Paul Ehrlich and O.T. Avery: pathfinders in the search for immunity.

This paper is concerned with the use of passive immunity for the prevention and treatment of infections from a historic perspective, particularly in regard to the research of Paul Ehrlich on diphtheria and that of O.T. Avery on pneumonia. It is timely to reexamine this matter, particularly in regard to virus infections, in view of the difficulties in developing antiviral drugs. In addition, specific antibodies can serve as an alternative therapy for antibiotic resistant microbes. It is worth recalling in the history of vaccine development that success in passive immunity has been the key element in devising a successful strategy to develop a vaccine to produce humoral immunity.

Ivermectin: a positive allosteric effector of the alpha7 neuronal nicotinic acetylcholine receptor.

We report that preapplication of ivermectin, in the micromolar range, strongly enhances the subsequent acetylcholine-evoked current of the neuronal chick or human alpha7 nicotinic acetylcholine receptors reconstituted in Xenopus laevis oocytes and K-28 cells. This potentiation does not result from nonspecific Cl- currents. The concomitant increase in apparent affinity and cooperativity of the dose-response curve suggest that ivermectin acts as a positive allosteric effector. This interpretation is supported by the observation of an increase in efficiency of a partial agonist associated with the potentiation and by the differential effect of ivermectin on mutants within the M2 channel domain. Ivermectin effects reveal a novel allosteric site for pharmacological agents on neuronal alpha7 nicotinic acetylcholine receptors.

Summary of antibody workshop: The Role of Humoral Immunity in the Treatment and Prevention of Emerging and Extant Infectious Diseases.

In the era before antibiotics, human diseases were commonly treated with immune animal and human sera, often with life-saving results. With the advent of emerging infectious diseases, many of which cannot be adequately treated or prevented, attempts to develop antibody treatments have taken on new importance. The role of humoral immunity in treatment and prevention was the focus of discussion at a 1996 workshop. The cellular and molecular mechanisms of neutralization were examined in detail. It was noted that success in passive immunity has frequently been the key element in devising a successful strategy to develop a vaccine for active immunization. The workshop concluded on a cautious note of optimism that antibody-based treatment and prevention for diseases such as human immunodeficiency virus infection, Ebola fever, and others of clinical and public health importance deserve further development and clinical trial.

Electrophysiology: a method to investigate the functional properties of ligand-gated channels.

Ligand-gated channels (LGCs) play a fundamental role in the fast transmission of electrical activity from neuron to neuron and/or to effector cells. Studies of LGCs in isolation have become possible since the identification of genes coding for these membrane proteins together with the establishment of reconstitution techniques in host systems. Methods for electrophysiological investigations of LGCs reconstituted either in the Xenopus oocytes or stably tranfected in cell lines are discussed. Functional studies of reconstituted receptors enable fast determination of LGCs' pharmacological profiles and comparison of their physiological properties. Combination of molecular engineering with physiological measurements allows studies with unpreceeding resolution and it is now possible to examine at the amino-acid level the contribution of some residues in the formation of the ligand-binding site or the ionic channel domains.

Reflections on the first decade of the HIV/AIDS pandemic: opportunities and priorities for international behavioural research and interventions.

AIDS, a sexually transmitted disease, is unlikely to respond in the near term to either common sense or scientific innovation. Other forces are at play here that embrace the psychobiology of reproduction, addictive behaviour, and the social sciences. We must explore these matters within the historical and cultural context. We must push to the limit the science of behaviour and behaviour modification. To meet this challenge, the social and behavioural sciences and the natural sciences must form a new union in which the sum is greater than the parts. The integration of population biology into research strategies will require special attention. Planning the design and employment of interventions and the evaluation of their effects will require the use of mathematical models. Efforts to change individual and social behaviour must be tailored to the cultural circumstances of the afflicted populations. None of this will be easy. But the changing sexual behaviour among homosexual men and the increasing use of condoms by prostitutes and their customers in many societies stand as testimony to the power of public efforts.

Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

1. Fusion of myogenic cells is important for muscle growth and repair. The aim of this study was to examine the possible involvement of nicotinic acetylcholine receptors (nAChR) in the fusion process of myoblasts derived from postnatal human satellite cells. 2. Acetylcholine-activated currents (ACh currents) were characterized in pure preparations of freshly isolated satellite cells, proliferating myoblasts, myoblasts triggered to fuse and myotubes, using whole-cell and single-channel voltage clamp recordings. Also, the effect of cholinergic agonists on myoblast fusion was tested. 3. No nAChR were observed in freshly isolated satellite cells. nAChR were first observed in proliferating myoblasts, but ACh current densities increased markedly only just before fusion. At that time most mononucleated myoblasts had ACh current densities similar to those of myotubes. ACh channels had similar properties at all stages of myoblast maturation. 4. The fraction of myoblasts that did not fuse under fusion-promoting conditions had no ACh current and thus resembled freshly isolated satellite cells. 5. The rate of myoblast fusion was increased by carbachol, an effect antagonized by alpha-bungarotoxin, curare and decamethonium, but not by atropine, indicating that nAChR were involved. Even though a prolonged exposure to carbachol led to desensitization, a residual ACh current persisted after several days of exposure to the nicotinic agonist. 6. Our observations suggest that nAChR play a role in myoblast fusion and that part of this role is mediated by the flow of ions through open ACh channels.

Dynamics of emergence.

I have touched briefly here on the complex matrix of social, economic, political, and ecologic factors that have played a major role in the emergence of microbial diseases. But beyond these factors that contribute to the emergence of new infectious diseases, we must also recognize changes in microbial agents, human populations, insect vectors, and the ecologic relationships among them. Microbes and vectors swim in the evolutionary stream and they swim much faster than we do. Bacteria reproduce every 30 min; for them a millennium is compressed into a fortnight. Microbes were here, learning every trick for survival, 2 billion years before humans arrived, and it is likely that they will be here 2 billion years after we depart. Furthermore, science cannot halt the future occurrence of new microbes, which emerge from the evolutionary stream as a consequence of genetic events and selective pressures that favor the new over the old. It is nature's way. For all of these reasons, old and new infections will occur in the future as they have in the past. Surveillance efforts, both in the United States and other regions of the world, will be needed to blunt the emergence of such infections and to forestall epidemics and pandemics. But surveillance alone cannot detect the unexpected emergence of future microbes or prepare the defense against them. That will require a broadly based research effort to devise new methods of diagnosis, treatment, and prevention. We must swim with the microbes and study their survival and adaptation to new habitats.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium and potassium currents in freshly isolated and in proliferating human muscle satellite cells.

1. Human muscle satellite cells (SC) were studied either immediately after dissociation of muscle biopsies or later, as they proliferated in culture. A purification procedure combined with clonal cultures ensured that electrophysiological recordings were done in myogenic cells. Hoechst staining for the DNA attested that cells were mononucleated. 2. The goals of this study were to examine (i) whether the electrophysiological properties of freshly isolated SC resembled those of SC that proliferated in culture for several weeks, (ii) whether freezing and thawing affected these properties, and (iii) whether SC constituted a homogeneous population. 3. We found that there were only subtle differences between the electrophysiological results obtained in freshly isolated SC and in proliferating SC with or without previous freezing and thawing. Most SC expressed two voltage-gated currents, a TTX-resistant Na+ current and a calcium-activated potassium current (IK, Ca). 4. The level of expression of the Na+ current and of IK, Ca was affected in a different way by cellular proliferation; the normalized Na+ conductance (pS pF-1) of proliferating cells resembled that of freshly isolated SC, whereas the IK, Ca conductance increased 10 times. The analysis of the amplitude distributions of the Na+ current and of IK, Ca in the various SC preparations suggested that there was only one class of SC.

A voltage-dependent proton current in cultured human skeletal muscle myotubes.

1. A voltage-dependent proton current, IH, was studied in cultured myotubes obtained from biopsies of human muscle, using whole-cell recording with the patch-clamp technique. 2. With a pHo of 8.0 and a calculated pHi of 6.3, IH was activated at voltages more depolarized than -50 mV and its conductance reached its maximum value at voltages more depolarized than +10 mV. 3. Studies of the reversal potential of IH during substitution of K+, Na+, Ca2+, Cl-, Cs+ and H+ in the extracellular solution indicated that protons were the major charge carriers of IH. 4. IH was also activated during a voltage step to +22 mV with a pHo of 7.3 and a calculated pHi of 7.3. 5. Acidification of the extracellular solution led to a shift towards depolarized voltages of the conductance-voltage relationship. 6. Stationary noise analysis of IH suggested that the elementary event underlying IH was very small with a conductance of less than 0.09 pS. 7. Extracellular application of various divalent cations blocked IH. The block by divalent cations was voltage dependent, being more efficient at hyperpolarized than at depolarized voltages. For Cd2+, the Michaelis-Menten constant (Km) for the block was 0.6 microM at -28 mV and 10.4 microM at +12 mV. 8. Ca2+ was a less efficient blocker than Cd2+ but could block IH at physiological concentrations (the Km values for the block were 0.9 mM at -38 mV and 7.3 mM at -8 mV). 9. The voltage-dependent properties of IH and its ability to be affected by pH and Ca2+ suggest that IH might be used by skeletal muscle cells to extrude protons during action potentials. 10. A model of IH activation suggests that under extreme conditions, the conductance of IH can reach 40% of its maximum value after less than ten action potentials.

