An Interview with Professor Manuel Varela: Cholera and Complementing the Antibodies

Sep 5, 2018 by

Jules Bordet

Michael F. Shaughnessy –

1) Professor Varela, we have all heard the ominous word “cholera”.  What exactly does this word mean and what are the signs and symptoms?

The term cholera refers to a particular type of infectious disease which is caused by a microbe that is scientifically named Vibrio cholerae. The cholera-causing microbe is a bacterium.

The severity level of the cholera disease in patients can vary tremendously.  On the one hand, some patients may lack signs and symptoms, being asymptomatic, while others may experience mild or severe gastrointestinal disease.  On the other hand, cholera can be quite serious in that the disease may actually be lethal to the patient. Certain human patients are particularly susceptible to the cholera, such as the very young or the elderly. 

The vast majority of individuals who have encountered the Vibrio cholerae bacterium experience no symptoms or have mild diarrhea. However, if the disease is serious, the patient may rapidly take a downturn and experience severe symptomology or perhaps even die because of the cholera. 

Clinical disease involving cholera can commence typically around 2 to 3 days after the patient consumes the microbe, the cholera-causing Vibrio cholerae.  Sometimes, the symptoms can arise quickly, shortly after exposure to the bacteria, perhaps even just a few hours after the microbe-person encounter befalls.

The main classical symptom of the cholera is the so-called “rice water stool.” That is, the diarrhea has the appearance of the type of supernatant suspension that occurs when cooking rice.  Almost always, there is no actual rice in the cholera-laden rice water stool—it merely LOOKS like it does so, and it is nonetheless the classic hallmark of cholera. The stool is watery with bits of intestinal mucous in it plus the patient’s electrolytes. The loss of a patient’s water and electrolytes can be serious.  The patient can get dehydrated rather quickly, and their muscles may experience cramping, sometimes becoming severe in their intensities.

Additionally, the patient can suffer a condition known as metabolic acidosis as a result of the loss of their bicarbonate molecules, affecting their pH levels in the body. The loss of the important electrolyte ion, potassium, can lead to the patient suffering from hypokalemia (i.e., potassium levels in the blood are too low) and may even undergo hypovolemic shock. Eventually the patient can succumb to heart arrhythmia and kidney failure, especially if the patient does not receive prompt medical treatment. Untreated cases of severe cholera can result in almost 70% of the patients dying from the ailment.  However, with treatment, the cholera mortality rates can be drastically reduced to percentages on the level of about 1%.

2) How is cholera transmitted, and is there a standard treatment?

The principal mode of cholera transmission to patients is via the fecal-oral route.  This particular transmission mode involves the patient having consumed water or food that is contaminated with fecal matter harboring Vibrio cholerae bacteria, such as, perhaps, from another cholera patient, or from sewage, in certain cases.

Thus, cholera may be endemic in regions where public sanitation is sub-standard. Furthermore, the incidences of cholera may rise in epidemic proportions during natural disasters, such as during flooding or in earthquakes, in which sewage is released into adjacent areas in the affected communities.

The World Health Organization and the Centers for Disease Control and Prevention have estimated that on an annual basis approximately 3 – 5 million cholera cases occur with an estimated annual mortality numbering in the hundreds of thousands of lives lost.

Infrequently, worldwide occurrences of cholera cases have occurred, i.e., pandemics.  Historically speaking, the world has experienced at least seven major cholera pandemics. These great pandemics of cholera, in turn, have resulted in seriously profound alterations in human socioeconomic statuses.

Quite often, the cholera patient will have consumed a large quantity of the Vibrio cholerae bacteria in order to acquire the illness, because many of the microbes will have succumbed to the low pH effects of the stomach acid during their transit to the lower gastrointestinal tract of the patient. Therefore, the inoculum number of the cholera-causing microorganisms that are consumed prior to illness onset may need to be as large as 100 million individual bacterial cells! 

