Morphometric evidence of the trophic effect of L-carnitine on human skeletal muscle

We investigated the effect of long-term i.v. administration of L-carnitine on human muscle fibers using morphometric parameters. We administered 2g/day L-carnitine to patients undergoing hemodialysis for at least 12 months. At the end of this period a marked increase in serum and muscle carnitine levels was observed in all patients, together with hypertrophy and predominance of type 1 fibers. L-carnitine was withheld for 4 months, during which time serum and muscle levels gradually decreased and no changes were observed in muscle fibers. Subsequent addition of L-carnitine to dialysis fluid for another 4 months stabilized lower levels. At the end of this period reduction of diameter of type 1 fibers was observed. Type 2 fibers remained unchanged. Moreover, type 1 fibers remained predominant in all cases. Hence, we suggest that carnitine has a specific trophic effect on type 1 fibers which are characterized by an oxidative metabolism.     

Densitometric and morphometric study of immunocytochemical estrogen receptors detection in breast carcinomas.

Immunohistochemical quantitative evaluation of estrogen receptors (ER) detected in tissue sections from 30 breast tumors by monoclonal antibody was performed using a densitometric method. In particular, ER concentration was calculated by nuclear mean optical density (nMOD), while heterogeneity in ER content was calculated by the coefficient of variation (CV) of the nuclear optical density histogram. Tumors which showed more than 60% of positive cells had a mean value of ER-nMOD of 0.116 +/- 0.002 a.u. and of ER-CV of 33.74 +/- 0.68. Tumors which showed 30% to 60% of positive cells had a mean value of ER-nMOD of 0.082 +/- 0.006 a.u. (arbitrary units) and of ER-CV of 36.25 +/- 3.44. Tumors showing less than 30% of positive cells had ER-nMOD of 0.052 +/- 0.009 a.u. and ER-CV of 48.49 +/- 5.61. These results indicate that the greater the concentration the lower the ER heterogeneity within the tumor sample. No significant differences between ER-ICA results, nuclear size and form factors were found.     

Tyrosine kinase-dependent and -independent events induced by interleukin-2 stimulation: interleukin-2-mediated NO production required for the induction of lymphokine-activated killer cell activity in rat splenocytes is tyrosine kinase independent.

Nitric oxide (NO) has recently been shown to be an indispensable co-factor in the generation of lymphokine-activated killer (LAK) cells induced by interleukin-2 (IL-2). Upon stimulation with IL-2, cells endowed with specific receptors undergo phosphorylation of substrates mediated by protein tyrosine kinases (PTK). In this work we utilized a well-characterized PTK inhibitor, genistein (GEN), to address the role of PTK on NO-dependent LAK cell generation. The effects of GEN were tested on the expression of the inducible NO synthase (iNOS) gene, proliferation, generation of cytotoxic activity and production of NO upon IL-2 stimulation of rat splenocytes. We report here that GEN displays profound inhibitory effects on recombinant (r)IL-2 induced proliferation and on LAK cell generation, while only marginally affecting NO production, measured as NO2-. In contrast, a specific inhibitor of the NO synthetic pathway (NG-monomethyl-L-arginine; NMMA) blocked generation of LAK cells and NO production without affecting cell proliferation. If added directly to the cytotoxicity tests, GEN exerted minor inhibitory effects, not exceeding 25% of control tests, while NMMA was completely ineffective. Sodium nitroprusside (SNP), a non-enzymatic NO-releasing substance, restored LAK cell generation in cultures performed in the presence of NMMA, but not in those performed in the presence of GEN. These results indicate that IL-2-induced NO production is a PTK-independent event. IL-2-stimulated LAK cell generation obligatorily requires the concurrent activation of PTK dependent and independent signal transduction pathways.     

Cytokine gene expression in human peripheral blood mononuclear cells stimulated by mannoprotein constituents from Candida albicans.

The expression of cytokine genes in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with mannoprotein constituents (MP) of Candida albicans has been studied by means of S1 nuclease mapping analysis, polymerase chain reaction, and enzyme-linked immunosorbent assay. MP induced early, consistent, and long-lasting production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and IL-6 mRNAs. Similar results were obtained when the same PBMC cultures were stimulated with the purified protein derivative (PPD) from Mycobacterium tuberculosis or with IL-2, although lower levels of IL-6 mRNA were detected in IL-2-stimulated cells than in MP- or PPD-stimulated cells. MP, PPD, and IL-2 induced appreciable levels of granulocyte-macrophage colony-stimulating factor and gamma interferon, but only MP and PPD were able to induce IL-2 mRNA. MP were unable to stimulate a consistent expression of the genes encoding for IL-4, IL-5, and IL-10, while low, sometimes barely detectable levels of these cytokine mRNAs were observed in PPD- or IL-2-stimulated PBMC cultures. When protein synthesis of MP-stimulated PBMC was inhibited by cycloheximide, a superinduction of mRNAs for IL-4 and IL-10 and, more markedly, gamma interferon was observed. Overall, these results highlight the powerful, selective induction of cytokine gene expression by MP constituents of C. albicans in human PBMC cultures, thus providing some functional clues to explain the efficient state of the anticandidal response in normal human subjects.     

CD14 and tolerance to lipopolysaccharide: biochemical and functional analysis.

