Antonio Zicca

Reduction of cisplatin hepatotoxicity by procainamide hydrochloride in rats

In preceding papers, we proposed that procainamide hydrochloride, a class I antiarrhythmic agent, was able to protect mice and rats from cisplatin-induced nephrotoxicity and that it could exert its action through accumulation in kidneys followed by coordination with cisplatin (or its hydrolysis metabolites) and formation of a less toxic platinum compound similar to the new platinum(II) triamine complex cis-diamminechloro-[2-(diethylamino)ethyl 4-amino-benzoate, N4]-chlorideplatinum(II) monohydrochloride monohydrate, obtained by the reaction of cisplatin with procaine hydrochloride. Hepatotoxicity is not considered as a dose-limiting toxicity for cisplatin, but liver toxicity can occur when the antineoplastic drug is administered at high doses. Here, we report that procainamide hydrochloride, at an i.p. dose of 100 mg/kg, reduces cisplatin-induced hepatotoxicity, as evidenced by the normalization of plasma activity of glutamic oxalacetic transaminase and gamma-glutamyl transpeptidase, as well as by histological examination of the liver tissue. Twenty-four hours after i.p. treatment with the combination of 7.5 mg/kg cisplatin and 100 mg/kg procainamide, a significant increase of procainamide (+56%, P<0.05), total platinum (+31%, P<0.05), platinum-DNA adducts (+31%, P<0.05) and percent DNA-DNA interstrand cross-links (+69%, P<0.02) was found in liver tissue, as compared to animals treated with cisplatin alone. Moreover, in accordance with these findings, we also observed a slightly lower concentration and cumulative excretion of platinum in the feces. Since mitochondrial injury is considered a central event in the early stages of the nephrotoxic effect of cisplatin, the distribution of platinum in these subcellular organelles obtained from hepatocytes was determined after treatment with cisplatin with or without procainamide hydrochloride, together with platinum concentration in their cytosolic fraction. Our data show that the coadministration of procainamide hydrochloride produced a rearrangement of subcellular platinum distribution in hepatocytes with a slight decrease in mitochondria (-15%, P<0.10) and a slight increase in the cytosolic fraction (+40%, P<0.10) of platinum content, compared to the treatment with cisplatin alone. In analogy with our previous results in the kidney, confirmed here by our data in vitro, we suggest that the hepatoprotective activity of procainamide hydrochloride is linked to the formation of a less toxic platinum complex, which leads to inactivation of cisplatin itself and/or its highly toxic hydrolysis metabolites and to a different subcellular distribution of platinum.

HLA‐DR Schwann cell reactivity in peripheral neuropathies of different origins

HLA-DR antigens have been found on Schwann cells in peripheral neuropathies of different origins but not in normal control cases. Class II antigen reactivity was more intense in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and hereditary motor and sensory neuropathy type 1 (HMSN), but was also observed in toxic or metabolic neuropathies. The expression of HLA-DR antigen on Schwann cells does not appear to be related to the inflammatory or autoimmune origin of the disease.

A lymphoproliferative disorder of the large granular lymphocytes with natural killer activity

This paper reports the case of a patient with an abnormally expanded population of circulating lymphoid cells displaying the features of the so-called large granular lymphocytes (LGL). These cells were peroxidase negative and nonphagocytic, formed rosettes with sheep erythrocytes, had receptors for IgG, and contained azurophilic (electron-dense) granules. Like normal LGL, the patient cells were positive for two acid hydrolases (acid phosphatase and β-glucuronidase) but did not stain for α-naphthyl acetate esterase (ANAE), which is present in normal LGL. Ultrastructural studies revealed that the patient cells were rich in Golgi-derived vesicles, coated vesicles, multivesicular bodies, and immature granules, indicating that, unlike normal LGL, they were engaged in granulogenesis. These features, together with the absence of ANAE activity, are suggestive of some degree of cell immaturity. The patient cells displayed natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities comparable to those of normal peripheral blood mononuclear cells, or even higher, and did not respond to T-cell mitogens or allogeneic cells. Furthermore, they were incapable of suppressing normal T-cell proliferation or pokeweed mitogen-induced B-cell differentiation. Analysis of the NK activity at the single-cell level revealed that a large proportion of the patient cells bound to the K562 target cells but could not accomplish the entire lytic process. This finding supports further the possibility that the patient cells were immature LGL. The surface phenotype of the patient cells (as defined by a battery of monoclonal antibodies) was somewhat different from that usually observed in the majority of the normal LGL because, in addition to the HNK-1 marker, the cells were OKT3+, aLeul+, aLeu4+, OKT8+, aLeu2a+, and 3A1+ but were OKM1− and 4F2−. This phenotype could correspond to that of maturing LGL.

