Imre Zs.-Nagy

Neutralizing and enhancing antibodies measured in complement-restored serum samples from HIV-1-infected individuals correlate with immunosuppression and disease

ObjectiveTo study the association between the progression of HIV disease and HIV neutralization and enhancement measured in the presence of human complement. DesignTwo studies were performed: (1) longitudinal measurement of the complement-dependent enhancing antibodies in parallel with T-cell subset determination in 55 serum samples from seven HIV-infected patients, and (2) determination of the titres of neutralizing and enhancing antibodies in stored samples of 21 HIV-asymptomatic patients obtained between 1986 and 1987 and follow-up of the patients until October 1992. MethodsHIV-1 [human T-lymphotropic virus (HTLV)HIB strain, 100 median tissue culture infective dose (TCID50)] was incubated with twofold dilutions of sera in the presence of human complement (final dilution, 1 :4) and added to MT-4 cells. HIV growth was monitored daily for 5 days using the reclustering inhibition and p24 immunofluorescence assays. ResultsA significant negative correlation between the titres of enhancing antibodies and CD4+ cell count was found in longitudinal measurements. In the prospective studies, marked differences were observed between patients with undetectable, low, or high titres of enhancing antibodies in the clinical course of HIV disease: CD4+ cell counts and percentages decreased more rapidly in the high titre group within 3 years. After 5 years, AIDS developed in five out of six patients in the high titre group but only in five out of 15 of the low titre group (P<0.05). A similar difference was observed between patients with and without neutralizing antibodies. ConclusionsMeasurement of HIV neutralization and enhancement in complement-containing serum samples using a complement receptor carrying target may provide data of clinical relevance. Neutralization appears to be associated with a favourable prognosis whereas high titre enhancing antibodies predict rapid progression of HIV disease.

INDUCTION OF AGE PIGMENT ACCUMULATION IN THE BRAIN CELLS OF YOUNG MALE RATS THROUGH IRON-INJECTION INTO THE CEREBROSPINAL FLUID

The OH free radical formation can be increased in the brain by intralumbar iron injections into the cerebrospinal fluid (CSF) through the Fenton reaction. Ferrous ammonium sulphate was injected to male CFY rats of 3 months of age. The animals survived a single dose of 4 mumoles, and this treatment was repeated daily for 3 or 6 days. The total iron contents of the large brain and the cerebellum were measured by atomic absorption spectroscopy; 3 days of iron administration resulted in significant increases of iron contents only in the cerebellum, whereas after 6 days, both parts of the brain showed considerably higher iron contents. Electron microscopic point-counting morphometric analysis revealed a 65% increase of volume density of the lipofuscin in the large cells of parietal cortex after 6 days of iron-treatment. The Purkinje cells displayed a particularly frequent occurrence of an apoptosis-like structural alteration under the effect of iron. This kind of experimental approach is complicated by the facts that (i) the increase of OH radical flux above a certain level is lethal, and (ii) the young animals have a very active elimination mechanism of the waste products. Nevertheless, the enhancement of OH radical yield by an increased availability of iron for Fenton reaction resulted in an accumulation of lipofuscin even in young rats.

Cytochemical studies on the fibroblast-preadipocyte relationships in cultured fibroblast cell lines.

The theoretical basis of adipogenesis has always been a matter of debate. One concept suggests that all types of adipocytes are derived from undifferentiated connective tissue cells, whereas another concept suggests that adipocytes develop from specialized cells only that are able to accumulate fat. Many conflicting data have been published with respect to the transition of fibroblasts into preadipocytes. For example, this transition has been declared as impossible for dermal and perimysial fibroblasts. The present study analysed spontaneous accumulation of fat in various types of fibroblasts from different origin (retroocular, skin, NIH/3T3, and L929). It was found that intense Oil Red O-positive triglyceride-containing droplets accumulated in practically all types of fibroblasts provided that the cells were cultured on glass surface. When the cells were cultured on plastic surfaces, lipid staining was inhibited in a variable manner: inhibition was virtually complete in skin fibroblasts, whereas in other types of fibroblasts, inhibition was only partial. It is concluded that all types of fibroblasts can accumulate fat spontaneously, and thus can be considered as preadipocytes. Therefore, interpretations of data obtained with cultures of fibroblasts with respect to adipogenesis have to be reconsidered.

