocyte chemokinesis tests provided interesting data on leucocyte performance when challenged with metals. Therefore the present study adds new basic information on cell behaviour when metal products are present in the body, e.g. around devices implanted in human tissues.

Biocompatible Materials↗

PubMed1991

Commercial serum-free media: hybridoma growth and monoclonal antibody production.

Various growth factors, hormones and proteins present in serum are presumed to be responsible for its growth-stimulating activity in culture media. However, synthetic or serum substitute media supporting cell growth are advantageous when it is necessary to standardize culture conditions, particularly when cell products are used. In this study we have evaluated and compared the effects of some commercially available serum substitute media (10% NU Serum, 10% BMS, 2% Ultroser HY, 1% ITS + Premix, 1% Nutridoma, Ultradoma, FEB100, DCCM1 and DCCM2) on growth and immunoglobulin production in different hybridoma cell lines. Six different hybrids were studied: OKT3, OKT4 and OKT8 (producing monoclonal antibodies against CD3, CD4 and CD8 molecules), HB43 and HB57 (producing monoclonal antibodies against human IgG and IgM), and CRL 8019 (producing monoclonal antibodies against high-molecular weight CEA). Several parameters were evaluated, such as viability, doubling time, DNA synthesis, monoclonal antibody production and cell cycle under different culture conditions. Since not all of the hybridomas grew equally well in the same serum substitute media, one synthetic medium cannot be used for all the lines. We found that the serum-free media that best supported hybridoma growth were Nutridoma, DCCM1, DCCM2, NU Serum and FEB100.

Animals↗

PubMed1991

Natural killer function in flow cytometry: identification of human lymphoid subsets able to bind to the NK sensitive target K562.

NK cells are a phenotypically, morphologically and functionally heterogeneous population. This has led to the current thought that the non-MHC restricted cytotoxicity is a cellular function that can be associated to different phenotypes. The recognition of the target cell and the conjugate formation is always the first step that eventually leads to the lysis of target. Characterization of the phenotypical pattern of the cells able to bind to K562 targets is the purpose of this study. A multi-parametric flow cytometry binding assay has been employed to identify the different K562-bound lymphocyte subsets. In particular, cells that coexpress the CD16 and CD8 antigens (CD16+8dim+) showed a significantly higher binding capacity than their CD16+8- counterpart. Moreover, the highest binding values have been found in cells that did not express the CD16 antigen at all, but still expressed the CD8dim antigen, such as the small CD8dim+3+ population. These data show that, the NK lytic function being dependent on binding, minor subpopulations must be considered among effector cells, which might correspond to different lytic activities. None of the previously published methodologies that analyze conjugates by flow cytometry or fluorescence microscopy were able to measure the binding capacity of small, double stained, lymphocyte subsets.

Adult↗

PubMed1991

Reactivity of monoclonal antibody (mAb) GD9 with a cytoplasmic antigen preserved in formalin-fixed, paraffin-embedded gastrointestinal tumors.

In order to develop monoclonal antibodies to tumor associated antigens which are preserved in formalin-fixed, paraffin-embedded tissues, BALB/c mice were immunized with a formalin-fixed paraffin-embedded melanoma lesion. One of the resulting monoclonal antibodies, monoclonal antibody (mAb) GD9, stained formalin-fixed, paraffin-embedded melanoma lesions. When tested with a large panel of normal tissues from adults and from two fetuses of 4 and 16 week gestation and of benign and malignant tumors of different embryological origin, the mAb GD9 stained normal small bowel mucosa, areas of gastric intestinal metaplasia, the 6 pancreatic carcinoid tumors tested and a high percentage of gastrointestinal tumors. In the latter group, the reactivity with well differentiated tumors was higher than with poorly differentiated ones. Furthermore, the percentage of malignant cells stained by mAb GD9 in differentiated tumors was higher than in poorly differentiated ones. In the latter, the staining was diffuse and granular, while in the former, the staining was located in the cytoplasm close to the glandular lumen. The reactivity of mAb GD9 with colon carcinomas did not correlate with the Duke's staging and with the clinical course of the disease. The results of the immunohistochemical staining of normal tissues and benign and malignant lesions suggest that the specificity of mAb GD9 is different from that of previously described monoclonal antibodies which recognize tumor associated antigens expressed by tumors of the gastrointestinal tract.

Animals↗

PubMed1991

Human Achilles tendon: morphological and morphometric variations as a function of age.

Aging of human Achilles tendon results in changes in both cellular and fibrous components. Cells flatten and become less numerous. Their thin and long cytoplasmatic projections tend to shorten and diminish in number. Tendon fibers lose their typical undulating appearance and become quite straight. Collagen fibril diameter, small and uniform in the neonatal period, becomes large and extremely variable from adolescence onwards. Age related morphometric changes include a decrease in the average, maximum diameter and density of collagen fibrils and an increase of fibril concentration. In our opinion these morphological and morphometric variations are strictly related to functional requirements.

Achilles Tendon↗

PubMed1990

Complement activation products in idiopathic pulmonary fibrosis: relevance of fragment Ba to disease severity.

Immune complexes have been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a chronic inflammatory disease of unknown etiology. Using newly developed techniques to measure complement fragments, we assessed whether complement activation occurs in IPF and its relationship to circulating immune complexes (CIC), disease severity, and prognosis. We measured the fragments C3d, C4d, and Ba of the common, classical, and alternative pathway, respectively, along with the components C3, C4, and Factor B and CIC, in 18 patients and 19 healthy subjects. The fragment to parent molecule ratios C3d/C3, C4d/C4, and Ba/Factor B were derived. Complement determinations were repeated in 14 of the patients on a second occasion separated by 8-33 months (median 17 months) from the first. All parameters were higher in patients than controls, and the difference between the two groups was significant for C3d, C4d/C4, C3, CIC (P less than 0.05) and for C4d and Ba (P less than 0.01). Fragment Ba was found to correlate with disease duration and indices of disease severity, i.e., positively with the dyspnoea score (Rs = 0.44, P less than 0.04), the physiological score (Rs = 0.50, P less than 0.02), the fibrosis score (Rs = 0.45, P less than 0.04), and a composite clinical, radiological, and physiological score (Rs = 0.48, P less than 0.03) and negatively with the forced vital capacity (Rs = -0.47, P less than 0.03). Ba was higher in those patients who deteriorated on follow-up analysis than those who improved or remained unchanged (P less than 0.04). These results show that conversion products of all complement pathways are increased in IPF and demonstrate classical pathway activation. They also show that the measurement of Ba might prove a useful laboratory test of disease severity in this condition.

Adult↗

PubMed1990

Age-associated changes in CD8+ and CD16+ cell reactivity: clonal analysis.

A cloning technique was used to estimate the frequency of proliferating T cell precursors, the growth capacity of clone-forming cells and the functional activity of clones established in vitro from peripheral blood lymphocytes of young and old people. The mean frequency of proliferating precursors was lower in the elderly as was the proliferative capacity of CD8+ clones. In contrast, CD4+ and CD16+ clones showed a proliferation similar to that obtained from young subjects. When the clones were examined for their functional activity, CD4+ clones from both groups failed to show any cytolytic activity, while CD8+ clones exerted cytolysis against K562 and in antibody-dependent cell-mediated cytotoxicity but this function was reduced in clones derived from old subjects. Similarly, CD16+ clones from the elderly showed a decreased activity at some effector-to-target cell ratios. We conclude that the impaired functional activity (T or NK-dependent) found in the peripheral blood of aged subjects persists after in vitro selection when these cells are analysed at clonal level.

Adult↗