Homogentisic acid induces morphological and mechanical aberration of ochronotic cartilage in alkaptonuria

Alkaptonuria (AKU) is a disease caused by a deficient homogentisate 1,2-dioxygenase activity leading to systemic accumulation of homogentisic acid (HGA), that forms a melanin-like polymer that progressively deposits onto connective tissues causing a pigmentation called “ochronosis” and tissue degeneration. The effects of AKU and ochronotic pigment on the biomechanical properties of articular cartilage need further investigation. To this aim, AKU cartilage was studied using thermal (thermogravimetry and differential scanning calorimetry) and rheological analysis. We found that AKU cartilage had a doubled mesopore radius compared to healthy cartilage. Since the mesoporous structure is the main responsible for maintaining a correct hydrostatic pressure and tissue homoeostasis, drastic changes of thermal and rheological parameters were found in AKU. In particular, AKU tissue lost its capability to enhance chondrocytes metabolism (decreased heat capacity) and hence the production of proteoglycans. A drastic increase in stiffness and decrease in dissipative and lubricant role ensued in AKU cartilage. Multiphoton and scanning electron microscopies revealed destruction of cell–matrix microstructure and disruption of the superficial layer. Such observations on AKU specimens were confirmed in HGA-treated healthy cartilage, indicating that HGA is the toxic responsible of morphological and mechanical alterations of cartilage in AKU.     

Monoclonal antibody Leo Mel 3, which inhibits killing of human melanoma cells by anomalous killer cells, binds to a sugar sequence in GD2 (II3(NeuAc)2-GgOse3Cer) and several other gangliosides

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.     

In vitro targeting of erythrocytes to cytotoxic T-cells by coupling of Thy-1.2 monoclonal antibody.

A method was explored to develop a general means to target erythrocytes to T-cells in vitro. Mouse erythrocytes were coupled with an anti-Thy-1.2 monoclonal antibody by two methods. Chromic chloride coupling of antibody was preferred to biotinylation. The morphology, osmotic fragility, and the hematological values of treated cells were normal compared with those of control erythrocytes. Antibody-coupled erythrocytes were incubated with cytotoxic T-lymphocytes (CTLL) in vitro at a 20:1 ratio. Approximately 60-70% of the CTLL formed rosettes. The cell mixture was subjected to gradient centrifugation and separated into four fractions. THe rosettes were clearly identified only in the treated group containing anti-Thy-1.2-coupled erythrocytes. No rosettes were found when aspecific monoclonal antibody was coupled to erythrocytes. Examination by scanning and transmission electron microscopy revealed CTLL with 4-5 erythrocytes attached to them but did not show any evidence of membrane fusion. Rosettes of CTLL incubated in vitro proliferated as well as CTLL alone and maintained their dependency on interleukin-2. Targeting of erythrocyte carriers to lymphocytes offers the potential for delivery of molecules directly to the target cell.     

Red blood cells as an antigen-delivery system.

The use of adjuvants is usually required to induce strong immunological responses to protein antigens. However, in many cases these adjuvants cannot be extensively applied in human and veterinary vaccinations because of associated inflammatory reactions or granuloma formation. We show here that protein antigens (bovine serum albumin, hog liver uricase, and yeast hexokinase), coupled to autologous red blood cells by way of a biotin-avidin-biotin bridge, elicit an immunological response in mice similar to or higher than that obtained by the use of Freund's adjuvant. Quantities as low as 0.5 micrograms/mouse are high enough to generate these immunological responses. Furthermore, splenocytes of mice immunized by red blood cell-coupled antigens can be used to generate hybridomas secreting monoclonal antibodies. Thus, the delivery of antigens by autologous red blood cells is an effective way to avoid the use of adjuvants for producing anti-peptide antibodies and possibly to generate peptide vaccines.     

Human plasma glutathione oxidation in normal and pathological conditions

Reduced glutathione added to human plasma disappears rapidly, and it is concomitantly recovered in its oxidized form. This oxidation is not due to the plasma metal content since it is not inhibited by EDTA or by passage of plasma through Chelex columns. Furthermore, this oxidation is not due to the peroxidase activity of glutathione S-transferases, which are usually undetectable in normal human serum, and it does not correlate with the amount of plasma glutathione peroxidase. A significant increase in the rate of glutathione oxidation was observed in plasma of patients with increased gamma-glutamyltranspeptidase activity. It is concluded that the side-oxidase activity of gamma-glutamyltransferase is responsible for the oxidation of glutathione in human plasma.     