On the useful role of OH free radicals in differentiation of cultured human fibroblasts

The working hypothesis assuming that oxygen free radicals cannot be considered only as harmful by-products of the oxidative metabolism has been experimentally tested. Human fibroblasts were grown in culture from the following five types of tissues: (1) normal orbital fat; (2) orbital fat of patients with endocrine ophtalmopathy (EOP); (3) normal orbital muscle; (4) orbital muscle of EOP patients; (5) skin. These fibroblasts (second to 12th passages) were treated for 2x72 h with the Fenton reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.055 mM), final concentrations. This treatment caused a slowing down of the cell proliferation, induced various morphological signs of differentiation, and significantly increased (40-150%) the total superoxide dismutase (SOD) and catalase (CAT) activities of the fibroblasts. Authors suggest that the increased expression of these enzymes may play a general role in the cell differentiation mechanisms, meaning that the generation of oxygen free radicals is an essential, useful factor even during the early phases of development, and may not be taken only as a harmful process during aging.

Pharmacological Interventions against Aging through the Cell Plasma Membrane

As was shown in a recent review by this author (Ann. N.Y. Acad. Sci., 928:187‐199, 2001), oxyradicals cannot be considered only as harmful by‐products of the oxidative metabolism, but living cells and organisms implicitly require their production. This idea is supported by numerous facts and arguments, the most important of which is that the complete inhibition of the oxyradical production by KCN (or by any block of respiration) kills the living organisms long before the energy reserves would be exhausted. This new theoretical approach not only helps our understanding of the normal functions of the living organisms, such as the basic memory mechanisms in the brain cells, but also helps in identifying the site‐specific, radical‐induced damaging mechanisms that represent the undesirable side effects of oxygen free radicals. First of all, these effects make the cell plasma membrane vulnerable and cause a series of intracellular functional disorders, as described by the membrane hypothesis of aging (MHA). The logical way for any antiaging intervention therefore should be to increase the available number of loosely bound electrons inside the plasma membrane that are easily accessible for OH• free radical scavenging. The present review summarizes the available knowledge regarding the theory of the use of membrane‐related antiaging pharmaca, like centrophenoxine (CPH), tested in both animal experiments and human clinical trials. A modified, developed version of CPH coded as BCE‐001 is also reported.

Enzyme activities in the light of the membrane hypothesis of aging: [An answer to K. Kitani, Mech. Ageing Dev. 107 (1999) 299–322]

The paper of Kitani cited in the title has raised an apparent contradiction regarding the validity of certain aspects of the membrane hypothesis of aging (MHA). He collected data showing that a number of detoxifying liver enzyme activities, although decline with age in male Fischer 344 rats, remain at an unchanged level in females of the same strain. He concluded that the main assumption of the MHA, according to which intracellular enzyme activities generally decline with age, cannot be maintained, and invoked me (p. 312) ellipsis to provide in the future ample (and convincing) evidence in this respect. The present paper answers this criticism by showing that the apparent contradiction mentioned above is based on a misunderstanding on behalf of Kitani. Namely, MHA speaks about the general, density-dependent decline of the catalytic rate constant of any enzyme (k(cat)), i.e., activity per mole of enzyme, being the true specific activity of the enzymes. This parameter inevitably decreases at the increased physical density of the intracellular colloids during aging. This statement derives from the molecular enzyme kinetic models, and has extensively been proven experimentally, too. On the other hand, Kitani speaks about enzyme activities per mg total protein content of certain tissue extracts, which is a very illdefined parameter, since the concentration of the measured enzyme remains unknown. Therefore, this latter parameter is irrelevant from the point of view of MHA in any aspect.

The biological waste product formation in the light of the membrane hypothesis of aging.

The membrane hypothesis of aging (MHA) explains the biological waste product (lipofuscin) formation as a disbalance between the rates of protein synthesis and damage, as well as of elimination of the damaged components. Although, this concept has not been refuted on the basis of any experimental evidence, it has neither been widely accepted. During the last decade the general interest has turned toward the molecular genetics so intensely, that research aimed at clarifying cell biological mechanisms became so to say hibernated. Nowadays it is being recognized more and more that after the complete description of the human genetic code, attention has to be dedicated again to the cellular mechanisms explaining the function of the gene products (proteins). In this context, our experimental findings described during the recent years may become again the subject of interest. We have shown that the in vivo inhibition of the lysosomal thiol-proteinase functions by sublethal doses of leupeptin in young, adult and old mice results in a considerable increase (about 30%) of the immobile fraction of membrane proteins in hepatocyte plasma membrane, meanwhile the lateral diffusion constant of the still mobile membrane proteins increased. These observations were interpreted as signs of a general slowing down of protein turnover in the plasma membrane, just by inhibiting the elimination mechanisms in the lysosomes. This paper will discuss the theoretical conclusions and significance of these findings for the biological waste product formation, as a basic cell biological function.

