Abstract
The scientific community mourns the passing of an outstanding scientist, a wonderful human being, and a dear friend
With Chris, one of the great personalities of endotoxin research has left us.
Chris shaped, inspired and moved the field of endotoxins, the LPSs and O-antigens of Gram-negative bacteria, and their interaction with higher organisms in various dimensions. His profound scientific and methodological contributions had—and still have—an impact on almost everyone concerned with the field of bacterial infection, inflammation and immunity.
Chris Galanos was born on 23 April 1937 in Larnaca, Cyprus. After completing his education at the local lyceum, he moved to London, where he studied at the University of London, receiving a General Certificate of Education in Chemistry, Biology, Physics, and Mathematics in 1958. He was first employed by Martin R. Pollock at the National Institute for Medical Research in Mill Hill (London), working on the synthesis of new penicillins and the induction of penicillinases in Staphylococcus aureus.
During these early years of his career, Chris had to acknowledge that his professional status as a technician did not allow him to be scientifically independent and to publish his original data under his own name. He therefore looked for a laboratory that would grant him greater scientific freedom. For personal reasons, Chris wanted to work in Germany, where he found employment at the Max Planck Institute for Immunobiology in Freiburg, then headed by Otto Westphal, Otto Lüderitz and Herbert Fischer. The Max Planck Institute in general, and the laboratory of Otto Lüderitz in particular, were considered the ‘Mecca of endotoxin research’ and known for providing an atmosphere that embodied the motto of the French revolution—liberty, equality and fraternity.
Chris resumed his work in Freiburg on 1 January 1964.
Chris was a hands-on wet chemistry experimenter who loved to play with the system he worked on. Endotoxins were an ideal and challenging object for biochemical, biological, serological and physical experimentation because of their multifaced physicochemical properties, their manifold harmful, as well as beneficial, biological activities and, in particular, their complex chemical nature. Chemically, LPSs are amphipathic molecules that consist of a hydrophilic (polysaccharide) and lipophilic (lipid A) portion and, at the same time, they are amphoteric, carrying both negatively and positively charged groups.
For their isolation, the phenol–water procedure had been elaborated by the fathers of endotoxin research, Otto Westphal and Otto Lüderitz (the ‘two Ottos’) in the early 1950s. This procedure, however, was poorly suited for the extraction of LPS from rough (R) mutants. One of Chris's earliest pioneering activities after joining the laboratory of Otto Lüderitz was therefore the development of a new and powerful method for isolating R-form LPS from R mutants. This procedure, involving phenol–chloroform–light petroleum (PCP), turned out to yield LPS preparation largely free of protein and nucleic acids and exhibiting good solubility in water. It became a standard for the extraction of R-form LPS in the global endotoxin research community.
Solubility of LPS in water was generally a difficult to overcome problem for researchers working with endotoxin, particularly in the case of R-form LPS. EDTA dialysis helped occasionally, but not reproducibly. Chris reasoned that solubility was determined to a large extent by bivalent cations (Mg2+, Ca2+) and basic amines (putrescine, cadaverine) present in LPS samples. In an ingenious approach, he developed a procedure, termed electrodialysis, which removed ions from the original LPS preparation, yielding the respective LPS as a free acid. This acidic form of LPS could then, by adding a particular base, be converted to a defined salt form. Chris showed that the triethylamine salt of LPS was particularly soluble in water (in contrast to the Ca2+ salt), a finding causing researchers worldwide to add triethylamine to their milky (R-form) LPS samples, which miraculously cleared up and exhibited enhanced endotoxicity.
Using defined salt forms, Chris could differentiate various biological LPS effects. For example, the triethylamine salt of LPS exhibited potent acute toxicity and no complement-activating activity, whereas the Ca2+ form activated complement but was of low lethality. Chris thereby demonstrated that the physico-chemical properties were an important factor determining bioactivity of LPS.