Secretion and binding of HMG1 protein to the external surface of the membrane are required for murine erythroleukemia cell differentiation

We show here that murine erythroleukemia (MEL) cells, following induction with hexamethylene bisacetamide, accumulate high mobility group (HMG)1 protein onto the external surface of the cell in a membrane‐associated form detectable by immunostaining with a specific anti‐HMG1 protein antibody. This association is maximal at a time corresponding to cell commitment. At longer times, immunostainable cells are progressively reduced and become almost completely undetectable along with the appearance of hemoglobin molecules. Binding to MEL cells does not affect the native molecular structure of HMG1 protein. The type of functional correlation between HMG1 protein and MEL cell differentiation is suggested by the observation that if an anti‐HMG1 protein antibody is added at the same time of the inducer almost complete inhibition of cell differentiation is observed, whereas if the antibody is added within the time period in which cells undergo through irreversible commitment, inhibition progressively disappears. A correlation between MEL cell commitment and the biological effect of HMG1 protein can thus be consistently suggested.

GFAP expression of human Schwann cells in tissue culture

We have studied the expression of the intermediate filament (IF) proteins, vimentin and glial fibrillary acidic protein (GFAP), in cultured human Schwann cells (SC) from patients with different neuropathies and normal control cases. SC cultures from sural nerve biopsies of 8 subjects with axonal neuropathies, 8 with demyelinating neuropathies and 3 normal controls were included in this study and processed with double immunofluorescence technique, using anti-vimentin and anti-GFAP antibodies, during the 2nd, 4th and 6th week of culture. Five cultures incubated with anti-GFAP antibodies were also processed for immunoelectron microscopy. Specificity tests of the used antibodies were performed. We have found that: (1) cultured human SC constantly express vimentin; (2) SC from normal controls are GFAP-negative in the first period of culture; (3) SC from pathologic nerves can contain GFAP-immunoreactive IF and the percentage of GFAP-positive SC is higher in axonal than in demyelinating neuropathies; (4) during the permanence in culture human SC from both normal and pathologic cases acquire the ability to synthesize GFAP. The obtained data suggest that the removal from axonal contact and the resulting loss of myelinating function induce a cytoskeletal cellular response in human SC characterized by the cytoplasmic accumulation of GFAP-immunoreactive IF.

Schwann cell GFAP expression increases in axonal neuropathies

We studied the Schwann cell (SC) GFAP immunoreactivity in normal human peripheral nerves and in neuropathies of different origin. Immunofluorescence and immunocytochemistry were carried out on serial frozen sections of 58 peripheral nerve biopsies using monoclonal antibodies (mabs) antivimentin and anti GFAP, and antiserum anti S-100 and anti GFAP. To test the specificity of the mabs and antiserum used, proper competition controls on tissue sections of 2 selected cases, tissue cultures studies of human fibroblasts and immunoblotting of homogenates of human fibroblasts, 3 normal and 5 pathologic nerves were carried out. In order to evaluate a possible correlation between SC GFAP positivity and neuropathologic findings a quantitative study was performed, evaluating the SC GFAP reactivity in all the 58 cases, and relating the SC GFAP positivity to the index of nerve pathology (IP) in 9 selected cases, and to the percentage of teased fibers showing axonal degeneration or demyelination and remyelination in 25 representative cases. We demonstrate that in normal human sural nerves and in demyelinating neuropathies only a few scattered SC are recognized by the mabs or antiserum anti GFAP. On the contrary in axonal neuropathies the majority of SC gain the property to express intermediate filaments which show common antigenic properties with GFAP.