Liver fatty acid-binding protein in two cases of human lipid storage

Summary FABPs in the various tissues play an important role in the intracellular fatty acid transport and metabolism. Reye's syndrome (RS) and multisystemic lipid storage (MLS) are human disorders characterized by a disturbance of lipid metabolism of unknown etiology. We investigated for the first time L-FABP in these two conditions. Affinity purified antibodies against chicken L-FABP were raised in rabbits, and found to cross-react specifically with partially purified human L-FABP. L-FABP content in liver samples of two patients with RS and MLS was investigated by immuno-histochemistry, SDS-PAGE and ELISA. L-FABP immuno-histochemistry showed increased reactivity in the liver of RS patient and normal reactivity in MLS liver. L-FABP increase in RS liver was confirmed by densitometry of SDS-PAGE and ELISA method. By these two methods the increase amounted to 180% and 199% (p < 0.02), respectively, as compared to controls. A possible role of L-FABP in the pathogenesis of RS is discussed.     

Use of locally weighted scatterplot smoothing (LOWESS) regression to study selection signatures in Piedmontese and Italian Brown cattle breeds

Selection is the major force affecting local levels of genetic variation in species. The availability of dense marker maps offers new opportunities for a detailed understanding of genetic diversity distribution across the animal genome. Over the last 50 years, cattle breeds have been subjected to intense artificial selection. Consequently, regions controlling traits of economic importance are expected to exhibit selection signatures. The fixation index (F_st) is an estimate of population differentiation, based on genetic polymorphism data, and it is calculated using the relationship between inbreeding and heterozygosity. In the present study, locally weighted scatterplot smoothing (LOWESS) regression and a control chart approach were used to investigate selection signatures in two cattle breeds with different production aptitudes (dairy and beef). F_st was calculated for 42 514 SNP marker loci distributed across the genome in 749 Italian Brown and 364 Piedmontese bulls. The statistical significance of F_st values was assessed using a control chart. The LOWESS technique was efficient in removing noise from the raw data and was able to highlight selection signatures in chromosomes known to harbour genes affecting dairy and beef traits. Examples include the peaks detected for BTA2 in the region where the myostatin gene is located and for BTA6 in the region harbouring the ABCG2 locus. Moreover, several loci not previously reported in cattle studies were detected.     

Effect of phenylhydrazine on red blood cell metabolism

In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1-14C]-glucose showed a maximum value at 2mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine-induced anemia is discussed.     

Identification of an Interstitial 18p11.32-p11.31 Duplication Including the EMILIN2 Gene in a Family with Porokeratosis of Mibelli

Porokeratosis is a rare disease of epidermal keratinization characterized by the histopathological feature of the cornoid lamella, a column of tightly fitted parakeratocytic cells, whose etiology is still unclear. Porokeratosis of Mibelli is a subtype of porokeratosis presenting a single plaque or a small number of plaques of variable size located unilaterally on limbs. It frequently appears in childhood and occurs with a higher incidence in males. Cytogenetic analyses were performed in all members of the family on lesioned and uninvolved skin. An array-CGH analysis was also performed utilizing the Human Genome CGH Microarray Kit G3 400 with 5.3 KB overall median probe spacing. Gene expression was performed on skin fibroblasts. In this study, we describe a Caucasian healthy 4-year-old child and his father showing features of porokeratosis of Mibelli. Array-CGH analysis revealed an interstitial 429.5 Kb duplication of chromosome 18p11.32-p11.3 containing four genes, namely: SMCHD1, EMILIN2, LPIN2, and MYOM1 both in patient and his father. EMILIN2 resulted overexpressed on skin fibroblasts. Also other members of this family, without evident signs of porokeratosis, carried the same duplication. Among these genes, we focused our attention on elastin microfibril interfacer 2 (EMILIN2) gene. Apoptosis plays a fundamental role in maintaining epidermal homeostasis, balancing keratinocytes proliferation, and forming the stratum corneum. EMILIN2 is known to trigger the apoptosis of different cell lines negatively affecting cell survival. It is expressed in the skin. We could speculate that the duplication and overexpression of EMILIN2 cause an abnormal apoptosis of epidermal keratinocytes and alter the process of keratinization, even if other epigenetic and genetic factors could also be involved. Our results could contribute to a better understanding of the pathogenesis of porokeratosis of Mibelli.