With respect to the medical treatment of cholera, there are two approaches.  The first treatment approach is the preferred one in which the fluids and electrolytes that are lost by the patient are resupplied back into the patient.  This first-choice method of treatment is often referred to as replacement therapy. This replenishment of fluids and electrolytes requires that it be invoked immediately or as soon as is possible, before the more serious conditions involving cardiac and kidney systems commence.

A second-choice method of cholera treatment, less preferred, involves antimicrobial agents.  Modern antibiotic therapy involves the medication called azithromycin, an inhibitor of bacterial protein synthesis. This antimicrobial agent is usually the first medicine of choice, especially in patients who are young. Other medicines may involve doxycycline or ciprofloxacin. The doxycycline is also a protein synthesis inhibitor. The ciprofloxacin agent is known to inhibit the synthesis of bacterial DNA.

3) Now what does cholera have to do with complementing antibodies? And Jules Bordet?

In short, Dr. Jules Bordet discovered that antibodies and complement work together to kill the causative agent of cholera, the so-called Vibrio cholerae bacterium.

In the laboratory, Dr. Bordet found that blood serum antibodies taken from laboratory animals that had been inoculated with cholera bacteria could destroy these same bacteria.  Importantly, he discovered that the antibodies in the blood serum needed help in order to do so. That is, Dr. Bordet found that when the body produces specific antibodies in response to the cholera bacteria, these so-called anti-cholera antibodies needed assistance from another immune system-based process, called the complement system, in order for the antibodies to neutralize or destroy the invading cholera bacteria.

Dr. Bordet had originally called this antibody-complementing system “alexine,” or alexin, after learning that Dr. Hans Buchner had coined the term from Greek, meaning “to defend.”  Dr. Paul Ehrlich later changed the term alexin to the word “complement,” a term that is still used in modern times.

Additionally, Dr. Bordet found that if the blood serum came from a laboratory animal that had not been inoculated with the cholera bacteria, the cholera antibody complementation could still function to result in the lysis (killing by bursting) of the cholera microbe cells, as long as the blood serum was not heat-treated beforehand.  The complement factors were susceptible to heat, whereas the antibodies were heat-resistant but could not kill the cholera bacteria without an intact complement system in place. 

Bordet even found that the heat-susceptible complement could come from an animal that had not even been purposefully exposed to the cholera bacteria. The antibodies could kill the bacteria with the assistance of Bordet’s complement factor, and it did not seem to matter whether the complement had come from cholera-injected animals.  The main requirement, of course, was that the complement was not heated beforehand. Otherwise, complement that was treated by heat destroyed the antibody-assisting effect.

While not completely understood in molecular and cellular terms, even in modern times, we do know basically, however, that the complement system presently has three starting points. The complement initiating pathways are called the classical system, the alternative (or properdin) system, and the lectin system.  Independent of its initiation mechanism pathway, one of the main end-results is the lysis of the invading pathogenic bacteria, including the microbes of cholera. 

We also know that the complement system uses a cascade amplification system during its process. In the complement cascade mechanism, a given intact and inactive complement component will become activated by its breakage into two active parts.  Each of these active parts, in turn, will act on other inactive components by cleaving them, as well, to turn them on. Each of these sliced complement factors have certain physiological functions of their own.

In the complement activation series, the cascading process reaches a crescendo in which the infecting bacteria are burst open, killing them. The assembly of a large molecular based membrane-attack complex, known as the MAC, can accomplish the bacterial killing process. I help my students to remember this mechanism by calling it the “big MAC!”

The MAC structure is a giant pore, or opening, that is made in the cell wall of the bacterium and which allows the leakage of the bacterial cytoplasmic contents. An intact cytoplasm is required for the life of the bacteria. Without its internal contents, the bacterium will die.

In a related fashion, if the target cells of the MAC are eukaryotic in nature, the pores can allow entry into the cytoplasm certain death factors, called apoptotic or programmed cell death molecules that result in the self-killing of the target eukaryotic cells. This intrinsic cell death mechanism can occur for cells that are damaged or infected with pathogens and in cancer cells.