Forebrain white matter in spontaneously hypertensive rats: a quantitative image analysis study.

The volume and the morphology of brain white matter as well as the number and the size of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were investigated in 6-month-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats. The volume of frontal and occipital cortex and of hippocampus was decreased in SHR in comparison with normotensive rats, whereas the volume of neostriatum was unchanged. A remarkable decrease of the volume of internal capsule and striosomes, a moderate reduction of that of corpus callosum and no changes of the volume of external capsule and of white matter of hippocampus were also observed in SHR. In SHR the number of astrocytes was higher in the frontal and occipital cortex and in the white matter of the CA1 and CA3 subfields of the hippocampus, but not in the corpus callosum or in the grey matter of the CA1 and CA3 subfields. Staining for myelin did not reveal alterations in single fibre sheath morphology. These findings indicate the occurrence of changes of forebrain white matter in SHR, consisting in the reduction of it without qualitative modifications of myelinated fibres. The development of gliosis apparently not related with changes of volume of white matter was also found.

Receptors for the Third Complement Component on a Proportion of Large Granular Lymphocytes from Human Peripheral Blood

Large granular lymphocytes (LGL) are nonadherent cells with cytoplasmic azurophilic granules, avid receptors for the Fc portion of IgG, and a paranuclear localization of alpha‐naphthyl acid esterase or acid phosphatasc. LGL constitute the bulk of TG cells (cells with receptors for sheep erythrocytes and for IgG molecules) and null cells (non‐T, non‐B cells). In the present study we demonstrate that 20–33% of the circulating human LGL express receptors for the third complement component (C3R). When TG cell or null cell fractions from normal individuals or non‐T cells from a patient with infantile agammaglobulinaemia (which contained almost exclusively LGL) were rosetted with erythro cytes coated with antibody and complement, a variable number of C3R‐bearing cells were detected. Such cells were isolated and analysed further; the great majority of them displayed the cytochemical and ultrastructural features of LGL.

Class II antigen expression on human cultured Schwann cells from patients with Charcot-Marie-Tooth disease

T lymphocytes control the extent of the immune reaction by recognizing the antigen in connection with class II histocompatibility surface molecules, coded by genes located on the HLA-D locus. The expression of HLA-DR antigens is confined to a few antigen presenting cells, like lymphocytes and macrophages, which can therefore induce the initial phase of the immune reaction. We report that also Schwann cells (SC) from patients with Charcot-Marie-Tooth disease (CMT), an hereditary disorder of the peripheral nervous system, are able to express HLA-DR antigens. Human SC cultures were carried out from sural nerve biopsies of CMT and normal control cases. Cultures were tested on day 7, 14, 21 and 28, with double immunofluorescence technique using rabbit antiserum anti-S-100 and mouse anti-HLA-DR monoclonal antibody. SC from CMT were HLA-DR positive since the first few days, continuing to express class II antigens for all the duration of the culture. The presence of class II antigens on cultured SC from CMT disease suggests that immune-mediated mechanisms may be relevant in the pathogenesis of this degenerative disorder of the peripheral nervous system.

Elutriation of human keratinocytes and melanocytes fromin vitro cultured epithelium

SummaryHuman epidermal keratinocytes grown in culture and at different stages of differentiation are shown to be viably separated by elutriation. A specific fraction enriched in melanocytes was obtained. Elutriation of cells obtained fromin vitro cultured epithlium could prove useful in studies concerning the biochemistry and molecular markers of cells isolated from normal epithelium and from different pathologies.