The involvement of hydroxyl free radicals in differentiation of the PC-12 rat pheochromocytoma cell line.

These experiments tested the differentiation properties of the PC-12 cell line under conditions of in vitro generation of OH&z.rad; free radicals by Fenton reaction. This involves the simultaneous addition of the following reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.025 mM), final concentrations. Superoxide dismutase activity, the increase of which is considered as a marker of differentiation, catalase and glutathione peroxidase enzyme activities were investigated, which all displayed significant increases after single and repeated interventions with hydroxyl free radicals, while the cell number remained nearly at the starting-value. It is known that the differentiation takes place when the cell number has reached a plateau. These data, therefore, suggest that hydroxyl free radicals can induce in vitro cell differentiation, and that they play a more complex role in cell physiology than simply causing oxidative damages. It is interesting that the cells can maintain high levels of these enzyme activities for a relatively long time (2 or 4 days) after a very short flux of hydroxyl free radicals.

Comparative study of antibodies that are associated with disease progression in HIV disease

Two types of antibodies which previously were found to be inversely associated with CD4+ cell counts and which may contribute to the progression of HIV disease were measured in parallel in 55 serum samples of 7 longitudinally tested HIV-infected patients (4 homosexual men, 3 haemophilic men) and in 15 serum samples from 15 patients with advanced AIDS. HIV-infection enhancing antibodies were determined in the presence of near-physiologic human complement concentration using a complement receptor type 2 (CR2) carrying HIV-target cell line. IgG and IgA class autoantibodies directed against human IgG-Fab fragments were measured in specific ELISA assays. In agreement with our previous studies obtained in HIV-seropositive haemophilic patients, significant negative correlations were found between CD4+ cell counts and IgG anti-Fab and IgA anti-Fab antibodies (Spearman correlation coefficient r = -0.587, P < 0.0001; and r = -0.269, P = 0.024, respectively). A significant positive correlation was observed between complement-dependent enhancing antibodies and IgA anti-Fab antibodies (r = 0.408, P = 0.003), whereas the correlation with IgG anti-Fab antibodies was only weak (r = 0.288, P = 0.034). Serum samples with high titres of complement-dependent enhancing antibodies had almost 3 times higher IgA anti-Fab autoantibody activity than sera with low titres (P = 0.0038). Our findings indicate that the two disease markers in HIV disease, enhancing antibodies and autoantibodies directed against the Fab moiety of IgG, are not identical. However, anti-Fab antibodies may contribute to complement-dependent HIV infection enhancement.

Is consensus in anti-aging medical intervention an elusive expectation or a realistic goal?

One of the biggest scandals of the recent history of medicine is the conflict of views between the gerontological establishment and the American Academy of Anti-Aging Medicine (A4M). The style used in that discussion was really rough and unusual. On the one hand, according to some representatives of the American Medical Associations (AMA), the use of human growth hormone (hGH) for anti-aging medical interventions is illegal, criminal, and requires persecution. On the other hand, A4M is of the opinion that all this is ...filled with incorrect, misplaced references and studies, and multiple basic scientific errors, in an apparent attempt to damage the anti-aging medical profession.... It is evident that in the frame of a short article is impossible to treat all the relevant aspects of this complicated story. Nevertheless, this Editorial attempts to point out the main results obtained so far, together with the most important issues of theoretical feasibility of the hGH replacement therapy (hGHRT). The comprehensive explanation of the aging process called membrane hypothesis of aging (MHA) offers a solid basis for the interpretation of the observed beneficial effects of the hGH through its practically ubiquitous membrane receptors, and the species specificity of this peptide hormone. The specific activation of these receptors stimulates the membrane transport functions, rehydrates the intracellular colloids, allowing to speed up the protein synthesis and turnover, and activates a great number of cellular functions, all observed so far. The facts known about the adult growth hormone deficiency (AGHD) syndrome, and the beneficial effects of hGHRT in all aspects of this pathology suggest that aging may generally be considered as an AGHD syndrome. If this concept is accepted by most of the gerontologists, we can resolve practically all problems involved in the above outlined controversies. All this requires an independent, open-minded approach to the problem, and pushes us to a better understanding of the results of theoretical aging research. This approach may open a new, realistic way to the development of efficient anti-aging medical interventions.