To evaluate the role of the high-affinity monocyte receptor for lipopolysaccharide (LPS), CD14, in the process of tolerance to LPS, the human monocytic cell line Mono-Mac-6 was cultured in the absence or presence of different amounts of LPS. The kinetics of CD14 modulation in these cells showed an initial 4-day period characterized by increased cell-surface expression, rate of biosynthesis (peaking at 48 hr) and release of its soluble forms (sCD14) which correlated with the amount of LPS in the culture. At this time, tolerance to LPS was already established, as measured by tumour necrosis factor-alpha (TNF-alpha) induction, it was LPS dose dependent and persisted up to 15 days. LPS also reduced the cell proliferation rate in a dose-dependent manner. After 8 days and up to 15 days, the CD14 biosynthesis, cell-surface expression and release of sCD14 inversely correlated with the level of LPS in the culture. The 48-hr LPS-pretreated cells showed a slightly decreased CD14 affinity for LPS, a relative high number of CD14 molecules per cells, and desensitization also to a phorbol 12-myristate 13-acetate (PMA) challenge. An anti-CD14 monoclonal antibody (mAb) protected the cells from tolerization when added at the beginning of culture, as revealed by challenge with LPS and PMA. The data indicate that in this model tolerization to LPS (1) precedes CD14 down-modulation, (2) operates by alteration of the receptor affinity for LPS and by a mechanism which affects a protein kinase C (PKC)-dependent signalling pathway, and (3) that CD14 plays a critical role in the establishment of tolerance to LPS. In addition, analysis of the data suggests the existence of a PKC-independent signalling pathway for LPS tolerization and a CD14-independent mechanism for establishing tolerance.     

Italian version of the organic brain syndrome and the depression scales from the CARE: evaluation of their performance in geriatric institutions

The Organic Brain Syndrome (OBS) and the Depression (D) scales derived from the Comprehensive Assessment and Referral Evaluation (CARE) were translated into Italian and used in a survey of geriatric institutions in Milan. During the survey validity and reliability tests of the scales were conducted. Inter-rater reliability (total score weighted kappa) was highly satisfactory for both scales (0.96 for OBS and 0.83 for D scale). Reliability was assessed three times during the survey and showed good stability for both scales, with a slight but significant trend towards reduction over time for the D scale. Reliability of the D scale was significantly lower when the subjects interviewed scored highly on the OBS scale (severe cognitive impairment). Criterion validity was highly satisfactory both for the OBS scale (cut-off point 4/5: sensitivity 77%, specificity 96%, positive predictive value 91%) and the D scale (cut-off point 10/11: sensitivity 95%, specificity 92%, positive predictive value 84%). Results are discussed with special reference to longitudinal assessment of reliability, the choice of the cut-off point, and the context-dependent properties of questionnaires.     

"INTERMED": a method to assess health service needs. II. Results on its validity and clinical use

The validity and clinical use of a recently developed instrument to assess health care needs of patients with a physical illness, called INTERMED, is investigated. The INTERMED combines data reflecting patients' biological, psychological, and social characteristics with information on health care utilization characteristics. An example of a patient population in which such an integral assessment can contribute to the appropriateness of care, are patients with low back pain of degenerative or unknown origin. It supports the validity and the clinical usefulness of the INTERMED when clinically relevant subgroups in this heterogeneous population can be identified and described based on their INTERMED scores. The INTERMED was utilized in a group of patients (N = 108) having low back pain who vary on the chronicity of complaints, functional status, and associated disability. All patients underwent a medical examination and responded to a battery of validated questionnaires assessing biological, psychological, and social aspects of their life. In addition, the patients were assessed by the INTERMED. It was studied whether it proved to be possible to form clinically meaningful groups of patients based on their INTERMED scores; for this, a hierarchical cluster analysis was performed. In order to clinically describe them, the groups of patients were compared with the data from the questionnaires. The cluster analysis on the INTERMED scores revealed three distinguishable groups of patients. Comparison with the questionnaires assessing biological, psychological, and social aspects of disease showed that one group can be characterized as complex patients with chronic complaints and reduced capacity to work who apply for a disability compensation. The other groups differed explicitly with regard to chronicity, but also on other variables. By means of the INTERMED, clinically relevant groups of patients can be identified, which supports its use in clinical practice and its use as a method to describe case mix for scientific or health care policy purposes. In addition, the INTERMED is easy to implement in daily clinical practice and can be of help to ease the operationalization of the biopychosocial model of disease. More information on its validity in different patient populations is necessary.     

Nitric oxide-independent inhibitory effects of L-arginine analog NG-monomethy-L-arginine on the generation of interleukin-2 activated cytotoxic activity in humans

Nitric oxide (NO) derived intracellularly from L-arginine (Arg) is indispensable for optimalgeneration of lymphokine-activated killer (LAK) cell activity in rodents. Still unclear, however, is its role in humans. To address this question human peripheral blood mononuclear cells (PBMC) from healthy donors were cultured in L-arginine free medium supplemented with recombinant interleukin-2 (rIL-2) and in the presence of exogenous L-arginine analog NG-monomethyl-L-arginine (NMMA), a specific inhibitor of the NO synthetic pathway. Cultured PBMC were tested for cytotoxic activity, proliferative capacity, and expression of phenotypic and activation markers (CD3, CD4, CD8, CD16, CD56 and CD25). Culture supernatants were assayed for nitrite (NO2-) and tumor necrosis factor-alpha (TNF-alpha) production. We found that NMMA inhibits the generation of optimal LAK cell activity when no exogenous Arg is supplied. Similar effects were also observed on proliferation, expression of IL-2 receptor induced upon rIL-2 stimulation and on TNF-alpha production. Sodium nitroprusside (SNP), used as a source of exogenous NO could not overcome this effect of NMMA on LAK cell activity. NO2- production was virtually undetectable in culture supernatants. Thus, NMMA affects in an NO-independent manner rlL-2 induced LAK activity in human PBMC.