The anti-DNA story

The presence of DNA-binding antibodies in the serum of patients with lupus was reported by three independent groups in 1957, from Milan, New York and Paris. ^{1 – 4} Science does not operate in a vacuum, and there was a slender thread that linked the three discoveries: the youngest and least experienced of all the researchers involved. Still a medical student, I unwittingly played this role, and since I have a vivid memory of my first assignment, I have agreed to write down how all of this happened.

In Milan, third-year medical students could apply to become ‘interns’ in a University Department. In 1953 I filed my application to the Medical Clinic. The applicants exceeded the openings: when I was accepted I felt like I had won the lottery, and reported to the Laboratory of Hematology. Head of the Lab was Elio Polli, one of the senior Assistant Professors. A pleasant but strong personality, years later he would be called upon to succeed the legendary Luigi Villa as Director of the Clinic, and number one internist in town. However, his powerful influence on the first steps of my career came from a visionary conviction of his: the solution of the leukemia riddle was in the DNA, and the hopeless patients that crowded his infirmary could only be saved if he could determine what the difference was between normal and leukemic DNA; that is, what went wrong in the latter.

Each intern student had four beds to tend to in the morning, but immediately after the ‘visit’ (a procession-and-discussion of cases by the Professor and his Assistants) I ran to work in the lab. My assignment was to purify DNA, from any source we could lay our hands on: calf thymus from the slaughterhouse, leukocytes from the blood bank and, of course, and foremost, cells of different types of leukemia from patients bedded in the clinic. I worked singlehandedly and my equipment was state of the art, directly imported from the USA following the specifications of Erwin Chargaff ⁵ (to whom Polli had paid regular visits in Chicago): four Beckman centrifuges, looking like flying saucers on tripods (that had to be run in the cold room); a Waring Blender (which had been the most difficult piece to obtain because nobody in Italy knew what it was, nor that it could be replaced by a local piece of kitchenware); and a custom-made precipitation chamber with a rotating arm to collect threads of pure white DNA materializing in −10°C absolute ethanol. As soon as a preparation was done, I delivered it to a team of young doctors in the next room; they seemed very competent and a bit status conscious (several of them – DiMayorca, Semenza, Basilico – would become internationally known basic scientists), but their analysis was crude: they measured the viscosity of DNA solutions at different pH. The probability of discovering meaningful differences by this method was low, and reflected what was then known (this was the epoch of the tetranucleotide model of DNA, years before the double helix became the consensus paradigm): hopelessly little. Polli realized it: he struggled and searched for new, more discriminating ways to fulfill his mission.

In the Spring of 1955, the opportunity for a 2-month studentship from the French government landed in the University of Milan. The Rector sent it to Prof Villa, and Prof Villa asked his Assistants to find the student who had worked hardest: it was me. I was asked which Institution I wanted to be sent to. That week Polli was traveling in the States, so I said I needed some time to decide: the time allotted was short … Paris had many areas of excellence in medicine … Cardiology? Pharmacology? Psychiatry? Why not visit all three? Back from Chicago, Polli stormed in like a Fury: “You are going to Pasteur! I just saw some pictures where antibodies can distinguish between similar antigens! Antibodies are the answer, and you are going to learn all the techniques from Professor Grabar!” I saw his light. Broken-hearted (my girl had just left me), I was more than ready to go. Polli asked his colleague in Biology, Prof Ranzi, who had once invited Grabar to give a seminar, to write him a letter on my behalf, and at the end of October I was on the night train to ‘la Ville Lumière’.

My arrival at Pasteur was unexpected. Pierre (Piotr Petrovic) Grabar had not received any letter. “And had I received it, I would have answered no, we have no openings!” Then he looked at me, and must have seen a lot in my face. After a pause, he said: “But I realize it is not your fault, and I know by experience how difficult it is to begin … ” (I learned later that as an officer of the White Russian Army he had fled for his life in 1917, and had worked long years as a chemist in the mines of northern France before emerging at Pasteur.) “I will keep you, and you will work in the lab of Maxime Seligmann.” Young Doctor Seligmann accepted me unwillingly: he resented being coerced by his boss, and made this point perfectly clear for an entire month, before we finally became friends.