Complement activation has other physiological consequences that occur in the body. In addition to bacterial lysis by the MACs mentioned above, the complement system will turn on a process called opsonization, which is a molecular coating system that makes certain coated-antigens become targets for enhanced phagocytosis. Another physiological consequence of complement activation is inflammation of affected tissues or organ systems. A last physiological outcome considered here is a cellular activity called chemotaxis, in which specific mobile cells will migrate to certain body locations that have been invaded by a pathogen or are perhaps damaged in some traumatic way.

4) Who exactly WAS Jules Bordet, when did he live and what kinds of discoveries and experiments was he involved with?

Jules Jean Baptiste Vincent Bordet was born on the 13th day of the month of June, in 1870, in a small village called Soignies, Belgium. His parents were Charles-Henri Thomas Joseph Bordet and Marie Therese Celestine Vandenabeele, his mother. The child Jules attended elementary school where his father had been a schoolteacher at the Ecole Moyenne de Schaerbeek, located in a suburban neighborhood of Brussels.

In 1880, Jules moved to the Royal Athenaeum of Brussels, a secondary school where it is reported that he had had a keen interest in chemistry. Apparently, he was known to have constructed a homemade makeshift chemistry laboratory for himself. His parents and teachers had early on recognized Jules as being extremely intelligent.

Then, at the age of 16 years, Jules entered medical school at the University of Brussels, in the Faculty of Medicine, earning his medical doctorate degree with honors in 1892, at the age of 22 years. During the years spent in his medical studies, Bordet had worked in the research laboratories of Profs. Paul Heger and Abraham Leo Errera, the latter laboratory of which he studied the microbe known as Vibrio metschnikovii, in which he found that the microorganisms could be made increasingly pathogenic in animals when repeatedly passaged during culturing in the laboratory.

While working as a staff physician in Middelkerke, Dr. Bordet received a research grant in 1894 to continue his studies at the prestigious Pasteur Institute in Paris, France. Upon his arrival to the Institute of Pasteur, Dr. Bordet began working under the famous Prof. Elie (Ilya) Ilyich Metchnikoff, discoverer of the phagocytes. It is here at the Pasteur Institute where Dr. Bordet conducted his studies of alexine and cholera antibodies.

In 1899, Dr. Bordet married Marthe Lovoz, and together they had three children. One of their progeny, named Paul, in later years went on to become the director at the Pasteur Institute in Brussels, picking up the post left by his father, Jules.

In 1901, Dr. Jules Bordet moved back to Brussels in order to establish an Institute of Pasteur there and become its first director. Then, in 1907, he became professor of bacteriology and pathology at the University of Brussels, also known as the Free University.

After his arrival, Dr. Bordet studied children who suffered from the malady called whooping cough. The respiratory disease, now known as pertussis, was a serious ailment in Bordet’s time. He was able to isolate and culture the causative bacterial agent from the young patients who had the illness. One source reported that one of Bordet’s whooping cough patients was his own son Paul, who had come down with the disease in 1906.

In any case, Dr. Bordet followed up on the causative agent discovery and noticed that when repeatedly cultured in the laboratory the microbe tended to lose its dependence on blood for its growth. The pathogenic microbe of pertussis has been named Bordetella pertussis, in honor of its famous discoverer.

Other work by Dr. Bordet includes the observation he made in 1905 of a spirochete-shaped bacterium located within skin lesions of syphilis.  However, Drs. Eric Hoffmann and Fritz Schaudinn had beaten Bordet by a few months, publishing their largely similar work first. In 1906, Dr. Bordet also discovered the bacterium that causes the bird version of the diphtheria.

In another project, he had discovered a new substance, a type of anaphylatoxin, that conferred a serious medical condition called anaphylaxis. He had speculated that the substance may have something to do with his complement system.  Indeed, in the early 1990s, Dr. Bordet’s prediction came true with the discovery of several such complement-specific agents, called in modern times, C3a and C5a. These complement proteins play roles in the cascade system inherent the physiological activation and in mediating anaphylaxis.

In 1919, Dr. Bordet became a Nobel Laureate in the fields of medicine or physiology for his work with complement and immunity. Dr. Bordet passed away on the 6th of April, in 1961, at the age of 90 years.