Solubility, or rather the state of aggregation of LPS, was also a problem in the case of the lipoidal component of LPS, i.e. free lipid A devoid of the LPS polysaccharide portion. The lipid A molecule was a particular focus of scientific research, as it had been postulated by Westphal and Lüderitz to represent the endotoxic principle of LPS. It was difficult to prove this hypothesis because of the poor solubility of free lipid A in water. Chris succeeded in overcoming this problem by complexing free lipid A with a water-soluble carrier such as albumin. Lipid A/albumin complex then proved to be of similar potency as LPS, thereby supporting the concept of lipid A representing the toxic component of LPS. This concept was finally proven through the chemical synthesis of lipid A by Shoichi Kusumoto and Tetsuo Shiba (Osaka, Japan), and the subsequent biological analysis of synthetic preparations. In these analyses, Chris played an essential role as he showed, for the first time, that bacterial and synthetic lipid A exhibited identical bioactivity in various endotoxin test systems. These data unequivocally showed that lipid A and not some other molecule possibly present in bacterial LPS or lipid A preparations was responsible for endotoxicity.
This result was of utmost importance as it indicated that, in the search for a cellular receptor of endotoxin, one had to look for a structure interacting with the lipid A portion of LPS. This cellular receptor was later identified by Bruce Beutler (La Jolla, CA, USA) as TLR4, a finding that won him the Nobel Prize for Medicine in 2011. Chris kept a close scientific relationship and personal friendship with Bruce. Together, they published the essential data on the identification of the LPS/lipid A receptor as TLR4 in a highly cited paper (Science 1998; 282: 2085–2088).
They had found that the TLR4 gene was mutated in the C3H/HeN mouse strain (somewhat resistant to LPS toxicity) and deleted in the C57BL/10ScCr strain, which had been shown by Chris to be completely resistant to LPS toxicity. The latter strain was used to generate transgenic mice carrying only mouse or human TLR4. Experiments with transgenic mice confirmed the essential role of TLR4 in the induction of endotoxicity. Furthermore, these strains helped to recognize that Ni2+ is a ligand of human but not of murine TLR4 and that this property explains why Ni2+ is a highly potent contact allergen for humans.
This instrumental mouse strain (C57BL/10 ScCr) also played an important role in work on the presensitization of animals towards endotoxin or free lipid A. It was known from earlier work by Louis Chedid (Paris) that the sensitivity of mice to LPS could be enhanced by pretreatment with BCG, but Chris extended this observation by showing that infections, in general, whether bacterial, viral or parasitic in origin, significantly lowered the dose necessary for (lethal) LPS toxicity. The most dramatic sensitizing effect, however, was caused by pretreating the animals with
Antibodies to lipid A, the toxic center of LPS, were a highly attractive research topic in the early 1970s, as it could be expected that they would neutralize endotoxic effects. In these years, antibodies to lipids were rarely described, and the immunogenicity of lipids, as well as their antigenicity, remained doubtful. Thus, the chances of generating antibodies to lipid A were estimated to be low. Chris remained undeterred by this pessimistic view and used the experimental approach of immunizing rabbits with free lipid A adsorbed to erythrocytes. He thereby engendered antibodies to lipid A, as determined by passive hemolysis. Later, he used acid-treated Gram-negative bacteria, which expose lipid A as an immunogen, and obtained even higher titers of lipid A antibodies. The discovery of lipid A antibodies was, at the time, a notable accomplishment. Unfortunately, lipid A antibodies reacted only with (polysaccharide deprived) free lipid A, i.e. they did not cross-react with LPS. Furthermore, they showed protective activity against lipid A bioactivity only under peculiar experimental conditions. Lipid A serology, however, turned out to be a powerful tool in immunochemistry, where, for example, it allowed the identification of unknown samples as LPS after removal of the polysaccharide part by hydrolysis. Lipid A antibodies were also increasingly used in studies concerning the biosynthesis of lipid A and LPS. Interestingly, although the production of lipid A antibodies is not induced by intact LPS, low titers of anti-lipid A are present in sera of healthy humans and higher titers in sera of patients with urinary tract infections. In collaboration with Martin Westenfelder (Freiburg), Chris demonstrated the damaging properties of lipid A antibodies experimentally in dogs. Such antibodies may contribute to inflammatory lesions in patients with chronic bacterial infections.