Human skeletal muscle has a voltage-gated proton current.

A voltage-gated proton current, IH, was studied with the whole-cell patch-clamp technique in human myotubes obtained from biopsies of human muscle. Studies of the reversal potential of IH during substitution of K+, Na+, Ca2+, Cl-, Cs+, and H+ in the extracellular solution indicated that protons were the major charge carriers of IH. This current is similar in many respects, but not identical, to the proton currents already described in other cell types. IH is activated by depolarization and it can be affected by extracellular pH. IH can be blocked by external divalent cations including Ca2+. This block is voltage-dependent, being more efficient at hyperpolarized than at depolarized voltages. The voltage-dependent properties of IH and its ability to be affected by pH and extracellular Ca2+ suggest that IH might be used by muscle cells to extrude protons during action potentials.

Purification of human muscle satellite cells by flow cytometry.

To purify satellite cells directly from human muscle biopsies, we have developed a method based on size separation of dissociated cells by flow cytometry. Immediately after tryptic dissociation of human muscle biopsies and elimination of erythrocytes, microscopic observation and flow cytometry analysis of cell suspensions revealed two populations of cells differing in size and nucleocytoplasmic ratio. Clonal cultures of these two cell types with a manual procedure demonstrated that only the small cells were myogenic satellite cells. Flow cytometry-sorting and analysis of the small cell population showed that (1) all sorted cells contained desmin immediately after dissociation and plating; (2) more than 98% of the cells expressed the 5.1.H11 epitope after 2 weeks of proliferation in culture; and (3) 90% of the sorted cells were able to form myotubes when cultivated at low density or in clonal cultures. Thus, human muscle satellite cells can be directly purified from human muscle samples using flow cytometry.

The origin of plagues: old and new.

Viruses and bacteria emerge in new and old forms to cause disease epidemics. Some microorganisms recur when changing life-styles (including increased international travel) offer new opportunities; others arise from new genetic variations. These various epidemics connect the future with the past, offering lessons for guarding the health of generations to come--lessons learned from diseases such as tuberculosis, toxic shock syndrome, Lyme disease, streptococcal infection, influenza, and acquired immunodeficiency syndrome (AIDS). The public must be vigilant to the possibility of new epidemics, learn more about the biology and epidemiology of microbes, and strengthen systems of surveillance and detection.

Syphilis during 1900-1910: similarities to present-day AIDS.

At the beginning of the 20th century, despite 20 years of intensive bacteriologic research, the cause of syphilis was unknown; no diagnostic test and no treatment had been found. Syphilis was one of the leading causes of morbidity and mortality, and those who had the disease were burdened with a social stigma. It was considered a disease of "bad blood." But success was soon to follow. In only 10 years, from 1900 to 1910, the Treponema pallidum was discovered as the cause of syphilis. Animal models were developed for research. The Wassermann test was "invented" for serologic diagnosis, and Paul Ehrlich proved that salvarsan, or 606, was effective for the treatment of syphilis. This success was preceded by 300 failures with related arsenical compounds. The scientific, medical, social, ethical, and economic issues of that day have recurred again with the AIDS epidemic. This earlier drama, therefore, is reflected in the current decade, but the success of Ehrlich, Wassermann, and others in the fight against syphilis is an optimistic omen that researchers will be equally successful in the fight against acquired immune deficiency syndrome (AIDS).

"The beginning of health is to know the disease".

There is public impatience over the pace of medical progress. Some say prevention and health have not been well served by the research community. Rather than devising extended investigations, scientists should apply now what we know now. Activists argue that, although research on a better understanding of disease must continue, a companion effort to develop strategies for health promotion and disease prevention should exist. The national effort should emphasize "health" and not "disease," as the names of the various NIH Institutes would imply. I disagree with that proposed direction of prevention research. It is not possible to divorce research on health from research on disease. Are the secrets of nature open to us through mere observation? Does not research require the perturbation of a system in order to make valid observations on the nature of that system? This, after all, is the nature of the scientific method. Disease is, itself, a perturbation of the state of health and it is through our research on disease that we learn how to prevent it. I believe that the National Institutes of Health does devote equal time to the study of health. For it is my thesis that by studying disease we have, in fact, given our total time to the study of health. "The beginning of health is to know the disease."