5) What exactly is the disease known as the whooping cough or pertussis, and how is it treated or prevented?

Pertussis is a serious airborne respiratory illness with significant morbidity and mortality rates worldwide, affecting mainly children but also recently adults. The signs and symptoms in patients of the whooping cough occur in three phases. 

The first phase is called the catarrhal stage, and it involves classic common cold-like symptoms, lasting 1 or 2 weeks in duration.

The second phase of pertussis is called the paroxysmal stage. This second stage involves primarily the classic whooping cough phenomenon, lasting about 2 to 4 weeks. Here, the patient coughs repeatedly for an extended period of time, making the patient feel that he or she has depleted the air from the lungs. The patient will then attempt to bring their lungs back to their normal air-filled state by inhaling quite rapidly, making a loud “whooping” sound that is characteristic of the ailment. This paroxysm phenomenon can be a disturbingly dramatic situation for parents to witness in their children with the affliction. 

The third and last phase of pertussis is called the convalescent stage, and it involves largely a recovery period lasting approximately 3 to 4 weeks, sometimes longer. While this latter phase involves a recovery from the disease with diminished symptomology, it can sometimes also involve a risk from secondary infections with other pathogenic microbes.

Modern treatment consists of hospitalization with supportive nursing care and anti-bacterial medicines. The prime choice currently includes the so-called macrolide antibiotics called azithromycin or clarithromycin.

Prevention includes two vaccines, one for the young and the other for adults. Both vaccines contain a disabled form of the pertussis toxin, plus other disarmed bacterial components called filamentous hemagglutinin and pertactin. 

6) Why is the work of Jules Bordet so important? And how does his work live on, so to speak?

Dr. Bordet’s work indeed lives on today in all major textbooks that deal with medical infectious disease, microbiology and immunology. First, his complement system is considered a major arm of the innate immune system.  The topic is often given its very own chapter in medical textbooks. The system involves major proteins, cell systems, physiological processes, and helps the other arm of the immune system, called specific or adaptive immunity. Without Bordet’s complement, the antibody response might very well be impossible to take place.  It plays a major role in protection from many pathogenic microbes, such as the cholera or the pertussis bacteria, as well as from cancer.

Because of Dr. Bordet’s development of the complement fixation test, clinicians are able to readily detect microbial infections in patients. Dr. Bordet’s work on this invention is still given wide consideration in clinical laboratories worldwide. The test is used both in basic and applied biomedical science research in modern times.

In addition, Dr. Bordet’s discovery of the causative microbe for the whooping cough has led to the development of a vaccine for the illness. Therefore, the pertussis remains a relatively rare occurrence in developed countries, but outbreaks may occur due to recent loss of enthusiasm for vaccination. In 2012, it was reported that over 40,000 cases of pertussis occurred in the U.S. On a worldwide basis, pertussis is endemic in many areas, especially where vaccination is lacking. Current data estimates that over 16 million cases of whooping cough occur annually on a global scale, with about 200,000 yearly fatalities. 

Clearly, much work remains if cholera and pertussis are ever to be controlled.  Education of the populace will be key.

7) What have I neglected to ask about these topics?

Because of Dr. Bordet’s work with cholera and pertussis, modern investigators have worked out the cellular mechanisms of the cholera toxin and of the pertussis toxin. Furthermore, as I mentioned above the basic cellular and physiological mechanisms for the functioning and regulation of the complement system have been elucidated. Lastly, much work remains to be conducted with respect to modulation of antibody activity and in learning how anti-microbe specific antibodies work to neutralize and eliminate infectious pathogens and cancer.

It may interest our readers to learn that Dr. Bordet occasionally clashed with another prominent investigator, Dr. Paul Ehrlich, whom you’ll recall was famous for having formulated the magic bullet theory. It seems that Dr. Bordet was at odds with Ehrlich’s incorrect side-chain theory, and Dr. Ehrlich was equally at odds with Bordet’s incorrect colloidal theory of antibody-antigen binding.  

Print Friendly, PDF & Email

Leave a Reply

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.