There are many other fields in which Chris contributed to our knowledge on LPS biology and biochemistry, including MDP-mediated sensitization of mice, clinical application of LPS to tumor-bearing patients, B-cell mitogenicity (together with Fritz Melchers, Basel), and the pathogenesis of contact allergies. Here, the remarkable discovery was made (together with Stefan Martin and Matthias Goebeller, Freiburg) that TLR4 and TLR2 are involved in the sensitization to contact allergens and that Ni2+ directly activates TLR4, thereby leading to TNF-α release and local inflammation.
On 5 February 1974, Chris defended his doctoral thesis, entitled ‘Activité biologique des lipopolysaccharides et du lipid A’, at the Faculté des Sciences d'Orsay, Université de Paris-Sud, France. The three professors taking the final examination were Edgar Lederer, Louis Chedid and Otto Lüderitz. They rated his thesis ‘summa cum laude’, the highest grade to be awarded. The doctoral title he received at the time symbolized the transformation of a technician, whose work was perhaps mentioned in an acknowledgement of a publication, to an internationally recognized scientist who published his data under his own name. Over 250 original peer reviewed papers and reviews now carry his name.
Also in 1974, Marina Freudenberg, a postdoc at the time and herself a passionate researcher, joined Chris's team. From then on, the two worked together as an inseparable unit. They built an ideal partnership of a biochemically and a medically oriented research-team, in addition to becoming life partners and eventually spouses.
Chris was honored for his various scientific contributions by receiving the Frederic Bang Award, a lifelong honorary membership of the International Endotoxin Society, visiting professorships at the University of Bari (Italy) and the University of Sao Paolo (Brazil), as well as an honorary doctorate by the Second Military Medical University of Shanghai (China).
Chris had the characteristics of an experimental genius who generously shared his knowledge and data with everyone who showed an interest in his work. He liked to question and re-examine his own scientific results as much as those of others, and gave little regard to supposedly established theories and mainstream scientific trends. His enthusiasm for science was contagious. He engaged his many doctoral students and postdoctoral fellows from all over the world in discussions, often at late-night hours, usually smoking one of his beloved cigarettes, patiently suggesting approaches to experimentation without, however, insisting on a particular methodology or strategy.
For his co-workers, Chris was a master, but he was a partner, a modest person who rarely pushed to be in the spotlight. In those days at the Max Planck Institute, selling oneself was considered unnecessary to be recognized as a remarkable scientist in daily scientific life. Rather, a hands-on style of doing science prevailed, which lived off the free exchange of ideas, social openness, collegial respect and active friendship. The three directors of the Institute themselves set an example by creating an atmosphere that motivated scientists to deliver exceptional results—an atmosphere that spurred competition but at the same time cooperation and unity among the members of the Institute. It was this environment that allowed Chris to express his social qualities and to finally develop his intellectual capabilities to their full potential. ‘Amicable science’ was the secret of the Max Planck Institute and of the success of its researchers, including Chris.
Chris kept a vivid interest in the science of endotoxins and innate immunity long after he had retired from his position at the Institute in 2002. He continued working experimentally as a highly welcome ‘volunteer’ in Marina’s lab until 2012 and for 2 more years at the ‘Staatliche Weinbauinstitut’ in Freiburg. Until his last days, he discussed with Marina ongoing research and new data from her laboratory.
Chris carried his illness, an advanced, metastasized cancer, with patience and dignity. He passed away peacefully on 9 July 2015.
Berlin, 18.8.2015
Ernst Th. Rietschel