I learned all of the immunoprecipitation techniques, from the simple ring test, to Oudin’s linear diffusion in gel (which allowed counting of the immune complexes formed), to the Ouchterlony two-dimensional precipitation plates (revealing different antibodies and different antigens), and finally to Grabar’s own immunoelectrophoresis. It took four months instead of two. My generous father thought it was a good investment. And four months at Pasteur was sufficient to catch an incurable virus: I would become an immunologist at any cost, even if I had to give up a doctor’s career. (Both events eventually happened.)

On the last day of my stay, Françoise Seligmann told her husband to bring me to dinner à la maison. Maxime drove me in his 2-Chevaux, but before reaching home he stopped at the roadside and asked a lot of questions, which I answered as best I could: “What is this DNA that you always mention?” (Well … ) “Why is it important?” (Polli says … ) “Are you sure it will elicit antibodies?” (I wasn’t … ) “How can one purify it?” (I am the expert … ) “What is the Molecular Weight?” (Many millions … ). Lupus was not mentioned then, but we were both aware that it was a goldmine for autoantibodies, and that antibodies against the nucleus had been described in lupus sera.

Back in Milan, I needed an animal facility to house the rabbits I was going to inject with DNA. The medical clinic was not the right place. Polli called human geneticist Ruggero Ceppellini: he agreed to let me work afternoons at the Istituto Sieroterapico Milanese, a private institution on the edge of town.

I traveled miles on my faithful 48cc 4-stroke Motom (a jewel for its time), and injected into eighteen rabbits DNA of various sources, with and without Freund’s adjuvant. Months passed. My girl called me. Her art studio was halfway between the clinic and the lab. The Motom began making an extra stop. Some inconsistent shadows appeared and disappeared in the agar gels, and the conclusion became slowly clear: DNA was not immunogenic., perhaps because it was perceived as ‘self.' A great deception: I had invested a lot in this project on which I planned to write my thesis.

But one morning a new patient appeared by chance in one of my four beds. I took his history. It was a severe lupus. That afternoon I brought a sample of his serum to the Sieroterapico. The same evening I tried a ring test: layering a DNA solution on a series of dilutions of the serum. The white precipitation at the interface looked like a slow-motion explosion in the side-illuminated tubes.

Over the following days I tried all variations, and immediately realized that all DNAs were precipitated, including calf, dog and bacterial DNAs. This killed any hope of applying this serum to the leukemia project. Another impossibility was to utilize my sophisticated gel precipitation methods: the DNA had a molecular weight in the millions, way too large to diffuse in the agar gel! Ceppellini, who had been skeptical about the rabbit work, became instantly interested when he saw my lupus precipitations. He ordered his faithful technician, Mario Turri, to perform complement fixation, which turned out positive, thus corroborating the immune nature of the reaction.

Very few days had passed when Ceppellini came to see me and introduced “a famous immunologist of New York!”. I was just looking at a ring test, and was happy and proud to show it to Henry Kunkel. He smiled approvingly, patting me on the shoulder, and said “Did you try DNase?” – moving his fingers to mime the distribution of salt crystals on food. “No, not yet, but it is an excellent idea … ”

DNase-treated DNA was not precipitated by my lupus serum. Kunkel presented our paper ¹ (a translation of our first account in an Italian Bulletin ² ) to the Proceedings of the Society of Experimental Biology and Medicine, where it appeared back-to-back with the account from Kunkel’s group. ³ Seligmann sent his paper to Compte Rendus de l’Academie des Sciences de Paris. ⁴ Most protagonists of this story are no longer with us. However, Polli – active and well at age 95 – read the manuscript, and we had an emotional meeting in Milano on 19 November 2010.